In-depth proteomic analysis of Varroa destructor: Detection of DWV-complex, ABPV, VdMLV and honeybee proteins in the mite.

PubMed

Erban, Tomas; Harant, Karel; Hubalek, Martin; Vitamvas, Pavel; Kamler, Martin; Poltronieri, Palmiro; Tyl, Jan; Markovic, Martin; Titera, Dalibor

2015-09-11

We investigated pathogens in the parasitic honeybee mite Varroa destructor using nanoLC-MS/MS (TripleTOF) and 2D-E-MS/MS proteomics approaches supplemented with affinity-chromatography to concentrate trace target proteins. Peptides were detected from the currently uncharacterized Varroa destructor Macula-like virus (VdMLV), the deformed wing virus (DWV)-complex and the acute bee paralysis virus (ABPV). Peptide alignments revealed detection of complete structural DWV-complex block VP2-VP1-VP3, VDV-1 helicase and single-amino-acid substitution A/K/Q in VP1, the ABPV structural block VP1-VP4-VP2-VP3 including uncleaved VP4/VP2, and VdMLV coat protein. Isoforms of viral structural proteins of highest abundance were localized via 2D-E. The presence of all types of capsid/coat proteins of a particular virus suggested the presence of virions in Varroa. Also, matches between the MWs of viral structural proteins on 2D-E and their theoretical MWs indicated that viruses were not digested. The absence/scarce detection of non-structural proteins compared with high-abundance structural proteins suggest that the viruses did not replicate in the mite; hence, virions accumulate in the Varroa gut via hemolymph feeding. Hemolymph feeding also resulted in the detection of a variety of honeybee proteins. The advantages of MS-based proteomics for pathogen detection, false-positive pathogen detection, virus replication, posttranslational modifications, and the presence of honeybee proteins in Varroa are discussed.

In-depth proteomic analysis of Varroa destructor: Detection of DWV-complex, ABPV, VdMLV and honeybee proteins in the mite

PubMed Central

Erban, Tomas; Harant, Karel; Hubalek, Martin; Vitamvas, Pavel; Kamler, Martin; Poltronieri, Palmiro; Tyl, Jan; Markovic, Martin; Titera, Dalibor

2015-01-01

We investigated pathogens in the parasitic honeybee mite Varroa destructor using nanoLC-MS/MS (TripleTOF) and 2D-E-MS/MS proteomics approaches supplemented with affinity-chromatography to concentrate trace target proteins. Peptides were detected from the currently uncharacterized Varroa destructor Macula-like virus (VdMLV), the deformed wing virus (DWV)-complex and the acute bee paralysis virus (ABPV). Peptide alignments revealed detection of complete structural DWV-complex block VP2-VP1-VP3, VDV-1 helicase and single-amino-acid substitution A/K/Q in VP1, the ABPV structural block VP1-VP4-VP2-VP3 including uncleaved VP4/VP2, and VdMLV coat protein. Isoforms of viral structural proteins of highest abundance were localized via 2D-E. The presence of all types of capsid/coat proteins of a particular virus suggested the presence of virions in Varroa. Also, matches between the MWs of viral structural proteins on 2D-E and their theoretical MWs indicated that viruses were not digested. The absence/scarce detection of non-structural proteins compared with high-abundance structural proteins suggest that the viruses did not replicate in the mite; hence, virions accumulate in the Varroa gut via hemolymph feeding. Hemolymph feeding also resulted in the detection of a variety of honeybee proteins. The advantages of MS-based proteomics for pathogen detection, false-positive pathogen detection, virus replication, posttranslational modifications, and the presence of honeybee proteins in Varroa are discussed. PMID:26358842

Accumulation of sediment-associated viruses in shellfish.

PubMed Central

Landry, E F; Vaughn, J M; Vicale, T J; Mann, R

1983-01-01

The present study focused on the importance of contaminated sediments in shellfish accumulation of human viruses. Epifaunal (Crassostrea virginica) and infaunal (Mercenaria mercenaria) shellfish, placed on or in cores, were exposed to either resuspended or undisturbed sediments containing bound poliovirus type 1 (LSc 2ab). Consistent bioaccumulation by oysters (four of five trials) was only noted when sediment-bound viruses occurred in the water column. Virus accumulation was observed in a single instance where sediments remained in an undisturbed state. While the incidence of bioaccumulation was higher with resuspended rather than undisturbed contaminated sediment, the actual concentration of accumulated viruses was not significantly different. The accumulation of viruses from oysters residing on uninoculated sediments. When clams were exposed to undisturbed, virus-contaminated sediments, two of five shellfish pools yielded viral isolates. Bioaccumulation of undisturbed sediments by these bivalves was considered marginal when related to the concentration of virus in contaminated sediments; they would only represent a significant threat when suspended in the water column. Arguments were advanced for water-column sampling in the region of the water-sediment interface to provide an accurate determination of the virological quality of shellfish harvesting waters. PMID:6297392

Subcellular targeting and interactions among the Potato virus X TGB proteins.

PubMed

Samuels, Timmy D; Ju, Ho-Jong; Ye, Chang-Ming; Motes, Christy M; Blancaflor, Elison B; Verchot-Lubicz, Jeanmarie

2007-10-25

Potato virus X (PVX) encodes three proteins named TGBp1, TGBp2, and TGBp3 which are required for virus cell-to-cell movement. To determine whether PVX TGB proteins interact during virus cell-cell movement, GFP was fused to each TGB coding sequence within the viral genome. Confocal microscopy was used to study subcellular accumulation of each protein in virus-infected plants and protoplasts. GFP:TGBp2 and TGBp3:GFP were both seen in the ER, ER-associated granular vesicles, and perinuclear X-bodies suggesting that these proteins interact in the same subdomains of the endomembrane network. When plasmids expressing CFP:TGBp2 and TGBp3:GFP were co-delivered to tobacco leaf epidermal cells, the fluorescent signals overlapped in ER-associated granular vesicles indicating that these proteins colocalize in this subcellular compartment. GFP:TGBp1 was seen in the nucleus, cytoplasm, rod-like inclusion bodies, and in punctate sites embedded in the cell wall. The puncta were reminiscent of previous reports showing viral proteins in plasmodesmata. Experiments using CFP:TGBp1 and YFP:TGBp2 or TGBp3:GFP showed CFP:TGBp1 remained in the cytoplasm surrounding the endomembrane network. There was no evidence that the granular vesicles contained TGBp1. Yeast two hybrid experiments showed TGBp1 self associates but failed to detect interactions between TGBp1 and TGBp2 or TGBp3. These experiments indicate that the PVX TGB proteins have complex subcellular accumulation patterns and likely cooperate across subcellular compartments to promote virus infection.

Ludwik Gross, Sarah Stewart, and the 1950s discoveries of Gross murine leukemia virus and polyoma virus.

PubMed

Morgan, Gregory J

2014-12-01

The Polish-American scientist Ludwik Gross made two important discoveries in the early 1950s. He showed that two viruses - murine leukemia virus and parotid tumor virus - could cause cancer when they were injected into susceptible animals. At first, Gross's discoveries were greeted with skepticism: it seemed implausible that viruses could cause a disease as complex as cancer. Inspired by Gross's initial experiments, similar results were obtained by Sarah Stewart and Bernice Eddy who later renamed the parotid tumor virus SE polyoma virus after finding it could cause many different types of tumors in mice, hamsters, and rats. Eventually the "SE" was dropped and virologists adopted the name "polyoma virus." After Gross's work was published, additional viruses capable of causing solid tumors or blood-borne tumors in mice were described by Arnold Graffi, Charlotte Friend, John Moloney and others. By 1961, sufficient data had been accumulated for Gross to confidently publish an extensive monograph--Oncogenic Viruses--the first history of tumor virology, which became a standard reference work and marked the emergence of tumor virology as a distinct, legitimate field of study. Copyright © 2014 Elsevier Ltd. All rights reserved.

Viral Infection Induces Expression of Novel Phased MicroRNAs from Conserved Cellular MicroRNA Precursors

PubMed Central

Zhang, Jiayao; Zhao, Shuqi; Zheng, Hong; Gao, Ge; Wei, Liping; Li, Yi

2011-01-01

RNA silencing, mediated by small RNAs including microRNAs (miRNAs) and small interfering RNAs (siRNAs), is a potent antiviral or antibacterial mechanism, besides regulating normal cellular gene expression critical for development and physiology. To gain insights into host small RNA metabolism under infections by different viruses, we used Solexa/Illumina deep sequencing to characterize the small RNA profiles of rice plants infected by two distinct viruses, Rice dwarf virus (RDV, dsRNA virus) and Rice stripe virus (RSV, a negative sense and ambisense RNA virus), respectively, as compared with those from non-infected plants. Our analyses showed that RSV infection enhanced the accumulation of some rice miRNA*s, but not their corresponding miRNAs, as well as accumulation of phased siRNAs from a particular precursor. Furthermore, RSV infection also induced the expression of novel miRNAs in a phased pattern from several conserved miRNA precursors. In comparison, no such changes in host small RNA expression was observed in RDV-infected rice plants. Significantly RSV infection elevated the expression levels of selective OsDCLs and OsAGOs, whereas RDV infection only affected the expression of certain OsRDRs. Our results provide a comparative analysis, via deep sequencing, of changes in the small RNA profiles and in the genes of RNA silencing machinery induced by different viruses in a natural and economically important crop host plant. They uncover new mechanisms and complexity of virus-host interactions that may have important implications for further studies on the evolution of cellular small RNA biogenesis that impact pathogen infection, pathogenesis, as well as organismal development. PMID:21901091

Internal Disequilibria and Phenotypic Diversification during Replication of Hepatitis C Virus in a Noncoevolving Cellular Environment

PubMed Central

Moreno, Elena; Gallego, Isabel; Gregori, Josep; Lucía-Sanz, Adriana; Soria, María Eugenia; Castro, Victoria; Beach, Nathan M.; Manrubia, Susanna; Quer, Josep; Esteban, Juan Ignacio; Rice, Charles M.; Gómez, Jordi; Gastaminza, Pablo

2017-01-01

ABSTRACT Viral quasispecies evolution upon long-term virus replication in a noncoevolving cellular environment raises relevant general issues, such as the attainment of population equilibrium, compliance with the molecular-clock hypothesis, or stability of the phenotypic profile. Here, we evaluate the adaptation, mutant spectrum dynamics, and phenotypic diversification of hepatitis C virus (HCV) in the course of 200 passages in human hepatoma cells in an experimental design that precluded coevolution of the cells with the virus. Adaptation to the cells was evidenced by increase in progeny production. The rate of accumulation of mutations in the genomic consensus sequence deviated slightly from linearity, and mutant spectrum analyses revealed a complex dynamic of mutational waves, which was sustained beyond passage 100. The virus underwent several phenotypic changes, some of which impacted the virus-host relationship, such as enhanced cell killing, a shift toward higher virion density, and increased shutoff of host cell protein synthesis. Fluctuations in progeny production and failure to reach population equilibrium at the genomic level suggest internal instabilities that anticipate an unpredictable HCV evolution in the complex liver environment. IMPORTANCE Long-term virus evolution in an unperturbed cellular environment can reveal features of virus evolution that cannot be explained by comparing natural viral isolates. In the present study, we investigate genetic and phenotypic changes that occur upon prolonged passage of hepatitis C virus (HCV) in human hepatoma cells in an experimental design in which host cell evolutionary change is prevented. Despite replication in a noncoevolving cellular environment, the virus exhibited internal population disequilibria that did not decline with increased adaptation to the host cells. The diversification of phenotypic traits suggests that disequilibria inherent to viral populations may provide a selective advantage to viruses that can be fully exploited in changing environments. PMID:28275194

Water deficit enhances the transmission of plant viruses by insect vectors

PubMed Central

Yvon, Michel; Vile, Denis; Dader, Beatriz; Fereres, Alberto

2017-01-01

Drought is a major threat to crop production worldwide and is accentuated by global warming. Plant responses to this abiotic stress involve physiological changes overlapping, at least partially, the defense pathways elicited both by viruses and their herbivore vectors. Recently, a number of theoretical and empirical studies anticipated the influence of climate changes on vector-borne viruses of plants and animals, mainly addressing the effects on the virus itself or on the vector population dynamics, and inferring possible consequences on virus transmission. Here, we directly assess the effect of a severe water deficit on the efficiency of aphid-transmission of the Cauliflower mosaic virus (CaMV) or the Turnip mosaic virus (TuMV). For both viruses, our results demonstrate that the rate of vector-transmission is significantly increased from water-deprived source plants: CaMV transmission reproducibly increased by 34% and that of TuMV by 100%. In both cases, the enhanced transmission rate could not be explained by a higher virus accumulation, suggesting a more complex drought-induced process that remains to be elucidated. The evidence that infected plants subjected to drought are much better virus sources for insect vectors may have extensive consequences for viral epidemiology, and should be investigated in a wide range of plant-virus-vector systems. PMID:28467423

Water deficit enhances the transmission of plant viruses by insect vectors.

PubMed

van Munster, Manuella; Yvon, Michel; Vile, Denis; Dader, Beatriz; Fereres, Alberto; Blanc, Stéphane

2017-01-01

Drought is a major threat to crop production worldwide and is accentuated by global warming. Plant responses to this abiotic stress involve physiological changes overlapping, at least partially, the defense pathways elicited both by viruses and their herbivore vectors. Recently, a number of theoretical and empirical studies anticipated the influence of climate changes on vector-borne viruses of plants and animals, mainly addressing the effects on the virus itself or on the vector population dynamics, and inferring possible consequences on virus transmission. Here, we directly assess the effect of a severe water deficit on the efficiency of aphid-transmission of the Cauliflower mosaic virus (CaMV) or the Turnip mosaic virus (TuMV). For both viruses, our results demonstrate that the rate of vector-transmission is significantly increased from water-deprived source plants: CaMV transmission reproducibly increased by 34% and that of TuMV by 100%. In both cases, the enhanced transmission rate could not be explained by a higher virus accumulation, suggesting a more complex drought-induced process that remains to be elucidated. The evidence that infected plants subjected to drought are much better virus sources for insect vectors may have extensive consequences for viral epidemiology, and should be investigated in a wide range of plant-virus-vector systems.

A murine retrovirus co-Opts YB-1, a translational regulator and stress granule-associated protein, to facilitate virus assembly.

PubMed

Bann, Darrin V; Beyer, Andrea R; Parent, Leslie J

2014-04-01

The Gag protein of the murine retrovirus mouse mammary tumor virus (MMTV) orchestrates the assembly of immature virus particles in the cytoplasm which are subsequently transported to the plasma membrane for release from the cell. The morphogenetic pathway of MMTV assembly is similar to that of Saccharomyces cerevisiae retrotransposons Ty1 and Ty3, which assemble virus-like particles (VLPs) in intracytoplasmic ribonucleoprotein (RNP) complexes. Assembly of Ty1 and Ty3 VLPs depends upon cellular mRNA processing factors, prompting us to examine whether MMTV utilizes a similar set of host proteins to facilitate viral capsid assembly. Our data revealed that MMTV Gag colocalized with YB-1, a translational regulator found in stress granules and P bodies, in intracytoplasmic foci. The association of MMTV Gag and YB-1 in cytoplasmic granules was not disrupted by cycloheximide treatment, suggesting that these sites were not typical stress granules. However, the association of MMTV Gag and YB-1 was RNA dependent, and an MMTV RNA reporter construct colocalized with Gag and YB-1 in cytoplasmic RNP complexes. Knockdown of YB-1 resulted in a significant decrease in MMTV particle production, indicating that YB-1 plays a role in MMTV capsid formation. Analysis by live-cell imaging with fluorescence recovery after photobleaching (FRAP) revealed that the population of Gag proteins localized within YB-1 complexes was relatively immobile, suggesting that Gag forms stable complexes in association with YB-1. Together, our data imply that the formation of intracytoplasmic Gag-RNA complexes is facilitated by YB-1, which promotes MMTV virus assembly. Cellular mRNA processing factors regulate the posttranscriptional fates of mRNAs, affecting localization and utilization of mRNAs under normal conditions and in response to stress. RNA viruses such as retroviruses interact with cellular mRNA processing factors that accumulate in ribonucleoprotein complexes known as P bodies and stress granules. This report shows for the first time that mouse mammary tumor virus (MMTV), a mammalian retrovirus that assembles intracytoplasmic virus particles, commandeers the cellular factor YB-1, a key regulator of translation involved in the cellular stress response. YB-1 is essential for the efficient production of MMTV particles, a process directed by the viral Gag protein. We found that Gag and YB-1 localize together in cytoplasmic granules. Functional studies of Gag/YB-1 granules suggest that they may be sites where virus particles assemble. These studies provide significant insights into the interplay between mRNA processing factors and retroviruses.

The Potyviral P3 Protein Targets Eukaryotic Elongation Factor 1A to Promote the Unfolded Protein Response and Viral Pathogenesis1[OPEN

PubMed Central

Shine, M.B.; Cui, Xiaoyan; Chen, Xin; Ma, Na; Kachroo, Pradeep; Zhi, Haijan; Kachroo, Aardra

2016-01-01

The biochemical function of the potyviral P3 protein is not known, although it is known to regulate virus replication, movement, and pathogenesis. We show that P3, the putative virulence determinant of soybean mosaic virus (SMV), targets a component of the translation elongation complex in soybean. Eukaryotic elongation factor 1A (eEF1A), a well-known host factor in viral pathogenesis, is essential for SMV virulence and the associated unfolded protein response (UPR). Silencing GmEF1A inhibits accumulation of SMV and another ER-associated virus in soybean. Conversely, endoplasmic reticulum (ER) stress-inducing chemicals promote SMV accumulation in wild-type, but not GmEF1A-knockdown, plants. Knockdown of genes encoding the eEF1B isoform, which is important for eEF1A function in translation elongation, has similar effects on UPR and SMV resistance, suggesting a link to translation elongation. P3 and GmEF1A promote each other’s nuclear localization, similar to the nuclear-cytoplasmic transport of eEF1A by the Human immunodeficiency virus 1 Nef protein. Our results suggest that P3 targets host elongation factors resulting in UPR, which in turn facilitates SMV replication and place eEF1A upstream of BiP in the ER stress response during pathogen infection. PMID:27356973

UL31 and UL34 Proteins of Herpes Simplex Virus Type 1 Form a Complex That Accumulates at the Nuclear Rim and Is Required for Envelopment of Nucleocapsids

PubMed Central

Reynolds, Ashley E.; Ryckman, Brent J.; Baines, Joel D.; Zhou, Yuping; Liang, Li; Roller, Richard J.

2001-01-01

The herpes simplex virus type 1 (HSV-1) UL34 protein is likely a type II membrane protein that localizes within the nuclear membrane and is required for efficient envelopment of progeny virions at the nuclear envelope, whereas the UL31 gene product of HSV-1 is a nuclear matrix-associated phosphoprotein previously shown to interact with UL34 protein in HSV-1-infected cell lysates. For these studies, polyclonal antisera directed against purified fusion proteins containing UL31 protein fused to glutathione-S-transferase (UL31-GST) and UL34 protein fused to GST (UL34-GST) were demonstrated to specifically recognize the UL31 and UL34 proteins of approximately 34,000 and 30,000 Da, respectively. The UL31 and UL34 gene products colocalized in a smooth pattern throughout the nuclear rim of infected cells by 10 h postinfection. UL34 protein also accumulated in pleiomorphic cytoplasmic structures at early times and associated with an altered nuclear envelope late in infection. Localization of UL31 protein at the nuclear rim required the presence of UL34 protein, inasmuch as cells infected with a UL34 null mutant virus contained UL31 protein primarily in central intranuclear domains separate from the nuclear rim, and to a lesser extent in the cytoplasm. Conversely, localization of UL34 protein exclusively at the nuclear rim required the presence of the UL31 gene product, inasmuch as UL34 protein was detectable at the nuclear rim, in replication compartments, and in the cytoplasm of cells infected with a UL31 null virus. When transiently expressed in the absence of other viral factors, UL31 protein localized diffusely in the nucleoplasm, whereas UL34 protein localized primarily in the cytoplasm and at the nuclear rim. In contrast, coexpression of the UL31 and UL34 proteins was sufficient to target both proteins exclusively to the nuclear rim. The proteins were also shown to directly interact in vitro in the absence of other viral proteins. In cells infected with a virus lacking the US3-encoded protein kinase, previously shown to phosphorylate the UL34 gene product, UL31 and UL34 proteins colocalized in small punctate areas that accumulated on the nuclear rim. Thus, US3 kinase is required for even distribution of UL31 and UL34 proteins throughout the nuclear rim. Taken together with the similar phenotypes of the UL31 and UL34 deletion mutants, these data strongly suggest that the UL31 and UL34 proteins form a complex that accumulates at the nuclear membrane and plays an important role in nucleocapsid envelopment at the inner nuclear membrane. PMID:11507225

Ebola virus requires phosphatidylinositol (3,5) bisphosphate production for efficient viral entry.

PubMed

Qiu, Shirley; Leung, Anders; Bo, Yuxia; Kozak, Robert A; Anand, Sai Priya; Warkentin, Corina; Salambanga, Fabiola D R; Cui, Jennifer; Kobinger, Gary; Kobasa, Darwyn; Côté, Marceline

2018-01-01

For entry, Ebola virus (EBOV) requires the interaction of its viral glycoprotein with the cellular protein Niemann-Pick C1 (NPC1) which resides in late endosomes and lysosomes. How EBOV is trafficked and delivered to NPC1 and whether this is positively regulated during entry remain unclear. Here, we show that the PIKfyve-ArPIKfyve-Sac3 cellular complex, which is involved in the metabolism of phosphatidylinositol (3,5) bisphosphate (PtdIns(3,5)P 2 ), is critical for EBOV infection. Although the expression of all subunits of the complex was required for efficient entry, PIKfyve kinase activity was specifically critical for entry by all pathogenic filoviruses. Inhibition of PIKfyve prevented colocalization of EBOV with NPC1 and led to virus accumulation in intracellular vesicles with characteristics of early endosomes. Importantly, genetically-encoded phosphoinositide probes revealed an increase in PtdIns(3,5)P 2 -positive vesicles in cells during EBOV entry. Taken together, our studies suggest that EBOV requires PtdIns(3,5)P 2 production in cells to promote efficient delivery to NPC1. Copyright © 2017 Elsevier Inc. All rights reserved.

Vertical Transmission Selects for Reduced Virulence in a Plant Virus and for Increased Resistance in the Host

PubMed Central

Pagán, Israel; Montes, Nuria; Milgroom, Michael G.; García-Arenal, Fernando

2014-01-01

For the last three decades, evolutionary biologists have sought to understand which factors modulate the evolution of parasite virulence. Although theory has identified several of these modulators, their effect has seldom been analysed experimentally. We investigated the role of two such major factors—the mode of transmission, and host adaptation in response to parasite evolution—in the evolution of virulence of the plant virus Cucumber mosaic virus (CMV) in its natural host Arabidopsis thaliana. To do so, we serially passaged three CMV strains under strict vertical and strict horizontal transmission, alternating both modes of transmission. We quantified seed (vertical) transmission rate, virus accumulation, effect on plant growth and virulence of evolved and non-evolved viruses in the original plants and in plants derived after five passages of vertical transmission. Our results indicated that vertical passaging led to adaptation of the virus to greater vertical transmission, which was associated with reductions of virus accumulation and virulence. On the other hand, horizontal serial passages did not significantly modify virus accumulation and virulence. The observed increases in CMV seed transmission, and reductions in virus accumulation and virulence in vertically passaged viruses were due also to reciprocal host adaptation during vertical passages, which additionally reduced virulence and multiplication of vertically passaged viruses. This result is consistent with plant-virus co-evolution. Host adaptation to vertically passaged viruses was traded-off against reduced resistance to the non-evolved viruses. Thus, we provide evidence of the key role that the interplay between mode of transmission and host-parasite co-evolution has in determining the evolution of virulence. PMID:25077948

Viruses Roll the Dice: The Stochastic Behavior of Viral Genome Molecules Accelerates Viral Adaptation at the Cell and Tissue Levels

PubMed Central

Miyashita, Shuhei; Ishibashi, Kazuhiro; Kishino, Hirohisa; Ishikawa, Masayuki

2015-01-01

Recent studies on evolutionarily distant viral groups have shown that the number of viral genomes that establish cell infection after cell-to-cell transmission is unexpectedly small (1–20 genomes). This aspect of viral infection appears to be important for the adaptation and survival of viruses. To clarify how the number of viral genomes that establish cell infection is determined, we developed a simulation model of cell infection for tomato mosaic virus (ToMV), a positive-strand RNA virus. The model showed that stochastic processes that govern the replication or degradation of individual genomes result in the infection by a small number of genomes, while a large number of infectious genomes are introduced in the cell. It also predicted two interesting characteristics regarding cell infection patterns: stochastic variation among cells in the number of viral genomes that establish infection and stochastic inequality in the accumulation of their progenies in each cell. Both characteristics were validated experimentally by inoculating tobacco cells with a library of nucleotide sequence–tagged ToMV and analyzing the viral genomes that accumulated in each cell using a high-throughput sequencer. An additional simulation model revealed that these two characteristics enhance selection during tissue infection. The cell infection model also predicted a mechanism that enhances selection at the cellular level: a small difference in the replication abilities of coinfected variants results in a large difference in individual accumulation via the multiple-round formation of the replication complex (i.e., the replication machinery). Importantly, this predicted effect was observed in vivo. The cell infection model was robust to changes in the parameter values, suggesting that other viruses could adopt similar adaptation mechanisms. Taken together, these data reveal a comprehensive picture of viral infection processes including replication, cell-to-cell transmission, and evolution, which are based on the stochastic behavior of the viral genome molecules in each cell. PMID:25781391

Rewiring of cellular membrane homeostasis by picornaviruses.

PubMed

Belov, George A; Sztul, Elizabeth

2014-09-01

Viruses are obligatory intracellular parasites and utilize host elements to support key viral processes, including penetration of the plasma membrane, initiation of infection, replication, and suppression of the host's antiviral defenses. In this review, we focus on picornaviruses, a family of positive-strand RNA viruses, and discuss the mechanisms by which these viruses hijack the cellular machinery to form and operate membranous replication complexes. Studies aimed at revealing factors required for the establishment of viral replication structures identified several cellular-membrane-remodeling proteins and led to the development of models in which the virus used a preexisting cellular-membrane-shaping pathway "as is" for generating its replication organelles. However, as more data accumulate, this view is being increasingly questioned, and it is becoming clearer that viruses may utilize cellular factors in ways that are distinct from the normal functions of these proteins in uninfected cells. In addition, the proteincentric view is being supplemented by important new studies showing a previously unappreciated deep remodeling of lipid homeostasis, including extreme changes to phospholipid biosynthesis and cholesterol trafficking. The data on viral modifications of lipid biosynthetic pathways are still rudimentary, but it appears once again that the viruses may rewire existing pathways to generate novel functions. Despite remarkable progress, our understanding of how a handful of viral proteins can completely overrun the multilayered, complex mechanisms that control the membrane organization of a eukaryotic cell remains very limited. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Complex modulation of the Aedes aegypti transcriptome in response to dengue virus infection.

PubMed

Bonizzoni, Mariangela; Dunn, W Augustine; Campbell, Corey L; Olson, Ken E; Marinotti, Osvaldo; James, Anthony A

2012-01-01

Dengue fever is the most important arboviral disease world-wide, with Aedes aegypti being the major vector. Interactions between the mosquito host and dengue viruses (DENV) are complex and vector competence varies among geographically-distinct Ae. aegypti populations. Additionally, dengue is caused by four antigenically-distinct viral serotypes (DENV1-4), each with multiple genotypes. Each virus genotype interacts differently with vertebrate and invertebrate hosts. Analyses of alterations in mosquito transcriptional profiles during DENV infection are expected to provide the basis for identifying networks of genes involved in responses to viruses and contribute to the molecular-genetic understanding of vector competence. In addition, this knowledge is anticipated to support the development of novel disease-control strategies. RNA-seq technology was used to assess genome-wide changes in transcript abundance at 1, 4 and 14 days following DENV2 infection in carcasses, midguts and salivary glands of the Ae. aegypti Chetumal strain. DENV2 affected the expression of 397 Ae. aegypti genes, most of which were down-regulated by viral infection. Differential accumulation of transcripts was mainly tissue- and time-specific. Comparisons of our data with other published reports reveal conservation of functional classes, but limited concordance of specific mosquito genes responsive to DENV2 infection. These results indicate the necessity of additional studies of mosquito-DENV interactions, specifically those focused on recently-derived mosquito strains with multiple dengue virus serotypes and genotypes.

Phosphatidic Acid Produced by Phospholipase D Promotes RNA Replication of a Plant RNA Virus

PubMed Central

Hyodo, Kiwamu; Taniguchi, Takako; Manabe, Yuki; Kaido, Masanori; Mise, Kazuyuki; Sugawara, Tatsuya; Taniguchi, Hisaaki; Okuno, Tetsuro

2015-01-01

Eukaryotic positive-strand RNA [(+)RNA] viruses are intracellular obligate parasites replicate using the membrane-bound replicase complexes that contain multiple viral and host components. To replicate, (+)RNA viruses exploit host resources and modify host metabolism and membrane organization. Phospholipase D (PLD) is a phosphatidylcholine- and phosphatidylethanolamine-hydrolyzing enzyme that catalyzes the production of phosphatidic acid (PA), a lipid second messenger that modulates diverse intracellular signaling in various organisms. PA is normally present in small amounts (less than 1% of total phospholipids), but rapidly and transiently accumulates in lipid bilayers in response to different environmental cues such as biotic and abiotic stresses in plants. However, the precise functions of PLD and PA remain unknown. Here, we report the roles of PLD and PA in genomic RNA replication of a plant (+)RNA virus, Red clover necrotic mosaic virus (RCNMV). We found that RCNMV RNA replication complexes formed in Nicotiana benthamiana contained PLDα and PLDβ. Gene-silencing and pharmacological inhibition approaches showed that PLDs and PLDs-derived PA are required for viral RNA replication. Consistent with this, exogenous application of PA enhanced viral RNA replication in plant cells and plant-derived cell-free extracts. We also found that a viral auxiliary replication protein bound to PA in vitro, and that the amount of PA increased in RCNMV-infected plant leaves. Together, our findings suggest that RCNMV hijacks host PA-producing enzymes to replicate. PMID:26020241

Subcellular distribution of swine vesicular disease virus proteins and alterations induced in infected cells: A comparative study with foot-and-mouth disease virus and vesicular stomatitis virus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Martin-Acebes, Miguel A.; Gonzalez-Magaldi, Monica; Rosas, Maria F.

2008-05-10

The intracellular distribution of swine vesicular disease virus (SVDV) proteins and the induced reorganization of endomembranes in IBRS-2 cells were analyzed. Fluorescence to new SVDV capsids appeared first upon infection, concentrated in perinuclear circular structures and colocalized to dsRNA. As in foot-and-mouth disease virus (FMDV)-infected cells, a vesicular pattern was predominantly found in later stages of SVDV capsid morphogenesis that colocalized with those of non-structural proteins 2C, 2BC and 3A. These results suggest that assembly of capsid proteins is associated to the replication complex. Confocal microscopy showed a decreased fluorescence to ER markers (calreticulin and protein disulfide isomerase), and disorganizationmore » of cis-Golgi gp74 and trans-Golgi caveolin-1 markers in SVDV- and FMDV-, but not in vesicular stomatitis virus (VSV)-infected cells. Electron microscopy of SVDV-infected cells at an early stage of infection revealed fragmented ER cisternae with expanded lumen and accumulation of large Golgi vesicles, suggesting alterations of vesicle traffic through Golgi compartments. At this early stage, FMDV induced different patterns of ER fragmentation and Golgi alterations. At later stages of SVDV cytopathology, cells showed a completely vacuolated cytoplasm containing vesicles of different sizes. Cell treatment with brefeldin A, which disrupts the Golgi complex, reduced SVDV ({approx} 5 log) and VSV ({approx} 4 log) titers, but did not affect FMDV growth. Thus, three viruses, which share target tissues and clinical signs in natural hosts, induce different intracellular effects in cultured cells.« less

Plant viral synergism: the potyviral genome encodes a broad-range pathogenicity enhancer that transactivates replication of heterologous viruses.

PubMed Central

Pruss, G; Ge, X; Shi, X M; Carrington, J C; Bowman Vance, V

1997-01-01

Synergistic viral diseases of higher plants are caused by the interaction of two independent viruses in the same host and are characterized by dramatic increases in symptoms and in accumulation of one of the coinfecting viruses. In potato virus X (PVX)/potyviral synergism, increased pathogenicity and accumulation of PVX are mediated by the expression of potyviral 5' proximal sequences encoding P1, the helper component proteinase (HC-Pro), and a fraction of P3. Here, we report that the same potyviral sequence (termed P1/HC-Pro) enhances the pathogenicity and accumulation of two other heterologous viruses: cucumber mosaic virus and tobacco mosaic virus. In the case of PVX-potyviral synergism, we show that the expression of the HC-Pro gene product, but not the RNA sequence itself, is sufficient to induce the increase in PVX pathogenicity and that both P1 and P3 coding sequences are dispensable for this aspect of the synergistic interaction. In protoplasts, expression of the potyviral P1/HC-Pro region prolongs the accumulation of PVX (-) strand RNA and transactivates expression of a reporter gene from a PVX subgenomic promoter. Unlike the synergistic enhancement of PVX pathogenicity, which requires only expression of HC-Pro, the enhancement of PVX (-) strand RNA accumulation in protoplasts is significantly greater when the entire P1/HC-Pro sequence is expressed. These results indicate that the potyviral P1/HC-Pro region affects a step in disease development that is common to a broad range of virus infections and suggest a mechanism involving transactivation of viral replication. PMID:9212462

Intergenotypic replacement of lyssavirus matrix proteins demonstrates the role of lyssavirus M proteins in intracellular virus accumulation.

PubMed

Finke, Stefan; Granzow, Harald; Hurst, Jose; Pollin, Reiko; Mettenleiter, Thomas C

2010-02-01

Lyssavirus assembly depends on the matrix protein (M). We compared lyssavirus M proteins from different genotypes for their ability to support assembly and egress of genotype 1 rabies virus (RABV). Transcomplementation of M-deficient RABV with M from European bat lyssavirus (EBLV) types 1 and 2 reduced the release of infectious virus. Stable introduction of the heterogenotypic M proteins into RABV led to chimeric viruses with reduced virus release and intracellular accumulation of virus genomes. Although the chimeras indicated genotype-specific evolution of M, rapid selection of a compensatory mutant suggested conserved mechanisms of lyssavirus assembly and the requirement for only few adaptive mutations to fit the heterogenotypic M to a RABV backbone. Whereas the compensatory mutant replicated to similar infectious titers as RABV M-expressing virus, ultrastructural analysis revealed that both nonadapted EBLV M chimeras and the compensatory mutant differed from RABV M expressing viruses in the lack of intracellular viruslike structures that are enveloped and accumulate in cisterna of the degranulated and dilated rough endoplasmic reticulum compartment. Moreover, all viruses were able to bud at the plasma membrane. Since the lack of the intracellular viruslike structures correlated with the type of M protein but not with the efficiency of virus release, we hypothesize that the M proteins of EBLV-1 and RABV differ in their target membranes for virus assembly. Although the biological function of intracellular assembly and accumulation of viruslike structures in the endoplasmic reticulum remain unclear, the observed differences could contribute to diverse host tropism or pathogenicity.

The presence of anti-Tat antibodies in HIV-infected individuals is associated with containment of CD4+ T-cell decay and viral load, and with delay of disease progression: results of a 3-year cohort study

PubMed Central

2014-01-01

Background Tat is a key HIV-1 virulence factor, which plays pivotal roles in virus gene expression, replication, transmission and disease progression. After release, extracellular Tat accumulates in tissues and exerts effects on both the virus and the immune system, promoting immune activation and virus spreading while disabling the host immune defense. In particular, Tat binds Env spikes on virus particles forming a virus entry complex, which favors infection of dendritic cells and efficient transmission to T cells via RGD-binding integrins. Tat also shields the CCR5-binding sites of Env rendering ineffective virus neutralization by anti-Env antibodies (Abs). This is reversed by the anti-Tat Abs present in natural infection or induced by vaccination. Findings Here we present the results of a cohort study, showing that the presence of anti-Tat Abs in asymptomatic and treatment-naïve HIV-infected subjects is associated with containment of CD4+ T-cell loss and viral load and with a delay of disease progression. In fact, no subjects with high anti-Tat Ab titers initiated antiretroviral therapy during the three years of follow-up. In contrast, no significant effects were seen for anti-Env and anti-Gag Abs. The increase of anti-Env Ab titers was associated with a reduced risk of starting therapy only in the presence of anti-Tat Abs, suggesting an effect of combined anti-Tat and anti-Env Abs on the Tat/Env virus entry complex and on virus neutralization. Conclusions Anti-Tat immunity may help delay HIV disease progression, thus, targeting Tat may offer a novel therapeutic intervention to postpone antiretroviral treatment or to increase its efficacy. PMID:24961156

A parapoxviral virion protein inhibits NF-κB signaling early in infection

PubMed Central

Khatiwada, Sushil; Delhon, Gustavo; Nagendraprabhu, Ponnuraj; Chaulagain, Sabal; Luo, Shuhong; Diel, Diego G.; Flores, Eduardo F.

2017-01-01

Poxviruses have evolved unique proteins and mechanisms to counteract the nuclear factor κB (NF-κB) signaling pathway, which is an essential regulatory pathway of host innate immune responses. Here, we describe a NF-κB inhibitory virion protein of orf virus (ORFV), ORFV073, which functions very early in infected cells. Infection with ORFV073 gene deletion virus (OV-IA82Δ073) led to increased accumulation of NF-κB essential modulator (NEMO), marked phosphorylation of IκB kinase (IKK) subunits IKKα and IKKβ, IκBα and NF-κB subunit p65 (NF-κB-p65), and to early nuclear translocation of NF-κB-p65 in virus-infected cells (≤ 30 min post infection). Expression of ORFV073 alone was sufficient to inhibit TNFα induced activation of the NF-κB signaling in uninfected cells. Consistent with observed inhibition of IKK complex activation, ORFV073 interacted with the regulatory subunit of the IKK complex NEMO. Infection of sheep with OV-IA82Δ073 led to virus attenuation, indicating that ORFV073 is a virulence determinant in the natural host. Notably, ORFV073 represents the first poxviral virion-associated NF-κB inhibitor described, highlighting the significance of viral inhibition of NF-κB signaling very early in infection. PMID:28787456

Internal Disequilibria and Phenotypic Diversification during Replication of Hepatitis C Virus in a Noncoevolving Cellular Environment.

PubMed

Moreno, Elena; Gallego, Isabel; Gregori, Josep; Lucía-Sanz, Adriana; Soria, María Eugenia; Castro, Victoria; Beach, Nathan M; Manrubia, Susanna; Quer, Josep; Esteban, Juan Ignacio; Rice, Charles M; Gómez, Jordi; Gastaminza, Pablo; Domingo, Esteban; Perales, Celia

2017-05-15

Viral quasispecies evolution upon long-term virus replication in a noncoevolving cellular environment raises relevant general issues, such as the attainment of population equilibrium, compliance with the molecular-clock hypothesis, or stability of the phenotypic profile. Here, we evaluate the adaptation, mutant spectrum dynamics, and phenotypic diversification of hepatitis C virus (HCV) in the course of 200 passages in human hepatoma cells in an experimental design that precluded coevolution of the cells with the virus. Adaptation to the cells was evidenced by increase in progeny production. The rate of accumulation of mutations in the genomic consensus sequence deviated slightly from linearity, and mutant spectrum analyses revealed a complex dynamic of mutational waves, which was sustained beyond passage 100. The virus underwent several phenotypic changes, some of which impacted the virus-host relationship, such as enhanced cell killing, a shift toward higher virion density, and increased shutoff of host cell protein synthesis. Fluctuations in progeny production and failure to reach population equilibrium at the genomic level suggest internal instabilities that anticipate an unpredictable HCV evolution in the complex liver environment. IMPORTANCE Long-term virus evolution in an unperturbed cellular environment can reveal features of virus evolution that cannot be explained by comparing natural viral isolates. In the present study, we investigate genetic and phenotypic changes that occur upon prolonged passage of hepatitis C virus (HCV) in human hepatoma cells in an experimental design in which host cell evolutionary change is prevented. Despite replication in a noncoevolving cellular environment, the virus exhibited internal population disequilibria that did not decline with increased adaptation to the host cells. The diversification of phenotypic traits suggests that disequilibria inherent to viral populations may provide a selective advantage to viruses that can be fully exploited in changing environments. Copyright © 2017 American Society for Microbiology.

A single U/C nucleotide substitution changing alanine to valine in the beet necrotic yellow vein virus P25 protein promotes increased virus accumulation in roots of mechanically inoculated, partially resistant sugar beet seedlings.

PubMed

Koenig, R; Loss, S; Specht, J; Varrelmann, M; Lüddecke, P; Deml, G

2009-03-01

Beet necrotic yellow vein virus (BNYVV) A type isolates E12 and S8, originating from areas where resistance-breaking had or had not been observed, respectively, served as starting material for studying the influence of sequence variations in BNYVV RNA 3 on virus accumulation in partially resistant sugar beet varieties. Sub-isolates containing only RNAs 1 and 2 were obtained by serial local lesion passages; biologically active cDNA clones were prepared for RNAs 3 which differed in their coding sequences for P25 aa 67, 68 and 129. Sugar beet seedlings were mechanically inoculated with RNA 1+2/RNA 3 pseudorecombinants. The origin of RNAs 1+2 had little influence on virus accumulation in rootlets. E12 RNA 3 coding for V(67)C(68)Y(129) P25, however, enabled a much higher virus accumulation than S8 RNA 3 coding for A(67)H(68)H(129) P25. Mutants revealed that this was due only to the V(67) 'GUU' codon as opposed to the A(67) 'GCU' codon.

Sorafenib Impedes Rift Valley Fever Virus Egress by Inhibiting Valosin-Containing Protein Function in the Cellular Secretory Pathway.

PubMed

Brahms, Ashwini; Mudhasani, Rajini; Pinkham, Chelsea; Kota, Krishna; Nasar, Farooq; Zamani, Rouzbeh; Bavari, Sina; Kehn-Hall, Kylene

2017-11-01

There is an urgent need for therapeutic development to combat infections caused by Rift Valley fever virus (RVFV), which causes devastating disease in both humans and animals. In an effort to repurpose drugs for RVFV treatment, our previous studies screened a library of FDA-approved drugs. The most promising candidate identified was the hepatocellular and renal cell carcinoma drug sorafenib. Mechanism-of-action studies indicated that sorafenib targeted a late stage in virus infection and caused a buildup of virions within cells. In addition, small interfering RNA (siRNA) knockdown studies suggested that nonclassical targets of sorafenib are important for the propagation of RVFV. Here we extend our previous findings to identify the mechanism by which sorafenib inhibits the release of RVFV virions from the cell. Confocal microscopy imaging revealed that glycoprotein Gn colocalizes and accumulates within the endoplasmic reticulum (ER) and the transport of Gn from the Golgi complex to the host cell membrane is reduced. Transmission electron microscopy demonstrated that sorafenib caused virions to be present inside large vacuoles inside the cells. p97/valosin-containing protein (VCP), which is involved in membrane remodeling in the secretory pathway and a known target of sorafenib, was found to be important for RVFV egress. Knockdown of VCP resulted in decreased RVFV replication, reduced Gn Golgi complex localization, and increased Gn ER accumulation. The intracellular accumulation of RVFV virions was also observed in cells transfected with siRNA targeting VCP. Collectively, these data indicate that sorafenib causes a disruption in viral egress by targeting VCP and the secretory pathway, resulting in a buildup of virions within dilated ER vesicles. IMPORTANCE In humans, symptoms of RVFV infection mainly include a self-limiting febrile illness. However, in some cases, infected individuals can also experience hemorrhagic fever, neurological disorders, liver failure, and blindness, which could collectively be lethal. The ability of RVFV to expand geographically outside sub-Saharan Africa is of concern, particularly to the Americas, where native mosquito species are capable of virus transmission. Currently, there are no FDA-approved therapeutics to treat RVFV infection, and thus, there is an urgent need to understand the mechanisms by which the virus hijacks the host cell machinery to replicate. The significance of our research is in identifying the cellular target of sorafenib that inhibits RVFV propagation, so that this information can be used as a tool for the further development of therapeutics used to treat RVFV infection. Copyright © 2017 American Society for Microbiology.

In Vivo Delivery of Cytoplasmic RNA Virus-derived miRNAs

PubMed Central

Langlois, Ryan A; Shapiro, Jillian S; Pham, Alissa M; tenOever, Benjamin R

2012-01-01

The discovery of microRNAs (miRNAs) revealed an unappreciated level of post-transcriptional control used by the cell to maintain optimal protein levels. This process has represented an attractive strategy for therapeutics that is currently limited by in vivo delivery constraints. Here, we describe the generation of a single-stranded, cytoplasmic virus of negative polarity capable of producing functional miRNAs. Cytoplasmic RNA virus-derived miRNAs accumulated to high levels in vitro, generated significant amounts of miRNA star strand, associated with the RNA-induced silencing complex (RISC), and conferred post transcriptional gene silencing in a sequence-specific manner. Furthermore, we demonstrate that these vectors could deliver miRNAs to a wide range of tissues, and sustain prolonged expression capable of achieving measurable knockdown of physiological targets in vivo. Taken together, these results validate noncanonical processing of cytoplasmic-derived miRNAs and provide a novel platform for small RNA delivery. PMID:22086233

Disruption of chloroplast function through downregulation of phytoene desaturase enhances the systemic accumulation of an aphid-borne, phloem-restricted virus.

PubMed

DeBlasio, Stacy L; Rebelo, Ana Rita; Parks, Katherine; Gray, Stewart; Cilia, Michelle

2018-05-16

Chloroplasts play a central role in pathogen defense in plants. However, the majority of studies explaining the relationship between pathogens and chloroplasts have focused on pathogens that infect mesophyll cells. In contrast, the family Luteoviridae comprises RNA viruses that replicate and traffic exclusively in the phloem. Recently, our lab has shown that Potato leafroll virus (PLRV), the type species in the genus Polerovirus, forms an extensive interaction network with chloroplast-localized proteins, which is partially dependent on the PLRV capsid readthrough domain (RTD). In this study, we used virus-induced gene silencing to disrupt chloroplast function and assess the effects on PLRV accumulation in two host species. Silencing of phytoene desaturase (PDS), a key enzyme in carotenoid, chlorophyll and gibberellic acid (GA) biosynthesis, resulted in a substantial increase in the systemic accumulation of PLRV. This increased accumulation was attenuated when plants were infected with a viral mutant that does not express the RTD. Application of GA partially suppressed the increase in virus accumulation in PDS-silenced plants, suggesting that GA signaling also plays a role in limiting PLRV infection. In addition, the fecundity of the aphid vector of PLRV was increased when fed on PDS-silenced plants relative to PLRV-infected plants.

Antibody responses to avian influenza viruses in wild birds broaden with age

PubMed Central

Manvell, Ruth J.; Schulenburg, Bodo; Shell, Wendy; Wikramaratna, Paul S.; Perrins, Christopher; Sheldon, Ben C.; Brown, Ian H.; Pybus, Oliver G.

2016-01-01

For viruses such as avian influenza, immunity within a host population can drive the emergence of new strains by selecting for viruses with novel antigens that avoid immune recognition. The accumulation of acquired immunity with age is hypothesized to affect how influenza viruses emerge and spread in species of different lifespans. Despite its importance for understanding the behaviour of avian influenza viruses, little is known about age-related accumulation of immunity in the virus's primary reservoir, wild birds. To address this, we studied the age structure of immune responses to avian influenza virus in a wild swan population (Cygnus olor), before and after the population experienced an outbreak of highly pathogenic H5N1 avian influenza in 2008. We performed haemagglutination inhibition assays on sampled sera for five avian influenza strains and show that breadth of response accumulates with age. The observed age-related distribution of antibody responses to avian influenza strains may explain the age-dependent mortality observed during the highly pathogenic H5N1 outbreak. Age structures and species lifespan are probably important determinants of viral epidemiology and virulence in birds. PMID:28003449

The molecular biology of Bluetongue virus replication.

PubMed

Patel, Avnish; Roy, Polly

2014-03-01

The members of Orbivirus genus within the Reoviridae family are arthropod-borne viruses which are responsible for high morbidity and mortality in ruminants. Bluetongue virus (BTV) which causes disease in livestock (sheep, goat, cattle) has been in the forefront of molecular studies for the last three decades and now represents the best understood orbivirus at a molecular and structural level. The complex nature of the virion structure has been well characterised at high resolution along with the definition of the virus encoded enzymes required for RNA replication; the ordered assembly of the capsid shell as well as the protein and genome sequestration required for it; and the role of host proteins in virus entry and virus release. More recent developments of Reverse Genetics and Cell-Free Assembly systems have allowed integration of the accumulated structural and molecular knowledge to be tested at meticulous level, yielding higher insight into basic molecular virology, from which the rational design of safe efficacious vaccines has been possible. This article is centred on the molecular dissection of BTV with a view to understanding the role of each protein in the virus replication cycle. These areas are important in themselves for BTV replication but they also indicate the pathways that related viruses, which includes viruses that are pathogenic to man and animals, might also use providing an informed starting point for intervention or prevention. Copyright © 2014 Elsevier B.V. All rights reserved.

Provirophages and transpovirons as the diverse mobilome of giant viruses.

PubMed

Desnues, Christelle; La Scola, Bernard; Yutin, Natalya; Fournous, Ghislain; Robert, Catherine; Azza, Saïd; Jardot, Priscilla; Monteil, Sonia; Campocasso, Angélique; Koonin, Eugene V; Raoult, Didier

2012-10-30

A distinct class of infectious agents, the virophages that infect giant viruses of the Mimiviridae family, has been recently described. Here we report the simultaneous discovery of a giant virus of Acanthamoeba polyphaga (Lentille virus) that contains an integrated genome of a virophage (Sputnik 2), and a member of a previously unknown class of mobile genetic elements, the transpovirons. The transpovirons are linear DNA elements of ~7 kb that encompass six to eight protein-coding genes, two of which are homologous to virophage genes. Fluorescence in situ hybridization showed that the free form of the transpoviron replicates within the giant virus factory and accumulates in high copy numbers inside giant virus particles, Sputnik 2 particles, and amoeba cytoplasm. Analysis of deep-sequencing data showed that the virophage and the transpoviron can integrate in nearly any place in the chromosome of the giant virus host and that, although less frequently, the transpoviron can also be linked to the virophage chromosome. In addition, integrated fragments of transpoviron DNA were detected in several giant virus and Sputnik genomes. Analysis of 19 Mimivirus strains revealed three distinct transpovirons associated with three subgroups of Mimiviruses. The virophage, the transpoviron, and the previously identified self-splicing introns and inteins constitute the complex, interconnected mobilome of the giant viruses and are likely to substantially contribute to interviral gene transfer.

Provirophages and transpovirons as the diverse mobilome of giant viruses

PubMed Central

Desnues, Christelle; La Scola, Bernard; Yutin, Natalya; Fournous, Ghislain; Robert, Catherine; Azza, Saïd; Jardot, Priscilla; Monteil, Sonia; Campocasso, Angélique; Koonin, Eugene V.; Raoult, Didier

2012-01-01

A distinct class of infectious agents, the virophages that infect giant viruses of the Mimiviridae family, has been recently described. Here we report the simultaneous discovery of a giant virus of Acanthamoeba polyphaga (Lentille virus) that contains an integrated genome of a virophage (Sputnik 2), and a member of a previously unknown class of mobile genetic elements, the transpovirons. The transpovirons are linear DNA elements of ∼7 kb that encompass six to eight protein-coding genes, two of which are homologous to virophage genes. Fluorescence in situ hybridization showed that the free form of the transpoviron replicates within the giant virus factory and accumulates in high copy numbers inside giant virus particles, Sputnik 2 particles, and amoeba cytoplasm. Analysis of deep-sequencing data showed that the virophage and the transpoviron can integrate in nearly any place in the chromosome of the giant virus host and that, although less frequently, the transpoviron can also be linked to the virophage chromosome. In addition, integrated fragments of transpoviron DNA were detected in several giant virus and Sputnik genomes. Analysis of 19 Mimivirus strains revealed three distinct transpovirons associated with three subgroups of Mimiviruses. The virophage, the transpoviron, and the previously identified self-splicing introns and inteins constitute the complex, interconnected mobilome of the giant viruses and are likely to substantially contribute to interviral gene transfer. PMID:23071316

Maize Elongin C interacts with the viral genome-linked protein, VPg, of Sugarcane mosaic virus and facilitates virus infection

PubMed Central

Zhu, Min; Chen, Yuting; Ding, Xin Shun; Webb, Stephen L; Zhou, Tao; Nelson, Richard S; Fan, Zaifeng

2014-01-01

The viral genome-linked protein, VPg, of potyviruses is involved in viral genome replication and translation. To determine host proteins that interact with Sugarcane mosaic virus (SCMV) VPg, a yeast two-hybrid screen was used and a maize (Zea mays) Elongin C (ZmElc) protein was identified. ZmELC transcript was observed in all maize organs, but most highly in leaves and pistil extracts, and ZmElc was present in the cytoplasm and nucleus of maize cells in the presence or absence of SCMV. ZmELC expression was increased in maize tissue at 4 and 6 d post SCMV inoculation. When ZmELC was transiently overexpressed in maize protoplasts the accumulation of SCMV RNA was approximately doubled compared with the amount of virus in control protoplasts. Silencing ZmELC expression using a Brome mosaic virus-based gene silencing vector (virus-induced gene silencing) did not influence maize plant growth and development, but did decrease RNA accumulation of two isolates of SCMV and host transcript encoding ZmeIF4E during SCMV infection. Interestingly, Maize chlorotic mottle virus, from outside the Potyviridae, was increased in accumulation after silencing ZmELC expression. Our results describe both the location of ZmElc expression in maize and a new activity associated with an Elc: support of potyvirus accumulation. PMID:24954157

Maize Elongin C interacts with the viral genome-linked protein, VPg, of Sugarcane mosaic virus and facilitates virus infection.

PubMed

Zhu, Min; Chen, Yuting; Ding, Xin Shun; Webb, Stephen L; Zhou, Tao; Nelson, Richard S; Fan, Zaifeng

2014-09-01

The viral genome-linked protein, VPg, of potyviruses is involved in viral genome replication and translation. To determine host proteins that interact with Sugarcane mosaic virus (SCMV) VPg, a yeast two-hybrid screen was used and a maize (Zea mays) Elongin C (ZmElc) protein was identified. ZmELC transcript was observed in all maize organs, but most highly in leaves and pistil extracts, and ZmElc was present in the cytoplasm and nucleus of maize cells in the presence or absence of SCMV. ZmELC expression was increased in maize tissue at 4 and 6 d post SCMV inoculation. When ZmELC was transiently overexpressed in maize protoplasts the accumulation of SCMV RNA was approximately doubled compared with the amount of virus in control protoplasts. Silencing ZmELC expression using a Brome mosaic virus-based gene silencing vector (virus-induced gene silencing) did not influence maize plant growth and development, but did decrease RNA accumulation of two isolates of SCMV and host transcript encoding ZmeIF4E during SCMV infection. Interestingly, Maize chlorotic mottle virus, from outside the Potyviridae, was increased in accumulation after silencing ZmELC expression. Our results describe both the location of ZmElc expression in maize and a new activity associated with an Elc: support of potyvirus accumulation. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

A protein kinase binds the C-terminal domain of the readthrough protein of Turnip yellows virus and regulates virus accumulation.

PubMed

Rodriguez-Medina, Caren; Boissinot, Sylvaine; Chapuis, Sophie; Gereige, Dalya; Rastegar, Maryam; Erdinger, Monique; Revers, Frédéric; Ziegler-Graff, Véronique; Brault, Véronique

2015-12-01

Turnip yellows virus (TuYV), a phloem-limited virus, encodes a 74kDa protein known as the readthrough protein (RT) involved in virus movement. We show here that a TuYV mutant deleted of the C-terminal part of the RT protein (TuYV-∆RTCter) was affected in long-distance trafficking in a host-specific manner. By using the C-terminal domain of the RT protein as a bait in a yeast two-hybrid screen of a phloem cDNA library from Arabidopsis thaliana we identified the calcineurin B-like protein-interacting protein kinase-7 (AtCIPK7). Transient expression of a GFP:CIPK7 fusion protein in virus-inoculated Nicotiana benthamiana leaves led to local increase of wild-type TuYV accumulation, but not that of TuYV-∆RTCter. Surprisingly, elevated virus titer in inoculated leaves did not result in higher TuYV accumulation in systemic leaves, which indicates that virus long-distance movement was not affected. Since GFP:CIPK7 was localized in or near plasmodesmata, CIPK7 could negatively regulate TuYV export from infected cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Complex Modulation of the Aedes aegypti Transcriptome in Response to Dengue Virus Infection

PubMed Central

Bonizzoni, Mariangela; Dunn, W. Augustine; Campbell, Corey L.; Olson, Ken E.; Marinotti, Osvaldo; James, Anthony A.

2012-01-01

Dengue fever is the most important arboviral disease world-wide, with Aedes aegypti being the major vector. Interactions between the mosquito host and dengue viruses (DENV) are complex and vector competence varies among geographically-distinct Ae. aegypti populations. Additionally, dengue is caused by four antigenically-distinct viral serotypes (DENV1–4), each with multiple genotypes. Each virus genotype interacts differently with vertebrate and invertebrate hosts. Analyses of alterations in mosquito transcriptional profiles during DENV infection are expected to provide the basis for identifying networks of genes involved in responses to viruses and contribute to the molecular-genetic understanding of vector competence. In addition, this knowledge is anticipated to support the development of novel disease-control strategies. RNA-seq technology was used to assess genome-wide changes in transcript abundance at 1, 4 and 14 days following DENV2 infection in carcasses, midguts and salivary glands of the Ae. aegypti Chetumal strain. DENV2 affected the expression of 397 Ae. aegypti genes, most of which were down-regulated by viral infection. Differential accumulation of transcripts was mainly tissue- and time-specific. Comparisons of our data with other published reports reveal conservation of functional classes, but limited concordance of specific mosquito genes responsive to DENV2 infection. These results indicate the necessity of additional studies of mosquito-DENV interactions, specifically those focused on recently-derived mosquito strains with multiple dengue virus serotypes and genotypes. PMID:23209765

Differential Roles of AC2 and AC4 of Cassava Geminiviruses in Mediating Synergism and Suppression of Posttranscriptional Gene Silencing

PubMed Central

Vanitharani, Ramachandran; Chellappan, Padmanabhan; Pita, Justin S.; Fauquet, Claude M.

2004-01-01

Posttranscriptional gene silencing (PTGS) in plants is a natural defense mechanism against virus infection. In mixed infections, virus synergism is proposed to result from suppression of the host defense mechanism by the viruses. Synergistic severe mosaic disease caused by simultaneous infection with isolates of the Cameroon strain of African cassava mosaic virus (ACMV-[CM]) and East African cassava mosaic Cameroon virus (EACMCV) in cassava and tobacco is characterized by a dramatic increase in symptom severity and a severalfold increase in viral-DNA accumulation by both viruses compared to that in singly infected plants. Here, we report that synergism between ACMV-[CM] and EACMCV is a two-way process, as the presence of the DNA-A component of ACMV-[CM] or EACMCV in trans enhanced the accumulation of viral DNA of EACMCV and ACMV-[CM], respectively, in tobacco BY-2 protoplasts. Furthermore, transient expression of ACMV-[CM] AC4 driven by the Cauliflower mosaic virus 35S promoter (p35S-AC4) enhanced EACMCV DNA accumulation by ∼8-fold in protoplasts, while p35S-AC2 of EACMCV enhanced ACMV-[CM] DNA accumulation, also by ∼8-fold. An Agrobacterium-based leaf infiltration assay determined that ACMV-[CM] AC4 and EACMCV AC2, the putative synergistic genes, were able to suppress PTGS induced by green fluorescent protein (GFP) and eliminated the short interfering RNAs associated with PTGS, with a correlated increase in GFP mRNA accumulation. In addition, we have identified AC4 of Sri Lankan cassava mosaic virus and AC2 of Indian cassava mosaic virus as suppressors of PTGS, indicating that geminiviruses evolved differently in regard to interaction with the host. The specific and different roles played by these AC2 and AC4 proteins of cassava geminiviruses in regulating anti-PTGS activity and their relation to synergism are discussed. PMID:15308741

Herpes Simplex Virus 2 Infection Impacts Stress Granule Accumulation

PubMed Central

Finnen, Renée L.; Pangka, Kyle R.

2012-01-01

Interference with stress granule (SG) accumulation is gaining increased appreciation as a common strategy used by diverse viruses to facilitate their replication and to cope with translational arrest. Here, we examined the impact of infection by herpes simplex virus 2 (HSV-2) on SG accumulation by monitoring the localization of the SG components T cell internal antigen 1 (TIA-1), Ras-GTPase-activating SH3-domain-binding protein (G3BP), and poly(A)-binding protein (PABP). Our results indicate that SGs do not accumulate in HSV-2-infected cells and that HSV-2 can interfere with arsenite-induced SG accumulation early after infection. Surprisingly, SG accumulation was inhibited despite increased phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), implying that HSV-2 encodes previously unrecognized activities designed to maintain translation initiation downstream of eIF2α. SG accumulation was not inhibited in HSV-2-infected cells treated with pateamine A, an inducer that works independently of eIF2α phosphorylation. The SGs that accumulated following pateamine A treatment of infected cells contained G3BP and PABP but were largely devoid of TIA-1. We also identified novel nuclear structures containing TIA-1 that form late in infection. These structures contain the RNA binding protein 68-kDa Src-associated in mitosis (Sam68) and were noticeably absent in infected cells treated with inhibitors of viral DNA replication, suggesting that they arise as a result of late events in the virus replicative cycle. PMID:22623775

A Recombinant Potato virus Y Infectious Clone Tagged with the Rosea1 Visual Marker (PVY-Ros1) Facilitates the Analysis of Viral Infectivity and Allows the Production of Large Amounts of Anthocyanins in Plants

PubMed Central

Cordero, Teresa; Mohamed, Mohamed A.; López-Moya, Juan-José; Daròs, José-Antonio

2017-01-01

Potato virus Y (PVY) is a major threat to the cultivation of potato and other solanaceous plants. By inserting a cDNA coding for the Antirrhinum majus Rosea1 transcription factor into a PVY infectious clone, we created a biotechnological tool (PVY-Ros1) that allows infection by this relevant plant virus to be tracked by the naked eye with no need for complex instrumentation. Rosea1 is an MYB-type transcription factor whose expression activates the biosynthesis of anthocyanin pigments in a dose-specific and cell-autonomous manner. Our experiments showed that the mechanical inoculation of solanaceous plants with PVY-Ros1 induced the formation of red infection foci in inoculated tissue and solid dark red pigmentation in systemically infected tissue, which allows disease progression to be easily monitored. By using silver nanoparticles, a nanomaterial with exciting antimicrobial properties, we proved the benefits of PVY-Ros1 to analyze novel antiviral treatments in plants. PVY-Ros1 was also helpful for visually monitoring the virus transmission process by an aphid vector. Most importantly, the anthocyanin analysis of infected tobacco tissues demonstrated that PVY-Ros1 is an excellent biotechnological tool for molecular farming because it induces the accumulation of larger amounts of anthocyanins, antioxidant compounds of nutritional, pharmaceutical and industrial interest, than those that naturally accumulate in some fruits and vegetables well known for their high anthocyanin content. Hence these results support the notion that the virus-mediated expression of regulatory factors and enzymes in plants facilitates easy quick plant metabolism engineering. PMID:28428782

Plant Virus Infection and the Ubiquitin Proteasome Machinery: Arms Race along the Endoplasmic Reticulum.

PubMed

Verchot, Jeanmarie

2016-11-19

The endoplasmic reticulum (ER) is central to plant virus replication, translation, maturation, and egress. Ubiquitin modification of ER associated cellular and viral proteins, alongside the actions of the 26S proteasome, are vital for the regulation of infection. Viruses can arrogate ER associated ubiquitination as well as cytosolic ubiquitin ligases with the purpose of directing the ubiquitin proteasome system (UPS) to new targets. Such targets include necessary modification of viral proteins which may stabilize certain complexes, or modification of Argonaute to suppress gene silencing. The UPS machinery also contributes to the regulation of effector triggered immunity pattern recognition receptor immunity. Combining the results of unrelated studies, many positive strand RNA plant viruses appear to interact with cytosolic Ub-ligases to provide novel avenues for controlling the deleterious consequences of disease. Viral interactions with the UPS serve to regulate virus infection in a manner that promotes replication and movement, but also modulates the levels of RNA accumulation to ensure successful biotrophic interactions. In other instances, the UPS plays a central role in cellular immunity. These opposing roles are made evident by contrasting studies where knockout mutations in the UPS can either hamper viruses or lead to more aggressive diseases. Understanding how viruses manipulate ER associated post-translational machineries to better manage virus-host interactions will provide new targets for crop improvement.

Interaction of the Tobacco mosaic virus movement protein with microtubules during the cell cycle in tobacco BY-2 cells.

PubMed

Boutant, Emmanuel; Fitterer, Chantal; Ritzenthaler, Christophe; Heinlein, Manfred

2009-10-01

Cell-to-cell movement of Tobacco mosaic virus (TMV) involves the interaction of virus-encoded 30-kDa movement protein (MP) with microtubules. In cells behind the infection front that accumulate high levels of MP, this activity is reflected by the formation of stabilized MP/microtubule complexes. The ability of MP to bind along and stabilize microtubules is conserved upon expression in mammalian cells. In mammalian cells, the protein also leads to inhibition of mitosis and cell division through a microtubule-independent process correlated with the loss of centrosomal gamma-tubulin and of centrosomal microtubule-nucleation activity. Since MP has the capacity to interact with plant factors involved in microtubule nucleation and dynamics, we used inducible expression in BY-2 cells to test whether MP expression inhibits mitosis and cell division also in plants. We demonstrate that MP:GFP associates with all plant microtubule arrays and, unlike in mammalian cells, does not interfere with mitosis. Thus, MP function and the interaction of MP with factors of the cytoskeleton do not entail an inhibition of mitosis in plants. We also report that the protein targets primary plasmodesmata in BY-2 cells immediately upon or during cytokinesis and that the accumulation of MP in plasmodesmata occurs in the presence of inhibitors of the cytoskeleton and the secretory pathway.

Alfalfa dwarf cytorhabdovirus P protein is a local and systemic RNA silencing supressor which inhibits programmed RISC activity and prevents transitive amplification of RNA silencing.

PubMed

Bejerman, Nicolás; Mann, Krin S; Dietzgen, Ralf G

2016-09-15

Plants employ RNA silencing as an innate defense mechanism against viruses. As a counter-defense, plant viruses have evolved to express RNA silencing suppressor proteins (RSS), which target one or more steps of the silencing pathway. In this study, we show that the phosphoprotein (P) encoded by the negative-sense RNA virus alfalfa dwarf virus (ADV), a species of the genus Cytorhabdovirus, family Rhabdoviridae, is a suppressor of RNA silencing. ADV P has a relatively weak local RSS activity, and does not prevent siRNA accumulation. On the other hand, ADV P strongly suppresses systemic RNA silencing, but does not interfere with the short-distance spread of silencing, which is consistent with its lack of inhibition of siRNA accumulation. The mechanism of suppression appears to involve ADV P binding to RNA-induced silencing complex proteins AGO1 and AGO4 as shown in protein-protein interaction assays when ectopically expressed. In planta, we demonstrate that ADV P likely functions by inhibiting miRNA-guided AGO1 cleavage and prevents transitive amplification by repressing the production of secondary siRNAs. As recently described for lettuce necrotic yellows cytorhabdovirus P, but in contrast to other viral RSS known to disrupt AGO activity, ADV P sequence does not contain any recognizable GW/WG or F-box motifs, which suggests that cytorhabdovirus P proteins may use alternative motifs to bind to AGO proteins. Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.

Pharmacokinetics of the Antiviral Lectin Griffithsin Administered by Different Routes Indicates Multiple Potential Uses.

PubMed

Barton, Christopher; Kouokam, J Calvin; Hurst, Harrell; Palmer, Kenneth E

2016-12-17

Griffithsin (GRFT) is a red alga-derived lectin with demonstrated broad spectrum antiviral activity against enveloped viruses, including severe acute respiratory syndrome-Coronavirus (SARS-CoV), Japanese encephalitis virus (JEV), hepatitis C virus (HCV), and herpes simplex virus-2 (HSV-2). However, its pharmacokinetic profile remains largely undefined. Here, Sprague Dawley rats were administered a single dose of GRFT at 10 or 20 mg/kg by intravenous, oral, and subcutaneous routes, respectively, and serum GRFT levels were measured at select time points. In addition, the potential for systemic accumulation after oral dosing was assessed in rats after 10 daily treatments with GRFT (20 or 40 mg/kg). We found that parenterally-administered GRFT in rats displayed a complex elimination profile, which varied according to administration routes. However, GRFT was not orally bioavailable, even after chronic treatment. Nonetheless, active GRFT capable of neutralizing HIV-Env pseudoviruses was detected in rat fecal extracts after chronic oral dosing. These findings support further evaluation of GRFT for pre-exposure prophylaxis against emerging epidemics for which specific therapeutics are not available, including systemic and enteric infections caused by susceptible enveloped viruses. In addition, GRFT should be considered for antiviral therapy and the prevention of rectal transmission of HIV-1 and other susceptible viruses.

Accumulation of Herpes Simplex Virus Type 1 Early and Leaky-Late Proteins Correlates with Apoptosis Prevention in Infected Human HEp-2 Cells

PubMed Central

Aubert, Martine; Rice, Stephen A.; Blaho, John A.

2001-01-01

We previously reported that a recombinant ICP27-null virus stimulated, but did not prevent, apoptosis in human HEp-2 cells during infection (M. Aubert and J. A. Blaho, J. Virol. 73:2803–2813, 1999). In the present study, we used a panel of 15 recombinant ICP27 mutant viruses to determine which features of herpes simplex virus type 1 (HSV-1) replication are required for the apoptosis-inhibitory activity. Each virus was defined experimentally as either apoptotic, partially apoptotic, or nonapoptotic based on infected HEp-2 cell morphologies, percentages of infected cells with condensed chromatin, and patterns of specific cellular death factor processing. Viruses d27-1, d1-5, d1-2, M11, M15, M16, n504R, n406R, n263R, and n59R are apoptotic or partially apoptotic in HEp-2 cells and severely defective for growth in Vero cells. Viruses d2-3, d3-4, d4-5, d5-6, and d6-7 are nonapoptotic, demonstrating that ICP27 contains a large amino-terminal region, including its RGG box RNA binding domain, which is not essential for apoptosis prevention. Accumulations of viral TK, VP16, and gD but not gC, ICP22, or ICP4 proteins correlated with prevention of apoptosis during the replication of these viruses. Of the nonapoptotic viruses, d4-5 did not produce gC, indicating that accumulation of true late gene products is not necessary for the prevention process. Analyses of viral DNA synthesis in HEp-2 cells indicated that apoptosis prevention by HSV-1 requires that the infection proceeds to the stage in which viral DNA replication takes place. Infections performed in the presence of the drug phosphonoacetic acid confirmed that the process of viral DNA synthesis and the accumulation of true late (γ2) proteins are not required for apoptosis prevention. Based on our results, we conclude that the accumulation of HSV-1 early (β) and leaky-late (γ1) proteins correlates with the prevention of apoptosis in infected HEp-2 cells. PMID:11134315

Profiling of Genes Related to Cross Protection and Competition for NbTOM1 by HLSV and TMV

PubMed Central

Wen, Yi; Lim, Grace Xiao-Yun; Wong, Sek-Man

2013-01-01

Cross protection is the phenomenon through which a mild strain virus suppresses symptoms induced by a closely related severe strain virus in infected plants. Hibiscus latent Singapore virus (HLSV) and Tobacco mosaic virus (TMV) are species within the genus tobamovirus. HLSV can protect Nicotiana benthamiana against TMV-U1 strain, resulting in mild symptoms instead of severe systemic necrosis. The mechanism of cross protection between HLSV and TMV is unknown. In the past, some researchers suggest that the protecting virus strain might occupy virus-specific replication sites within a cell leaving no room for the challenge virus. Quantitative real-time RT-PCR was performed to detect viral RNA levels during cross protection. HLSV accumulation increased in cross protected plants compared with that of single HLSV infected plants, while TMV decreased in cross protected plants. This suggests that there is a competition for host factors between HLSV and TMV for replication. To investigate the mechanism under the cross protection between HLSV and TMV, microarray analysis was conducted to examine the transcriptional levels of global host genes during cross protection, using Tobacco Gene Expression Microarray, 4x44 k slides. The transcriptional level of some host genes corresponded to accumulation level of TMV. Some host genes were up-regulated only by HLSV. Tobamovirus multiplication gene 1 (TOM1), essential for tobamovirus multiplication, was involved in competition for replication by HLSV and TMV during cross protection. Both HLSV and TMV accumulation decreased when NbTOM1 was silenced. A large quantity of HLSV resulted in decreased TMV accumulation in HLSV+TMV (100:1) co-infection. These results indicate that host genes involved in the plant defense response and virus multiplication are up-regulated by challenge virus TMV but not by protecting virus HLSV during cross protection. PMID:24023899

Recursion-based depletion of human immunodeficiency virus-specific naive CD4(+) T cells may facilitate persistent viral replication and chronic viraemia leading to acquired immunodeficiency syndrome.

PubMed

Tsukamoto, Tetsuo; Yamamoto, Hiroyuki; Okada, Seiji; Matano, Tetsuro

2016-09-01

Although antiretroviral therapy has made human immunodeficiency virus (HIV) infection a controllable disease, it is still unclear how viral replication persists in untreated patients and causes CD4(+) T-cell depletion leading to acquired immunodeficiency syndrome (AIDS) in several years. Theorists tried to explain it with the diversity threshold theory in which accumulated mutations in the HIV genome make the virus so diverse that the immune system will no longer be able to recognize all the variants and fail to control the viraemia. Although the theory could apply to a number of cases, macaque AIDS models using simian immunodeficiency virus (SIV) have shown that failed viral control at the set point is not always associated with T-cell escape mutations. Moreover, even monkeys without a protective major histocompatibility complex (MHC) allele can contain replication of a super infected SIV following immunization with a live-attenuated SIV vaccine, while those animals are not capable of fighting primary SIV infection. Here we propose a recursion-based virus-specific naive CD4(+) T-cell depletion hypothesis through thinking on what may happen in individuals experiencing primary immunodeficiency virus infection. This could explain the mechanism for impairment of virus-specific immune response in the course of HIV infection. Copyright © 2016 Elsevier Ltd. All rights reserved.

Establishing RNA virus resistance in plants by harnessing CRISPR immune system.

PubMed

Zhang, Tong; Zheng, Qiufeng; Yi, Xin; An, Hong; Zhao, Yaling; Ma, Siqi; Zhou, Guohui

2018-01-11

Recently, CRISPR-Cas (clustered, regularly interspaced short palindromic repeats-CRISPR-associated proteins) system has been used to produce plants resistant to DNA virus infections. However, there is no RNA virus control method in plants that uses CRISPR-Cas system to target the viral genome directly. Here, we reprogrammed the CRISPR-Cas9 system from Francisella novicida to confer molecular immunity against RNA viruses in Nicotiana benthamiana and Arabidopsis plants. Plants expressing FnCas9 and sgRNA specific for the cucumber mosaic virus (CMV) or tobacco mosaic virus (TMV) exhibited significantly attenuated virus infection symptoms and reduced viral RNA accumulation. Furthermore, in the transgenic virus-targeting plants, the resistance was inheritable and the progenies showed significantly less virus accumulation. These data reveal that the CRISPR/Cas9 system can be used to produce plant that stable resistant to RNA viruses, thereby broadening the use of such technology for virus control in agricultural field. © 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

A protein kinase binds the C-terminal domain of the readthrough protein of Turnip yellows virus and regulates virus accumulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rodriguez-Medina, Caren; Boissinot, Sylvaine; Chapuis, Sophie

Turnip yellows virus (TuYV), a phloem-limited virus, encodes a 74 kDa protein known as the readthrough protein (RT) involved in virus movement. We show here that a TuYV mutant deleted of the C-terminal part of the RT protein (TuYV-∆RT{sub Cter}) was affected in long-distance trafficking in a host-specific manner. By using the C-terminal domain of the RT protein as a bait in a yeast two-hybrid screen of a phloem cDNA library from Arabidopsis thaliana we identified the calcineurin B-like protein-interacting protein kinase-7 (AtCIPK7). Transient expression of a GFP:CIPK7 fusion protein in virus-inoculated Nicotiana benthamiana leaves led to local increase ofmore » wild-type TuYV accumulation, but not that of TuYV-∆RT{sub Cter}. Surprisingly, elevated virus titer in inoculated leaves did not result in higher TuYV accumulation in systemic leaves, which indicates that virus long-distance movement was not affected. Since GFP:CIPK7 was localized in or near plasmodesmata, CIPK7 could negatively regulate TuYV export from infected cells. - Highlights: • The C-terminal domain of TuYV-RT is required for long-distance movement. • CIPK7 from Arabidopsis interacts with RT{sub Cter} in yeast and in plants. • CIPK7 overexpression increases virus titer locally but not virus systemic movement. • CIPK7 localizes to plasmodesmata. • CIPK7 could be a defense protein regulating virus export.« less

The degree of attenuation of tick-borne encephalitis virus depends on the cumulative effects of point mutations.

PubMed

Gritsun, T S; Desai, A; Gould, E A

2001-07-01

An infectious clone (pGGVs) of the tick-borne encephalitis complex virus Vasilchenko (Vs) was constructed previously. Virus recovered from pGGVs produced slightly smaller plaques than the Vs parental virus. Sequence analysis demonstrated five nucleotide differences between the original Vs virus and pGGVs; four of these mutations resulted in amino acid substitutions, while the fifth mutation was located in the 3' untranslated region (3'UTR). Two mutations were located in conserved regions and three mutations were located in variable regions of the virus genome. Reverse substitutions from the conserved regions of the genome, R(496)-->H in the envelope (E) gene and C(10884)-->T in the 3'UTR, were introduced both separately and together into the infectious clone and their biological effect on virus phenotype was evaluated. The engineered viruses with R(496) in the E protein produced plaques of smaller size than viruses with H(496) at this position. This mutation also affected the growth and neuroinvasiveness of the virus. In contrast, the consequence of a T(10884)-->C substitution within the 3'UTR was noticeable only in cytotoxicity and neuroinvasiveness tests. However, all virus mutants engineered by modification of the infectious clone, including one with two wild-type mutations, H(496) and T(10884), showed reduced neuroinvasiveness in comparison with the Vs parental virus. Therefore, although the H(496)-->R and T(10884)-->C substitutions clearly reduce virus virulence, the other mutations within the variable regions of the capsid (I(45)-->F) and the NS5 (T(2688)-->A and M(3385)-->I) genes also contribute to the process of attenuation. In terms of developing flavivirus vaccines, the impact of accumulating apparently minor mutations should be assessed in detail.

Prevailing PA Mutation K356R in Avian Influenza H9N2 Virus Increases Mammalian Replication and Pathogenicity.

PubMed

Xu, Guanlong; Zhang, Xuxiao; Gao, Weihua; Wang, Chenxi; Wang, Jinliang; Sun, Honglei; Sun, Yipeng; Guo, Lu; Zhang, Rui; Chang, Kin-Chow; Liu, Jinhua; Pu, Juan

2016-09-15

Adaptation of the viral polymerase complex comprising PB1, PB2, and PA is necessary for efficient influenza A virus replication in new host species. We found that PA mutation K356R (PA-K356R) has become predominant since 2014 in avian H9N2 viruses in China as with seasonal human H1N1 viruses. The same mutation is also found in most human isolates of emergent avian H7N9 and H10N8 viruses whose six internal gene segments are derived from the H9N2 virus. We further demonstrated the mammalian adaptive functionality of the PA-K356R mutation. Avian H9N2 virus with the PA-K356R mutation in human A549 cells showed increased nuclear accumulation of PA and increased viral polymerase activity that resulted in elevated levels of viral transcription and virus output. The same mutant virus in mice also enhanced virus replication and caused lethal infection. In addition, combined mutation of PA-K356R and PB2-E627K, a well-known mammalian adaptive marker, in the H9N2 virus showed further cooperative increases in virus production and severity of infection in vitro and in vivo In summary, PA-K356R behaves as a novel mammalian tropism mutation, which, along with other mutations such as PB2-E627K, might render avian H9N2 viruses adapted for human infection. Mutations of the polymerase complex (PB1, PB2, and PA) of influenza A virus are necessary for viral adaptation to new hosts. This study reports a novel and predominant mammalian adaptive mutation, PA-K356R, in avian H9N2 viruses and human isolates of emergent H7N9 and H10N8 viruses. We found that PA-356R in H9N2 viruses causes significant increases in virus replication and severity of infection in human cells and mice and that PA-K356R cooperates with the PB2-E627K mutation, a well-characterized human adaptive marker, to exacerbate mammalian infection in vitro and in vivo Therefore, the PA-K356R mutation is a significant adaptation in H9N2 viruses and related H7N9 and H10N8 reassortants toward human infectivity. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Prevailing PA Mutation K356R in Avian Influenza H9N2 Virus Increases Mammalian Replication and Pathogenicity

PubMed Central

Xu, Guanlong; Zhang, Xuxiao; Gao, Weihua; Wang, Chenxi; Wang, Jinliang; Sun, Honglei; Sun, Yipeng; Guo, Lu; Zhang, Rui; Chang, Kin-Chow; Liu, Jinhua

2016-01-01

ABSTRACT Adaptation of the viral polymerase complex comprising PB1, PB2, and PA is necessary for efficient influenza A virus replication in new host species. We found that PA mutation K356R (PA-K356R) has become predominant since 2014 in avian H9N2 viruses in China as with seasonal human H1N1 viruses. The same mutation is also found in most human isolates of emergent avian H7N9 and H10N8 viruses whose six internal gene segments are derived from the H9N2 virus. We further demonstrated the mammalian adaptive functionality of the PA-K356R mutation. Avian H9N2 virus with the PA-K356R mutation in human A549 cells showed increased nuclear accumulation of PA and increased viral polymerase activity that resulted in elevated levels of viral transcription and virus output. The same mutant virus in mice also enhanced virus replication and caused lethal infection. In addition, combined mutation of PA-K356R and PB2-E627K, a well-known mammalian adaptive marker, in the H9N2 virus showed further cooperative increases in virus production and severity of infection in vitro and in vivo. In summary, PA-K356R behaves as a novel mammalian tropism mutation, which, along with other mutations such as PB2-E627K, might render avian H9N2 viruses adapted for human infection. IMPORTANCE Mutations of the polymerase complex (PB1, PB2, and PA) of influenza A virus are necessary for viral adaptation to new hosts. This study reports a novel and predominant mammalian adaptive mutation, PA-K356R, in avian H9N2 viruses and human isolates of emergent H7N9 and H10N8 viruses. We found that PA-356R in H9N2 viruses causes significant increases in virus replication and severity of infection in human cells and mice and that PA-K356R cooperates with the PB2-E627K mutation, a well-characterized human adaptive marker, to exacerbate mammalian infection in vitro and in vivo. Therefore, the PA-K356R mutation is a significant adaptation in H9N2 viruses and related H7N9 and H10N8 reassortants toward human infectivity. PMID:27384648

Cytomegalovirus (CMV) research in immune senescence comes of age: overview of the 6th International Workshop on CMV and Immunosenescence.

PubMed

Nikolich-Žugich, Janko; van Lier, René A W

2017-06-01

Cytomegalovirus (CMV) is one of the most complex and most ubiquitous latent persistent viruses, with a considerable ability to evade and manipulate the immune system. Following an early-life infection, most immunocompetent humans spend several decades living with CMV, and, because the virus in these hosts does not cause manifest disease, CMV can be considered part of normal aging for more than half of humanity. However, there is accumulating evidence that CMV carriage is not a null event and that both potentially harmful and potentially beneficial outcomes emanate from the interaction of CMV with its mammalian hosts. This article provides an overview of the 6th International Workshop on CMV and Immunosenescence, highlighting the advances in the field made in the past two years, as related to CMV epidemiology/geroscience, CMV virology with an accent on latency, and CMV immune evasion and immune recognition of the virus and its antigens.

Structural and functional analysis of virus factories purified from Rabbit vesivirus-infected Vero cells.

PubMed

Casais, Rosa; Molleda, Lorenzo González; Machín, Angeles; del Barrio, Gloria; Manso, Alberto García; Dalton, Kevin P; Coto, Ana; Alonso, José Manuel Martín; Prieto, Miguel; Parra, Francisco

2008-10-01

Rabbit vesivirus infection induces membrane modifications and accumulation of vesicular structures in the cytoplasm of infected Vero cells. Crude RaV replication complexes (RCs) have been purified and their structural and functional properties have been characterized. We show that calnexin, an ER-resident protein, RaV non-structural proteins 2AB-, 2C-, 3A-, 3B- and 3CD-like as well as viral RNAs co-localize within membranous structures which are able to replicate the endogenous RNA templates. The purified virus factories protected their viral RNA contents from microccocal nuclease degradation and were inaccessible to exogenously added synthetic transcripts. In addition, we have shown that RCs can be used to investigate uridylylation of native endogenous VPg. In contrast to the observation that the virus factories were inaccessible to RNAs, RCs were accessible to added recombinant VPg which was subsequently nucleotidylylated. Nevertheless no elongation of an RNA chain attached to native or recombinant VPg could be demonstrated.

Hepatitis C Virus-Induced Cytoplasmic Organelles Use the Nuclear Transport Machinery to Establish an Environment Conducive to Virus Replication

PubMed Central

Neufeldt, Christopher J.; Joyce, Michael A.; Levin, Aviad; Steenbergen, Rineke H.; Pang, Daniel; Shields, Justin; Tyrrell, D. Lorne J.; Wozniak, Richard W.

2013-01-01

Hepatitis C virus (HCV) infection induces formation of a membranous web structure in the host cell cytoplasm where the viral genome replicates and virions assemble. The membranous web is thought to concentrate viral components and hide viral RNA from pattern recognition receptors. We have uncovered a role for nuclear pore complex proteins (Nups) and nuclear transport factors (NTFs) in the membranous web. We show that HCV infection leads to increased levels of cytoplasmic Nups that accumulate at sites enriched for HCV proteins. Moreover, we detected interactions between specific HCV proteins and both Nups and NTFs. We hypothesize that cytoplasmically positioned Nups facilitate formation of the membranous web and contribute to the compartmentalization of viral replication. Accordingly, we show that transport cargo proteins normally targeted to the nucleus are capable of entering regions of the membranous web, and that depletion of specific Nups or Kaps inhibits HCV replication and assembly. PMID:24204278

Synthetic Protocells Interact with Viral Nanomachinery and Inactivate Pathogenic Human Virus

PubMed Central

Moscona, Anne; LaVan, David A.

2011-01-01

We present a new antiviral strategy and research tool that could be applied to a wide range of enveloped viruses that infect human beings via membrane fusion. We test this strategy on two emerging zoonotic henipaviruses that cause fatal encephalitis in humans, Nipah (NiV) and Hendra (HeV) viruses. In the new approach, artificial cell-like particles (protocells) presenting membrane receptors in a biomimetic manner were developed and found to attract and inactivate henipavirus envelope glycoprotein pseudovirus particles, preventing infection. The protocells do not accumulate virus during the inactivation process. The use of protocells that interact with, but do not accumulate, viruses may provide significant advantages over current antiviral drugs, and this general approach may have wide potential for antiviral development. PMID:21390296

Functional Compensation of a Detrimental Amino Acid Substitution in a Cytotoxic-T-Lymphocyte Epitope of Influenza A Viruses by Comutations

PubMed Central

Rimmelzwaan, G. F.; Berkhoff, E. G. M.; Nieuwkoop, N. J.; Fouchier, R. A. M.; Osterhaus, A. D. M. E.

2004-01-01

Influenza A viruses accumulate amino acid substitutions in cytotoxic-T-lymphocyte (CTL) epitopes, allowing these viruses to escape from CTL immunity. The arginine-to-glycine substitution at position 384 of the viral nucleoprotein is associated with escape from CTLs. Introduction of the R384G substitution in the nucleoprotein gene segment of influenza virus A/Hong Kong/2/68 by site-directed mutagenesis was detrimental to viral fitness. Introduction of one of the comutations associated with R384G, E375G, partially restored viral fitness and nucleoprotein functionality. We hypothesized that influenza A viruses need to overcome functional constraints to accumulate mutations in CTL epitopes and escape from CTLs. PMID:15280506

Model of OSBP-Mediated Cholesterol Supply to Aichi Virus RNA Replication Sites Involving Protein-Protein Interactions among Viral Proteins, ACBD3, OSBP, VAP-A/B, and SAC1.

PubMed

Ishikawa-Sasaki, Kumiko; Nagashima, Shigeo; Taniguchi, Koki; Sasaki, Jun

2018-04-15

Positive-strand RNA viruses, including picornaviruses, utilize cellular machinery for genome replication. Previously, we reported that each of the 2B, 2BC, 2C, 3A, and 3AB proteins of Aichi virus (AiV), a picornavirus, forms a complex with the Golgi apparatus protein ACBD3 and phosphatidylinositol 4-kinase IIIβ (PI4KB) at viral RNA replication sites (replication organelles [ROs]), enhancing PI4KB-dependent phosphatidylinositol 4-phosphate (PI4P) production. Here, we demonstrate AiV hijacking of the cellular cholesterol transport system involving oxysterol-binding protein (OSBP), a PI4P-binding cholesterol transfer protein. AiV RNA replication was inhibited by silencing cellular proteins known to be components of this pathway, OSBP, the ER membrane proteins VAPA and VAPB (VAP-A/B), the PI4P-phosphatase SAC1, and PI-transfer protein β. OSBP, VAP-A/B, and SAC1 were present at RNA replication sites. We also found various previously unknown interactions among the AiV proteins (2B, 2BC, 2C, 3A, and 3AB), ACBD3, OSBP, VAP-A/B, and SAC1, and the interactions were suggested to be involved in recruiting the component proteins to AiV ROs. Importantly, the OSBP-2B interaction enabled PI4P-independent recruitment of OSBP to AiV ROs, indicating preferential recruitment of OSBP among PI4P-binding proteins. Protein-protein interaction-based OSBP recruitment has not been reported for other picornaviruses. Cholesterol was accumulated at AiV ROs, and inhibition of OSBP-mediated cholesterol transfer impaired cholesterol accumulation and AiV RNA replication. Electron microscopy showed that AiV-induced vesicle-like structures were close to ER membranes. Altogether, we conclude that AiV directly recruits the cholesterol transport machinery through protein-protein interactions, resulting in formation of membrane contact sites between the ER and AiV ROs and cholesterol supply to the ROs. IMPORTANCE Positive-strand RNA viruses utilize host pathways to modulate the lipid composition of viral RNA replication sites for replication. Previously, we demonstrated that Aichi virus (AiV), a picornavirus, forms a complex comprising certain proteins of AiV, the Golgi apparatus protein ACBD3, and the lipid kinase PI4KB to synthesize PI4P lipid at the sites for AiV RNA replication. Here, we confirmed cholesterol accumulation at the AiV RNA replication sites, which are established by hijacking the host cholesterol transfer machinery mediated by a PI4P-binding cholesterol transfer protein, OSBP. We showed that the component proteins of the machinery, OSBP, VAP, SAC1, and PITPNB, are all essential host factors for AiV replication. Importantly, the machinery is directly recruited to the RNA replication sites through previously unknown interactions of VAP/OSBP/SAC1 with the AiV proteins and with ACBD3. Consequently, we propose a specific strategy employed by AiV to efficiently accumulate cholesterol at the RNA replication sites via protein-protein interactions. Copyright © 2018 American Society for Microbiology.

Functional organization of cytoplasmic inclusion bodies in cells infected by respiratory syncytial virus.

PubMed

Rincheval, Vincent; Lelek, Mickael; Gault, Elyanne; Bouillier, Camille; Sitterlin, Delphine; Blouquit-Laye, Sabine; Galloux, Marie; Zimmer, Christophe; Eleouet, Jean-François; Rameix-Welti, Marie-Anne

2017-09-15

Infection of cells by respiratory syncytial virus induces the formation of cytoplasmic inclusion bodies (IBs) where all the components of the viral RNA polymerase complex are concentrated. However, the exact organization and function of these IBs remain unclear. In this study, we use conventional and super-resolution imaging to dissect the internal structure of IBs. We observe that newly synthetized viral mRNA and the viral transcription anti-terminator M2-1 concentrate in IB sub-compartments, which we term "IB-associated granules" (IBAGs). In contrast, viral genomic RNA, the nucleoprotein, the L polymerase and its cofactor P are excluded from IBAGs. Live imaging reveals that IBAGs are highly dynamic structures. Our data show that IBs are the main site of viral RNA synthesis. They further suggest that shortly after synthesis in IBs, viral mRNAs and M2-1 transiently concentrate in IBAGs before reaching the cytosol and suggest a novel post-transcriptional function for M2-1.Respiratory syncytial virus (RSV) induces formation of inclusion bodies (IBs) sheltering viral RNA synthesis. Here, Rincheval et al. identify highly dynamic IB-associated granules (IBAGs) that accumulate newly synthetized viral mRNA and the viral M2-1 protein but exclude viral genomic RNA and RNA polymerase complexes.

EVM005: an ectromelia-encoded protein with dual roles in NF-κB inhibition and virulence.

PubMed

van Buuren, Nicholas; Burles, Kristin; Schriewer, Jill; Mehta, Ninad; Parker, Scott; Buller, R Mark; Barry, Michele

2014-08-01

Poxviruses contain large dsDNA genomes encoding numerous open reading frames that manipulate cellular signalling pathways and interfere with the host immune response. The NF-κB signalling cascade is an important mediator of innate immunity and inflammation, and is tightly regulated by ubiquitination at several key points. A critical step in NF-κB activation is the ubiquitination and degradation of the inhibitor of kappaB (IκBα), by the cellular SCFβ-TRCP ubiquitin ligase complex. We show here that upon stimulation with TNFα or IL-1β, Orthopoxvirus-infected cells displayed an accumulation of phosphorylated IκBα, indicating that NF-κB activation was inhibited during poxvirus infection. Ectromelia virus is the causative agent of lethal mousepox, a natural disease that is fatal in mice. Previously, we identified a family of four ectromelia virus genes (EVM002, EVM005, EVM154 and EVM165) that contain N-terminal ankyrin repeats and C-terminal F-box domains that interact with the cellular SCF ubiquitin ligase complex. Since degradation of IκBα is catalyzed by the SCFβ-TRCP ubiquitin ligase, we investigated the role of the ectromelia virus ankyrin/F-box protein, EVM005, in the regulation of NF-κB. Expression of Flag-EVM005 inhibited both TNFα- and IL-1β-stimulated IκBα degradation and p65 nuclear translocation. Inhibition of the NF-κB pathway by EVM005 was dependent on the F-box domain, and interaction with the SCF complex. Additionally, ectromelia virus devoid of EVM005 was shown to inhibit NF-κB activation, despite lacking the EVM005 open reading frame. Finally, ectromelia virus devoid of EVM005 was attenuated in both A/NCR and C57BL/6 mouse models, indicating that EVM005 is required for virulence and immune regulation in vivo.

EVM005: An Ectromelia-Encoded Protein with Dual Roles in NF-κB Inhibition and Virulence

PubMed Central

Schriewer, Jill; Mehta, Ninad; Parker, Scott; Buller, R. Mark; Barry, Michele

2014-01-01

Poxviruses contain large dsDNA genomes encoding numerous open reading frames that manipulate cellular signalling pathways and interfere with the host immune response. The NF-κB signalling cascade is an important mediator of innate immunity and inflammation, and is tightly regulated by ubiquitination at several key points. A critical step in NF-κB activation is the ubiquitination and degradation of the inhibitor of kappaB (IκBα), by the cellular SCFβ-TRCP ubiquitin ligase complex. We show here that upon stimulation with TNFα or IL-1β, Orthopoxvirus-infected cells displayed an accumulation of phosphorylated IκBα, indicating that NF-κB activation was inhibited during poxvirus infection. Ectromelia virus is the causative agent of lethal mousepox, a natural disease that is fatal in mice. Previously, we identified a family of four ectromelia virus genes (EVM002, EVM005, EVM154 and EVM165) that contain N-terminal ankyrin repeats and C-terminal F-box domains that interact with the cellular SCF ubiquitin ligase complex. Since degradation of IκBα is catalyzed by the SCFβ-TRCP ubiquitin ligase, we investigated the role of the ectromelia virus ankyrin/F-box protein, EVM005, in the regulation of NF-κB. Expression of Flag-EVM005 inhibited both TNFα- and IL-1β-stimulated IκBα degradation and p65 nuclear translocation. Inhibition of the NF-κB pathway by EVM005 was dependent on the F-box domain, and interaction with the SCF complex. Additionally, ectromelia virus devoid of EVM005 was shown to inhibit NF-κB activation, despite lacking the EVM005 open reading frame. Finally, ectromelia virus devoid of EVM005 was attenuated in both A/NCR and C57BL/6 mouse models, indicating that EVM005 is required for virulence and immune regulation in vivo. PMID:25122471

Identification and characterization of the host protein DNAJC14 as a broadly active flavivirus replication modulator.

PubMed

Yi, Zhigang; Sperzel, Lindsey; Nürnberger, Cindy; Bredenbeek, Peter J; Lubick, Kirk J; Best, Sonja M; Stoyanov, Cristina T; Law, Lok Man J; Yuan, Zhenghong; Rice, Charles M; MacDonald, Margaret R

2011-01-13

Viruses in the Flavivirus genus of the Flaviviridae family are arthropod-transmitted and contribute to staggering numbers of human infections and significant deaths annually across the globe. To identify cellular factors with antiviral activity against flaviviruses, we screened a cDNA library using an iterative approach. We identified a mammalian Hsp40 chaperone protein (DNAJC14) that when overexpressed was able to mediate protection from yellow fever virus (YFV)-induced cell death. Further studies revealed that DNAJC14 inhibits YFV at the step of viral RNA replication. Since replication of bovine viral diarrhea virus (BVDV), a member of the related Pestivirus genus, is also known to be modulated by DNAJC14, we tested the effect of this host factor on diverse Flaviviridae family members. Flaviviruses, including the pathogenic Asibi strain of YFV, Kunjin, and tick-borne Langat virus, as well as a Hepacivirus, hepatitis C virus (HCV), all were inhibited by overexpression of DNAJC14. Mutagenesis showed that both the J-domain and the C-terminal domain, which mediates self-interaction, are required for anti-YFV activity. We found that DNAJC14 does not block YFV nor HCV NS2-3 cleavage, and using non-inhibitory mutants demonstrate that DNAJC14 is recruited to YFV replication complexes. Immunofluorescence analysis demonstrated that endogenous DNAJC14 rearranges during infection and is found in replication complexes identified by dsRNA staining. Interestingly, silencing of endogenous DNAJC14 results in impaired YFV replication suggesting a requirement for DNAJC14 in YFV replication complex assembly. Finally, the antiviral activity of overexpressed DNAJC14 occurs in a time- and dose-dependent manner. DNAJC14 overexpression may disrupt the proper stoichiometry resulting in inhibition, which can be overcome upon restoration of the optimal ratios due to the accumulation of viral nonstructural proteins. Our findings, together with previously published work, suggest that the members of the Flaviviridae family have evolved in unique and important ways to interact with this host Hsp40 chaperone molecule.

Cholesterol and Fatty Acids Regulate Dynamic Caveolin Trafficking through the Golgi Complex and between the Cell Surface and Lipid BodiesV⃞

PubMed Central

Pol, Albert; Martin, Sally; Fernández, Manuel A.; Ingelmo-Torres, Mercedes; Ferguson, Charles; Enrich, Carlos; Parton, Robert G.

2005-01-01

Caveolins are a crucial component of plasma membrane (PM) caveolae but have also been localized to intracellular compartments, including the Golgi complex and lipid bodies. Mutant caveolins associated with human disease show aberrant trafficking to the PM and Golgi accumulation. We now show that the Golgi pool of mainly newly synthesized protein is detergent-soluble and predominantly in a monomeric state, in contrast to the surface pool. Caveolin at the PM is not recognized by specific caveolin antibodies unless PM cholesterol is depleted. Exit from the Golgi complex of wild-type caveolin-1 or -3, but not vesicular stomatitis virus-G protein, is modulated by changing cellular cholesterol levels. In contrast, a muscular dystrophy-associated mutant of caveolin-3, Cav3P104L, showed increased accumulation in the Golgi complex upon cholesterol treatment. In addition, we demonstrate that in response to fatty acid treatment caveolin can follow a previously undescribed pathway from the PM to lipid bodies and can move from lipid bodies to the PM in response to removal of fatty acids. The results suggest that cholesterol is a rate-limiting component for caveolin trafficking. Changes in caveolin flux through the exocytic pathway can therefore be an indicator of cellular cholesterol and fatty acid levels. PMID:15689493

Fusion between perinuclear virions and the outer nuclear membrane requires the fusogenic activity of herpes simplex virus gB.

PubMed

Wright, Catherine C; Wisner, Todd W; Hannah, Brian P; Eisenberg, Roselyn J; Cohen, Gary H; Johnson, David C

2009-11-01

Herpesviruses cross nuclear membranes (NMs) in two steps, as follows: (i) capsids assemble and bud through the inner NM into the perinuclear space, producing enveloped virus particles, and (ii) the envelopes of these virus particles fuse with the outer NM. Two herpes simplex virus (HSV) glycoproteins, gB and gH (the latter, likely complexed as a heterodimer with gL), are necessary for the second step of this process. Mutants lacking both gB and gH accumulate in the perinuclear space or in herniations (membrane vesicles derived from the inner NM). Both gB and gH/gL are also known to act directly in fusing the virion envelope with host cell membranes during HSV entry into cells, i.e., both glycoproteins appear to function directly in different aspects of the membrane fusion process. We hypothesized that HSV gB and gH/gL also act directly in the membrane fusion that occurs during virus egress from the nucleus. Previous studies of the role of gB and gH/gL in nuclear egress involved HSV gB and gH null mutants that could potentially also possess gross defects in the virion envelope. Here, we produced recombinant HSV-expressing mutant forms of gB with single amino acid substitutions in the hydrophobic "fusion loops." These fusion loops are thought to play a direct role in membrane fusion by insertion into cellular membranes. HSV recombinants expressing gB with any one of four fusion loop mutations (W174R, W174Y, Y179K, and A261D) were unable to enter cells. Moreover, two of the mutants, W174Y and Y179K, displayed reduced abilities to mediate HSV cell-to-cell spread, and W174R and A261D exhibited no spread. All mutant viruses exhibited defects in nuclear egress, enveloped virions accumulated in herniations and in the perinuclear space, and fewer enveloped virions were detected on cell surfaces. These results support the hypothesis that gB functions directly to mediate the fusion between perinuclear virus particles and the outer NM.

Association of Satellites with a Mastrevirus in Natural Infection: Complexity of Wheat Dwarf India Virus Disease

PubMed Central

Kumar, Jitendra; Kumar, Jitesh; Singh, Sudhir P.

2014-01-01

ABSTRACT In contrast to begomoviruses, mastreviruses have not previously been shown to interact with satellites. This study reports the first identification of the association of satellites with a mastrevirus in field-grown plants. Two alphasatellite species were detected in different field samples of wheat infected with Wheat Dwarf India Virus (WDIV), a Cotton leaf curl Multan alphasatellite (CLCuMA) and a Guar leaf curl alphasatellite (GLCuA). In addition to the alphasatellites, a betasatellite, Ageratum yellow leaf curl betasatellite (AYLCB), was also identified in the wheat samples. No begomovirus was detected in the wheat samples, thus establishing association of the above-named satellites with WDIV. Agrobacterium-mediated inoculation of WDIV in wheat, in the presence of either of the alphasatellites or the betasatellite, resulted in infections inducing more severe symptoms. WDIV efficiently maintained each of the alphasatellites and the betasatellite in wheat. The satellites enhanced the level of WDIV DNA in wheat. Inoculation of the satellites isolated from wheat with various begomoviruses into Nicotiana tabacum demonstrated that these remain capable of interacting with the viruses with which they were first identified. Virus-specific small RNAs accumulated in wheat upon infection with WDIV but were lower in abundance in plants coinfected with the satellites, suggesting that both the alphasatellites and the betasatellite suppress RNA silencing. These results suggest that the selective advantage for the maintenance of the alphasatellites and the betasatellite by WDIV in the field is in overcoming RNA silencing-mediated host defense. IMPORTANCE Wheat is the most widely cultivated cereal crop in the world. A number of viruses are important pathogens of wheat, including the viruses of the genus Mastrevirus, family Geminiviridae. This study reports the association of subgenomic components, called satellites (alpha- and betasatellites), with a mastrevirus, Wheat Dwarf India Virus (WDIV), isolated from two distant locations in India. This study reports the first identification of the satellites in a monocot plant. The satellites enhanced accumulation of WDIV and severity of disease symptoms. The satellites lowered the concentration of virus-specific small RNAs in wheat plants, indicating their silencing suppressor activity. The involvement of the satellites in symptom severity of the mastrevirus can have implications in the form of economic impact of the virus on crop yield. Understanding the role of the satellites in disease severity is important for developing disease management strategies. PMID:24719407

Biology, etiology, and control of virus diseases of banana and plantain.

PubMed

Kumar, P Lava; Selvarajan, Ramasamy; Iskra-Caruana, Marie-Line; Chabannes, Matthieu; Hanna, Rachid

2015-01-01

Banana and plantain (Musa spp.), produced in 10.3 million ha in the tropics, are among the world's top 10 food crops. They are vegetatively propagated using suckers or tissue culture plants and grown almost as perennial plantations. These are prone to the accumulation of pests and pathogens, especially viruses which contribute to yield reduction and are also barriers to the international exchange of germplasm. The most economically important viruses of banana and plantain are Banana bunchy top virus (BBTV), a complex of banana streak viruses (BSVs) and Banana bract mosaic virus (BBrMV). BBTV is known to cause the most serious economic losses in the "Old World," contributing to a yield reduction of up to 100% and responsible for a dramatic reduction in cropping area. The BSVs exist as episomal and endogenous forms are known to be worldwide in distribution. In India and the Philippines, BBrMV is known to be economically important but recently the virus was discovered in Colombia and Costa Rica, thus signaling its spread into the "New World." Banana and plantain are also known to be susceptible to five other viruses of minor significance, such as Abaca mosaic virus, Abaca bunchy top virus, Banana mild mosaic virus, Banana virus X, and Cucumber mosaic virus. Studies over the past 100 years have contributed to important knowledge on disease biology, distribution, and spread. Research during the last 25 years have led to a better understanding of the virus-vector-host interactions, virus diversity, disease etiology, and epidemiology. In addition, new diagnostic tools were developed which were used for surveillance and the certification of planting material. Due to a lack of durable host resistance in the Musa spp., phytosanitary measures and the use of virus-free planting material are the major methods of virus control. The state of knowledge on BBTV, BBrMV, and BSVs, and other minor viruses, disease spread, and control are summarized in this review. © 2015 Elsevier Inc. All rights reserved.

Modulation of Re-initiation of Measles Virus Transcription at Intergenic Regions by PXD to NTAIL Binding Strength.

PubMed

Bloyet, Louis-Marie; Brunel, Joanna; Dosnon, Marion; Hamon, Véronique; Erales, Jenny; Gruet, Antoine; Lazert, Carine; Bignon, Christophe; Roche, Philippe; Longhi, Sonia; Gerlier, Denis

2016-12-01

Measles virus (MeV) and all Paramyxoviridae members rely on a complex polymerase machinery to ensure viral transcription and replication. Their polymerase associates the phosphoprotein (P) and the L protein that is endowed with all necessary enzymatic activities. To be processive, the polymerase uses as template a nucleocapsid made of genomic RNA entirely wrapped into a continuous oligomer of the nucleoprotein (N). The polymerase enters the nucleocapsid at the 3'end of the genome where are located the promoters for transcription and replication. Transcription of the six genes occurs sequentially. This implies ending and re-initiating mRNA synthesis at each intergenic region (IGR). We explored here to which extent the binding of the X domain of P (XD) to the C-terminal region of the N protein (NTAIL) is involved in maintaining the P/L complex anchored to the nucleocapsid template during the sequential transcription. Amino acid substitutions introduced in the XD-binding site on NTAIL resulted in a wide range of binding affinities as determined by combining protein complementation assays in E. coli and human cells and isothermal titration calorimetry. Molecular dynamics simulations revealed that XD binding to NTAIL involves a complex network of hydrogen bonds, the disruption of which by two individual amino acid substitutions markedly reduced the binding affinity. Using a newly designed, highly sensitive dual-luciferase reporter minigenome assay, the efficiency of re-initiation through the five measles virus IGRs was found to correlate with NTAIL/XD KD. Correlatively, P transcript accumulation rate and F/N transcript ratios from recombinant viruses expressing N variants were also found to correlate with the NTAIL to XD binding strength. Altogether, our data support a key role for XD binding to NTAIL in maintaining proper anchor of the P/L complex thereby ensuring transcription re-initiation at each intergenic region.

Modulation of Re-initiation of Measles Virus Transcription at Intergenic Regions by PXD to NTAIL Binding Strength

PubMed Central

Hamon, Véronique; Erales, Jenny; Bignon, Christophe; Roche, Philippe

2016-01-01

Measles virus (MeV) and all Paramyxoviridae members rely on a complex polymerase machinery to ensure viral transcription and replication. Their polymerase associates the phosphoprotein (P) and the L protein that is endowed with all necessary enzymatic activities. To be processive, the polymerase uses as template a nucleocapsid made of genomic RNA entirely wrapped into a continuous oligomer of the nucleoprotein (N). The polymerase enters the nucleocapsid at the 3’end of the genome where are located the promoters for transcription and replication. Transcription of the six genes occurs sequentially. This implies ending and re-initiating mRNA synthesis at each intergenic region (IGR). We explored here to which extent the binding of the X domain of P (XD) to the C-terminal region of the N protein (NTAIL) is involved in maintaining the P/L complex anchored to the nucleocapsid template during the sequential transcription. Amino acid substitutions introduced in the XD-binding site on NTAIL resulted in a wide range of binding affinities as determined by combining protein complementation assays in E. coli and human cells and isothermal titration calorimetry. Molecular dynamics simulations revealed that XD binding to NTAIL involves a complex network of hydrogen bonds, the disruption of which by two individual amino acid substitutions markedly reduced the binding affinity. Using a newly designed, highly sensitive dual-luciferase reporter minigenome assay, the efficiency of re-initiation through the five measles virus IGRs was found to correlate with NTAIL/XD KD. Correlatively, P transcript accumulation rate and F/N transcript ratios from recombinant viruses expressing N variants were also found to correlate with the NTAIL to XD binding strength. Altogether, our data support a key role for XD binding to NTAIL in maintaining proper anchor of the P/L complex thereby ensuring transcription re-initiation at each intergenic region. PMID:27936158

Teratology Studies of Lewisite and Sulfur Mustard Agents: Effects of Lewisite in Rats and Rabbits

DTIC Science & Technology

1987-12-31

virus of mice (PCM), rat corona virus /sialodacryoadenitis virus (RCV/SDA), H-1 virus and Kilham rat virus (KRV) by Microbiological Associates...Pneumonia virus of mice RCV/SDA = Rat corona virus /sialodacryoadenitis virus RH = Relative humidity SC = Subcutaneous SD = Standard deviation SE = Standard... cat , rabbit and human but apparently did not cross the placental membranes readily. The accumulation of a sufficient quantity of arsenate to induce a

Melon Resistance to Cucurbit yellow stunting disorder virus Is Characterized by Reduced Virus Accumulation.

PubMed

Marco, Cristina F; Aguilar, Juan M; Abad, Jesús; Gómez-Guillamón, María Luisa; Aranda, Miguel A

2003-07-01

ABSTRACT The pattern of accumulation of Cucurbit yellow stunting disorder virus (CYSDV; genus Crinivirus, family Closteroviridae) RNA has been analyzed in several cucurbit accessions. In susceptible accessions of melon (Cucumis melo), cucumber (Cucumis sativus), marrow (Cucurbita maxima), and squash (Cucurbita pepo), CYSDV RNA accumulation peaked during the first to second week postinoculation in the first to third leaf above the inoculated one; younger leaves showed very low or undetectable levels of CYSDV. Three melon accessions previously shown to remain asymptomatic after CYSDV inoculation under natural conditions were also assayed for their susceptibility to CYSDV. Hybridization and reverse transcription-polymerase chain reaction (RT-PCR) analysis of noninoculated leaves showed that only one of these, C-105, remained virus-free for up to 6 weeks after whitefly inoculation. In this accession, very low CYSDV levels were detected by RT-PCR in whitefly-inoculated leaves, and therefore, multiplication or spread of CYSDV in C-105 plants appeared to remain restricted to the inoculated leaves. When C-105 plants were graft inoculated, CYSDV RNA could be detected in phloem tissues, but the systemic colonization of C-105 by CYSDV upon graft inoculation seemed to be seriously impeded. Additionally, in situ hybridization experiments showed that, after C-105 graft inoculation, only a portion of the vascular bundles in petioles and stems were colonized by CYSDV and virus could not be found in leaf veins. RT-PCR experiments using primers to specifically detect negative-sense CYSDV RNA were carried out and showed that CYSDV replication took place in graft-inoculated C-105 scions. Therefore, the resistance mechanism may involve a restriction of the virus movement in the vascular system of the plants and/or prevention of high levels of virus accumulation.

Viral carcinogenesis: revelation of molecular mechanisms and etiology of human disease

NASA Technical Reports Server (NTRS)

Butel, J. S.

2000-01-01

The RNA and DNA tumor viruses have made fundamental contributions to two major areas of cancer research. Viruses were vital, first, to the discovery and analysis of cellular growth control pathways and the synthesis of current concepts of cancer biology and, second, to the recognition of the etiology of some human cancers. Transforming retroviruses carry oncogenes derived from cellular genes that are involved in mitogenic signalling and growth control. DNA tumor viruses encode oncogenes of viral origin that are essential for viral replication and cell transformation; viral oncoproteins complex with cellular proteins to stimulate cell cycle progression and led to the discovery of tumor suppressors. Viral systems support the concept that cancer development occurs by the accumulation of multiple cooperating events. Viruses are now accepted as bona fide etiologic factors of human cancer; these include hepatitis B virus, Epstein-Barr virus, human papillomaviruses, human T-cell leukemia virus type I and hepatitis C virus, plus several candidate human cancer viruses. It is estimated that 15% of all human tumors worldwide are caused by viruses. The infectious nature of viruses distinguishes them from all other cancer-causing factors; tumor viruses establish long-term persistent infections in humans, with cancer an accidental side effect of viral replication strategies. Viruses are usually not complete carcinogens, and the known human cancer viruses display different roles in transformation. Many years may pass between initial infection and tumor appearance and most infected individuals do not develop cancer, although immunocompromised individuals are at elevated risk of viral-associated cancers. Variable factors that influence viral carcinogenesis are reviewed, including possible synergy between viruses and environmental cofactors. The difficulties in establishing an etiologic role for a virus in human cancer are discussed, as well as the different approaches that proved viral links to cancer. Future directions for tumor virus studies are considered.

Diverging affinity of tospovirus RNA silencing suppressor proteins, NSs, for various RNA duplex molecules.

PubMed

Schnettler, Esther; Hemmes, Hans; Huismann, Rik; Goldbach, Rob; Prins, Marcel; Kormelink, Richard

2010-11-01

The tospovirus NSs protein was previously shown to suppress the antiviral RNA silencing mechanism in plants. Here the biochemical analysis of NSs proteins from different tospoviruses, using purified NSs or NSs containing cell extracts, is described. The results showed that all tospoviral NSs proteins analyzed exhibited affinity to small double-stranded RNA molecules, i.e., small interfering RNAs (siRNAs) and micro-RNA (miRNA)/miRNA* duplexes. Interestingly, the NSs proteins from tomato spotted wilt virus (TSWV), impatiens necrotic spot virus (INSV), and groundnut ringspot virus (GRSV) also showed affinity to long double-stranded RNA (dsRNA), whereas tomato yellow ring virus (TYRV) NSs did not. The TSWV NSs protein was shown to be capable of inhibiting Dicer-mediated cleavage of long dsRNA in vitro. In addition, it suppressed the accumulation of green fluorescent protein (GFP)-specific siRNAs during coinfiltration with an inverted-repeat-GFP RNA construct in Nicotiana benthamiana. In vivo interference of TSWV NSs in the miRNA pathway was shown by suppression of an enhanced GFP (eGFP) miRNA sensor construct. The ability to stabilize miRNA/miRNA* by different tospovirus NSs proteins in vivo was demonstrated by increased accumulation and detection of both miRNA171c and miRNA171c* in tospovirus-infected N. benthamiana. All together, these data suggest that tospoviruses interfere in the RNA silencing pathway by sequestering siRNA and miRNA/miRNA* molecules before they are uploaded into their respective RNA-induced silencing complexes. The observed affinity to long dsRNA for only a subset of the tospoviruses studied is discussed in light of evolutional divergence and their ancestral relation to the animal-infecting members of the Bunyaviridae.

Rad51 Interacts with Non-structural 3 Protein of Hepatitis C Virus and Regulates Viral Production

PubMed Central

Son, Kidong; Nguyen, Tram T. T.; Choi, Jae-Woong; Pham, Long V.; Luong, Trang T. D.; Lim, Yun-Sook; Hwang, Soon B.

2017-01-01

Hepatitis C virus (HCV) is a leading cause of chronic liver disease affecting over 170 million people worldwide. Chronic infection with HCV progresses to liver fibrosis, cirrhosis, and hepatocellular carcinoma. HCV exploits host cellular factors for viral propagation. To investigate the cellular factors required for HCV propagation, we screened a siRNA library targeting human cell cycle genes using cell culture grown HCV-infected cells. In the present study, we selected and characterized a gene encoding Rad51. Rad51, a member of a conserved recombinase family, is an essential factor for homologous recombination and repair of double-strand DNA breaks. We demonstrated that siRNA-mediated knockdown of Rad51 significantly inhibited HCV propagation without affecting HCV RNA replication. Silencing of Rad51 impaired secretion of infectious HCV particles and thus intracellular viruses were accumulated. We showed that HCV NS3 specifically interacted with Rad51 and accumulated Rad51 in the cytosol. Furthermore, Rad51 was coprecipitated with NS3 and HCV RNA. By employing membrane flotation and protease protection assays, we also demonstrated that Rad51 was co-fractionated with HCV NS3 on the lipid raft. These data indicate that Rad51 may be a component of the HCV RNA replication complex. Collectively, these data suggest that HCV may exploit cellular Rad51 to promote viral propagation and thus Rad51 may be a potential therapeutic target for HCV. PMID:28729862

Resistance to Cucurbit aphid-borne yellows virus in Melon Accession TGR-1551.

PubMed

Kassem, Mona A; Gosalvez, Blanca; Garzo, Elisa; Fereres, Alberto; Gómez-Guillamón, Maria Luisa; Aranda, Miguel A

2015-10-01

The genetic control of resistance to Cucurbit aphid-borne yellows virus (CABYV; genus Polerovirus, family Luteoviridae) in the TGR-1551 melon accession was studied through agroinoculation of a genetic family obtained from the cross between this accession and the susceptible Spanish cultivar 'Bola de Oro'. Segregation analyses were consistent with the hypothesis that one dominant gene and at least two more modifier genes confer resistance; one of these additional genes is likely present in the susceptible parent 'Bola de Oro'. Local and systemic accumulation of the virus was analyzed in a time course experiment, showing that TGR-1551 resistance was expressed systemically as a significant reduction of virus accumulation compared with susceptible controls, but not locally in agroinoculated cotyledons. In aphid transmission experiments, CABYV inoculation by aphids was significantly reduced in TGR-1551 plants, although the virus was acquired at a similar rate from TGR-1551 as from susceptible plants. Results of feeding behavior studies using the DC electrical penetration graph technique suggested that viruliferous aphids can salivate and feed from the phloem of TGR-1551 plants and that the observed reduction in virus transmission efficiency is not related to reduced salivation by Aphis gossypii in phloem sieve elements. Since the virus is able to accumulate to normal levels in agroinoculated tissues, our results suggest that resistance of TGR-1551 plants to CABYV is related to impairment of virus movement or translocation after it reaches the phloem sieve elements.

The Epstein-Barr virus miR-BHRF1-1 targets RNF4 during productive infection to promote the accumulation of SUMO conjugates and the release of infectious virus.

PubMed

Li, Jinlin; Callegari, Simone; Masucci, Maria G

2017-04-01

Post-translational modification by the Small Ubiquitin-like Modifier (SUMO) regulates a variety of cellular functions, and is hijacked by viruses to remodel the host cell during latent and productive infection. Here we have monitored the activity of the SUMO conjugation machinery in cells productively infected with Epstein-Barr virus (EBV). We found that SUMO2/3 conjugates accumulate during the late phase of the productive virus cycle, and identified several viral proteins as bone fide SUMOylation substrates. Analysis of the mechanism involved in the accumulation of SUMOylated proteins revealed upregulation of several components of the SUMO-conjugation machinery and post-transcriptional downregulation of the SUMO-targeted ubiquitin ligase RNF4. The latter effect was mediated by selective inhibition of RNF4 protein expression by the viral miR-BHRF1-1. Reconstitution of RNF4 in cells expressing an inducible miR-BHRF1-1 sponge or a miR-BHRF1-1 resistant RNF4 was associated with reduced levels of early and late viral proteins and impaired virus release. These findings illustrate a novel strategy for viral interference with the SUMO pathway, and identify the EBV miR-BHRF1-1 and the cellular RNF4 as regulators of the productive virus cycle.

The Epstein-Barr virus miR-BHRF1-1 targets RNF4 during productive infection to promote the accumulation of SUMO conjugates and the release of infectious virus

PubMed Central

Li, Jinlin; Callegari, Simone

2017-01-01

Post-translational modification by the Small Ubiquitin-like Modifier (SUMO) regulates a variety of cellular functions, and is hijacked by viruses to remodel the host cell during latent and productive infection. Here we have monitored the activity of the SUMO conjugation machinery in cells productively infected with Epstein-Barr virus (EBV). We found that SUMO2/3 conjugates accumulate during the late phase of the productive virus cycle, and identified several viral proteins as bone fide SUMOylation substrates. Analysis of the mechanism involved in the accumulation of SUMOylated proteins revealed upregulation of several components of the SUMO-conjugation machinery and post-transcriptional downregulation of the SUMO-targeted ubiquitin ligase RNF4. The latter effect was mediated by selective inhibition of RNF4 protein expression by the viral miR-BHRF1-1. Reconstitution of RNF4 in cells expressing an inducible miR-BHRF1-1 sponge or a miR-BHRF1-1 resistant RNF4 was associated with reduced levels of early and late viral proteins and impaired virus release. These findings illustrate a novel strategy for viral interference with the SUMO pathway, and identify the EBV miR-BHRF1-1 and the cellular RNF4 as regulators of the productive virus cycle. PMID:28414785

Influenza A Virus Host Shutoff Disables Antiviral Stress-Induced Translation Arrest

PubMed Central

Khaperskyy, Denys A.; Emara, Mohamed M.; Johnston, Benjamin P.; Anderson, Paul; Hatchette, Todd F.; McCormick, Craig

2014-01-01

Influenza A virus (IAV) polymerase complexes function in the nucleus of infected cells, generating mRNAs that bear 5′ caps and poly(A) tails, and which are exported to the cytoplasm and translated by host machinery. Host antiviral defences include mechanisms that detect the stress of virus infection and arrest cap-dependent mRNA translation, which normally results in the formation of cytoplasmic aggregates of translationally stalled mRNA-protein complexes known as stress granules (SGs). It remains unclear how IAV ensures preferential translation of viral gene products while evading stress-induced translation arrest. Here, we demonstrate that at early stages of infection both viral and host mRNAs are sensitive to drug-induced translation arrest and SG formation. By contrast, at later stages of infection, IAV becomes partially resistant to stress-induced translation arrest, thereby maintaining ongoing translation of viral gene products. To this end, the virus deploys multiple proteins that block stress-induced SG formation: 1) non-structural protein 1 (NS1) inactivates the antiviral double-stranded RNA (dsRNA)-activated kinase PKR, thereby preventing eIF2α phosphorylation and SG formation; 2) nucleoprotein (NP) inhibits SG formation without affecting eIF2α phosphorylation; 3) host-shutoff protein polymerase-acidic protein-X (PA-X) strongly inhibits SG formation concomitant with dramatic depletion of cytoplasmic poly(A) RNA and nuclear accumulation of poly(A)-binding protein. Recombinant viruses with disrupted PA-X host shutoff function fail to effectively inhibit stress-induced SG formation. The existence of three distinct mechanisms of IAV-mediated SG blockade reveals the magnitude of the threat of stress-induced translation arrest during viral replication. PMID:25010204

Japanese encephalitis virus replication is negatively regulated by autophagy and occurs on LC3-I- and EDEM1-containing membranes.

PubMed

Sharma, Manish; Bhattacharyya, Sankar; Nain, Minu; Kaur, Manpreet; Sood, Vikas; Gupta, Vishal; Khasa, Renu; Abdin, Malik Z; Vrati, Sudhanshu; Kalia, Manjula

2014-09-01

Autophagy is a lysosomal degradative pathway that has diverse physiological functions and plays crucial roles in several viral infections. Here we examine the role of autophagy in the life cycle of JEV, a neurotropic flavivirus. JEV infection leads to induction of autophagy in several cell types. JEV replication was significantly enhanced in neuronal cells where autophagy was rendered dysfunctional by ATG7 depletion, and in Atg5-deficient mouse embryonic fibroblasts (MEFs), resulting in higher viral titers. Autophagy was functional during early stages of infection however it becomes dysfunctional as infection progressed resulting in accumulation of misfolded proteins. Autophagy-deficient cells were highly susceptible to virus-induced cell death. We also observed JEV replication complexes that are marked by nonstructural protein 1 (NS1) and dsRNA colocalized with endogenous LC3 but not with GFP-LC3. Colocalization of NS1 and LC3 was also observed in Atg5 deficient MEFs, which contain only the nonlipidated form of LC3. Viral replication complexes furthermore show association with a marker of the ER-associated degradation (ERAD) pathway, EDEM1 (ER degradation enhancer, mannosidase α-like 1). Our data suggest that virus replication occurs on ERAD-derived EDEM1 and LC3-I-positive structures referred to as EDEMosomes. While silencing of ERAD regulators EDEM1 and SEL1L suppressed JEV replication, LC3 depletion exerted a profound inhibition with significantly reduced RNA levels and virus titers. Our study suggests that while autophagy is primarily antiviral for JEV and might have implications for disease progression and pathogenesis of JEV, nonlipidated LC3 plays an important autophagy independent function in the virus life cycle.

Distribution of Potato virus Y in potato plant organs, tissues, and cells.

PubMed

Kogovšek, P; Kladnik, A; Mlakar, J; Znidarič, M Tušek; Dermastia, M; Ravnikar, M; Pompe-Novak, M

2011-11-01

The distribution of Potato virus Y (PVY) in the systemically infected potato (Solanum tuberosum) plants of the highly susceptible cultivar Igor was investigated. Virus presence and accumulation was analyzed in different plant organs and tissues using real-time polymerase chain reaction and transmission electron microscopy (TEM) negative staining methods. To get a complete insight into the location of viral RNA within the tissue, in situ hybridization was developed and optimized for the detection of PVY RNA at the cellular level. PVY was shown to accumulate in all studied leaf and stem tissues, in shoot tips, roots, and tubers; however, the level of virus accumulation was specific for each organ or tissue. The highest amounts of viral RNA and viral particles were found in symptomatic leaves and stem. By observing cell ultrastructure with TEM, viral cytoplasmic inclusion bodies were localized in close vicinity to the epidermis and in trichomes. Our results show that viral RNA, viral particles, and cytoplasmic inclusion bodies colocalize within the same type of cells or in close vicinity.

Modulation of DNA Damage and Repair Pathways by Human Tumour Viruses

PubMed Central

Hollingworth, Robert; Grand, Roger J

2015-01-01

With between 10% and 15% of human cancers attributable to viral infection, there is great interest, from both a scientific and clinical viewpoint, as to how these pathogens modulate host cell functions. Seven human tumour viruses have been identified as being involved in the development of specific malignancies. It has long been known that the introduction of chromosomal aberrations is a common feature of viral infections. Intensive research over the past two decades has subsequently revealed that viruses specifically interact with cellular mechanisms responsible for the recognition and repair of DNA lesions, collectively known as the DNA damage response (DDR). These interactions can involve activation and deactivation of individual DDR pathways as well as the recruitment of specific proteins to sites of viral replication. Since the DDR has evolved to protect the genome from the accumulation of deleterious mutations, deregulation is inevitably associated with an increased risk of tumour formation. This review summarises the current literature regarding the complex relationship between known human tumour viruses and the DDR and aims to shed light on how these interactions can contribute to genomic instability and ultimately the development of human cancers. PMID:26008701

Plants transformed with a tobacco mosaic virus nonstructural gene sequence are resistant to the virus.

PubMed Central

Golemboski, D B; Lomonossoff, G P; Zaitlin, M

1990-01-01

Nicotiana tabacum cv. Xanthi nn plants were transformed with nucleotides 3472-4916 of tobacco mosaic virus (TMV) strain U1. This sequence contains all but the three 3 terminal nucleotides of the TMV 54-kDa gene, which encodes a putative component of the replicase complex. These plants were resistant to infection when challenged with either TMV U1 virions or TMV U1 RNA at concentrations of up to 500 micrograms/ml or 300 micrograms/ml, respectively, the highest concentrations tested. Resistance was also exhibited when plants were inoculated at 100 micrograms/ml with the closely related TMV mutant YSI/1 but was not shown in plants challenged at the same concentrations with the more distantly related TMV strains U2 or L or cucumber mosaic virus. Although the copy number of the 54-kDa gene sequence varied in individual transformants from 1 to approximately 5, the level of resistance in plants was not dependent on the number of copies of the 54-kDa gene sequence integrated. The transformed plants accumulated a 54-kDa gene sequence-specific RNA transcript of the expected size, but no protein product was detected. Images PMID:2385595

Fluorescent protein-tagged Vpr dissociates from HIV-1 core after viral fusion and rapidly enters the cell nucleus.

PubMed

Desai, Tanay M; Marin, Mariana; Sood, Chetan; Shi, Jiong; Nawaz, Fatima; Aiken, Christopher; Melikyan, Gregory B

2015-10-29

HIV-1 Vpr is recruited into virions during assembly and appears to remain associated with the viral core after the reverse transcription and uncoating steps of entry. This feature has prompted the use of fluorescently labeled Vpr to visualize viral particles and to follow trafficking of post-fusion HIV-1 cores in the cytoplasm. Here, we tracked single pseudovirus entry and fusion and observed that fluorescently tagged Vpr gradually dissociates from post-fusion viral cores over the course of several minutes and accumulates in the nucleus. Kinetics measurements showed that fluorescent Vpr released from the cores very rapidly entered the cell nucleus. More than 10,000 Vpr molecules can be delivered into the cell nucleus within 45 min of infection by HIV-1 particles pseudotyped with the avian sarcoma and leukosis virus envelope glycoprotein. The fraction of Vpr from cell-bound viruses that accumulated in the nucleus was proportional to the extent of virus-cell fusion and was fully blocked by viral fusion inhibitors. Entry of virus-derived Vpr into the nucleus occurred independently of envelope glycoproteins or target cells. Fluorescence correlation spectroscopy revealed two forms of nuclear Vpr-monomers and very large complexes, likely involving host factors. The kinetics of viral Vpr entering the nucleus after fusion was not affected by point mutations in the capsid protein that alter the stability of the viral core. The independence of Vpr shedding of capsid stability and its relatively rapid dissociation from post-fusion cores suggest that this process may precede capsid uncoating, which appears to occur on a slower time scale. Our results thus demonstrate that a bulk of fluorescently labeled Vpr incorporated into HIV-1 particles is released shortly after fusion. Future studies will address the question whether the quick and efficient nuclear delivery of Vpr derived from incoming viruses can regulate subsequent steps of HIV-1 infection.

Rift Valley fever virus NS{sub S} gene expression correlates with a defect in nuclear mRNA export

DOE Office of Scientific and Technical Information (OSTI.GOV)

Copeland, Anna Maria; Van Deusen, Nicole M.; Schmaljohn, Connie S., E-mail: Connie.s.schmaljohn.civ@mail.mil

We investigated the localization of host mRNA during Rift Valley fever virus (RVFV) infection. Fluorescence in situ hybridization revealed that infection with RVFV altered the localization of host mRNA. mRNA accumulated in the nuclei of RVFV-infected but not mock-infected cells. Further, overexpression of the NS{sub S} gene, but not the N, G{sub N} or NS{sub M} genes correlated with mRNA nuclear accumulation. Nuclear accumulation of host mRNA was not observed in cells infected with a strain of RVFV lacking the gene encoding NS{sub S}, confirming that expression of NS{sub S} is likely responsible for this phenomenon. - Highlights: • Riftmore » Valley fever virus (RVFV) infection alters the localization of host mRNA. • mRNA accumulates in the nuclei of RVFV-infected but not mock-infected cells. • NS{sub S} is likely responsible for mRNA relocalization to the nucleus.« less

Nuclear-cytoplasmic partitioning of cucumber mosaic virus protein 2b determines the balance between its roles as a virulence determinant and an RNA-silencing suppressor.

PubMed

Du, Zhiyou; Chen, Aizhong; Chen, Wenhu; Liao, Qiansheng; Zhang, Hengmu; Bao, Yiming; Roossinck, Marilyn J; Carr, John P

2014-05-01

The Cucumber Mosaic Virus (CMV) 2b protein is an RNA-silencing suppressor that plays roles in CMV accumulation and virulence. The 2b proteins of subgroup IA CMV strains partition between the nucleus and cytoplasm, but the biological significance of this is uncertain. We fused an additional nuclear localization signal (NLS) to the 2b protein of subgroup IA strain Fny-CMV to create 2b-NLS and tested its effects on subcellular distribution, silencing, and virulence. The additional NLS enhanced 2b protein nuclear and nucleolar accumulation, but nuclear and nucleolar enrichment correlated with markedly diminished silencing suppressor activity in patch assays and abolished 2b protein-mediated disruption of microRNA activity in transgenic Arabidopsis. Nucleus/nucleolus-localized 2b protein possesses at least some ability to inhibit antiviral silencing, but this was not sufficient to prevent recovery from disease in younger, developing leaves in Arabidopsis. However, enhanced nuclear and nucleolar accumulation of 2b increased virulence and accelerated symptom appearance in older leaves. Experiments with Arabidopsis lines carrying mutant Dicer-like alleles demonstrated that compromised suppressor activity explained the diminished ability of 2b-NLS to enhance virus accumulation. Remarkably, the increased virulence that 2b-NLS engendered was unrelated to effects on microRNA- or short interfering RNA-regulated host functions. Thus, although nucleus- and nucleolus-localized 2b protein is less efficient at silencing suppression than cytoplasm-localized 2b, it enhances CMV virulence. We propose that partitioning of the 2b protein between the cytoplasmic and nuclear/nucleolar compartments allows CMV to regulate the balance between virus accumulation and damage to the host, presumably to maximize the benefit for the virus. In this work, the main finding is that nucleus/nucleolus-localized 2b protein is strongly associated with CMV virulence, which is independent of its effect on small RNA pathways. Moreover, this work supports the contention that the silencing suppressor activity of CMV 2b protein is predominantly exerted by that portion of the 2b protein residing in the cytoplasm. Thus, we propose that partitioning of the 2b protein between the cytoplasmic and nuclear/nucleolar compartments allows CMV to regulate the balance between virus accumulation and damage to the host, presumably to maximize the benefit for the virus.

Interaction between Cucumber mosaic virus 2b protein and plant catalase induces a specific necrosis in association with proteasome activity.

PubMed

Murota, Katsunori; Shimura, Hanako; Takeshita, Minoru; Masuta, Chikara

2017-01-01

Cucumber mosaic virus (CMV) can induce a specific necrosis on Arabidopsis through the interaction between the CMV 2b protein and host catalase, in which the ubiquitin-proteasome pathway may be involved. We previously reported that the CMV 2b protein, the viral RNA silencing suppressor, interacted with the H 2 O 2 scavenger catalase (CAT3), leading to necrosis on CMV-inoculated Arabidopsis leaves. We here confirmed that CMV could more abundantly accumulate in the CAT3-knockout mutant (cat3), and that CAT3 makes host plants a little more tolerant to CMV. We also found that the necrosis severity is not simply explained by a high level of H 2 O 2 given by the lack of CAT3, because the recombinant CMV, CMV-N, induced much milder necrosis in cat3 than in the wild type, suggesting some specific mechanism for the necrosis induction. To further characterize the 2b-inducing necrosis in relation to its binding to CAT3, we conducted the agroinfiltration experiments to overexpress CAT3 and 2b in N. benthamiana leaves. The accumulation levels of CAT3 were higher when co-expressed with the CMV-N 2b (N2b) than with CMV-Y 2b (Y2b). We infer that N2b made a more stable complex with CAT3 than Y2b did, and the longevity of the 2b-CAT3 complex seemed to be important to induce necrosis. By immunoprecipitation (IP) with an anti-ubiquitin antibody followed by the detection with anti-CAT3 antibodies, we detected a higher molecular-weight smear and several breakdown products of CAT3 among the IP-proteins. In addition, the proteasome inhibitor MG132 treatment could actually increase the accumulation levels of CAT3. This study suggests that the host proteasome pathway is, at least partially, responsible for the degradation of CAT3, which is manifested in CMV-infected tissues.

Polarized release of T-cell-receptor-enriched microvesicles at the immunological synapse.

PubMed

Choudhuri, Kaushik; Llodrá, Jaime; Roth, Eric W; Tsai, Jones; Gordo, Susana; Wucherpfennig, Kai W; Kam, Lance C; Stokes, David L; Dustin, Michael L

2014-03-06

The recognition events that mediate adaptive cellular immunity and regulate antibody responses depend on intercellular contacts between T cells and antigen-presenting cells (APCs). T-cell signalling is initiated at these contacts when surface-expressed T-cell receptors (TCRs) recognize peptide fragments (antigens) of pathogens bound to major histocompatibility complex molecules (pMHC) on APCs. This, along with engagement of adhesion receptors, leads to the formation of a specialized junction between T cells and APCs, known as the immunological synapse, which mediates efficient delivery of effector molecules and intercellular signals across the synaptic cleft. T-cell recognition of pMHC and the adhesion ligand intercellular adhesion molecule-1 (ICAM-1) on supported planar bilayers recapitulates the domain organization of the immunological synapse, which is characterized by central accumulation of TCRs, adjacent to a secretory domain, both surrounded by an adhesive ring. Although accumulation of TCRs at the immunological synapse centre correlates with T-cell function, this domain is itself largely devoid of TCR signalling activity, and is characterized by an unexplained immobilization of TCR-pMHC complexes relative to the highly dynamic immunological synapse periphery. Here we show that centrally accumulated TCRs are located on the surface of extracellular microvesicles that bud at the immunological synapse centre. Tumour susceptibility gene 101 (TSG101) sorts TCRs for inclusion in microvesicles, whereas vacuolar protein sorting 4 (VPS4) mediates scission of microvesicles from the T-cell plasma membrane. The human immunodeficiency virus polyprotein Gag co-opts this process for budding of virus-like particles. B cells bearing cognate pMHC receive TCRs from T cells and initiate intracellular signals in response to isolated synaptic microvesicles. We conclude that the immunological synapse orchestrates TCR sorting and release in extracellular microvesicles. These microvesicles deliver transcellular signals across antigen-dependent synapses by engaging cognate pMHC on APCs.

Polarized release of T-cell-receptor-enriched microvesicles at the immunological synapse

NASA Astrophysics Data System (ADS)

Choudhuri, Kaushik; Llodrá, Jaime; Roth, Eric W.; Tsai, Jones; Gordo, Susana; Wucherpfennig, Kai W.; Kam, Lance C.; Stokes, David L.; Dustin, Michael L.

2014-03-01

The recognition events that mediate adaptive cellular immunity and regulate antibody responses depend on intercellular contacts between T cells and antigen-presenting cells (APCs). T-cell signalling is initiated at these contacts when surface-expressed T-cell receptors (TCRs) recognize peptide fragments (antigens) of pathogens bound to major histocompatibility complex molecules (pMHC) on APCs. This, along with engagement of adhesion receptors, leads to the formation of a specialized junction between T cells and APCs, known as the immunological synapse, which mediates efficient delivery of effector molecules and intercellular signals across the synaptic cleft. T-cell recognition of pMHC and the adhesion ligand intercellular adhesion molecule-1 (ICAM-1) on supported planar bilayers recapitulates the domain organization of the immunological synapse, which is characterized by central accumulation of TCRs, adjacent to a secretory domain, both surrounded by an adhesive ring. Although accumulation of TCRs at the immunological synapse centre correlates with T-cell function, this domain is itself largely devoid of TCR signalling activity, and is characterized by an unexplained immobilization of TCR-pMHC complexes relative to the highly dynamic immunological synapse periphery. Here we show that centrally accumulated TCRs are located on the surface of extracellular microvesicles that bud at the immunological synapse centre. Tumour susceptibility gene 101 (TSG101) sorts TCRs for inclusion in microvesicles, whereas vacuolar protein sorting 4 (VPS4) mediates scission of microvesicles from the T-cell plasma membrane. The human immunodeficiency virus polyprotein Gag co-opts this process for budding of virus-like particles. B cells bearing cognate pMHC receive TCRs from T cells and initiate intracellular signals in response to isolated synaptic microvesicles. We conclude that the immunological synapse orchestrates TCR sorting and release in extracellular microvesicles. These microvesicles deliver transcellular signals across antigen-dependent synapses by engaging cognate pMHC on APCs.

A poliovirus-induced cytoplasmic membrane complex is exploited by the RNA polymerase of superinfecting Mouse Elberfeld (ME) virus.

PubMed

Zeichhardt, H; Habermehl, K O; Wetz, K

1983-04-01

The preexistence of a cytoplasmic membrane complex in HEp-2 cells, induced by poliovirus when inhibited in its reproduction by guanidine, was a prerequisite for accelerated reproduction of superinfecting Mouse Elberfeld (ME) virus. Guanidine-inhibited poliovirus induced a membrane complex of 470S that was successively modified into a faster sedimenting membrane complex (up to 700S) by superinfecting ME virus and exploited for ME virus reproduction. The modified membrane complex was the site for ME virus-specific RNA polymerization characterized by the existence of in vivo and in vitro activity of ME virus RNA polymerase associated with the modified membrane complex. Proof of membrane-bound RNA polymerase and newly synthesized ME virus RNA including replicative intermediate led to the conclusion that superinfecting ME virus exploits the 'poliovirus/guanidine'-induced complex as the site of action of its replication complex.

tRNAs promote nuclear import of HIV-1 intracellular reverse transcription complexes.

PubMed

Zaitseva, Lyubov; Myers, Richard; Fassati, Ariberto

2006-10-01

Infection of non-dividing cells is a biological property of HIV-1 crucial for virus transmission and AIDS pathogenesis. This property depends on nuclear import of the intracellular reverse transcription and pre-integration complexes (RTCs/PICs). To identify cellular factors involved in nuclear import of HIV-1 RTCs, cytosolic extracts were fractionated by chromatography and import activity examined by the nuclear import assay. A near-homogeneous fraction was obtained, which was active in inducing nuclear import of purified and labeled RTCs. The active fraction contained tRNAs, mostly with defective 3' CCA ends. Such tRNAs promoted HIV-1 RTC nuclear import when synthesized in vitro. Active tRNAs were incorporated into and recovered from virus particles. Mutational analyses indicated that the anticodon loop mediated binding to the viral complex whereas the T-arm may interact with cellular factors involved in nuclear import. These tRNA species efficiently accumulated into the nucleus on their own in a energy- and temperature-dependent way. An HIV-1 mutant containing MLV gag did not incorporate tRNA species capable of inducing HIV-1 RTC nuclear import and failed to infect cell cycle-arrested cells. Here we provide evidence that at least some tRNA species can be imported into the nucleus of human cells and promote HIV-1 nuclear import.

Suppression of cell wall-related genes associated with stunting of Oryza glaberrima infected with Rice tungro spherical virus

PubMed Central

Budot, Bernard O.; Encabo, Jaymee R.; Ambita, Israel Dave V.; Atienza-Grande, Genelou A.; Satoh, Kouji; Kondoh, Hiroaki; Ulat, Victor J.; Mauleon, Ramil; Kikuchi, Shoshi; Choi, Il-Ryong

2014-01-01

Rice tungro disease is a complex disease caused by the interaction between Rice tungro bacilliform virus and Rice tungro spherical virus (RTSV). RTSV alone does not cause recognizable symptoms in most Asian rice (Oryza sativa) plants, whereas some African rice (O. glaberrima) plants were found to become stunted by RTSV. Stunting of rice plants by virus infections usually accompanies the suppression of various cell wall-related genes. The expression of cell wall-related genes was examined in O. glaberrima and O. sativa infected with RTSV to see the relationship between the severity of stunting and the suppression of cell wall-related genes by RTSV. The heights of four accessions of O. glaberrima were found to decline by 14–34% at 28 days post-inoculation (dpi) with RTSV, whereas the height reduction of O. sativa plants by RTSV was not significant. RTSV accumulated more in O. glaberrima plants than in O. sativa plants, but the level of RTSV accumulation was not correlated with the degree of height reduction among the four accessions of O. glaberrima. Examination for expression of genes for cellulose synthase A5 (CESA5) and A6 (CESA6), cellulose synthase-like A9 (CSLA9) and C7, and α-expansin 1 (expansin 1) and 15 precursors in O. glaberrima and O. sativa plants between 7 and 28 dpi with RTSV showed that the genes such as those for CESA5, CESA6, CSLA9, and expansin 1were more significantly suppressed in stunted plants of O. glaberrima at 14 dpi with RTSV than in O. sativa, suggesting that stunting of O. glaberrima might be associated with these cell wall-related genes suppressed by RTSV. Examination for expression of these genes in O. sativa plants infected with other rice viruses in previous studies indicated that the suppression of the expansin 1 gene is likely to be a signature response commonly associated with virus-induced stunting of Oryza species. These results suggest that stunting of O. glaberrima by RTSV infection might be associated with the suppression of these cell wall-related genes at the early stage of infection with RTSV. PMID:24550897

Chronic or accidental exposure of oysters to norovirus: is there any difference in contamination?

PubMed

Ventrone, Iole; Schaeffer, Julien; Ollivier, Joanna; Parnaudeau, Sylvain; Pepe, Tiziana; Le Pendu, Jacques; Le Guyader, Françoise S

2013-03-01

Bivalve molluscan shellfish such as oysters may be contaminated by human pathogens. Currently, the primary pathogens associated with shellfish-related outbreaks are noroviruses. This study was conducted to improve understanding of oyster bioaccumulation when oysters were exposed to daily contamination or one accidental contamination event, i.e., different modes of contamination. Oysters were contaminated with two representative strains of norovirus (GI.1 and GII.3) and then analyzed with real-time reverse transcription PCR. Exposure to a repeated virus dose for 9 days (mimicking a growing area subjected to frequent sewage contamination) led to an additive accumulation that was not significantly different from that obtained when the same total dose of virus was added all at once (as may happen after accidental sewage discharge). Similarly, bioaccumulation tests performed with mixed strains revealed additive accumulation of both viruses. Depuration may not be efficient for eliminating viruses; therefore, to prevent contaminated shellfish from being put onto the market, continuous sanitary monitoring must be considered. All climatic events or sewage failures occurring in production areas must be recorded, because repeated low-dose exposure or abrupt events may lead to similar levels of accumulation. This study contributes to an understanding of norovirus accumulation in oysters and provides suggestions for risk management strategies.

Soybean mosaic virus infection and helper component-protease enhance accumulation of Bean pod mottle virus-specific siRNAs

USDA-ARS?s Scientific Manuscript database

Soybean plants infected with Bean pod mottle virus (BPMV) develop acute symptoms that usually decrease in severity over time. In other plant-virus interactions, this type of symptom recovery has been associated with degradation of viral RNAs by post-transcriptional gene silencing (PTGS), which is ac...

Beet yellow stunt virus in cells of Sonchus oleraceus L. and its relation to host mitochondria.

PubMed

Esau, K

1979-10-15

In Sonchus oleraceus L. (Asteraceae) infected with the beet yellow stunt virus (BYSV) the virions are found in phloem cells, including the sieve elements. In parenchymatous phloem cells, the virus is present mainly in the cytoplasm. In some parenchymatous cells, containing massive accumulations of virus, the flexuous rodlike virus particles are found partly inserted into mitochondrial cristae. The mitochondrial envelope is absent where virus is present in the cristae. A similar relation between virus and host mitochondria apparently has not been recorded for any other plant virus.

Association of satellites with a mastrevirus in natural infection: complexity of Wheat dwarf India virus disease.

PubMed

Kumar, Jitendra; Kumar, Jitesh; Singh, Sudhir P; Tuli, Rakesh

2014-06-01

In contrast to begomoviruses, mastreviruses have not previously been shown to interact with satellites. This study reports the first identification of the association of satellites with a mastrevirus in field-grown plants. Two alphasatellite species were detected in different field samples of wheat infected with Wheat Dwarf India Virus (WDIV), a Cotton leaf curl Multan alphasatellite (CLCuMA) and a Guar leaf curl alphasatellite (GLCuA). In addition to the alphasatellites, a betasatellite, Ageratum yellow leaf curl betasatellite (AYLCB), was also identified in the wheat samples. No begomovirus was detected in the wheat samples, thus establishing association of the above-named satellites with WDIV. Agrobacterium-mediated inoculation of WDIV in wheat, in the presence of either of the alphasatellites or the betasatellite, resulted in infections inducing more severe symptoms. WDIV efficiently maintained each of the alphasatellites and the betasatellite in wheat. The satellites enhanced the level of WDIV DNA in wheat. Inoculation of the satellites isolated from wheat with various begomoviruses into Nicotiana tabacum demonstrated that these remain capable of interacting with the viruses with which they were first identified. Virus-specific small RNAs accumulated in wheat upon infection with WDIV but were lower in abundance in plants coinfected with the satellites, suggesting that both the alphasatellites and the betasatellite suppress RNA silencing. These results suggest that the selective advantage for the maintenance of the alphasatellites and the betasatellite by WDIV in the field is in overcoming RNA silencing-mediated host defense. Wheat is the most widely cultivated cereal crop in the world. A number of viruses are important pathogens of wheat, including the viruses of the genus Mastrevirus, family Geminiviridae. This study reports the association of subgenomic components, called satellites (alpha- and betasatellites), with a mastrevirus, Wheat Dwarf India Virus (WDIV), isolated from two distant locations in India. This study reports the first identification of the satellites in a monocot plant. The satellites enhanced accumulation of WDIV and severity of disease symptoms. The satellites lowered the concentration of virus-specific small RNAs in wheat plants, indicating their silencing suppressor activity. The involvement of the satellites in symptom severity of the mastrevirus can have implications in the form of economic impact of the virus on crop yield. Understanding the role of the satellites in disease severity is important for developing disease management strategies. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

The Cellular Autophagy Pathway Modulates Human T-Cell Leukemia Virus Type 1 Replication

PubMed Central

Tang, Sai-Wen; Chen, Chia-Yen; Klase, Zachary; Zane, Linda

2013-01-01

Autophagy, a general homeostatic process for degradation of cytosolic proteins or organelles, has been reported to modulate the replication of many viruses. The role of autophagy in human T-cell leukemia virus type 1 (HTLV-1) replication has, however, been uncharacterized. Here, we report that HTLV-1 infection increases the accumulation of autophagosomes and that this accumulation increases HTLV-1 production. We found that the HTLV-1 Tax protein increases cellular autophagosome accumulation by acting to block the fusion of autophagosomes to lysosomes, preventing the degradation of the former by the latter. Interestingly, the inhibition of cellular autophagosome-lysosome fusion using bafilomycin A increased the stability of the Tax protein, suggesting that cellular degradation of Tax occurs in part through autophagy. Our current findings indicate that by interrupting the cell's autophagic process, Tax exerts a positive feedback on its own stability. PMID:23175371

Whitefly transmission of Sweet potato leaf curl virus in sweetpotato germplasm

USDA-ARS?s Scientific Manuscript database

Sweetpotato, Ipomoea batatas (L.) Lam., is among an extensive number of plant species attacked by Bemisia tabaci (Gennadius). Because this important world food crop is vegetatively propagated, it can conveniently accumulate infections by several viruses. Sweet potato leaf curl virus (SPLCV) (ssDNA...

Proviruses with Long-Term Stable Expression Accumulate in Transcriptionally Active Chromatin Close to the Gene Regulatory Elements: Comparison of ASLV-, HIV- and MLV-Derived Vectors

PubMed Central

Miklík, Dalibor; Šenigl, Filip; Hejnar, Jiří

2018-01-01

Individual groups of retroviruses and retroviral vectors differ in their integration site preference and interaction with the host genome. Hence, immediately after infection genome-wide distribution of integrated proviruses is non-random. During long-term in vitro or persistent in vivo infection, the genomic position and chromatin environment of the provirus affects its transcriptional activity. Thus, a selection of long-term stably expressed proviruses and elimination of proviruses, which have been gradually silenced by epigenetic mechanisms, helps in the identification of genomic compartments permissive for proviral transcription. We compare here the extent and time course of provirus silencing in single cell clones of the K562 human myeloid lymphoblastoma cell line that have been infected with retroviral reporter vectors derived from avian sarcoma/leukosis virus (ASLV), human immunodeficiency virus type 1 (HIV) and murine leukaemia virus (MLV). While MLV proviruses remain transcriptionally active, ASLV proviruses are prone to rapid silencing. The HIV provirus displays gradual silencing only after an extended time period in culture. The analysis of integration sites of long-term stably expressed proviruses shows a strong bias for some genomic features—especially integration close to the transcription start sites of active transcription units. Furthermore, complex analysis of histone modifications enriched at the site of integration points to the accumulation of proviruses of all three groups in gene regulatory segments, particularly close to the enhancer loci. We conclude that the proximity to active regulatory chromatin segments correlates with stable provirus expression in various retroviral species. PMID:29517993

The movement protein of cucumber mosaic virus traffics into sieve elements in minor veins of nicotiana clevelandii

PubMed Central

Blackman, LM; Boevink, P; Cruz, SS; Palukaitis, P; Oparka, KJ

1998-01-01

The location of the 3a movement protein (MP) of cucumber mosaic virus (CMV) was studied by quantitative immunogold labeling of the wild-type 3a MP in leaves of Nicotiana clevelandii infected by CMV as well as by using a 3a-green fluorescent protein (GFP) fusion expressed from a potato virus X (PVX) vector. Whether expressed from CMV or PVX, the 3a MP targeted plasmodesmata and accumulated in the central cavity of the pore. Within minor veins, the most extensively labeled plasmodesmata were those connecting sieve elements and companion cells. In addition to targeting plasmodesmata, the 3a MP accumulated in the parietal layer of mature sieve elements. Confocal imaging of cells expressing the 3a-GFP fusion protein showed that the 3a MP assembled into elaborate fibrillar formations in the sieve element parietal layer. The ability of 3a-GFP, expressed from PVX rather than CMV, to enter sieve elements demonstrates that neither the CMV RNA nor the CMV coat protein is required for trafficking of the 3a MP into sieve elements. CMV virions were not detected in plasmodesmata from CMV-infected tissue, although large CMV aggregates were often found in the parietal layer of sieve elements and were usually surrounded by 3a MP. These data suggest that CMV traffics into minor vein sieve elements as a ribonucleoprotein complex that contains the viral RNA, coat protein, and 3a MP, with subsequent viral assembly occurring in the sieve element parietal layer. PMID:9548980

Reduced accumulation of defective viral genomes contributes to severe outcome in influenza virus infected patients

PubMed Central

Zamarreño, Noelia; Oliveros, Juan Carlos; Rodriguez, Guadalupe; Rey, Sonia; Barba, Isabel; Nieto, Amelia

2017-01-01

Influenza A virus (IAV) infection can be severe or even lethal in toddlers, the elderly and patients with certain medical conditions. Infection of apparently healthy individuals nonetheless accounts for many severe disease cases and deaths, suggesting that viruses with increased pathogenicity co-circulate with pandemic or epidemic viruses. Looking for potential virulence factors, we have identified a polymerase PA D529N mutation detected in a fatal IAV case, whose introduction into two different recombinant virus backbones, led to reduced defective viral genomes (DVGs) production. This mutation conferred low induction of antiviral response in infected cells and increased pathogenesis in mice. To analyze the association between low DVGs production and pathogenesis in humans, we performed a genomic analysis of viruses isolated from a cohort of previously healthy individuals who suffered highly severe IAV infection requiring admission to Intensive Care Unit and patients with fatal outcome who additionally showed underlying medical conditions. These viruses were compared with those isolated from a cohort of mild IAV patients. Viruses with fewer DVGs accumulation were observed in patients with highly severe/fatal outcome than in those with mild disease, suggesting that low DVGs abundance constitutes a new virulence pathogenic marker in humans. PMID:29023600

Reduced accumulation of defective viral genomes contributes to severe outcome in influenza virus infected patients.

PubMed

Vasilijevic, Jasmina; Zamarreño, Noelia; Oliveros, Juan Carlos; Rodriguez-Frandsen, Ariel; Gómez, Guillermo; Rodriguez, Guadalupe; Pérez-Ruiz, Mercedes; Rey, Sonia; Barba, Isabel; Pozo, Francisco; Casas, Inmaculada; Nieto, Amelia; Falcón, Ana

2017-10-01

Influenza A virus (IAV) infection can be severe or even lethal in toddlers, the elderly and patients with certain medical conditions. Infection of apparently healthy individuals nonetheless accounts for many severe disease cases and deaths, suggesting that viruses with increased pathogenicity co-circulate with pandemic or epidemic viruses. Looking for potential virulence factors, we have identified a polymerase PA D529N mutation detected in a fatal IAV case, whose introduction into two different recombinant virus backbones, led to reduced defective viral genomes (DVGs) production. This mutation conferred low induction of antiviral response in infected cells and increased pathogenesis in mice. To analyze the association between low DVGs production and pathogenesis in humans, we performed a genomic analysis of viruses isolated from a cohort of previously healthy individuals who suffered highly severe IAV infection requiring admission to Intensive Care Unit and patients with fatal outcome who additionally showed underlying medical conditions. These viruses were compared with those isolated from a cohort of mild IAV patients. Viruses with fewer DVGs accumulation were observed in patients with highly severe/fatal outcome than in those with mild disease, suggesting that low DVGs abundance constitutes a new virulence pathogenic marker in humans.

Rift Valley fever virus NSS gene expression correlates with a defect in nuclear mRNA export.

PubMed

Copeland, Anna Maria; Van Deusen, Nicole M; Schmaljohn, Connie S

2015-12-01

We investigated the localization of host mRNA during Rift Valley fever virus (RVFV) infection. Fluorescence in situ hybridization revealed that infection with RVFV altered the localization of host mRNA. mRNA accumulated in the nuclei of RVFV-infected but not mock-infected cells. Further, overexpression of the NSS gene, but not the N, GN or NSM genes correlated with mRNA nuclear accumulation. Nuclear accumulation of host mRNA was not observed in cells infected with a strain of RVFV lacking the gene encoding NSS, confirming that expression of NSS is likely responsible for this phenomenon. Published by Elsevier Inc.

Detection of Animal Viruses in Coastal Seawater and Sediments

PubMed Central

De Flora, Silvio; De Renzi, Giuseppe P.; Badolati, Giuseppe

1975-01-01

Animal viruses, predominantly enteroviruses, were detected in shallow waters at bottom depths and in clastic marine sediments. Viruses accumulated in sandy and slimy deposits of the sea bottom near the shore and could be easily released into water by means of simple mechanical shaking. Images PMID:170859

Identification and Characterization of the Host Protein DNAJC14 as a Broadly Active Flavivirus Replication Modulator

PubMed Central

Yi, Zhigang; Sperzel, Lindsey; Nürnberger, Cindy; Bredenbeek, Peter J.; Lubick, Kirk J.; Best, Sonja M.; Stoyanov, Cristina T.; Law, Lok Man J.; Yuan, Zhenghong; Rice, Charles M.; MacDonald, Margaret R.

2011-01-01

Viruses in the Flavivirus genus of the Flaviviridae family are arthropod-transmitted and contribute to staggering numbers of human infections and significant deaths annually across the globe. To identify cellular factors with antiviral activity against flaviviruses, we screened a cDNA library using an iterative approach. We identified a mammalian Hsp40 chaperone protein (DNAJC14) that when overexpressed was able to mediate protection from yellow fever virus (YFV)-induced cell death. Further studies revealed that DNAJC14 inhibits YFV at the step of viral RNA replication. Since replication of bovine viral diarrhea virus (BVDV), a member of the related Pestivirus genus, is also known to be modulated by DNAJC14, we tested the effect of this host factor on diverse Flaviviridae family members. Flaviviruses, including the pathogenic Asibi strain of YFV, Kunjin, and tick-borne Langat virus, as well as a Hepacivirus, hepatitis C virus (HCV), all were inhibited by overexpression of DNAJC14. Mutagenesis showed that both the J-domain and the C-terminal domain, which mediates self-interaction, are required for anti-YFV activity. We found that DNAJC14 does not block YFV nor HCV NS2-3 cleavage, and using non-inhibitory mutants demonstrate that DNAJC14 is recruited to YFV replication complexes. Immunofluorescence analysis demonstrated that endogenous DNAJC14 rearranges during infection and is found in replication complexes identified by dsRNA staining. Interestingly, silencing of endogenous DNAJC14 results in impaired YFV replication suggesting a requirement for DNAJC14 in YFV replication complex assembly. Finally, the antiviral activity of overexpressed DNAJC14 occurs in a time- and dose-dependent manner. DNAJC14 overexpression may disrupt the proper stoichiometry resulting in inhibition, which can be overcome upon restoration of the optimal ratios due to the accumulation of viral nonstructural proteins. Our findings, together with previously published work, suggest that the members of the Flaviviridae family have evolved in unique and important ways to interact with this host Hsp40 chaperone molecule. PMID:21249176

Branched-chain amino acids reduce hepatic iron accumulation and oxidative stress in hepatitis C virus polyprotein-expressing mice

PubMed Central

Korenaga, Masaaki; Nishina, Sohji; Korenaga, Keiko; Tomiyama, Yasuyuki; Yoshioka, Naoko; Hara, Yuichi; Sasaki, Yusuke; Shimonaka, Yasushi; Hino, Keisuke

2015-01-01

Background & Aims Branched-chain amino acids (BCAA) reduce the incidence of hepatocellular carcinoma (HCC) in patients with cirrhosis. However, the mechanisms that underlie these effects remain unknown. Previously, we reported that oxidative stress in male transgenic mice that expressed hepatitis C virus polyprotein (HCVTgM) caused hepatic iron accumulation by reducing hepcidin transcription, thereby leading to HCC development. This study investigated whether long-term treatment with BCAA reduced hepatic iron accumulation and oxidative stress in iron-overloaded HCVTgM and in patients with HCV-related advanced fibrosis. Methods Male HCVTgM were fed an excess-iron diet that comprised either casein or 3.0% BCAA, or a control diet, for 6 months. Results For HCVTgM, BCAA supplementation increased the serum hepcidin-25 levels and antioxidant status [ratio of biological antioxidant potential (BAP) relative to derivatives of reactive oxygen metabolites (dROM)], decreased the hepatic iron contents, attenuated reactive oxygen species generation, and restored mitochondrial superoxide dismutase expression and mitochondrial complex I activity in the liver compared with mice fed the control diet. After 48 weeks of BCAA supplementation in patients with HCV-related advanced fibrosis, BAP/dROM and serum hepcidin-25 increased and serum ferritin decreased compared with the pretreatment levels. Conclusions BCAA supplementation reduced oxidative stress by restoring mitochondrial function and improved iron metabolism by increasing hepcidin-25 in both iron-overloaded HCVTgM and patients with HCV-related advanced fibrosis. These activities of BCAA may partially account for their inhibitory effects on HCC development in cirrhosis patients. PMID:25156780

Proteomic analysis of Frankliniella occidentalis and differentially expressed proteins in response to tomato spotted wilt virus infection.

PubMed

Badillo-Vargas, I E; Rotenberg, D; Schneweis, D J; Hiromasa, Y; Tomich, J M; Whitfield, A E

2012-08-01

Tomato spotted wilt virus (TSWV) is transmitted by Frankliniella occidentalis in a persistent propagative manner. Despite the extensive replication of TSWV in midgut and salivary glands, there is little to no pathogenic effect on F. occidentalis. We hypothesize that the first-instar larva (L1) of F. occidentalis mounts a response to TSWV that protects it from pathogenic effects caused by virus infection and replication in various insect tissues. A partial thrips transcriptome was generated using 454-Titanium sequencing of cDNA generated from F. occidentalis exposed to TSWV. Using these sequences, the L1 thrips proteome that resolved on a two-dimensional gel was characterized. Forty-seven percent of the resolved protein spots were identified using the thrips transcriptome. Real-time quantitative reverse transcriptase PCR (RT-PCR) analysis of virus titer in L1 thrips revealed a significant increase in the normalized abundance of TSWV nucleocapsid RNA from 2 to 21 h after a 3-h acquisition access period on virus-infected plant tissue, indicative of infection and accumulation of virus. We compared the proteomes of infected and noninfected L1s to identify proteins that display differential abundances in response to virus. Using four biological replicates, 26 spots containing 37 proteins were significantly altered in response to TSWV. Gene ontology assignments for 32 of these proteins revealed biological roles associated with the infection cycle of other plant- and animal-infecting viruses and antiviral defense responses. Our findings support the hypothesis that L1 thrips display a complex reaction to TSWV infection and provide new insights toward unraveling the molecular basis of this interaction.

Proteomic Analysis of Frankliniella occidentalis and Differentially Expressed Proteins in Response to Tomato Spotted Wilt Virus Infection

PubMed Central

Badillo-Vargas, I. E.; Rotenberg, D.; Schneweis, D. J.; Hiromasa, Y.; Tomich, J. M.

2012-01-01

Tomato spotted wilt virus (TSWV) is transmitted by Frankliniella occidentalis in a persistent propagative manner. Despite the extensive replication of TSWV in midgut and salivary glands, there is little to no pathogenic effect on F. occidentalis. We hypothesize that the first-instar larva (L1) of F. occidentalis mounts a response to TSWV that protects it from pathogenic effects caused by virus infection and replication in various insect tissues. A partial thrips transcriptome was generated using 454-Titanium sequencing of cDNA generated from F. occidentalis exposed to TSWV. Using these sequences, the L1 thrips proteome that resolved on a two-dimensional gel was characterized. Forty-seven percent of the resolved protein spots were identified using the thrips transcriptome. Real-time quantitative reverse transcriptase PCR (RT-PCR) analysis of virus titer in L1 thrips revealed a significant increase in the normalized abundance of TSWV nucleocapsid RNA from 2 to 21 h after a 3-h acquisition access period on virus-infected plant tissue, indicative of infection and accumulation of virus. We compared the proteomes of infected and noninfected L1s to identify proteins that display differential abundances in response to virus. Using four biological replicates, 26 spots containing 37 proteins were significantly altered in response to TSWV. Gene ontology assignments for 32 of these proteins revealed biological roles associated with the infection cycle of other plant- and animal-infecting viruses and antiviral defense responses. Our findings support the hypothesis that L1 thrips display a complex reaction to TSWV infection and provide new insights toward unraveling the molecular basis of this interaction. PMID:22696645

Metabolic and miRNA Profiling of TMV Infected Plants Reveals Biphasic Temporal Changes

PubMed Central

Bazzini, Ariel A.; Manacorda, Carlos A.; Tohge, Takayuki; Conti, Gabriela; Rodriguez, Maria C.; Nunes-Nesi, Adriano; Villanueva, Sofía; Fernie, Alisdair R.; Carrari, Fernando; Asurmendi, Sebastian

2011-01-01

Plant viral infections induce changes including gene expression and metabolic components. Identification of metabolites and microRNAs (miRNAs) differing in abundance along infection may provide a broad view of the pathways involved in signaling and defense that orchestrate and execute the response in plant-pathogen interactions. We used a systemic approach by applying both liquid and gas chromatography coupled to mass spectrometry to determine the relative level of metabolites across the viral infection, together with a miRs profiling using a micro-array based procedure. Systemic changes in metabolites were characterized by a biphasic response after infection. The first phase, detected at one dpi, evidenced the action of a systemic signal since no virus was detected systemically. Several of the metabolites increased at this stage were hormone-related. miRs profiling after infection also revealed a biphasic alteration, showing miRs alteration at 5 dpi where no virus was detected systemically and a late phase correlating with virus accumulation. Correlation analyses revealed a massive increase in the density of correlation networks after infection indicating a complex reprogramming of the regulatory pathways, either in response to the plant defense mechanism or to the virus infection itself. Our data propose the involvement of a systemic signaling on early miRs alteration. PMID:22174812

Nipah and Hendra Virus Nucleoproteins Inhibit Nuclear Accumulation of Signal Transducer and Activator of Transcription 1 (STAT1) and STAT2 by Interfering with Their Complex Formation

PubMed Central

Sugai, Akihiro; Sato, Hiroki; Takayama, Ikuyo; Yoneda, Misako

2017-01-01

ABSTRACT Henipaviruses, such as Nipah (NiV) and Hendra (HeV) viruses, are highly pathogenic zoonotic agents within the Paramyxoviridae family. The phosphoprotein (P) gene products of the paramyxoviruses have been well characterized for their interferon (IFN) antagonist activity and their contribution to viral pathogenicity. In this study, we demonstrated that the nucleoprotein (N) of henipaviruses also prevents the host IFN signaling response. Reporter assays demonstrated that the NiV and HeV N proteins (NiV-N and HeV-N, respectively) dose-dependently suppressed both type I and type II IFN responses and that the inhibitory effect was mediated by their core domains. Additionally, NiV-N prevented the nuclear transport of signal transducer and activator of transcription 1 (STAT1) and STAT2. However, NiV-N did not associate with Impα5, Impβ1, or Ran, which are members of the nuclear transport system for STATs. Although P protein is known as a binding partner of N protein and actively retains N protein in the cytoplasm, the IFN antagonist activity of N protein was not abolished by the coexpression of P protein. This suggests that the IFN inhibition by N protein occurs in the cytoplasm. Furthermore, we demonstrated that the complex formation of STATs was hampered in the N protein-expressing cells. As a result, STAT nuclear accumulation was reduced, causing a subsequent downregulation of interferon-stimulated genes (ISGs) due to low promoter occupancy by STAT complexes. This novel route for preventing host IFN responses by henipavirus N proteins provides new insight into the pathogenesis of these viruses. IMPORTANCE Paramyxoviruses are well known for suppressing interferon (IFN)-mediated innate immunity with their phosphoprotein (P) gene products, and the henipaviruses also possess P, V, W, and C proteins for evading host antiviral responses. There are numerous studies providing evidence for the relationship between viral pathogenicity and antagonistic activities against IFN responses by P gene products. Meanwhile, little attention has been paid to the influence of nucleoprotein (N) on host innate immune responses. In this study, we demonstrated that both the NiV and HeV N proteins have antagonistic activity against the JAK/STAT signaling pathway by preventing the nucleocytoplasmic trafficking of STAT1 and STAT2. This inhibitory effect is due to an impairment of the ability of STATs to form complexes. These results provide new insight into the involvement of N protein in viral pathogenicity via its IFN antagonism. PMID:28835499

Nipah and Hendra Virus Nucleoproteins Inhibit Nuclear Accumulation of Signal Transducer and Activator of Transcription 1 (STAT1) and STAT2 by Interfering with Their Complex Formation.

PubMed

Sugai, Akihiro; Sato, Hiroki; Takayama, Ikuyo; Yoneda, Misako; Kai, Chieko

2017-11-01

Henipaviruses, such as Nipah (NiV) and Hendra (HeV) viruses, are highly pathogenic zoonotic agents within the Paramyxoviridae family. The phosphoprotein (P) gene products of the paramyxoviruses have been well characterized for their interferon (IFN) antagonist activity and their contribution to viral pathogenicity. In this study, we demonstrated that the nucleoprotein (N) of henipaviruses also prevents the host IFN signaling response. Reporter assays demonstrated that the NiV and HeV N proteins (NiV-N and HeV-N, respectively) dose-dependently suppressed both type I and type II IFN responses and that the inhibitory effect was mediated by their core domains. Additionally, NiV-N prevented the nuclear transport of signal transducer and activator of transcription 1 (STAT1) and STAT2. However, NiV-N did not associate with Impα5, Impβ1, or Ran, which are members of the nuclear transport system for STATs. Although P protein is known as a binding partner of N protein and actively retains N protein in the cytoplasm, the IFN antagonist activity of N protein was not abolished by the coexpression of P protein. This suggests that the IFN inhibition by N protein occurs in the cytoplasm. Furthermore, we demonstrated that the complex formation of STATs was hampered in the N protein-expressing cells. As a result, STAT nuclear accumulation was reduced, causing a subsequent downregulation of interferon-stimulated genes (ISGs) due to low promoter occupancy by STAT complexes. This novel route for preventing host IFN responses by henipavirus N proteins provides new insight into the pathogenesis of these viruses. IMPORTANCE Paramyxoviruses are well known for suppressing interferon (IFN)-mediated innate immunity with their phosphoprotein (P) gene products, and the henipaviruses also possess P, V, W, and C proteins for evading host antiviral responses. There are numerous studies providing evidence for the relationship between viral pathogenicity and antagonistic activities against IFN responses by P gene products. Meanwhile, little attention has been paid to the influence of nucleoprotein (N) on host innate immune responses. In this study, we demonstrated that both the NiV and HeV N proteins have antagonistic activity against the JAK/STAT signaling pathway by preventing the nucleocytoplasmic trafficking of STAT1 and STAT2. This inhibitory effect is due to an impairment of the ability of STATs to form complexes. These results provide new insight into the involvement of N protein in viral pathogenicity via its IFN antagonism. Copyright © 2017 American Society for Microbiology.

Retention of secretory proteins in an intermediate compartment and disappearance of the Golgi complex in an END4 mutant of Chinese hamster ovary cells

PubMed Central

1992-01-01

Mutant V.24.1, a member of the End4 complementation group of temperature-sensitive CHO cells, is defective in secretion at the restrictive temperature (Wang, R.-H., P. A. Colbaugh, C.-Y. Kao, E. A. Rutledge, and R. K. Draper. 1990. J. Biol. Chem. 265:20179-20187; Presley, J. F., R. K. Draper, and D. T. Brown. 1991. J. Virol. 65:1332- 1339). We have further investigated the secretory lesion and report three main findings. First, the block in secretion is not due to aberrant folding or oligomerization of secretory proteins in the endoplasmic reticulum because the hemagglutinin of influenza virus folded and oligomerized at the same rate in mutant and parental cells at the restrictive temperature. Second, secretory proteins accumulated in a compartment intermediate between the ER and the Golgi. Several lines of evidence support this conclusion, the most direct being the colocalization by immunofluorescence microscopy of influenza virus hemagglutinin with a 58-kD protein that is known to reside in an intermediate compartment. Third, at the resolution of fluorescence microscopy, the Golgi complex in the mutant cells vanished at the restrictive temperature. PMID:1577851

Mutations that alter a conserved element upstream of the potato virus X triple block and coat protein genes affect subgenomic RNA accumulation.

PubMed

Kim, K H; Hemenway, C

1997-05-26

The putative subgenomic RNA (sgRNA) promoter regions upstream of the potato virus X (PVX) triple block and coat protein (CP) genes contain sequences common to other potexviruses. The importance of these sequences to PVX sgRNA accumulation was determined by inoculation of Nicotiana tabacum NT1 cell suspension protoplasts with transcripts derived from wild-type and modified PVX cDNA clones. Analyses of RNA accumulation by S1 nuclease digestion and primer extension indicated that a conserved octanucleotide sequence element and the spacing between this element and the start-site for sgRNA synthesis are critical for accumulation of the two major sgRNA species. The impact of mutations on CP sgRNA levels was also reflected in the accumulation of CP. In contrast, genomic minus- and plus-strand RNA accumulation were not significantly affected by mutations in these regions. Studies involving inoculation of tobacco plants with the modified transcripts suggested that the conserved octanucleotide element functions in sgRNA accumulation and some other aspect of the infection process.

SUN1 Regulates HIV-1 Nuclear Import in a Manner Dependent on the Interaction between the Viral Capsid and Cellular Cyclophilin A.

PubMed

Luo, Xinlong; Yang, Wei; Gao, Guangxia

2018-07-01

Human immunodeficiency virus type 1 (HIV-1) can infect nondividing cells via passing through the nuclear pore complex. The nuclear membrane-imbedded protein SUN2 was recently reported to be involved in the nuclear import of HIV-1. Whether SUN1, which shares many functional similarities with SUN2, is involved in this process remained to be explored. Here we report that overexpression of SUN1 specifically inhibited infection by HIV-1 but not that by simian immunodeficiency virus (SIV) or murine leukemia virus (MLV). Overexpression of SUN1 did not affect reverse transcription but led to reduced accumulation of the 2-long-terminal-repeat (2-LTR) circular DNA and integrated viral DNA, suggesting a block in the process of nuclear import. HIV-1 CA was mapped as a determinant for viral sensitivity to SUN1. Treatment of SUN1-expressing cells with cyclosporine (CsA) significantly reduced the sensitivity of the virus to SUN1, and an HIV-1 mutant containing CA-G89A, which does not interact with cyclophilin A (CypA), was resistant to SUN1 overexpression. Downregulation of endogenous SUN1 inhibited the nuclear entry of the wild-type virus but not that of the G89A mutant. These results indicate that SUN1 participates in the HIV-1 nuclear entry process in a manner dependent on the interaction of CA with CypA. IMPORTANCE HIV-1 infects both dividing and nondividing cells. The viral preintegration complex (PIC) can enter the nucleus through the nuclear pore complex. It has been well known that the viral protein CA plays an important role in determining the pathways by which the PIC enters the nucleus. In addition, the interaction between CA and the cellular protein CypA has been reported to be important in the selection of nuclear entry pathways, though the underlying mechanisms are not very clear. Here we show that both SUN1 overexpression and downregulation inhibited HIV-1 nuclear entry. CA played an important role in determining the sensitivity of the virus to SUN1: the regulatory activity of SUN1 toward HIV-1 relied on the interaction between CA and CypA. These results help to explain how SUN1 is involved in the HIV-1 nuclear entry process. Copyright © 2018 American Society for Microbiology.

Accumulation of CD11b+Gr-1+ cells in the lung, blood and bone marrow of mice infected with highly pathogenic H5N1 and H1N1 influenza viruses

PubMed Central

Kotur, Mark S.; Stark, Gregory V.; Warren, Richard L.; Kasoji, Manjula; Craft, Jeremy L.; Albrecht, Randy A.; García-Sastre, Adolfo; Katze, Michael G.; Waters, Katrina M.; Vasconcelos, Daphne; Sabourin, Patrick J.; Bresler, Herbert S.; Sabourin, Carol L.

2017-01-01

Infection with pathogenic influenza viruses isassociated with intense inflammatory disease. Here, we investigated the innate immune response in mice infectedwith H5N1 A/Vietnam/1203/04 and with reassortanthuman H1N1 A/Texas/36/91 viruse s containing the virulence genes hemagglutinin (HA), neuraminidase (NA) and NS1 of the 1918 pandemic virus. Inclusion of the 1918 HAand NA glycoproteins rendered a seasonal H1N1 virus capable of inducing an exacerbated host innate immuneresponse similar to that observed for highly pathogenicA/Vietnam/1203/04 virus. Infection with 1918 HA/NA:Tx/91 and A/Vietnam/1203/04 were associated with severelung pathology, increased cytokine and chemokine production, and significant immune cell changes, including the presence of CD11b+Gr-1+cells in the blood, lung and bone marrow. Significant differential gene expression in the lung included pathways for cell death, apoptosis, production and response to reactive oxygen radicals, as well asarginine and proline meta bolism and chemokines associated with monocyte and neutrophil/granulocyte accumulation and/or activation. Arginase was produced in the lungof animals infected with A/Vietnam/1204. These results demonstrate that the innate immune cell response resultsin t he accumulation of CD11b+Gr-1+cells and products that have previously been shown to contribute to T cellsuppression. PMID:23397329

Cell-Specific Establishment of Poliovirus Resistance to an Inhibitor Targeting a Cellular Protein

PubMed Central

Viktorova, Ekaterina G.; Nchoutmboube, Jules; Ford-Siltz, Lauren A.

2015-01-01

ABSTRACT It is hypothesized that targeting stable cellular factors involved in viral replication instead of virus-specific proteins may raise the barrier for development of resistant mutants, which is especially important for highly adaptable small (+)RNA viruses. However, contrary to this assumption, the accumulated evidence shows that these viruses easily generate mutants resistant to the inhibitors of cellular proteins at least in some systems. We investigated here the development of poliovirus resistance to brefeldin A (BFA), an inhibitor of the cellular protein GBF1, a guanine nucleotide exchange factor for the small cellular GTPase Arf1. We found that while resistant viruses can be easily selected in HeLa cells, they do not emerge in Vero cells, in spite that in the absence of the drug both cultures support robust virus replication. Our data show that the viral replication is much more resilient to BFA than functioning of the cellular secretory pathway, suggesting that the role of GBF1 in the viral replication is independent of its Arf activating function. We demonstrate that the level of recruitment of GBF1 to the replication complexes limits the establishment and expression of a BFA resistance phenotype in both HeLa and Vero cells. Moreover, the BFA resistance phenotype of poliovirus mutants is also cell type dependent in different cells of human origin and results in a fitness loss in the form of reduced efficiency of RNA replication in the absence of the drug. Thus, a rational approach to the development of host-targeting antivirals may overcome the superior adaptability of (+)RNA viruses. IMPORTANCE Compared to the number of viral diseases, the number of available vaccines is miniscule. For some viruses vaccine development has not been successful after multiple attempts, and for many others vaccination is not a viable option. Antiviral drugs are needed for clinical practice and public health emergencies. However, viruses are highly adaptable and can easily generate mutants resistant to practically any compounds targeting viral proteins. An alternative approach is to target stable cellular factors recruited for the virus-specific functions. In the present study, we analyzed the factors permitting and restricting the establishment of the resistance of poliovirus, a small (+)RNA virus, to brefeldin A (BFA), a drug targeting a cellular component of the viral replication complex. We found that the emergence and replication potential of resistant mutants is cell type dependent and that BFA resistance reduces virus fitness. Our data provide a rational approach to the development of antiviral therapeutics targeting host factors. PMID:25653442

Herpes Simplex Virus Type 1 and Other Pathogens are Key Causative Factors in Sporadic Alzheimer’s Disease

PubMed Central

Harris, Steven A.; Harris, Elizabeth A.

2015-01-01

Abstract This review focuses on research in epidemiology, neuropathology, molecular biology, and genetics regarding the hypothesis that pathogens interact with susceptibility genes and are causative in sporadic Alzheimer’s disease (AD). Sporadic AD is a complex multifactorial neurodegenerative disease with evidence indicating coexisting multi-pathogen and inflammatory etiologies. There are significant associations between AD and various pathogens, including Herpes simplex virus type 1 (HSV-1), Cytomegalovirus, and other Herpesviridae, Chlamydophila pneumoniae, spirochetes, Helicobacter pylori, and various periodontal pathogens. These pathogens are able to evade destruction by the host immune system, leading to persistent infection. Bacterial and viral DNA and RNA and bacterial ligands increase the expression of pro-inflammatory molecules and activate the innate and adaptive immune systems. Evidence demonstrates that pathogens directly and indirectly induce AD pathology, including amyloid-β (Aβ) accumulation, phosphorylation of tau protein, neuronal injury, and apoptosis. Chronic brain infection with HSV-1, Chlamydophila pneumoniae, and spirochetes results in complex processes that interact to cause a vicious cycle of uncontrolled neuroinflammation and neurodegeneration. Infections such as Cytomegalovirus, Helicobacter pylori, and periodontal pathogens induce production of systemic pro-inflammatory cytokines that may cross the blood-brain barrier to promote neurodegeneration. Pathogen-induced inflammation and central nervous system accumulation of Aβ damages the blood-brain barrier, which contributes to the pathophysiology of AD. Apolipoprotein E4 (ApoE4) enhances brain infiltration by pathogens including HSV-1 and Chlamydophila pneumoniae. ApoE4 is also associated with an increased pro-inflammatory response by the immune system. Potential antimicrobial treatments for AD are discussed, including the rationale for antiviral and antibiotic clinical trials. PMID:26401998

The vaccinia virus I3L gene product is localized to a complex endoplasmic reticulum-associated structure that contains the viral parental DNA.

PubMed

Welsch, Sonja; Doglio, Laura; Schleich, Sibylle; Krijnse Locker, Jacomine

2003-05-01

The vaccinia virus (VV) I3L gene product is a single-stranded DNA-binding protein made early in infection that localizes to the cytoplasmic sites of viral DNA replication (S. C. Rochester and P. Traktman, J. Virol. 72:2917-2926, 1998). Surprisingly, when replication was blocked, the protein localized to distinct cytoplasmic spots (A. Domi and G. Beaud, J. Gen. Virol. 81:1231-1235, 2000). Here these I3L-positive spots were characterized in more detail. By using an anti-I3L peptide antibody we confirmed that the protein localized to the cytoplasmic sites of viral DNA replication by both immunofluorescence and electron microscopy (EM). Before replication had started or when replication was inhibited with hydroxyurea or cytosine arabinoside, I3L localized to distinct cytoplasmic punctate structures of homogeneous size. We show that these structures are not incoming cores or cytoplasmic sites of VV early mRNA accumulation. Instead, morphological and quantitative data indicate that they are specialized sites where the parental DNA accumulates after its release from incoming viral cores. By EM, these sites appeared as complex, electron-dense structures that were intimately associated with the cellular endoplasmic reticulum (ER). By double labeling of cryosections we show that they contain DNA and a viral early protein, the gene product of E8R. Since E8R is a membrane protein that is able to bind to DNA, the localization of this protein to the I3L puncta suggests that they are composed of membranes. The results are discussed in relation to our previous data showing that the process of viral DNA replication also occurs in close association with the ER.

Cyclophilins facilitate dissociation of the human papillomavirus type 16 capsid protein L1 from the L2/DNA complex following virus entry.

PubMed

Bienkowska-Haba, Malgorzata; Williams, Carlyn; Kim, Seong Man; Garcea, Robert L; Sapp, Martin

2012-09-01

Human papillomaviruses (HPV) are composed of the major and minor capsid proteins, L1 and L2, that encapsidate a chromatinized, circular double-stranded DNA genome. At the outset of infection, the interaction of HPV type 16 (HPV16) (pseudo)virions with heparan sulfate proteoglycans triggers a conformational change in L2 that is facilitated by the host cell chaperone cyclophilin B (CyPB). This conformational change results in exposure of the L2 N terminus, which is required for infectious internalization. Following internalization, L2 facilitates egress of the viral genome from acidified endosomes, and the L2/DNA complex accumulates at PML nuclear bodies. We recently described a mutant virus that bypasses the requirement for cell surface CyPB but remains sensitive to cyclosporine for infection, indicating an additional role for CyP following endocytic uptake of virions. We now report that the L1 protein dissociates from the L2/DNA complex following infectious internalization. Inhibition and small interfering RNA (siRNA)-mediated knockdown of CyPs blocked dissociation of L1 from the L2/DNA complex. In vitro, purified CyPs facilitated the dissociation of L1 pentamers from recombinant HPV11 L1/L2 complexes in a pH-dependent manner. Furthermore, CyPs released L1 capsomeres from partially disassembled HPV16 pseudovirions at slightly acidic pH. Taken together, these data suggest that CyPs mediate the dissociation of HPV L1 and L2 capsid proteins following acidification of endocytic vesicles.

Cyclophilins Facilitate Dissociation of the Human Papillomavirus Type 16 Capsid Protein L1 from the L2/DNA Complex following Virus Entry

PubMed Central

Bienkowska-Haba, Malgorzata; Williams, Carlyn; Kim, Seong Man; Garcea, Robert L.

2012-01-01

Human papillomaviruses (HPV) are composed of the major and minor capsid proteins, L1 and L2, that encapsidate a chromatinized, circular double-stranded DNA genome. At the outset of infection, the interaction of HPV type 16 (HPV16) (pseudo)virions with heparan sulfate proteoglycans triggers a conformational change in L2 that is facilitated by the host cell chaperone cyclophilin B (CyPB). This conformational change results in exposure of the L2 N terminus, which is required for infectious internalization. Following internalization, L2 facilitates egress of the viral genome from acidified endosomes, and the L2/DNA complex accumulates at PML nuclear bodies. We recently described a mutant virus that bypasses the requirement for cell surface CyPB but remains sensitive to cyclosporine for infection, indicating an additional role for CyP following endocytic uptake of virions. We now report that the L1 protein dissociates from the L2/DNA complex following infectious internalization. Inhibition and small interfering RNA (siRNA)-mediated knockdown of CyPs blocked dissociation of L1 from the L2/DNA complex. In vitro, purified CyPs facilitated the dissociation of L1 pentamers from recombinant HPV11 L1/L2 complexes in a pH-dependent manner. Furthermore, CyPs released L1 capsomeres from partially disassembled HPV16 pseudovirions at slightly acidic pH. Taken together, these data suggest that CyPs mediate the dissociation of HPV L1 and L2 capsid proteins following acidification of endocytic vesicles. PMID:22761365

UL74 of Human Cytomegalovirus Contributes to Virus Release by Promoting Secondary Envelopment of Virions▿ †

PubMed Central

Jiang, Xiao Jing; Adler, Barbara; Sampaio, Kerstin Laib; Digel, Margarete; Jahn, Gerhard; Ettischer, Nicole; Stierhof, York-Dieter; Scrivano, Laura; Koszinowski, Ulrich; Mach, Michael; Sinzger, Christian

2008-01-01

The glycoprotein (g) complex gH/gL represents an essential part of the herpesvirus fusion machinery mediating entry of cell-free virions and cell-associated viral spread. In some herpesviruses additional proteins are associated with gH/gL contributing to the cell tropism of the respective virus. Human cytomegalovirus (HCMV) gH/gL forms complexes with either gO (UL74) or proteins of the UL128-131A gene locus. While a contribution of UL128-131A to endothelial cell tropism is known, the role of gO is less clear. We studied the role of gH/gL-associated proteins in HCMV replication in human foreskin fibroblasts (HFF) and human umbilical vein endothelial cells (HUVEC). Deletions of UL74 alone or in combination with mutations of the UL128-131A gene region were introduced into bacterial artificial chromosome vectors derived from the endotheliotropic strain TB40/E. Deletion of UL74 caused a profound defect regarding virus release from infected HFF and HUVEC. Large numbers of capsids accumulated in the cytoplasm of infected HFF but failed to acquire an envelope. Clear cell type differences were observed in the cell-associated spread of the UL74-defective virus. In HFF, focal growth was severely impaired, whereas it was normal in HUVEC. Deletion of UL131A abolished focal growth in endothelial cells. UL74/UL128-131A dual mutants showed severely impaired reconstitution efficiency. Our data suggest that gO plays a critical role in secondary envelopment and release of cell-free virions independent of the cell type but affects cell-associated growth specifically in HFF, whereas UL128-131A contributes to cell-associated spread in HFF and HUVEC. PMID:18184717

EFFECTS OF X RADIATION ON THE REPRODUCTION OF SMALLPOX VACCINE VIRUS IN TISSUE CULTURE (in Russian)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kamalyan, L.A.; Ter-Pogosyan, R.A.

1963-01-01

Infectious and hemagglutinic properties of smallpox vaccine virus in chick embryo cutaneous-muscular tissue irradiated with 20-kr of x radiation were investigated. It was found that the radiation induced increased virus reproduction. Accumulation of hemagglutinin did not differ in irradiated and nonirradiated cultures. (R.V.J.)

Impact of curly top host plants on accumulation, competitiveness, and durability of curtovirus species.

USDA-ARS?s Scientific Manuscript database

Curly top disease, caused by viruses in the genus, Curtovirus, has affected sugarbeet production throughout much of the West for over a century; however, over that period the viruses responsible for causing the disease have changed. The two curly top virus species currently affecting production, Bee...

RPLP1 and RPLP2 Are Essential Flavivirus Host Factors That Promote Early Viral Protein Accumulation

PubMed Central

Campos, Rafael K.; Wong, Benjamin; Lu, Yi-Fan; Shi, Pei-Yong; Pompon, Julien

2016-01-01

ABSTRACT The Flavivirus genus contains several arthropod-borne viruses that pose global health threats, including dengue viruses (DENV), yellow fever virus (YFV), and Zika virus (ZIKV). In order to understand how these viruses replicate in human cells, we previously conducted genome-scale RNA interference screens to identify candidate host factors. In these screens, we identified ribosomal proteins RPLP1 and RPLP2 (RPLP1/2) to be among the most crucial putative host factors required for DENV and YFV infection. RPLP1/2 are phosphoproteins that bind the ribosome through interaction with another ribosomal protein, RPLP0, to form a structure termed the ribosomal stalk. RPLP1/2 were validated as essential host factors for DENV, YFV, and ZIKV infection in two human cell lines: A549 lung adenocarcinoma and HuH-7 hepatoma cells, and for productive DENV infection of Aedes aegypti mosquitoes. Depletion of RPLP1/2 caused moderate cell-line-specific effects on global protein synthesis, as determined by metabolic labeling. In A549 cells, global translation was increased, while in HuH-7 cells it was reduced, albeit both of these effects were modest. In contrast, RPLP1/2 knockdown strongly reduced early DENV protein accumulation, suggesting a requirement for RPLP1/2 in viral translation. Furthermore, knockdown of RPLP1/2 reduced levels of DENV structural proteins expressed from an exogenous transgene. We postulate that these ribosomal proteins are required for efficient translation elongation through the viral open reading frame. In summary, this work identifies RPLP1/2 as critical flaviviral host factors required for translation. IMPORTANCE Flaviviruses cause important diseases in humans. Examples of mosquito-transmitted flaviviruses include dengue, yellow fever and Zika viruses. Viruses require a plethora of cellular factors to infect cells, and the ribosome plays an essential role in all viral infections. The ribosome is a complex macromolecular machine composed of RNA and proteins and it is responsible for protein synthesis. We identified two specific ribosomal proteins that are strictly required for flavivirus infection of human cells and mosquitoes: RPLP1 and RPLP2 (RPLP1/2). These proteins are part of a structure known as the ribosomal stalk and help orchestrate the elongation phase of translation. We show that flaviviruses are particularly dependent on the function of RPLP1/2. Our findings suggest that ribosome composition is an important factor for virus translation and may represent a regulatory layer for translation of specific cellular mRNAs. PMID:27974556

Accumulation of Human-Adapting Mutations during Circulation of A(H1N1)pdm09 Influenza Virus in Humans in the United Kingdom

PubMed Central

Elderfield, Ruth A.; Watson, Simon J.; Godlee, Alexandra; Adamson, Walt E.; Thompson, Catherine I.; Dunning, Jake; Fernandez-Alonso, Mirian; Blumenkrantz, Deena; Hussell, Tracy; Zambon, Maria; Openshaw, Peter; Kellam, Paul

2014-01-01

ABSTRACT The influenza pandemic that emerged in 2009 provided an unprecedented opportunity to study adaptation of a virus recently acquired from an animal source during human transmission. In the United Kingdom, the novel virus spread in three temporally distinct waves between 2009 and 2011. Phylogenetic analysis of complete viral genomes showed that mutations accumulated over time. Second- and third-wave viruses replicated more rapidly in human airway epithelial (HAE) cells than did the first-wave virus. In infected mice, weight loss varied between viral isolates from the same wave but showed no distinct pattern with wave and did not correlate with viral load in the mouse lungs or severity of disease in the human donor. However, second- and third-wave viruses induced less alpha interferon in the infected mouse lungs. NS1 protein, an interferon antagonist, had accumulated several mutations in second- and third-wave viruses. Recombinant viruses with the third-wave NS gene induced less interferon in human cells, but this alone did not account for increased virus fitness in HAE cells. Mutations in HA and NA genes in third-wave viruses caused increased binding to α-2,6-sialic acid and enhanced infectivity in human mucus. A recombinant virus with these two segments replicated more efficiently in HAE cells. A mutation in PA (N321K) enhanced polymerase activity of third-wave viruses and also provided a replicative advantage in HAE cells. Therefore, multiple mutations allowed incremental changes in viral fitness, which together may have contributed to the apparent increase in severity of A(H1N1)pdm09 influenza virus during successive waves. IMPORTANCE Although most people infected with the 2009 pandemic influenza virus had mild or unapparent symptoms, some suffered severe and devastating disease. The reasons for this variability were unknown, but the numbers of severe cases increased during successive waves of human infection in the United Kingdom. To determine the causes of this variation, we studied genetic changes in virus isolates from individual hospitalized patients. There were no consistent differences between these viruses and those circulating in the community, but we found multiple evolutionary changes that in combination over time increased the virus's ability to infect human cells. These adaptations may explain the remarkable ability of A(H1N1)pdm09 virus to continue to circulate despite widespread immunity and the apparent increase in severity of influenza over successive waves of infection. PMID:25210166

Accumulation of human-adapting mutations during circulation of A(H1N1)pdm09 influenza virus in humans in the United Kingdom.

PubMed

Elderfield, Ruth A; Watson, Simon J; Godlee, Alexandra; Adamson, Walt E; Thompson, Catherine I; Dunning, Jake; Fernandez-Alonso, Mirian; Blumenkrantz, Deena; Hussell, Tracy; Zambon, Maria; Openshaw, Peter; Kellam, Paul; Barclay, Wendy S

2014-11-01

The influenza pandemic that emerged in 2009 provided an unprecedented opportunity to study adaptation of a virus recently acquired from an animal source during human transmission. In the United Kingdom, the novel virus spread in three temporally distinct waves between 2009 and 2011. Phylogenetic analysis of complete viral genomes showed that mutations accumulated over time. Second- and third-wave viruses replicated more rapidly in human airway epithelial (HAE) cells than did the first-wave virus. In infected mice, weight loss varied between viral isolates from the same wave but showed no distinct pattern with wave and did not correlate with viral load in the mouse lungs or severity of disease in the human donor. However, second- and third-wave viruses induced less alpha interferon in the infected mouse lungs. NS1 protein, an interferon antagonist, had accumulated several mutations in second- and third-wave viruses. Recombinant viruses with the third-wave NS gene induced less interferon in human cells, but this alone did not account for increased virus fitness in HAE cells. Mutations in HA and NA genes in third-wave viruses caused increased binding to α-2,6-sialic acid and enhanced infectivity in human mucus. A recombinant virus with these two segments replicated more efficiently in HAE cells. A mutation in PA (N321K) enhanced polymerase activity of third-wave viruses and also provided a replicative advantage in HAE cells. Therefore, multiple mutations allowed incremental changes in viral fitness, which together may have contributed to the apparent increase in severity of A(H1N1)pdm09 influenza virus during successive waves. Although most people infected with the 2009 pandemic influenza virus had mild or unapparent symptoms, some suffered severe and devastating disease. The reasons for this variability were unknown, but the numbers of severe cases increased during successive waves of human infection in the United Kingdom. To determine the causes of this variation, we studied genetic changes in virus isolates from individual hospitalized patients. There were no consistent differences between these viruses and those circulating in the community, but we found multiple evolutionary changes that in combination over time increased the virus's ability to infect human cells. These adaptations may explain the remarkable ability of A(H1N1)pdm09 virus to continue to circulate despite widespread immunity and the apparent increase in severity of influenza over successive waves of infection. Copyright © 2014 Elderfield et al.

Nuclear export of human hepatitis B virus core protein and pregenomic RNA depends on the cellular NXF1-p15 machinery.

PubMed

Yang, Ching-Chun; Huang, Er-Yi; Li, Hung-Cheng; Su, Pei-Yi; Shih, Chiaho

2014-01-01

Hepatitis B virus (HBV) core protein (HBc) can shuttle between nucleus and cytoplasm. Cytoplasm-predominant HBc is clinically associated with severe liver inflammation. Previously, we found that HBc arginine-rich domain (ARD) can associate with a host factor NXF1 (TAP) by coimmunoprecipitation. It is well known that NXF1-p15 heterodimer can serve as a major export receptor of nuclear mRNA as a ribonucleoprotein complex (RNP). In the NXF1-p15 pathway, TREX (transcription/export) complex plays an important role in coupling nuclear pre-mRNA processing with mRNA export in mammalian cells. Here, we tested the hypothesis whether HBc and HBV specific RNA can be exported via the TREX and NXF1-p15 mediated pathway. We demonstrated here that HBc can physically and specifically associate with TREX components, and the NXF1-p15 export receptor by coimmunoprecipitation. Accumulation of HBc protein in the nucleus can be induced by the interference with TREX and NXF1-p15 mediated RNA export machinery. HBV transcripts encodes a non-spliced 3.5 kb pregenomic RNA (pgRNA) which can serve as a template for reverse transcription. Cytoplasmic HBV pgRNA appeared to be reduced by siRNA treatment specific for the NXF1-p15 complex by quantitative RT-qPCR and Northern blot analyses. This result suggests that the pgRNA was also exported via the NXF1-p15 machinery. We entertain the hypothesis that HBc protein can be exported as an RNP cargo via the mRNA export pathway by hijacking the TREX and NXF1-p15 complex. In our current and previous studies, HBc is not required for pgRNA accumulation in the cytoplasm. Furthermore, HBc ARD can mediate nuclear export of a chimeric protein containing HBc ARD in a pgRNA-independent manner. Taken together, it suggests that while both pgRNA and HBc protein exports are dependent on NXF1-p15, they are using the same export machinery in a manner independent of each other.

Sampling strategies and biodiversity of influenza A subtypes in wild birds

USGS Publications Warehouse

Olson, Sarah H.; Parmley, Jane; Soos, Catherine; Gilbert, Martin; Latore-Margalef, Neus; Hall, Jeffrey S.; Hansbro, Phillip M.; Leighton, Frank; Munster, Vincent; Joly, Damien

2014-01-01

Wild aquatic birds are recognized as the natural reservoir of avian influenza A viruses (AIV), but across high and low pathogenic AIV strains, scientists have yet to rigorously identify most competent hosts for the various subtypes. We examined 11,870 GenBank records to provide a baseline inventory and insight into patterns of global AIV subtype diversity and richness. Further, we conducted an extensive literature review and communicated directly with scientists to accumulate data from 50 non-overlapping studies and over 250,000 birds to assess the status of historic sampling effort. We then built virus subtype sample-based accumulation curves to better estimate sample size targets that capture a specific percentage of virus subtype richness at seven sampling locations. Our study identifies a sampling methodology that will detect an estimated 75% of circulating virus subtypes from a targeted bird population and outlines future surveillance and research priorities that are needed to explore the influence of host and virus biodiversity on emergence and transmission.

The prion protein has RNA binding and chaperoning properties characteristic of nucleocapsid protein NCP7 of HIV-1.

PubMed

Gabus, C; Derrington, E; Leblanc, P; Chnaiderman, J; Dormont, D; Swietnicki, W; Morillas, M; Surewicz, W K; Marc, D; Nandi, P; Darlix, J L

2001-06-01

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases associated with the accumulation of a protease-resistant form of the prion protein (PrP). Although PrP is conserved in vertebrates, its function remains to be identified. In vitro PrP binds large nucleic acids causing the formation of nucleoprotein complexes resembling human immunodeficiency virus type 1 (HIV-1) nucleocapsid-RNA complexes and in vivo MuLV replication accelerates the scrapie infectious process, suggesting possible interactions between retroviruses and PrP. Retroviruses, including HIV-1 encode a major nucleic acid binding protein (NC protein) found within the virus where 2000 NC protein molecules coat the dimeric genome. NC is required in virus assembly and infection to chaperone RNA dimerization and packaging and in proviral DNA synthesis by reverse transcriptase (RT). In HIV-1, 5'-leader RNA/NC interactions appear to control these viral processes. This prompted us to compare and contrast the interactions of human and ovine PrP and HIV-1 NCp7 with HIV-1 5'-leader RNA. Results show that PrP has properties characteristic of NCp7 with respect to viral RNA dimerization and proviral DNA synthesis by RT. The NC-like properties of huPrP map to the N-terminal region of huPrP. Interestingly, PrP localizes in the membrane and cytoplasm of PrP-expressing cells. These findings suggest that PrP is a multifunctional protein possibly participating in nucleic acid metabolism.

Expression and assembly of a fully active antibody in algae

NASA Astrophysics Data System (ADS)

Mayfield, Stephen P.; Franklin, Scott E.; Lerner, Richard A.

2003-01-01

Although combinatorial antibody libraries have solved the problem of access to large immunological repertoires, efficient production of these complex molecules remains a problem. Here we demonstrate the efficient expression of a unique large single-chain (lsc) antibody in the chloroplast of the unicellular, green alga, Chlamydomonas reinhardtii. We achieved high levels of protein accumulation by synthesizing the lsc gene in chloroplast codon bias and by driving expression of the chimeric gene using either of two C. reinhardtii chloroplast promoters and 5' and 3' RNA elements. This lsc antibody, directed against glycoprotein D of the herpes simplex virus, is produced in a soluble form by the alga and assembles into higher order complexes in vivo. Aside from dimerization by disulfide bond formation, the antibody undergoes no detectable posttranslational modification. We further demonstrate that accumulation of the antibody can be modulated by the specific growth regime used to culture the alga, and by the choice of 5' and 3' elements used to drive expression of the antibody gene. These results demonstrate the utility of alga as an expression platform for recombinant proteins, and describe a new type of single chain antibody containing the entire heavy chain protein, including the Fc domain.

Emerging role of lipid droplets in Aedes aegypti immune response against bacteria and Dengue virus

PubMed Central

Barletta, Ana Beatriz Ferreira; Alves, Liliane Rosa; Nascimento Silva, Maria Clara L.; Sim, Shuzhen; Dimopoulos, George; Liechocki, Sally; Maya-Monteiro, Clarissa M.; Sorgine, Marcos H. Ferreira

2016-01-01

In mammals, lipid droplets (LDs) are ubiquitous organelles that modulate immune and inflammatory responses through the production of lipid mediators. In insects, it is unknown whether LDs play any role during the development of immune responses. We show that Aedes aegypti Aag2 cells – an immune responsive cell lineage – accumulates LDs when challenged with Enterobacter cloacae, Sindbis, and Dengue viruses. Microarray analysis of Aag2 challenged with E.cloacae or infected with Dengue virus revealed high transcripts levels of genes associated with lipid storage and LDs biogenesis, correlating with the increased LDs numbers in those conditions. Similarly, in mosquitoes, LDs accumulate in midgut cells in response to Serratia marcescens and Sindbis virus or when the native microbiota proliferates, following a blood meal. Also, constitutive activation of Toll and IMD pathways by knocking-down their respective negative modulators (Cactus and Caspar) increases LDs numbers in the midgut. Our results show for the first time an infection-induced LDs accumulation in response to both bacterial and viral infections in Ae. Aegypti, and we propose a role for LDs in mosquito immunity. These findings open new venues for further studies in insect immune responses associated with lipid metabolism. PMID:26887863

Deletion of the Vaccinia Virus I2 Protein Interrupts Virion Morphogenesis, Leading to Retention of the Scaffold Protein and Mislocalization of Membrane-Associated Entry Proteins

PubMed Central

Hyun, Seong-In; Weisberg, Andrea

2017-01-01

ABSTRACT The I2L open reading frame of vaccinia virus (VACV) encodes a conserved 72-amino-acid protein with a putative C-terminal transmembrane domain. Previous studies with a tetracycline-inducible mutant demonstrated that I2-deficient virions are defective in cell entry. The purpose of the present study was to determine the step of replication or entry that is affected by loss of the I2 protein. Fluorescence microscopy experiments showed that I2 colocalized with a major membrane protein of immature and mature virions. We generated a cell line that constitutively expressed I2 and allowed construction of the VACV I2L deletion mutant vΔI2. As anticipated, vΔI2 was unable to replicate in cells that did not express I2. Unexpectedly, morphogenesis was interrupted at a stage after immature virion formation, resulting in the accumulation of dense spherical particles instead of brick-shaped mature virions with well-defined core structures. The abnormal particles retained the D13 scaffold protein of immature virions, were severely deficient in the transmembrane proteins that comprise the entry fusion complex (EFC), and had increased amounts of unprocessed membrane and core proteins. Total lysates of cells infected with vΔI2 also had diminished EFC proteins due to instability attributed to their hydrophobicity and failure to be inserted into viral membranes. A similar instability of EFC proteins had previously been found with unrelated mutants blocked earlier in morphogenesis that also accumulated viral membranes retaining the D13 scaffold. We concluded that I2 is required for virion morphogenesis, release of the D13 scaffold, and the association of EFC proteins with viral membranes. IMPORTANCE Poxviruses comprise a large family that infect vertebrates and invertebrates, cause disease in both in humans and in wild and domesticated animals, and are being engineered as vectors for vaccines and cancer therapy. In addition, investigations of poxviruses have provided insights into many aspects of cell biology. The I2 protein is conserved in all poxviruses that infect vertebrates, suggesting an important role. The present study revealed that this protein is essential for vaccinia virus morphogenesis and that its absence results in an accumulation of deformed virus particles retaining the scaffold protein and deficient in surface proteins needed for cell entry. PMID:28490596

Persistent Simian Immunodeficiency Virus Infection Drives Differentiation, Aberrant Accumulation, and Latent Infection of Germinal Center Follicular T Helper Cells

PubMed Central

Xu, Huanbin; Wang, Xiaolei; Malam, Naomi; Aye, Pyone P.; Alvarez, Xavier; Lackner, Andrew A.

2015-01-01

ABSTRACT CD4+ follicular T helper (Tfh) cells play a prominent role in humoral immune responses, but the mechanisms of their accumulation and infection in AIDS remain unclear. Here we found that germinal center (GC) Tfh cells, defined here as CXCR5+ PD-1HIGH CD4+ T cells, do not express the HIV coreceptor CCR5 yet serve as a latent reservoir in GCs. With disease progression, an expansion of GC Tfh cells is accompanied by increases in dysfunctional CD8+ T cells. In contrast, Tfh precursor (CXCR5− CD4+ T) cells in lymph nodes do express CCR5 and differentiate into GC Tfh cells following interleukin-6 (IL-6) and IL-21 stimulation, and viral DNA is detectable in fully differentiated GC Tfh cells ex vivo. This suggests that SIV-infected GC Tfh cells may be derived from Tfh precursor cell subsets that become infected in marginal zones and then migrate into GCs as fully mature GC Tfh cells that serve as persistent virus reservoirs. These findings suggest that viral persistence in lymph nodes drives compensatory differentiation, aberrant accumulation, and latent infection of GC Tfh cells, resulting in marked impairment of humoral immune responses. IMPORTANCE Generation of antibodies that can effectively eliminate viruses requires interactions of B cells with highly specialized T cells in GCs of lymphoid tissues called follicular T helper cells. Here we show that in simian immunodeficiency virus infection, these cells are initially infected in a precursor stage that leads to alterations in their homing, accumulation, and function that may be responsible for the inability of human immunodeficiency virus-infected patients to generate effective antibody responses. PMID:26608323

Persistent Simian Immunodeficiency Virus Infection Drives Differentiation, Aberrant Accumulation, and Latent Infection of Germinal Center Follicular T Helper Cells.

PubMed

Xu, Huanbin; Wang, Xiaolei; Malam, Naomi; Aye, Pyone P; Alvarez, Xavier; Lackner, Andrew A; Veazey, Ronald S

2016-02-01

CD4(+) follicular T helper (Tfh) cells play a prominent role in humoral immune responses, but the mechanisms of their accumulation and infection in AIDS remain unclear. Here we found that germinal center (GC) Tfh cells, defined here as CXCR5(+) PD-1(HIGH) CD4(+) T cells, do not express the HIV coreceptor CCR5 yet serve as a latent reservoir in GCs. With disease progression, an expansion of GC Tfh cells is accompanied by increases in dysfunctional CD8(+) T cells. In contrast, Tfh precursor (CXCR5(-) CD4(+) T) cells in lymph nodes do express CCR5 and differentiate into GC Tfh cells following interleukin-6 (IL-6) and IL-21 stimulation, and viral DNA is detectable in fully differentiated GC Tfh cells ex vivo. This suggests that SIV-infected GC Tfh cells may be derived from Tfh precursor cell subsets that become infected in marginal zones and then migrate into GCs as fully mature GC Tfh cells that serve as persistent virus reservoirs. These findings suggest that viral persistence in lymph nodes drives compensatory differentiation, aberrant accumulation, and latent infection of GC Tfh cells, resulting in marked impairment of humoral immune responses. Generation of antibodies that can effectively eliminate viruses requires interactions of B cells with highly specialized T cells in GCs of lymphoid tissues called follicular T helper cells. Here we show that in simian immunodeficiency virus infection, these cells are initially infected in a precursor stage that leads to alterations in their homing, accumulation, and function that may be responsible for the inability of human immunodeficiency virus-infected patients to generate effective antibody responses. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Deinococcus Mn2+-peptide complex: A novel approach to alphavirus vaccine development.

PubMed

Gayen, Manoshi; Gupta, Paridhi; Morazzani, Elaine M; Gaidamakova, Elena K; Knollmann-Ritschel, Barbara; Daly, Michael J; Glass, Pamela J; Maheshwari, Radha K

2017-06-22

Over the last ten years, Chikungunya virus (CHIKV), an Old World alphavirus has caused numerous outbreaks in Asian and European countries and the Americas, making it an emerging pathogen of great global health importance. Venezuelan equine encephalitis virus (VEEV), a New World alphavirus, on the other hand, has been developed as a bioweapon in the past due to its ease of preparation, aerosol dispersion and high lethality in aerosolized form. Currently, there are no FDA approved vaccines against these viruses. In this study, we used a novel approach to develop inactivated vaccines for VEEV and CHIKV by applying gamma-radiation together with a synthetic Mn-decapeptide-phosphate complex (MnDpPi), based on manganous-peptide-orthophosphate antioxidants accumulated in the extremely radiation-resistant bacterium Deinococcus radiodurans. Classical gamma-irradiated vaccine development approaches are limited by immunogenicity-loss due to oxidative damage to the surface proteins at the high doses of radiation required for complete virus-inactivation. However, addition of MnDpPi during irradiation process selectively protects proteins, but not the nucleic acids, from the radiation-induced oxidative damage, as required for safe and efficacious vaccine development. Previously, this approach was used to develop a bacterial vaccine. In the present study, we show that this approach can successfully be applied to protecting mice against viral infections. Irradiation of VEEV and CHIKV in the presence of MnDpPi resulted in substantial epitope preservation even at supra-lethal doses of gamma-rays (50,000Gy). Irradiated viruses were found to be completely inactivated and safe in vivo (neonatal mice). Upon immunization, VEEV inactivated in the presence of MnDpPi resulted in drastically improved protective efficacy. Thus, the MnDpPi-based gamma-inactivation approach described here can readily be applied to developing vaccines against any pathogen of interest in a fast and cost-effective manner. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

High-level rapid production of full-size monoclonal antibodies in plants by a single-vector DNA replicon system

PubMed Central

Huang, Zhong; Phoolcharoen, Waranyoo; Lai, Huafang; Piensook, Khanrat; Cardineau, Guy; Zeitlin, Larry; Whaley, Kevin J.; Arntzen, Charles J.

2010-01-01

Plant viral vectors have great potential in rapid production of important pharmaceutical proteins. However, high-yield production of heterooligomeric proteins that require the expression and assembly of two or more protein subunits often suffers problems due to the “competing” nature of viral vectors derived from the same virus. Previously we reported that a bean yellow dwarf virus (BeYDV)-derived, three-component DNA replicon system allows rapid production of single recombinant proteins in plants (Huang et al. 2009). In this article, we report further development of this expression system for its application in high-yield production of oligomeric protein complexes including monoclonal antibodies (mAbs) in plants. We showed that the BeYDV replicon system permits simultaneous efficient replication of two DNA replicons and thus, high-level accumulation of two recombinant proteins in the same plant cell. We also demonstrated that a single vector that contains multiple replicon cassettes was as efficient as the three-component system in driving the expression of two distinct proteins. Using either the non-competing, three-vector system or the multi-replicon single vector, we produced both the heavy and light chain subunits of a protective IgG mAb 6D8 against Ebola virus GP1 (Wilson et al. 2000) at 0.5 mg of mAb per gram leaf fresh weight within 4 days post infiltration of Nicotiana benthamiana leaves. We further demonstrated that full-size tetrameric IgG complex containing two heavy and two light chains was efficiently assembled and readily purified, and retained its functionality in specific binding to inactivated Ebola virus. Thus, our single-vector replicon system provides high-yield production capacity for heterooligomeric proteins, yet eliminates the difficult task of identifying non-competing virus and the need for co-infection of multiple expression modules. The multi-replicon vector represents a significant advance in transient expression technology for antibody production in plants. PMID:20047189

Pepino mosaic virus RNA-Dependent RNA Polymerase POL Domain Is a Hypersensitive Response-Like Elicitor Shared by Necrotic and Mild Isolates.

PubMed

Sempere, Raquel N; Gómez-Aix, Cristina; Ruíz-Ramón, Fabiola; Gómez, Pedro; Hasiów-Jaroszewska, Beata; Sánchez-Pina, María Amelia; Aranda, Miguel A

2016-04-01

Pepino mosaic virus (PepMV) is an emerging pathogen that represents a serious threat to tomato production worldwide. PepMV-induced diseases manifest with a wide range of symptoms, including systemic necrosis. Our results showed that PepMV accumulation depends on the virus isolate, tomato cultivar, and environmental conditions, and associates with the development of necrosis. Substitution of lysine for glutamic acid at position 67 in the triple gene block 3 (TGB3) protein, previously described as a necrosis determinant, led to increased virus accumulation and was necessary but not sufficient to induce systemic necrosis. Systemic necrosis both in tomato and Nicotiana benthamiana shared hypersensitive response (HR) features, allowing the assessment of the role of different genomic regions on necrosis induction. Overexpression of both TGB3 and the polymerase domain (POL) of the RNA-dependent RNA polymerase (RdRp) resulted in necrosis, although only local expression of POL triggered HR-like symptoms. Our results also indicated that the necrosis-eliciting activity of POL resides in its highly conserved "palm" domain, and that necrosis was jasmonic acid-dependent but not salicylic acid-dependent. Altogether, our data suggest that the RdRp-POL domain plays an important role in PepMV necrosis induction, with necrosis development depending on the virus accumulation level, which can be modulated by the nature of TGB3, host genotype and environmental conditions.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ju, H.-J.; Ye, C.-M.; Verchot-Lubicz, Jeanmarie

Potato virus X (PVX) TGBp3 is required for virus cell-to-cell transport, has an N-terminal transmembrane domain, and a C-terminal cytosolic domain. In the absence of virus infection TGBp3:GFP is seen in the cortical and perinuclear ER. In PVX infected cells the TGBp3:GFP fusion is also seen in the nucleoplasm indicating that events during PVX infection trigger entry into the nucleus. Mutational analysis failed to identify a nuclear targeting domain. Mutations inhibiting TGBp3 association with the ER and inhibiting virus movement did not block TGBp3:GFP in the nucleoplasm. A mutation disrupting the N-terminal transmembrane domain of TGBp3 caused the fusion tomore » accumulate in the nucleus indicating that nuclear import is regulated by ER interactions. Tunicamycin, an ER-stress inducing chemical, caused lower levels of GFP and TGBp3:GFP to accumulate in virus infected protoplasts. MG115 and MG132 were used to demonstrate that wild-type and mutant TGBp3:GFP fusions were degraded by the 26S proteasome. These observations are consistent with an ER-associated protein degradation (ERAD) pathway suggesting that PVX TGBp3, similar to aberrant ER proteins, is translocate to the cytoplasm for degradation. Nuclear accumulation of mutant and wild-type TGBp3:GFP is independent of other PVX proteins and may be another feature of an ERAD pathway.« less

An efficient plant viral expression system generating orally immunogenic Norwalk virus-like particles.

PubMed

Santi, Luca; Batchelor, Lance; Huang, Zhong; Hjelm, Brooke; Kilbourne, Jacquelyn; Arntzen, Charles J; Chen, Qiang; Mason, Hugh S

2008-03-28

Virus-like particles (VLPs) derived from enteric pathogens like Norwalk virus (NV) are well suited to study oral immunization. We previously described stable transgenic plants that accumulate recombinant NV-like particles (rNVs) that were orally immunogenic in mice and humans. The transgenic approach suffers from long generation time and modest level of antigen accumulation. We now overcome these constraints with an efficient tobacco mosaic virus (TMV)-derived transient expression system using leaves of Nicotiana benthamiana. We produced properly assembled rNV at 0.8 mg/g leaf 12 days post-infection (dpi). Oral immunization of CD1 mice with 100 or 250 microg/dose of partially purified rNV elicited systemic and mucosal immune responses. We conclude that the plant viral transient expression system provides a robust research tool to generate abundant quantities of rNV as enriched, concentrated VLP preparations that are orally immunogenic.

An efficient plant viral expression system generating orally immunogenic Norwalk virus-like particles

PubMed Central

Santi, Luca; Batchelor, Lance; Huang, Zhong; Hjelm, Brooke; Kilbourne, Jacquelyn; Arntzen, Charles J.; Chen, Qiang; Mason, Hugh S.

2009-01-01

Virus like particles (VLPs) derived from enteric pathogens like Norwalk virus (NV) are well suited to study oral immunization. We previously described stable transgenic plants that accumulate recombinant NV-like particles (rNV) that were orally immunogenic in mice and humans. The transgenic approach suffers from long generation time and modest level of antigen accumulation. We now overcome these constraints with an efficient tobacco mosaic virus (TMV)-derived transient expression system using leaves of Nicotiana benthamiana. We produced properly assembled rNV at 0.8 mg/g leaf 12 days post infection. Oral immunization of CD1 mice with 100 or 250 μg/dose of partially purified rNV elicited systemic and mucosal immune responses. We conclude that the plant viral transient expression system provides a robust research tool to generate abundant quantities of rNV as enriched, concentrated VLP preparations that are orally immunogenic. PMID:18325641

Next Generation Sequencing of Elite Berry Germplasm and Data Analysis Using a Bioinformatics Pipeline for Virus Detection and Discovery

USDA-ARS?s Scientific Manuscript database

Berry crops (members of the genera Fragaria, Ribes, Rubus, Sambucus and Vaccinium) are known hosts for more than 70 viruses and new ones are identified continually. In modern berry cultivars, viruses tend to be be asymptomatic in single infections and symptoms only develop after plants accumulate m...

Next-Generation Sequencing of Elite Berry Germplasm and Data Analysis Using a Bioinformatics Pipeline for Virus Detection and Discovery

USDA-ARS?s Scientific Manuscript database

Berry crops (members of the genera Fragaria, Ribes, Rubus, Sambucus and Vaccinium) are known hosts for more than 70 viruses and new ones are identified frequently. In modern berry cultivars, viruses tend to be asymptomatic in single infections and symptoms only develop after plants accumulate multip...

Virus-Induced Silencing of Key Genes Leads to Differential Impact on Withanolide Biosynthesis in the Medicinal Plant, Withania somnifera.

PubMed

Agarwal, Aditya Vikram; Singh, Deeksha; Dhar, Yogeshwar Vikram; Michael, Rahul; Gupta, Parul; Chandra, Deepak; Trivedi, Prabodh Kumar

2018-02-01

Withanolides are a collection of naturally occurring, pharmacologically active, secondary metabolites synthesized in the medicinally important plant, Withania somnifera. These bioactive molecules are C28-steroidal lactone triterpenoids and their synthesis is proposed to take place via the mevalonate (MVA) and 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways through the sterol pathway using 24-methylene cholesterol as substrate flux. Although the phytochemical profiles as well as pharmaceutical activities of Withania extracts have been well studied, limited genomic information and difficult genetic transformation have been a major bottleneck towards understanding the participation of specific genes in withanolide biosynthesis. In this study, we used the Tobacco rattle virus (TRV)-mediated virus-induced gene silencing (VIGS) approach to study the participation of key genes from MVA, MEP and triterpenoid biosynthesis for their involvement in withanolide biosynthesis. TRV-infected W. somnifera plants displayed unique phenotypic characteristics and differential accumulation of total Chl as well as carotenoid content for each silenced gene suggesting a reduction in overall isoprenoid synthesis. Comprehensive expression analysis of putative genes of withanolide biosynthesis revealed transcriptional modulations conferring the presence of complex regulatory mechanisms leading to withanolide biosynthesis. In addition, silencing of genes exhibited modulated total and specific withanolide accumulation at different levels as compared with control plants. Comparative analysis also suggests a major role for the MVA pathway as compared with the MEP pathway in providing substrate flux for withanolide biosynthesis. These results demonstrate that transcriptional regulation of selected Withania genes of the triterpenoid biosynthetic pathway critically affects withanolide biosynthesis, providing new horizons to explore this process further, in planta.

Early detection of a two-long-terminal-repeat junction molecule in the cytoplasm of recombinant murine leukemia virus-infected cells.

PubMed

Serhan, Fatima; Penaud, Magalie; Petit, Caroline; Leste-Lasserre, Thierry; Trajcevski, Stéphane; Klatzmann, David; Duisit, Ghislaine; Sonigo, Pierre; Moullier, Philippe

2004-06-01

We showed that a U5-U3 junction was reproducibly detected by a PCR assay as early as 1 to 2 h postinfection with a DNase-treated murine leukemia virus (MLV)-containing supernatant in aphidicolin-arrested NIH 3T3 cells, as well as in nonarrested cells. Such detection is azidothymidine sensitive and corresponded to neosynthesized products of the reverse transcriptase. This observation was confirmed in two additional human cell lines, TE671 and ARPE-19. Using cell fractionation combined with careful controls, we found that a two-long-terminal-repeat (two-LTR) junction molecule was detectable in the cytoplasm as early as 2 h post virus entry. Altogether, our data indicated that the neosynthesized retroviral DNA led to the early formation of structures including true two-LTR junctions in the cytoplasm of MLV-infected cells. Thus, the classical assumption that two-LTR circles are a mitosis-dependent dead-end product accumulating in the nucleus must be reconsidered. MLV-derived products containing a two-LTR junction can no longer be used as an exclusive surrogate for the preintegration complex nuclear translocation event.

GOLDEN2-LIKE transcription factors coordinate the tolerance to Cucumber mosaic virus in Arabidopsis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Xue-Ying; Li, Peng-Xu; Zou, Li-Juan

Arabidopsis thaliana GOLDEN2-LIKE (GLKs) transcription factors play important roles in regulation of photosynthesis-associated nuclear genes, as well as participate in chloroplast development. However, the involvement of GLKs in plants resistance to virus remains largely unknown. Here, the relationship between GLKs and Cucumber mosaic virus (CMV) stress response was investigated. Our results showed that the Arabidopsis glk1glk2 double-mutant was more susceptible to CMV infection and suffered more serious damages (such as higher oxidative damages, more compromised in PSII photochemistry and more reactive oxygen species accumulation) when compared with the wild-type plants. Interestingly, there was little difference between single mutant (glk1 ormore » glk2) and wild-type plants in response to CMV infection, suggesting GLK1 and GLK2 might function redundant in virus resistance in Arabidopsis. Furthermore, the induction of antioxidant system and defense-associated genes expression in the double mutant were inhibited when compared with single mutant or wild-type plants after CMV infection. Further evidences showed that salicylic acid (SA) and jasmonic acid (JA) might be involved in GLKs-mediated virus resistance, as SA or JA level and synthesis-related genes transcription were impaired in glk1glk2 mutant. Taken together, our results indicated that GLKs played a positively role in virus resistance in Arabidopsis. - Highlights: • GLKs play a positive role in CMV resistance in Arabidopsis. • Defective of GLKs suffered more ROS accumulation. • Arabidopsis lacking GLKs have damaged photosynthesis. • Arabidopsis lacking GLKs show low SA and JA accumulation.« less

Experimental Transmission of Karshi and Langat (Tick-Borne Encephalitis Virus Complex) Viruses by Ornithodoros Ticks (Acari: Argasidae)

DTIC Science & Technology

2004-01-01

mosquitoes and Ornithodoros ticks were evaluated for their potential to transmit Karshi and Langat (tick-borne encephalitis virus complex) viruses in the...orally exposed to Langat virus, were able to transmit this virus after more than 3 years, the longest interval tested. Therefore, Ornithodoros spp

Preliminary Observations on the Uptake of Poliovirus by West Coast Shore Crabs

PubMed Central

DiGirolamo, Rudolph; Wiczynski, Leokadia; Daley, Michael; Miranda, Florencio

1972-01-01

West Coast shore crabs (Pachygrapsus sp. and Hemigrapsus sp.), when in seawater contaminated with poliovirus or allowed to feed on virus-contaminated mussels (Mytilus californianus), were found to accumulate high titers of virus. PMID:4333894

Daclatasvir inhibits hepatitis C virus NS5A motility and hyper-accumulation of phosphoinositides

PubMed Central

Chukkapalli, Vineela; Berger, Kristi L.; Kelly, Sean M.; Thomas, Meryl; Deiters, Alexander; Randall, Glenn

2014-01-01

Combinations of direct-acting antivirals (DAAs) against the hepatitis C virus (HCV) have the potential to revolutionize the HCV therapeutic regime. An integral component of DAA combination therapies are HCV NS5A inhibitors. It has previously been proposed that NS5A DAAs inhibit two functions of NS5A: RNA replication and virion assembly. In this study, we characterize the impact of a prototype NS5A DAA, daclatasvir (DCV), on HCV replication compartment formation. DCV impaired HCV replicase localization and NS5A motility. In order to characterize the mechanism behind altered HCV replicase localization, we examined the impact of DCV on the interaction of NS5A with its essential cellular cofactor, phosphatidylinositol-4-kinase III α (PI4KA). We observed that DCV does not inhibit PI4KA directly, nor does it impair early events of the NS5A-PI4KA interaction that can occur when NS5A is expressed alone. NS5A functions that are unaffected by DCV include PI4KA binding, as determined by co-immunoprecipitation, and a basal accumulation of the PI4KA product, PI4P. However, DCV impairs late steps in PI4KA activation that requires NS5A expressed in the context of the HCV polyprotein. These NS5A functions include hyper-stimulation of PI4P levels and appropriate replication compartment formation. The data are most consistent with a model wherein DCV inhibits conformational changes in the NS5A protein or protein complex formations that occur in the context of HCV polyprotein expression and stimulate PI4P hyper-accumulation and replication compartment formation. PMID:25546252

The Bean Pod Mottle Virus RNA2-Encoded 58-Kilodalton Protein P58 Is Required in cis for RNA2 Accumulation

PubMed Central

Lin, Junyan; Guo, Jiangbo; Finer, John; Dorrance, Anne E.; Redinbaugh, Margaret G.

2014-01-01

ABSTRACT Bean pod mottle virus (BPMV) is a bipartite, positive-sense (+) RNA plant virus in the Secoviridae family. Its RNA1 encodes proteins required for genome replication, whereas RNA2 primarily encodes proteins needed for virion assembly and cell-to-cell movement. However, the function of a 58-kDa protein (P58) encoded by RNA2 has not been resolved. P58 and the movement protein (MP) of BPMV are two largely identical proteins differing only at their N termini, with P58 extending MP upstream by 102 amino acid residues. In this report, we unveil a unique role for P58. We show that BPMV RNA2 accumulation in infected cells was abolished when the start codon of P58 was eliminated. The role of P58 does not require the region shared by MP, as RNA2 accumulation in individual cells remained robust even when most of the MP coding sequence was removed. Importantly, the function of P58 required the P58 protein, rather than its coding RNA, as compensatory mutants could be isolated that restored RNA2 accumulation by acquiring new start codons upstream of the original one. Most strikingly, loss of P58 function could not be complemented by P58 provided in trans, suggesting that P58 functions in cis to selectively promote the accumulation of RNA2 copies that encode a functional P58 protein. Finally, we found that all RNA1-encoded proteins are cis-acting relative to RNA1. Together, our results suggest that P58 probably functions by recruiting the RNA1-encoded polyprotein to RNA2 to enable RNA2 reproduction. IMPORTANCE Bean pod mottle virus (BPMV) is one of the most important pathogens of the crop plant soybean, yet its replication mechanism is not well understood, hindering the development of knowledge-based control measures. The current study examined the replication strategy of BPMV RNA2, one of the two genomic RNA segments of this virus, and established an essential role for P58, one of the RNA2-encoded proteins, in the process of RNA2 replication. Our study demonstrates for the first time that P58 functions preferentially with the very RNA from which it is translated, thus greatly advancing our understanding of the replication mechanisms of this and related viruses. Furthermore, this study is important because it provides a potential target for BPMV-specific control, and hence could help to mitigate soybean production losses caused by this virus. PMID:24390330

Virus inactivation under the photodynamic effect of phthalocyanine zinc(II) complexes.

PubMed

Remichkova, Mimi; Mukova, Luchia; Nikolaeva-Glomb, Lubomira; Nikolova, Nadya; Doumanova, Lubka; Mantareva, Vanya; Angelov, Ivan; Kussovski, Veselin; Galabov, Angel S

2017-03-01

Various metal phthalocyanines have been studied for their capacity for photodynamic effects on viruses. Two newly synthesized water-soluble phthalocyanine Zn(II) complexes with different charges, cationic methylpyridyloxy-substituted Zn(II)- phthalocyanine (ZnPcMe) and anionic sulfophenoxy-substituted Zn(II)-phthalocyanine (ZnPcS), were used for photoinactivation of two DNA-containing enveloped viruses (herpes simplex virus type 1 and vaccinia virus), two RNA-containing enveloped viruses (bovine viral diarrhea virus and Newcastle disease virus) and two nude viruses (the enterovirus Coxsackie B1, a RNA-containing virus, and human adenovirus 5, a DNA virus). These two differently charged phthalocyanine complexes showed an identical marked virucidal effect against herpes simplex virus type 1, which was one and the same at an irradiation lasting 5 or 20 min (Δlog=3.0 and 4.0, respectively). Towards vaccinia virus this effect was lower, Δlog=1.8 under the effect of ZnPcMe and 2.0 for ZnPcS. Bovine viral diarrhea virus manifested a moderate sensitivity to ZnPcMe (Δlog=1.8) and a pronounced one to ZnPcS at 5- and 20-min irradiation (Δlog=5.8 and 5.3, respectively). The complexes were unable to inactivate Newcastle disease virus, Coxsackievirus B1 and human adenovirus type 5.

The Bean pod mottle virus RNA2-encoded 58-kilodalton protein P58 is required in cis for RNA2 accumulation

USDA-ARS?s Scientific Manuscript database

Bean pod mottle virus (BPMV) is a bipartite, positive sense (+) RNA plant virus in the Secoviridae family. Its RNA1 encodes proteins required for genome replication, whereas RNA2 primarily encodes proteins needed for virion assembly and cell-to-cell movement. However, the function of a 58 kilo-dalto...

The HEX1 Gene of Fusarium graminearum Is Required for Fungal Asexual Reproduction and Pathogenesis and for Efficient Viral RNA Accumulation of Fusarium graminearum Virus 1

PubMed Central

Son, Moonil; Lee, Kyung-Mi; Yu, Jisuk; Kang, Minji; Park, Jin Man; Kwon, Sun-Jung

2013-01-01

The accumulation of viral RNA depends on many host cellular factors. The hexagonal peroxisome (Hex1) protein is a fungal protein that is highly expressed when the DK21 strain of Fusarium graminearum virus 1 (FgV1) infects its host, and Hex1 affects the accumulation of FgV1 RNA. The Hex1 protein is the major constituent of the Woronin body (WB), which is a peroxisome-derived electron-dense core organelle that seals the septal pore in response to hyphal wounding. To clarify the role of Hex1 and the WB in the relationship between FgV1 and Fusarium graminearum, we generated targeted gene deletion and overexpression mutants. Although neither HEX1 gene deletion nor overexpression substantially affected vegetative growth, both changes reduced the production of asexual spores and reduced virulence on wheat spikelets in the absence of FgV1 infection. However, the vegetative growth of deletion and overexpression mutants was increased and decreased, respectively, upon FgV1 infection compared to that of an FgV1-infected wild-type isolate. Viral RNA accumulation was significantly decreased in deletion mutants but was significantly increased in overexpression mutants compared to the viral RNA accumulation in the virus-infected wild-type control. Overall, these data indicate that the HEX1 gene plays a direct role in the asexual reproduction and virulence of F. graminearum and facilitates viral RNA accumulation in the FgV1-infected host fungus. PMID:23864619

Intrinsically disordered region of influenza A NP regulates viral genome packaging via interactions with viral RNA and host PI(4,5)P2.

PubMed

Kakisaka, Michinori; Yamada, Kazunori; Yamaji-Hasegawa, Akiko; Kobayashi, Toshihide; Aida, Yoko

2016-09-01

To be incorporated into progeny virions, the viral genome must be transported to the inner leaflet of the plasma membrane (PM) and accumulate there. Some viruses utilize lipid components to assemble at the PM. For example, simian virus 40 (SV40) targets the ganglioside GM1 and human immunodeficiency virus type 1 (HIV-1) utilizes phosphatidylinositol (4,5) bisphosphate [PI(4,5)P2]. Recent studies clearly indicate that Rab11-mediated recycling endosomes are required for influenza A virus (IAV) trafficking of vRNPs to the PM but it remains unclear how IAV vRNP localized or accumulate underneath the PM for viral genome incorporation into progeny virions. In this study, we found that the second intrinsically disordered region (IDR2) of NP regulates two binding steps involved in viral genome packaging. First, IDR2 facilitates NP oligomer binding to viral RNA to form vRNP. Secondly, vRNP assemble by interacting with PI(4,5)P2 at the PM via IDR2. These findings suggest that PI(4,5)P2 functions as the determinant of vRNP accumulation at the PM. Copyright © 2016 Elsevier Inc. All rights reserved.

Toward a method for tracking virus evolutionary trajectory applied to the pandemic H1N1 2009 influenza virus.

PubMed

Squires, R Burke; Pickett, Brett E; Das, Sajal; Scheuermann, Richard H

2014-12-01

In 2009 a novel pandemic H1N1 influenza virus (H1N1pdm09) emerged as the first official influenza pandemic of the 21st century. Early genomic sequence analysis pointed to the swine origin of the virus. Here we report a novel computational approach to determine the evolutionary trajectory of viral sequences that uses data-driven estimations of nucleotide substitution rates to track the gradual accumulation of observed sequence alterations over time. Phylogenetic analysis and multiple sequence alignments show that sequences belonging to the resulting evolutionary trajectory of the H1N1pdm09 lineage exhibit a gradual accumulation of sequence variations and tight temporal correlations in the topological structure of the phylogenetic trees. These results suggest that our evolutionary trajectory analysis (ETA) can more effectively pinpoint the evolutionary history of viruses, including the host and geographical location traversed by each segment, when compared against either BLAST or traditional phylogenetic analysis alone. Copyright © 2014 Elsevier B.V. All rights reserved.

Disruption of a stem-loop structure located upstream of pseudoknot domain in Tobacco mosaic virus enhanced its infectivity and viral RNA accumulation.

PubMed

Guo, Song; Wong, Sek-Man

2018-06-01

A predicted stem-loop structure of 25 nucleotides, located in the coat protein (CP) gene and 3'-UTR sequences of Tobacco mosaic virus (TMV), was validated previously (Guo et al., 2015). In this study, both disrupted stem-loop and nucleotide deletion mutants of TMV replicated more rapidly in Nicotiana benthamiana protoplasts. The TMV mutant with a complete mirrored stem-loop structure showed similar level of viral RNA accumulation as TMV. Recovering the stem-loop structure also resulted in a similar replication level as TMV. All these mutants induced necrosis in N. benthamiana and assembled into typical rigid rod-shaped virions. TMV mutant without the stem-loop structure induced more local lesions in Chenopodium quinoa. When the putative stem-loop structure in Tomato mosaic virus (ToMV) was disrupted, the mutant also showed an enhanced virus replication. This suggests that the stem-loop structure of TMV is a new cis-acting element with a role in virus replication. Copyright © 2018 Elsevier Inc. All rights reserved.

Identification of amino acids of the beet necrotic yellow vein virus p25 protein required for induction of the resistance response in leaves of Beta vulgaris plants.

PubMed

Chiba, Soutaro; Miyanishi, Masaki; Andika, Ida Bagus; Kondo, Hideki; Tamada, Tetsuo

2008-05-01

The RNA3-encoded p25 protein of beet necrotic yellow vein virus (BNYVV) is responsible for the production of rhizomania symptoms of sugar beet roots (Beta vulgaris subsp. vulgaris). Here, it was found that the presence of the p25 protein is also associated with the resistance response in rub-inoculated leaves of sugar beet and wild beet (Beta vulgaris subsp. maritima) plants. The resistance phenotype displayed a range of symptoms from no visible lesions to necrotic or greyish lesions at the inoculation site, and only very low levels of virus and viral RNA accumulated. The susceptible phenotype showed large, bright yellow lesions and developed high levels of virus accumulation. In roots after Polymyxa betae vector inoculation, however, no drastic differences in virus and viral RNA accumulation levels were found between plants with susceptible and resistant phenotypes, except at an early stage of infection. There was a genotype-specific interaction between BNYVV strains and two selected wild beet lines (MR1 and MR2) and sugar beet cultivars. Sequence analysis of natural BNYVV isolates and site-directed mutagenesis of the p25 protein revealed that 3 aa residues at positions 68, 70 and 179 are important in determining the resistance phenotype, and that host-genotype specificity is controlled by single amino acid changes at position 68. The mechanism of the occurrence of resistance-breaking BNYVV strains is discussed.

Cucumber Necrosis Virus Recruits Cellular Heat Shock Protein 70 Homologs at Several Stages of Infection

PubMed Central

Alam, Syed Benazir

2015-01-01

ABSTRACT RNA viruses often depend on host factors for multiplication inside cells due to the constraints of their small genome size and limited coding capacity. One such factor that has been exploited by several plant and animal viruses is heat shock protein 70 (HSP70) family homologs which have been shown to play roles for different viruses in viral RNA replication, viral assembly, disassembly, and cell-to-cell movement. Using next generation sequence analysis, we reveal that several isoforms of Hsp70 and Hsc70 transcripts are induced to very high levels during cucumber necrosis virus (CNV) infection of Nicotiana benthamiana and that HSP70 proteins are also induced by at least 10-fold. We show that HSP70 family protein homologs are co-opted by CNV at several stages of infection. We have found that overexpression of Hsp70 or Hsc70 leads to enhanced CNV genomic RNA, coat protein (CP), and virion accumulation, whereas downregulation leads to a corresponding decrease. Hsc70-2 was found to increase solubility of CNV CP in vitro and to increase accumulation of CNV CP independently of viral RNA replication during coagroinfiltration in N. benthamiana. In addition, virus particle assembly into virus-like particles in CP agroinfiltrated plants was increased in the presence of Hsc70-2. HSP70 was found to increase the targeting of CNV CP to chloroplasts during infection, reinforcing the role of HSP70 in chloroplast targeting of host proteins. Hence, our findings have led to the discovery of a highly induced host factor that has been co-opted to play multiple roles during several stages of the CNV infection cycle. IMPORTANCE Because of the small size of its RNA genome, CNV is dependent on interaction with host cellular components to successfully complete its multiplication cycle. We have found that CNV induces HSP70 family homologs to a high level during infection, possibly as a result of the host response to the high levels of CNV proteins that accumulate during infection. Moreover, we have found that CNV co-opts HSP70 family homologs to facilitate several aspects of the infection process such as viral RNA, coat protein and virus accumulation. Chloroplast targeting of the CNV CP is also facilitated, which may aid in CNV suppression of host defense responses. Several viruses have been shown to induce HSP70 during infection and others to utilize HSP70 for specific aspects of infection such as replication, assembly, and disassembly. We speculate that HSP70 may play multiple roles in the infection processes of many viruses. PMID:26719261

Accumulation of MS2, GA, and Qβ phages on high density polyethylene (HDPE) and drinking water biofilms under flow/non-flow conditions.

PubMed

Pelleïeux, Sandra; Bertrand, Isabelle; Skali-Lami, Salaheddine; Mathieu, Laurence; Francius, Grégory; Gantzer, Christophe

2012-12-01

Accumulation of enteric viruses on surfaces within a drinking water distribution system was investigated in a reactor using three F-specific RNA bacteriophages (MS2, GA, and Qβ) as models of human pathogenic viruses. The influence of hydrodynamic versus hydrostatic conditions and the effect of the colonization of HDPE surfaces with two-month-old biofilms were assessed for virus accumulation on surfaces. In order to work under controlled laminar conditions and to study various wall shear stresses at the same time, a new rotating disc reactor was designed. Among the wall shear rates applied in the reactor (450 to 1640 s(-1)) no significant differences were observed concerning both the total number of bacteria, which was found to be around 1.7 × 10(7) cells/cm(2) and the virus concentrations on surfaces were about 3 × 10(4), 5 × 10(5) and 3 × 10(5) eq PFU/cm(2) for MS2, GA and Qβ phages, respectively. Comparison between static versus dynamic conditions revealed that both Brownian diffusion and convective diffusion were involved in the transport of these soft colloidal particles and an increase reaching about 1 log in virus concentrations measured on surfaces appeared when hydrodynamic conditions where applied. Our results also showed the influence of the colonization by two-month-old drinking water biofilms which led to a change in the level of virus adhesion. The implication of the physico-chemical properties was also underlined since different adhesion profiles were obtained for the three bacteriophages and MS2 phage was found to be the less adherent one whatever the conditions applied. Copyright © 2012 Elsevier Ltd. All rights reserved.

Maize Iranian mosaic virus shows a descending transcript accumulation order in plant and insect hosts.

PubMed

Hortamani, Mozhgan; Massah, Amir; Izadpanah, Keramat

2018-04-01

Maize Iranian mosaic virus (MIMV) is a distinct member of the genus Nucleorhabdovirus. In this study, expression of all MIMV genes in maize for four weeks after inoculation and in inoculative planthoppers was examined using a quantitative RT-PCR (RT-qPCR) assay. Accumulation of MIMV P, gene 3, M, G and L transcripts relative to N transcripts was measured and normalized to 18S rRNA in maize plants and to the ribosomal protein S13 gene (RPS13) in planthoppers using the comparative C T method. In plants, higher levels of MIMV N transcripts were found relative to other transcripts, while MIMV L transcripts were at the lowest levels. The highest accumulation of MIMV transcripts was found at 14 days postinoculation (dpi). At 21 dpi, we found the lowest transcript levels for all genes, which increased again at 28 dpi, although in lower amounts than at 14 dpi. In Laodelphax striatellus, MIMV M, G and L transcripts accumulated at lower levels than other transcripts. The gene 3 transcript level was high in both plants and planthoppers. Our results showed that transcript accumulation for the MIMV genes was similar in both hosts and followed the pattern of sequential transcriptional attenuation from the 3' to the 5' end of the genome, similar to vertebrate rhabdoviruses. These results indicate that the regulation of virus gene transcription for this plant-infecting rhabdovirus is similar to that of some vertebrate-infecting rhabdoviruses.

Influenza A Virus Hemagglutinin is Required for the Assembly of Viral Components Including Bundled vRNPs at the Lipid Raft.

PubMed

Takizawa, Naoki; Momose, Fumitaka; Morikawa, Yuko; Nomoto, Akio

2016-09-10

The influenza glycoproteins, hemagglutinin (HA) and neuraminidase (NA), which are associated with the lipid raft, have the potential to initiate virion budding. However, the role of these viral proteins in infectious virion assembly is still unclear. In addition, it is not known how the viral ribonucleoprotein complex (vRNP) is tethered to the budding site. Here, we show that HA is necessary for the efficient progeny virion production and vRNP packaging in the virion. We also found that the level of HA does not affect the bundling of the eight vRNP segments, despite reduced virion production. Detergent solubilization and a subsequent membrane flotation analysis indicated that the accumulation of nucleoprotein, viral polymerases, NA, and matrix protein 1 (M1) in the lipid raft fraction was delayed without HA. Based on our results, we inferred that HA plays a role in the accumulation of viral components, including bundled vRNPs, at the lipid raft.

Clinical Investigation Program RCS MED-300 (R1).

DTIC Science & Technology

1985-09-30

of Group A Streptococcus ......................................... 193 Work Unit No. 85/17 (FY85,T) Serological Survey of Epstein - Barr Virus ...Prot No: 85/17 Status: Terminated Title: Serological Survey of Epstein - Barr Virus Infections Followed Over a 12-Month Period Start Date: Est Comp Date...Principal Investigator: Facility: CPT C.A. Borchert, MC Dept/Sec: Pediatrics Assoc Investigators Key Words: Epstein - Barr Virus Accumulative MEDCASE Est

A stem–loop structure in the 59 untranslated region of bean pod mottle virus RNA2 is specifically required for RNA2 accumulation

USDA-ARS?s Scientific Manuscript database

Bean pod mottle virus (BPMV) is a bipartite, positive-sense (+) RNA plant virus of the family Secoviridae. Its RNA1 encodes all proteins needed for genome replication and is capable of autonomous replication. By contrast, BPMV RNA2 must utilize RNA1-encoded proteins for replication. Here, we sought ...

Plum pox virus accumulates mutations in different genome parts during a long-term maintenance in Prunus host plants and passage in Nicotiana benthamiana.

PubMed

Vozárová, Z; Kamencayová, M; Glasa, M; Subr, Z

2013-01-01

Plum pox virus (PPV) isolates of the strain PPV-M prevalently infect peaches under natural conditions in Middle Europe. Comparison of complete genome sequences obtained from subisolates of a PPV-M isolate maintained experimentally over a 6-year period in different Prunus host species and passaged in Nicotiana benthamiana was performed with the aim to highlight the mutations potentially connected with the virus-host adaptation. The results showed that the lowest number of non-silent mutations was accumulated in PPV-M maintained in peach (original host species), approximately two times higher diversity was recorded in plum, apricot and N. benthamiana, indicating the genetic determination of the PPV host preference. The sequence variability of Prunus subisolates was distributed more or less evenly along the PPV genome and no amino acid motif could be outlined as responsible for the host adaptation. In N. benthamiana the mutations were accumulated notably in the P1 and P3 genes indicating their non-essentiality in the infection of this experimental host plant.

Scion on a Stock Producing siRNAs of Potato Spindle Tuber Viroid (PSTVd) Attenuates Accumulation of the Viroid

PubMed Central

Kasai, Atsushi; Sano, Teruo; Harada, Takeo

2013-01-01

Plants can attenuate the replication of plant viruses and viroids by RNA silencing induced by virus and viroid infection. In higher plants, silencing signals such as small interfering RNAs (siRNAs) produced by RNA silencing can be transported systemically through phloem, so it is anticipated that antiviral siRNA signals produced in a stock would have the potential to attenuate propagation of viruses or viroids in the scion. To test whether this is indeed the case, we prepared transgenic tobacco (Nicotiana benthamiana) expressing a hairpin RNA (hpRNA) of Potato spindle tuber viroid (PSTVd) in companion cells by using a strong companion cell-specific promoter. A grafting experiment of the wild type tobacco scion on the top of the transgenic tobacco stock revealed that accumulation of PSTVd challenge-inoculated into the scion was apparently attenuated compared to the control grafted plants. These results indicate that genetically modified rootstock expressing viroid-specific siRNAs can attenuate viroid accumulation in a non-genetically modified scion grafted on the stock. PMID:23469061

Uptake and elimination of poliovirus by West Coast oysters.

PubMed

Di Girolamo, R; Liston, J; Matches, J

1975-02-01

Accumulation of poliovirus Lsc-2ab by West Coast oysters was determined by using a stationary seawater system, and depuration was determined by using both stationary and free-flow systems. Results indicate that these shellfish have the same pattern of accumulation and localization of viruses as do East Coast species. However, uptake appeared to occur more rapidly than described for East Coast shellfish. There appeared to be a gradual diffusion of virus from the digestive area into the body. Depuration was found to occur more rapidly and completely under free-flow conditions than in a stationary system.

Uptake and Elimination of Poliovirus by West Coast Oysters

PubMed Central

Girolamo, Rudolph Di; Liston, John; Matches, J.

1975-01-01

Accumulation of poliovirus Lsc-2ab by West Coast oysters was determined by using a stationary seawater system, and depuration was determined by using both stationary and free-flow systems. Results indicate that these shellfish have the same pattern of accumulation and localization of viruses as do East Coast species. However, uptake appeared to occur more rapidly than described for East Coast shellfish. There appeared to be a gradual diffusion of virus from the digestive area into the body. Depuration was found to occur more rapidly and completely under free-flow conditions than in a stationary system. PMID:163615

Characterization of mutant tobacco mosaic virus coat protein that interferes with virus cell-to-cell movement.

PubMed

Bendahmane, Mohammed; Szecsi, Judit; Chen, Iju; Berg, R Howard; Beachy, Roger N

2002-03-19

Expression of tobacco mosaic virus (TMV) coat protein (CP) in plants confers resistance to infection by TMV and related tobamoviruses. Certain mutants of the CP (CP(T42W)) provide much greater levels of resistance than wild-type (wt) CP. In the present work, infection induced by RNA transcripts of TMV clones that contain wt CP or mutant CP(T42W) fused to the green fluorescent protein (GFP) (TMV-CP:GFP, TMV-CP(T42W):GFP) and clones harboring TMV movement protein (MP):GFP were followed in nontransgenic and transgenic tobacco BY-2 protoplasts and Nicotiana tabaccum Xanthi-nn plants that express wt CP or CP(T42W). On nontransgenic and wt CP transgenic plants, TMV-CP:GFP produced expanding, highly fluorescent disk-shaped areas. On plants expressing CP(T42W), infection by TMV-CP:GFP or TMV-MP:GFP-CP produced infection sites of smaller size that were characterized by low fluorescence, reflecting reduced levels of virus spread and reduced accumulation of both CP:GFP and MP:GFP. TMV-CP(T42W):GFP failed to produce visible infection sites on nontransgenic plants, yet produced normal infection sites on MP-transgenic plants that produce MP. TMV infection of transgenic BY-CP(T42W) protoplasts resulted in very low levels of MP accumulation, whereas on BY-CP protoplasts (containing wt CP), infection produced higher levels of MP than in nontransgenic BY-2 cells. The results suggest that wt CP has a positive effect on the production of MP, whereas the CP(T42W) has a negative effect on MP accumulation and/or function. This effect results in very high levels of resistance to TMV infection in plants containing CP(T42W). This report shows that the CP of a plant virus regulates production of the MP, and that a mutant CP interferes with MP accumulation and cell-to-cell movement of infection.

[Ribonucleic acids and proteins of influenza A/USSR/90/77 viruses].

PubMed

Vorkunova, G K; Dotsenko, G N; Bukrinskaia, A G; Zhdanov, V M

1979-01-01

The "return" of influenza A (HINI) virus after 20 years of absence raised the question of the sources and mechanisms of emergence of epidemic influenza viruses and, particularly, of a new HINI virus (A/USSR/90/77). Two alternative hypotheses answer this question differently: the new HINI virus is the progeny of old HINI viruses retained in the human population or is a newly arising recombinant between numerous human and animal influenza viruses circulation in the biosphere. For the acceptance of one or the other hypothesis further accumulation of facts is required and, first of all, comparative investigations of RNAs and proteins of various influenza viruses. This paper presents the results of comparative studies of RNAs and proteins of old and new influenza A (HINI) viruses.

Two key arginine residues in the coat protein of Bamboo mosaic virus differentially affect the accumulation of viral genomic and subgenomic RNAs.

PubMed

Hung, Chien-Jen; Hu, Chung-Chi; Lin, Na-Sheng; Lee, Ya-Chien; Meng, Menghsiao; Tsai, Ching-Hsiu; Hsu, Yau-Heiu

2014-02-01

The interactions between viral RNAs and coat proteins (CPs) are critical for the efficient completion of infection cycles of RNA viruses. However, the specificity of the interactions between CPs and genomic or subgenomic RNAs remains poorly understood. In this study, Bamboo mosaic virus (BaMV) was used to analyse such interactions. Using reversible formaldehyde cross-linking and mass spectrometry, two regions in CP, each containing a basic amino acid (R99 and R227, respectively), were identified to bind directly to the 5' untranslated region of BaMV genomic RNA. Analyses of the alanine mutations of R99 and R227 revealed that the secondary structures of CP were not affected significantly, whereas the accumulation of BaMV genomic, but not subgenomic, RNA was severely decreased at 24 h post-inoculation in the inoculated protoplasts. In the absence of CP, the accumulation levels of genomic and subgenomic RNAs were decreased to 1.1%-1.5% and 33%-40% of that of the wild-type (wt), respectively, in inoculated leaves at 5 days post-inoculation (dpi). In contrast, in the presence of mutant CPs, the genomic RNAs remained about 1% of that of wt, whereas the subgenomic RNAs accumulated to at least 87%, suggesting that CP might increase the accumulation of subgenomic RNAs. The mutations also restricted viral movement and virion formation in Nicotiana benthamiana leaves at 5 dpi. These results demonstrate that R99 and R227 of CP play crucial roles in the accumulation, movement and virion formation of BaMV RNAs, and indicate that genomic and subgenomic RNAs interact differently with BaMV CP. © 2013 BSPP AND JOHN WILEY & SONS LTD.

Light and electron microscope study of the neurotropism of Powassan virus strain P-40.

PubMed

Isachkova, L M; Shestopalova, N M; Frolova, M P; Reingold, V N

1979-01-01

Brains of adult white mice inoculated with the P-40 strain of Powassan virus isolated in Primorsky Krai (U.S.S.R) from ticks were studied by light and electron microscopy. Accumulations of virus particles were found in neurons and their dendrites and axons, in glial cells, and in intercellular spaces. In the nerve cells, most prevalent were changes of the type of chromatolysis and formation of small vacuoles, associated with the alteration of the endoplasmic reticulum induced by virus morphogenesis. In virus-affected cells, multilayer dense membranes were found.

Compatible GLRaV-3 viral infections affect berry ripening decreasing sugar accumulation and anthocyanin biosynthesis in Vitis vinifera.

PubMed

Vega, Andrea; Gutiérrez, Rodrigo A; Peña-Neira, Alvaro; Cramer, Grant R; Arce-Johnson, Patricio

2011-10-01

Virus infections in grapevine cause important economic losses and affect fruit quality worldwide. Although the phenotypic symptoms associated to viral infections have been described, the molecular plant response triggered by virus infection is still poorly understood in Vitis vinifera. As a first step to understand the fruit changes and mechanisms involved in the compatible grapevine-virus interaction, we analyzed the berry transcriptome in two stages of development in the red wine cultivar Cabernet Sauvignon infected with Grapevine leaf-roll-associated virus-3 (GLRaV-3). Analysis of global gene expression patterns indicate incomplete berry maturation in infected berries as compared to uninfected fruit suggesting viral infection interrupts the normal berry maturation process. Genes with altered expression in berries harvested from GLRaV-3-infected vines as compared to uninfected tissue include anthocyanin biosynthesis and sugar metabolism genes. The reduction in transcript accumulation for sugar and anthocyanin metabolism during fruit development is consistent with a dramatic reduction in anthocyanin biosynthesis as well as reduced sugar levels in berries, a hallmark phenotypic change observed in virus infected grapevines. Analysis of key regulatory factors provides a mechanism for the observed gene expression changes. Our results provide insight into commonly observed phenotypic alterations in virus infected vines and the molecular mechanisms associated with the plant response to the virus during berry ripening.

Evaluation of methods using celite to concentrate norovirus, adenovirus and enterovirus from wastewater

EPA Science Inventory

Enteroviruses, noroviruses and adenoviruses are among the most common viruses infecting humans worldwide. These viruses are shed in the feces of infected individuals and can accumulate in wastewater. Therefore, wastewater is a source of a potentially diverse group of enteric viru...

TYLCV-Is movement in planta does not require V2 protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hak, Hagit; Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem; Levy, Yael

Tomato yellow leaf curl virus (TYLCV), a major tomato pathogen causing extensive crop losses, is a whitefly-transmitted geminivirus. V2 mutants of TYLCV-Is and related viruses tend to induce symptomless infection with attenuated viral DNA levels, while accumulating close to wild-type DNA levels in protoplasts, suggesting V2 as a movement protein. The discovery of plant-silencing mechanisms and viral silencing suppressors, V2 included, led us to reconsider V2's involvement in viral movement. We studied two mutant versions of the virus, one impaired in V2 silencing-suppression activity, and another carrying a non-translatable V2. While both mutant viruses spread in the infected plant tomore » newly emerged leaves at the same rate as the wild-type virus, their DNA-accumulation levels were tenfold lower than in the wild-type virus. Thus, we suggest that the setback in virus proliferation, previously ascribed to a movement impediment, is due to lack of silencing-suppression activity. - Highlights: • TYLCV-Is V2 protein is localized in distinct microbodies throughout the cell cytoplasm, around the nucleus and in association with cytoplasmic strands but is not associated with the plasmodesmata. • Disruption of RNA-silencing suppression activity of TYLCV-Is V2 protein causes low titer of the virus in the infected plants. • The movement of TYLCV-Is in planta does not require a functional V2 protein.« less

Disruption of Microtubules Post-Virus Entry Enhances Adeno-Associated Virus Vector Transduction

PubMed Central

Xiao, Ping-Jie; Mitchell, Angela M.; Huang, Lu; Li, Chengwen; Samulski, R. Jude

2016-01-01

Perinuclear retention of viral particles is a poorly understood phenomenon observed during many virus infections. In this study, we investigated whether perinuclear accumulation acts as a barrier to limit recombinant adeno-associated virus (rAAV) transduction. After nocodazole treatment to disrupt microtubules at microtubule-organization center (MT-MTOC) after virus entry, we observed higher rAAV transduction. To elucidate the role of MT-MTOC in rAAV infection and study its underlying mechanisms, we demonstrated that rAAV's perinuclear localization was retained by MT-MTOC with fluorescent analysis, and enhanced rAAV transduction from MT-MTOC disruption was dependent on the rAAV capsid's nuclear import signals. Interestingly, after knocking down RhoA or inhibiting its downstream effectors (ROCK and Actin), MT-MTOC disruption failed to increase rAAV transduction or nuclear entry. These data suggest that enhancement of rAAV transduction is the result of increased trafficking to the nucleus via the RhoA-ROCK-Actin pathway. Ten-fold higher rAAV transduction was also observed by disrupting MT-MTOC in brain, liver, and tumor in vivo. In summary, this study indicates that virus perinuclear accumulation at MT-MTOC is a barrier-limiting parameter for effective rAAV transduction and defines a novel defense mechanism by which host cells restrain viral invasion. PMID:26942476

Deletion of the Vaccinia Virus I2 Protein Interrupts Virion Morphogenesis, Leading to Retention of the Scaffold Protein and Mislocalization of Membrane-Associated Entry Proteins.

PubMed

Hyun, Seong-In; Weisberg, Andrea; Moss, Bernard

2017-08-01

The I2L open reading frame of vaccinia virus (VACV) encodes a conserved 72-amino-acid protein with a putative C-terminal transmembrane domain. Previous studies with a tetracycline-inducible mutant demonstrated that I2-deficient virions are defective in cell entry. The purpose of the present study was to determine the step of replication or entry that is affected by loss of the I2 protein. Fluorescence microscopy experiments showed that I2 colocalized with a major membrane protein of immature and mature virions. We generated a cell line that constitutively expressed I2 and allowed construction of the VACV I2L deletion mutant vΔI2. As anticipated, vΔI2 was unable to replicate in cells that did not express I2. Unexpectedly, morphogenesis was interrupted at a stage after immature virion formation, resulting in the accumulation of dense spherical particles instead of brick-shaped mature virions with well-defined core structures. The abnormal particles retained the D13 scaffold protein of immature virions, were severely deficient in the transmembrane proteins that comprise the entry fusion complex (EFC), and had increased amounts of unprocessed membrane and core proteins. Total lysates of cells infected with vΔI2 also had diminished EFC proteins due to instability attributed to their hydrophobicity and failure to be inserted into viral membranes. A similar instability of EFC proteins had previously been found with unrelated mutants blocked earlier in morphogenesis that also accumulated viral membranes retaining the D13 scaffold. We concluded that I2 is required for virion morphogenesis, release of the D13 scaffold, and the association of EFC proteins with viral membranes. IMPORTANCE Poxviruses comprise a large family that infect vertebrates and invertebrates, cause disease in both in humans and in wild and domesticated animals, and are being engineered as vectors for vaccines and cancer therapy. In addition, investigations of poxviruses have provided insights into many aspects of cell biology. The I2 protein is conserved in all poxviruses that infect vertebrates, suggesting an important role. The present study revealed that this protein is essential for vaccinia virus morphogenesis and that its absence results in an accumulation of deformed virus particles retaining the scaffold protein and deficient in surface proteins needed for cell entry. Copyright © 2017 American Society for Microbiology.

Cross-neutralisation of viruses of the tick-borne encephalitis complex following tick-borne encephalitis vaccination and/or infection.

PubMed

McAuley, Alexander J; Sawatsky, Bevan; Ksiazek, Thomas; Torres, Maricela; Korva, Miša; Lotrič-Furlan, Stanka; Avšič-Županc, Tatjana; von Messling, Veronika; Holbrook, Michael R; Freiberg, Alexander N; Beasley, David W C; Bente, Dennis A

2017-01-01

The tick-borne encephalitis complex contains a number of flaviviruses that share close genetic homology, and are responsible for significant human morbidity and mortality with widespread geographical range. Although many members of this complex have been recognised for decades, licenced human vaccines with broad availability are only available for tick-borne encephalitis virus. While tick-borne encephalitis virus vaccines have been demonstrated to induce significant protective immunity, as determined by virus-neutralisation titres, vaccine breakthrough (clinical infection following complete vaccination), has been described. The aim of this study was to confirm the cross-neutralisation of tick-borne flaviviruses using mouse immune ascitic fluids, and to determine the magnitude of cross-neutralising antibody titres in sera from donors following tick-borne encephalitis vaccination, infection, and vaccine breakthrough. The results demonstrate that there is significant cross-neutralisation of representative members of the tick-borne encephalitis complex following vaccination and/or infection, and that the magnitude of immune responses varies based upon the exposure type. Donor sera successfully neutralised most of the viruses tested, with 85% of vaccinees neutralising Kyasanur forest disease virus and 73% of vaccinees neutralising Alkhumra virus. By contrast, only 63% of vaccinees neutralised Powassan virus, with none of these neutralisation titres exceeding 1:60. Taken together, the data suggest that tick-borne encephalitis virus vaccination may protect against most of the members of the tick-borne encephalitis complex including Kyasanur forest disease virus and Alkhumra virus, but that the neutralisation of Powassan virus following tick-borne encephalitis vaccination is minimal.

A large scale laboratory cage trial of Aedes densonucleosis virus (AeDNV).

PubMed

Wise de Valdez, Megan R; Suchman, Erica L; Carlson, Jonathan O; Black, William C

2010-05-01

Aedes aegypti (L.) (Diptera: Culicidae) the primary vector of dengue viruses (DENV1-4), oviposit in and around human dwellings, including sites difficult to locate, making control of this mosquito challenging. We explored the efficacy and sustainability of Aedes Densonucleosis Virus (AeDNV) as a biocontrol agent for Ae. aegypti in and among oviposition sites in large laboratory cages (> 92 m3) as a prelude to field trials. Select cages were seeded with AeDNV in a single oviposition site (OPS) with unseeded OPSs established at varied distances. Quantitative real-time polymerase chain reaction was used to track dispersal and accumulation of AeDNV among OPSs. All eggs were collected weekly from each cage and counted. We asked: (1) Is AeDNV dispersed over varying distances and can it accumulate and persist in novel OPSs? (2) Are egg densities reduced in AeDNV treated populations? AeDNV was dispersed to and sustained in novel OPSs. Virus accumulation in OPSs was positively correlated with egg densities and proximity to the initial infection source affected the timing of dispersal and maintenance of viral titers. AeDNV did not significantly reduce Ae. aegypti egg densities. The current study documents that adult female Ae. aegypti oviposition behavior leads to successful viral dispersal from treated to novel containers in large-scale cages; however, the AeDNV titers reached were not sufficient to reduce egg densities.

Ubiquitin-like modifier FAT10 attenuates RIG-I mediated antiviral signaling by segregating activated RIG-I from its signaling platform

PubMed Central

Nguyen, Nhung T.H.; Now, Hesung; Kim, Woo-Jong; Kim, Nari; Yoo, Joo-Yeon

2016-01-01

RIG-I is a key cytosolic RNA sensor that mediates innate immune defense against RNA virus. Aberrant RIG-I activity leads to severe pathological states such as autosomal dominant multi-system disorder, inflammatory myophathies and dermatomyositis. Therefore, identification of regulators that ensure efficient defense without harmful immune-pathology is particularly critical to deal with RIG-I-associated diseases. Here, we presented the inflammatory inducible FAT10 as a novel negative regulator of RIG-I-mediated inflammatory response. In various cell lines, FAT10 protein is undetectable unless it is induced by pro-inflammatory cytokines. FAT10 non-covalently associated with the 2CARD domain of RIG-I, and inhibited viral RNA-induced IRF3 and NF-kB activation through modulating the RIG-I protein solubility. We further demonstrated that FAT10 was recruited to RIG-I-TRIM25 to form an inhibitory complex where FAT10 was stabilized by E3 ligase TRIM25. As the result, FAT10 inhibited the antiviral stress granules formation contains RIG-I and sequestered the active RIG-I away from the mitochondria. Our study presented a novel mechanism to dampen RIG-I activity. Highly accumulated FAT10 is observed in various cancers with pro-inflammatory environment, therefore, our finding which uncovered the suppressive effect of the accumulated FAT10 during virus-mediated inflammatory response may also provide molecular clue to understand the carcinogenesis related with infection and inflammation. PMID:26996158

Ubiquitin-like modifier FAT10 attenuates RIG-I mediated antiviral signaling by segregating activated RIG-I from its signaling platform.

PubMed

Nguyen, Nhung T H; Now, Hesung; Kim, Woo-Jong; Kim, Nari; Yoo, Joo-Yeon

2016-03-21

RIG-I is a key cytosolic RNA sensor that mediates innate immune defense against RNA virus. Aberrant RIG-I activity leads to severe pathological states such as autosomal dominant multi-system disorder, inflammatory myophathies and dermatomyositis. Therefore, identification of regulators that ensure efficient defense without harmful immune-pathology is particularly critical to deal with RIG-I-associated diseases. Here, we presented the inflammatory inducible FAT10 as a novel negative regulator of RIG-I-mediated inflammatory response. In various cell lines, FAT10 protein is undetectable unless it is induced by pro-inflammatory cytokines. FAT10 non-covalently associated with the 2CARD domain of RIG-I, and inhibited viral RNA-induced IRF3 and NF-kB activation through modulating the RIG-I protein solubility. We further demonstrated that FAT10 was recruited to RIG-I-TRIM25 to form an inhibitory complex where FAT10 was stabilized by E3 ligase TRIM25. As the result, FAT10 inhibited the antiviral stress granules formation contains RIG-I and sequestered the active RIG-I away from the mitochondria. Our study presented a novel mechanism to dampen RIG-I activity. Highly accumulated FAT10 is observed in various cancers with pro-inflammatory environment, therefore, our finding which uncovered the suppressive effect of the accumulated FAT10 during virus-mediated inflammatory response may also provide molecular clue to understand the carcinogenesis related with infection and inflammation.

Influenza A Virus Polymerase Recruits the RNA Helicase DDX19 to Promote the Nuclear Export of Viral mRNAs

PubMed Central

Diot, Cédric; Fournier, Guillaume; Dos Santos, Mélanie; Magnus, Julie; Komarova, Anastasia; van der Werf, Sylvie; Munier, Sandie; Naffakh, Nadia

2016-01-01

Enhancing the knowledge of host factors that are required for efficient influenza A virus (IAV) replication is essential to address questions related to pathogenicity and to identify targets for antiviral drug development. Here we focused on the interplay between IAV and DExD-box RNA helicases (DDX), which play a key role in cellular RNA metabolism by remodeling RNA-RNA or RNA-protein complexes. We performed a targeted RNAi screen on 35 human DDX proteins to identify those involved in IAV life cycle. DDX19 was a major hit. In DDX19-depleted cells the accumulation of viral RNAs and proteins was delayed, and the production of infectious IAV particles was strongly reduced. We show that DDX19 associates with intronless, unspliced and spliced IAV mRNAs and promotes their nuclear export. In addition, we demonstrate an RNA-independent association between DDX19 and the viral polymerase, that is modulated by the ATPase activity of DDX19. Our results provide a model in which DDX19 is recruited to viral mRNAs in the nucleus of infected cells to enhance their nuclear export. Information gained from this virus-host interaction improves the understanding of both the IAV replication cycle and the cellular function of DDX19. PMID:27653209

Tobacco etch virus protein P1 traffics to the nucleolus and associates with the host 60S ribosomal subunits during infection.

PubMed

Martínez, Fernando; Daròs, José-Antonio

2014-09-01

The genus Potyvirus comprises a large group of positive-strand RNA plant viruses whose genome encodes a large polyprotein processed by three viral proteinases. P1 protein, the most amino-terminal product of the polyprotein, is an accessory factor stimulating viral genome amplification whose role during infection is not well understood. We infected plants with Tobacco etch virus (TEV; genus Potyvirus) clones in which P1 was tagged with a fluorescent protein to track its expression and subcellular localization or with an affinity tag to identify host proteins involved in complexes in which P1 also takes part during infection. Our results showed that TEV P1 exclusively accumulates in infected cells at an early stage of infection and that the protein displays a dynamic subcellular localization, trafficking in and out of the nucleus and nucleolus during infection. Inside the nucleolus, P1 particularly targets the dense granular component. Consistently, we found functional nucleolar localization and nuclear export signals in TEV P1 sequence. Our results also indicated that TEV P1 physically interacts with the host 80S cytoplasmic ribosomes and specifically binds to the 60S ribosomal subunits during infection. In vitro translation assays of reporter proteins suggested that TEV P1 stimulates protein translation, particularly when driven from the TEV internal ribosome entry site. These in vitro assays also suggested that TEV helper-component proteinase (HC-Pro) inhibits protein translation. Based on these findings, we propose that TEV P1 stimulates translation of viral proteins in infected cells. In this work, we researched the role during infection of tobacco etch virus P1 protease. P1 is the most mysterious protein of potyviruses, a relevant group of RNA viruses infecting plants. Our experiments showed that the viral P1 protein exclusively accumulates in infected cells at an early stage of infection and moves in and out of the nucleus of infected cells, particularly targeting the nucleolus. Our experiments also showed that P1 protein binds host ribosomes during infection. Based on these findings and other in vitro experiments we propose that P1 protein stimulates translation of viral proteins during infection. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Identification of a spliced gene from duck enteritis virus encoding a protein homologous to UL15 of herpes simplex virus 1.

PubMed

Zhu, Hongwei; Li, Huixin; Han, Zongxi; Shao, Yuhao; Wang, Yu; Kong, Xiangang

2011-04-06

In herpesviruses, UL15 homologue is a subunit of terminase complex responsible for cleavage and packaging of the viral genome into pre-assembled capsids. However, for duck enteritis virus (DEV), the causative agent of duck viral enteritis (DVE), the genomic sequence was not completely determined until most recently. There is limited information of this putative spliced gene and its encoding protein. DEV UL15 consists of two exons with a 3.5 kilobases (kb) inron and transcribes into two transcripts: the full-length UL15 and an N-terminally truncated UL15.5. The 2.9 kb UL15 transcript encodes a protein of 739 amino acids with an approximate molecular mass of 82 kiloDaltons (kDa), whereas the UL15.5 transcript is 1.3 kb in length, containing a putative 888 base pairs (bp) ORF that encodes a 32 kDa product. We also demonstrated that UL15 gene belonged to the late kinetic class as its expression was sensitive to cycloheximide and phosphonoacetic acid. UL15 is highly conserved within the Herpesviridae, and contains Walker A and B motifs homologous to the catalytic subunit of the bacteriophage terminase as revealed by sequence analysis. Phylogenetic tree constructed with the amino acid sequences of 23 herpesvirus UL15 homologues suggests a close relationship of DEV to the Mardivirus genus within the Alphaherpesvirinae. Further, the UL15 and UL15.5 proteins can be detected in the infected cell lysate but not in the sucrose density gradient-purified virion when reacting with the antiserum against UL15. Within the CEF cells, the UL15 and/or UL15.5 localize(s) in the cytoplasm at 6 h post infection (h p. i.) and mainly in the nucleus at 12 h p. i. and at 24 h p. i., while accumulate(s) in the cytoplasm in the absence of any other viral protein. DEV UL15 is a spliced gene that encodes two products encoded by 2.9 and 1.3 kb transcripts respectively. The UL15 is expressed late during infection. The coding sequences of DEV UL15 are very similar to those of alphaherpesviruses and most similar to the genus Mardivirus. The UL15 and/or UL15.5 accumulate(s) in the cytoplasm during early times post-infection and then are translocated to the nucleus at late times.

Accumulation of 24 nucleotide transgene-derived siRNAs is associated with crinivirus immunity in transgenic plants.

PubMed

Qiao, Wenjie; Zarzyńska-Nowak, Aleksandra; Nerva, Luca; Kuo, Yen-Wen; Falk, Bryce W

2018-04-28

RNA silencing is a conserved antiviral defense mechanism that has been used to develop robust resistance against plant virus infections. Previous efforts have been made to develop RNA silencing-mediated resistance to criniviruses, yet none have given immunity. In this study, transgenic Nicotiana benthamiana plants harboring a hairpin construct of the Lettuce infectious yellows virus (LIYV) RdRp sequence exhibited immunity to systemic LIYV infection. Deep-sequencing analysis was performed to characterize virus-derived siRNAs (vsiRNAs) generated upon systemic LIYV infection in non-transgenic N. benthamiana plants as well as transgene-derived siRNAs (t-siRNAs) derived from the immune transgenic plants before and after LIYV inoculation. Interestingly, a similar sequence distribution pattern was obtained with t-siRNAs and vsiRNAs mapped to the transgene region in both immune and susceptible plants except a significant increase of t-siRNAs of 24 nt in length, which was consistent with small RNA northern blot results that showed the abundance of t-siRNAs of 21-, 22-, and 24- nt in length. The accumulated 24-nt sequences haven't yet been reported in transgenic plants partially resistant to criniviruses, thus may indicate their correlation with crinivirus immunity. To further test this hypothesis, we developed transgenic melon (Cucumis melo) plants immune to systemic infection of another crinivirus, Cucurbit yellow stunting disorder virus (CYSDV). As predicted, the accumulation of 24-nt t-siRNAs was detected in transgenic melon plants by northern blot. Together with our findings and previous studies on crinivirus resistance, we propose that the accumulation of 24 nt t-siRNAs is associated with crinivirus immunity in transgenic plants. This article is protected by copyright. All rights reserved. © 2018 BSPP and John Wiley & Sons Ltd.

Hepatitis C virus core protein induces hepatic steatosis via Sirt1-dependent pathway.

PubMed

Zhang, Chuanhai; Wang, Jingjing; Zhang, Hanlin; Liu, Shunai; Lee, Hyuek Jong; Jin, Wanzhu; Cheng, Jun

2018-05-01

Hepatic steatosis is a common feature of patients with chronic hepatitis C. Previous reports have shown that the overexpression of hepatitis C virus core-encoding sequences (hepatitis C virus genotypes 3a and 1b) significantly induces intracellular triglyceride accumulation. However, the underlying mechanism has not yet been revealed. To investigate whether Sirt1 is involved in hepatitis C virus-mediated hepatic steatosis, the overexpression of hepatitis C virus core 1b protein and Sirt1 and the knockdown of Sirt1 in HepG2 cells were performed. To confirm the results of the cellular experiment liver-specific Sirt1 KO mice with lentivirus-mediated hepatitis C virus core 1b overexpression were studied. Our results show that hepatitis C virus core 1b protein overexpression led to the accumulation of triglycerides in HepG2 cells. Notably the expression of PPARγ2 was dramatically increased at both the mRNA and protein levels by hepatitis C virus core 1b overexpression. The protein expression of Sirt1 is an upstream regulator of PPARγ2 and was also significantly increased after core 1b overexpression. In addition, the overexpression or knockdown of Sirt1 expression alone was sufficient to modulate p300-mediated PPARγ2 deacetylation. In vivo studies showed that hepatitis C virus core protein 1b-induced hepatic steatosis was attenuated in liver-specific Sirt1 KO mice by downregulation of PPARγ2 expression. Sirt1 mediates hepatitis C virus core protein 1b-induced hepatic steatosis by regulation of PPARγ2 expression. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Immunopathology of highly virulent pathogens: insights from Ebola virus.

PubMed

Zampieri, Carisa A; Sullivan, Nancy J; Nabel, Gary J

2007-11-01

Ebola virus is a highly virulent pathogen capable of inducing a frequently lethal hemorrhagic fever syndrome. Accumulating evidence indicates that the virus actively subverts both innate and adaptive immune responses and triggers harmful inflammatory responses as it inflicts direct tissue damage. The host immune system is ultimately overwhelmed by a combination of inflammatory factors and virus-induced cell damage, particularly in the liver and vasculature, often leading to death from septic shock. We summarize the mechanisms of immune dysregulation and virus-mediated cell damage in Ebola virus-infected patients. Future approaches to prevention and treatment of infection will be guided by answers to unresolved questions about interspecies transmission, molecular mechanisms of pathogenesis, and protective adaptive and innate immune responses to Ebola virus.

Epstein–Barr virus glycoprotein gM can interact with the cellular protein p32 and knockdown of p32 impairs virus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Changotra, Harish; Turk, Susan M.; Artigues, Antonio

The Epstein–Barr virus glycoprotein complex gMgN has been implicated in assembly and release of fully enveloped virus, although the precise role that it plays has not been elucidated. We report here that the long predicted cytoplasmic tail of gM is not required for complex formation and that it interacts with the cellular protein p32, which has been reported to be involved in nuclear egress of human cytomegalovirus and herpes simplex virus. Although redistribution of p32 and colocalization with gM was not observed in virus infected cells, knockdown of p32 expression by siRNA or lentivirus-delivered shRNA recapitulated the phenotype of amore » virus lacking expression of gNgM. A proportion of virus released from cells sedimented with characteristics of virus lacking an intact envelope and there was an increase in virus trapped in nuclear condensed chromatin. The observations suggest the possibility that p32 may also be involved in nuclear egress of Epstein–Barr virus. - Highlights: • The predicted cytoplasmic tail of gM is not required to complex with gN. • Cellular p32 can interact with the predicted cytoplasmic tail of EBV gM. • Knockdown of p32 recapitulates the phenotype of virus lacking the gNgM complex.« less

Establishment of an in vitro transcription system for Peste des petits ruminant virus.

PubMed

Yunus, Mohammad; Shaila, Melkote S

2012-12-05

Peste-des-petits ruminants virus (PPRV) is a non segmented negative strand RNA virus of the genus Morbillivirus within Paramyxoviridae family. Negative strand RNA viruses are known to carry nucleocapsid (N) protein, phospho (P) protein and RNA polymerase (L protein) packaged within the virion which possess all activities required for transcription, post-transcriptional modification of mRNA and replication. In order to understand the mechanism of transcription and replication of the virus, an in vitro transcription reconstitution system is required. In the present work, an in vitro transcription system has been developed with ribonucleoprotein (RNP) complex purified from virus infected cells as well as partially purified recombinant polymerase (L-P) complex from insect cells along with N-RNA (genomic RNA encapsidated by N protein) template isolated from virus infected cells. RNP complex isolated from virus infected cells and recombinant L-P complex purified from insect cells was used to reconstitute transcription on N-RNA template. The requirement for this transcription reconstitution has been defined. Transcription of viral genes in the in vitro system was confirmed by PCR amplification of cDNAs corresponding to individual transcripts using gene specific primers. In order to measure the relative expression level of viral transcripts, real time PCR analysis was carried out. qPCR analysis of the transcription products made in vitro showed a gradient of polarity of transcription from 3' end to 5' end of the genome similar to that exhibited by the virus in infected cells. This report describes for the first time, the development of an in vitro transcription reconstitution system for PPRV with RNP complex purified from infected cells and recombinant L-P complex expressed in insect cells. Both the complexes were able to synthesize all the mRNA species in vitro, exhibiting a gradient of polarity in transcription.

Determinants of virulence of influenza A virus

PubMed Central

Schrauwen, Eefje J.A.; de Graaf, Miranda; Herfst, Sander; Rimmelzwaan, Guus F.; Osterhaus, Albert D.M.E.; Fouchier, Ron A.M.

2013-01-01

Influenza A viruses cause yearly seasonal epidemics and occasional global pandemics in humans. In the last century, four human influenza A virus pandemics have occured. Ocasionally, influenza A viruses that circulate in other species, cross the species barrier and infect humans. Virus re-assortment (i.e. mixing of gene segments of multiple viruses) and the accumulation of mutations contribute to the emergence of new influenza A virus variants. Fortunately, most of these variants do not have the ability to spread among humans and subsequently cause a pandemic. In this review we focus on the threat of animal influenza A viruses which have shown the ability to infect humans. In addition, genetic factors which could alter the virulence of influenza A viruses are discussed. Identification and characterization of these factors may provide insights into genetic traits which change virulence and help us to understand which genetic determinants are of importance for the pandemic potential of animal influenza A viruses. PMID:24078062

Plant Response to TSWV and Seed Accumulation of Resveratrol in Peanut

USDA-ARS?s Scientific Manuscript database

Biotic and abiotic stress may induce peanut plants to produce a high amount of resveratrol. The relationship of plant response to tomato spotted wilt virus (TSWV) and seed accumulation of resveratrol was investigated. Twenty peanut accessions and six wild relatives were selected from the US peanut g...

Trafficking of bluetongue virus visualized by recovery of tetracysteine-tagged virion particles.

PubMed

Du, Junzheng; Bhattacharya, Bishnupriya; Ward, Theresa H; Roy, Polly

2014-11-01

Bluetongue virus (BTV), a member of the Orbivirus genus in the Reoviridae family, is a double-capsid insect-borne virus enclosing a genome of 10 double-stranded RNA segments. Like those of other members of the family, BTV virions are nonenveloped particles containing two architecturally complex capsids. The two proteins of the outer capsid, VP2 and VP5, are involved in BTV entry and in the delivery of the transcriptionally active core to the cell cytoplasm. Although the importance of the endocytic pathway in BTV entry has been reported, detailed analyses of entry and the role of each protein in virus trafficking have not been possible due to the lack of availability of a tagged virus. Here, for the first time, we report on the successful manipulation of a segmented genome of a nonenveloped capsid virus by the introduction of tags that were subsequently fluorescently visualized in infected cells. The genetically engineered fluorescent BTV particles were observed to enter live cells immediately after virus adsorption. Further, we showed the separation of VP2 from VP5 during virus entry and confirmed that while VP2 is shed from virions in early endosomes, virus particles still consisting of VP5 were trafficked sequentially from early to late endosomes. Since BTV infects both mammalian and insect cells, the generation of tagged viruses will allow visualization of the trafficking of BTV farther downstream in different host cells. In addition, the tagging technology has potential for transferable application to other nonenveloped complex viruses. Live-virus trafficking in host cells has been highly informative on the interactions between virus and host cells. Although the insertion of fluorescent markers into viral genomes has made it possible to study the trafficking of enveloped viruses, the physical constraints of architecturally complex capsid viruses have imposed practical limitations. In this study, we have successfully genetically engineered the segmented RNA genome of bluetongue virus (BTV), a complex nonenveloped virus belonging to the Reoviridae family. The resulting fluorescent virus particles could be visualized in virus entry studies of both live and fixed cells. This is the first time a structurally complex capsid virus has been successfully genetically manipulated to generate virus particles that could be visualized in infected cells. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Isolation and characterization of a virus infecting the freshwater algae Chrysochromulina parva

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mirza, S.F.; Staniewski, M.A.; Short, C.M.

Water samples from Lake Ontario, Canada were tested for lytic activity against the freshwater haptophyte algae Chrysochromulina parva. A filterable lytic agent was isolated and identified as a virus via transmission electron microscopy and molecular methods. The virus, CpV-BQ1, is icosahedral, ca. 145 nm in diameter, assembled within the cytoplasm, and has a genome size of ca. 485 kb. Sequences obtained through PCR-amplification of DNA polymerase (polB) genes clustered among sequences from the family Phycodnaviridae, whereas major capsid protein (MCP) sequences clustered among sequences from either the Phycodnaviridae or Mimiviridae. Based on quantitative molecular assays, C. parva's abundance in Lakemore » Ontario was relatively stable, yet CpV-BQ1's abundance was variable suggesting complex virus-host dynamics. This study demonstrates that CpV-BQ1 is a member of the proposed order Megavirales with characteristics of both phycodnaviruses and mimiviruses indicating that, in addition to its complex ecological dynamics, it also has a complex evolutionary history. - Highlights: • A virus infecting the algae C. parva was isolated from Lake Ontario. • Virus characteristics demonstrated that this novel virus is an NCLDV. • The virus's polB sequence suggests taxonomic affiliation with the Phycodnaviridae. • The virus's capsid protein sequences also suggest Mimiviridae ancestry. • Surveys of host and virus natural abundances revealed complex host–virus dynamics.« less

Suppression of Murine Retrovirus Polypeptide Termination: Effect of Amber Suppressor tRNA on the Cell-Free Translation of Rauscher Murine Leukemia Virus, Moloney Murine Leukemia Virus, and Moloney Murine Sarcoma Virus 124 RNA

PubMed Central

Murphy, Edwin C.; Wills, Norma; Arlinghaus, Ralph B.

1980-01-01

The effect of suppressor tRNA's on the cell-free translation of several leukemia and sarcoma virus RNAs was examined. Yeast amber suppressor tRNA (amber tRNA) enhanced the synthesis of the Rauscher murine leukemia virus and clone 1 Moloney murine leukemia virus Pr200gag-pol polypeptides by 10- to 45-fold, but at the same time depressed the synthesis of Rauscher murine leukemia virus Pr65gag and Moloney murine leukemia virus Pr63gag. Under suppressor-minus conditions, Moloney murine leukemia virus Pr70gag was present as a closely spaced doublet. Amber tRNA stimulated the synthesis of the “upper” Moloney murine leukemia virus Pr70gag polypeptide. Yeast ochre suppressor tRNA appeared to be ineffective. Quantitative analyses of the kinetics of viral precursor polypeptide accumulation in the presence of amber tRNA showed that during linear protein synthesis, the increase in accumulated Moloney murine leukemia virus Pr200gag-pol coincided closely with the molar loss of Pr63gag. Enhancement of Pr200gag-pol and Pr70gag by amber tRNA persisted in the presence of pactamycin, a drug which blocks the initiation of protein synthesis, thus arguing for the addition of amino acids to the C terminus of Pr63gag as the mechanism behind the amber tRNA effect. Moloney murine sarcoma virus 124 30S RNA was translated into four major polypeptides, Pr63gag, P42, P38, and P23. In the presence of amber tRNA, a new polypeptide, Pr67gag, appeared, whereas Pr63gag synthesis was decreased. Quantitative estimates indicated that for every 1 mol of Pr67gag which appeared, 1 mol of Pr63gag was lost. Images PMID:7373716

Immunity to Rice black streaked dwarf virus, a plant reovirus, can be achieved in rice plants by RNA silencing against the gene for the viroplasm component protein.

PubMed

Shimizu, Takumi; Nakazono-Nagaoka, Eiko; Akita, Fusamichi; Uehara-Ichiki, Tamaki; Omura, Toshihiro; Sasaya, Takahide

2011-09-01

The nonstructural protein P9-1 of Rice black streaked dwarf virus has been confirmed to accumulate in viroplasms, the putative sites of viral replication, in infected plants and insects. We transformed rice plants by introducing an RNA interference construct against the P9-1-encoding gene. The resultant transgenic plants accumulated short interfering RNAs specific to the construct. All progenies produced by self-fertilization of these transgenic plants with induced RNA interference against the gene for P9-1 were resistant to infection by the virus. Our results demonstrated that interfering with the expression of a viroplasm component protein of plant reoviruses, which plays an important role in viral proliferation, might be a practical and effective way to control plant reovirus infection in crop plants. Copyright © 2011 Elsevier B.V. All rights reserved.

In vivo replication of an ICP34.5 second-site suppressor mutant following corneal infection correlates with in vitro regulation of eIF2 alpha phosphorylation.

PubMed

Ward, Stephen L; Scheuner, Donalyn; Poppers, Jeremy; Kaufman, Randal J; Mohr, Ian; Leib, David A

2003-04-01

In animal models of herpes simplex virus type 1 (HSV-1) infection, ICP34.5-null viruses are avirulent and also fail to grow in a variety of cultured cells due to their inability to prevent RNA-dependent protein kinase (PKR)-mediated inhibition of protein synthesis. We show here that the inability of ICP34.5 mutants to grow in vitro is due specifically to the accumulation of phosphorylated eIF2 alpha. Mutations suppressing the in vitro phenotype of ICP34.5-null mutants have been described which map to the unique short region of the HSV-1 genome, resulting in dysregulated expression of the US11 gene. Despite the inability of the suppressor mutation to suppress the avirulent phenotype of the ICP34.5-null parental virus following intracranial inoculation, the suppressor mutation enhanced virus growth in the cornea, trigeminal ganglia, and periocular skin following corneal infection compared to that with the ICP34.5-null virus. The phosphorylation state of eIF2 alpha following in vitro infection with the suppressor virus was examined to determine if in vivo differences could be attributed to differential regulation of eIF2 alpha phosphorylation. The suppressor virus prevented accumulation of phosphorylated eIF2 alpha, while the wild-type virus substantially reduced eIF2 alpha phosphorylation levels. These data suggest that US11 functions as a PKR antagonist in vivo, although its activity may be modulated by tissue-specific differences in translation regulation.

In Vivo Replication of an ICP34.5 Second-Site Suppressor Mutant following Corneal Infection Correlates with In Vitro Regulation of eIF2α Phosphorylation

PubMed Central

Ward, Stephen L.; Scheuner, Donalyn; Poppers, Jeremy; Kaufman, Randal J.; Mohr, Ian; Leib, David A.

2003-01-01

In animal models of herpes simplex virus type 1 (HSV-1) infection, ICP34.5-null viruses are avirulent and also fail to grow in a variety of cultured cells due to their inability to prevent RNA-dependent protein kinase (PKR)-mediated inhibition of protein synthesis. We show here that the inability of ICP34.5 mutants to grow in vitro is due specifically to the accumulation of phosphorylated eIF2α. Mutations suppressing the in vitro phenotype of ICP34.5-null mutants have been described which map to the unique short region of the HSV-1 genome, resulting in dysregulated expression of the US11 gene. Despite the inability of the suppressor mutation to suppress the avirulent phenotype of the ICP34.5-null parental virus following intracranial inoculation, the suppressor mutation enhanced virus growth in the cornea, trigeminal ganglia, and periocular skin following corneal infection compared to that with the ICP34.5-null virus. The phosphorylation state of eIF2α following in vitro infection with the suppressor virus was examined to determine if in vivo differences could be attributed to differential regulation of eIF2α phosphorylation. The suppressor virus prevented accumulation of phosphorylated eIF2α, while the wild-type virus substantially reduced eIF2α phosphorylation levels. These data suggest that US11 functions as a PKR antagonist in vivo, although its activity may be modulated by tissue-specific differences in translation regulation. PMID:12663769

Microarray hybridization for assessment of the genetic stability of chimeric West Nile/dengue 4 virus.

PubMed

Laassri, Majid; Bidzhieva, Bella; Speicher, James; Pletnev, Alexander G; Chumakov, Konstantin

2011-05-01

Genetic stability is an important characteristic of live viral vaccines because an accumulation of mutants can cause reversion to a virulent phenotype as well as a loss of immunogenic properties. This study was aimed at evaluating the genetic stability of a live attenuated West Nile (WN) virus vaccine candidate that was generated by replacing the pre-membrane and envelope protein genes of dengue 4 virus with those from WN. Chimeric virus was serially propagated in Vero, SH-SY5Y human neuroblastoma and HeLa cells and screened for point mutations using hybridization with microarrays of overlapping oligonucleotide probes covering the entire genome. The analysis revealed several spontaneous mutations that led to amino acid changes, most of which were located in the envelope (E) and non-structural NS4A, NS4B, and NS5 proteins. Viruses passaged in Vero and SH-SY5Y cells shared two common mutations: G(2337) C (Met(457) Ile) in the E gene and A(6751) G (Lys(125) Arg) in the NS4A gene. Quantitative assessment of the contents of these mutants in viral stocks indicated that they accumulated independently with different kinetics during propagation in cell cultures. Mutant viruses grew better in Vero cells compared to the parental virus, suggesting that they have a higher fitness. When tested in newborn mice, the cell culture-passaged viruses did not exhibit increased neurovirulence. The approach described in this article could be useful for monitoring the molecular consistency and quality control of vaccine strains. Copyright © 2011 Wiley-Liss, Inc.

Microarray Hybridization for Assessment of the Genetic Stability of Chimeric West Nile/Dengue 4 Virus

PubMed Central

Laassri, Majid; Bidzhieva, Bella; Speicher, James; Pletnev, Alexander G.; Chumakov, Konstantin

2012-01-01

Genetic stability is an important characteristic of live viral vaccines because an accumulation of mutants can cause reversion to a virulent phenotype as well as a loss of immunogenic properties. This study was aimed at evaluating the genetic stability of a live attenuated West Nile (WN) virus vaccine candidate that was generated by replacing the pre-membrane and envelope protein genes of dengue 4 virus with those from WN. Chimeric virus was serially propagated in Vero, SH-SY5Y human neuroblastoma and HeLa cells and screened for point mutations using hybridization with microarrays of overlapping oligonucleotide probes covering the entire genome. The analysis revealed several spontaneous mutations that led to amino acid changes, most of which were located in the envelope (E) and non-structural NS4A, NS4B, and NS5 proteins. Viruses passaged in Vero and SH-SY5Y cells shared two common mutations: G2337C (Met457Ile) in the E gene and A6751G (Lys125Arg) in the NS4A gene. Quantitative assessment of the contents of these mutants in viral stocks indicated that they accumulated independently with different kinetics during propagation in cell cultures. Mutant viruses grew better in Vero cells compared to the parental virus, suggesting that they have a higher fitness. When tested in newborn mice, the cell culture-passaged viruses did not exhibit increased neurovirulence. The approach described in this paper could be useful for monitoring the molecular consistency and quality control of vaccine strains. PMID:21360544

IL-2 complex treatment amplifies CD8+ T cell mediated immunity following herpes simplex virus-1 infection.

PubMed

Rajasagi, Naveen K; Rouse, Barry T

2016-12-01

CD8 + T cells play an important role in controlling numerous virus infections and some tumors and therefore several strategies have been adopted to modulate CD8 + T cell responses. One such approach includes treatment with IL-2 bound to a monoclonal antibody against IL-2 (IL-2 complex) which was shown to enhance CD8 + T cell responses and provide protection against some cancers and pathogens. This report analyses the value of IL-2 complex therapy to protect against a cutaneous virus infection as occurs with herpes simplex virus-1 (HSV-1) infection. Treatment with IL-2 complex after infection reduced virus levels and lesion severity in a zosteriform model of HSV infection in mice. Furthermore, IL-2 complex treatment expanded HSV-1-gB epitope-specific CD8 + T cells, IFN-γ and TNF-α producing CD8 + T cells as well as cells that produced more than one cytokine. In addition, IL-2 complex therapy recipients showed enhanced cytolytic activity of CD8 + T cells as shown by increased granzyme B expression and lytic granule release. Taken, together, these studies demonstrate that IL-2 complex therapy can be useful to boost protection against a cutaneous virus infection. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Ectromelia virus accumulates less double-stranded RNA compared to vaccinia virus in BS-C-1 cells.

PubMed

Frey, Tiffany R; Lehmann, Michael H; Ryan, Colton M; Pizzorno, Marie C; Sutter, Gerd; Hersperger, Adam R

2017-09-01

Most orthopoxviruses, including vaccinia virus (VACV), contain genes in the E3L and K3L families. The protein products of these genes have been shown to combat PKR, a host defense pathway. Interestingly, ectromelia virus (ECTV) contains an E3L ortholog but does not possess an intact K3L gene. Here, we gained insight into how ECTV can still efficiently evade PKR despite lacking K3L. Relative to VACV, we found that ECTV-infected BS-C-1 cells accumulated considerably less double-stranded (ds) RNA, which was due to lower mRNA levels and less transcriptional read-through of some genes by ECTV. The abundance of dsRNA in VACV-infected cells, detected using a monoclonal antibody, was able to activate the RNase L pathway at late time points post-infection. Historically, the study of transcription by orthopoxviruses has largely focused on VACV as a model. Our data suggest that there could be more to learn by studying other members of this genus. Copyright © 2017 Elsevier Inc. All rights reserved.

Differential Contribution of RNA Interference Components in Response to Distinct Fusarium graminearum Virus Infections.

PubMed

Yu, Jisuk; Lee, Kyung-Mi; Cho, Won Kyong; Park, Ju Yeon; Kim, Kook-Hyung

2018-05-01

The mechanisms of RNA interference (RNAi) as a defense response against viruses remain unclear in many plant-pathogenic fungi. In this study, we used reverse genetics and virus-derived small RNA profiling to investigate the contributions of RNAi components to the antiviral response against Fusarium graminearum viruses 1 to 3 (FgV1, -2, and -3). Real-time reverse transcription-quantitative PCR (qRT-PCR) indicated that infection of Fusarium graminearum by FgV1, -2, or -3 differentially induces the gene expression of RNAi components in F. graminearum Transcripts of the DICER-2 and AGO-1 genes of F. graminearum ( FgDICER-2 and FgAGO-1 ) accumulated at lower levels following FgV1 infection than following FgV2 or FgV3 infection. We constructed gene disruption and overexpression mutants for each of the Argonaute and dicer genes and for two RNA-dependent RNA polymerase (RdRP) genes and generated virus-infected strains of each mutant. Interestingly, mycelial growth was significantly faster for the FgV1-infected FgAGO-1 overexpression mutant than for the FgV1-infected wild type, while neither FgV2 nor FgV3 infection altered the colony morphology of the gene deletion and overexpression mutants. FgV1 RNA accumulation was significantly decreased in the FgAGO-1 overexpression mutant. Furthermore, the levels of induction of FgAGO-1 , FgDICER-2 , and some of the FgRdRP genes caused by FgV2 and FgV3 infection were similar to those caused by hairpin RNA-induced gene silencing. Using small RNA sequencing analysis, we documented different patterns of virus-derived small interfering RNA (vsiRNA) production in strains infected with FgV1, -2, and -3. Our results suggest that the Argonaute protein encoded by FgAGO-1 is required for RNAi in F. graminearum , that FgAGO-1 induction differs in response to FgV1, -2, and -3, and that FgAGO-1 might contribute to the accumulation of vsiRNAs in FgV1-infected F. graminearum IMPORTANCE To increase our understanding of how RNAi components in Fusarium graminearum react to mycovirus infections, we characterized the role(s) of RNAi components involved in the antiviral defense response against Fusarium graminearum viruses (FgVs). We observed differences in the levels of induction of RNA silencing-related genes, including FgDICER-2 and FgAGO-1 , in response to infection by three different FgVs. FgAGO-1 can efficiently induce a robust RNAi response against FgV1 infection, but FgDICER genes might be relatively redundant to FgAGO-1 with respect to antiviral defense. However, the contribution of this gene in the response to the other FgV infections might be small. Compared to previous studies of Cryphonectria parasitica , which showed dicer-like protein 2 and Argonaute-like protein 2 to be important in antiviral RNA silencing, our results showed that F. graminearum developed a more complex and robust RNA silencing system against mycoviruses and that FgDICER-1 and FgDICER-2 and FgAGO-1 and FgAGO-2 had redundant roles in antiviral RNA silencing. Copyright © 2018 American Society for Microbiology.

Antivirion Effects of Streptovaricin Complex Against Friend Virus

PubMed Central

Horoszewicz, Julius S.; Leong, Susan S.; Byrd, Daniel M.; Carter, William A.

1974-01-01

The in vitro antivirion activities of five different streptovaricin complex lots against the polycythemic strain of the Friend virus were evaluated. The assay system was based on the inhibition of the Friend virus-induced spleen foci. The virus inactivation process was shown to be susceptible to variation in temperature, pH, and time. The antivirion activity and the acute toxicity for mice, as well as the optical properties of these streptovaricin complexes, do not co-vary; this suggests that their biological activities are not associated with a single molecular structure. In addition, the antivirion activity of the five preparations of streptovaricin complex differs about 30-fold, indicating that this activity does not reside in a major component of the complex. PMID:15825311

Pinpointing retrovirus entry sites in cells expressing alternatively spliced receptor isoforms by single virus imaging.

PubMed

Padilla-Parra, Sergi; Marin, Mariana; Kondo, Naoyuki; Melikyan, Gregory B

2014-06-16

The majority of viruses enter host cells via endocytosis. Current knowledge of viral entry pathways is largely based upon infectivity measurements following genetic and/or pharmacological interventions that disrupt vesicular trafficking and maturation. Imaging of single virus entry in living cells provides a powerful means to delineate viral trafficking pathways and entry sites under physiological conditions. Here, we visualized single avian retrovirus co-trafficking with markers for early (Rab5) and late (Rab7) endosomes, acidification of endosomal lumen and the resulting viral fusion measured by the viral content release into the cytoplasm. Virus-carrying vesicles either merged with the existing Rab5-positive early endosomes or slowly accumulated Rab5. The Rab5 recruitment to virus-carrying endosomes correlated with acidification of their lumen. Viral fusion occurred either in early (Rab5-positive) or intermediate (Rab5- and Rab7-positive) compartments. Interestingly, different isoforms of the cognate receptor directed virus entry from distinct endosomes. In cells expressing the transmembrane receptor, viruses preferentially entered and fused with slowly maturing early endosomes prior to accumulation of Rab7. By comparison, in cells expressing the GPI-anchored receptor, viruses entered both slowly and quickly maturing endosomes and fused with early (Rab5-positive) and intermediate (Rab5- and Rab7-positive) compartments. Since the rate of low pH-triggered fusion was independent of the receptor isoform, we concluded that the sites of virus entry are determined by the kinetic competition between endosome maturation and viral fusion. Our findings demonstrate the ability of this retrovirus to enter cells via alternative endocytic pathways and establish infection by releasing its content from distinct endosomal compartments.

Mutation of a Short Variable Region in HCpro Protein of Potato virus A Affects Interactions with a Microtubule-Associated Protein and Induces Necrotic Responses in Tobacco.

PubMed

Haikonen, Tuuli; Rajamäki, Minna-Liisa; Tian, Yan-Ping; Valkonen, Jari P T

2013-07-01

Helper component proteinase (HCpro) is a multifunctional protein of potyviruses (genus Potyvirus). HCpro of Potato virus A (PVA) interacts with the microtubule-associated protein HIP2 in host cells, and depletion of HIP2 reduces virus accumulation. This study shows that HCpro of Potato virus Y and Tobacco etch virus also interact with HIP2. The C-proximal portion of PVA HCpro determines the interaction with HIP2 and was found to contain a stretch of six residues comprising a highly variable region (HVR) in potyviruses. Mutations in HVR reduced PVA accumulation in tobacco plants and induced necrotic symptoms novel to PVA. Microarray and quantitative reverse transcription polymerase chain reaction analyses revealed induction of many defense-related genes including ethylene- and jasmonic acid-inducible pathways in systemically infected leaves at necrosis onset. Salicylic acid-mediated signaling was dispensable for the response. Genes related to microtubule functions were down-regulated. Structural modeling of HCpro suggested that all mutations in HVR caused conformational changes in adjacent regions containing functionally important motifs conserved in potyviruses. Those mutations, which also caused conformational changes in HVR, led to the greatest reduction of fitness. Our results implicate HVR in the regulation of HCpro conformation and virus-host interactions and suggest that mutation of HVR induces host defense.

Back to the future: revisiting HIV-1 lethal mutagenesis

PubMed Central

Dapp, Michael J.; Patterson, Steven E.; Mansky, Louis M.

2012-01-01

The concept of eliminating HIV-1 infectivity by elevating the viral mutation rate was first proposed over a decade ago, even though the general concept had been conceived earlier for RNA viruses. Lethal mutagenesis was originally viewed as a novel chemotherapeutic approach for treating HIV-1 infection in which use of a viral mutagen would over multiple rounds of replication lead to the lethal accumulation of mutations, rendering the virus population non infectious – known as the slow mutation accumulation model. There have been limitations in obtaining good efficacy data with drug leads, leaving some doubt into clinical translation. More recent studies of the APOBEC3 proteins as well as new progress in the use of nucleoside analogs for inducing lethal mutagenesis have helped to refocus attention on rapid induction of HIV-1 lethal mutagenesis in a single or limited number of replication cycles leading to a rapid mutation accumulation model. PMID:23195922

ONE-HELIX PROTEIN 2 (OHP2) is required for the stability of OHP1 and assembly factor HCF244 and is functionally linked to PSII biogenesis.

PubMed

Hey, Daniel; Grimm, Bernhard

2018-06-21

The members of the light-harvesting-complex protein (LHCP) family, which include the one-helix proteins (OHPs), are characterized by one to four membrane-spanning helices. These pro-teins function in light absorption and energy dissipation, sensing light intensity, and triggering photomorphogenesis or binding of chlorophyll and intermediates of chlorophyll biosynthesis. Arabidopsis thaliana contains two OHPs, while four homologs (named high-light-induced pro-teins, Hlips) exist in Synechocystis PCC6803. Various functions have been assigned to Hlips, ranging from photoprotection and assembly of photosystem (PS) I and PSII to regulation of the early steps of chlorophyll biosynthesis, but little is known about the function of the two plant OHPs. Here, we show that the two Arabidopsis OHPs form heterodimers and that the stromal part of OHP2 interacts with the plastid-localized PSII assembly factor HIGH CHLOROPHYLL FLUORESCENCE 244 (HCF244). Moreover, concurrent accumulation of the two OHPs and HCF244 is critical for the stability of all three proteins. In particular, the absence of OHP2 leads to the complete loss of OHP1 and HCF244. We used a virus-induced gene silencing approach to minimize the expression of OHP1 or OHP2 in adult Arabidopsis plants and revealed that OHP2 is essential for the accumulation of the PSII core subunits, while the other photosynthetic com-plexes and the major LHCPs remained unaffected. We examined the potential functions of the OHP1-OHP2-HCF244 complex in the assembly and/or repair of PSII and propose a role for this heterotrimeric complex in thylakoid membrane biogenesis. {copyright, serif} 2018 American Society of Plant Biologists. All rights reserved.

Involvement of potato (Solanum tuberosum L.) MKK6 in response to potato virus Y.

PubMed

Lazar, Ana; Coll, Anna; Dobnik, David; Baebler, Spela; Bedina-Zavec, Apolonija; Zel, Jana; Gruden, Kristina

2014-01-01

Mitogen-activated protein kinase (MAPK) cascades have crucial roles in the regulation of plant development and in plant responses to stress. Plant recognition of pathogen-associated molecular patterns or pathogen-derived effector proteins has been shown to trigger activation of several MAPKs. This then controls defence responses, including synthesis and/or signalling of defence hormones and activation of defence related genes. The MAPK cascade genes are highly complex and interconnected, and thus the precise signalling mechanisms in specific plant-pathogen interactions are still not known. Here we investigated the MAPK signalling network involved in immune responses of potato (Solanum tuberosum L.) to Potato virus Y, an important potato pathogen worldwide. Sequence analysis was performed to identify the complete MAPK kinase (MKK) family in potato, and to identify those regulated in the hypersensitive resistance response to Potato virus Y infection. Arabidopsis has 10 MKK family members, of which we identified five in potato and tomato (Solanum lycopersicum L.), and eight in Nicotiana benthamiana. Among these, StMKK6 is the most strongly regulated gene in response to Potato virus Y. The salicylic acid treatment revealed that StMKK6 is regulated by the hormone that is in agreement with the salicylic acid-regulated domains found in the StMKK6 promoter. The involvement of StMKK6 in potato defence response was confirmed by localisation studies, where StMKK6 accumulated strongly only in Potato-virus-Y-infected plants, and predominantly in the cell nucleus. Using a yeast two-hybrid method, we identified three StMKK6 targets downstream in the MAPK cascade: StMAPK4_2, StMAPK6 and StMAPK13. These data together provide further insight into the StMKK6 signalling module and its involvement in plant defence.

Involvement of Potato (Solanum tuberosum L.) MKK6 in Response to Potato virus Y

PubMed Central

Lazar, Ana; Coll, Anna; Dobnik, David; Baebler, Špela; Bedina-Zavec, Apolonija; Žel, Jana; Gruden, Kristina

2014-01-01

Mitogen-activated protein kinase (MAPK) cascades have crucial roles in the regulation of plant development and in plant responses to stress. Plant recognition of pathogen-associated molecular patterns or pathogen-derived effector proteins has been shown to trigger activation of several MAPKs. This then controls defence responses, including synthesis and/or signalling of defence hormones and activation of defence related genes. The MAPK cascade genes are highly complex and interconnected, and thus the precise signalling mechanisms in specific plant–pathogen interactions are still not known. Here we investigated the MAPK signalling network involved in immune responses of potato (Solanum tuberosum L.) to Potato virus Y, an important potato pathogen worldwide. Sequence analysis was performed to identify the complete MAPK kinase (MKK) family in potato, and to identify those regulated in the hypersensitive resistance response to Potato virus Y infection. Arabidopsis has 10 MKK family members, of which we identified five in potato and tomato (Solanum lycopersicum L.), and eight in Nicotiana benthamiana. Among these, StMKK6 is the most strongly regulated gene in response to Potato virus Y. The salicylic acid treatment revealed that StMKK6 is regulated by the hormone that is in agreement with the salicylic acid-regulated domains found in the StMKK6 promoter. The involvement of StMKK6 in potato defence response was confirmed by localisation studies, where StMKK6 accumulated strongly only in Potato-virus-Y-infected plants, and predominantly in the cell nucleus. Using a yeast two-hybrid method, we identified three StMKK6 targets downstream in the MAPK cascade: StMAPK4_2, StMAPK6 and StMAPK13. These data together provide further insight into the StMKK6 signalling module and its involvement in plant defence. PMID:25111695

Virus variants with differences in the P1 protein coexist in a Plum pox virus population and display particular host-dependent pathogenicity features.

PubMed

Maliogka, Varvara I; Salvador, Beatriz; Carbonell, Alberto; Sáenz, Pilar; León, David San; Oliveros, Juan Carlos; Delgadillo, Ma Otilia; García, Juan Antonio; Simón-Mateo, Carmen

2012-10-01

Subisolates segregated from an M-type Plum pox virus (PPV) isolate, PPV-PS, differ widely in pathogenicity despite their high degree of sequence similarity. A single amino acid substitution, K109E, in the helper component proteinase (HCPro) protein of PPV caused a significant enhancement of symptom severity in herbaceous hosts, and notably modified virus infectivity in peach seedlings. The presence of this substitution in certain subisolates that induced mild symptoms in herbaceous hosts and did not infect peach seedlings suggested the existence of uncharacterized attenuating factors in these subisolates. In this study, we show that two amino acid changes in the P1 protein are specifically associated with the mild pathogenicity exhibited by some PS subisolates. Site-directed mutagenesis studies demonstrated that both substitutions, W29R and V139E, but especially W29R, resulted in lower levels of virus accumulation and symptom severity in a woody host, Prunus persica. Furthermore, when W29R and V139E mutations were expressed concomitantly, PPV infectivity was completely abolished in this host. In contrast, the V139E substitution, but not W29R, was found to be responsible for symptom attenuation in herbaceous hosts. Deep sequencing analysis demonstrated that the W29R and V139E heterogeneities already existed in the original PPV-PS isolate before its segregation in different subisolates by local lesion cloning. These results highlight the potential complexity of potyviral populations and the relevance of the P1 protein of potyviruses in pathogenesis and viral adaptation to the host. © 2012 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2012 BSPP AND BLACKWELL PUBLISHING LTD.

Mutational Analysis of Influenza A Virus Nucleoprotein: Identification of Mutations That Affect RNA Replication

PubMed Central

Mena, Ignacio; Jambrina, Enrique; Albo, Carmen; Perales, Beatriz; Ortín, Juan; Arrese, Marta; Vallejo, Dolores; Portela, Agustín

1999-01-01

The influenza A virus nucleoprotein (NP) is a multifunctional polypeptide which plays a pivotal role in virus replication. To get information on the domains and specific residues involved in the different NP activities, we describe here the preparation and characterization of 20 influenza A virus mutant NPs. The mutations, mostly single-amino-acid substitutions, were introduced in a cDNA copy of the A/Victoria/3/75 NP gene and, in most cases, affected residues located in regions that were highly conserved across the NPs of influenza A, B, and C viruses. The mutant NPs were characterized (i) in vivo (cell culture) by analyzing their intracellular localization and their functionality in replication, transcription, and expression of model RNA templates; and (ii) in vitro by analyzing their RNA-binding and sedimentation properties. The results obtained allowed us to identify both a mutant protein that accumulated in the cytoplasm and mutations that altered the functionality and/or the oligomerization state of the NP polypeptide. Among the mutations that reduced the NP capability to express chloramphenicol acetyltransferase protein from a model viral RNA (vRNA) template, some displayed a temperature-sensitive phenotype. Interestingly, four mutant NPs, which showed a reduced functionality in synthesizing cRNA molecules from a vRNA template, were fully competent to reconstitute complementary ribonucleoproteins (cRNPs) capable of synthesizing vRNAs, which in turn yielded mRNA molecules. Based on the phenotype of these mutants and on previously published observations, it is proposed that these mutant NPs have a reduced capability to interact with the polymerase complex and that this NP-polymerase interaction is responsible for making vRNPs switch from mRNA to cRNA synthesis. PMID:9882320

IL-1β Signaling Promotes CNS-Intrinsic Immune Control of West Nile Virus Infection

PubMed Central

Ramos, Hilario J.; Lanteri, Marion C.; Blahnik, Gabriele; Negash, Amina; Suthar, Mehul S.; Brassil, Margaret M.; Sodhi, Khushbu; Treuting, Piper M.; Busch, Michael P.; Norris, Philip J.; Gale, Michael

2012-01-01

West Nile virus (WNV) is an emerging flavivirus capable of infecting the central nervous system (CNS) and mediating neuronal cell death and tissue destruction. The processes that promote inflammation and encephalitis within the CNS are important for control of WNV disease but, how inflammatory signaling pathways operate to control CNS infection is not defined. Here, we identify IL-1β signaling and the NLRP3 inflammasome as key host restriction factors involved in viral control and CNS disease associated with WNV infection. Individuals presenting with acute WNV infection displayed elevated levels of IL-1β in their plasma over the course of infection, suggesting a role for IL-1β in WNV immunity. Indeed, we found that in a mouse model of infection, WNV induced the acute production of IL-1β in vivo, and that animals lacking the IL-1 receptor or components involved in inflammasome signaling complex exhibited increased susceptibility to WNV pathogenesis. This outcome associated with increased accumulation of virus within the CNS but not peripheral tissues and was further associated with altered kinetics and magnitude of inflammation, reduced quality of the effector CD8+ T cell response and reduced anti-viral activity within the CNS. Importantly, we found that WNV infection triggers production of IL-1β from cortical neurons. Furthermore, we found that IL-1β signaling synergizes with type I IFN to suppress WNV replication in neurons, thus implicating antiviral activity of IL-1β within neurons and control of virus replication within the CNS. Our studies thus define the NLRP3 inflammasome pathway and IL-1β signaling as key features controlling WNV infection and immunity in the CNS, and reveal a novel role for IL-1β in antiviral action that restricts virus replication in neurons. PMID:23209411

A Virus-Derived Stacked RNAi Construct Confers Robust Resistance to Cassava Brown Streak Disease

PubMed Central

Beyene, Getu; Chauhan, Raj Deepika; Ilyas, Muhammad; Wagaba, Henry; Fauquet, Claude M.; Miano, Douglas; Alicai, Titus; Taylor, Nigel J.

2017-01-01

Cassava brown streak disease (CBSD) threatens food and economic security for smallholder farmers throughout East and Central Africa, and poses a threat to cassava production in West Africa. CBSD is caused by two whitefly-transmitted virus species: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) (Genus: Ipomovirus, Family Potyviridae). Although varying levels of tolerance have been achieved through conventional breeding, to date, effective resistance to CBSD within East African cassava germplasm has not been identified. RNAi technology was utilized to integrate CBSD resistance into the Ugandan farmer-preferred cassava cultivar TME 204. Transgenic plant lines were generated expressing an inverted repeat construct (p5001) derived from coat-protein (CP) sequences of CBSV and UCBSV fused in tandem. Northern blots using probes specific for each CP sequence were performed to characterize 169 independent transgenic lines for accumulation of CP-derived siRNAs. Transgenic plant lines accumulating low, medium and high levels of siRNAs were bud graft challenged with the virulent CBSV Naliendele isolate alone or in combination with UCBSV. Resistance to CBSD in the greenhouse directly correlated to levels of CP-derived siRNAs as determined by visual assessment of leaf and storage root symptoms, and RT-PCR diagnosis for presence of the pathogens. Low expressing lines were found to be susceptible to CBSV and UCBSV, while medium to high accumulating plant lines were resistant to both virus species. Absence of detectable virus in the best performing p5001 transgenic lines was further confirmed by back-inoculation via sap or graft challenge to CBSD susceptible Nicotiana benthamiana and cassava cultivar 60444, respectively. Data presented shows robust resistance of transgenic p5001 TME 204 lines to both CBSV and UCBSV under greenhouse conditions. Levels of resistance correlated directly with levels of transgene derived siRNA expression such that the latter can be used as predictor of resistance to CBSD. PMID:28149300

Rice Stripe Tenuivirus Nonstructural Protein 3 Hijacks the 26S Proteasome of the Small Brown Planthopper via Direct Interaction with Regulatory Particle Non-ATPase Subunit 3

PubMed Central

Xu, Yi; Wu, Jianxiang; Fu, Shuai; Li, Chenyang; Zhu, Zeng-Rong

2015-01-01

ABSTRACT The ubiquitin/26S proteasome system plays a vital role in regulating host defenses against pathogens. Previous studies have highlighted different roles for the ubiquitin/26S proteasome in defense during virus infection in both mammals and plants, but their role in the vectors that transmit those viruses is still unclear. In this study, we determined that the 26S proteasome is present in the small brown planthopper (SBPH) (Laodelphgax striatellus) and has components similar to those in plants and mammals. There was an increase in the accumulation of Rice stripe virus (RSV) in the transmitting vector SBPH after disrupting the 26S proteasome, indicating that the SBPH 26S proteasome plays a role in defense against RSV infection by regulating RSV accumulation. Yeast two-hybrid analysis determined that a subunit of the 26S proteasome, named RPN3, could interact with RSV NS3. Transient overexpression of RPN3 had no effect on the RNA silencing suppressor activity of RSV NS3. However, NS3 could inhibit the ability of SBPH rpn3 to complement an rpn3 mutation in yeast. Our findings also indicate that the direct interaction between RPN3 and NS3 was responsible for inhibiting the complementation ability of RPN3. In vivo, we found an accumulation of ubiquitinated protein in SBPH tissues where the RSV titer was high, and silencing of rpn3 resulted in malfunction of the SBPH proteasome-mediated proteolysis. Consequently, viruliferous SBPH in which RPN3 was repressed transmitted the virus more effectively as a result of higher accumulation of RSV. Our results suggest that the RSV NS3 protein is able to hijack the 26S proteasome in SBPH via a direct interaction with the RPN3 subunit to attenuate the host defense response. IMPORTANCE We show, for the first time, that the 26S proteasome components are present in the small brown planthopper and play a role in defense against its vectored plant virus (RSV). In turn, RSV encodes a protein that subverts the SBPH 26S proteasome via direct interaction with the 26S proteasome subunit RPN3. Our results imply that the molecular arms race observed in plant hosts can be extended to the insect vector that transmits those viruses. PMID:25653432

Functional analysis of dengue virus (DENV) type 2 envelope protein domain 3 type-specific and DENV complex-reactive critical epitope residues.

PubMed

Pitcher, Trevor J; Sarathy, Vanessa V; Matsui, Kiyohiko; Gromowski, Gregory D; Huang, Claire Y-H; Barrett, Alan D T

2015-02-01

The dengue virus (DENV) envelope protein domain 3 (ED3) is the target of potent virus neutralizing antibodies. The DENV-2 ED3 contains adjacent type-specific and DENV complex-reactive antigenic sites that are composed of a small number of residues that were previously demonstrated to be critical for antibody binding. Site-directed mutagenesis of a DENV-2 16681 infectious clone was used to mutate critical residues in the DENV-2 type-specific (K305A and P384A) and DENV complex-reactive (K310A) antigenic sites. The K305A mutant virus multiplied like the parent virus in mosquito and mammalian cells, as did the P384A mutant virus, which required a compensatory mutation (G330D) for viability. However, the K310A mutant virus could not be recovered. The DENV-2 type-specific critical residue mutations K305A and P384A+G330D reduced the ability of DENV-2 type-specific, but not DENV complex-reactive, mAbs to neutralize virus infectivity and this was directly correlated with mAb binding affinity to the rED3 mutants. © 2015 The Authors.

Changing patterns of localization of the tobacco mosaic virus movement protein and replicase to the endoplasmic reticulum and microtubules during infection

NASA Technical Reports Server (NTRS)

Heinlein, M.; Padgett, H. S.; Gens, J. S.; Pickard, B. G.; Casper, S. J.; Epel, B. L.; Beachy, R. N.; Evans, M. L. (Principal Investigator)

1998-01-01

Tobacco mosaic virus (TMV) derivatives that encode movement protein (MP) as a fusion to the green fluorescent protein (MP:GFP) were used in combination with antibody staining to identify host cell components to which MP and replicase accumulate in cells of infected Nicotiana benthamiana leaves and in infected BY-2 protoplasts. MP:GFP and replicase colocalized to the endoplasmic reticulum (ER; especially the cortical ER) and were present in large, irregularly shaped, ER-derived structures that may represent "viral factories." The ER-derived structures required an intact cytoskeleton, and microtubules appeared to redistribute MP:GFP from these sites during late stages of infection. In leaves, MP:GFP accumulated in plasmodesmata, whereas in protoplasts, the MP:GFP was targeted to distinct, punctate sites near the plasma membrane. Treating protoplasts with cytochalasin D and brefeldin A at the time of inoculation prevented the accumulation of MP:GFP at these sites. It is proposed that the punctate sites anchor the cortical ER to plasma membrane and are related to sites at which plasmodesmata form in walled cells. Hairlike structures containing MP:GFP appeared on the surface of some of the infected protoplasts and are reminiscent of similar structures induced by other plant viruses. We present a model that postulates the role of the ER and cytoskeleton in targeting the MP and viral ribonucleoprotein from sites of virus synthesis to the plasmodesmata through which infection is spread.

Inefficient Mechanical Transmission of Langat (Tick-Borne Encephalitis Virus Complex) Virus by Blood-Feeding Mites (Acari) to Laboratory Mice

DTIC Science & Technology

1993-05-01

AD--A269 706 SPSSHORT C:OMMNUNICATION 8 Inefficient Mechanical Transmission of Langat (Tick-Bornee Encephalitis Virus Complex) Virus by Blood-Feeding...I d after a . iremic blood meal. but onhv immediatelIy after the vi re muo. LANGAT (LGT) VIRUS is a member of the tick- No isolations of LCT virus...Use of ulatus collected in the Ulu Langat Forest re- Laboratory Animals." as promulgated by the Committee on serve, Malaysia. in l959’(Struth 1956

Asynchronous accumulation of lettuce infectious yellows virus RNAs 1 and 2 and identification of an RNA 1 trans enhancer of RNA 2 accumulation.

PubMed

Yeh, H H; Tian, T; Rubio, L; Crawford, B; Falk, B W

2000-07-01

Time course and mutational analyses were used to examine the accumulation in protoplasts of progeny RNAs of the bipartite Crinivirus, Lettuce infectious yellow virus (LIYV; family Closteroviridae). Hybridization analyses showed that simultaneous inoculation of LIYV RNAs 1 and 2 resulted in asynchronous accumulation of progeny LIYV RNAs. LIYV RNA 1 progeny genomic and subgenomic RNAs could be detected in protoplasts as early as 12 h postinoculation (p.i.) and accumulated to high levels by 24 h p.i. The LIYV RNA 1 open reading frame 2 (ORF 2) subgenomic RNA was the most abundant of all LIYV RNAs detected. In contrast, RNA 2 progeny were not readily detected until ca. 36 h p.i. Mutational analyses showed that in-frame stop codons introduced into five of seven RNA 2 ORFs did not affect accumulation of progeny LIYV RNA 1 or RNA 2, confirming that RNA 2 does not encode proteins necessary for LIYV RNA replication. Mutational analyses also supported that LIYV RNA 1 encodes proteins necessary for replication of LIYV RNAs 1 and 2. A mutation introduced into the LIYV RNA 1 region encoding the overlapping ORF 1B and ORF 2 was lethal. However, mutations introduced into only LIYV RNA 1 ORF 2 resulted in accumulation of progeny RNA 1 near or equal to wild-type RNA 1. In contrast, the RNA 1 ORF 2 mutants did not efficiently support the trans accumulation of LIYV RNA 2. Three distinct RNA 1 ORF 2 mutants were analyzed and all exhibited a similar phenotype for progeny LIYV RNA accumulation. These data suggest that the LIYV RNA 1 ORF 2 encodes a trans enhancer for RNA 2 accumulation.

Asynchronous Accumulation of Lettuce Infectious Yellows Virus RNAs 1 and 2 and Identification of an RNA 1 trans Enhancer of RNA 2 Accumulation

PubMed Central

Yeh, Hsin-Hung; Tian, Tongyan; Rubio, Luis; Crawford, Brett; Falk, Bryce W.

2000-01-01

Time course and mutational analyses were used to examine the accumulation in protoplasts of progeny RNAs of the bipartite Crinivirus, Lettuce infectious yellow virus (LIYV; family Closteroviridae). Hybridization analyses showed that simultaneous inoculation of LIYV RNAs 1 and 2 resulted in asynchronous accumulation of progeny LIYV RNAs. LIYV RNA 1 progeny genomic and subgenomic RNAs could be detected in protoplasts as early as 12 h postinoculation (p.i.) and accumulated to high levels by 24 h p.i. The LIYV RNA 1 open reading frame 2 (ORF 2) subgenomic RNA was the most abundant of all LIYV RNAs detected. In contrast, RNA 2 progeny were not readily detected until ca. 36 h p.i. Mutational analyses showed that in-frame stop codons introduced into five of seven RNA 2 ORFs did not affect accumulation of progeny LIYV RNA 1 or RNA 2, confirming that RNA 2 does not encode proteins necessary for LIYV RNA replication. Mutational analyses also supported that LIYV RNA 1 encodes proteins necessary for replication of LIYV RNAs 1 and 2. A mutation introduced into the LIYV RNA 1 region encoding the overlapping ORF 1B and ORF 2 was lethal. However, mutations introduced into only LIYV RNA 1 ORF 2 resulted in accumulation of progeny RNA 1 near or equal to wild-type RNA 1. In contrast, the RNA 1 ORF 2 mutants did not efficiently support the trans accumulation of LIYV RNA 2. Three distinct RNA 1 ORF 2 mutants were analyzed and all exhibited a similar phenotype for progeny LIYV RNA accumulation. These data suggest that the LIYV RNA 1 ORF 2 encodes a trans enhancer for RNA 2 accumulation. PMID:10846054

[The clinico-laboratory characteristics of cases of diseases connected with viruses of the California encephalitis complex in the inhabitants of Moscow].

PubMed

Kolobukhina, L V; L'vov, D K; Butenko, A M; Kuznetsov, A A; Galkina, I V

1989-10-01

To study the role of viruses of the California encephalitis virus complex (the family Bunyaviridae) in infectious pathology, 187 fever patients admitted to the Clinical Infectious Hospital in May-September 1986 were examined. In 10 of these patients the neutralization test revealed the presence of diagnostically significant changes in neutralizing antibodies (neutralization indices), which was indicative of the role played by Tahyna virus or other related viruses belonging to the California encephalitis virus complex in the etiology of the diseases. The analysis of the clinical picture showed that in all patients the disease took an acute course in its initial stage, starting with shivering and characterized by high fever, headache, pronounced toxicosis, the possibility of the formation of intracerebral hypertension and pneumonia.

Regulation of ROS in transmissible gastroenteritis virus-activated apoptotic signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ding, Li; College of Life Sciences, Hainan Normal University, Haikou, Hainan 571158; Zhao, Xiaomin

Highlights: •TGEV infection induced ROS accumulation. •ROS accumulation is involved in TGEV-induced mitochondrial integrity impairment. •ROS is associated with p53 activation and apoptosis occurrence in TGEV-infected cells. -- Abstract: Transmissible gastroenteritis virus (TGEV), an enteropathogenic coronavirus, causes severe lethal watery diarrhea and dehydration in piglets. Previous studies indicate that TGEV infection induces cell apoptosis in host cells. In this study, we investigated the roles and regulation of reactive oxygen species (ROS) in TGEV-activated apoptotic signaling. The results showed that TGEV infection induced ROS accumulation, whereas UV-irradiated TGEV did not promote ROS accumulation. In addition, TGEV infection lowered mitochondrial transmembrane potentialmore » in PK-15 cell line, which could be inhibited by ROS scavengers, pyrrolidinedithiocarbamic (PDTC) and N-acetyl-L-cysteine (NAC). Furthermore, the two scavengers significantly inhibited the activation of p38 MAPK and p53 and further blocked apoptosis occurrence through suppressing the TGEV-induced Bcl-2 reduction, Bax redistribution, cytochrome c release and caspase-3 activation. These results suggest that oxidative stress pathway might be a key element in TGEV-induced apoptosis and TGEV pathogenesis.« less

RNAi and retroviruses: are they in RISC?

PubMed

Vasselon, Thierry; Bouttier, Manuella; Saumet, Anne; Lecellier, Charles-Henri

2013-02-01

RNA interference (RNAi) is a potent cellular system against viruses in various organisms. Although common traits are observed in plants, insects, and nematodes, the situation observed in mammals appears more complex. In mammalian somatic cells, RNAi is implicated in endonucleolytic cleavage mediated by artificially delivered small interfering RNAs (siRNAs) as well as in translation repression mediated by microRNAs (miRNAs). Because siRNAs and miRNAs recognize viral mRNAs, RNAi inherently limits virus production and participates in antiviral defense. However, several observations made in the cases of hepatitis C virus and retroviruses (including the human immunodeficiency virus and the primate foamy virus) bring evidence that this relationship is much more complex and that certain components of the RNAi effector complex [called the RNA-induced silencing complex (RISC)], such as AGO2, are also required for viral replication. Here, we summarize recent discoveries that have revealed this dual implication in virus biology. We further discuss their potential implications for the functions of RNAi-related proteins, with special emphasis on retrotransposition and genome stability.

A new look at an old virus: patterns of mutation accumulation in the human H1N1 influenza virus since 1918

PubMed Central

2012-01-01

Background The H1N1 influenza A virus has been circulating in the human population for over 95 years, first manifesting itself in the pandemic of 1917–1918. Initial mortality was extremely high, but dropped exponentially over time. Influenza viruses have high mutation rates, and H1N1 has undergone significant genetic changes since 1918. The exact nature of H1N1 mutation accumulation over time has not been fully explored. Methods We have made a comprehensive historical analysis of mutational changes within H1N1 by examining over 4100 fully-sequenced H1N1 genomes. This has allowed us to examine the genetic changes arising within H1N1 from 1918 to the present. Results We document multiple extinction events, including the previously known extinction of the human H1N1 lineage in the 1950s, and an apparent second extinction of the human H1N1 lineage in 2009. These extinctions appear to be due to a continuous accumulation of mutations. At the time of its disappearance in 2009, the human H1N1 lineage had accumulated over 1400 point mutations (more than 10% of the genome), including approximately 330 non-synonymous changes (7.4% of all codons). The accumulation of both point mutations and non-synonymous amino acid changes occurred at constant rates (μ = 14.4 and 2.4 new mutations/year, respectively), and mutations accumulated uniformly across the entire influenza genome. We observed a continuous erosion over time of codon-specificity in H1N1, including a shift away from host (human, swine, and bird [duck]) codon preference patterns. Conclusions While there have been numerous adaptations within the H1N1 genome, most of the genetic changes we document here appear to be non-adaptive, and much of the change appears to be degenerative. We suggest H1N1 has been undergoing natural genetic attenuation, and that significant attenuation may even occur during a single pandemic. This process may play a role in natural pandemic cessation and has apparently contributed to the exponential decline in mortality rates over time, as seen in all major human influenza strains. These findings may be relevant to the development of strategies for managing influenza pandemics and strain evolution. PMID:23062055

Vector-virus interactions and transmission dynamics of West Nile virus.

PubMed

Ciota, Alexander T; Kramer, Laura D

2013-12-09

West Nile virus (WNV; Flavivirus; Flaviviridae) is the cause of the most widespread arthropod-borne viral disease in the world and the largest outbreak of neuroinvasive disease ever observed. Mosquito-borne outbreaks are influenced by intrinsic (e.g., vector and viral genetics, vector and host competence, vector life-history traits) and extrinsic (e.g., temperature, rainfall, human land use) factors that affect virus activity and mosquito biology in complex ways. The concept of vectorial capacity integrates these factors to address interactions of the virus with the arthropod host, leading to a clearer understanding of their complex interrelationships, how they affect transmission of vector-borne disease, and how they impact human health. Vertebrate factors including host competence, population dynamics, and immune status also affect transmission dynamics. The complexity of these interactions are further exacerbated by the fact that not only can divergent hosts differentially alter the virus, but the virus also can affect both vertebrate and invertebrate hosts in ways that significantly alter patterns of virus transmission. This chapter concentrates on selected components of the virus-vector-vertebrate interrelationship, focusing specifically on how interactions between vector, virus, and environment shape the patterns and intensity of WNV transmission.

Vector-Virus Interactions and Transmission Dynamics of West Nile Virus

PubMed Central

Ciota, Alexander T.; Kramer, Laura D.

2013-01-01

West Nile virus (WNV; Flavivirus; Flaviviridae) is the cause of the most widespread arthropod-borne viral disease in the world and the largest outbreak of neuroinvasive disease ever observed. Mosquito-borne outbreaks are influenced by intrinsic (e.g., vector and viral genetics, vector and host competence, vector life-history traits) and extrinsic (e.g., temperature, rainfall, human land use) factors that affect virus activity and mosquito biology in complex ways. The concept of vectorial capacity integrates these factors to address interactions of the virus with the arthropod host, leading to a clearer understanding of their complex interrelationships, how they affect transmission of vector-borne disease, and how they impact human health. Vertebrate factors including host competence, population dynamics, and immune status also affect transmission dynamics. The complexity of these interactions are further exacerbated by the fact that not only can divergent hosts differentially alter the virus, but the virus also can affect both vertebrate and invertebrate hosts in ways that significantly alter patterns of virus transmission. This chapter concentrates on selected components of the virus-vector-vertebrate interrelationship, focusing specifically on how interactions between vector, virus, and environment shape the patterns and intensity of WNV transmission. PMID:24351794

An Improved Brome mosaic virus Silencing Vector: Greater Insert Stability and More Extensive VIGS1[OPEN

PubMed Central

2018-01-01

Virus-induced gene silencing (VIGS) is used extensively for gene function studies in plants. VIGS is inexpensive and rapid compared with silencing conducted through stable transformation, but many virus-silencing vectors, especially in grasses, induce only transient silencing phenotypes. A major reason for transient phenotypes is the instability of the foreign gene fragment (insert) in the vector during VIGS. Here, we report the development of a Brome mosaic virus (BMV)-based vector that better maintains inserts through modification of the original BMV vector RNA sequence. Modification of the BMV RNA3 sequence yielded a vector, BMVCP5, that better maintained phytoene desaturase and heat shock protein70-1 (HSP70-1) inserts in Nicotiana benthamiana and maize (Zea mays). Longer maintenance of inserts was correlated with greater target gene silencing and more extensive visible silencing phenotypes displaying greater tissue penetration and involving more leaves. The modified vector accumulated similarly to the original vector in N. benthamiana after agroinfiltration, thus maintaining a high titer of virus in this intermediate host used to produce virus inoculum for grass hosts. For HSP70, silencing one family member led to a large increase in the expression of another family member, an increase likely related to the target gene knockdown and not a general effect of virus infection. The cause of the increased insert stability in the modified vector is discussed in relationship to its recombination and accumulation potential. The modified vector will improve functional genomic studies in grasses, and the conceptual methods used to improve the vector may be applied to other VIGS vectors. PMID:29127260

Inferring HIV Escape Rates from Multi-Locus Genotype Data

DOE PAGES

Kessinger, Taylor A.; Perelson, Alan S.; Neher, Richard A.

2013-09-03

Cytotoxic T-lymphocytes (CTLs) recognize viral protein fragments displayed by major histocompatibility complex molecules on the surface of virally infected cells and generate an anti-viral response that can kill the infected cells. Virus variants whose protein fragments are not efficiently presented on infected cells or whose fragments are presented but not recognized by CTLs therefore have a competitive advantage and spread rapidly through the population. We present a method that allows a more robust estimation of these escape rates from serially sampled sequence data. The proposed method accounts for competition between multiple escapes by explicitly modeling the accumulation of escape mutationsmore » and the stochastic effects of rare multiple mutants. Applying our method to serially sampled HIV sequence data, we estimate rates of HIV escape that are substantially larger than those previously reported. The method can be extended to complex escapes that require compensatory mutations. We expect our method to be applicable in other contexts such as cancer evolution where time series data is also available.« less

The vaccinia virus E6 protein influences virion protein localization during virus assembly

DOE Office of Scientific and Technical Information (OSTI.GOV)

Condit, Richard C., E-mail: condit@mgm.ufl.edu; Moussatche, Nissin

2015-08-15

Vaccinia virus mutants in which expression of the virion core protein gene E6R is repressed are defective in virion morphogenesis. E6 deficient infections fail to properly package viroplasm into viral membranes, resulting in an accumulation of empty immature virions and large aggregates of viroplasm. We have used immunogold electron microscopy and immunofluorescence confocal microscopy to assess the intracellular localization of several virion structural proteins and enzymes during E6R mutant infections. We find that during E6R mutant infections virion membrane proteins and virion transcription enzymes maintain a normal localization within viral factories while several major core and lateral body proteins accumulatemore » in aggregated virosomes. The results support a model in which vaccinia virions are assembled from at least three substructures, the membrane, the viroplasm and a “pre-nucleocapsid”, and that the E6 protein is essential for maintaining proper localization of the seven-protein complex and the viroplasm during assembly. - Highlights: • Mutation of E6 disrupts association of viral membranes with viral core proteins • Mutation of E6 does not perturb viral membrane biosynthesis • Mutation of E6 does not perturb localization of viral transcription enzymes • Mutation of E6 causes mis-localization and aggregation of viral core proteins • Vaccinia assembly uses three subassemblies: membranes, viroplasm, prenucleocapsid.« less

Dinuclear polypyridylruthenium(II) complexes: flow cytometry studies of their accumulation in bacteria and the effect on the bacterial membrane.

PubMed

Li, Fangfei; Feterl, Marshall; Warner, Jeffrey M; Keene, F Richard; Collins, J Grant

2013-12-01

To determine the energy dependency of and the contribution of the membrane potential to the cellular accumulation of the dinuclear complexes [{Ru(phen)2}2{μ-bbn}](4+) (Rubbn) and the mononuclear complexes [Ru(Me4phen)3](2+) and [Ru(phen)2(bb7)](2+) in Staphylococcus aureus and Escherichia coli, and to examine their effect on the bacterial membrane. The accumulation of the ruthenium complexes in bacteria was determined using flow cytometry at a range of temperatures. The cellular accumulation of the ruthenium complexes was also determined in cells that had been incubated with the metal complexes in the presence or absence of metabolic stimulators or inhibitors and/or commercial dyes to determine the membrane potential or membrane permeability. The accumulation of ruthenium complexes in the two bacterial strains was shown to increase with increasing incubation temperature, with the relative increase in accumulation greater with E. coli, particularly for Rubb12 and Rubb16. No decrease in accumulation was observed for Rubb12 in ATP-inhibited cells. While carbonyl cyanide m-chlorophenyl hydrazone (CCCP) did depolarize the cell membrane, no reduction in the accumulation of Rubb12 was observed; however, all ruthenium complexes, when incubated with S. aureus at concentrations twice their MIC, depolarized the membrane to a similar extent to CCCP. Except for the mononuclear complex [Ru(Me4phen)3](2+), incubation of any of the other ruthenium complexes allowed a greater quantity of the membrane-impermeable dye TO-PRO-3 to be taken up by S. aureus. The results indicate that the potential new antimicrobial Rubbn complexes enter the cell in an energy-independent manner, depolarize the cell membrane and significantly permeabilize the cellular membrane.

The virulence–transmission trade-off in vector-borne plant viruses: a review of (non-)existing studies

PubMed Central

Froissart, R.; Doumayrou, J.; Vuillaume, F.; Alizon, S.; Michalakis, Y.

2010-01-01

The adaptive hypothesis invoked to explain why parasites harm their hosts is known as the trade-off hypothesis, which states that increased parasite transmission comes at the cost of shorter infection duration. This correlation arises because both transmission and disease-induced mortality (i.e. virulence) are increasing functions of parasite within-host density. There is, however, a glaring lack of empirical data to support this hypothesis. Here, we review empirical investigations reporting to what extent within-host viral accumulation determines the transmission rate and the virulence of vector-borne plant viruses. Studies suggest that the correlation between within-plant viral accumulation and transmission rate of natural isolates is positive. Unfortunately, results on the correlation between viral accumulation and virulence are very scarce. We found only very few appropriate studies testing such a correlation, themselves limited by the fact that they use symptoms as a proxy for virulence and are based on very few viral genotypes. Overall, the available evidence does not allow us to confirm or refute the existence of a transmission–virulence trade-off for vector-borne plant viruses. We discuss the type of data that should be collected and how theoretical models can help us refine testable predictions of virulence evolution. PMID:20478886

Topology of evolving, mutagenized viral populations: quasispecies expansion, compression, and operation of negative selection.

PubMed

Ojosnegros, Samuel; Agudo, Rubén; Sierra, Macarena; Briones, Carlos; Sierra, Saleta; González-López, Claudia; Domingo, Esteban; Cristina, Juan

2008-07-17

The molecular events and evolutionary forces underlying lethal mutagenesis of virus (or virus extinction through an excess of mutations) are not well understood. Here we apply for the first time phylogenetic methods and Partition Analysis of Quasispecies (PAQ) to monitor genetic distances and intra-population structures of mutant spectra of foot-and-mouth disease virus (FMDV) quasispecies subjected to mutagenesis by base and nucleoside analogues. Phylogenetic and PAQ analyses have revealed a highly dynamic variation of intrapopulation diversity of FMDV quasispecies. The population diversity first suffers striking expansions in the presence of mutagens and then compressions either when the presence of the mutagenic analogue was discontinued or when a mutation that decreased sensitivity to a mutagen was selected. The pattern of mutations found in the populations was in agreement with the behavior of the corresponding nucleotide analogues with FMDV in vitro. Mutations accumulated at preferred genomic sites, and dn/ds ratios indicate the operation of negative (or purifying) selection in populations subjected to mutagenesis. No evidence of unusually elevated genetic distances has been obtained for FMDV populations approaching extinction. Phylogenetic and PAQ analysis provide adequate procedures to describe the evolution of viral sequences subjected to lethal mutagenesis. These methods define the changes of intra-population structure more precisely than mutation frequencies and Shannon entropies. PAQ is very sensitive to variations of intrapopulation genetic distances. Strong negative (or purifying) selection operates in FMDV populations subjected to enhanced mutagenesis. The quantifications provide evidence that extinction does not imply unusual increases of intrapopulation complexity, in support of the lethal defection model of virus extinction.

Plant Resistance to TSWV and Seed Accumulation of Resveratrol within Peanut Germplasm and Its Wild Relatives in the US Collection

USDA-ARS?s Scientific Manuscript database

Biotic and abiotic stress may induce peanut plants to produce a high amount of resveratrol. The relationship of plant response to tomato spotted wilt virus (TSWV) and seed accumulation of resveratrol was investigated. Twenty peanut accessions and six wild relatives were selected from the US peanut g...

The NSs protein of tomato spotted wilt virus is required for persistent infection and transmission by Frankliniella occidentalis.

PubMed

Margaria, P; Bosco, L; Vallino, M; Ciuffo, M; Mautino, G C; Tavella, L; Turina, M

2014-05-01

Tomato spotted wilt virus (TSWV) is the type member of tospoviruses (genus Tospovirus), plant-infecting viruses that cause severe damage to ornamental and vegetable crops. Tospoviruses are transmitted by thrips in the circulative propagative mode. We generated a collection of NSs-defective TSWV isolates and showed that TSWV coding for truncated NSs protein could not be transmitted by Frankliniella occidentalis. Quantitative reverse transcription (RT)-PCR and immunostaining of individual insects detected the mutant virus in second-instar larvae and adult insects, demonstrating that insects could acquire and accumulate the NSs-defective virus. Nevertheless, adults carried a significantly lower viral load, resulting in the absence of transmission. Genome sequencing and analyses of reassortant isolates showed genetic evidence of the association between the loss of competence in transmission and the mutation in the NSs coding sequence. Our findings offer new insight into the TSWV-thrips interaction and Tospovirus pathogenesis and highlight, for the first time in the Bunyaviridae family, a major role for the S segment, and specifically for the NSs protein, in virulence and efficient infection in insect vector individuals. Our work is the first to show a role for the NSs protein in virus accumulation in the insect vector in the Bunyaviridae family: demonstration was obtained for the system TSWV-F. occidentalis, arguably one of the most damaging combination for vegetable crops. Genetic evidence of the involvement of the NSs protein in vector transmission was provided with multiple approaches.

The NSs Protein of Tomato spotted wilt virus Is Required for Persistent Infection and Transmission by Frankliniella occidentalis

PubMed Central

Margaria, P.; Bosco, L.; Vallino, M.; Ciuffo, M.; Mautino, G. C.; Tavella, L.

2014-01-01

ABSTRACT Tomato spotted wilt virus (TSWV) is the type member of tospoviruses (genus Tospovirus), plant-infecting viruses that cause severe damage to ornamental and vegetable crops. Tospoviruses are transmitted by thrips in the circulative propagative mode. We generated a collection of NSs-defective TSWV isolates and showed that TSWV coding for truncated NSs protein could not be transmitted by Frankliniella occidentalis. Quantitative reverse transcription (RT)-PCR and immunostaining of individual insects detected the mutant virus in second-instar larvae and adult insects, demonstrating that insects could acquire and accumulate the NSs-defective virus. Nevertheless, adults carried a significantly lower viral load, resulting in the absence of transmission. Genome sequencing and analyses of reassortant isolates showed genetic evidence of the association between the loss of competence in transmission and the mutation in the NSs coding sequence. Our findings offer new insight into the TSWV-thrips interaction and Tospovirus pathogenesis and highlight, for the first time in the Bunyaviridae family, a major role for the S segment, and specifically for the NSs protein, in virulence and efficient infection in insect vector individuals. IMPORTANCE Our work is the first to show a role for the NSs protein in virus accumulation in the insect vector in the Bunyaviridae family: demonstration was obtained for the system TSWV-F. occidentalis, arguably one of the most damaging combination for vegetable crops. Genetic evidence of the involvement of the NSs protein in vector transmission was provided with multiple approaches. PMID:24623427

Inhibition of the Host Proteasome Facilitates Papaya Ringspot Virus Accumulation and Proteosomal Catalytic Activity Is Modulated by Viral Factor HcPro

PubMed Central

Sahana, Nandita; Kaur, Harpreet; Basavaraj; Tena, Fatima; Jain, Rakesh Kumar; Palukaitis, Peter; Canto, Tomas; Praveen, Shelly

2012-01-01

The ubiquitin/26S proteasome system plays an essential role not only in maintaining protein turnover, but also in regulating many other plant responses, including plant–pathogen interactions. Previous studies highlighted different roles of the 20S proteasome in plant defense during virus infection, either indirectly through viral suppressor-mediated degradation of Argonaute proteins, affecting the RNA interference pathway, or directly through modulation of the proteolytic and RNase activity of the 20S proteasome, a component of the 20S proteasome, by viral proteins, affecting the levels of viral proteins and RNAs. Here we show that MG132, a cell permeable proteasomal inhibitor, caused an increase in papaya ringspot virus (PRSV) accumulation in its natural host papaya (Carica papaya). We also show that the PRSV HcPro interacts with the papaya homologue of the Arabidopsis PAA (α1 subunit of the 20S proteasome), but not with the papaya homologue of Arabidopsis PAE (α5 subunit of the 20S proteasome), associated with the RNase activity, although the two 20S proteasome subunits interacted with each other. Mutated forms of PRSV HcPro showed that the conserved KITC54 motif in the N-terminal domain of HcPro was necessary for its binding to PAA. Co-agroinfiltration assays demonstrated that HcPro expression mimicked the action of MG132, and facilitated the accumulation of bothtotal ubiquitinated proteins and viral/non-viral exogenous RNA in Nicotiana benthamiana leaves. These effects were not observed by using an HcPro mutant (KITS54), which impaired the HcPro – PAA interaction. Thus, the PRSV HcPro interacts with a proteasomal subunit, inhibiting the action of the 20S proteasome, suggesting that HcPro might be crucial for modulating its catalytic activities in support of virus accumulation. PMID:23300704

Inhibition of the host proteasome facilitates papaya ringspot virus accumulation and proteosomal catalytic activity is modulated by viral factor HcPro.

PubMed

Sahana, Nandita; Kaur, Harpreet; Basavaraj; Tena, Fatima; Jain, Rakesh Kumar; Palukaitis, Peter; Canto, Tomas; Praveen, Shelly

2012-01-01

The ubiquitin/26S proteasome system plays an essential role not only in maintaining protein turnover, but also in regulating many other plant responses, including plant-pathogen interactions. Previous studies highlighted different roles of the 20S proteasome in plant defense during virus infection, either indirectly through viral suppressor-mediated degradation of Argonaute proteins, affecting the RNA interference pathway, or directly through modulation of the proteolytic and RNase activity of the 20S proteasome, a component of the 20S proteasome, by viral proteins, affecting the levels of viral proteins and RNAs. Here we show that MG132, a cell permeable proteasomal inhibitor, caused an increase in papaya ringspot virus (PRSV) accumulation in its natural host papaya (Carica papaya). We also show that the PRSV HcPro interacts with the papaya homologue of the Arabidopsis PAA (α1 subunit of the 20S proteasome), but not with the papaya homologue of Arabidopsis PAE (α5 subunit of the 20S proteasome), associated with the RNase activity, although the two 20S proteasome subunits interacted with each other. Mutated forms of PRSV HcPro showed that the conserved KITC54 motif in the N-terminal domain of HcPro was necessary for its binding to PAA. Co-agroinfiltration assays demonstrated that HcPro expression mimicked the action of MG132, and facilitated the accumulation of bothtotal ubiquitinated proteins and viral/non-viral exogenous RNA in Nicotiana benthamiana leaves. These effects were not observed by using an HcPro mutant (KITS54), which impaired the HcPro - PAA interaction. Thus, the PRSV HcPro interacts with a proteasomal subunit, inhibiting the action of the 20S proteasome, suggesting that HcPro might be crucial for modulating its catalytic activities in support of virus accumulation.

Molecular determinants of dengue virus 2 envelope protein important for virus entry in FcγRIIA-mediated antibody-dependent enhancement of infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chotiwan, Nunya; Roehrig, John T.; Schlesinger, Jacob J.

Antibody-dependent enhancement (ADE) of infection may cause severe illness in patients suffering a secondary infection by a heterologous dengue virus (DENV) serotype. During ADE of infection, cross-reactive non- or poorly-neutralizing antibodies form infectious virus-Ab complexes with the newly infecting serotype and enhance virus infection by binding to the Fcγ receptors (FcγR) on FcγR-bearing cells. In this study, we determined that molecular determinants of DENV2 envelope protein critical for virus entry during non-ADE infection are also required for ADE infection mediated by FcγRIIA, and binding of virus-Ab complexes with FcγRIIA alone is not sufficient for ADE of infection. The FcγRIIA mainlymore » plays an auxiliary role in concentrating the virus–Ab complex to the cell surface, and other primary cellular receptors are required for virus entry. Understanding the viral entry pathway in ADE of DENV infection will greatly facilitate rational designs of anti-viral therapeutics against severe dengue disease associated with ADE. - Highlights: • KKK305/307/310 in DENV2 E-DIII is critical for virus attachment in ADE and non-ADE infection. • Binding of DENV2–Ab complex with FcγRII alone is not sufficient for virus entry in ADE infection. • Other primary receptors were required for DENV2 internalization during FcγRII–mediated ADE. • G104 and L135 of DENV2 E are critical for virus-mediated membrane fusion. • DENV2 virus-mediated membrane fusion is required for both ADE and non-ADE infection.« less

A complex adenovirus vaccine against chikungunya virus provides complete protection against viraemia and arthritis

PubMed Central

Wang, Danher; Suhrbier, Andreas; Penn-Nicholson, Adam; Woraratanadharm, Jan; Gardner, Joy; Luo, Min; Le, Thuy T.; Anraku, Itaru; Sakalian, Michael; Einfeld, David; Dong, John Y.

2011-01-01

Chikungunya virus, a mosquito-borne alphavirus, recently caused the largest epidemic ever seen for this virus. Chikungunya disease primarily manifests as a painful and debilitating arthralgia/arthritis, and no effective drug or vaccine is currently available. Here we describe a recombinant chikungunya virus vaccine comprising a non-replicating complex adenovirus vector encoding the structural polyprotein cassette of chikungunya virus. A single immunisation with this vaccine consistently induced high titres of anti-chikungunya virus antibodies that neutralised both an old Asian isolate and a Réunion Island isolate from the recent epidemic. The vaccine also completely protected mice against viraemia and arthritic disease caused by both virus isolates. PMID:21320541

Accumulation of a 5’ proximal subgenomic RNA of Citrus tristeza virus is correlated with encapsidation by the minor coat protein

USDA-ARS?s Scientific Manuscript database

During replication, Citrus tristeza virus (CTV) produces large amounts of two unusual subgenomic (sg) RNAs that are positive-stranded and 5' -coterminal. Although these RNAs are produced in similar amounts and are similar in size, with LMT1 (~750 nt) only slightly larger than LMT2 (~650), we found ...

The N-terminal region of the Plantago asiatica mosaic virus coat protein is required for cell-to-cell movement but is dispensable for virion assembly.

PubMed

Ozeki, Johji; Hashimoto, Masayoshi; Komatsu, Ken; Maejima, Kensaku; Himeno, Misako; Senshu, Hiroko; Kawanishi, Takeshi; Kagiwada, Satoshi; Yamaji, Yasuyuki; Namba, Shigetou

2009-06-01

Potexvirus cell-to-cell movement requires coat protein (CP) and movement proteins. In this study, mutations in two conserved in-frame AUG codons in the 5' region of the CP open reading frame of Plantago asiatica mosaic virus (PlAMV) were introduced, and virus accumulation of these mutants was analyzed in inoculated and upper noninoculated leaves. When CP was translated only from the second AUG codon, virus accumulation in inoculated leaves was lower than that of wild-type PlAMV, and the viral spread was impaired. Trans-complementation analysis showed that the leucine residue at the third position (Leu-3) of CP is important for cell-to-cell movement of PlAMV. The 14-amino-acid N-terminal region of CP was dispensable for virion formation. Immunoprecipitation assays conducted with an anti-TGBp1 antibody indicated that PlAMV CP interacts with TGBp1 in vivo and that this interaction is not affected by alanine substitution at Leu-3. These results support the concept that the N-terminal region of potexvirus CP can be separated into two distinct functional domains.

The ubiquitin-proteasome system is required for African swine fever replication.

PubMed

Barrado-Gil, Lucía; Galindo, Inmaculada; Martínez-Alonso, Diego; Viedma, Sergio; Alonso, Covadonga

2017-01-01

Several viruses manipulate the ubiquitin-proteasome system (UPS) to initiate a productive infection. Determined viral proteins are able to change the host's ubiquitin machinery and some viruses even encode their own ubiquitinating or deubiquitinating enzymes. African swine fever virus (ASFV) encodes a gene homologous to the E2 ubiquitin conjugating (UBC) enzyme. The viral ubiquitin-conjugating enzyme (UBCv1) is expressed throughout ASFV infection and accumulates at late times post infection. UBCv is also present in the viral particle suggesting that the ubiquitin-proteasome pathway could play an important role at early ASFV infection. We determined that inhibition of the final stage of the ubiquitin-proteasome pathway blocked a post-internalization step in ASFV replication in Vero cells. Under proteasome inhibition, ASF viral genome replication, late gene expression and viral production were severely reduced. Also, ASFV enhanced proteasome activity at late times and the accumulation of polyubiquitinated proteins surrounding viral factories. Core-associated and/or viral proteins involved in DNA replication may be targets for the ubiquitin-proteasome pathway that could possibly assist virus uncoating at final core breakdown and viral DNA release. At later steps, polyubiquitinated proteins at viral factories could exert regulatory roles in cell signaling.

Timing Is Everything: Coordinated Control of Host Shutoff by Influenza A Virus NS1 and PA-X Proteins

PubMed Central

Khaperskyy, Denys A.

2015-01-01

Like all viruses, influenza viruses (IAVs) use host translation machinery to decode viral mRNAs. IAVs ensure efficient translation of viral mRNAs through host shutoff, a process whereby viral proteins limit the accumulation of host proteins through subversion of their biogenesis. Despite its small genome, the virus deploys multiple host shutoff mechanisms at different stages of infection, thereby ensuring successful replication while limiting the communication of host antiviral responses. In this Gem, we review recent data on IAV host shutoff proteins, frame the outstanding questions in the field, and propose a temporally coordinated model of IAV host shutoff. PMID:25878098

New Insights into the Hendra Virus Attachment and Entry Process from Structures of the Virus G Glycoprotein and Its Complex with Ephrin-B2

DTIC Science & Technology

2012-11-05

organization of measles virus fusion complexes. J Virol 83: 10480–10493. 24. Bishop KA, Hickey AC, Khetawat D, Patch JR, Bossart KN, et al. (2008...its receptor, sialyllactose. Structure 13: 803–815. 38. Colf LA, Juo ZS, Garcia KC (2007) Structure of the measles virus hemagglutinin. Nat Struct Mol...Biol 14: 1227–1228. 39. Hashiguchi T, Kajikawa M, Maita N, Takeda M, Kuroki K, et al. (2007) Crystal structure of measles virus hemagglutinin provides

Genome characterization, prevalence and distribution of a Macula-like virus from Apis mellifera and Varroa destructor

USDA-ARS?s Scientific Manuscript database

Numerous viruses have been detected in honeybees, which can be roughly divided into 14 unique and distinct species-complexes, each with one or more strains or sub-species. Here we present the initial characterization of an entirely new virus species-complex discovered in honeybee (Apis mellifera L.)...

WDR5 is essential for assembly of the VISA-associated signaling complex and virus-triggered IRF3 and NF-kappaB activation.

PubMed

Wang, Yan-Yi; Liu, Li-Juan; Zhong, Bo; Liu, Tian-Tian; Li, Ying; Yang, Yan; Ran, Yong; Li, Shu; Tien, Po; Shu, Hong-Bing

2010-01-12

Viral infection causes activation of the transcription factors NF-kappaB and IRF3, which collaborate to induce type I interferons (IFNs) and cellular antiviral response. The mitochondrial outer membrane protein VISA acts as a critical adapter for assembling a virus-induced complex that signals NF-kappaB and IRF3 activation. Using a biochemical purification approach, we identified the WD repeat protein WDR5 as a VISA-associated protein. WDR5 was recruited to VISA in a viral infection dependent manner. Viral infection also caused translocation of WDR5 from the nucleus to mitochondria. Knockdown of WDR5 impaired the formation of virus-induced VISA-associated complex. Consistently, knockdown of WDR5 inhibited virus-triggered activation of IRF3 and NF-kappaB as well as transcription of the IFNB1 gene. These findings suggest that WDR5 is essential in assembling a virus-induced VISA-associated complex and plays an important role in virus-triggered induction of type I IFNs.

Sensitization with vaccinia virus encoding H5N1 hemagglutinin restores immune potential against H5N1 influenza virus.

PubMed

Yasui, Fumihiko; Itoh, Yasushi; Ikejiri, Ai; Kitabatake, Masahiro; Sakaguchi, Nobuo; Munekata, Keisuke; Shichinohe, Shintaro; Hayashi, Yukiko; Ishigaki, Hirohito; Nakayama, Misako; Sakoda, Yoshihiro; Kida, Hiroshi; Ogasawara, Kazumasa; Kohara, Michinori

2016-11-28

H5N1 highly pathogenic avian influenza (H5N1 HPAI) virus causes elevated mortality compared with seasonal influenza viruses like H1N1 pandemic influenza (H1N1 pdm) virus. We identified a mechanism associated with the severe symptoms seen with H5N1 HPAI virus infection. H5N1 HPAI virus infection induced a decrease of dendritic cell number in the splenic extrafollicular T-cell zone and impaired formation of the outer layers of B-cell follicles, resulting in insufficient levels of antibody production after infection. However, in animals vaccinated with a live recombinant vaccinia virus expressing the H5 hemagglutinin, infection with H5N1 HPAI virus induced parafollicular dendritic cell accumulation and efficient antibody production. These results indicate that a recombinant vaccinia encoding H5 hemagglutinin gene does not impair dendritic cell recruitment and can be a useful vaccine candidate.

Editing plants for virus resistance using CRISPR-Cas.

PubMed

Green, J C; Hu, J S

This minireview summarizes recent advancements using the clustered regularly interspaced palindromic repeats-associated nuclease systems (CRISPR-Cas) derived from prokaryotes to breed plants resistant to DNA and RNA viruses. The CRISPR-Cas system represents a powerful tool able to edit and insert novel traits into plants precisely at chosen loci offering enormous advantages to classical breeding. Approaches to engineering plant virus resistance in both transgenic and non-transgenic plants are discussed. Iterations of the CRISPR-Cas system, FnCas9 and C2c2 capable of editing RNA in eukaryotic cells offer a particular advantage for providing resistance to RNA viruses which represent the great majority of known plant viruses. Scientists have obtained conflicting results using gene silencing technology to produce transgenic plants resistant to geminiviruses. CRISPR-Cas systems engineered in plants to target geminiviruses have consistently reduced virus accumulation providing increased resistance to virus infection. CRISPR-Cas may provide novel and reliable approaches to control geminiviruses and other ssDNA viruses such as Banana bunchy top virus (BBTV).

Generation of Marker-free Transgenic Plants Concurrently Resistant to a DNA Geminivirus and a RNA Tospovirus

PubMed Central

Yang, Ching-Fu; Chen, Kuan-Chun; Cheng, Ying-Hui; Raja, Joseph A. J.; Huang, Ya-Ling; Chien, Wan-Chu; Yeh, Shyi-Dong

2014-01-01

Global threats of ssDNA geminivirus and ss(-)RNA tospovirus on crops necessitate the development of transgenic resistance. Here, we constructed a two-T DNA vector carrying a hairpin of the intergenic region (IGR) of Ageratum yellow vein virus (AYVV), residing in an intron inserted in an untranslatable nucleocapsid protein (NP) fragment of Melon yellow spot virus (MYSV). Transgenic tobacco lines highly resistant to AYVV and MYSV were generated. Accumulation of 24-nt siRNA, higher methylation levels on the IGR promoters of the transgene, and suppression of IGR promoter activity of invading AYVV indicate that AYVV resistance is mediated by transcriptional gene silencing. Lack of NP transcript and accumulation of corresponding siRNAs indicate that MYSV resistance is mediated through post-transcriptional gene silencing. Marker-free progenies with concurrent resistance to both AYVV and MYSV, stably inherited as dominant nuclear traits, were obtained. Hence, we provide a novel way for concurrent control of noxious DNA and RNA viruses with less biosafety concerns. PMID:25030413

Role for herpes simplex virus 1 ICP27 in the inhibition of type I interferon signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Johnson, Karen E.; Song, Byeongwoon; Knipe, David M.

2008-05-10

Host cells respond to viral infection by many mechanisms, including the production of type I interferons which act in a paracrine and autocrine manner to induce the expression of antiviral interferon-stimulated genes (ISGs). Viruses have evolved means to inhibit interferon signaling to avoid induction of the innate immune response. Herpes simplex virus 1 (HSV-1) has several mechanisms to inhibit type I interferon production, the activities of ISGs, and the interferon signaling pathway itself. We report that the inhibition of the Jak/STAT pathway by HSV-1 requires viral gene expression and that viral immediate-early protein ICP27 plays a role in downregulating STAT-1more » phosphorylation and in preventing the accumulation of STAT-1 in the nucleus. We also show that expression of ICP27 by transfection causes an inhibition of IFN-induced STAT-1 nuclear accumulation. Therefore, ICP27 is necessary and sufficient for at least some of the effects of HSV infection on STAT-1.« less

The length of an internal poly(A) tract of hibiscus latent Singapore virus is crucial for its replication.

PubMed

Niu, Shengniao; Cao, Shishu; Huang, Li-Jing; Tan, Kelvin Chee-Leong; Wong, Sek-Man

2015-01-01

Hibiscus latent Singapore virus (HLSV) mutants were constructed to study roles of its internal poly(A) tract (IPAT) in viral replication and coat protein (CP) expression. Shortening of the IPAT resulted in reduced HLSV RNA accumulation and its minimal length required for HLSV CP expression in plants was 24 nt. Disruption of a putative long range RNA-RNA interacting structure between 5' and 3' untranslated regions of HLSV-22A and -24A resulted in reduced viral RNA and undetectable CP accumulation in inoculated leaves. Replacement of the IPAT in HLSV with an upstream pseudoknot domain (UPD) of Tobacco mosaic virus (TMV) or insertion of the UPD to the immediate downstream of a 24 nt IPAT in HLSV resulted in drastically reduced viral RNA replication. Plants infected with a TMV mutant by replacement of the UPD with 43 nt IPAT exhibited milder mosaic symptoms without necrosis. We have proposed a model for HLSV replication. Copyright © 2014 Elsevier Inc. All rights reserved.

Isolation and characterization of a virus-specific ribonucleoprotein complex from reticuloendotheliosis virus-transformed chicken bone marrow cells.

PubMed Central

Wong, T C; Kang, C Y

1978-01-01

Chicken bone marrow cells transformed by reticuloendotheliosis virus (REV) produce in the cytoplasm a ribonucleoprotein (RNP) complex which has a sedimentation value of approximately 80 to 100S and a density of 1.23 g/cm3. This RNP complex is not derived from the mature virion. An endogenous RNA-directed DNA polymerase activity is associated with the RNP complex. The enzyme activity was completely neutralized by anti-REV DNA polymerase antibody but not by anti-avian myeloblastosis virus DNA polymerase antibody. The DNA product from the endogenous RNA-directed DNA polymerase reaction of the RNP complex hybridized to REV RNA but not to avian leukosis virus RNA. The RNA extracted from the RNP hybridized only to REV-specific complementary DNA synthesized from an endogenous DNA polymerase reaction of purified REV. The size of the RNA in the RNP is 30 to 35S, which represents the subunit size of the genomic RNA. No 60S mature genomic RNA was found within the RNP complex. The significance of finding the endogenous DNA polymerase activity in the viral RNP in infected cells and the maturation process of 60S virion RNA of REV are discussed. PMID:81319

Giant Viruses of Amoebae: A Journey Through Innovative Research and Paradigm Changes.

PubMed

Colson, Philippe; La Scola, Bernard; Raoult, Didier

2017-09-29

Giant viruses of amoebae were discovered serendipitously in 2003; they are visible via optical microscopy, making them bona fide microbes. Their lifestyle, structure, and genomes break the mold of classical viruses. Giant viruses of amoebae are complex microorganisms. Their genomes harbor between 444 and 2,544 genes, including many that are unique to viruses, and encode translation components; their virions contain >100 proteins as well as mRNAs. Mimiviruses have a specific mobilome, including virophages, provirophages, and transpovirons, and can resist virophages through a system known as MIMIVIRE (mimivirus virophage resistance element). Giant viruses of amoebae bring upheaval to the definition of viruses and tend to separate the current virosphere into two categories: very simple viruses and viruses with complexity similar to that of other microbes. This new paradigm is propitious for enhanced detection and characterization of giant viruses of amoebae, and a particular focus on their role in humans is warranted.

Formation of infectious dengue virus-antibody immune complex in vivo in marmosets (Callithrix jacchus) after passive transfer of anti-dengue virus monoclonal antibodies and infection with dengue virus.

PubMed

Moi, Meng Ling; Ami, Yasushi; Shirai, Kenji; Lim, Chang-Kweng; Suzaki, Yuriko; Saito, Yuka; Kitaura, Kazutaka; Saijo, Masayuki; Suzuki, Ryuji; Kurane, Ichiro; Takasaki, Tomohiko

2015-02-01

Infection with a dengue virus (DENV) serotype induces cross-reactive, weakly neutralizing antibodies to different dengue serotypes. It has been postulated that cross-reactive antibodies form a virus-antibody immune complex and enhance DENV infection of Fc gamma receptor (FcγR)-bearing cells. We determined whether infectious DENV-antibody immune complex is formed in vivo in marmosets after passive transfer of DENV-specific monoclonal antibody (mAb) and DENV inoculation and whether infectious DENV-antibody immune complex is detectable using FcγR-expressing cells. Marmosets showed that DENV-antibody immune complex was exclusively infectious to FcγR-expressing cells on days 2, 4, and 7 after passive transfer of each of the mAbs (mAb 4G2 and mAb 6B6C) and DENV inoculation. Although DENV-antibody immune complex was detected, contribution of the passively transferred antibody to overall viremia levels was limited in this study. The results indicate that DENV cross-reactive antibodies form DENV-antibody immune complex in vivo, which is infectious to FcγR-bearing cells but not FcγR-negative cells. © The American Society of Tropical Medicine and Hygiene.

Dual Function of the pUL7-pUL51 Tegument Protein Complex in Herpes Simplex Virus 1 Infection.

PubMed

Albecka, Anna; Owen, Danielle J; Ivanova, Lyudmila; Brun, Juliane; Liman, Rukayya; Davies, Laura; Ahmed, M Firoz; Colaco, Susanna; Hollinshead, Michael; Graham, Stephen C; Crump, Colin M

2017-01-15

The tegument of herpesviruses is a highly complex structural layer between the nucleocapsid and the envelope of virions. Tegument proteins play both structural and regulatory functions during replication and spread, but the interactions and functions of many of these proteins are poorly understood. Here we focus on two tegument proteins from herpes simplex virus 1 (HSV-1), pUL7 and pUL51, which have homologues in all other herpesviruses. We have now identified that HSV-1 pUL7 and pUL51 form a stable and direct protein-protein interaction, their expression levels rely on the presence of each other, and they function as a complex in infected cells. We demonstrate that expression of the pUL7-pUL51 complex is important for efficient HSV-1 assembly and plaque formation. Furthermore, we also discovered that the pUL7-pUL51 complex localizes to focal adhesions at the plasma membrane in both infected cells and in the absence of other viral proteins. The expression of pUL7-pUL51 is important to stabilize focal adhesions and maintain cell morphology in infected cells and cells infected with viruses lacking pUL7 and/or pUL51 round up more rapidly than cells infected with wild-type HSV-1. Our data suggest that, in addition to the previously reported functions in virus assembly and spread for pUL51, the pUL7-pUL51 complex is important for maintaining the attachment of infected cells to their surroundings through modulating the activity of focal adhesion complexes. Herpesviridae is a large family of highly successful human and animal pathogens. Virions of these viruses are composed of many different proteins, most of which are contained within the tegument, a complex structural layer between the nucleocapsid and the envelope within virus particles. Tegument proteins have important roles in assembling virus particles as well as modifying host cells to promote virus replication and spread. However, little is known about the function of many tegument proteins during virus replication. Our study focuses on two tegument proteins from herpes simplex virus 1 that are conserved in all herpesviruses: pUL7 and pUL51. We demonstrate that these proteins directly interact and form a functional complex that is important for both virus assembly and modulation of host cell morphology. Further, we identify for the first time that these conserved herpesvirus tegument proteins localize to focal adhesions in addition to cytoplasmic juxtanuclear membranes within infected cells. Copyright © 2017 Albecka et al.

The human immunodeficiency virus type 1 long terminal repeat specifies two different transcription complexes, only one of which is regulated by Tat.

PubMed Central

Lu, X; Welsh, T M; Peterlin, B M

1993-01-01

The human immunodeficiency virus type 1 long terminal repeat sets up two different transcription complexes, which have been called processive and nonprocessive complexes. By mutating and substituting cis-acting sequences, we mapped elements of the human immunodeficiency virus long terminal repeat that are responsible for creating each transcription complex. Whereas processive complexes are efficiently assembled by upstream promoter elements in the absence of the TATA box, nonprocessive complexes absolutely require the TATA box. Moreover, the TATA box alone can set up these nonprocessive complexes, and nonprocessive but not processive complexes are trans activated by Tat. Finally, a strong DNA-binding site between the TATA box and trans-activation-responsive region interferes with either the assembly or movement of these nonprocessive complexes and diminishes the effects of Tat. Thus, Tat affects a critical step in the formation of elongation-competent transcription complexes. Images PMID:8445708

Activation of Paramyxovirus Membrane Fusion and Virus Entry

PubMed Central

Jardetzky, Theodore S.; Lamb, Robert A.

2014-01-01

The paramyxoviruses represent a diverse virus family responsible for a wide range of human and animal diseases. In contrast to other viruses, such as HIV and influenza virus, which use a single glycoprotein to mediate host receptor binding and virus entry, the paramyxoviruses require two distinct proteins. One of these is an attachment glycoprotein that binds receptor, while the second is a fusion glycoprotein, which undergoes conformational changes that drive virus-cell membrane fusion and virus entry. The details of how receptor binding by one protein activates the second to undergo conformational changes have been poorly understood until recently. Over the past couple of years, structural and functional data have accumulated on representative members of this family, including parainfluenza virus 5, Newcastle disease virus, measles virus, Nipah virus and others, which suggest a mechanistic convergence of activation models. Here we review the data indicating that paramyxovirus attachment glycoproteins shield activating residues within their N-terminal stalk domains, which are then exposed upon receptor binding, leading to the activation of the fusion protein by a ‘provocateur’ mechanism. PMID:24530984

3D structure of the influenza virus polymerase complex: Localization of subunit domains

PubMed Central

Area, Estela; Martín-Benito, Jaime; Gastaminza, Pablo; Torreira, Eva; Valpuesta, José M.; Carrascosa, José L.; Ortín, Juan

2004-01-01

The 3D structure of the influenza virus polymerase complex was determined by electron microscopy and image processing of recombinant ribonucleoproteins (RNPs). The RNPs were generated by in vivo amplification using cDNAs of the three polymerase subunits, the nucleoprotein, and a model virus-associated RNA containing 248 nt. The polymerase structure obtained is very compact, with no apparent boundaries among subunits. The position of specific regions of the PB1, PB2, and PA subunits was determined by 3D reconstruction of either RNP–mAb complexes or tagged RNPs. This structural model is available for the polymerase of a negative-stranded RNA virus and provides a general delineation of the complex and its interaction with the template-associated nucleoprotein monomers in the RNP. PMID:14691253

In planta expression of HIV-1 p24 protein using an RNA plant virus-based expression vector.

PubMed

Zhang, G; Leung, C; Murdin, L; Rovinski, B; White, K A

2000-02-01

Plant viruses show significant potential as expression vectors for the production of foreign proteins (e.g., antigens) in plants. The HIV-1 p24 nucleocapsid protein is an important early marker of HIV infection and has been used as an antigen in the development of HIV vaccines. Toward developing a plant-based expression system for the production of p24, we have investigated the use of a (positive)-strand RNA plant virus, tomato bushy stunt virus (TBSV), as an expression vector. The HIV p24 open reading frame (ORF) was introduced into a cloned cDNA copy of the TBSV genome as an in-frame fusion with a 5'-terminal portion of the TBSV coat protein ORF. In vitro-generated RNA transcripts corresponding to the engineered virus vector were infectious when inoculated into plant protoplasts; Northern and Western blot analyses verified the accumulation of a predicted p24-encoding viral subgenomic mRNA and the production of p24 fusion product. Whole-plant infections with the viral vector led to the accumulation of p24 fusion protein in inoculated leaves, which cross-reacted with p24-specific antibodies, thus confirming the maintenance of key antigenic determinants. This study is the first to demonstrate that TBSV can be engineered to express a complete foreign protein of clinical importance. Strategies for optimizing protein yield from this viral vector are discussed.

Renilla luciferase-based quantitation of Potato virus A infection initiated with Agrobacterium infiltration of N. benthamiana leaves.

PubMed

Eskelin, K; Suntio, T; Hyvärinen, S; Hafren, A; Mäkinen, K

2010-03-01

A quantitation method based on the sensitive detection of Renilla luciferase (Rluc) activity was developed and optimized for Potato virus A (PVA; genus Potyviridae) gene expression. This system is based on infections initiated by Agrobacterium infiltration and subsequent detection of the translation of PVA::Rluc RNA, which is enhanced by viral replication, first within the cells infected initially and later by translation and replication within new cells after spread of the virus. Firefly luciferase (Fluc) was used as an internal control to normalize the Rluc activity. An approximately 10-fold difference in the Rluc/Fluc activity ratio between a movement-deficient and a replication-deficient mutant was observed starting from 48h post Agrobacterium infiltration (h.p.i.). The Rluc activity derived from wild type (wt) PVA increased significantly between 48 and 72h.p.i. and the Rluc/Fluc activity deviated clearly from that of the mutant viruses. Quantitation of the Rluc and Fluc mRNAs by semi-quantitative RT-PCR indicated that increases and decreases in the Renillareniformis luciferase (rluc) mRNA levels coincided with changes in Rluc activity. However, a subtle increase in the mRNA level led to pronounced changes in Rluc activity. PVA CP accumulation was quantitated by enzyme-linked immunosorbent assay. The increase in Rluc activity correlated closely with virus accumulation. Copyright (c) 2009 Elsevier B.V. All rights reserved.

Activation of DNA damage repair pathways by murine polyomavirus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Heiser, Katie; Nicholas, Catherine; Garcea, Robert

Nuclear replication of DNA viruses activates DNA damage repair (DDR) pathways, which are thought to detect and inhibit viral replication. However, many DNA viruses also depend on these pathways in order to optimally replicate their genomes. We investigated the relationship between murine polyomavirus (MuPyV) and components of DDR signaling pathways including CHK1, CHK2, H2AX, ATR, and DNAPK. We found that recruitment and retention of DDR proteins at viral replication centers was independent of H2AX, as well as the viral small and middle T-antigens. Additionally, infectious virus production required ATR kinase activity, but was independent of CHK1, CHK2, or DNAPK signaling.more » ATR inhibition did not reduce the total amount of viral DNA accumulated, but affected the amount of virus produced, indicating a defect in virus assembly. These results suggest that MuPyV may utilize a subset of DDR proteins or non-canonical DDR signaling pathways in order to efficiently replicate and assemble. -- Highlights: •Murine polyomavirus activates and recruits DNA damage repair (DDR) proteins to replication centers. •Large T-antigen mediates recruitment of DDR proteins to viral replication centers. •Inhibition or knockout of CHK1, CHK2, DNA-PK or H2AX do not affect viral titers. •Inhibition of ATR activity reduces viral titers, but not viral DNA accumulation.« less

A Viral Pilot for HCMV Navigation?

PubMed

Adler, Barbara

2015-07-15

gH/gL virion envelope glycoprotein complexes of herpesviruses serve as entry complexes and mediate viral cell tropism. By binding additional viral proteins, gH/gL forms multimeric complexes which bind to specific host cell receptors. Both Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV) express alternative multimeric gH/gL complexes. Relative amounts of these alternative complexes in the viral envelope determine which host cells are preferentially infected. Host cells of EBV can modulate the gH/gL complex complement of progeny viruses by cell type-dependent degradation of one of the associating proteins. Host cells of HCMV modulate the tropism of their virus progenies by releasing or not releasing virus populations with a specific gH/gL complex complement out of a heterogeneous pool of virions. The group of Jeremy Kamil has recently shown that the HCMV ER-resident protein UL148 controls integration of one of the HCMV gH/gL complexes into virions and thus creates a pool of virions which can be routed by different host cells. This first mechanistic insight into regulation of the gH/gL complex complement of HCMV progenies presents UL148 as a pilot candidate for HCMV navigation in its infected host.

Dynamics of Small RNA Profiles of Virus and Host Origin in Wheat Cultivars Synergistically Infected by Wheat Streak Mosaic Virus and Triticum Mosaic Virus: Virus Infection Caused a Drastic Shift in the Endogenous Small RNA Profile

PubMed Central

Tatineni, Satyanarayana; Riethoven, Jean-Jack M.; Graybosch, Robert A.; French, Roy; Mitra, Amitava

2014-01-01

Co-infection of wheat (Triticum aestivum L.) by Wheat streak mosaic virus (WSMV, a Tritimovirus) and Triticum mosaic virus (TriMV, a Poacevirus) of the family Potyviridae causes synergistic interaction. In this study, the effects of the synergistic interaction between WSMV and TriMV on endogenous and virus-derived small interfering RNAs (vsiRNAs) were examined in susceptible (‘Arapahoe’) and temperature-sensitive resistant (‘Mace’) wheat cultivars at 18°C and 27°C. Single and double infections in wheat caused a shift in the profile of endogenous small RNAs from 24 nt being the most predominant in healthy plants to 21 nt in infected wheat. Massive amounts of 21 and 22 nt vsiRNAs accumulated in singly and doubly infected Arapahoe at both temperatures and in Mace at 27°C but not 18°C. The plus- and minus-sense vsiRNAs were distributed throughout the genomic RNAs in Arapahoe at both temperature regimens and in Mace at 27°C, although some regions served as hot-spots, spawning an excessive number of vsiRNAs. The vsiRNA peaks were conserved among cultivars, suggesting that the Dicer-like enzymes in susceptible and resistant cultivars similarly accessed the genomic RNAs of WSMV or TriMV. Accumulation of large amounts of vsiRNAs in doubly infected plants suggests that the silencing suppressor proteins encoded by TriMV and WSMV do not prevent the formation of vsiRNAs; thus, the synergistic effect observed is independent from RNA-silencing mediated vsiRNA biogenesis. The high-resolution map of endogenous and vsiRNAs from WSMV- and/or TriMV-infected wheat cultivars may form a foundation for understanding the virus-host interactions, the effect of synergistic interactions on host defense, and virus resistance mechanisms in wheat. PMID:25365307

Dynamics of small RNA profiles of virus and host origin in wheat cultivars synergistically infected by Wheat streak mosaic virus and Triticum mosaic virus: virus infection caused a drastic shift in the endogenous small RNA profile.

PubMed

Tatineni, Satyanarayana; Riethoven, Jean-Jack M; Graybosch, Robert A; French, Roy; Mitra, Amitava

2014-01-01

Co-infection of wheat (Triticum aestivum L.) by Wheat streak mosaic virus (WSMV, a Tritimovirus) and Triticum mosaic virus (TriMV, a Poacevirus) of the family Potyviridae causes synergistic interaction. In this study, the effects of the synergistic interaction between WSMV and TriMV on endogenous and virus-derived small interfering RNAs (vsiRNAs) were examined in susceptible ('Arapahoe') and temperature-sensitive resistant ('Mace') wheat cultivars at 18°C and 27°C. Single and double infections in wheat caused a shift in the profile of endogenous small RNAs from 24 nt being the most predominant in healthy plants to 21 nt in infected wheat. Massive amounts of 21 and 22 nt vsiRNAs accumulated in singly and doubly infected Arapahoe at both temperatures and in Mace at 27°C but not 18°C. The plus- and minus-sense vsiRNAs were distributed throughout the genomic RNAs in Arapahoe at both temperature regimens and in Mace at 27°C, although some regions served as hot-spots, spawning an excessive number of vsiRNAs. The vsiRNA peaks were conserved among cultivars, suggesting that the Dicer-like enzymes in susceptible and resistant cultivars similarly accessed the genomic RNAs of WSMV or TriMV. Accumulation of large amounts of vsiRNAs in doubly infected plants suggests that the silencing suppressor proteins encoded by TriMV and WSMV do not prevent the formation of vsiRNAs; thus, the synergistic effect observed is independent from RNA-silencing mediated vsiRNA biogenesis. The high-resolution map of endogenous and vsiRNAs from WSMV- and/or TriMV-infected wheat cultivars may form a foundation for understanding the virus-host interactions, the effect of synergistic interactions on host defense, and virus resistance mechanisms in wheat.

Herpes simplex virus-1 evasion of CD8+ T cell accumulation contributes to viral encephalitis.

PubMed

Koyanagi, Naoto; Imai, Takahiko; Shindo, Keiko; Sato, Ayuko; Fujii, Wataru; Ichinohe, Takeshi; Takemura, Naoki; Kakuta, Shigeru; Uematsu, Satoshi; Kiyono, Hiroshi; Maruzuru, Yuhei; Arii, Jun; Kato, Akihisa; Kawaguchi, Yasushi

2017-10-02

Herpes simplex virus-1 (HSV-1) is the most common cause of sporadic viral encephalitis, which can be lethal or result in severe neurological defects even with antiviral therapy. While HSV-1 causes encephalitis in spite of HSV-1-specific humoral and cellular immunity, the mechanism by which HSV-1 evades the immune system in the central nervous system (CNS) remains unknown. Here we describe a strategy by which HSV-1 avoids immune targeting in the CNS. The HSV-1 UL13 kinase promotes evasion of HSV-1-specific CD8+ T cell accumulation in infection sites by downregulating expression of the CD8+ T cell attractant chemokine CXCL9 in the CNS of infected mice, leading to increased HSV-1 mortality due to encephalitis. Direct injection of CXCL9 into the CNS infection site enhanced HSV-1-specific CD8+ T cell accumulation, leading to marked improvements in the survival of infected mice. This previously uncharacterized strategy for HSV-1 evasion of CD8+ T cell accumulation in the CNS has important implications for understanding the pathogenesis and clinical treatment of HSV-1 encephalitis.

Intracellular Transport of Vaccinia Virus in HeLa Cells Requires WASH-VPEF/FAM21-Retromer Complexes and Recycling Molecules Rab11 and Rab22

PubMed Central

Hsiao, Jye-Chian; Chu, Li-Wei; Lo, Yung-Tsun; Lee, Sue-Ping; Chen, Tzu-Jung; Huang, Cheng-Yen

2015-01-01

ABSTRACT Vaccinia virus, the prototype of the Orthopoxvirus genus in the family Poxviridae, infects a wide range of cell lines and animals. Vaccinia mature virus particles of the WR strain reportedly enter HeLa cells through fluid-phase endocytosis. However, the intracellular trafficking process of the vaccinia mature virus between cellular uptake and membrane fusion remains unknown. We used live imaging of single virus particles with a combination of various cellular vesicle markers, to track fluorescent vaccinia mature virus particle movement in cells. Furthermore, we performed functional interference assays to perturb distinct vesicle trafficking processes in order to delineate the specific route undertaken by vaccinia mature virus prior to membrane fusion and virus core uncoating in cells. Our results showed that vaccinia virus traffics to early endosomes, where recycling endosome markers Rab11 and Rab22 are recruited to participate in subsequent virus trafficking prior to virus core uncoating in the cytoplasm. Furthermore, we identified WASH-VPEF/FAM21-retromer complexes that mediate endosome fission and sorting of virus-containing vesicles prior to virus core uncoating in the cytoplasm. IMPORTANCE Vaccinia mature virions of the WR strain enter HeLa cells through fluid phase endocytosis. We previously demonstrated that virus-containing vesicles are internalized into phosphatidylinositol 3-phosphate positive macropinosomes, which are then fused with Rab5-positive early endosomes. However, the subsequent process of sorting the virion-containing vesicles prior to membrane fusion remains unclear. We dissected the intracellular trafficking pathway of vaccinia mature virions in cells up to virus core uncoating in cytoplasm. We show that vaccinia mature virions first travel to early endosomes. Subsequent trafficking events require the important endosome-tethered protein VPEF/FAM21, which recruits WASH and retromer protein complexes to the endosome. There, the complex executes endosomal membrane fission and cargo sorting to the Rab11-positive and Rab22-positive recycling pathway, resulting in membrane fusion and virus core uncoating in the cytoplasm. PMID:26041286

Identification and Characterization of Two Novel RNA Viruses from Anopheles gambiae Species Complex Mosquitoes

PubMed Central

Carissimo, Guillaume; Eiglmeier, Karin; Reveillaud, Julie; Holm, Inge; Diallo, Mawlouth; Diallo, Diawo; Vantaux, Amélie; Kim, Saorin; Ménard, Didier; Siv, Sovannaroth; Belda, Eugeni; Bischoff, Emmanuel; Antoniewski, Christophe; Vernick, Kenneth D.

2016-01-01

Mosquitoes of the Anopheles gambiae complex display strong preference for human bloodmeals and are major malaria vectors in Africa. However, their interaction with viruses or role in arbovirus transmission during epidemics has been little examined, with the exception of O’nyong-nyong virus, closely related to Chikungunya virus. Deep-sequencing has revealed different RNA viruses in natural insect viromes, but none have been previously described in the Anopheles gambiae species complex. Here, we describe two novel insect RNA viruses, a Dicistrovirus and a Cypovirus, found in laboratory colonies of An. gambiae taxa using small-RNA deep sequencing. Sequence analysis was done with Metavisitor, an open-source bioinformatic pipeline for virus discovery and de novo genome assembly. Wild-collected Anopheles from Senegal and Cambodia were positive for the Dicistrovirus and Cypovirus, displaying high sequence identity to the laboratory-derived virus. Thus, the Dicistrovirus (Anopheles C virus, AnCV) and Cypovirus (Anopheles Cypovirus, AnCPV) are components of the natural virome of at least some anopheline species. Their possible influence on mosquito immunity or transmission of other pathogens is unknown. These natural viruses could be developed as models for the study of Anopheles-RNA virus interactions in low security laboratory settings, in an analogous manner to the use of rodent malaria parasites for studies of mosquito anti-parasite immunity. PMID:27138938

An Improved Brome mosaic virus Silencing Vector: Greater Insert Stability and More Extensive VIGS.

PubMed

Ding, Xin Shun; Mannas, Stephen W; Bishop, Bethany A; Rao, Xiaolan; Lecoultre, Mitchell; Kwon, Soonil; Nelson, Richard S

2018-01-01

Virus-induced gene silencing (VIGS) is used extensively for gene function studies in plants. VIGS is inexpensive and rapid compared with silencing conducted through stable transformation, but many virus-silencing vectors, especially in grasses, induce only transient silencing phenotypes. A major reason for transient phenotypes is the instability of the foreign gene fragment (insert) in the vector during VIGS. Here, we report the development of a Brome mosaic virus (BMV)-based vector that better maintains inserts through modification of the original BMV vector RNA sequence. Modification of the BMV RNA3 sequence yielded a vector, BMVCP5, that better maintained phytoene desaturase and heat shock protein70-1 ( HSP70-1 ) inserts in Nicotiana benthamiana and maize ( Zea mays ). Longer maintenance of inserts was correlated with greater target gene silencing and more extensive visible silencing phenotypes displaying greater tissue penetration and involving more leaves. The modified vector accumulated similarly to the original vector in N. benthamiana after agroinfiltration, thus maintaining a high titer of virus in this intermediate host used to produce virus inoculum for grass hosts. For HSP70 , silencing one family member led to a large increase in the expression of another family member, an increase likely related to the target gene knockdown and not a general effect of virus infection. The cause of the increased insert stability in the modified vector is discussed in relationship to its recombination and accumulation potential. The modified vector will improve functional genomic studies in grasses, and the conceptual methods used to improve the vector may be applied to other VIGS vectors. © 2018 American Society of Plant Biologists. All Rights Reserved.

A Trio of Viral Proteins Tunes Aphid-Plant Interactions in Arabidopsis thaliana

PubMed Central

Du, Zhiyou; Murphy, Alex M.; Anggoro, Damar Tri; Tungadi, Trisna; Luang-In, Vijitra; Lewsey, Mathew G.; Rossiter, John T.; Powell, Glen; Smith, Alison G.; Carr, John P.

2013-01-01

Background Virus-induced deterrence to aphid feeding is believed to promote plant virus transmission by encouraging migration of virus-bearing insects away from infected plants. We investigated the effects of infection by an aphid-transmitted virus, cucumber mosaic virus (CMV), on the interaction of Arabidopsis thaliana, one of the natural hosts for CMV, with Myzus persicae (common names: ‘peach-potato aphid’, ‘green peach aphid’). Methodology/Principal Findings Infection of Arabidopsis (ecotype Col-0) with CMV strain Fny (Fny-CMV) induced biosynthesis of the aphid feeding-deterrent 4-methoxy-indol-3-yl-methylglucosinolate (4MI3M). 4MI3M inhibited phloem ingestion by aphids and consequently discouraged aphid settling. The CMV 2b protein is a suppressor of antiviral RNA silencing, which has previously been implicated in altering plant-aphid interactions. Its presence in infected hosts enhances the accumulation of CMV and the other four viral proteins. Another viral gene product, the 2a protein (an RNA-dependent RNA polymerase), triggers defensive signaling, leading to increased 4MI3M accumulation. The 2b protein can inhibit ARGONAUTE1 (AGO1), a host factor that both positively-regulates 4MI3M biosynthesis and negatively-regulates accumulation of substance(s) toxic to aphids. However, the 1a replicase protein moderated 2b-mediated inhibition of AGO1, ensuring that aphids were deterred from feeding but not poisoned. The LS strain of CMV did not induce feeding deterrence in Arabidopsis ecotype Col-0. Conclusions/Significance Inhibition of AGO1 by the 2b protein could act as a booby trap since this will trigger antibiosis against aphids. However, for Fny-CMV the interplay of three viral proteins (1a, 2a and 2b) appears to balance the need of the virus to inhibit antiviral silencing, while inducing a mild resistance (antixenosis) that is thought to promote transmission. The strain-specific effects of CMV on Arabidopsis-aphid interactions, and differences between the effects of Fny-CMV on this plant and those seen previously in tobacco (inhibition of resistance to aphids) may have important epidemiological consequences. PMID:24349433

Characterization of Staufen1 Ribonucleoproteins by Mass Spectrometry and Biochemical Analyses Reveal the Presence of Diverse Host Proteins Associated with Human Immunodeficiency Virus Type 1

PubMed Central

Milev, Miroslav P.; Ravichandran, Mukunthan; Khan, Morgan F.; Schriemer, David C.; Mouland, Andrew J.

2012-01-01

The human immunodeficiency virus type 1 (HIV-1) unspliced, 9 kb genomic RNA (vRNA) is exported from the nucleus for the synthesis of viral structural proteins and enzymes (Gag and Gag/Pol) and is then transported to sites of virus assembly where it is packaged into progeny virions. vRNA co-exists in the cytoplasm in the context of the HIV-1 ribonucleoprotein (RNP) that is currently defined by the presence of Gag and several host proteins including the double-stranded RNA-binding protein, Staufen1. In this study we isolated Staufen1 RNP complexes derived from HIV-1-expressing cells using tandem affinity purification and have identified multiple host protein components by mass spectrometry. Four viral proteins, including Gag, Gag/Pol, Env and Nef as well as >200 host proteins were identified in these RNPs. Moreover, HIV-1 induces both qualitative and quantitative differences in host protein content in these RNPs. 22% of Staufen1-associated factors are virion-associated suggesting that the RNP could be a vehicle to achieve this. In addition, we provide evidence on how HIV-1 modulates the composition of cytoplasmic Staufen1 RNPs. Biochemical fractionation by density gradient analyses revealed new facets on the assembly of Staufen1 RNPs. The assembly of dense Staufen1 RNPs that contain Gag and several host proteins were found to be entirely RNA-dependent but their assembly appeared to be independent of Gag expression. Gag-containing complexes fractionated into a lighter and another, more dense pool. Lastly, Staufen1 depletion studies demonstrated that the previously characterized Staufen1 HIV-1-dependent RNPs are most likely aggregates of smaller RNPs that accumulate at juxtanuclear domains. The molecular characterization of Staufen1 HIV-1 RNPs will offer important information on virus-host cell interactions and on the elucidation of the function of these RNPs for the transport of Gag and the fate of the unspliced vRNA in HIV-1-producing cells. PMID:23125841

RNA Viruses: ROS-Mediated Cell Death

PubMed Central

Reshi, Mohammad Latif; Su, Yi-Che; Hong, Jiann-Ruey

2014-01-01

Reactive oxygen species (ROS) are well known for being both beneficial and deleterious. The main thrust of this review is to investigate the role of ROS in ribonucleic acid (RNA) virus pathogenesis. Much evidences has accumulated over the past decade, suggesting that patients infected with RNA viruses are under chronic oxidative stress. Changes to the body's antioxidant defense system, in relation to SOD, ascorbic acid, selenium, carotenoids, and glutathione, have been reported in various tissues of RNA-virus infected patients. This review focuses on RNA viruses and retroviruses, giving particular attention to the human influenza virus, Hepatitis c virus (HCV), human immunodeficiency virus (HIV), and the aquatic Betanodavirus. Oxidative stress via RNA virus infections can contribute to several aspects of viral disease pathogenesis including apoptosis, loss of immune function, viral replication, inflammatory response, and loss of body weight. We focus on how ROS production is correlated with host cell death. Moreover, ROS may play an important role as a signal molecule in the regulation of viral replication and organelle function, potentially providing new insights in the prevention and treatment of RNA viruses and retrovirus infections. PMID:24899897

A Herpesviral Immediate Early Protein Promotes Transcription Elongation of Viral Transcripts.

PubMed

Fox, Hannah L; Dembowski, Jill A; DeLuca, Neal A

2017-06-13

Herpes simplex virus 1 (HSV-1) genes are transcribed by cellular RNA polymerase II (RNA Pol II). While four viral immediate early proteins (ICP4, ICP0, ICP27, and ICP22) function in some capacity in viral transcription, the mechanism by which ICP22 functions remains unclear. We observed that the FACT complex (comprised of SSRP1 and Spt16) was relocalized in infected cells as a function of ICP22. ICP22 was also required for the association of FACT and the transcription elongation factors SPT5 and SPT6 with viral genomes. We further demonstrated that the FACT complex interacts with ICP22 throughout infection. We therefore hypothesized that ICP22 recruits cellular transcription elongation factors to viral genomes for efficient transcription elongation of viral genes. We reevaluated the phenotype of an ICP22 mutant virus by determining the abundance of all viral mRNAs throughout infection by transcriptome sequencing (RNA-seq). The accumulation of almost all viral mRNAs late in infection was reduced compared to the wild type, regardless of kinetic class. Using chromatin immunoprecipitation sequencing (ChIP-seq), we mapped the location of RNA Pol II on viral genes and found that RNA Pol II levels on the bodies of viral genes were reduced in the ICP22 mutant compared to wild-type virus. In contrast, the association of RNA Pol II with transcription start sites in the mutant was not reduced. Taken together, our results indicate that ICP22 plays a role in recruiting elongation factors like the FACT complex to the HSV-1 genome to allow for efficient viral transcription elongation late in viral infection and ultimately infectious virion production. IMPORTANCE HSV-1 interacts with many cellular proteins throughout productive infection. Here, we demonstrate the interaction of a viral protein, ICP22, with a subset of cellular proteins known to be involved in transcription elongation. We determined that ICP22 is required to recruit the FACT complex and other transcription elongation factors to viral genomes and that in the absence of ICP22 viral transcription is globally reduced late in productive infection, due to an elongation defect. This insight defines a fundamental role of ICP22 in HSV-1 infection and elucidates the involvement of cellular factors in HSV-1 transcription. Copyright © 2017 Fox et al.

NXT1, a Novel Influenza A NP Binding Protein, Promotes the Nuclear Export of NP via a CRM1-Dependent Pathway.

PubMed

Chutiwitoonchai, Nopporn; Aida, Yoko

2016-07-28

Influenza remains a serious worldwide public health problem. After infection, viral genomic RNA is replicated in the nucleus and packed into viral ribonucleoprotein, which will then be exported to the cytoplasm via a cellular chromosome region maintenance 1 (CRM1)-dependent pathway for further assembly and budding. However, the nuclear export mechanism of influenza virus remains controversial. Here, we identify cellular nuclear transport factor 2 (NTF2)-like export protein 1 (NXT1) as a novel binding partner of nucleoprotein (NP) that stimulates NP-mediated nuclear export via the CRM1-dependent pathway. NXT1-knockdown cells exhibit decreased viral replication kinetics and nuclear accumulated viral RNA and NP. By contrast, NXT1 overexpression promotes nuclear export of NP in a CRM1-dependent manner. Pull-down assays suggest the formation of an NXT1, NP, and CRM1 complex, and demonstrate that NXT1 binds to the C-terminal region of NP. These findings reveal a distinct mechanism for nuclear export of the influenza virus and identify the NXT1/NP interaction as a potential target for antiviral drug development.

Herpes Simplex Virus DNA Packaging without Measurable DNA Synthesis

PubMed Central

Church, Geoffrey A.; Dasgupta, Anindya; Wilson, Duncan W.

1998-01-01

Herpes simplex virus (HSV) type 1 DNA synthesis and packaging occur within the nuclei of infected cells; however, the extent to which the two processes are coupled remains unclear. Correct packaging is thought to be dependent upon DNA debranching or other repair processes, and such events commonly involve new DNA synthesis. Furthermore, the HSV UL15 gene product, essential for packaging, nevertheless localizes to sites of active DNA replication and may link the two events. It has previously been difficult to determine whether packaging requires concomitant DNA synthesis due to the complexity of these processes and of the viral life cycle; however, we have recently described a model system which simplifies the study of HSV assembly. Cells infected with HSV strain tsProt.A accumulate unpackaged capsids at the nonpermissive temperature of 39°C. Following release of the temperature block, these capsids proceed to package viral DNA in a single, synchronous wave. Here we report that, when DNA replication was inhibited prior to release of the temperature block, DNA packaging and later events in viral assembly nevertheless occurred at near-normal levels. We conclude that, under our conditions, HSV DNA packaging does not require detectable levels of DNA synthesis. PMID:9525593

Infectious Agents in Atherosclerotic Cardiovascular Diseases through Oxidative Stress

PubMed Central

Di Pietro, Marisa; Filardo, Simone; Falasca, Francesca; Turriziani, Ombretta; Sessa, Rosa

2017-01-01

Accumulating evidence demonstrates that vascular oxidative stress is a critical feature of atherosclerotic process, potentially triggered by several infectious agents that are considered as risk co-factors for the atherosclerotic cardiovascular diseases (CVDs). C. pneumoniae has been shown to upregulate multiple enzymatic systems capable of producing reactive oxygen species (ROS) such as NADPH oxidase (NOX) and cyclooxygenase in vascular endothelial cells, NOX and cytochrome c oxidase in macrophages as well as nitric oxide synthase and lipoxygenase in platelets contributing to both early and late stages of atherosclerosis. P. gingivalis seems to be markedly involved in the atherosclerotic process as compared to A. actinomycetemcomitans contributing to LDL oxidation and foam cell formation. Particularly interesting is the evidence describing the NLRP3 inflammasome activation as a new molecular mechanism underlying P. gingivalis-induced oxidative stress and inflammation. Amongst viral agents, immunodeficiency virus-1 and hepatitis C virus seem to have a major role in promoting ROS production, contributing, hence, to the early stages of atherosclerosis including endothelial dysfunction and LDL oxidation. In conclusion, oxidative mechanisms activated by several infectious agents during the atherosclerotic process underlying CVDs are very complex and not well-known, remaining, thus, an attractive target for future research. PMID:29156574

Infectious Agents in Atherosclerotic Cardiovascular Diseases through Oxidative Stress.

PubMed

Di Pietro, Marisa; Filardo, Simone; Falasca, Francesca; Turriziani, Ombretta; Sessa, Rosa

2017-11-18

Accumulating evidence demonstrates that vascular oxidative stress is a critical feature of atherosclerotic process, potentially triggered by several infectious agents that are considered as risk co-factors for the atherosclerotic cardiovascular diseases (CVDs). C. pneumoniae has been shown to upregulate multiple enzymatic systems capable of producing reactive oxygen species (ROS) such as NADPH oxidase (NOX) and cyclooxygenase in vascular endothelial cells, NOX and cytochrome c oxidase in macrophages as well as nitric oxide synthase and lipoxygenase in platelets contributing to both early and late stages of atherosclerosis. P. gingivalis seems to be markedly involved in the atherosclerotic process as compared to A. actinomycetemcomitans contributing to LDL oxidation and foam cell formation. Particularly interesting is the evidence describing the NLRP3 inflammasome activation as a new molecular mechanism underlying P. gingivalis -induced oxidative stress and inflammation. Amongst viral agents, immunodeficiency virus-1 and hepatitis C virus seem to have a major role in promoting ROS production, contributing, hence, to the early stages of atherosclerosis including endothelial dysfunction and LDL oxidation. In conclusion, oxidative mechanisms activated by several infectious agents during the atherosclerotic process underlying CVDs are very complex and not well-known, remaining, thus, an attractive target for future research.

Overexpression of Promyelocytic Leukemia Protein Precludes the Dispersal of ND10 Structures and Has No Effect on Accumulation of Infectious Herpes Simplex Virus 1 or Its Proteins

PubMed Central

Lopez, Pascal; Jacob, Robert J.; Roizman, Bernard

2002-01-01

A key early event in the replication of herpes simplex virus 1 (HSV-1) is the localization of infected-cell protein no. 0 (ICP0) in nuclear structures knows as ND10 or promyelocytic leukemia oncogenic domains (PODs). This is followed by dispersal of ND10 constituents such as the promyelocytic leukemia protein (PML), CREB-binding protein (CBP), and Daxx. Numerous experiments have shown that this dispersal is mediated by ICP0. PML is thought to be the organizing structural component of ND10. To determine whether the virus targets PML because it is inimical to viral replication, telomerase-immortalized human foreskin fibroblasts and HEp-2 cells were transduced with wild-type baculovirus or a baculovirus expressing the Mr 69,000 form of PML. The transduced cultures were examined for expression and localization of PML in mock-infected and HSV-1-infected cells. The results obtained from studies of cells overexpressing PML were as follows. (i) Transduced cells accumulate large amounts of unmodified and SUMO-I-modified PML. (ii) Mock-infected cells exhibited enlarged ND10 structures containing CBP and Daxx in addition to PML. (iii) In infected cells, ICP0 colocalized with PML in ND10 early in infection, but the two proteins did not overlap or were juxtaposed in orderly structures. (iv) The enlarged ND10 structures remained intact at least until 12 h after infection and retained CBP and Daxx in addition to PML. (v) Overexpression of PML had no effect on the accumulation of viral proteins representative of α, β, or γ groups and had no effect on the accumulation of infectious virus in cells infected with wild-type virus or a mutant (R7910) from which the α0 genes had been deleted. These results indicate the following: (i) PML overexpressed in transduced cells cannot be differentiated from endogenous PML with respect to sumoylation and localization in ND10 structures. (ii) PML does not affect viral replication or the changes in the localization of ICP0 through infection. (iii) Disaggregation of ND10 structures is not an obligatory event essential for viral replication. PMID:12186918

Horizon scanning for emergence of new viruses: from constructing complex scenarios to online games.

PubMed

Gale, P; Breed, A C

2013-10-01

Horizon scanning techniques can be developed to identify novel routes and sources for the emergence of viruses in the medium to long term. Central to horizon scanning is prediction of the complex scenarios through which viruses could emerge before they occur. One approach involves 'spidergrams' in which complex scenarios are generated by combining factors randomly selected from different categories of events. Spidergrams provide a framework for how different factors could interact, irrespective of the virus, and also enable testing of combinations not previously considered but which would be 'tested' in nature by a virus. The emergence of viruses through new routes is often related to changes, for example, in environmental and social factors, and the Internet will undoubtedly be used to identify long-term trends for consideration. In addition, online games may provide horizon scanners with suggestions for new routes and strategies that could be used by emerging viruses. © 2012 Crown copyright Reproduced with the permission of the Controller of Her Majesty's Stationery Office and Animal Health and Veterinary Laboratories Agency.

Preliminary development of a live attenuated canine parvovirus vaccine from an isolate of British origin.

PubMed

Churchill, A E

1987-04-04

Canine parvovirus isolated from a case of haemorrhagic enteritis in a breeding kennel in England was passaged and cloned in cultured feline and canine cells. No significant evidence of pathogenicity was found during six serial passages of the modified virus back through young dogs. The attenuated virus was excreted by inoculated animals and spread rapidly to uninoculated animals held in contact. When high titre attenuated virus was given to the six-week-old offspring of a seropositive dam a prompt seroconversion was observed. When the attenuated virus was used as an experimental vaccine in 108 pups in an infected breeding colony a highly significant improvement was obtained in the accumulated morbidity and mortality compared with a parallel group vaccinated with modified live feline panleucopenia virus.

Characterization of a proposed dichorhavirus associated with the citrus leprosis disease and analysis of the host response.

PubMed

Cruz-Jaramillo, José Luis; Ruiz-Medrano, Roberto; Rojas-Morales, Lourdes; López-Buenfil, José Abel; Morales-Galván, Oscar; Chavarín-Palacio, Claudio; Ramírez-Pool, José Abrahán; Xoconostle-Cázares, Beatriz

2014-07-07

The causal agents of Citrus leprosis are viruses; however, extant diagnostic methods to identify them have failed to detect known viruses in orange, mandarin, lime and bitter orange trees with severe leprosis symptoms in Mexico, an important citrus producer. Using high throughput sequencing, a virus associated with citrus leprosis was identified, belonging to the proposed Dichorhavirus genus. The virus was termed Citrus Necrotic Spot Virus (CNSV) and contains two negative-strand RNA components; virions accumulate in the cytoplasm and are associated with plasmodesmata-channels interconnecting neighboring cells-suggesting a mode of spread within the plant. The present study provides insights into the nature of this pathogen and the corresponding plant response, which is likely similar to other pathogens that do not spread systemically in plants.

Visual Tracking of Plant Virus Infection and Movement Using a Reporter MYB Transcription Factor That Activates Anthocyanin Biosynthesis1[W

PubMed Central

Bedoya, Leonor C.; Martínez, Fernando; Orzáez, Diego; Daròs, José-Antonio

2012-01-01

Insertion of reporter genes into plant virus genomes is a common experimental strategy to research many aspects of the viral infection dynamics. Their numerous advantages make fluorescent proteins the markers of choice in most studies. However, the use of fluorescent proteins still has some limitations, such as the need of specialized material and facilities to detect the fluorescence. Here, we demonstrate a visual reporter marker system to track virus infection and movement through the plant. The reporter system is based on expression of Antirrhinum majus MYB-related Rosea1 (Ros1) transcription factor (220 amino acids; 25.7 kD) that activates a series of biosynthetic genes leading to accumulation of colored anthocyanins. Using two different tobacco etch potyvirus recombinant clones tagged with Ros1, we show that infected tobacco (Nicotiana tabacum) tissues turn bright red, demonstrating that in this context, the sole expression of Ros1 is sufficient to induce pigment accumulation to a level readily detectable to the naked eye. This marker system also reports viral load qualitatively and quantitatively by means of a very simple extraction process. The Ros1 marker remained stable within the potyvirus genome through successive infectious passages from plant to plant. The main limitation of this marker system is that color output will depend on each particular plant host-virus combination and must be previously tested. However, our experiments demonstrate accurate tracking of turnip mosaic potyvirus infecting Arabidopsis (Arabidopsis thaliana) and either tobacco mosaic virus or potato X virus infecting Nicotiana benthamiana, stressing the general applicability of the method. PMID:22238422

Bimolecular Complementation to Visualize Filovirus VP40-Host Complexes in Live Mammalian Cells: Toward the Identification of Budding Inhibitors

DTIC Science & Technology

2011-01-01

of virus particles; however, mechanistic details of the forma - tion, dynamics, and trafficking of these virus-host complexes in the natural...17, pp. 9173–9182, 2003. [18] T. Irie , J. M. Licata, and R. N. Harty, “Functional character- ization of Ebola virus L-domains using VSV recombinants

The paramyxovirus polymerase complex as a target for next-generation anti-paramyxovirus therapeutics

PubMed Central

Cox, Robert; Plemper, Richard K.

2015-01-01

The paramyxovirus family includes major human and animal pathogens, including measles virus, mumps virus, and human respiratory syncytial virus (RSV), as well as the emerging zoonotic Hendra and Nipah viruses. In the U.S., RSV is the leading cause of infant hospitalizations due to viral infectious disease. Despite their clinical significance, effective drugs for the improved management of paramyxovirus disease are lacking. The development of novel anti-paramyxovirus therapeutics is therefore urgently needed. Paramyxoviruses contain RNA genomes of negative polarity, necessitating a virus-encoded RNA-dependent RNA polymerase (RdRp) complex for replication and transcription. Since an equivalent enzymatic activity is absent in host cells, the RdRp complex represents an attractive druggable target, although structure-guided drug development campaigns are hampered by the lack of high-resolution RdRp crystal structures. Here, we review the current structural and functional insight into the paramyxovirus polymerase complex in conjunction with an evaluation of the mechanism of activity and developmental status of available experimental RdRp inhibitors. Our assessment spotlights the importance of the RdRp complex as a premier target for therapeutic intervention and examines how high-resolution insight into the organization of the complex will pave the path toward the structure-guided design and optimization of much-needed next-generation paramyxovirus RdRp blockers. PMID:26029193

The paramyxovirus polymerase complex as a target for next-generation anti-paramyxovirus therapeutics.

PubMed

Cox, Robert; Plemper, Richard K

2015-01-01

The paramyxovirus family includes major human and animal pathogens, including measles virus, mumps virus, and human respiratory syncytial virus (RSV), as well as the emerging zoonotic Hendra and Nipah viruses. In the U.S., RSV is the leading cause of infant hospitalizations due to viral infectious disease. Despite their clinical significance, effective drugs for the improved management of paramyxovirus disease are lacking. The development of novel anti-paramyxovirus therapeutics is therefore urgently needed. Paramyxoviruses contain RNA genomes of negative polarity, necessitating a virus-encoded RNA-dependent RNA polymerase (RdRp) complex for replication and transcription. Since an equivalent enzymatic activity is absent in host cells, the RdRp complex represents an attractive druggable target, although structure-guided drug development campaigns are hampered by the lack of high-resolution RdRp crystal structures. Here, we review the current structural and functional insight into the paramyxovirus polymerase complex in conjunction with an evaluation of the mechanism of activity and developmental status of available experimental RdRp inhibitors. Our assessment spotlights the importance of the RdRp complex as a premier target for therapeutic intervention and examines how high-resolution insight into the organization of the complex will pave the path toward the structure-guided design and optimization of much-needed next-generation paramyxovirus RdRp blockers.

Exploration of sequence space as the basis of viral RNA genome segmentation.

PubMed

Moreno, Elena; Ojosnegros, Samuel; García-Arriaza, Juan; Escarmís, Cristina; Domingo, Esteban; Perales, Celia

2014-05-06

The mechanisms of viral RNA genome segmentation are unknown. On extensive passage of foot-and-mouth disease virus in baby hamster kidney-21 cells, the virus accumulated multiple point mutations and underwent a transition akin to genome segmentation. The standard single RNA genome molecule was replaced by genomes harboring internal in-frame deletions affecting the L- or capsid-coding region. These genomes were infectious and killed cells by complementation. Here we show that the point mutations in the nonstructural protein-coding region (P2, P3) that accumulated in the standard genome before segmentation increased the relative fitness of the segmented version relative to the standard genome. Fitness increase was documented by intracellular expression of virus-coded proteins and infectious progeny production by RNAs with the internal deletions placed in the sequence context of the parental and evolved genome. The complementation activity involved several viral proteins, one of them being the leader proteinase L. Thus, a history of genetic drift with accumulation of point mutations was needed to allow a major variation in the structure of a viral genome. Thus, exploration of sequence space by a viral genome (in this case an unsegmented RNA) can reach a point of the space in which a totally different genome structure (in this case, a segmented RNA) is favored over the form that performed the exploration.

Novel aspects of defensins' involvement in virus-induced autoimmunity in the central nervous system.

PubMed

Kazakos, Evangelos I; Kountouras, Jannis; Polyzos, Stergios A; Deretzi, Georgia

2017-05-01

Recent research on re-circulation of interstitial fluid from the brain parenchyma to the periphery and its inferred importance in immune surveillance dysregulation are changing our conceptualization of the pathophysiology of virus-induced autoimmunity. In this context, it is necessary to reassess the immunomodulatory properties of human defensins that are variably expressed by cerebral microglia, astrocytes and choroid plexus epithelial cells and exhibit complex and often confounding roles in neuroinflammatory processes. Therefore, in this review we describe current contributions in this field and we propose novel hypotheses regarding the potential impact of defensin-related pathways on virus-driven autoimmune neurodegeneration. In this regard, we have previously proposed that abnormal expression of defensins by penetrating the blood-brain barrier (BBB) may contribute to the pathophysiology of Helicobacter pylori-related brain neurodegenerative disorders through variable modulations of innate and adaptive immune responses. We hereby propose that impaired expression of defensins by structural components of the BBB may impede glymphatic circulation and disrupt receptor signalling in pericytes that is essential for microvascular stability, thereby retaining blood-derived toxins and bystander activated T-cells in the brain and further impairing BBB integrity and hampering viral clearance. Autoreactive T-cell infiltrates in neuronaxonal lesions characteristic of chronic central nervous system diseases, such as multiple sclerosis, are directed against both, myelin and non-myelin, antigens the precise nature of which remains enigmatic. Inadequate expression of the autoimmune regulator (AIRE), a gene expressed in medullary thymic epithelial cells, induces the recruitment of defensin-specific T-cells. These cells may access the brain, thereby causing a decrease in defensin expression and subsequent down-regulation of CD91/LRP1-mediated clearance of amyloid-β that ultimately accumulates as protein deposits. It should be highlighted that β-amyloid brain deposits are a hallmark of many neurodegenerative diseases. During human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infections, selective elicitation of the VH1-69 antibody subfamily directed against specific, viral glycoprotein epitopes, often correlates with a higher likelihood of developing autoimmune disorders. In addition, we propose that recently described defensin affinity towards the same epitopes may prevent proper viral fusion, but at the expense of augmenting autoimmune reactivity. Moreover, in the context of complex and largely unknown symbiotic relations between host microbiota and pathogenic viruses, we propose that co-stimulation of defensin expression may modulate adaptive immune responses thereby enhancing inflammatory cascades responsible for autoimmunity. Finally, we describe current evidence that supports the hypothesis of a possible involvement of defensins in HCV-related autoimmunity. Exploring further potential implications of human defensins in virus-related autoimmunity may lead to improved strategies for the treatment of neurodegenerative disorders. Copyright © 2017 Elsevier Ltd. All rights reserved.

Structural Studies of the Parainfluenza Virus 5 Hemagglutinin-Neuraminidase Tetramer in Complex with Its Receptor, Sialyllactose

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yuan, Ping; Thompson, Thomas B.; Wurzburg, Beth A.

2010-03-08

The paramyxovirus hemagglutinin-neuraminidase (HN) functions in virus attachment to cells, cleavage of sialic acid from oligosaccharides, and stimulating membrane fusion during virus entry into cells. The structural basis for these diverse functions remains to be fully understood. We report the crystal structures of the parainfluenza virus 5 (SV5) HN and its complexes with sialic acid, the inhibitor DANA, and the receptor sialyllactose. SV5 HN shares common structural features with HN of Newcastle disease virus (NDV) and human parainfluenza 3 (HPIV3), but unlike the previously determined HN structures, the SV5 HN forms a tetramer in solution, which is thought to bemore » the physiological oligomer. The sialyllactose complex reveals intact receptor within the active site, but no major conformational changes in the protein. The SV5 HN structures do not support previously proposed models for HN action in membrane fusion and suggest alternative mechanisms by which HN may promote virus entry into cells.« less

Managing thrips and tospoviruses in tomato

USDA-ARS?s Scientific Manuscript database

Tomato spotted wilt virus and more recently emerged Tomato chlorotic spot virus and Groundnut ringspot virus are all transmitted by thrips, making managment complex. All three viruses and the thrips vector are major pests of tomato in Florida. Current management tools for these viruses and the th...

Culicoides-virus interactions: infection barriers and possible factors underlying vector competence

USDA-ARS?s Scientific Manuscript database

In the United States, Culicoides midges vector arboviruses of economic importance such as Bluetongue Virus and Epizootic Hemorrhagic Disease Virus. A limited number of studies have demonstrated the complexities of midge-virus interactions, including dynamic changes in virus titer and prevalence over...

Suppression of immunodeficiency virus-associated neural damage by the p75 neurotrophin receptor ligand, LM11A-31, in an in vitro feline model.

PubMed

Meeker, Rick B; Poulton, Winona; Feng, Wen-hai; Hudson, Lola; Longo, Frank M

2012-06-01

Feline immunodeficiency virus (FIV) infection like human immunodeficiency virus (HIV), produces systemic and central nervous system disease in its natural host, the domestic cat, that parallels the pathogenesis seen in HIV-infected humans. The ability to culture feline nervous system tissue affords the unique opportunity to directly examine interactions of infectious virus with CNS cells for the development of models and treatments that can then be translated to a natural infectious model. To explore the therapeutic potential of a new p75 neurotrophin receptor ligand, LM11A-31, we evaluated neuronal survival, neuronal damage and calcium homeostasis in cultured feline neurons following inoculation with FIV. FIV resulted in the gradual appearance of dendritic beading, pruning of processes and shrinkage of neuronal perikarya in the neurons. Astrocytes developed a more activated appearance and there was an enhanced accumulation of microglia, particularly at longer times post-inoculation. Addition of 10 nM LM11A-31, to the cultures greatly reduced or eliminated the neuronal pathology as well as the FIV effects on astrocytes and microglia. LM11A-31 also, prevented the development of delayed calcium deregulation in feline neurons exposed to conditioned medium from FIV treated macrophages. The suppression of calcium accumulation prevented the development of foci of calcium accumulation and beading in the dendrites. FIV replication was unaffected by LM11A-31. The strong neuroprotection afforded by LM11A-31 in an infectious in vitro model indicates that LM11A-31 may have excellent potential for the treatment of HIV-associated neurodegeneration.

Mutations that alter a repeated ACCA element located at the 5' end of the Potato virus X genome affect RNA accumulation.

PubMed

Park, Mi-Ri; Kwon, Sun-Jung; Choi, Hong-Soo; Hemenway, Cynthia L; Kim, Kook-Hyung

2008-08-15

The repeated ACCA or AC-rich sequence and structural (SL1) elements in the 5' non-translated region (NTR) of the Potato virus X (PVX) RNA play vital roles in the PVX life cycle by controlling translation, RNA replication, movement, and assembly. It has already been shown that the repeated ACCA or AC-rich sequence affect both gRNA and sgRNA accumulation, while not affecting minus-strand RNA accumulation, and are also required for host protein binding. The functional significance of the repeated ACCA sequence elements in the 5' NTR region was investigated by analyzing the effects of deletion and site-directed mutations on PVX replication in Nicotiana benthamiana plants and NT1 protoplasts. Substitution (ACCA into AAAA or UUUU) mutations introduced in the first (nt 10-13) element in the 5' NTR of the PVX RNA significantly affected viral replication, while mutations introduced in the second (nt 17-20) and third (nt 20-23) elements did not. The fourth (nt 29-32) ACCA element weakly affected virus replication, whereas mutations in the fifth (nt 38-41) significantly reduced virus replication due to the structure disruption of SL1 by AAAA and/or UUUU substitutions. Further characterization of the first ACCA element indicated that duplication of ACCA at nt 10-13 (nt 10-17, ACCAACCA) caused severe symptom development as compared to that of wild type, while deletion of the single element (nt 10-13), DeltaACCA) or tripling of this element caused reduced symptom development. Single- and double-nucleotide substitutions introduced into the first ACCA element revealed the importance of CC located at nt positions 11 and 12. Altogether, these results indicate that the first ACCA element is important for PVX replication.

Isolation of Highly Pathogenic Avian Influenza H5N1 Virus from Saker Falcons (Falco cherrug) in the Middle East

PubMed Central

Marjuki, Henju; Wernery, Ulrich; Yen, Hui-Ling; Franks, John; Seiler, Patrick; Walker, David; Krauss, Scott; Webster, Robert G.

2009-01-01

There is accumulating evidence that birds of prey are susceptible to fatal infection with highly pathogenic avian influenza (HPAI) virus. We studied the antigenic, molecular, phylogenetic, and pathogenic properties of 2 HPAI H5N1 viruses isolated from dead falcons in Saudi Arabia and Kuwait in 2005 and 2007, respectively. Phylogenetic and antigenic analyses grouped both isolates in clade 2.2 (Qinghai-like viruses). However, the viruses appeared to have spread westward via different flyways. It remains unknown how these viruses spread so rapidly from Qinghai after the 2005 outbreak and how they were introduced into falcons in these two countries. The H5N1 outbreaks in the Middle East are believed by some to be mediated by wild migratory birds. However, sporting falcons may be at additional risk from the illegal import of live quail to feed them. PMID:20148178

Hantavirus pulmonary syndrome.

PubMed

Macneil, Adam; Nichol, Stuart T; Spiropoulou, Christina F

2011-12-01

Hantavirus pulmonary syndrome (HPS) is a severe disease characterized by a rapid onset of pulmonary edema followed by respiratory failure and cardiogenic shock. The HPS associated viruses are members of the genus Hantavirus, family Bunyaviridae. Hantaviruses have a worldwide distribution and are broadly split into the New World hantaviruses, which includes those causing HPS, and the Old World hantaviruses [including the prototype Hantaan virus (HTNV)], which are associated with a different disease, hemorrhagic fever with renal syndrome (HFRS). Sin Nombre virus (SNV) and Andes virus (ANDV) are the most common causes of HPS in North and South America, respectively. Case fatality of HPS is approximately 40%. Pathogenic New World hantaviruses infect the lung microvascular endothelium without causing any virus induced cytopathic effect. However, virus infection results in microvascular leakage, which is the hallmark of HPS. This article briefly reviews the knowledge on HPS-associated hantaviruses accumulated since their discovery, less than 20 years ago. Published by Elsevier B.V.

Functional diversification upon leader protease domain duplication in the Citrus tristeza virus genome: Role of RNA sequences and the encoded proteins.

PubMed

Kang, Sung-Hwan; Atallah, Osama O; Sun, Yong-Duo; Folimonova, Svetlana Y

2018-01-15

Viruses from the family Closteroviridae show an example of intra-genome duplications of more than one gene. In addition to the hallmark coat protein gene duplication, several members possess a tandem duplication of papain-like leader proteases. In this study, we demonstrate that domains encoding the L1 and L2 proteases in the Citrus tristeza virus genome underwent a significant functional divergence at the RNA and protein levels. We show that the L1 protease is crucial for viral accumulation and establishment of initial infection, whereas its coding region is vital for virus transport. On the other hand, the second protease is indispensable for virus infection of its natural citrus host, suggesting that L2 has evolved an important adaptive function that mediates virus interaction with the woody host. Copyright © 2017 Elsevier Inc. All rights reserved.

A Viral Pilot for HCMV Navigation?

PubMed Central

Adler, Barbara

2015-01-01

gH/gL virion envelope glycoprotein complexes of herpesviruses serve as entry complexes and mediate viral cell tropism. By binding additional viral proteins, gH/gL forms multimeric complexes which bind to specific host cell receptors. Both Epstein–Barr virus (EBV) and human cytomegalovirus (HCMV) express alternative multimeric gH/gL complexes. Relative amounts of these alternative complexes in the viral envelope determine which host cells are preferentially infected. Host cells of EBV can modulate the gH/gL complex complement of progeny viruses by cell type-dependent degradation of one of the associating proteins. Host cells of HCMV modulate the tropism of their virus progenies by releasing or not releasing virus populations with a specific gH/gL complex complement out of a heterogeneous pool of virions. The group of Jeremy Kamil has recently shown that the HCMV ER-resident protein UL148 controls integration of one of the HCMV gH/gL complexes into virions and thus creates a pool of virions which can be routed by different host cells. This first mechanistic insight into regulation of the gH/gL complex complement of HCMV progenies presents UL148 as a pilot candidate for HCMV navigation in its infected host. PMID:26184287

Self-assembled virus-membrane complexes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Lihua; Liang, Hongjun; Angelini, Thomas

Anionic polyelectrolytes and cationic lipid membranes can self-assemble into lamellar structures ranging from alternating layers of membranes and polyelectrolytes to 'missing layer' superlattice structures. We show that these structural differences can be understood in terms of the surface-charge-density mismatch between the polyelectrolyte and membrane components by examining complexes between cationic membranes and highly charged M13 viruses, a system that allowed us to vary the polyelectrolyte diameter independently of the charge density. Such virus-membrane complexes have pore sizes that are about ten times larger in area than DNA-membrane complexes, and can be used to package and organize large functional molecules; correlatedmore » arrays of Ru(bpy){sub 3}{sup 2+} macroionic dyes have been directly observed within the virus-membrane complexes using an electron-density reconstruction. These observations elucidate fundamental design rules for rational control of self-assembled polyelectrolyte-membrane structures, which have applications ranging from non-viral gene therapy to biomolecular templates for nanofabrication.« less

Topology of evolving, mutagenized viral populations: quasispecies expansion, compression, and operation of negative selection

PubMed Central

2008-01-01

Background The molecular events and evolutionary forces underlying lethal mutagenesis of virus (or virus extinction through an excess of mutations) are not well understood. Here we apply for the first time phylogenetic methods and Partition Analysis of Quasispecies (PAQ) to monitor genetic distances and intra-population structures of mutant spectra of foot-and-mouth disease virus (FMDV) quasispecies subjected to mutagenesis by base and nucleoside analogues. Results Phylogenetic and PAQ analyses have revealed a highly dynamic variation of intrapopulation diversity of FMDV quasispecies. The population diversity first suffers striking expansions in the presence of mutagens and then compressions either when the presence of the mutagenic analogue was discontinued or when a mutation that decreased sensitivity to a mutagen was selected. The pattern of mutations found in the populations was in agreement with the behavior of the corresponding nucleotide analogues with FMDV in vitro. Mutations accumulated at preferred genomic sites, and dn/ds ratios indicate the operation of negative (or purifying) selection in populations subjected to mutagenesis. No evidence of unusually elevated genetic distances has been obtained for FMDV populations approaching extinction. Conclusion Phylogenetic and PAQ analysis provide adequate procedures to describe the evolution of viral sequences subjected to lethal mutagenesis. These methods define the changes of intra-population structure more precisely than mutation frequencies and Shannon entropies. PAQ is very sensitive to variations of intrapopulation genetic distances. Strong negative (or purifying) selection operates in FMDV populations subjected to enhanced mutagenesis. The quantifications provide evidence that extinction does not imply unusual increases of intrapopulation complexity, in support of the lethal defection model of virus extinction. PMID:18637173

Electron microscopy and antigenic studies of uncharacterized viruses. I. Evidence suggesting the placement of viruses in families Arenaviridae, Paramyxoviridae, or Poxviridae.

PubMed

Zeller, H G; Karabatsos, N; Calisher, C H; Digoutte, J P; Murphy, F A; Shope, R E

1989-01-01

During approximately 35 years, investigators in various laboratories studying arbovirus ecology and epidemiology accumulated many virus isolates, more than 60 of which were not characterized or placed in taxa. By a combination of electron microscopic and antigenic studies we collected information sufficient to provisionally classify 60 isolates. Electron microscopic observations suggest that 20 are members of the virus family Bunyaviridae, 20 Rhabdoviridae, 14 Reoviridae, one Togaviridae, one Paramyxoviridae (Mapuera virus, from a bat), and one Poxviridae (Yoka virus, from mosquitoes). Serologic studies provided evidence sufficient to place some of these viruses in recognized antigenic groups, within families and genera, and to establish new antigenic groups and taxa for others. Three viruses were found to have morphologic and morphogenetic characteristics consistent with those of members of the family Arenaviridae: Quaranfil virus, a human pathogen, Johnston Atoll virus, isolated from birds and ticks, and Araguari virus, isolated from an opossum. This, the first in a series of three papers, described methods used for these investigations and also presents descriptions of viruses provisionally placed in the families Arenaviridae, Paramyxoviridae, or Poxviridae. Descriptions of viruses provisionally placed in families Bunyaviridae and Reoviridae are published in the second and third papers, respectively. Viruses of the family Rhabdoviridae have been described separately.

Induction of cell-cell fusion by ectromelia virus is not inhibited by its fusion inhibitory complex.

PubMed

Erez, Noam; Paran, Nir; Maik-Rachline, Galia; Politi, Boaz; Israely, Tomer; Schnider, Paula; Fuchs, Pinhas; Melamed, Sharon; Lustig, Shlomo

2009-09-29

Ectromelia virus, a member of the Orthopox genus, is the causative agent of the highly infectious mousepox disease. Previous studies have shown that different poxviruses induce cell-cell fusion which is manifested by the formation of multinucleated-giant cells (polykaryocytes). This phenomenon has been widely studied with vaccinia virus in conditions which require artificial acidification of the medium. We show that Ectromelia virus induces cell-cell fusion under neutral pH conditions and requires the presence of a sufficient amount of viral particles on the plasma membrane of infected cells. This could be achieved by infection with a replicating virus and its propagation in infected cells (fusion "from within") or by infection with a high amount of virus particles per cell (fusion "from without"). Inhibition of virus maturation or inhibition of virus transport on microtubules towards the plasma membrane resulted in a complete inhibition of syncytia formation. We show that in contrast to vaccinia virus, Ectromelia virus induces cell-cell fusion irrespectively of its hemagglutination properties and cell-surface expression of the orthologs of the fusion inhibitory complex, A56 and K2. Additionally, cell-cell fusion was also detected in mice lungs following lethal respiratory infection. Ectromelia virus induces spontaneous cell-cell fusion in-vitro and in-vivo although expressing an A56/K2 fusion inhibitory complex. This syncytia formation property cannot be attributed to the 37 amino acid deletion in ECTV A56.

Membrane and inclusion body targeting of lyssavirus matrix proteins.

PubMed

Pollin, Reiko; Granzow, Harald; Köllner, Bernd; Conzelmann, Karl-Klaus; Finke, Stefan

2013-02-01

Lyssavirus matrix proteins (M) support virus budding and have accessory functions that may contribute to host cell manipulation and adaptation to specific hosts. Here, we show that rabies virus (RABV) and European Bat Lyssavirus Type 1 (EBLV-1) M proteins differ in targeting and accumulation at cellular membranes. In contrast to RABV M, EBLV-1 M expressed from authentic EBLV-1 or chimeric RABV accumulated at the Golgi apparatus. Chimeric M proteins revealed that Golgi association depends on the integrity of the entire EBLV-1 M protein. Since RABV and EBLV-1 M differ in the use of cellular membranes for particle formation, differential membrane targeting and transport of M might determine the site of virus production. Moreover, both RABV and EBLV-1 M were for the first time detected within the nucleus and in Negri body-like inclusions bodies. Whereas nuclear M may imply hitherto unknown functions of lyssavirus M in host cell manipulation, the presence of M in inclusion bodies may correlate with regulatory functions of M in virus RNA synthesis. The data strongly support a model in which targeting of lyssavirus M proteins to distinctintracellular sites is a key determinant of diverse features in lyssavirus replication, host adaptation and pathogenesis. © 2012 Blackwell Publishing Ltd.

Comparative analysis among the small RNA populations of source, sink and conductive tissues in two different plant-virus pathosystems.

PubMed

Herranz, Mari Carmen; Navarro, Jose Antonio; Sommen, Evelien; Pallas, Vicente

2015-02-22

In plants, RNA silencing plays a fundamental role as defence mechanism against viruses. During last years deep-sequencing technology has allowed to analyze the sRNA profile of a large variety of virus-infected tissues. Nevertheless, the majority of these studies have been restricted to a unique tissue and no comparative analysis between phloem and source/sink tissues has been conducted. In the present work, we compared the sRNA populations of source, sink and conductive (phloem) tissues in two different plant virus pathosystems. We chose two cucurbit species infected with two viruses very different in genome organization and replication strategy; Melon necrotic spot virus (MNSV) and Prunus necrotic ringspot virus (PNRSV). Our findings showed, in both systems, an increase of the 21-nt total sRNAs together with a decrease of those with a size of 24-nt in all the infected tissues, except for the phloem where the ratio of 21/24-nt sRNA species remained constant. Comparing the vsRNAs, both PNRSV- and MNSV-infected plants share the same vsRNA size distribution in all the analyzed tissues. Similar accumulation levels of sense and antisense vsRNAs were observed in both systems except for roots that showed a prevalence of (+) vsRNAs in both pathosystems. Additionally, the presence of overrepresented discrete sites along the viral genome, hot spots, were identified and validated by stem-loop RT-PCR. Despite that in PNRSV-infected plants the presence of vsRNAs was scarce both viruses modulated the host sRNA profile. We compare for the first time the sRNA profile of four different tissues, including source, sink and conductive (phloem) tissues, in two plant-virus pathosystems. Our results indicate that antiviral silencing machinery in melon and cucumber acts mainly through DCL4. Upon infection, the total sRNA pattern in phloem remains unchanged in contrast to the rest of the analyzed tissues indicating a certain tissue-tropism to this polulation. Independently of the accumulation level of the vsRNAs both viruses were able to modulate the host sRNA pattern.

Quantifying the risk of pandemic influenza virus evolution by mutation and re-assortment.

PubMed

Reperant, Leslie A; Grenfell, Bryan T; Osterhaus, Albert D M E

2015-12-08

Large outbreaks of zoonotic influenza A virus (IAV) infections may presage an influenza pandemic. However, the likelihood that an airborne-transmissible variant evolves upon zoonotic infection or co-infection with zoonotic and seasonal IAVs remains poorly understood, as does the relative importance of accumulating mutations versus re-assortment in this process. Using discrete-time probabilistic models, we determined quantitative probability ranges that transmissible variants with 1-5 mutations and transmissible re-assortants evolve after a given number of zoonotic IAV infections. The systematic exploration of a large population of model parameter values was designed to account for uncertainty and variability in influenza virus infection, epidemiological and evolutionary processes. The models suggested that immunocompromised individuals are at high risk of generating IAV variants with pandemic potential by accumulation of mutations. Yet, both immunocompetent and immunocompromised individuals could generate high viral loads of single and double mutants, which may facilitate their onward transmission and the subsequent accumulation of additional 1-2 mutations in newly-infected individuals. This may result in the evolution of a full transmissible genotype along short chains of contact transmission. Although co-infection with zoonotic and seasonal IAVs was shown to be a rare event, it consistently resulted in high viral loads of re-assortants, which may facilitate their onward transmission among humans. The prevention or limitation of zoonotic IAV infection in immunocompromised and contact individuals, including health care workers, as well as vaccination against seasonal IAVs-limiting the risk of co-infection-should be considered fundamental tools to thwart the evolution of a novel pandemic IAV by accumulation of mutations and re-assortment. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Environmental and Biological Factors Influencing Culex pipiens quinquefasciatus Say (Diptera: Culicidae) Vector Competence for Saint Louis Encephalitis Virus

PubMed Central

Richards, Stephanie L.; Lord, Cynthia C.; Pesko, Kendra; Tabachnick, Walter J.

2009-01-01

Complex interactions between environmental and biological factors influence the susceptibility of Culex pipiens quinquefasciatus to St. Louis encephalitis virus and could affect the epidemiology of virus transmission. Similar interactions could have epidemiologic implications for other vector-virus systems. We conducted an experiment to examine four such factors in combination: mosquito age, extrinsic incubation temperature (EIT), virus dose, and colony. The proportion of mosquitoes with body infections or disseminated infections varied between colonies, and was dependant on age, EIT, and dose. We also show that the probability of a body or leg infection interacted in complex ways between colonies, ages, EITs, and doses. The complex interactive effects of environmental and biological factors must be taken into account for studies of vector competence and epidemiology, especially when laboratory studies are used to generalize to natural transmission dynamics where the extent of variation is largely unknown. PMID:19635881

Environmental and biological factors influencing Culex pipiens quinquefasciatus Say (Diptera: Culicidae) vector competence for Saint Louis encephalitis virus.

PubMed

Richards, Stephanie L; Lord, Cynthia C; Pesko, Kendra; Tabachnick, Walter J

2009-08-01

Complex interactions between environmental and biological factors influence the susceptibility of Culex pipiens quinquefasciatus to St. Louis encephalitis virus and could affect the epidemiology of virus transmission. Similar interactions could have epidemiologic implications for other vector-virus systems. We conducted an experiment to examine four such factors in combination: mosquito age, extrinsic incubation temperature (EIT), virus dose, and colony. The proportion of mosquitoes with body infections or disseminated infections varied between colonies, and was dependant on age, EIT, and dose. We also show that the probability of a body or leg infection interacted in complex ways between colonies, ages, EITs, and doses. The complex interactive effects of environmental and biological factors must be taken into account for studies of vector competence and epidemiology, especially when laboratory studies are used to generalize to natural transmission dynamics where the extent of variation is largely unknown.

Digital sensing and sizing of vesicular stomatitis virus pseudotypes in complex media: a model for Ebola and Marburg detection.

PubMed

Daaboul, George G; Lopez, Carlos A; Chinnala, Jyothsna; Goldberg, Bennett B; Connor, John H; Ünlü, M Selim

2014-06-24

Rapid, sensitive, and direct label-free capture and characterization of nanoparticles from complex media such as blood or serum will broadly impact medicine and the life sciences. We demonstrate identification of virus particles in complex samples for replication-competent wild-type vesicular stomatitis virus (VSV), defective VSV, and Ebola- and Marburg-pseudotyped VSV with high sensitivity and specificity. Size discrimination of the imaged nanoparticles (virions) allows differentiation between modified viruses having different genome lengths and facilitates a reduction in the counting of nonspecifically bound particles to achieve a limit-of-detection (LOD) of 5 × 10(3) pfu/mL for the Ebola and Marburg VSV pseudotypes. We demonstrate the simultaneous detection of multiple viruses in a single sample (composed of serum or whole blood) for screening applications and uncompromised detection capabilities in samples contaminated with high levels of bacteria. By employing affinity-based capture, size discrimination, and a "digital" detection scheme to count single virus particles, we show that a robust and sensitive virus/nanoparticle sensing assay can be established for targets in complex samples. The nanoparticle microscopy system is termed the Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) and is capable of high-throughput and rapid sizing of large numbers of biological nanoparticles on an antibody microarray for research and diagnostic applications.

The eIF4AIII RNA helicase is a critical determinant of human cytomegalovirus replication

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ziehr, Ben; Lenarcic, Erik; Cecil, Chad

Human cytomegalovirus (HCMV) was recently shown to encode a large number of spliced mRNAs. While the nuclear export of unspliced viral transcripts has been extensively studied, the role of host mRNA export factors in HCMV mRNA trafficking remains poorly defined. We found that the eIF4AIII RNA helicase, a component of the exon junction complex, was necessary for efficient virus replication. Depletion of eIF4AIII limited viral DNA accumulation, export of viral mRNAs from the nucleus, and the production of progeny virus. However eIF4AIII was dispensable for the association of viral transcripts with ribosomes. We found that pateamine A, a natural compoundmore » that inhibits both eIF4AI/II and eIF4AIII, has potent antiviral activity and inhibits HCMV replication throughout the virus lytic cycle. Our results demonstrate that eIF4AIII is required for efficient HCMV replication, and suggest that eIF4A family helicases may be a new class of targets for the development of host-directed antiviral therapeutics. - Highlights: • The host eIF4AIII RNA helicase is required for efficient HCMV replication. • Depleting eIF4AIII inhibited the nuclear export of HCMV mRNAs. • HCMV mRNAs did not require eIF4AIII to associate with polyribosomes. • The eIF4A family helicases may be new targets for host-directed antiviral drugs.« less

A DNA replicon system for rapid high-level production of virus-like particles in plants.

PubMed

Huang, Zhong; Chen, Qiang; Hjelm, Brooke; Arntzen, Charles; Mason, Hugh

2009-07-01

Recombinant virus-like particles (VLPs) represent a safe and effective vaccine strategy. We previously described a stable transgenic plant system for inexpensive production and oral delivery of VLP vaccines. However, the relatively low-level antigen accumulation and long-time frame to produce transgenic plants are the two major roadblocks in the practical development of plant-based VLP production. In this article, we describe the optimization of geminivirus-derived DNA replicon vectors for rapid, high-yield plant-based production of VLPs. Co-delivery of bean yellow dwarf virus (BeYDV)-derived vector and Rep/RepA-supplying vector by agroinfiltration of Nicotiana benthamiana leaves resulted in efficient replicon amplification and robust protein production within 5 days. Co-expression of the P19 protein of tomato bush stunt virus, a gene silencing inhibitor, further enhanced VLP accumulation by stabilizing the mRNA. With this system, hepatitis B core antigen (HBc) and Norwalk virus capsid protein (NVCP) were produced at 0.80 and 0.34 mg/g leaf fresh weight, respectively. Sedimentation analysis and electron microscopy of transiently expressed antigens verified the efficient assembly of VLPs. Furthermore, a single replicon vector containing a built-in Rep/RepA cassette without P19 drove protein expression at similar levels as the three-component system. These results demonstrate the advantages of fast and high-level production of VLP-based vaccines using the BeYDV-derived DNA replicon system for transient expression in plants. (c) 2009 Wiley Periodicals, Inc.

A DNA replicon system for rapid high-level production of virus-like particles in plants

PubMed Central

Huang, Zhong; Chen, Qiang; Hjelm, Brooke; Arntzen, Charles

2009-01-01

Recombinant virus-like particles (VLPs) represent a safe and effective vaccine strategy. We previously described a stable transgenic plant system for inexpensive production and oral delivery of VLP vaccines. However, the relatively low level antigen accumulation and long time frame to produce transgenic plants are the two major roadblocks in the practical development of plant-based VLP production. In this paper, we describe the optimization of geminivirus-derived DNA replicon vectors for rapid, high-yield plant-based production of VLPs. Co-delivery of bean yellow dwarf virus (BeYDV)-derived vector and Rep/RepA-supplying vector by agroinfiltration of Nicotiana benthamiana leaves resulted in efficient replicon amplification and robust protein production within five days. Co-expression of the P19 protein of tomato bush stunt virus, a gene silencing inhibitor, further enhanced VLP accumulation by stabilizing the mRNA. With this system, hepatitis B core antigen (HBc) and Norwalk virus capsid protein (NVCP) were produced at 0.80 and 0.34 mg/g leaf fresh weight, respectively. Sedimentation analysis and electron microscopy of transiently expressed antigens verified the efficient assembly of VLPs. Furthermore, a single replicon vector containing a built-in Rep/RepA cassette without p19 drove protein expression at similar levels as the three-component system. These results demonstrate the advantages of fast and high-level production of VLP-based vaccines using the BeYDV-derived DNA replicon system for transient expression in plants. PMID:19309755

TIM-family proteins inhibit HIV-1 release

PubMed Central

Li, Minghua; Ablan, Sherimay D.; Miao, Chunhui; Zheng, Yi-Min; Fuller, Matthew S.; Rennert, Paul D.; Maury, Wendy; Johnson, Marc C.; Freed, Eric O.; Liu, Shan-Lu

2014-01-01

Accumulating evidence indicates that T-cell immunoglobulin (Ig) and mucin domain (TIM) proteins play critical roles in viral infections. Herein, we report that the TIM-family proteins strongly inhibit HIV-1 release, resulting in diminished viral production and replication. Expression of TIM-1 causes HIV-1 Gag and mature viral particles to accumulate on the plasma membrane. Mutation of the phosphatidylserine (PS) binding sites of TIM-1 abolishes its ability to block HIV-1 release. TIM-1, but to a much lesser extent PS-binding deficient mutants, induces PS flipping onto the cell surface; TIM-1 is also found to be incorporated into HIV-1 virions. Importantly, TIM-1 inhibits HIV-1 replication in CD4-positive Jurkat cells, despite its capability of up-regulating CD4 and promoting HIV-1 entry. In addition to TIM-1, TIM-3 and TIM-4 also block the release of HIV-1, as well as that of murine leukemia virus (MLV) and Ebola virus (EBOV); knockdown of TIM-3 in differentiated monocyte-derived macrophages (MDMs) enhances HIV-1 production. The inhibitory effects of TIM-family proteins on virus release are extended to other PS receptors, such as Axl and RAGE. Overall, our study uncovers a novel ability of TIM-family proteins to block the release of HIV-1 and other viruses by interaction with virion- and cell-associated PS. Our work provides new insights into a virus-cell interaction that is mediated by TIMs and PS receptors. PMID:25136083

Generation of transgenic papaya with double resistance to Papaya ringspot virus and Papaya leaf-distortion mosaic virus.

PubMed

Kung, Yi-Jung; Bau, Huey-Jiunn; Wu, Yi-Ling; Huang, Chiung-Huei; Chen, Tsui-Miao; Yeh, Shyi-Dong

2009-11-01

During the field tests of coat protein (CP)-transgenic papaya lines resistant to Papaya ringspot virus (PRSV), another Potyvirus sp., Papaya leaf-distortion mosaic virus (PLDMV), appeared as an emerging threat to the transgenic papaya. In this investigation, an untranslatable chimeric construct containing the truncated CP coding region of the PLDMV P-TW-WF isolate and the truncated CP coding region with the complete 3' untranslated region of PRSV YK isolate was transferred into papaya (Carica papaya cv. Thailand) via Agrobacterium-mediated transformation to generate transgenic plants with resistance to PLDMV and PRSV. Seventy-five transgenic lines were obtained and challenged with PRSV YK or PLDMV P-TW-WF by mechanical inoculation under greenhouse conditions. Thirty-eight transgenic lines showing no symptoms 1 month after inoculation were regarded as highly resistant lines. Southern and Northern analyses revealed that four weakly resistant lines have one or two inserts of the construct and accumulate detectable amounts of transgene transcript, whereas nine resistant lines contain two or three inserts without significant accumulation of transgene transcript. The results indicated that double virus resistance in transgenic lines resulted from double or more copies of the insert through the mechanism of RNA-mediated posttranscriptional gene silencing. Furthermore, three of nine resistant lines showed high levels of resistance to heterologous PRSV strains originating from Hawaii, Thailand, and Mexico. Our transgenic lines have great potential for controlling a number of PRSV strains and PLDMV in Taiwan and elsewhere.

Imbalanced Oxidative Stress Causes Chlamydial Persistence during Non-Productive Human Herpes Virus Co-Infection

PubMed Central

Prusty, Bhupesh K.; Böhme, Linda; Bergmann, Birgit; Siegl, Christine; Krause, Eva; Mehlitz, Adrian; Rudel, Thomas

2012-01-01

Both human herpes viruses and Chlamydia are highly prevalent in the human population and are detected together in different human disorders. Here, we demonstrate that co-infection with human herpes virus 6 (HHV6) interferes with the developmental cycle of C. trachomatis and induces persistence. Induction of chlamydial persistence by HHV6 is independent of productive virus infection, but requires the interaction and uptake of the virus by the host cell. On the other hand, viral uptake is strongly promoted under co-infection conditions. Host cell glutathione reductase activity was suppressed by HHV6 causing NADPH accumulation, decreased formation of reduced glutathione and increased oxidative stress. Prevention of oxidative stress restored infectivity of Chlamydia after HHV6-induced persistence. We show that co-infection with Herpes simplex virus 1 or human Cytomegalovirus also induces chlamydial persistence by a similar mechanism suggesting that Chlamydia -human herpes virus co-infections are evolutionary shaped interactions with a thus far unrecognized broad significance. PMID:23077614

Effect of caffeine on the ultraviolet light induction of SV40 virus from transformed hamster cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zamansky, G.B.; Kleinman, L.F.; Little, J.B.

1976-01-01

The effect of caffeine on the uv light induction of SV40 virus from two transformed hamster cell lines heterogeneous for the induction of infectious virus was studied. The amount of virus induced was significantly increased in both cell lines when exposure to uv light was followed by treatment with caffeine. Caffeine in the absence of uv irradiation did not stimulate virus induction, nor did it stimulate SV40 replication in a lytic infection. There was an apparent difference in the concentrations of caffeine which maximally stimulated SV40 virus induction in the two cell lines. This effect could not be explained bymore » differences in cell survival after exposure to uv light and caffeine. Since caffeine is known to cause the accumulation of gaps formed in DNA during postreplication repair of uv-irradiated rodent cells, our results support the hypothesis that the formation of gaps or breaks in DNA is an important early step in virus induction.« less

Tick-borne flavivirus infection in Ixodes scapularis larvae: development of a novel method for synchronous viral infection of ticks

PubMed Central

Mitzel, Dana N.; Wolfinbarger, James B.; Daniel Long, R.; Masnick, Max; Best, Sonja M.; Bloom, Marshall E.

2007-01-01

Following a bite from an infected tick, tick-borne flaviviruses cause encephalitis, meningitis and hemorrhagic fever in humans. Although these viruses spend most of their time in the tick, little is known regarding the virus-vector interactions. We developed a simple method for synchronously infecting Ixodes scapularis larvae with Langat virus (LGTV) by immersion in media containing the virus. This technique resulted in approximately 96% of ticks becoming infected. LGTV infection and replication were demonstrated by both viral antigen expression and the accumulation of viral RNA. Furthermore, ticks transmitted LGTV to 100% of the mice and maintained the virus through molting into the next life stage. This technique circumvents limitations present in the current methods by mimicking the natural route of infection and by using attenuated virus strains to infect ticks; thereby, making this technique a powerful tool to study both virus and tick determinants of replication, pathogenesis and transmission. PMID:17490700

Subcellular distribution and life cycle of Epstein-Barr virus in keratinocytes of oral hairy leukoplakia.

PubMed Central

Rabanus, J. P.; Greenspan, D.; Petersen, V.; Leser, U.; Wolf, H.; Greenspan, J. S.

1991-01-01

The authors investigated the life cycle of Epstein-Barr virus (EBV) in keratinocytes of oral hairy leukoplakia by combining immunohistochemistry. DNA in situ hybridization, and lectin histochemistry with electron microscopy. Diffuse-staining components of the EBV early antigen complex (EA-D), EBV 150-kd capsid antigen (VCA), EBV membrane antigen (gp350/220), and double-stranded DNA were labeled with monoclonal antibodies. An EBV-DNA probe was used to locate EBV DNA. Wheat-germ agglutinin (WGA) was employed to distinguish Golgi-associated compartments. The authors found EBV proteins and EBV DNA only in keratinocytes with apparent viral assembly. In situ hybridization showed EBV DNA in free corelike material and in electron-dense cores of mature nucleocapsids. Monoclonal antibodies to nonspecific double-stranded DNA attached to the same structures and to marginated chromatin. Components of EA-D were dispersed throughout the nuclei but accumulated near condensed chromatin and in 'punched-out' regions of the chromatin. Epstein-Barr virus 150-kd capsid antigen was found only in the nuclei, where it appeared preferentially on mature nucleocapsids. As yet unexplained arrays of intranuclear particles that remained unlabeled with all EBV-specific probes reacted intensely with an antiserum against common papillomavirus antigen. Gp350/220 was detectable in various cellular membrane compartments and was highly concentrated on EBV envelopes in peripheral Golgi-associated secretory vesicles. It was less abundant on the extracellular EBV, indicating that viral membrane antigen partly dissociates from the mature virus. Combined lectin-binding histochemistry and electron microscopy demonstrated for the first time that EBV is processed in the Golgi apparatus, which eventually releases the virus by fusion with the plasma membrane. These results provide insight into the biologic events that occur during complete EBV replication in vivo. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 PMID:1649554

In vivo virulence of MHC-adapted AIDS virus serially-passaged through MHC-mismatched hosts

PubMed Central

Yamamoto, Hiroyuki; Ishii, Hiroshi; Matsuoka, Saori; Mizuta, Kazuta; Sakawaki, Hiromi; Miura, Tomoyuki; Naruse, Taeko K.; Kimura, Akinori

2017-01-01

CD8+ T-cell responses exert strong suppressive pressure on HIV replication and select for viral escape mutations. Some of these major histocompatibility complex class I (MHC-I)-associated mutations result in reduction of in vitro viral replicative capacity. While these mutations can revert after viral transmission to MHC-I-disparate hosts, recent studies have suggested that these MHC-I-associated mutations accumulate in populations and make viruses less pathogenic in vitro. Here, we directly show an increase in the in vivo virulence of an MHC-I-adapted virus serially-passaged through MHC-I-mismatched hosts in a macaque AIDS model despite a reduction in in vitro viral fitness. The first passage simian immunodeficiency virus (1pSIV) obtained 1 year after SIVmac239 infection in a macaque possessing a protective MHC-I haplotype 90-120-Ia was transmitted into 90-120-Ia- macaques, whose plasma 1 year post-infection was transmitted into other 90-120-Ia- macaques to obtain the third passage SIV (3pSIV). Most of the 90-120-Ia-associated mutations selected in 1pSIV did not revert even in 3pSIV. 3pSIV showed lower in vitro viral fitness but induced persistent viremia in 90-120-Ia- macaques. Remarkably, 3pSIV infection in 90-120-Ia+ macaques resulted in significantly higher viral loads and reduced survival compared to wild-type SIVmac239. These results indicate that MHC-I-adapted SIVs serially-transmitted through MHC-I-mismatched hosts can have higher virulence in MHC-I-matched hosts despite their lower in vitro viral fitness. This study suggests that multiply-passaged HIVs could result in loss of HIV-specific CD8+ T cell responses in human populations and the in vivo pathogenic potential of these escaped viruses may be enhanced. PMID:28931083

Natural Killer Cell Recruitment to the Lung During Influenza A Virus Infection Is Dependent on CXCR3, CCR5, and Virus Exposure Dose

PubMed Central

Carlin, Lindsey E.; Hemann, Emily A.; Zacharias, Zeb R.; Heusel, Jonathan W.; Legge, Kevin L.

2018-01-01

Natural killer (NK) cells are vital components of the antiviral immune response, but their contributions in defense against influenza A virus (IAV) are not well understood. To better understand NK cell responses during IAV infections, we examined the magnitude, kinetics, and contribution of NK cells to immunity and protection during high- and low-dose IAV infections. Herein, we demonstrate an increased accumulation of NK cells in the lung in high-dose vs. low-dose infections. In part, this increase is due to the local proliferation of pulmonary NK cells. However, the majority of NK cell accumulation within the lungs and airways during an IAV infection is due to recruitment that is partially dependent upon CXCR3 and CCR5, respectively. Therefore, altogether, our results demonstrate that NK cells are actively recruited to the lungs and airways during IAV infection and that the magnitude of the recruitment may relate to the inflammatory environment found within the tissues during high- and low-dose IAV infections. PMID:29719539

SR proteins SRp20 and 9G8 contribute to efficient export of herpes simplex virus 1 mRNAs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Escudero-Paunetto, Laurimar; Li Ling; Hernandez, Felicia P.

2010-06-05

Herpes simplex virus 1 (HSV-1) mRNAs are exported to the cytoplasm through the export receptor TAP/NFX1. HSV-1 multifunctional protein ICP27 interacts with TAP/NXF1, binds viral RNAs, and is required for efficient viral RNA export. In ICP27 mutant infections, viral RNA export is reduced but not ablated, indicating that other export adaptors can aid in viral RNA export. Export adaptor protein Aly/REF is recruited to viral replication compartments, however, Aly/REF knockdown has little effect on viral RNA export. SR proteins SRp20 and 9G8 interact with TAP/NXF1 and mediate export of some cellular RNAs. We report that siRNA knockdown of SRp20 ormore » 9G8 resulted in about a 10 fold decrease in virus yields and in nuclear accumulation of poly(A+) RNA. In infected cells depleted of SRp20, newly transcribed Bromouridine-labeled RNA also accumulated in the nucleus. We conclude that SRp20 and 9G8 contribute to HSV-1 RNA export.« less

Molecular characterization of two sweepoviruses from China and evaluation of the infectivity of cloned SPLCV-JS in Nicotiana benthamiana.

PubMed

Bi, Huiping; Zhang, Peng

2012-03-01

Sweepoviruses are important begomoviruses that infect Ipomoea plants worldwide and cause sweet potato yield losses and cultivar decline. Two sweepoviruses, sweet potato leaf curl virus-Jiangsu (SPLCV-JS) and sweet potato leaf curl China virus-Zhejiang (SPLCCNV-ZJ), were cloned from diseased sweet potato plants collected in the Jiangsu and Zhejiang provinces of China. Sequence characterization and phylogenetic analysis demonstrated that both are typical monopartite begomoviruses and have close relationships to several reported SPLCV and SPLCCNV isolates, respectively, from Asian countries. Analysis of the protein alignments and subcellular localizations of the six SPLCV-JS proteins was also conducted to verify their putative functions. In Nicotiana benthamiana, an infectivity assay of the infectious SPLCV-JS clone resulted in mild symptoms and weak viral DNA accumulation. Interestingly, SPLCV-JS, together with a heterologous betasatellite DNA (tomato yellow leaf curl China virus isolate Y10 [TYLCCNV-Y10] DNA-β), showed a synergistic effect on enhanced symptom severity and viral DNA accumulation. This is the first reported infectious SPLCV clone.

Accumulation of Succinate in Cardiac Ischemia Primarily Occurs via Canonical Krebs Cycle Activity.

PubMed

Zhang, Jimmy; Wang, Yves T; Miller, James H; Day, Mary M; Munger, Joshua C; Brookes, Paul S

2018-05-29

Succinate accumulates during ischemia, and its oxidation at reperfusion drives injury. The mechanism of ischemic succinate accumulation is controversial and is proposed to involve reversal of mitochondrial complex II. Herein, using stable-isotope-resolved metabolomics, we demonstrate that complex II reversal is possible in hypoxic mitochondria but is not the primary succinate source in hypoxic cardiomyocytes or ischemic hearts. Rather, in these intact systems succinate primarily originates from canonical Krebs cycle activity, partly supported by aminotransferase anaplerosis and glycolysis from glycogen. Augmentation of canonical Krebs cycle activity with dimethyl-α-ketoglutarate both increases ischemic succinate accumulation and drives substrate-level phosphorylation by succinyl-CoA synthetase, improving ischemic energetics. Although two-thirds of ischemic succinate accumulation is extracellular, the remaining one-third is metabolized during early reperfusion, wherein acute complex II inhibition is protective. These results highlight a bifunctional role for succinate: its complex-II-independent accumulation being beneficial in ischemia and its complex-II-dependent oxidation being detrimental at reperfusion. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Coat Protein Regulation by CK2, CPIP, HSP70, and CHIP Is Required for Potato Virus A Replication and Coat Protein Accumulation

PubMed Central

Lõhmus, Andres; Hafrén, Anders

2016-01-01

ABSTRACT We demonstrate here that both coat protein (CP) phosphorylation by protein kinase CK2 and a chaperone system formed by two heat shock proteins, CP-interacting protein (CPIP) and heat shock protein 70 (HSP70), are essential for potato virus A (PVA; genus Potyvirus) replication and that all these host proteins have the capacity to contribute to the level of PVA CP accumulation. An E3 ubiquitin ligase called carboxyl terminus Hsc70-interacting protein (CHIP), which may participate in the CPIP-HSP70-mediated CP degradation, is also needed for robust PVA gene expression. Residue Thr243 within the CK2 consensus sequence of PVA CP was found to be essential for viral replication and to regulate CP protein stability. Substitution of Thr243 either with a phosphorylation-mimicking Asp (CPADA) or with a phosphorylation-deficient Ala (CPAAA) residue in CP expressed from viral RNA limited PVA gene expression to the level of nonreplicating PVA. We found that both the CPAAA mutant and CK2 silencing inhibited, whereas CPADA mutant and overexpression of CK2 increased, PVA translation. From our previous studies, we know that phosphorylation reduces the RNA binding capacity of PVA CP and an excess of CP fully blocks viral RNA translation. Together, these findings suggest that binding by nonphosphorylated PVA CP represses viral RNA translation, involving further CP phosphorylation and CPIP-HSP70 chaperone activities as prerequisites for PVA replication. We propose that this mechanism contributes to shifting potyvirus RNA from translation to replication. IMPORTANCE Host protein kinase CK2, two host chaperones, CPIP and HSP70, and viral coat protein (CP) phosphorylation at Thr243 are needed for potato virus A (PVA) replication. Our results show that nonphosphorylated CP blocks viral translation, likely via binding to viral RNA. We propose that this translational block is needed to allow time and space for the formation of potyviral replication complex around the 3′ end of viral RNA. Progression into replication involves CP regulation by both CK2 phosphorylation and chaperones CPIP and HSP70. PMID:27852853

Characterization of a Proposed Dichorhavirus Associated with the Citrus Leprosis Disease and Analysis of the Host Response

PubMed Central

Cruz-Jaramillo, José Luis; Ruiz-Medrano, Roberto; Rojas-Morales, Lourdes; López-Buenfil, José Abel; Morales-Galván, Oscar; Chavarín-Palacio, Claudio; Ramírez-Pool, José Abrahán; Xoconostle-Cázares, Beatriz

2014-01-01

The causal agents of Citrus leprosis are viruses; however, extant diagnostic methods to identify them have failed to detect known viruses in orange, mandarin, lime and bitter orange trees with severe leprosis symptoms in Mexico, an important citrus producer. Using high throughput sequencing, a virus associated with citrus leprosis was identified, belonging to the proposed Dichorhavirus genus. The virus was termed Citrus Necrotic Spot Virus (CNSV) and contains two negative-strand RNA components; virions accumulate in the cytoplasm and are associated with plasmodesmata—channels interconnecting neighboring cells—suggesting a mode of spread within the plant. The present study provides insights into the nature of this pathogen and the corresponding plant response, which is likely similar to other pathogens that do not spread systemically in plants. PMID:25004279

The Role of Culex pipiens L. (Diptera: Culicidae) in Virus Transmission in Europe

PubMed Central

Hernández-Triana, Luis M.; Medlock, Jolyon M.; Fooks, Anthony R.; Carpenter, Simon; Johnson, Nicholas

2018-01-01

Over the past three decades, a range of mosquito-borne viruses that threaten public and veterinary health have emerged or re-emerged in Europe. Mosquito surveillance activities have highlighted the Culex pipiens species complex as being critical for the maintenance of a number of these viruses. This species complex contains morphologically similar forms that exhibit variation in phenotypes that can influence the probability of virus transmission. Critical amongst these is the choice of host on which to feed, with different forms showing different feeding preferences. This influences the ability of the mosquito to vector viruses and facilitate transmission of viruses to humans and domestic animals. Biases towards blood-feeding on avian or mammalian hosts have been demonstrated for different Cx. pipiens ecoforms and emerging evidence of hybrid populations across Europe adds another level of complexity to virus transmission. A range of molecular methods based on DNA have been developed to enable discrimination between morphologically indistinguishable forms, although this remains an active area of research. This review provides a comprehensive overview of developments in the understanding of the ecology, behaviour and genetics of Cx. pipiens in Europe, and how this influences arbovirus transmission. PMID:29473903

Rapid evolution of hosts begets species diversity at the cost of intraspecific diversity.

PubMed

Frickel, Jens; Theodosiou, Loukas; Becks, Lutz

2017-10-17

Ecosystems are complex food webs in which multiple species interact and ecological and evolutionary processes continuously shape populations and communities. Previous studies on eco-evolutionary dynamics have shown that the presence of intraspecific diversity affects community structure and function, and that eco-evolutionary feedback dynamics can be an important driver for its maintenance. Within communities, feedbacks are, however, often indirect, and they can feed back over many generations. Here, we studied eco-evolutionary feedbacks in evolving communities over many generations and compared two-species systems (virus-host and prey-predator) with a more complex three-species system (virus-host-predator). Both indirect density- and trait-mediated effects drove the dynamics in the complex system, where host-virus coevolution facilitated coexistence of predator and virus, and where coexistence, in return, lowered intraspecific diversity of the host population. Furthermore, ecological and evolutionary dynamics were significantly altered in the three-species system compared with the two-species systems. We found that the predator slowed host-virus coevolution in the complex system and that the virus' effect on the overall population dynamics was negligible when the three species coexisted. Overall, we show that a detailed understanding of the mechanism driving eco-evolutionary feedback dynamics is necessary for explaining trait and species diversity in communities, even in communities with only three species.

ONYX-015: mechanisms of action and clinical potential of a replication-selective adenovirus

PubMed Central

Ries, S; Korn, W M

2002-01-01

Accumulated knowledge in the molecular processes of tumour development combined with the availability of genetically modified viruses resemble the basis for new promising cancer therapeutics. The main advantages of employing replication-competent viruses are achievement of tumour selective killing and amplification of their oncolytic potential within the tumour mass. In this review, we describe the development of ONYX-015, one of the first and most advanced replication-competent viruses for cancer therapy. We discuss the molecular biology of this therapeutic approach and the interesting results obtained with this virus in clinical trials. British Journal of Cancer (2002) 86, 5–11. DOI: 10.1038/sj/bjc/6600006 www.bjcancer.com © 2002 The Cancer Research Campaign PMID:11857003

Hemozoin Differentially Regulates Proinflammatory Cytokine Production in Human Immunodeficiency Virus-Seropositive and -Seronegative Women with Placental Malaria

PubMed Central

Moore, Julie M.; Chaisavaneeyakorn, Sujittra; Perkins, Douglas J.; Othoro, Caroline; Otieno, Juliana; Nahlen, Bernard L.; Shi, Ya Ping; Udhayakumar, Venkatachalam

2004-01-01

Pregnant women are at an increased risk for malarial infection. Plasmodium falciparum accumulates in the placenta and is associated with dysregulated immune function and poor birth outcomes. Malarial pigment (hemozoin) also accumulates in the placenta and may modulate local immune function. In this study, the impact of hemozoin on cytokine production by intervillous blood mononuclear cells from malaria-infected placentas was investigated. There was a dose-dependent, suppressive effect of hemozoin on production of gamma interferon (IFN-γ), with less of an effect on tumor necrosis factor alpha (TNF-α) and interleukin-10, in human immunodeficiency virus-seronegative (HIV−) women. In contrast, IFN-γ and TNF-α production tended to increase in HIV-seropositive women with increasing hemozoin levels. Production patterns of cytokines, especially IFN-γ in HIV− women, followed different trends as a function of parasite density and hemozoin level. The findings suggest that the influences of hemozoin accumulation and high-density parasitemia on placental cytokine production are not equivalent and may involve different mechanisms, all of which may operate differently in the context of HIV infection. Cytokine production dysregulated by accumulation of hemozoin or high-density parasitemia may induce pathology and impair protective immunity in HIV-infected and -uninfected women. PMID:15557625

Viral control of phytoplankton populations--a review.

PubMed

Brussaard, Corina P D

2004-01-01

Phytoplankton population dynamics are the result of imbalances between reproduction and losses. Losses include grazing, sinking, and natural mortality. As the importance of microbes in aquatic ecology has been recognized, so has the potential significance of viruses as mortality agents for phytoplankton. The field of algal virus ecology is steadily changing and advancing as new viruses are isolated and new methods are developed for quantifying the impact of viruses on phytoplankton dynamics and diversity. With this development, evidence is accumulating that viruses can control phytoplankton dynamics through reduction of host populations, or by preventing algal host populations from reaching high levels. The identification of highly specific host ranges of viruses is changing our understanding of population dynamics. Viral-mediated mortality may not only affect algal species succession, but may also affect intraspecies succession. Through cellular lysis, viruses indirectly affect the fluxes of energy, nutrients, and organic matter, especially during algal bloom events when biomass is high. Although the importance of viruses is presently recognized, it is apparent that many aspects of viral-mediated mortality of phytoplankton are still poorly understood. It is imperative that future research addresses the mechanisms that regulate virus infectivity, host resistance, genotype richness, abundance, and the fate of viruses over time and space.

The Heterologous Expression of the p22 RNA Silencing Suppressor of the Crinivirus Tomato Chlorosis Virus from Tobacco Rattle Virus and Potato Virus X Enhances Disease Severity but Does Not Complement Suppressor-Defective Mutant Viruses.

PubMed

Landeo-Ríos, Yazmín; Navas-Castillo, Jesús; Moriones, Enrique; Cañizares, M. Carmen

2017-11-24

To counteract host antiviral RNA silencing, plant viruses express suppressor proteins that function as pathogenicity enhancers. The genome of the Tomato chlorosis virus (ToCV) (genus Crinivirus , family Closteroviridae ) encodes an RNA silencing suppressor, the protein p22, that has been described as having one of the longest lasting local suppressor activities when assayed in Nicotiana benthamiana . Since suppression of RNA silencing and the ability to enhance disease severity are closely associated, we analyzed the effect of expressing p22 in heterologous viral contexts. Thus, we studied the effect of the expression of ToCV p22 from viral vectors Tobacco rattle virus (TRV) and Potato virus X (PVX), and from attenuated suppressor mutants in N. benthamiana plants. Our results show that although an exacerbation of disease symptoms leading to plant death was observed in the heterologous expression of ToCV p22 from both viruses, only in the case of TRV did increased viral accumulation occur. The heterologous expression of ToCV p22 could not complement suppressor-defective mutant viruses.

Type 1 immune mechanisms driven by the response to infection with attenuated rabies virus result in changes in the immune bias of the tumor microenvironment and necrosis of mouse GL261 brain tumors

PubMed Central

Bongiorno, Emily K.; Garcia, Samantha A.; Sauma, Sami; Hooper, D. Craig

2017-01-01

Immunotherapeutic strategies for malignant glioma have to overcome the immunomodulatory activities of M2 monocytes that appear in the circulation and as tumor associated macrophages (TAM). M2 cell products contribute to the growth-promoting attributes of the tumor microenvironment (TME) and bias immunity towards type 2, away from the type 1 mechanisms with anti-tumor properties. To drive type 1 immunity in CNS tissues we infected GL261 tumor-bearing mice with attenuated rabies virus (RABV). These neurotropic viruses spread to CNS tissues trans-axonally where they induce a strong type 1 immune response that involves Th1, CD8 and B cell entry across the blood brain barrier and virus clearance in the absence of overt sequelae. Intranasal infection with attenuated RABV prolonged the survival of mice bearing established GL261 brain tumors. Despite the failure of virus spread to the tumor, infection resulted in significantly enhanced tumor necrosis, extensive CD4 T cell accumulation and high levels of the proinflammatory factors IFNy, TNFa, and iNOS in the TME merely 4 days after infection, before significant virus spread or the appearance of RABV-specific immune mechanisms in CNS tissues. While the majority of g CD4 cells appeared functionally inactive, the proinflammatory changes in the TME later resulted in the loss of accumulating M2 and increased M1 TAM. Mice deficient in the Th1 transcription factor Tbet did not gain any survival advantage from RABV infection, exhibiting only limited tumor necrosis and no change in TME cytokines or TAM phenotype, highlighting the importance of type 1 mechanisms in this process. PMID:28461570

Genetic insights into Graminella nigrifrons Competence for maize fine streak virus infection and transmission.

PubMed

Cassone, Bryan J; Cisneros Carter, Fiorella M; Michel, Andrew P; Stewart, Lucy R; Redinbaugh, Margaret G

2014-01-01

Most plant-infecting rhabdoviruses are transmitted by one or a few closely related insect species. Additionally, intraspecific differences in transmission efficacy often exist among races/biotypes within vector species and among strains within a virus species. The black-faced leafhopper, Graminella nigrifrons, is the only known vector of the persistent propagative rhabdovirus Maize fine streak virus (MFSV). Only a small percentage of leafhoppers are capable of transmitting the virus, although the mechanisms underlying vector competence are not well understood. RNA-Seq was carried out to explore transcript expression changes and sequence variation in G. nigrifrons and MFSV that may be associated with the ability of the vector to acquire and transmit the virus. RT-qPCR assays were used to validate differential transcript accumulation. Feeding on MFSV-infected maize elicited a considerable transcriptional response in G. nigrifrons, with increased expression of cytoskeleton organization and immunity transcripts in infected leafhoppers. Differences between leafhoppers capable of transmitting MFSV, relative to non-transmitting but infected leafhoppers were more limited, which may reflect difficulties discerning between the two groups and/or the likelihood that the transmitter phenotype results from one or a few genetic differences. The ability of infected leafhoppers to transmit MFSV did not appear associated with virus transcript accumulation in the infected leafhoppers or sequence polymorphisms in the viral genome. However, the non-structural MFSV 3 gene was expressed at unexpectedly high levels in infected leafhoppers, suggesting it plays an active role in the infection of the insect host. The results of this study begin to define the functional roles of specific G. nigrifrons and MFSV genes in the viral transmission process.

Genetic Insights into Graminella nigrifrons Competence for Maize fine streak virus Infection and Transmission

PubMed Central

Michel, Andrew P.; Stewart, Lucy R.; Redinbaugh, Margaret G.

2014-01-01

Background Most plant-infecting rhabdoviruses are transmitted by one or a few closely related insect species. Additionally, intraspecific differences in transmission efficacy often exist among races/biotypes within vector species and among strains within a virus species. The black-faced leafhopper, Graminella nigrifrons, is the only known vector of the persistent propagative rhabdovirus Maize fine streak virus (MFSV). Only a small percentage of leafhoppers are capable of transmitting the virus, although the mechanisms underlying vector competence are not well understood. Methodology RNA-Seq was carried out to explore transcript expression changes and sequence variation in G. nigrifrons and MFSV that may be associated with the ability of the vector to acquire and transmit the virus. RT-qPCR assays were used to validate differential transcript accumulation. Results/Significance Feeding on MFSV-infected maize elicited a considerable transcriptional response in G. nigrifrons, with increased expression of cytoskeleton organization and immunity transcripts in infected leafhoppers. Differences between leafhoppers capable of transmitting MFSV, relative to non-transmitting but infected leafhoppers were more limited, which may reflect difficulties discerning between the two groups and/or the likelihood that the transmitter phenotype results from one or a few genetic differences. The ability of infected leafhoppers to transmit MFSV did not appear associated with virus transcript accumulation in the infected leafhoppers or sequence polymorphisms in the viral genome. However, the non-structural MFSV 3 gene was expressed at unexpectedly high levels in infected leafhoppers, suggesting it plays an active role in the infection of the insect host. The results of this study begin to define the functional roles of specific G. nigrifrons and MFSV genes in the viral transmission process. PMID:25420026

Effects of Point Mutations in the Major Capsid Protein of Beet Western Yellows Virus on Capsid Formation, Virus Accumulation, and Aphid Transmission

PubMed Central

Brault, V.; Bergdoll, M.; Mutterer, J.; Prasad, V.; Pfeffer, S.; Erdinger, M.; Richards, K. E.; Ziegler-Graff, V.

2003-01-01

Point mutations were introduced into the major capsid protein (P3) of cloned infectious cDNA of the polerovirus beet western yellows virus (BWYV) by manipulation of cloned infectious cDNA. Seven mutations targeted sites on the S domain predicted to lie on the capsid surface. An eighth mutation eliminated two arginine residues in the R domain, which is thought to extend into the capsid interior. The effects of the mutations on virus capsid formation, virus accumulation in protoplasts and plants, and aphid transmission were tested. All of the mutants replicated in protoplasts. The S-domain mutant W166R failed to protect viral RNA from RNase attack, suggesting that this particular mutation interfered with stable capsid formation. The R-domain mutant R7A/R8A protected ∼90% of the viral RNA strand from RNase, suggesting that lower positive-charge density in the mutant capsid interior interfered with stable packaging of the complete strand into virions. Neither of these mutants systemically infected plants. The six remaining mutants properly packaged viral RNA and could invade Nicotiana clevelandii systemically following agroinfection. Mutant Q121E/N122D was poorly transmitted by aphids, implicating one or both targeted residues in virus-vector interactions. Successful transmission of mutant D172N was accompanied either by reversion to the wild type or by appearance of a second-site mutation, N137D. This finding indicates that D172 is also important for transmission but that the D172N transmission defect can be compensated for by a “reverse” substitution at another site. The results have been used to evaluate possible structural models for the BWYV capsid. PMID:12584348

Elimination of antiviral defense by viral RNase III

PubMed Central

Cuellar, Wilmer J.; Kreuze, Jan F.; Rajamäki, Minna-Liisa; Cruzado, Karin R.; Untiveros, Milton; Valkonen, Jari P. T.

2009-01-01

Sweet potato (Ipomoea batatas) is an important subsistence and famine reserve crop grown in developing countries where Sweet potato chlorotic stunt virus (SPCSV; Closteroviridae), a single-stranded RNA (ssRNA) crinivirus, synergizes unrelated viruses in co-infected sweet potato plants. The most severe disease and yield losses are caused by co-infection with SPCSV and a potyvirus, Sweet potato feathery mottle virus (SPFMV; Potyviridae). Potyviruses synergize unrelated viruses by suppression of RNA silencing with the P1/HC-Pro polyprotein; however, the SPCSV-SPFMV synergism is unusual in that the potyvirus is the beneficiary. Our data show that transformation of an SPFMV-resistant sweet potato variety with the double-stranded RNA (dsRNA)-specific class 1 RNA endoribonuclease III (RNase3) of SPCSV broke down resistance to SPFMV, leading to high accumulation of SPFMV antigen and severe disease symptoms similar to the synergism in plants co-infected with SPCSV and SPFMV. RNase3-transgenic sweet potatoes also accumulated higher concentrations of 2 other unrelated viruses and developed more severe symptoms than non-transgenic plants. In leaves, RNase3 suppressed ssRNA-induced gene silencing (RNAi) in an endonuclease activity-dependent manner. It cleaved synthetic double-stranded small interfering RNAs (siRNAs) of 21, 22, and 24 bp in vitro to products of approximately 14 bp that are inactive in RNAi. It also affected total siRNA isolated from SPFMV-infected sweet potato plants, suggesting a viral mechanism for suppression of RNAi by cleavage of siRNA. Results implicate RNase3 in suppression of antiviral defense in sweet potato plants and reveal RNase3 as a protein that mediates viral synergism with several unrelated viruses, a function previously described only for P1/HC-Pro. PMID:19515815

Cryo-Electron Microscopy of Viruses Infecting Bacterium

NASA Astrophysics Data System (ADS)

Chiu, Wah

2010-03-01

Single particle cryo-EM can yield structures of infectious bacterial viruses with and without imposed icosahedral symmetry at subnanometer resolution. Reconstructions of infectious and empty phage particles show substantial differences in the portal vertex protein complex at one of the 12 pentameric vertices in the icosahedral virus particle through which the viral genomes are packaged or released. In addition, electron cryo-tomography of viruses during infecting its bacterial host cell displayed multiple conformations of the tail fiber of the virus. Our structural observations by single particle and tomographic reconstructions suggest a mechanism whereby the viral tail fibers, upon binding to the host cell, induce a cascade of structural alterations of the portal vertex protein complex that triggers DNA release.

Motion-Based Immunological Detection of Zika Virus Using Pt-Nanomotors and a Cellphone.

PubMed

Draz, Mohamed Shehata; Lakshminaraasimulu, Nivethitha Kota; Krishnakumar, Sanchana; Battalapalli, Dheerendranath; Vasan, Anish; Kanakasabapathy, Manoj Kumar; Sreeram, Aparna; Kallakuri, Shantanu; Thirumalaraju, Prudhvi; Li, Yudong; Hua, Stephane; Yu, Xu G; Kuritzkes, Daniel R; Shafiee, Hadi

2018-05-16

Zika virus (ZIKV) infection is an emerging pandemic threat to humans that can be fatal in newborns. Advances in digital health systems and nanoparticles can facilitate the development of sensitive and portable detection technologies for timely management of emerging viral infections. Here we report a nanomotor-based bead-motion cellphone (NBC) system for the immunological detection of ZIKV. The presence of virus in a testing sample results in the accumulation of platinum (Pt)-nanomotors on the surface of beads, causing their motion in H 2 O 2 solution. Then the virus concentration is detected in correlation with the change in beads motion. The developed NBC system was capable of detecting ZIKV in samples with virus concentrations as low as 1 particle/μL. The NBC system allowed a highly specific detection of ZIKV in the presence of the closely related dengue virus and other neurotropic viruses, such as herpes simplex virus type 1 and human cytomegalovirus. The NBC platform technology has the potential to be used in the development of point-of-care diagnostics for pathogen detection and disease management in developed and developing countries.

Evidence that the respiratory syncytial virus polymerase complex associates with lipid rafts in virus-infected cells: a proteomic analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

McDonald, Terence P.; Pitt, Andrew R.; Brown, Gaie

2004-12-05

The interaction between the respiratory syncytial virus (RSV) polymerase complex and lipid rafts was examined in HEp2 cells. Lipid-raft membranes were prepared from virus-infected cells and their protein content was analysed by Western blotting and mass spectrometry. This analysis revealed the presence of the N, P, L, M2-1 and M proteins. However, these proteins appeared to differ from one another in their association with these structures, with the M2-1 protein showing a greater partitioning into raft membranes compared to that of the N, P or M proteins. Determination of the polymerase activity profile of the gradient fractions revealed that 95%more » of the detectable viral enzyme activity was associated with lipid-raft membranes. Furthermore, analysis of virus-infected cells by confocal microscopy suggested an association between these proteins and the raft-lipid, GM1. Together, these results provide evidence that the RSV polymerase complex is able to associate with lipid rafts in virus-infected cells.« less

Structural and biochemical characterization of the inhibitor complexes of xenotropic murine leukemia virus-related virus protease.

PubMed

Li, Mi; Gustchina, Alla; Matúz, Krisztina; Tözsér, Jozsef; Namwong, Sirilak; Goldfarb, Nathan E; Dunn, Ben M; Wlodawer, Alexander

2011-11-01

Interactions between the protease (PR) encoded by the xenotropic murine leukemia virus-related virus and a number of potential inhibitors have been investigated by biochemical and structural techniques. It was observed that several inhibitors used clinically against HIV PR exhibit nanomolar or even subnanomolar values of K(i) , depending on the exact experimental conditions. Both TL-3, a universal inhibitor of retroviral PRs, and some inhibitors originally shown to inhibit plasmepsins were also quite potent, whereas inhibition by pepstatin A was considerably weaker. Crystal structures of the complexes of xenotropic murine leukemia virus-related virus PR with TL-3, amprenavir and pepstatin A were solved at high resolution and compared with the structures of complexes of these inhibitors with other retropepsins. Whereas TL-3 and amprenavir bound in a predictable manner, spanning the substrate-binding site of the enzyme, two molecules of pepstatin A bound simultaneously in an unprecedented manner, leaving the catalytic water molecule in place. Journal compilation © 2011 FEBS. No claim to original US government works.

Protection of Nonhuman Primates against Two Species of Ebola Virus Infection with a Single Complex Adenovirus Vector▿

PubMed Central

Pratt, William D.; Wang, Danher; Nichols, Donald K.; Luo, Min; Woraratanadharm, Jan; Dye, John M.; Holman, David H.; Dong, John Y.

2010-01-01

Ebola viruses are highly pathogenic viruses that cause outbreaks of hemorrhagic fever in humans and other primates. To meet the need for a vaccine against the several types of Ebola viruses that cause human diseases, we developed a multivalent vaccine candidate (EBO7) that expresses the glycoproteins of Zaire ebolavirus (ZEBOV) and Sudan ebolavirus (SEBOV) in a single complex adenovirus-based vector (CAdVax). We evaluated our vaccine in nonhuman primates against the parenteral and aerosol routes of lethal challenge. EBO7 vaccine provided protection against both Ebola viruses by either route of infection. Significantly, protection against SEBOV given as an aerosol challenge, which has not previously been shown, could be achieved with a boosting vaccination. These results demonstrate the feasibility of creating a robust, multivalent Ebola virus vaccine that would be effective in the event of a natural virus outbreak or biological threat. PMID:20181765

Protection of nonhuman primates against two species of Ebola virus infection with a single complex adenovirus vector.

PubMed

Pratt, William D; Wang, Danher; Nichols, Donald K; Luo, Min; Woraratanadharm, Jan; Dye, John M; Holman, David H; Dong, John Y

2010-04-01

Ebola viruses are highly pathogenic viruses that cause outbreaks of hemorrhagic fever in humans and other primates. To meet the need for a vaccine against the several types of Ebola viruses that cause human diseases, we developed a multivalent vaccine candidate (EBO7) that expresses the glycoproteins of Zaire ebolavirus (ZEBOV) and Sudan ebolavirus (SEBOV) in a single complex adenovirus-based vector (CAdVax). We evaluated our vaccine in nonhuman primates against the parenteral and aerosol routes of lethal challenge. EBO7 vaccine provided protection against both Ebola viruses by either route of infection. Significantly, protection against SEBOV given as an aerosol challenge, which has not previously been shown, could be achieved with a boosting vaccination. These results demonstrate the feasibility of creating a robust, multivalent Ebola virus vaccine that would be effective in the event of a natural virus outbreak or biological threat.

Binding of a neutralizing antibody to dengue virus alters the arrangement of surface glycoproteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lok, Shee-Mei; Kostyuchenko, Victor; Nybakken, Grant E.

The monoclonal antibody 1A1D-2 has been shown to strongly neutralize dengue virus serotypes 1, 2 and 3, primarily by inhibiting attachment to host cells. A crystal structure of its antigen binding fragment (Fab) complexed with domain III of the viral envelope glycoprotein, E, showed that the epitope would be partially occluded in the known structure of the mature dengue virus. Nevertheless, antibody could bind to the virus at 37 degrees C, suggesting that the virus is in dynamic motion making hidden epitopes briefly available. A cryo-electron microscope image reconstruction of the virus:Fab complex showed large changes in the organization ofmore » the E protein that exposed the epitopes on two of the three E molecules in each of the 60 icosahedral asymmetric units of the virus. The changes in the structure of the viral surface are presumably responsible for inhibiting attachment to cells.« less

A Novel Antiviral Target Structure Involved in the RNA Binding, Dimerization, and Nuclear Export Functions of the Influenza A Virus Nucleoprotein

PubMed Central

Yamada, Kazunori; Kondoh, Yasumitsu; Hikono, Hirokazu; Osada, Hiroyuki; Tomii, Kentaro; Saito, Takehiko; Aida, Yoko

2015-01-01

Developing antiviral therapies for influenza A virus (IAV) infection is an ongoing process because of the rapid rate of antigenic mutation and the emergence of drug-resistant viruses. The ideal strategy is to develop drugs that target well-conserved, functionally restricted, and unique surface structures without affecting host cell function. We recently identified the antiviral compound, RK424, by screening a library of 50,000 compounds using cell-based infection assays. RK424 showed potent antiviral activity against many different subtypes of IAV in vitro and partially protected mice from a lethal dose of A/WSN/1933 (H1N1) virus in vivo. Here, we show that RK424 inhibits viral ribonucleoprotein complex (vRNP) activity, causing the viral nucleoprotein (NP) to accumulate in the cell nucleus. In silico docking analysis revealed that RK424 bound to a small pocket in the viral NP. This pocket was surrounded by three functionally important domains: the RNA binding groove, the NP dimer interface, and nuclear export signal (NES) 3, indicating that it may be involved in the RNA binding, oligomerization, and nuclear export functions of NP. The accuracy of this binding model was confirmed in a NP-RK424 binding assay incorporating photo-cross-linked RK424 affinity beads and in a plaque assay evaluating the structure-activity relationship of RK424. Surface plasmon resonance (SPR) and pull-down assays showed that RK424 inhibited both the NP-RNA and NP-NP interactions, whereas size exclusion chromatography showed that RK424 disrupted viral RNA-induced NP oligomerization. In addition, in vitro nuclear export assays confirmed that RK424 inhibited nuclear export of NP. The amino acid residues comprising the NP pocket play a crucial role in viral replication and are highly conserved in more than 7,000 NP sequences from avian, human, and swine influenza viruses. Furthermore, we found that the NP pocket has a surface structure different from that of the pocket in host molecules. Taken together, these results describe a promising new approach to developing influenza virus drugs that target a novel pocket structure within NP. PMID:26222066

RNAi-derived transgenic resistance to Mungbean yellow mosaic India virus in cowpea.

PubMed

Kumar, Sanjeev; Tanti, Bhaben; Patil, Basavaprabhu L; Mukherjee, Sunil Kumar; Sahoo, Lingaraj

2017-01-01

Cowpea is an important grain legume crop of Africa, Latin America, and Southeast Asia. Leaf curl and golden mosaic diseases caused by Mungbean yellow mosaic India virus (MYMIV) have emerged as most devastating viral diseases of cowpea in Southeast Asia. In this study, we employed RNA interference (RNAi) strategy to control cowpea-infecting MYMIV. For this, we generated transgenic cowpea plants harbouring three different intron hairpin RNAi constructs, containing the AC2, AC4 and fusion of AC2 and AC4 (AC2+AC4) of seven cowpea-infecting begomoviruses. The T0 and T1 transgenic cowpea lines of all the three constructs accumulated transgene-specific siRNAs. Transgenic plants were further assayed up to T1 generations, for resistance to MYMIV using agro-infectious clones. Nearly 100% resistance against MYMIV infection was observed in transgenic lines, expressing AC2-hp and AC2+AC4-hp RNA, when compared with untransformed controls and plants transformed with empty vectors, which developed severe viral disease symptoms within 3 weeks. The AC4-hp RNA expressing lines displayed appearance of milder symptoms after 5 weeks of MYMIV-inoculation. Northern blots revealed a positive correlation between the level of transgene-specific siRNAs accumulation and virus resistance. The MYMIV-resistant transgenic lines accumulated nearly zero or very low titres of viral DNA. The transgenic cowpea plants had normal phenotype with no yield penalty in greenhouse conditions. This is the first demonstration of RNAi-derived resistance to MYMIV in cowpea.

RNAi-derived transgenic resistance to Mungbean yellow mosaic India virus in cowpea

PubMed Central

Kumar, Sanjeev; Tanti, Bhaben; Patil, Basavaprabhu L.; Mukherjee, Sunil Kumar

2017-01-01

Cowpea is an important grain legume crop of Africa, Latin America, and Southeast Asia. Leaf curl and golden mosaic diseases caused by Mungbean yellow mosaic India virus (MYMIV) have emerged as most devastating viral diseases of cowpea in Southeast Asia. In this study, we employed RNA interference (RNAi) strategy to control cowpea-infecting MYMIV. For this, we generated transgenic cowpea plants harbouring three different intron hairpin RNAi constructs, containing the AC2, AC4 and fusion of AC2 and AC4 (AC2+AC4) of seven cowpea-infecting begomoviruses. The T0 and T1 transgenic cowpea lines of all the three constructs accumulated transgene-specific siRNAs. Transgenic plants were further assayed up to T1 generations, for resistance to MYMIV using agro-infectious clones. Nearly 100% resistance against MYMIV infection was observed in transgenic lines, expressing AC2-hp and AC2+AC4-hp RNA, when compared with untransformed controls and plants transformed with empty vectors, which developed severe viral disease symptoms within 3 weeks. The AC4-hp RNA expressing lines displayed appearance of milder symptoms after 5 weeks of MYMIV-inoculation. Northern blots revealed a positive correlation between the level of transgene-specific siRNAs accumulation and virus resistance. The MYMIV-resistant transgenic lines accumulated nearly zero or very low titres of viral DNA. The transgenic cowpea plants had normal phenotype with no yield penalty in greenhouse conditions. This is the first demonstration of RNAi-derived resistance to MYMIV in cowpea. PMID:29077738

Genomic segments RNA1 and RNA2 of Prunus necrotic ringspot virus codetermine viral pathogenicity to adapt to alternating natural Prunus hosts.

PubMed

Cui, Hongguang; Hong, Ni; Wang, Guoping; Wang, Aiming

2013-05-01

Prunus necrotic ringspot virus (PNRSV) affects Prunus fruit production worldwide. To date, numerous PNRSV isolates with diverse pathological properties have been documented. To study the pathogenicity of PNRSV, which directly or indirectly determines the economic losses of infected fruit trees, we have recently sequenced the complete genome of peach isolate Pch12 and cherry isolate Chr3, belonging to the pathogenically aggressive PV32 group and mild PV96 group, respectively. Here, we constructed the Chr3- and Pch12-derived full-length cDNA clones that were infectious in the experimental host cucumber and their respective natural Prunus hosts. Pch12-derived clones induced much more severe symptoms than Chr3 in cucumber, and the pathogenicity discrepancy between Chr3 and Pch12 was associated with virus accumulation. By reassortment of genomic segments, swapping of partial genomic segments, and site-directed mutagenesis, we identified the 3' terminal nucleotide sequence (1C region) in RNA1 and amino acid K at residue 279 in RNA2-encoded P2 as the severe virulence determinants in Pch12. Gain-of-function experiments demonstrated that both the 1C region and K279 of Pch12 were required for severe virulence and high levels of viral accumulation. Our results suggest that PNRSV RNA1 and RNA2 codetermine viral pathogenicity to adapt to alternating natural Prunus hosts, likely through mediating viral accumulation.

Virus interaction with the apical junctional complex.

PubMed

Gonzalez-Mariscal, Lorenza; Garay, Erika; Lechuga, Susana

2009-01-01

In order to infect pathogens must breach the epithelial barriers that separate the organism from the external environment or that cover the internal cavities and ducts of the body. Epithelia seal the passage through the paracellular pathway with the apical junctional complex integrated by tight and adherens junctions. In this review we describe how viruses like coxsackie, swine vesicular disease virus, adenovirus, reovirus, feline calcivirus, herpes viruses 1 and 2, pseudorabies, bovine herpes virus 1, poliovirus and hepatitis C use as cellular receptors integral proteins present at the AJC of epithelial cells. Interaction with these proteins contributes in a significant manner in defining the particular tropism of each virus. Besides these proteins, viruses exhibit a wide range of cellular co-receptors among which proteins present in the basolateral cell surface like integrins are often found. Therefore targeting proteins of the AJC constitutes a strategy that might allow viruses to bypass the physical barrier that blocks their access to receptors expressed on the basolateral surface of epithelial cells.

Chicken and Duck Myotubes Are Highly Susceptible and Permissive to Influenza Virus Infection

PubMed Central

Baquero-Perez, Belinda; Kuchipudi, Suresh V.; Ho, Jemima; Sebastian, Sujith; Puranik, Anita; Howard, Wendy; Brookes, Sharon M.; Brown, Ian H.

2014-01-01

ABSTRACT Skeletal muscle, at 30 to 40% of body mass, is the most abundant soft tissue in the body. Besides its primary function in movement and posture, skeletal muscle is a significant innate immune organ with the capacity to produce cytokines and chemokines and respond to proinflammatory cytokines. Little is known about the role of skeletal muscle during systemic influenza A virus infection in any host and particularly avian species. Here we used primary chicken and duck multinucleated myotubes to examine their susceptibility and innate immune response to influenza virus infections. Both chicken and duck myotubes expressed avian and human sialic acid receptors and were readily susceptible to low-pathogenicity (H2N3 A/mallard duck/England/7277/06) and high-pathogenicity (H5N1 A/turkey/England/50-92/91 and H5N1 A/turkey/Turkey/1/05) avian and human H1N1 (A/USSR/77) influenza viruses. Both avian host species produced comparable levels of progeny H5N1 A/turkey/Turkey/1/05 virus. Notably, the rapid accumulation of viral nucleoprotein and matrix (M) gene RNA in chicken and duck myotubes was accompanied by extensive cytopathic damage with marked myotube apoptosis (widespread microscopic blebs, caspase 3/7 activation, and annexin V binding at the plasma membrane). Infected chicken myotubes produced significantly higher levels of proinflammatory cytokines than did the corresponding duck cells. Additionally, in chicken myotubes infected with H5N1 viruses, the induction of interferon beta (IFN-β) and IFN-inducible genes, including the melanoma differentiation-associated protein 5 (MDA-5) gene, was relatively weak compared to infection with the corresponding H2N3 virus. Our findings highlight that avian skeletal muscle fibers are capable of productive influenza virus replication and are a potential tissue source of infection. IMPORTANCE Infection with high-pathogenicity H5N1 viruses in ducks is often asymptomatic, and skeletal muscle from such birds could be a source of infection of humans and animals. Little is known about the ability of influenza A viruses to replicate in avian skeletal muscle fibers. We show here that cultured chicken and duck myotubes were highly susceptible to infection with both low- and high-pathogenicity avian influenza viruses. Infected myotubes of both avian species displayed rapid virus accumulation, apoptosis, and extensive cellular damage. Our results indicate that avian skeletal muscle fibers of chicken and duck could be significant contributors to progeny production of highly pathogenic H5N1 viruses. PMID:25540384

Chicken and duck myotubes are highly susceptible and permissive to influenza virus infection.

PubMed

Baquero-Perez, Belinda; Kuchipudi, Suresh V; Ho, Jemima; Sebastian, Sujith; Puranik, Anita; Howard, Wendy; Brookes, Sharon M; Brown, Ian H; Chang, Kin-Chow

2015-03-01

Skeletal muscle, at 30 to 40% of body mass, is the most abundant soft tissue in the body. Besides its primary function in movement and posture, skeletal muscle is a significant innate immune organ with the capacity to produce cytokines and chemokines and respond to proinflammatory cytokines. Little is known about the role of skeletal muscle during systemic influenza A virus infection in any host and particularly avian species. Here we used primary chicken and duck multinucleated myotubes to examine their susceptibility and innate immune response to influenza virus infections. Both chicken and duck myotubes expressed avian and human sialic acid receptors and were readily susceptible to low-pathogenicity (H2N3 A/mallard duck/England/7277/06) and high-pathogenicity (H5N1 A/turkey/England/50-92/91 and H5N1 A/turkey/Turkey/1/05) avian and human H1N1 (A/USSR/77) influenza viruses. Both avian host species produced comparable levels of progeny H5N1 A/turkey/Turkey/1/05 virus. Notably, the rapid accumulation of viral nucleoprotein and matrix (M) gene RNA in chicken and duck myotubes was accompanied by extensive cytopathic damage with marked myotube apoptosis (widespread microscopic blebs, caspase 3/7 activation, and annexin V binding at the plasma membrane). Infected chicken myotubes produced significantly higher levels of proinflammatory cytokines than did the corresponding duck cells. Additionally, in chicken myotubes infected with H5N1 viruses, the induction of interferon beta (IFN-β) and IFN-inducible genes, including the melanoma differentiation-associated protein 5 (MDA-5) gene, was relatively weak compared to infection with the corresponding H2N3 virus. Our findings highlight that avian skeletal muscle fibers are capable of productive influenza virus replication and are a potential tissue source of infection. Infection with high-pathogenicity H5N1 viruses in ducks is often asymptomatic, and skeletal muscle from such birds could be a source of infection of humans and animals. Little is known about the ability of influenza A viruses to replicate in avian skeletal muscle fibers. We show here that cultured chicken and duck myotubes were highly susceptible to infection with both low- and high-pathogenicity avian influenza viruses. Infected myotubes of both avian species displayed rapid virus accumulation, apoptosis, and extensive cellular damage. Our results indicate that avian skeletal muscle fibers of chicken and duck could be significant contributors to progeny production of highly pathogenic H5N1 viruses. Copyright © 2015, Baquero-Perez et al.

Bovine respiratory disease model based on dual infections with infection with bovine viral diarrhea virus and bovine corona virus

USDA-ARS?s Scientific Manuscript database

Bovine respiratory disease complex (BRDC) is the leading cause of economic loss in the U.S. cattle industry. BRDC likely results from simultaneous or sequential infections with multiple pathogens including both viruses and bacteria. Bovine viral diarrhea virus (BVDV) and bovine corona virus (BoCV...

Genetic characterization of North American populations of the wheat curl mite (Aceria tosichella) and dry bulb mite (Aceria tulipae)

USDA-ARS?s Scientific Manuscript database

The wheat curl mite, Aceria tosichella Keifer, transmits at least three harmful viruses, wheat streak mosaic virus (WSMV), high plains virus (HPV), and Triticum mosaic virus (TriMV) to wheat (Triticum aestivum L.) throughout the Great Plains. This virus complex is considered to be the most serious d...

Intracellular Transport of Plant Viruses: Finding the Door out of the Cell

PubMed Central

Schoelz, James E.; Harries, Phillip A.; Nelson, Richard S.

2011-01-01

Plant viruses are a class of plant pathogens that specialize in movement from cell to cell. As part of their arsenal for infection of plants, every virus encodes a movement protein (MP), a protein dedicated to enlarging the pore size of plasmodesmata (PD) and actively transporting the viral nucleic acid into the adjacent cell. As our knowledge of intercellular transport has increased, it has become apparent that viruses must also use an active mechanism to target the virus from their site of replication within the cell to the PD. Just as viruses are too large to fit through an unmodified plasmodesma, they are also too large to be freely diffused through the cytoplasm of the cell. Evidence has accumulated now for the involvement of other categories of viral proteins in intracellular movement in addition to the MP, including viral proteins originally associated with replication or gene expression. In this review, we will discuss the strategies that viruses use for intracellular movement from the replication site to the PD, in particular focusing on the role of host membranes for intracellular transport and the coordinated interactions between virus proteins within cells that are necessary for successful virus spread. PMID:21896501

Apoptosis in fatal Ebola infection. Does the virus toll the bell for immune system?

PubMed

Baize, S; Leroy, E M; Mavoungou, E; Fisher-Hoch, S P

2000-02-01

In fatal Ebola virus hemorrhagic fever massive intravascular apoptosis develops rapidly following infection and progressing relentlessly until death. While data suggest that T lymphocytes are mainly deleted by apoptosis in PBMC of human fatal cases, experimental Ebola infection in animal models have shown some evidence of destruction of lymphocytes in spleen and lymph nodes probably involving both T and B cells. Nevertheless, we are able to conclude from the accumulated evidence that early interactions between Ebola virus and the immune system, probably via macrophages, main targets for viral replication, lead to massive destruction of immune cells in fatal cases.

Clinical Sequencing Uncovers Origins and Evolution of Lassa Virus.

PubMed

Andersen, Kristian G; Shapiro, B Jesse; Matranga, Christian B; Sealfon, Rachel; Lin, Aaron E; Moses, Lina M; Folarin, Onikepe A; Goba, Augustine; Odia, Ikponmwonsa; Ehiane, Philomena E; Momoh, Mambu; England, Eleina M; Winnicki, Sarah; Branco, Luis M; Gire, Stephen K; Phelan, Eric; Tariyal, Ridhi; Tewhey, Ryan; Omoniwa, Omowunmi; Fullah, Mohammed; Fonnie, Richard; Fonnie, Mbalu; Kanneh, Lansana; Jalloh, Simbirie; Gbakie, Michael; Saffa, Sidiki; Karbo, Kandeh; Gladden, Adrianne D; Qu, James; Stremlau, Matthew; Nekoui, Mahan; Finucane, Hilary K; Tabrizi, Shervin; Vitti, Joseph J; Birren, Bruce; Fitzgerald, Michael; McCowan, Caryn; Ireland, Andrea; Berlin, Aaron M; Bochicchio, James; Tazon-Vega, Barbara; Lennon, Niall J; Ryan, Elizabeth M; Bjornson, Zach; Milner, Danny A; Lukens, Amanda K; Broodie, Nisha; Rowland, Megan; Heinrich, Megan; Akdag, Marjan; Schieffelin, John S; Levy, Danielle; Akpan, Henry; Bausch, Daniel G; Rubins, Kathleen; McCormick, Joseph B; Lander, Eric S; Günther, Stephan; Hensley, Lisa; Okogbenin, Sylvanus; Schaffner, Stephen F; Okokhere, Peter O; Khan, S Humarr; Grant, Donald S; Akpede, George O; Asogun, Danny A; Gnirke, Andreas; Levin, Joshua Z; Happi, Christian T; Garry, Robert F; Sabeti, Pardis C

2015-08-13

The 2013-2015 West African epidemic of Ebola virus disease (EVD) reminds us of how little is known about biosafety level 4 viruses. Like Ebola virus, Lassa virus (LASV) can cause hemorrhagic fever with high case fatality rates. We generated a genomic catalog of almost 200 LASV sequences from clinical and rodent reservoir samples. We show that whereas the 2013-2015 EVD epidemic is fueled by human-to-human transmissions, LASV infections mainly result from reservoir-to-human infections. We elucidated the spread of LASV across West Africa and show that this migration was accompanied by changes in LASV genome abundance, fatality rates, codon adaptation, and translational efficiency. By investigating intrahost evolution, we found that mutations accumulate in epitopes of viral surface proteins, suggesting selection for immune escape. This catalog will serve as a foundation for the development of vaccines and diagnostics. VIDEO ABSTRACT. Copyright © 2015 Elsevier Inc. All rights reserved.

Clinical sequencing uncovers origins and evolution of Lassa virus

PubMed Central

Andersen, Kristian G.; Shapiro, B. Jesse; Matranga, Christian B.; Sealfon, Rachel; Lin, Aaron E.; Moses, Lina M.; Folarin, Onikepe A.; Goba, Augustine; Odia, Ikponmwonsa; Ehiane, Philomena E.; Momoh, Mambu; England, Eleina M.; Winnicki, Sarah; Branco, Luis M.; Gire, Stephen K.; Phelan, Eric; Tariyal, Ridhi; Tewhey, Ryan; Omoniwa, Omowunmi; Fullah, Mohammed; Fonnie, Richard; Fonnie, Mbalu; Kanneh, Lansana; Jalloh, Simbirie; Gbakie, Michael; Saffa, Sidiki; Karbo, Kandeh; Gladden, Adrianne D.; Qu, James; Stremlau, Matthew; Nekoui, Mahan; Finucane, Hilary K.; Tabrizi, Shervin; Vitti, Joseph J.; Birren, Bruce; Fitzgerald, Michael; McCowan, Caryn; Ireland, Andrea; Berlin, Aaron M.; Bochicchio, James; Tazon-Vega, Barbara; Lennon, Niall J.; Ryan, Elizabeth M.; Bjornson, Zach; Milner, Danny A.; Lukens, Amanda K.; Broodie, Nisha; Rowland, Megan; Heinrich, Megan; Akdag, Marjan; Schieffelin, John S.; Levy, Danielle; Akpan, Henry; Bausch, Daniel G.; Rubins, Kathleen; McCormick, Joseph B.; Lander, Eric S.; Günther, Stephan; Hensley, Lisa; Okogbenin, Sylvanus; Schaffner, Stephen F.; Okokhere, Peter O.; Khan, S. Humarr; Grant, Donald S.; Akpede, George O.; Asogun, Danny A.; Gnirke, Andreas; Levin, Joshua Z.; Happi, Christian T.; Garry, Robert F.; Sabeti, Pardis C.

2015-01-01

Summary The 2013-2015 West African epidemic of Ebola virus disease (EVD) reminds us how little is known about biosafety level-4 viruses. Like Ebola virus, Lassa virus (LASV) can cause hemorrhagic fever with high case fatality rates. We generated a genomic catalog of almost 200 LASV sequences from clinical and rodent reservoir samples. We show that whereas the 2013-2015 EVD epidemic is fueled by human-to-human transmissions, LASV infections mainly result from reservoir-to-human infections. We elucidated the spread of LASV across West Africa and show that this migration was accompanied by changes in LASV genome abundance, fatality rates, codon adaptation, and translational efficiency. By investigating intrahost evolution, we found that mutations accumulate in epitopes of viral surface proteins, suggesting selection for immune escape. This catalog will serve as a foundation for the development of vaccines and diagnostics. PMID:26276630

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dinh, Phat X.; Panda, Debasis; Das, Phani B.

Using a recombinant vesicular stomatitis virus encoding eGFP fused in-frame with an essential viral replication protein, the phosphoprotein P, we show that during passage in culture, the virus mutates the nucleotide C289 within eGFP of the fusion protein PeGFP to A or T, resulting in R97S/C amino acid substitution and loss of fluorescence. The resultant non-fluorescent virus exhibits increased fitness and growth advantage over its fluorescent counterpart. The growth advantage of the non-fluorescent virus appears to be due to increased transcription and replication activities of the PeGFP protein carrying the R97S/C substitution. Further, our results show that the R97S/C mutationmore » occurs prior to accumulation of mutations that can result in loss of expression of the gene inserted at the G-L gene junction. These results suggest that fitness gain is more important for the recombinant virus than elimination of expression of the heterologous gene.« less

In silico mutation analysis of non-structural protein-5 (NS5) dengue virus

NASA Astrophysics Data System (ADS)

Puspitasari, R. D.; Tambunan, U. S. F.

2017-04-01

Dengue fever is a world disease. It is endemic in more than 100 countries. Information about the effect of mutations in the virus is important in drug design and development. In this research, we studied the effect of mutation on NS5 dengue virus. NS5 is the large protein containing 67% amino acid similarity in DENV 1-4 and has multifunctional enzymatic activities. Dengue virus is an RNA virus that has very high mutation frequency with an average of 100 times higher than DNA mutations, and the accumulation of mutations will be possible to generate the new serotype. In this study, we report that mutation occurs in NS5 of DENV serotype 3, glutamine mutates into methionine at position 10 and threonine mutates into isoleucine at position 55. These residues are part of the domain named S-Adenosyl-L-Methionine-Dependent Methyltransferase (IPR029063).

The contrasting phylodynamics of human influenza B viruses.

PubMed

Vijaykrishna, Dhanasekaran; Holmes, Edward C; Joseph, Udayan; Fourment, Mathieu; Su, Yvonne C F; Halpin, Rebecca; Lee, Raphael T C; Deng, Yi-Mo; Gunalan, Vithiagaran; Lin, Xudong; Stockwell, Timothy B; Fedorova, Nadia B; Zhou, Bin; Spirason, Natalie; Kühnert, Denise; Bošková, Veronika; Stadler, Tanja; Costa, Anna-Maria; Dwyer, Dominic E; Huang, Q Sue; Jennings, Lance C; Rawlinson, William; Sullivan, Sheena G; Hurt, Aeron C; Maurer-Stroh, Sebastian; Wentworth, David E; Smith, Gavin J D; Barr, Ian G

2015-01-16

A complex interplay of viral, host, and ecological factors shapes the spatio-temporal incidence and evolution of human influenza viruses. Although considerable attention has been paid to influenza A viruses, a lack of equivalent data means that an integrated evolutionary and epidemiological framework has until now not been available for influenza B viruses, despite their significant disease burden. Through the analysis of over 900 full genomes from an epidemiological collection of more than 26,000 strains from Australia and New Zealand, we reveal fundamental differences in the phylodynamics of the two co-circulating lineages of influenza B virus (Victoria and Yamagata), showing that their individual dynamics are determined by a complex relationship between virus transmission, age of infection, and receptor binding preference. In sum, this work identifies new factors that are important determinants of influenza B evolution and epidemiology.

Modeling the Propagation of Mobile Phone Virus under Complex Network

PubMed Central

Yang, Wei; Wei, Xi-liang; Guo, Hao; An, Gang; Guo, Lei

2014-01-01

Mobile phone virus is a rogue program written to propagate from one phone to another, which can take control of a mobile device by exploiting its vulnerabilities. In this paper the propagation model of mobile phone virus is tackled to understand how particular factors can affect its propagation and design effective containment strategies to suppress mobile phone virus. Two different propagation models of mobile phone viruses under the complex network are proposed in this paper. One is intended to describe the propagation of user-tricking virus, and the other is to describe the propagation of the vulnerability-exploiting virus. Based on the traditional epidemic models, the characteristics of mobile phone viruses and the network topology structure are incorporated into our models. A detailed analysis is conducted to analyze the propagation models. Through analysis, the stable infection-free equilibrium point and the stability condition are derived. Finally, considering the network topology, the numerical and simulation experiments are carried out. Results indicate that both models are correct and suitable for describing the spread of two different mobile phone viruses, respectively. PMID:25133209

Purification and crystallization of dengue and West Nile virus NS2B–NS3 complexes

DOE Office of Scientific and Technical Information (OSTI.GOV)

D’Arcy, Allan, E-mail: allan.darcy@novartis.com; Chaillet, Maxime; Schiering, Nikolaus

Crystals of dengue serotype 2 and West Nile virus NS2B–NS3 protease complexes have been obtained and the crystals of both diffract to useful resolution. Sample homogeneity was essential for obtaining X-ray-quality crystals of the dengue protease. Controlled proteolysis produced a crystallizable fragment of the apo West Nile virus NS2B–NS3 and crystals were also obtained in the presence of a peptidic inhibitor. Both dengue and West Nile virus infections are an increasing risk to humans, not only in tropical and subtropical areas, but also in North America and parts of Europe. These viral infections are generally transmitted by mosquitoes, but maymore » also be tick-borne. Infection usually results in mild flu-like symptoms, but can also cause encephalitis and fatalities. Approximately 2799 severe West Nile virus cases were reported this year in the United States, resulting in 102 fatalities. With this alarming increase in the number of West Nile virus infections in western countries and the fact that dengue virus already affects millions of people per year in tropical and subtropical climates, there is a real need for effective medicines. A possible therapeutic target to combat these viruses is the protease, which is essential for virus replication. In order to provide structural information to help to guide a lead identification and optimization program, crystallizations of the NS2B–NS3 protease complexes from both dengue and West Nile viruses have been initiated. Crystals that diffract to high resolution, suitable for three-dimensional structure determinations, have been obtained.« less

The presence of Epstein-Barr virus (EBV) in diffuse large B-cell lymphomas (DLBCLs) in Turkey: special emphasis on 'EBV-positive DLBCL of the elderly'.

PubMed

Uner, Aysegul; Akyurek, Nalan; Saglam, Arzu; Abdullazade, Samir; Uzum, Nuket; Onder, Sevgen; Barista, Ibrahim; Benekli, Mustafa

2011-04-01

Accumulated evidence has shown the importance of Epstein-Barr virus in the pathogenesis of various lymphomas. This study aimed to determine the prevalence of Epstein-Barr virus expression and its effect on survival amongst diffuse large B-cell lymphoma (DLBCL) cases from two large tertiary care centres in Turkey with a particular interest in identifying cases of 'Epstein-Barr virus-positive DLBCL of the elderly'. Diffuse large B-cell lymphoma cases diagnosed between 1999 and 2009 were retrieved and 340 cases were used to construct tissue microarrays. The presence of Epstein-Barr virus small ribonucleic acids was examined by in situ hybridization using Epstein-Barr virus (EBV)-encoded small RNA (EBER) oligonucleotides. A total of 18 cases (5.3%) showed Epstein-Barr virus expression. Twelve cases were classified as Epstein-Barr virus-positive DLBCL of the elderly. Epstein-Barr virus-positive DLBCL cases showed a significantly inferior overall survival as compared with Epstein-Barr virus-negative cases (p < 0.001). In our study group Epstein-Barr virus expression is not prevalent in DLBCLs. Epstein-Barr virus-positive DLBCL of the elderly is also rare in the Turkish population. The presence of Epstein-Barr virus, however, is associated with poor prognosis. © 2011 The Authors. APMIS © 2011 APMIS.

The resistance of sour orange to Citrus tristeza virus is mediated by both the salicylic acid and RNA silencing defence pathways.

PubMed

Gómez-Muñoz, Neus; Velázquez, Karelia; Vives, María Carmen; Ruiz-Ruiz, Susana; Pina, José Antonio; Flores, Ricardo; Moreno, Pedro; Guerri, José

2017-12-01

Citrus tristeza virus (CTV) induces in the field the decline and death of citrus varieties grafted on sour orange (SO) rootstock, which has forced the use of alternative decline-tolerant rootstocks in affected countries, despite the highly desirable agronomic features of the SO rootstock. Declining citrus plants display phloem necrosis below the bud union. In addition, SO is minimally susceptible to CTV compared with other citrus varieties, suggesting partial resistance of SO to CTV. Here, by silencing different citrus genes with a Citrus leaf blotch virus-based vector, we have examined the implication of the RNA silencing and salicylic acid (SA) defence pathways in the resistance of SO to CTV. Silencing of the genes RDR1, NPR1 and DCL2/DCL4, associated with these defence pathways, enhanced virus spread and accumulation in SO plants in comparison with non-silenced controls, whereas silencing of the genes NPR3/NPR4, associated with the hypersensitive response, produced a slight decrease in CTV accumulation and reduced stunting of SO grafted on CTV-infected rough lemon plants. We also found that the CTV RNA silencing suppressors p20 and p23 also suppress the SA signalling defence, with the suppressor activity being higher in the most virulent isolates. © 2016 BSPP AND JOHN WILEY & SONS LTD.

Effect of interferon on the replication of mink cell focus-inducing virus in murine cells: synthesis, processing, assembly, and release of viral proteins.

PubMed Central

Bilello, J A; Wivel, N A; Pitha, P M

1982-01-01

Treatment of mink cell focus-inducing (MCF) virus (isolate AK-13) producing SC-1 cells with mouse fibroblast interferon (150 to 600 U/ml) led to a 100-fold decrease in the release of infectious virus, whereas there was a 2.5- to 10-fold decrease in various parameters of virus particle release. Analysis of labeled virion proteins indicated that a temporal change in virion protein composition occurred after interferon treatment. After a 24-h exposure of chronically infected cells to interferon, the virions produced contained a 85,000-dalton glycoprotein (apparently of nonviral origin) which was in excess of the virus envelope glycoprotein gp70. Particles produced from cells treated with interferon for 32 to 48 h were nearly devoid of gp70 and contained substantially lower quantities of p30. Intracellular processing of viral precursor polyproteins to the mature virion structural proteins was not altered in the presence of interferon. However, an accumulation of the viral p30 and p12E proteins was observed in interferon-treated cells, consistent with an increase in cell-associated virions. Immunoprecipitation analysis of the tissue culture fluids from [35S]methionine-labeled control and interferon-treated cells revealed marked decrease in p30 and p15E/p12E released after interferon treatment. In contrast, gp70 did not accumulate in interferon-treated cells, but was released into the culture medium in a form that was neither pelletable nor associated with p15E/p12E. Images PMID:6180173

Synthesis of viral DNA forms in Nicotiana plumbaginifolia protoplasts inoculated with cassava latent virus (CLV); evidence for the independent replication of one component of the CLV genome.

PubMed Central

Townsend, R; Watts, J; Stanley, J

1986-01-01

Totipotent leaf mesophyll protoplasts of Nicotiana plumbaginifolia, Viviani were inoculated with cassava latent virus (CLV) or with full length copies of CLV genomic DNAs 1 and 2 excised from replicative forms of M13 clones. Virus specific DNAs began to appear 48-72h after inoculation with virus or cloned DNAs, coincident with the onset of host cell division. Infected cells accumulated supercoiled forms of DNAs 1 and 2 as well as progeny single-stranded (ss) virion (+) sense DNAs representing each component of the genome. Both supercoiled and ss molecules were synthesised by cells inoculated with cloned DNA 1 alone but DNA 2 failed to replicate independently. Images PMID:3951986

Lateral Organization of Influenza Virus Proteins in the Budozone Region of the Plasma Membrane.

PubMed

Leser, George P; Lamb, Robert A

2017-05-01

Influenza virus assembles and buds at the plasma membrane of virus-infected cells. The viral proteins assemble at the same site on the plasma membrane for budding to occur. This involves a complex web of interactions among viral proteins. Some proteins, like hemagglutinin (HA), NA, and M2, are integral membrane proteins. M1 is peripherally membrane associated, whereas NP associates with viral RNA to form an RNP complex that associates with the cytoplasmic face of the plasma membrane. Furthermore, HA and NP have been shown to be concentrated in cholesterol-rich membrane raft domains, whereas M2, although containing a cholesterol binding motif, is not raft associated. Here we identify viral proteins in planar sheets of plasma membrane using immunogold staining. The distribution of these proteins was examined individually and pairwise by using the Ripley K function, a type of nearest-neighbor analysis. Individually, HA, NA, M1, M2, and NP were shown to self-associate in or on the plasma membrane. HA and M2 are strongly coclustered in the plasma membrane; however, in the case of NA and M2, clustering depends upon the expression system used. Despite both proteins being raft resident, HA and NA occupy distinct but adjacent membrane domains. M2 and M1 strongly cocluster, but the association of M1 with HA or NA is dependent upon the means of expression. The presence of HA and NP at the site of budding depends upon the coexpression of other viral proteins. Similarly, M2 and NP occupy separate compartments, but an association can be bridged by the coexpression of M1. IMPORTANCE The complement of influenza virus proteins necessary for the budding of progeny virions needs to accumulate at budozones. This is complicated by HA and NA residing in lipid raft-like domains, whereas M2, although an integral membrane protein, is not raft associated. Other necessary protein components such as M1 and NP are peripherally associated with the membrane. Our data define spatial relationships between viral proteins in the plasma membrane. Some proteins, such as HA and M2, inherently cocluster within the membrane, although M2 is found mostly at the periphery of regions of HA, consistent with the proposed role of M2 in scission at the end of budding. The association between some pairs of influenza virus proteins, such as M2 and NP, appears to be brokered by additional influenza virus proteins, in this case M1. HA and NA, while raft associated, reside in distinct domains, reflecting their distributions in the viral membrane. Copyright © 2017 American Society for Microbiology.

Viral hemorrhagic fevers of animals caused by DNA viruses

USDA-ARS?s Scientific Manuscript database

Here we outline serious diseases of food and fiber animals that cause damaging economic effect on products all over the world. The only vector-borne DNA virus is included here, such as African swine fever virus, and the herpes viruses discussed have a complex epidemiology characterized by outbreak...

Immune evasion by pathogens of bovine respiratory disease complex.

PubMed

Srikumaran, Subramaniam; Kelling, Clayton L; Ambagala, Aruna

2007-12-01

Bovine respiratory tract disease is a multi-factorial disease complex involving several viruses and bacteria. Viruses that play prominent roles in causing the bovine respiratory disease complex include bovine herpesvirus-1, bovine respiratory syncytial virus, bovine viral diarrhea virus and parinfluenza-3 virus. Bacteria that play prominent roles in this disease complex are Mannheimia haemolytica and Mycoplasma bovis. Other bacteria that infect the bovine respiratory tract of cattle are Histophilus (Haemophilus) somni and Pasteurella multocida. Frequently, severe respiratory tract disease in cattle is associated with concurrent infections of these pathogens. Like other pathogens, the viral and bacterial pathogens of this disease complex have co-evolved with their hosts over millions of years. As much as the hosts have diversified and fine-tuned the components of their immune system, the pathogens have also evolved diverse and sophisticated strategies to evade the host immune responses. These pathogens have developed intricate mechanisms to thwart both the innate and adaptive arms of the immune responses of their hosts. This review presents an overview of the strategies by which the pathogens suppress host immune responses, as well as the strategies by which the pathogens modify themselves or their locations in the host to evade host immune responses. These immune evasion strategies likely contribute to the failure of currently-available vaccines to provide complete protection to cattle against these pathogens.

Complete inactivation of Venezuelan equine encephalitis virus by 1,5-iodonaphthylazide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sharma, Anuj; Birla Institute of Technology and Science, Pilani; Raviv, Yossef

2007-06-29

Hydrophobic alkylating compounds like 1,5-iodonaphthylazide (INA) partitions into biological membranes and accumulates selectively into the hydrophobic domain of the lipid bilayer. Upon irradiation with far UV light, INA binds selectively to transmembrane proteins in the viral envelope and renders them inactive. Such inactivation does not alter the ectodomains of the membrane proteins thus preserving the structural and conformational integrity of immunogens on the surface of the virus. In this study, we have used INA to inactivate Venezuelan equine encephalitis virus (VEEV). Treatment of VEEV with INA followed by irradiation with UV light resulted in complete inactivation of the virus. Immuno-fluorescencemore » for VEEV and virus titration showed no virus replication in-vitro. Complete loss of infectivity was also achieved in mice infected with INA treated plus irradiated preparations of VEEV. No change in the structural integrity of VEEV particles were observed after treatment with INA plus irradiation as assessed by electron microscopy. This data suggest that such inactivation strategies can be used for developing vaccine candidates for VEEV and other enveloped viruses.« less

A New Strain of Drosophila That Inhibits the Development of Symptoms in Imagoes Infected with Sigma Virus

PubMed Central

Herforth, R. S.

1978-01-01

A strain of Drosophila melanogaster bearing the mutant gene ebony has been found to slow the development of symptoms (carbon dioxide sensitivity) in adult flies inoculated with sigma virus, a member of the rhabdovirus group. This inhibition is made evident by comparing mean incubation times of the virus in ebony and wild-type (Oregon) flies. The increase in mean incubation time in ebony flies has ranged from about 3 to 8 days, depending on the virus strain, amount of virus injected, and the age of the flies at the time of inoculation. This delay in development of symptoms appears to be due to a dominant autosomal gene, although further work is needed to confirm this. When accumulation of infectious virus after inoculation is compared in ebony and Oregon flies, there seems to be no inhibition of multiplication in ebony at the level of the entire fly. The relationship of this work to current theories on the mechanism of symptom production by sigma virus is discussed. PMID:17248809

Exploring the Human-Nipah Virus Protein-Protein Interactome

PubMed Central

Vera-Velasco, Natalia M.; Mingarro, Ismael

2017-01-01

ABSTRACT Nipah virus is an emerging, highly pathogenic, zoonotic virus of the Paramyxoviridae family. Human transmission occurs by close contact with infected animals, the consumption of contaminated food, or, occasionally, via other infected individuals. Currently, we lack therapeutic or prophylactic treatments for Nipah virus. To develop these agents we must now improve our understanding of the host-virus interactions that underpin a productive infection. This aim led us to perform the present work, in which we identified 101 human-Nipah virus protein-protein interactions (PPIs), most of which (88) are novel. This data set provides a comprehensive view of the host complexes that are manipulated by viral proteins. Host targets include the PRP19 complex and the microRNA (miRNA) processing machinery. Furthermore, we explored the biologic consequences of the interaction with the PRP19 complex and found that the Nipah virus W protein is capable of altering p53 control and gene expression. We anticipate that these data will help in guiding the development of novel interventional strategies to counter this emerging viral threat. IMPORTANCE Nipah virus is a recently discovered virus that infects a wide range of mammals, including humans. Since its discovery there have been yearly outbreaks, and in some of them the mortality rate has reached 100% of the confirmed cases. However, the study of Nipah virus has been largely neglected, and currently we lack treatments for this infection. To develop these agents we must now improve our understanding of the host-virus interactions that underpin a productive infection. In the present work, we identified 101 human-Nipah virus protein-protein interactions using an affinity purification approach coupled with mass spectrometry. Additionally, we explored the cellular consequences of some of these interactions. Globally, this data set offers a comprehensive and detailed view of the host machinery's contribution to the Nipah virus's life cycle. Furthermore, our data present a large number of putative drug targets that could be exploited for the treatment of this infection. PMID:28904190

Exploring the Human-Nipah Virus Protein-Protein Interactome.

PubMed

Martinez-Gil, Luis; Vera-Velasco, Natalia M; Mingarro, Ismael

2017-12-01

Nipah virus is an emerging, highly pathogenic, zoonotic virus of the Paramyxoviridae family. Human transmission occurs by close contact with infected animals, the consumption of contaminated food, or, occasionally, via other infected individuals. Currently, we lack therapeutic or prophylactic treatments for Nipah virus. To develop these agents we must now improve our understanding of the host-virus interactions that underpin a productive infection. This aim led us to perform the present work, in which we identified 101 human-Nipah virus protein-protein interactions (PPIs), most of which (88) are novel. This data set provides a comprehensive view of the host complexes that are manipulated by viral proteins. Host targets include the PRP19 complex and the microRNA (miRNA) processing machinery. Furthermore, we explored the biologic consequences of the interaction with the PRP19 complex and found that the Nipah virus W protein is capable of altering p53 control and gene expression. We anticipate that these data will help in guiding the development of novel interventional strategies to counter this emerging viral threat. IMPORTANCE Nipah virus is a recently discovered virus that infects a wide range of mammals, including humans. Since its discovery there have been yearly outbreaks, and in some of them the mortality rate has reached 100% of the confirmed cases. However, the study of Nipah virus has been largely neglected, and currently we lack treatments for this infection. To develop these agents we must now improve our understanding of the host-virus interactions that underpin a productive infection. In the present work, we identified 101 human-Nipah virus protein-protein interactions using an affinity purification approach coupled with mass spectrometry. Additionally, we explored the cellular consequences of some of these interactions. Globally, this data set offers a comprehensive and detailed view of the host machinery's contribution to the Nipah virus's life cycle. Furthermore, our data present a large number of putative drug targets that could be exploited for the treatment of this infection. Copyright © 2017 American Society for Microbiology.

Inhibition of herpesvirus and influenza virus replication by blocking polymerase subunit interactions.

PubMed

Palù, Giorgio; Loregian, Arianna

2013-09-01

Protein-protein interactions (PPIs) play a key role in many biological processes, including virus replication in the host cell. Since most of the PPIs are functionally essential, a possible strategy to inhibit virus replication is based on the disruption of viral protein complexes by peptides or small molecules that interfere with subunit interactions. In particular, an attractive target for antiviral drugs is the binding between the subunits of essential viral enzymes. This review describes the development of new antiviral compounds that inhibit herpesvirus and influenza virus replication by blocking interactions between subunit proteins of their polymerase complexes. Copyright © 2013 Elsevier B.V. All rights reserved.

Activity of Pure Streptovaricins and Fractionated Streptovaricin Complex Against Friend Virus

PubMed Central

Horoszewicz, Julius S.; Rinehart, Kenneth L.; Leong, Susan S.; Carter, William A.

1975-01-01

Chromatographic fractionation of streptovaricin complex yields two stable components enriched (4- to 16-fold) in activity directed against the polycythemic strain of Friend virus; both components apparently contain no streptovaricins. When compared with their unfractionated parent streptovaricin complex, eight individual intact streptovaricins (A through G and J) show at least a 30-fold reduction in antiviral activity. These results further support the conclusion that the diversified biological properties of streptovaricin complex probably reside in different molecular structures. PMID:237470

Ultrastructure of the replication sites of positive-strand RNA viruses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Harak, Christian; Lohmann, Volker, E-mail: volker_lohmann@med.uni-heidelberg.de

2015-05-15

Positive strand RNA viruses replicate in the cytoplasm of infected cells and induce intracellular membranous compartments harboring the sites of viral RNA synthesis. These replication factories are supposed to concentrate the components of the replicase and to shield replication intermediates from the host cell innate immune defense. Virus induced membrane alterations are often generated in coordination with host factors and can be grouped into different morphotypes. Recent advances in conventional and electron microscopy have contributed greatly to our understanding of their biogenesis, but still many questions remain how viral proteins capture membranes and subvert host factors for their need. Inmore » this review, we will discuss different representatives of positive strand RNA viruses and their ways of hijacking cellular membranes to establish replication complexes. We will further focus on host cell factors that are critically involved in formation of these membranes and how they contribute to viral replication. - Highlights: • Positive strand RNA viruses induce massive membrane alterations. • Despite the great diversity, replication complexes share many similarities. • Host factors play a pivotal role in replication complex biogenesis. • Use of the same host factors by several viruses hints to similar functions.« less

Genetic variability and evolutionary dynamics of viruses of the family Closteroviridae

PubMed Central

Rubio, Luis; Guerri, José; Moreno, Pedro

2013-01-01

RNA viruses have a great potential for genetic variation, rapid evolution and adaptation. Characterization of the genetic variation of viral populations provides relevant information on the processes involved in virus evolution and epidemiology and it is crucial for designing reliable diagnostic tools and developing efficient and durable disease control strategies. Here we performed an updated analysis of sequences available in Genbank and reviewed present knowledge on the genetic variability and evolutionary processes of viruses of the family Closteroviridae. Several factors have shaped the genetic structure and diversity of closteroviruses. (I) A strong negative selection seems to be responsible for the high genetic stability in space and time for some viruses. (2) Long distance migration, probably by human transport of infected propagative plant material, have caused that genetically similar virus isolates are found in distant geographical regions. (3) Recombination between divergent sequence variants have generated new genotypes and plays an important role for the evolution of some viruses of the family Closteroviridae. (4) Interaction between virus strains or between different viruses in mixed infections may alter accumulation of certain strains. (5) Host change or virus transmission by insect vectors induced changes in the viral population structure due to positive selection of sequence variants with higher fitness for host-virus or vector-virus interaction (adaptation) or by genetic drift due to random selection of sequence variants during the population bottleneck associated to the transmission process. PMID:23805130

Systematics of Aedes Mosquito Project.

DTIC Science & Technology

1987-03-12

17 lots of eggs of Aedes (Stegomyia) africanus complex and Aedes (Stegomyia) simpsoni complex from Uganda (Dr. L. G. Mukwaya, Uganda Virus Research...Heme and M. Valade. 1978. Isolement au Senegal oriental d’une souche de virus amaril a partir d’un lot d’ Aedes du sous-genre Diceromyia. C. R. Acad...transmis par les Aedes , en particulier du virus amaril. Cah. O.R.S.T.O.M., Ser. Entomol. Med. Parasitol. 17:149-163. Edwards, F. W. 1941. Mosquitoes of

The contrasting phylodynamics of human influenza B viruses

PubMed Central

Vijaykrishna, Dhanasekaran; Holmes, Edward C; Joseph, Udayan; Fourment, Mathieu; Su, Yvonne CF; Halpin, Rebecca; Lee, Raphael TC; Deng, Yi-Mo; Gunalan, Vithiagaran; Lin, Xudong; Stockwell, Timothy B; Fedorova, Nadia B; Zhou, Bin; Spirason, Natalie; Kühnert, Denise; Bošková, Veronika; Stadler, Tanja; Costa, Anna-Maria; Dwyer, Dominic E; Huang, Q Sue; Jennings, Lance C; Rawlinson, William; Sullivan, Sheena G; Hurt, Aeron C; Maurer-Stroh, Sebastian; Wentworth, David E; Smith, Gavin JD; Barr, Ian G

2015-01-01

A complex interplay of viral, host, and ecological factors shapes the spatio-temporal incidence and evolution of human influenza viruses. Although considerable attention has been paid to influenza A viruses, a lack of equivalent data means that an integrated evolutionary and epidemiological framework has until now not been available for influenza B viruses, despite their significant disease burden. Through the analysis of over 900 full genomes from an epidemiological collection of more than 26,000 strains from Australia and New Zealand, we reveal fundamental differences in the phylodynamics of the two co-circulating lineages of influenza B virus (Victoria and Yamagata), showing that their individual dynamics are determined by a complex relationship between virus transmission, age of infection, and receptor binding preference. In sum, this work identifies new factors that are important determinants of influenza B evolution and epidemiology. DOI: http://dx.doi.org/10.7554/eLife.05055.001 PMID:25594904

Cis-drivers and trans-drivers of bovine leukemia virus oncogenesis.

PubMed

Safari, Roghaiyeh; Hamaidia, Malik; de Brogniez, Alix; Gillet, Nicolas; Willems, Luc

2017-10-01

The bovine leukemia virus (BLV) is a retrovirus inducing an asymptomatic and persistent infection in ruminants and leading in a minority of cases to the accumulation of B-lymphocytes (lymphocytosis, leukemia or lymphoma). Although the mechanisms of oncogenesis are still largely unknown, there is clear experimental evidence showing that BLV infection drastically modifies the pattern of gene expression of the host cell. This alteration of the transcriptome in infected B-lymphocytes results first, from a direct activity of viral proteins (i.e. transactivation of gene promoters, protein-protein interactions), second, from insertional mutagenesis by proviral integration (cis-activation) and third, from gene silencing by microRNAs. Expression of viral proteins stimulates a vigorous immune response that indirectly modifies gene transcription in other cell types (e.g. cytotoxic T-cells, auxiliary T-cells, macrophages). In principle, insertional mutagenesis and microRNA-associated RNA interference can modify the cell fate without inducing an antiviral immunity. Despite a tight control by the immune response, the permanent attempts of the virus to replicate ultimately induce mutations in the infected cell. Accumulation of these genomic lesions and Darwinian selection of tumor clones are predicted to lead to cancer. Copyright © 2017 Elsevier B.V. All rights reserved.

Label-free Proteomic Reveals that Cowpea Severe Mosaic Virus Transiently Suppresses the Host Leaf Protein Accumulation During the Compatible Interaction with Cowpea (Vigna unguiculata [L.] Walp.).

PubMed

Paiva, Ana L S; Oliveira, Jose T A; de Souza, Gustavo A; Vasconcelos, Ilka M

2016-12-02

Viruses are important plant pathogens that threaten diverse crops worldwide. Diseases caused by Cowpea severe mosaic virus (CPSMV) have drawn attention because of the serious damages they cause to economically important crops including cowpea. This work was undertaken to quantify and identify the responsive proteins of a susceptible cowpea genotype infected with CPSMV, in comparison with mock-inoculated controls, using label-free quantitative proteomics and databanks, aiming at providing insights on the molecular basis of this compatible interaction. Cowpea leaves were mock- or CPSMV-inoculated and 2 and 6 days later proteins were extracted and analyzed. More than 3000 proteins were identified (data available via ProteomeXchange, identifier PXD005025) and 75 and 55 of them differentially accumulated in response to CPSMV, at 2 and 6 DAI, respectively. At 2 DAI, 76% of the proteins decreased in amount and 24% increased. However, at 6 DAI, 100% of the identified proteins increased. Thus, CPSMV transiently suppresses the synthesis of proteins involved particularly in the redox homeostasis, protein synthesis, defense, stress, RNA/DNA metabolism, signaling, and other functions, allowing viral invasion and spread in cowpea tissues.

Antiviral activity of the exopolysaccharide produced by Serratia sp. strain Gsm01 against Cucumber mosaic virus.

PubMed

Ipper, Nagesh S; Cho, Saeyoull; Lee, Seon Hwa; Cho, Jun Mo; Hur, Jang Hyun; Lim, Chun Keun

2008-01-01

The potential of the exopolysaccharide (EPS) from a Serratia sp. strain Gsm01 as an antiviral agent against a yellow strain of Cucumber mosaic virus (CMV-Y) was evaluated in tobacco plants (Nicotiana tabacum cv. Xanthi-nc). The spray treatment of plants using an EPS preparation, 72 before CMV-Y inoculation, protected them against symptom appearance. Fifteen days after challenge inoculation with CMVY, 33.33% of plants showed mosaic symptoms in EPS-treated plants compared with 100% in the control plants. The EPS-treated plants, which showed mosaic symptoms, appeared three days later than the controls. The enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase polymerase chain reaction (RT-PCR) analyses of the leaves of the protected plants revealed that the EPS treatment affected virus accumulation in those plants. Analysis of phenylalanine ammonia lyase, peroxidase, and phenols in protected plants revealed enhanced accumulation of these substances. The pathogenesis-related (PR) genes expression represented by PR-1b was increased in EPS-treated plants. This is the first report of a systemic induction of protection triggered by EPS produced by Serratia sp. against CMV-Y.

Research on computer virus database management system

NASA Astrophysics Data System (ADS)

Qi, Guoquan

2011-12-01

The growing proliferation of computer viruses becomes the lethal threat and research focus of the security of network information. While new virus is emerging, the number of viruses is growing, virus classification increasing complex. Virus naming because of agencies' capture time differences can not be unified. Although each agency has its own virus database, the communication between each other lacks, or virus information is incomplete, or a small number of sample information. This paper introduces the current construction status of the virus database at home and abroad, analyzes how to standardize and complete description of virus characteristics, and then gives the information integrity, storage security and manageable computer virus database design scheme.

Modeling HSV-1 Latency in Human Embryonic Stem Cell-Derived Neurons

PubMed Central

Pourchet, Aldo; Modrek, Aram S.; Placantonakis, Dimitris G.; Mohr, Ian; Wilson, Angus C.

2017-01-01

Herpes simplex virus 1 (HSV-1) uses latency in peripheral ganglia to persist in its human host, however, recurrent reactivation from this reservoir can cause debilitating and potentially life-threatening disease. Most studies of latency use live-animal infection models, but these are complex, multilayered systems and can be difficult to manipulate. Infection of cultured primary neurons provides a powerful alternative, yielding important insights into host signaling pathways controlling latency. However, small animal models do not recapitulate all aspects of HSV-1 infection in humans and are limited in terms of the available molecular tools. To address this, we have developed a latency model based on human neurons differentiated in culture from an NIH-approved embryonic stem cell line. The resulting neurons are highly permissive for replication of wild-type HSV-1, but establish a non-productive infection state resembling latency when infected at low viral doses in the presence of the antivirals acyclovir and interferon-α. In this state, viral replication and expression of a late viral gene marker are not detected but there is an accumulation of the viral latency-associated transcript (LAT) RNA. After a six-day establishment period, antivirals can be removed and the infected cultures maintained for several weeks. Subsequent treatment with sodium butyrate induces reactivation and production of new infectious virus. Human neurons derived from stem cells provide the appropriate species context to study this exclusively human virus with the potential for more extensive manipulation of the progenitors and access to a wide range of preexisting molecular tools. PMID:28594343

The relative abundance of deer mice with antibody to Sin Nombre virus corresponds to the occurrence of hantavirus pulmonary syndrome in nearby humans.

PubMed

Calisher, Charles H; Mills, James N; Root, Jon Jeffrey; Doty, Jeffrey B; Beaty, Barry J

2011-05-01

Sin Nombre virus (SNV) is the principal cause of hantavirus pulmonary syndrome (HPS) in the United States and deer mice (Peromyscus maniculatus) are its principal rodent host, and thus the natural cycle of the virus is related to the occurrence of HPS. Prevalence of rodent infection appears to be associated with fluctuations in deer mouse populations and, indirectly, with timing and amount of precipitation, a complex of biologic events. Given that rodent population abundances fluctuate, often acutely, it is not unreasonable to assume a direct correlation between the numbers of infected rodents and the number of human infections, unless confounding factors are involved. During a 13-year longitudinal study at a site in southwestern Colorado, we accumulated data regarding deer mice and antibody to SNV and therefore had the opportunity to compare dynamics of deer mouse populations, seroprevalence of antibody to SNV in the rodents, and numbers of HPS cases in Durango and in the State of Colorado as a whole. If abundances of deer mouse populations are directly correlated with occurrence of HPS, it is reasonable to assume that low densities of deer mice and low prevalences of antibody to SNV would lead to fewer human cases than would high densities and high prevalences. Our results substantiate such an assumption and suggest that the risk of acquisition of HPS is likely related to both high numbers of infected deer mice and human activities, rather than being strictly related to prevalence of SNV in the host rodent.

High-level expression of human immunodeficiency virus antigens from the tobacco and tomato plastid genomes.

PubMed

Zhou, Fei; Badillo-Corona, Jesus A; Karcher, Daniel; Gonzalez-Rabade, Nuria; Piepenburg, Katrin; Borchers, A-M Inka; Maloney, Alan P; Kavanagh, Tony A; Gray, John C; Bock, Ralph

2008-12-01

Transgene expression from the plant's plastid genome represents a promising strategy in molecular farming because of the plastid's potential to accumulate foreign proteins to high levels and the increased biosafety provided by the maternal mode of organelle inheritance. In this article, we explore the potential of transplastomic plants to produce human immunodeficiency virus (HIV) antigens as potential components of an acquired immunodeficiency syndrome (AIDS) vaccine. It is shown that the HIV antigens p24 (the major target of T-cell-mediated immune responses in HIV-positive individuals) and Nef can be expressed to high levels in plastids of tobacco, a non-food crop, and tomato, a food crop with an edible fruit. Optimized p24-Nef fusion gene cassettes trigger antigen protein accumulation to up to approximately 40% of the plant's total protein, demonstrating the great potential of transgenic plastids to produce AIDS vaccine components at low cost and high yield.

Sequence of events in measles virus replication: role of phosphoprotein-nucleocapsid interactions.

PubMed

Brunel, Joanna; Chopy, Damien; Dosnon, Marion; Bloyet, Louis-Marie; Devaux, Patricia; Urzua, Erica; Cattaneo, Roberto; Longhi, Sonia; Gerlier, Denis

2014-09-01

The genome of nonsegmented negative-strand RNA viruses is tightly embedded within a nucleocapsid made of a nucleoprotein (N) homopolymer. To ensure processive RNA synthesis, the viral polymerase L in complex with its cofactor phosphoprotein (P) binds the nucleocapsid that constitutes the functional template. Measles virus P and N interact through two binding sites. While binding of the P amino terminus with the core of N (NCORE) prevents illegitimate encapsidation of cellular RNA, the interaction between their C-terminal domains, P(XD) and N(TAIL) is required for viral RNA synthesis. To investigate the binding dynamics between the two latter domains, the P(XD) F497 residue that makes multiple hydrophobic intramolecular interactions was mutated. Using a quantitative mammalian protein complementation assay and recombinant viruses, we found that an increase in P(XD)-to-N(TAIL) binding strength is associated with a slower transcript accumulation rate and that abolishing the interaction renders the polymerase nonfunctional. The use of a newly developed system allowing conditional expression of wild-type or mutated P genes, revealed that the loss of the P(XD)-N(TAIL) interaction results in reduced transcription by preformed transcriptases, suggesting reduced engagement on the genomic template. These intracellular data indicate that the viral polymerase entry into and progression along its genomic template relies on a protein-protein interaction that serves as a tightly controlled dynamic anchor. Mononegavirales have a unique machinery to replicate RNA. Processivity of their polymerase is only achieved when the genome template is entirely embedded into a helical homopolymer of nucleoproteins that constitutes the nucleocapsid. The polymerase binds to the nucleocapsid template through the phosphoprotein. How the polymerase complex enters and travels along the nucleocapsid template to ensure uninterrupted synthesis of up to ∼ 6,700-nucleotide messenger RNAs from six to ten consecutive genes is unknown. Using a quantitative protein complementation assay and a biGene-biSilencing system allowing conditional expression of two P genes copies, the role of the P-to-N interaction in polymerase function was further characterized. We report here a dynamic protein anchoring mechanism that differs from all other known polymerases that rely only onto a sustained and direct binding to their nucleic acid template. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Effect of Prior Influenza Virus Infection on Susceptibility of AKR/J Mice and Squirrel Monkeys to Respiratory Challenge with Legionella pneumophila.

DTIC Science & Technology

1980-07-30

Infection on Susceptibility of AKR/J Mice and Squirrel Monkeys to Respiratory Challenge with Legionella pneumophila RICHARD F. BERENDT U. S. Army Medical...influenza virus and Legionella pneumophila than to either agent alone. b 3 F As knowledge of Legionnaires’ disease has accumulated, the evidence...suggests that many infections occur in individuals with underlying disease. Since Legionella pneumophila appears to spread by the airborne route (5, 6, 8, 9

PML mediates the interferon-induced antiviral state against a complex retrovirus via its association with the viral transactivator

PubMed Central

Regad, Tarik; Saib, Ali; Lallemand-Breitenbach, Valérie; Pandolfi, Pier Paolo; de Thé, Hugues; Chelbi-Alix, Mounira K.

2001-01-01

The promyelocytic leukaemia (PML) protein localizes in the nucleus both in the nucleoplasm and in matrix-associated multiprotein complexes known as nuclear bodies (NBs). The number and the intensity of PML NBs increase in response to interferon (IFN). Overexpression of PML affects the replication of vesicular stomatitis virus and influenza virus. However, PML has a less powerful antiviral activity against these viruses than the IFN mediator MxA. Here, we show that overexpression of PML, but not that of Mx1 or MxA, leads to a drastic decrease of a complex retrovirus, the human foamy virus (HFV), gene expression. PML represses HFV transcription by complexing the HFV transactivator, Tas, preventing its direct binding to viral DNA. This physical interaction requires the N-terminal region of Tas and the RING finger of PML, but does not necessitate PML localization in NBs. Finally, we show that IFN treatment inhibits HFV replication in wild-type but not in PML–/– cells. These findings point to a role for PML in transcriptional repression and suggest that PML could play a key role in mediating an IFN-induced antiviral state against a complex retrovirus. PMID:11432836

Identification of an attenuated barley stripe mosaic virus for the virus-induced gene silencing of pathogenesis-related wheat genes.

PubMed

Buhrow, Leann M; Clark, Shawn M; Loewen, Michele C

2016-01-01

Virus-induced gene silencing (VIGS) has become an emerging technology for the rapid, efficient functional genomic screening of monocot and dicot species. The barley stripe mosaic virus (BSMV) has been described as an effective VIGS vehicle for the evaluation of genes involved in wheat and barley phytopathogenesis; however, these studies have been obscured by BSMV-induced phenotypes and defense responses. The utility of BSMV VIGS may be improved using a BSMV genetic background which is more tolerable to the host plant especially upon secondary infection of highly aggressive, necrotrophic pathogens such as Fusarium graminearum. BSMV-induced VIGS in Triticum aestivum (bread wheat) cv. 'Fielder' was assessed for the study of wheat genes putatively related to Fusarium Head Blight (FHB), the necrotrophism of wheat and other cereals by F. graminearum. Due to the lack of 'Fielder' spike viability and increased accumulation of Fusarium-derived deoxynivalenol contamination upon co-infection of BSMV and FHB, an attenuated BSMV construct was generated by the addition of a glycine-rich, C-terminal peptide to the BSMV γ b protein. This attenuated BSMV effectively silenced target wheat genes while limiting disease severity, deoxynivalenol contamination, and yield loss upon Fusarium co-infection compared to the original BSMV construct. The attenuated BSMV-infected tissue exhibited reduced abscisic, jasmonic, and salicylic acid defense phytohormone accumulation upon secondary Fusarium infection. Finally, the attenuated BSMV was used to investigate the role of the salicylic acid-responsive pathogenesis-related 1 in response to FHB. The use of an attenuated BSMV may be advantageous in characterizing wheat genes involved in phytopathogenesis, including Fusarium necrotrophism, where minimal viral background effects on defense are required. Additionally, the attenuated BSMV elicits reduced defense hormone accumulation, suggesting that this genotype may have applications for the investigation of phytohormone-related signaling, developmental responses, and pathogen defense.

The effect of infectious bursal disease virus induced immunosuppression on vaccination against highly pathogenic avian influenza virus

USDA-ARS?s Scientific Manuscript database

Poor efficacy of avian influenza virus (AIV) vaccines in chickens has been documented in the field in spite of good results in experimental settings. Although the causes are multi-factorial and complex, one contributing factor may be prior infection with immunosuppressive viruses. In an effort to ...

Replication of honey bee-associated RNA viruses across multiple bee species in apple orchards of Georgia, Germany and Kyrgyzstan.

PubMed

Radzevičiūtė, Rita; Theodorou, Panagiotis; Husemann, Martin; Japoshvili, George; Kirkitadze, Giorgi; Zhusupbaeva, Aigul; Paxton, Robert J

2017-06-01

The essential ecosystem service of pollination is provided largely by insects, which are considered threatened by diverse biotic and abiotic global change pressures. RNA viruses are one such pressure, and have risen in prominence as a major threat for honey bees (Apis mellifera) and global apiculture, as well as a risk factor for other bee species through pathogen spill-over between managed honey bees and sympatric wild pollinator communities. Yet despite their potential role in global bee decline, the prevalence of honey bee-associated RNA viruses in wild bees is poorly known from both geographic and taxonomic perspectives. We screened members of pollinator communities (honey bees, bumble bees and other wild bees belonging to four families) collected from apple orchards in Georgia, Germany and Kyrgyzstan for six common honey bee-associated RNA virus complexes encompassing nine virus targets. The Deformed wing virus complex (DWV genotypes A and B) had the highest prevalence across all localities and host species and was the only virus complex found in wild bee species belonging to all four studied families. Based on amplification of negative-strand viral RNA, we found evidence for viral replication in wild bee species of DWV-A/DWV-B (hosts: Andrena haemorrhoa and several Bombus spp.) and Black queen cell virus (hosts: Anthophora plumipes, several Bombus spp., Osmia bicornis and Xylocopa spp.). Viral amplicon sequences revealed that DWV-A and DWV-B are regionally distinct but identical in two or more bee species at any one site, suggesting virus is shared amongst sympatric bee taxa. This study demonstrates that honey bee associated RNA viruses are geographically and taxonomically widespread, likely infective in wild bee species, and shared across bee taxa. Copyright © 2017 Elsevier Inc. All rights reserved.

Effects of Inner Nuclear Membrane Proteins SUN1/UNC-84A and SUN2/UNC-84B on the Early Steps of HIV-1 Infection.

PubMed

Schaller, Torsten; Bulli, Lorenzo; Pollpeter, Darja; Betancor, Gilberto; Kutzner, Juliane; Apolonia, Luis; Herold, Nikolas; Burk, Robin; Malim, Michael H

2017-10-01

Human immunodeficiency virus type 1 (HIV-1) infection of dividing and nondividing cells involves regulatory interactions with the nuclear pore complex (NPC), followed by translocation to the nucleus and preferential integration into genomic areas in proximity to the inner nuclear membrane (INM). To identify host proteins that may contribute to these processes, we performed an overexpression screen of known membrane-associated NE proteins. We found that the integral transmembrane proteins SUN1/UNC84A and SUN2/UNC84B are potent or modest inhibitors of HIV-1 infection, respectively, and that suppression corresponds to defects in the accumulation of viral cDNA in the nucleus. While laboratory strains (HIV-1 NL4.3 and HIV-1 IIIB ) are sensitive to SUN1-mediated inhibition, the transmitted founder viruses RHPA and ZM247 are largely resistant. Using chimeric viruses, we identified the HIV-1 capsid (CA) protein as a major determinant of sensitivity to SUN1, and in vitro -assembled capsid-nucleocapsid (CANC) nanotubes captured SUN1 and SUN2 from cell lysates. Finally, we generated SUN1 -/- and SUN2 -/- cells by using CRISPR/Cas9 and found that the loss of SUN1 had no effect on HIV-1 infectivity, whereas the loss of SUN2 had a modest suppressive effect. Taken together, these observations suggest that SUN1 and SUN2 may function redundantly to modulate postentry, nuclear-associated steps of HIV-1 infection. IMPORTANCE HIV-1 causes more than 1 million deaths per year. The life cycle of HIV-1 has been studied extensively, yet important steps that occur between viral capsid release into the cytoplasm and the expression of viral genes remain elusive. We propose here that the INM components SUN1 and SUN2, two members of the linker of nucleoskeleton and cytoskeleton (LINC) complex, may interact with incoming HIV-1 replication complexes and affect key steps of infection. While overexpression of these proteins reduces HIV-1 infection, disruption of the individual SUN2 and SUN1 genes leads to a mild reduction or no effect on infectivity, respectively. We speculate that SUN1/SUN2 may function redundantly in early HIV-1 infection steps and therefore influence HIV-1 replication and pathogenesis. Copyright © 2017 Schaller et al.

Prasinoviruses reveal a complex evolutionary history and a patchy environmental distribution

NASA Astrophysics Data System (ADS)

Finke, J. F.; Suttle, C.

2016-02-01

Prasinophytes constitute a group of eukaryotic phytoplankton that has a global distribution and is a major component of coastal and oceanic communities. Members of this group are infected by large double-stranded DNA viruses that can be significant agents of mortality, and which show evidence of substantial horizontal transfer of genes from their hosts and other organisms. However, information on the genetic diversity of these viruses and their environmental distribution is limited. This study examines the genetic repertoire, phylogeny and environmental distribution of large double-stranded DNA viruses infecting Micromonas pusilla and other prasinophytes. The genomes of viruses infecting M. pusilla were sequenced and compared to those of viruses infecting other prasinophytes, revealing a relatively small set of core genes and a larger flexible pan genome. Comparing genomes among prasinoviruses highlights their variable genetic content and complex evolutionary history. While some of the pan genome is clearly host derived, many open reading frames are most similar to those found in other eukaryotes and bacteria. Gene content of the viruses is is congruent with phylogenetic analysis of viral DNA polymerase sequences and indicates that two clades of M. pusilla viruses are less related to each other than to other prasinoviruses. Moreover, the environmental distribution of prasinovirus DNA polymerase sequences indicates a complex pattern of virus-host interactions in nature. Ultimately, these patterns are influenced by the genetic repertoire encoded by prasinoviruses, and the distribution of the hosts they infect.

Antibodies to the Glycoprotein GP2 Subunit Cross-React between Old and New World Arenaviruses.

PubMed

Amanat, Fatima; Duehr, James; Oestereich, Lisa; Hastie, Kathryn M; Ollmann Saphire, Erica; Krammer, Florian

2018-01-01

Arenaviruses pose a major public health threat and cause numerous infections in humans each year. Although most viruses belonging to this family do not cause disease in humans, some arenaviruses, such as Lassa virus and Machupo virus, are the etiological agents of lethal hemorrhagic fevers. The absence of a currently licensed vaccine and the highly pathogenic nature of these viruses both make the necessity of developing viable vaccines and therapeutics all the more urgent. Arenaviruses have a single glycoprotein on the surface of virions, the glycoprotein complex (GPC), and this protein can be used as a target for vaccine development. Here, we describe immunization strategies to generate monoclonal antibodies (MAbs) that cross-react between the glycoprotein complexes of both Old World and New World arenaviruses. Several monoclonal antibodies isolated from immunized mice were highly cross-reactive, binding a range of Old World arenavirus glycoproteins, including that of Lassa virus. One such monoclonal antibody, KL-AV-2A1, bound to GPCs of both New World and Old World viruses, including Lassa and Machupo viruses. These cross-reactive antibodies bound to epitopes present on the glycoprotein 2 subunit of the glycoprotein complex, which is relatively conserved among arenaviruses. Monoclonal antibodies binding to these epitopes, however, did not inhibit viral entry as they failed to neutralize a replication-competent vesicular stomatitis virus pseudotyped with the Lassa virus glycoprotein complex in vitro In addition, no protection from virus challenge was observed in in vivo mouse models. Even so, these monoclonal antibodies might still prove to be useful in the development of clinical and diagnostic assays. IMPORTANCE Several viruses in the Arenaviridae family infect humans and cause severe hemorrhagic fevers which lead to high case fatality rates. Due to their pathogenicity and geographic tropisms, these viruses remain very understudied. As a result, an effective vaccine or therapy is urgently needed. Here, we describe efforts to produce cross-reactive monoclonal antibodies that bind to both New and Old World arenaviruses. All of our MAbs seem to be nonneutralizing and nonprotective and target subunit 2 of the glycoprotein. Due to the lack of reagents such as recombinant glycoproteins and antibodies for rapid detection assays, our MAbs could be beneficial as analytic and diagnostic tools. Copyright © 2018 Amanat et al.

Dynamic properties of epidemic spreading on finite size complex networks

NASA Astrophysics Data System (ADS)

Li, Ying; Liu, Yang; Shan, Xiu-Ming; Ren, Yong; Jiao, Jian; Qiu, Ben

2005-11-01

The Internet presents a complex topological structure, on which computer viruses can easily spread. By using theoretical analysis and computer simulation methods, the dynamic process of disease spreading on finite size networks with complex topological structure is investigated. On the finite size networks, the spreading process of SIS (susceptible-infected-susceptible) model is a finite Markov chain with an absorbing state. Two parameters, the survival probability and the conditional infecting probability, are introduced to describe the dynamic properties of disease spreading on finite size networks. Our results can help understanding computer virus epidemics and other spreading phenomena on communication and social networks. Also, knowledge about the dynamic character of virus spreading is helpful for adopting immunity policy.

Non-classical phase diagram for virus bacterial coevolution mediated by clustered regularly interspaced short palindromic repeats.

PubMed

Han, Pu; Deem, Michael W

2017-02-01

CRISPR is a newly discovered prokaryotic immune system. Bacteria and archaea with this system incorporate genetic material from invading viruses into their genomes, providing protection against future infection by similar viruses. The condition for coexistence of prokaryots and viruses is an interesting problem in evolutionary biology. In this work, we show an intriguing phase diagram of the virus extinction probability, which is more complex than that of the classical predator-prey model. As the CRISPR incorporates genetic material, viruses are under pressure to evolve to escape recognition by CRISPR. When bacteria have a small rate of deleting spacers, a new parameter region in which bacteria and viruses can coexist arises, and it leads to a more complex coexistence patten for bacteria and viruses. For example, when the virus mutation rate is low, the virus extinction probability changes non-montonically with the bacterial exposure rate. The virus and bacteria coevolution not only alters the virus extinction probability, but also changes the bacterial population structure. Additionally, we show that recombination is a successful strategy for viruses to escape from CRISPR recognition when viruses have multiple proto-spacers, providing support for a recombination-mediated escape mechanism suggested experimentally. Finally, we suggest that the re-entrant phase diagram, in which phages can progress through three phases of extinction and two phases of abundance at low spacer deletion rates as a function of exposure rate to bacteria, is an experimentally testable phenomenon. © 2017 The Author(s).

Arenavirus Stable Signal Peptide Is the Keystone Subunit for Glycoprotein Complex Organization

PubMed Central

Bederka, Lydia H.; Bonhomme, Cyrille J.; Ling, Emily L.

2014-01-01

ABSTRACT The rodent arenavirus glycoprotein complex encodes a stable signal peptide (SSP) that is an essential structural component of mature virions. The SSP, GP1, and GP2 subunits of the trimeric glycoprotein complex noncovalently interact to stud the surface of virions and initiate arenavirus infectivity. Nascent glycoprotein production undergoes two proteolytic cleavage events: first within the endoplasmic reticulum (ER) to cleave SSP from the remaining precursor GP1/2 (glycoprotein complex [GPC]) glycoprotein and second within the Golgi stacks by the cellular SKI-1/S1P for GP1/2 processing to yield GP1 and GP2 subunits. Cleaved SSP is not degraded but retained as an essential glycoprotein subunit. Here, we defined functions of the 58-amino-acid lymphocytic choriomeningitis virus (LCMV) SSP in regard to glycoprotein complex processing and maturation. Using molecular biology techniques, confocal microscopy, and flow cytometry, we detected SSP at the plasma membrane of transfected cells. Further, we identified a sorting signal (FLLL) near the carboxyl terminus of SSP that is required for glycoprotein maturation and trafficking. In the absence of SSP, the glycoprotein accumulated within the ER and was unable to undergo processing by SKI-1/S1P. Mutation of this highly conserved FLLL motif showed impaired glycoprotein processing and secretory pathway trafficking, as well as defective surface expression and pH-dependent membrane fusion. Immunoprecipitation of SSP confirmed an interaction between the signal peptide and the GP2 subunit; however, mutations within this FLLL motif disrupted the association of the GP1 subunit with the remaining glycoprotein complex. PMID:25352624

Possible viruses from outer space fall into the Earth's atmosphere

NASA Astrophysics Data System (ADS)

Steklov, A. F.; Dashkiev, G. N.; Vidmachenko, A. P.

2017-05-01

Statistical data show that sometime after the passage of the Earth along its orbit through the tail of a comet, a number of epidemics and pandemics occurred. This indicated a possible invasion of viruses, which could be in cometary dust. K.I. Churyumov proposed to develop special traps. They need to be placed under the wings of high-altitude aircraft. And with their help it is necessary to catch, accumulate and examine in the laboratories those particles, which are captured in traces of invasions. The main purpose of such experiments is to reveal, or prove the absence of cosmic viruses in the tracks from the intrusions of fragments of cometary nuclei.

An abyssal mobilome: viruses, plasmids and vesicles from deep-sea hydrothermal vents.

PubMed

Lossouarn, Julien; Dupont, Samuel; Gorlas, Aurore; Mercier, Coraline; Bienvenu, Nadege; Marguet, Evelyne; Forterre, Patrick; Geslin, Claire

2015-12-01

Mobile genetic elements (MGEs) such as viruses, plasmids, vesicles, gene transfer agents (GTAs), transposons and transpovirions, which collectively represent the mobilome, interact with cellular organisms from all three domains of life, including those thriving in the most extreme environments. While efforts have been made to better understand deep-sea vent microbial ecology, our knowledge of the mobilome associated with prokaryotes inhabiting deep-sea hydrothermal vents remains limited. Here we focus on the abyssal mobilome by reviewing accumulating data on viruses, plasmids and vesicles associated with thermophilic and hyperthermophilic Bacteria and Archaea present in deep-sea hydrothermal vents. Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

[Virus-like inclusions in the cells of the central nervous system in amyotrophic lateral sclerosis].

PubMed

Popova, L M; Sakharova, A V

1982-01-01

Electron microscopic examination of the frontal cortex of a patient with amyotrophic lateral sclerosis (ALS) with 14-year prolongation of life by artificial ventilation of the lungs revealed heretofore undescribed cytoplasmic virus-like inclusions in neurons and glial cells as well as changes in the granular endoplasmic reticulum in astrocytes consisting of large accumulations of ribosomes in which regular arrays were frequently observed. The discovered inclusions were not similar to any identified viral inclusions but were similar to virus-like particles found in the muscle in ALS and viral inclusions in experimental poliomyelitis. The role of the observed inclusions and their etiological importance remain obscure.

Transcriptome analysis provides insights into the delayed sticky disease symptoms in Carica papaya.

PubMed

Madroñero, Johana; Rodrigues, Silas P; Antunes, Tathiana F S; Abreu, Paolla M V; Ventura, José A; Fernandes, A Alberto R; Fernandes, Patricia Machado Bueno

2018-03-21

Global gene expression analysis indicates host stress responses, mainly those mediated by SA, associated to the tolerance to sticky disease symptoms at pre-flowering stage in Carica papaya. Carica papaya plants develop the papaya sticky disease (PSD) as a result of the combined infection of papaya meleira virus (PMeV) and papaya meleira virus 2 (PMeV2), or PMeV complex. PSD symptoms appear only after C. papaya flowers. To understand the mechanisms involved in this phenomenon, the global gene expression patterns of PMeV complex-infected C. papaya at pre-and post-flowering stages were assessed by RNA-Seq. The result was 633 and 88 differentially expressed genes at pre- and post-flowering stages, respectively. At pre-flowering stage, genes related to stress and transport were up-regulated while metabolism-related genes were down-regulated. It was observed that induction of several salicylic acid (SA)-activated genes, including PR1, PR2, PR5, WRKY transcription factors, ROS and callose genes, suggesting SA signaling involvement in the delayed symptoms. In fact, pre-flowering C. papaya treated with exogenous SA showed a tendency to decrease the PMeV and PMeV2 loads when compared to control plants. However, pre-flowering C. papaya also accumulated transcripts encoding a NPR1-inhibitor (NPR1-I/NIM1-I) candidate, genes coding for UDP-glucosyltransferases (UGTs) and several genes involved with ethylene pathway, known to be negative regulators of SA signaling. At post-flowering, when PSD symptoms appeared, the down-regulation of PR-1 encoding gene and the induction of BSMT1 and JA metabolism-related genes were observed. Hence, SA signaling likely operates at the pre-flowering stage of PMeV complex-infected C. papaya inhibiting the development of PSD symptoms, but the induction of its negative regulators prevents the full-scale and long-lasting tolerance.

RNA-dependent RNA polymerase complex of Brome mosaic virus: analysis of the molecular structure with monoclonal antibodies.

PubMed

Dohi, Koji; Mise, Kazuyuki; Furusawa, Iwao; Okuno, Tetsuro

2002-11-01

Viral RNA-dependent RNA polymerase (RdRp) plays crucial roles in the genomic replication and subgenomic transcription of Brome mosaic virus (BMV), a positive-stranded RNA plant virus. BMV RdRp is a complex of virus-encoded 1a and 2a proteins and some cellular factors, and associates with the endoplasmic reticulum at an infection-specific structure in the cytoplasm of host cells. In this study, we investigate the gross structure of the active BMV RdRp complex using monoclonal antibodies raised against the 1a and 2a proteins. Immunoprecipitation experiments showed that the intermediate region between the N-terminal methyltransferase-like domain and the C-terminal helicase-like domain of 1a protein, and the N terminus region of 2a protein are exposed on the surface of the solubilized RdRp complex. Inhibition assays for membrane-bound RdRp suggested that the intermediate region between the methyltransferase-like and the helicase-like domains of 1a protein is located at the border of the region buried within a membrane structure or with membrane-associated material.

Monkeypox Virus Host Factor Screen Using Haploid Cells Identifies Essential Role of GARP Complex in Extracellular Virus Formation.

PubMed

Realegeno, Susan; Puschnik, Andreas S; Kumar, Amrita; Goldsmith, Cynthia; Burgado, Jillybeth; Sambhara, Suryaprakash; Olson, Victoria A; Carroll, Darin; Damon, Inger; Hirata, Tetsuya; Kinoshita, Taroh; Carette, Jan E; Satheshkumar, Panayampalli Subbian

2017-06-01

Monkeypox virus (MPXV) is a human pathogen that is a member of the Orthopoxvirus genus, which includes Vaccinia virus and Variola virus (the causative agent of smallpox). Human monkeypox is considered an emerging zoonotic infectious disease. To identify host factors required for MPXV infection, we performed a genome-wide insertional mutagenesis screen in human haploid cells. The screen revealed several candidate genes, including those involved in Golgi trafficking, glycosaminoglycan biosynthesis, and glycosylphosphatidylinositol (GPI)-anchor biosynthesis. We validated the role of a set of vacuolar protein sorting (VPS) genes during infection, VPS51 to VPS54 (VPS51-54), which comprise the Golgi-associated retrograde protein (GARP) complex. The GARP complex is a tethering complex involved in retrograde transport of endosomes to the trans -Golgi apparatus. Our data demonstrate that VPS52 and VPS54 were dispensable for mature virion (MV) production but were required for extracellular virus (EV) formation. For comparison, a known antiviral compound, ST-246, was used in our experiments, demonstrating that EV titers in VPS52 and VPS54 knockout (KO) cells were comparable to levels exhibited by ST-246-treated wild-type cells. Confocal microscopy was used to examine actin tail formation, one of the viral egress mechanisms for cell-to-cell dissemination, and revealed an absence of actin tails in VPS52KO- or VPS54KO-infected cells. Further evaluation of these cells by electron microscopy demonstrated a decrease in levels of wrapped viruses (WVs) compared to those seen with the wild-type control. Collectively, our data demonstrate the role of GARP complex genes in double-membrane wrapping of MVs necessary for EV formation, implicating the host endosomal trafficking pathway in orthopoxvirus infection. IMPORTANCE Human monkeypox is an emerging zoonotic infectious disease caused by Monkeypox virus (MPXV). Of the two MPXV clades, the Congo Basin strain is associated with severe disease, increased mortality, and increased human-to-human transmission relative to the West African strain. Monkeypox is endemic in regions of western and central Africa but was introduced into the United States in 2003 from the importation of infected animals. The threat of MPXV and other orthopoxviruses is increasing due to the absence of routine smallpox vaccination leading to a higher proportion of naive populations. In this study, we have identified and validated candidate genes that are required for MPXV infection, specifically, those associated with the Golgi-associated retrograde protein (GARP) complex. Identifying host targets required for infection that prevents extracellular virus formation such as the GARP complex or the retrograde pathway can provide a potential target for antiviral therapy. Copyright © 2017 American Society for Microbiology.

History matching of a complex epidemiological model of human immunodeficiency virus transmission by using variance emulation.

PubMed

Andrianakis, I; Vernon, I; McCreesh, N; McKinley, T J; Oakley, J E; Nsubuga, R N; Goldstein, M; White, R G

2017-08-01

Complex stochastic models are commonplace in epidemiology, but their utility depends on their calibration to empirical data. History matching is a (pre)calibration method that has been applied successfully to complex deterministic models. In this work, we adapt history matching to stochastic models, by emulating the variance in the model outputs, and therefore accounting for its dependence on the model's input values. The method proposed is applied to a real complex epidemiological model of human immunodeficiency virus in Uganda with 22 inputs and 18 outputs, and is found to increase the efficiency of history matching, requiring 70% of the time and 43% fewer simulator evaluations compared with a previous variant of the method. The insight gained into the structure of the human immunodeficiency virus model, and the constraints placed on it, are then discussed.

Expression of Functional Influenza Virus RNA Polymerase in the Methylotrophic Yeast Pichia pastoris

PubMed Central

Hwang, Jung-Shan; Yamada, Kazunori; Honda, Ayae; Nakade, Kohji; Ishihama, Akira

2000-01-01

Influenza virus RNA polymerase with the subunit composition PB1-PB2-PA is a multifunctional enzyme with the activities of both synthesis and cleavage of RNA and is involved in both transcription and replication of the viral genome. In order to produce large amounts of the functional viral RNA polymerase sufficient for analysis of its structure-function relationships, the cDNAs for RNA segments 1, 2, and 3 of influenza virus A/PR/8, each under independent control of the alcohol oxidase gene promoter, were integrated into the chromosome of the methylotrophic yeast Pichia pastoris. Simultaneous expression of all three P proteins in the yeast P. pastoris was achieved by the addition of methanol. To purify the P protein complexes, a sequence coding for a histidine tag was added to the PB2 protein gene at its N terminus. Starting from the induced P. pastoris cell lysate, we partially purified a 3P complex by Ni2+-agarose affinity column chromatography. The 3P complex showed influenza virus model RNA-directed and ApG-primed RNA synthesis in vitro but was virtually inactive without addition of template or primer. The kinetic properties of model template-directed RNA synthesis and the requirements for template sequence were analyzed using the 3P complex. Furthermore, the 3P complex showed capped RNA-primed RNA synthesis. Thus, we conclude that functional influenza virus RNA polymerase with the catalytic properties of a transcriptase is formed in the methylotrophic yeast P. pastoris. PMID:10756019

Ectromelia virus encodes a novel family of F-box proteins that interact with the SCF complex.

PubMed

van Buuren, Nick; Couturier, Brianne; Xiong, Yue; Barry, Michele

2008-10-01

Poxviruses are notorious for encoding multiple proteins that regulate cellular signaling pathways, including the ubiquitin-proteasome system. Bioinformatics indicated that ectromelia virus, the causative agent of lethal mousepox, encoded four proteins, EVM002, EVM005, EVM154, and EVM165, containing putative F-box domains. In contrast to cellular F-box proteins, the ectromelia virus proteins contain C-terminal F-box domains in conjunction with N-terminal ankyrin repeats, a combination that has not been previously reported for cellular proteins. These observations suggested that the ectromelia virus F-box proteins interact with SCF (Skp1, cullin-1, and F-box) ubiquitin ligases. We focused our studies on EVM005, since this protein had only one ortholog in cowpox virus. Using mass spectrometry, we identified cullin-1 as a binding partner for EVM005, and this interaction was confirmed by overexpression of hemagglutinin (HA)-cullin-1. During infection, Flag-EVM005 and HA-cullin-1 colocalized to distinct cellular bodies. Significantly, EVM005 coprecipitated with endogenous Skp1, cullin-1, and Roc1 and associated with conjugated ubiquitin, suggesting that EVM005 interacted with the components of a functional ubiquitin ligase. Interaction of EVM005 with cullin-1 and Skp1 was abolished upon deletion of the F-box, indicating that the F-box played a crucial role in interaction with the SCF complex. Additionally, EVM002 and EVM154 interacted with Skp1 and conjugated ubiquitin, suggesting that ectromelia virus encodes multiple F-box-containing proteins that regulate the SCF complex. Our results indicate that ectromelia virus has evolved multiple proteins that interact with the SCF complex.

Comparative analysis of chrysanthemum transcriptome in response to three RNA viruses: Cucumber mosaic virus, Tomato spotted wilt virus and Potato virus X.

PubMed

Choi, Hoseong; Jo, Yeonhwa; Lian, Sen; Jo, Kyoung-Min; Chu, Hyosub; Yoon, Ju-Yeon; Choi, Seung-Kook; Kim, Kook-Hyung; Cho, Won Kyong

2015-06-01

The chrysanthemum is one of popular flowers in the world and a host for several viruses. So far, molecular interaction studies between the chrysanthemum and viruses are limited. In this study, we carried out a transcriptome analysis of chrysanthemum in response to three different viruses including Cucumber mosaic virus (CMV), Tomato spotted wilt virus (TSWV) and Potato virus X (PVX). A chrysanthemum 135K microarray derived from expressed sequence tags was successfully applied for the expression profiles of the chrysanthemum at early stage of virus infection. Finally, we identified a total of 125, 70 and 124 differentially expressed genes (DEGs) for CMV, TSWV and PVX, respectively. Many DEGs were virus specific; however, 33 DEGs were commonly regulated by three viruses. Gene ontology (GO) enrichment analysis identified a total of 132 GO terms, and of them, six GO terms related stress response and MCM complex were commonly identified for three viruses. Several genes functioning in stress response such as chitin response and ethylene mediated signaling pathway were up-regulated indicating their involvement in establishment of host immune system. In particular, TSWV infection significantly down-regulated genes related to DNA metabolic process including DNA replication, chromatin organization, histone modification and cytokinesis, and they are mostly targeted to nucleosome and MCM complex. Taken together, our comparative transcriptome analysis revealed several genes related to hormone mediated viral stress response and DNA modification. The identified chrysanthemums genes could be good candidates for further functional study associated with resistant to various plant viruses.

Tinkering with Translation: Protein Synthesis in Virus-Infected Cells

PubMed Central

Walsh, Derek; Mathews, Michael B.; Mohr, Ian

2013-01-01

Viruses are obligate intracellular parasites, and their replication requires host cell functions. Although the size, composition, complexity, and functions encoded by their genomes are remarkably diverse, all viruses rely absolutely on the protein synthesis machinery of their host cells. Lacking their own translational apparatus, they must recruit cellular ribosomes in order to translate viral mRNAs and produce the protein products required for their replication. In addition, there are other constraints on viral protein production. Crucially, host innate defenses and stress responses capable of inactivating the translation machinery must be effectively neutralized. Furthermore, the limited coding capacity of the viral genome needs to be used optimally. These demands have resulted in complex interactions between virus and host that exploit ostensibly virus-specific mechanisms and, at the same time, illuminate the functioning of the cellular protein synthesis apparatus. PMID:23209131

Structure of an Antibody in Complex with Its Mucin Domain Linear Epitope That Is Protective against Ebola Virus

PubMed Central

Olal, Daniel; Kuehne, Ana I.; Bale, Shridhar; Halfmann, Peter; Hashiguchi, Takao; Fusco, Marnie L.; Lee, Jeffrey E.; King, Liam B.; Kawaoka, Yoshihiro; Dye, John M.

2012-01-01

Antibody 14G7 is protective against lethal Ebola virus challenge and recognizes a distinct linear epitope in the prominent mucin-like domain of the Ebola virus glycoprotein GP. The structure of 14G7 in complex with its linear peptide epitope has now been determined to 2.8 Å. The structure shows that this GP sequence forms a tandem β-hairpin structure that binds deeply into a cleft in the antibody-combining site. A key threonine at the apex of one turn is critical for antibody interaction and is conserved among all Ebola viruses. This work provides further insight into the mechanism of protection by antibodies that target the protruding, highly accessible mucin-like domain of Ebola virus and the structural framework for understanding and characterizing candidate immunotherapeutics. PMID:22171276

Structure of an antibody in complex with its mucin domain linear epitope that is protective against Ebola virus.

PubMed

Olal, Daniel; Kuehne, Ana I; Bale, Shridhar; Halfmann, Peter; Hashiguchi, Takao; Fusco, Marnie L; Lee, Jeffrey E; King, Liam B; Kawaoka, Yoshihiro; Dye, John M; Saphire, Erica Ollmann

2012-03-01

Antibody 14G7 is protective against lethal Ebola virus challenge and recognizes a distinct linear epitope in the prominent mucin-like domain of the Ebola virus glycoprotein GP. The structure of 14G7 in complex with its linear peptide epitope has now been determined to 2.8 Å. The structure shows that this GP sequence forms a tandem β-hairpin structure that binds deeply into a cleft in the antibody-combining site. A key threonine at the apex of one turn is critical for antibody interaction and is conserved among all Ebola viruses. This work provides further insight into the mechanism of protection by antibodies that target the protruding, highly accessible mucin-like domain of Ebola virus and the structural framework for understanding and characterizing candidate immunotherapeutics.

Whole body positron emission tomography imaging of simian immunodeficiency virus-infected rhesus macaques.

PubMed Central

Scharko, A M; Perlman, S B; Hinds PW2nd; Hanson, J M; Uno, H; Pauza, C D

1996-01-01

Pathogenesis of simian immunodeficiency virus (SIV) infection in rhesus macaques begins with acute viremia and then progresses to a distributed infection in the solid lymphoid tissues, which is followed by a process of cellular destruction leading to terminal disease and death. Blood and tissue specimens show the progress of infection at the cellular level but do not reveal the pattern of infection and host responses occurring throughout the body. The purpose of this investigation was to determine whether positron emission tomography (PET) imaging with intravenous 2-18F-2-deoxyglucose (FDG) could identify activated lymphoid tissues in a living animal and whether this pattern would reflect the extent of SIV infection. PET images from SIV-infected animals were distinguishable from uninfected controls and revealed a pattern consistent with widespread lymphoid tissue activation. Significant FDG accumulation in colon along with mesenteric and ileocaecal lymph nodes was found in SIV infection, especially during terminal disease stages. Areas of elevated FDG uptake in the PET images were correlated with productive SIV infection using in situ hybridization as a test for virus replication. PET-FDG images of SIV-infected animals correlated sites of virus replication with high FDG accumulation. These data show that the method can be used to evaluate the distribution and activity of infected tissues in a living animal without biopsy. Fewer tissues had high FDG uptake in terminal animals than midstage animals, and both were clearly distinguishable from uninfected animal scans. Images Fig. 1 Fig. 2 Fig. 3 PMID:8692831

Viruses of parasites as actors in the parasite-host relationship: A "ménage à trois".

PubMed

Gómez-Arreaza, Amaranta; Haenni, Anne-Lise; Dunia, Irene; Avilán, Luisana

2017-02-01

The complex parasite-host relationship involves multiple mechanisms. Moreover, parasites infected by viruses modify this relationship adding more complexity to the system that now comprises three partners. Viruses infecting parasites were described several decades ago. However, until recently little was known about the viruses involved and their impact on the resulting disease caused to the hosts. To clarify this situation, we have concentrated on parasitic diseases caused to humans and on how virus-infected parasites could alter the symptoms inflicted on the human host. It is clear that the effect caused to the human host depends on the virus and on the parasite it has infected. Consequently, the review is divided as follows: Viruses with a possible effect on the virulence of the parasite. This section reviews pertinent articles showing that infection of parasites by viruses might increase the detrimental effect of the tandem virus-parasite on the human host (hypervirulence) or decrease virulence of the parasite (hypovirulence). Parasites as vectors affecting the transmission of viruses. In some cases, the virus-infected parasite might facilitate the transfer of the virus to the human host. Parasites harboring viruses with unidentified effects on their host. In spite of recently renewed interest in parasites in connection with their viruses, there still remains a number of cases in which the effect of the virus of a given parasite on the human host remains ambiguous. The triangular relationship between the virus, the parasite and the host, and the modulation of the pathogenicity and virulence of the parasites by viruses should be taken into account in the rationale of fighting against parasites. Copyright © 2016 Elsevier B.V. All rights reserved.

The HSP terminator of Arabidopsis thaliana induces a high level of miraculin accumulation in transgenic tomatoes.

PubMed

Hirai, Tadayoshi; Kurokawa, Natsuko; Duhita, Narendra; Hiwasa-Tanase, Kyoko; Kato, Kazuhisa; Kato, Ko; Ezura, Hiroshi

2011-09-28

High-level accumulation of the target recombinant protein is a significant issue in heterologous protein expression using transgenic plants. Miraculin, a taste-modifying protein, was accumulated in transgenic tomatoes using an expression cassette in which the miraculin gene was expressed by the cauliflower mosaic virus (CaMV) 35S promoter and the heat shock protein (HSP) terminator (MIR-HSP). The HSP terminator was derived from heat shock protein 18.2 in Arabidopsis thaliana . Using this HSP-containing cassette, the miraculin concentration in T0 transgenic tomato lines was 1.4-13.9% of the total soluble protein (TSP), and that in the T1 transgenic tomato line homozygous for the miraculin gene reached 17.1% of the TSP. The accumulation level of the target protein was comparable to levels observed with chloroplast transformation. The high-level accumulation of miraculin in T0 transgenic tomato lines achieved by the HSP terminator was maintained in the successive T1 generation, demonstrating the genetic stability of this accumulation system.

Detection of enteric viruses in shellfish

USDA-ARS?s Scientific Manuscript database

Norovirus and hepatitis A virus contamination are significant threats to the safety of shellfish and other foods. Methods for the extraction and assay of these viruses from shellfish are complex, time consuming, and technically challenging. Here, we itemize some of the salient points in extracting...

Involvement of the Reparative DNA Polymerase Pol X of African Swine Fever Virus in the Maintenance of Viral Genome Stability In Vivo

PubMed Central

Redrejo-Rodríguez, Modesto; Rodríguez, Javier M.; Suárez, Cristina; Salas, José

2013-01-01

The function of the African swine fever virus (ASFV) reparative DNA polymerase, Pol X, was investigated in the context of virus infection. Pol X is a late structural protein that localizes at cytoplasmic viral factories during DNA replication. Using an ASFV deletion mutant lacking the Pol X gene, we have shown that Pol X is not required for virus growth in Vero cells or swine macrophages under one-step growth conditions. However, at a low multiplicity of infection, when multiple rounds of replication occur, the growth of the mutant virus is impaired in swine macrophages but not in Vero cells, suggesting that Pol X is needed to repair the accumulated DNA damage. The replication of the mutant virus in Vero cells presents sensitivity to oxidative damage, and mutational analysis of viral DNA shows that deletion of Pol X results in an increase in the mutation frequency in macrophages. Therefore, our data reveal a biological role for ASFV Pol X in the context of the infected cell in the preservation of viral genetic information. PMID:23824796

A peptide that binds the pea aphid gut impedes entry of Pea enation mosaic virus into the aphid hemocoel

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu Sijun, E-mail: sliu@iastate.ed; Sivakumar, S., E-mail: sivaento@iastate.ed; Sparks, Wendy O., E-mail: wosparks@iastate.ed

2010-05-25

Development of ways to block virus transmission by aphids could lead to novel and broad-spectrum means of controlling plant viruses. Viruses in the Luteoviridae enhanced are obligately transmitted by aphids in a persistent manner that requires virion accumulation in the aphid hemocoel. To enter the hemocoel, the virion must bind and traverse the aphid gut epithelium. By screening a phage display library, we identified a 12-residue gut binding peptide (GBP3.1) that binds to the midgut and hindgut of the pea aphid Acyrthosiphon pisum. Binding was confirmed by labeling the aphid gut with a GBP3.1-green fluorescent protein fusion. GBP3.1 reduced uptakemore » of Pea enation mosaic virus (Luteoviridae) from the pea aphid gut into the hemocoel. GBP3.1 also bound to the gut epithelia of the green peach aphid and the soybean aphid. These results suggest a novel strategy for inhibiting plant virus transmission by at least three major aphid pest species.« less

Paenibacillus lentimorbus Inoculation Enhances Tobacco Growth and Extenuates the Virulence of Cucumber mosaic virus

PubMed Central

Agrawal, Lalit; Raj, Rashmi; Srivastava, Ashish; Gupta, Swati; Mishra, Shashank Kumar; Yadav, Sumit; Singh, Poonam C.; Raj, Shri Krishna; Nautiyal, Chandra Shekhar

2016-01-01

Previous studies with Paenibacillus lentimorbus B-30488” (hereafter referred as B-30488), a plant growth promoting rhizobacteria (PGPR) isolated from cow’s milk, revealed its capabilities to improve plant quality under normal and stress conditions. Present study investigates its potential as a biocontrol agent against an economically important virus, Cucumber mosaic virus (CMV), in Nicotiana tabacum cv. White Burley plants and delineates the physical, biophysical, biochemical and molecular perturbations due to the trilateral interactions of PGPR-host-CMV. Soil inoculation of B-30488 enhanced the plant vigor while significantly decreased the virulence and virus RNA accumulation by ~12 fold (91%) in systemic leaves of CMV infected tobacco plants as compared to the control ones. Histology of these leaves revealed the improved tissue’s health and least aging signs in B-30488 inoculated tobacco plants, with or without CMV infection, and showed lesser intercellular spaces between collenchyma cells, reduced amount of xyloglucans and pectins in connecting primary cells, and higher polyphenol accumulation in hypodermis layer extending to collenchyma cells. B-30488 inoculation has favorably maneuvered the essential biophysical (ion leakage and photosynthetic efficiency) and biochemical (sugar, proline, chlorophyll, malondialdehyde, acid phosphatase and alkaline phosphatase) attributes of tobacco plants to positively regulate and release the virus stress. Moreover, activities of defense related enzymes (ascorbate peroxidase, guaiacol peroxidase, superoxide dismutase and catalase) induced due to CMV-infection were ameliorated with inoculation of B-30488, suggesting systemic induced resistance mediated protection against CMV in tobacco. The quantitative RT-PCR analyses of the genes related to normal plant development, stress and pathogenesis also corroborate well with the biochemical data and revealed the regulation (either up or down) of these genes in favor of plant to combat the CMV mediated stress. These improvements led tobacco plant to produce more flowers and seeds with no negative impact on plant health. The present study may advocate the applicability of B-30488 for crop yield improvement in virus infested areas. PMID:26934600

Herpes Simplex Virus Selectively Induces Expression of the CC Chemokine RANTES/CCL5 in Macrophages through a Mechanism Dependent on PKR and ICP0

PubMed Central

Melchjorsen, Jesper; Pedersen, Finn S.; Mogensen, Søren C.; Paludan, Søren R.

2002-01-01

Recruitment of leukocytes is essential for eventual control of virus infections. Macrophages represent a leukocyte population involved in the first line of defense against many infections, including herpes simplex virus (HSV) infection. Through presentation of antigens to T cells and production of cytokines and chemokines, macrophages also constitute an important link between the innate and adaptive immune systems. Here, we have investigated the chemokine expression profile of macrophages after HSV infection and the virus-cell interactions involved. By reverse transcription-PCR and cDNA arrays, we found that HSV type 1 (HSV-1) and HSV-2 induced expression of the CC chemokine RANTES/CCL5 in murine macrophage cell lines and peritoneal cells. The CXC chemokine BCA-1/CXCL13 was also induced in peritoneal cells. Twenty-six other chemokines tested were not affected. Accumulation of RANTES mRNA was detectable after 5 h of infection, was sensitive to UV irradiation of the virus, and was preceded by accumulation of viral immediate-early mRNA and proteins. The viral components responsible for initiation of RANTES expression were examined with virus mutants and RAW 264.7 macrophage-like cells expressing a dominant negative mutant of the double-stranded-RNA-activated protein kinase (PKR). The PKR mutant cell line displayed reduced constitutive and HSV-inducible RANTES expression compared to the control cell line. HSV-1 mutants deficient in genes encoding the immediate-early proteins ICP4, ICP22, and ICP27 remained fully capable of inducing RANTES expression in macrophages. By contrast, the ability of an ICP0-deficient HSV-1 mutant to induce RANTES expression was compromised. Thus, HSV selectively induces expression of RANTES in macrophages through a mechanism dependent on cellular PKR and viral ICP0. PMID:11861845

Differential growth of U and M type infectious haematopoietic necrosis virus in a rainbow trout–derived cell line, RTG-2

USGS Publications Warehouse

Kurath, Gael; Purcell, Maureen K.; Wargo, Andrew; Park, Jeong Woo; Moon, Chang Hoon

2010-01-01

Infectious haematopoietic necrosis virus (IHNV) is one of the most important viral pathogens of salmonids. In rainbow trout, IHNV isolates in the M genogroup are highly pathogenic, while U genogroup isolates are significantly less pathogenic. We show here that, at a multiplicity of infection (MOI) of 1, a representative U type strain yielded 42-fold less infectious virus than an M type strain in the rainbow trout–derived RTG-2 cell line at 24 h post-infection (p.i.). However, at an MOI of 10, there was only fivefold difference in the yield of infectious virus between the U and M strains. Quantification of extracellular viral genomic RNA suggested that the number of virus particles released from cells infected with the U strain at a MOI of 1 was 47-fold lower than from M-infected cells, but U and M virions were equally infectious by particle to infectivity ratios. At an MOI of 1, U strain intracellular viral genome accumulation and transcription were 37- and 12-fold lower, respectively, than those of the M strain at 24 h p.i. Viral nucleocapsid (N) protein accumulation in U strain infections was fivefold lower than in M strain infections. These results suggest that the block in U type strain growth in RTG-2 cells was because of the effects of reduced genome replication and transcription. The reduced growth of the U strain does not seem to be caused by defective genes, because the U and M strains grew equally well in the permissive epithelioma papulosum cyprini cell line at an MOI of 1. This suggests that host-specific factors in RTG-2 cells control the growth of the IHNV U and M strains differently, leading to growth restriction of the U type virus during the RNA synthesis step.

Modulation of the virus-receptor interaction by mutations in the V5 loop of feline immunodeficiency virus (FIV) following in vivo escape from neutralising antibody.

PubMed

Willett, Brian J; Kraase, Martin; Logan, Nicola; McMonagle, Elizabeth L; Samman, Ayman; Hosie, Margaret J

2010-04-26

In the acute phase of infection with feline immunodeficiency virus (FIV), the virus targets activated CD4+ T cells by utilising CD134 (OX40) as a primary attachment receptor and CXCR4 as a co-receptor. The nature of the virus-receptor interaction varies between isolates; strains such as GL8 and CPGammer recognise a "complex" determinant on CD134 formed by cysteine-rich domains (CRDs) 1 and 2 of the molecule while strains such as PPR and B2542 require a more "simple" determinant comprising CRD1 only for infection. These differences in receptor recognition manifest as variations in sensitivity to receptor antagonists. In this study, we ask whether the nature of the virus-receptor interaction evolves in vivo. Following infection with a homogeneous viral population derived from a pathogenic molecular clone, a quasispecies emerged comprising variants with distinct sensitivities to neutralising antibody and displaying evidence of conversion from a "complex" to a "simple" interaction with CD134. Escape from neutralising antibody was mediated primarily by length and sequence polymorphisms in the V5 region of Env, and these alterations in V5 modulated the virus-receptor interaction as indicated by altered sensitivities to antagonism by both anti-CD134 antibody and soluble CD134. The FIV-receptor interaction evolves under the selective pressure of the host humoral immune response, and the V5 loop contributes to the virus-receptor interaction. Our data are consistent with a model whereby viruses with distinct biological properties are present in early versus late infection and with a shift from a "complex" to a "simple" interaction with CD134 with time post-infection.

Oxylipin Biosynthesis Genes Positively Regulate Programmed Cell Death during Compatible Infections with the Synergistic Pair Potato Virus X-Potato Virus Y and Tomato Spotted Wilt Virus

PubMed Central

García-Marcos, Alberto; Pacheco, Remedios; Manzano, Aranzazu; Aguilar, Emmanuel

2013-01-01

One of the most severe symptoms caused by compatible plant-virus interactions is systemic necrosis, which shares common attributes with the hypersensitive response to incompatible pathogens. Although several studies have identified viral symptom determinants responsible for systemic necrosis, mechanistic models of how they contribute to necrosis in infected plants remain scarce. Here, we examined the involvement of different branches of the oxylipin biosynthesis pathway in the systemic necrosis response caused either by the synergistic interaction of Potato virus X with Potato virus Y (PVX-PVY) or by Tomato spotted wilt virus (TSWV) in Nicotiana benthamiana. Silencing either 9-lipoxygenase (LOX), 13-LOX, or α-dioxygenase-1 (α-DOX-1) attenuated the programmed cell death (PCD)-associated symptoms caused by infection with either PVX-PVY or TSWV. In contrast, silencing of the jasmonic acid perception gene, COI1 (Coronatine insensitive 1), expedited cell death during infection with compatible viruses. This correlated with an enhanced expression of oxylipin biosynthesis genes and dioxygenase activity in PVX-PVY-infected plants. Moreover, the Arabidopsis thaliana double lox1 α-dox-1 mutant became less susceptible to TSWV infection. We conclude that oxylipin metabolism is a critical component that positively regulates the process of PCD during compatible plant-virus interactions but does not play a role in restraining virus accumulation in planta. PMID:23487466

CHLORELLA VIRUSES

PubMed Central

Yamada, Takashi; Onimatsu, Hideki; Van Etten, James L.

2007-01-01

Chlorella viruses or chloroviruses are large, icosahedral, plaque‐forming, double‐stranded‐DNA—containing viruses that replicate in certain strains of the unicellular green alga Chlorella. DNA sequence analysis of the 330‐kbp genome of Paramecium bursaria chlorella virus 1 (PBCV‐1), the prototype of this virus family (Phycodnaviridae), predict ∼366 protein‐encoding genes and 11 tRNA genes. The predicted gene products of ∼50% of these genes resemble proteins of known function, including many that are completely unexpected for a virus. In addition, the chlorella viruses have several features and encode many gene products that distinguish them from most viruses. These products include: (1) multiple DNA methyltransferases and DNA site‐specific endonucleases, (2) the enzymes required to glycosylate their proteins and synthesize polysaccharides such as hyaluronan and chitin, (3) a virus‐encoded K+ channel (called Kcv) located in the internal membrane of the virions, (4) a SET domain containing protein (referred to as vSET) that dimethylates Lys27 in histone 3, and (5) PBCV‐1 has three types of introns; a self‐splicing intron, a spliceosomal processed intron, and a small tRNA intron. Accumulating evidence indicates that the chlorella viruses have a very long evolutionary history. This review mainly deals with research on the virion structure, genome rearrangements, gene expression, cell wall degradation, polysaccharide synthesis, and evolution of PBCV‐1 as well as other related viruses. PMID:16877063

Disruption of Specific RNA-RNA Interactions in a Double-Stranded RNA Virus Inhibits Genome Packaging and Virus Infectivity

PubMed Central

Fajardo, Teodoro; Sung, Po-Yu; Roy, Polly

2015-01-01

Bluetongue virus (BTV) causes hemorrhagic disease in economically important livestock. The BTV genome is organized into ten discrete double-stranded RNA molecules (S1-S10) which have been suggested to follow a sequential packaging pathway from smallest to largest segment during virus capsid assembly. To substantiate and extend these studies, we have investigated the RNA sorting and packaging mechanisms with a new experimental approach using inhibitory oligonucleotides. Putative packaging signals present in the 3’untranslated regions of BTV segments were targeted by a number of nuclease resistant oligoribonucleotides (ORNs) and their effects on virus replication in cell culture were assessed. ORNs complementary to the 3’ UTR of BTV RNAs significantly inhibited virus replication without affecting protein synthesis. Same ORNs were found to inhibit complex formation when added to a novel RNA-RNA interaction assay which measured the formation of supramolecular complexes between and among different RNA segments. ORNs targeting the 3’UTR of BTV segment 10, the smallest RNA segment, were shown to be the most potent and deletions or substitution mutations of the targeted sequences diminished the RNA complexes and abolished the recovery of viable viruses using reverse genetics. Cell-free capsid assembly/RNA packaging assay also confirmed that the inhibitory ORNs could interfere with RNA packaging and further substitution mutations within the putative RNA packaging sequence have identified the recognition sequence concerned. Exchange of 3’UTR between segments have further demonstrated that RNA recognition was segment specific, most likely acting as part of the secondary structure of the entire genomic segment. Our data confirm that genome packaging in this segmented dsRNA virus occurs via the formation of supramolecular complexes formed by the interaction of specific sequences located in the 3’ UTRs. Additionally, the inhibition of packaging in-trans with inhibitory ORNs suggests this that interaction is a bona fide target for the design of compounds with antiviral activity. PMID:26646790

Disruption of Specific RNA-RNA Interactions in a Double-Stranded RNA Virus Inhibits Genome Packaging and Virus Infectivity.

PubMed

Fajardo, Teodoro; Sung, Po-Yu; Roy, Polly

2015-12-01

Bluetongue virus (BTV) causes hemorrhagic disease in economically important livestock. The BTV genome is organized into ten discrete double-stranded RNA molecules (S1-S10) which have been suggested to follow a sequential packaging pathway from smallest to largest segment during virus capsid assembly. To substantiate and extend these studies, we have investigated the RNA sorting and packaging mechanisms with a new experimental approach using inhibitory oligonucleotides. Putative packaging signals present in the 3'untranslated regions of BTV segments were targeted by a number of nuclease resistant oligoribonucleotides (ORNs) and their effects on virus replication in cell culture were assessed. ORNs complementary to the 3' UTR of BTV RNAs significantly inhibited virus replication without affecting protein synthesis. Same ORNs were found to inhibit complex formation when added to a novel RNA-RNA interaction assay which measured the formation of supramolecular complexes between and among different RNA segments. ORNs targeting the 3'UTR of BTV segment 10, the smallest RNA segment, were shown to be the most potent and deletions or substitution mutations of the targeted sequences diminished the RNA complexes and abolished the recovery of viable viruses using reverse genetics. Cell-free capsid assembly/RNA packaging assay also confirmed that the inhibitory ORNs could interfere with RNA packaging and further substitution mutations within the putative RNA packaging sequence have identified the recognition sequence concerned. Exchange of 3'UTR between segments have further demonstrated that RNA recognition was segment specific, most likely acting as part of the secondary structure of the entire genomic segment. Our data confirm that genome packaging in this segmented dsRNA virus occurs via the formation of supramolecular complexes formed by the interaction of specific sequences located in the 3' UTRs. Additionally, the inhibition of packaging in-trans with inhibitory ORNs suggests this that interaction is a bona fide target for the design of compounds with antiviral activity.

Discovery and Targeted LC-MS/MS of Purified Polerovirus Reveals Differences in the Virus-Host Interactome Associated with Altered Aphid Transmission

PubMed Central

Howe, Kevin; Fish, Tara; Smith, Dawn; Gildow, Fredrick; MacCoss, Michael J.; Thannhauser, Theodore W.; Gray, Stewart M.

2012-01-01

Circulative transmission of viruses in the Luteoviridae, such as cereal yellow dwarf virus (CYDV), requires a series of precisely orchestrated interactions between virus, plant, and aphid proteins. Natural selection has favored these viruses to be retained in the phloem to facilitate acquisition and transmission by aphids. We show that treatment of infected oat tissue homogenate with sodium sulfite reduces transmission of the purified virus by aphids. Transmission electron microscopy data indicated no gross change in virion morphology due to treatments. However, treated virions were not acquired by aphids through the hindgut epithelial cells and were not transmitted when injected directly into the hemocoel. Analysis of virus preparations using nanoflow liquid chromatography coupled to tandem mass spectrometry revealed a number of host plant proteins co-purifying with viruses, some of which were lost following sodium sulfite treatment. Using targeted mass spectrometry, we show data suggesting that several of the virus-associated host plant proteins accumulated to higher levels in aphids that were fed on CYDV-infected plants compared to healthy plants. We propose two hypotheses to explain these observations, and these are not mutually exclusive: (a) that sodium sulfite treatment disrupts critical virion-host protein interactions required for aphid transmission, or (b) that host infection with CYDV modulates phloem protein expression in a way that is favorable for virus uptake by aphids. Importantly, the genes coding for the plant proteins associated with virus may be examined as targets in breeding cereal crops for new modes of virus resistance that disrupt phloem-virus or aphid-virus interactions. PMID:23118947

Discovery and targeted LC-MS/MS of purified polerovirus reveals differences in the virus-host interactome associated with altered aphid transmission.

PubMed

Cilia, Michelle; Peter, Kari A; Bereman, Michael S; Howe, Kevin; Fish, Tara; Smith, Dawn; Gildow, Fredrick; MacCoss, Michael J; Thannhauser, Theodore W; Gray, Stewart M

2012-01-01

Circulative transmission of viruses in the Luteoviridae, such as cereal yellow dwarf virus (CYDV), requires a series of precisely orchestrated interactions between virus, plant, and aphid proteins. Natural selection has favored these viruses to be retained in the phloem to facilitate acquisition and transmission by aphids. We show that treatment of infected oat tissue homogenate with sodium sulfite reduces transmission of the purified virus by aphids. Transmission electron microscopy data indicated no gross change in virion morphology due to treatments. However, treated virions were not acquired by aphids through the hindgut epithelial cells and were not transmitted when injected directly into the hemocoel. Analysis of virus preparations using nanoflow liquid chromatography coupled to tandem mass spectrometry revealed a number of host plant proteins co-purifying with viruses, some of which were lost following sodium sulfite treatment. Using targeted mass spectrometry, we show data suggesting that several of the virus-associated host plant proteins accumulated to higher levels in aphids that were fed on CYDV-infected plants compared to healthy plants. We propose two hypotheses to explain these observations, and these are not mutually exclusive: (a) that sodium sulfite treatment disrupts critical virion-host protein interactions required for aphid transmission, or (b) that host infection with CYDV modulates phloem protein expression in a way that is favorable for virus uptake by aphids. Importantly, the genes coding for the plant proteins associated with virus may be examined as targets in breeding cereal crops for new modes of virus resistance that disrupt phloem-virus or aphid-virus interactions.

Activation of DNA Damage Repair Pathways by Murine Polyomavirus

PubMed Central

Heiser, Katie; Nicholas, Catherine; Garcea, Robert L.

2016-01-01

Nuclear replication of DNA viruses activates DNA damage repair (DDR) pathways, which are thought to detect and inhibit viral replication. However, many DNA viruses also depend on these pathways in order to optimally replicate their genomes. We investigated the relationship between murine polyomavirus (MuPyV) and components of DDR signaling pathways including CHK1, CHK2, H2AX, ATR, and DNAPK. We found that recruitment and retention of DDR proteins at viral replication centers was independent of H2AX, as well as the viral small and middle T-antigens. Additionally, infectious virus production required ATR kinase activity, but was independent of CHK1, CHK2, or DNAPK signaling. ATR inhibition did not reduce the total amount of viral DNA accumulated, but affected the amount of virus produced, indicating a defect in virus assembly. These results suggest that MuPyV may utilize a subset of DDR proteins or non-canonical DDR signaling pathways in order to efficiently replicate and assemble. PMID:27529739

Recognition of cis-acting sequences in RNA 3 of Prunus necrotic ringspot virus by the replicase of Alfalfa mosaic virus.

PubMed

Aparicio, F; Sánchez-Navarro, J A; Olsthoorn, R C; Pallás, V; Bol, J F

2001-04-01

Alfalfa mosaic virus (AMV) and Prunus necrotic ringspot virus (PNRSV) belong to the genera ALFAMOVIRUS: and ILARVIRUS:, respectively, of the family BROMOVIRIDAE: Initiation of infection by AMV and PNRSV requires binding of a few molecules of coat protein (CP) to the 3' termini of the inoculum RNAs and the CPs of the two viruses are interchangeable in this early step of the replication cycle. CIS:-acting sequences in PNRSV RNA 3 that are recognized by the AMV replicase were studied in in vitro replicase assays and by inoculation of AMV-PNRSV RNA 3 chimeras to tobacco plants and protoplasts transformed with the AMV replicase genes (P12 plants). The results showed that the AMV replicase recognized the promoter for minus-strand RNA synthesis in PNRSV RNA 3 but not the promoter for plus-strand RNA synthesis. A chimeric RNA with PNRSV movement protein and CP genes accumulated in tobacco, which is a non-host for PNRSV.

Quantification of RNA Content in Reconstituted Ebola Virus Nucleocapsids by Immunoprecipitation.

PubMed

Banadyga, Logan; Ebihara, Hideki

2017-01-01

Immunoprecipitations are commonly used to isolate proteins or protein complexes and assess protein-protein interactions; however, they can also be used to assess protein-RNA complexes. Here we describe an adapted RNA immunoprecipitation technique that permits the quantification of RNA content in Ebola virus nucleocapsids that have been reconstituted in vitro by transient transfection.

The Incredible Journey of Begomoviruses in Their Whitefly Vector

PubMed Central

Czosnek, Henryk; Hariton-Shalev, Aliza; Sobol, Iris; Gorovits, Rena; Ghanim, Murad

2017-01-01

Begomoviruses are vectored in a circulative persistent manner by the whitefly Bemisia tabaci. The insect ingests viral particles with its stylets. Virions pass along the food canal and reach the esophagus and the midgut. They cross the filter chamber and the midgut into the haemolymph, translocate into the primary salivary glands and are egested with the saliva into the plant phloem. Begomoviruses have to cross several barriers and checkpoints successfully, while interacting with would-be receptors and other whitefly proteins. The bulk of the virus remains associated with the midgut and the filter chamber. In these tissues, viral genomes, mainly from the tomato yellow leaf curl virus (TYLCV) family, may be transcribed and may replicate. However, at the same time, virus amounts peak, and the insect autophagic response is activated, which in turn inhibits replication and induces the destruction of the virus. Some begomoviruses invade tissues outside the circulative pathway, such as ovaries and fat cells. Autophagy limits the amounts of virus associated with these organs. In this review, we discuss the different sites begomoviruses need to cross to complete a successful circular infection, the role of the coat protein in this process and the sites that balance between virus accumulation and virus destruction. PMID:28946649

Measles virus, immune control and persistence

PubMed Central

Griffin, Diane E.; Lin, Wen-Hsuan; Pan, Chien-Hsiung

2012-01-01

Measles remains one of the most important causes of child morbidity and mortality worldwide with the greatest burden in the youngest children. Most acute measles deaths are due to secondary infections that result from a poorly understood measles-induced suppression of immune responses. Young children are also vulnerable to late development of subacute sclerosing panencephalitis, a progressive, uniformly fatal neurologic disease caused by persistent measles virus (MeV) infection. During acute infection, the rash marks the appearance of the adaptive immune response and CD8+ T cell-mediated clearance of infectious virus. However, after clearance of infectious virus, MeV RNA persists and can be detected in blood, respiratory secretions, urine and lymphoid tissue for many weeks to months. This prolonged period of virus clearance may help to explain measles immunosuppression and the development of lifelong immunity to re-infection, as well as occasional infection of the nervous system. Once MeV infects neurons, the virus can spread transynaptically and the envelope proteins needed to form infectious virus are unnecessary, accumulate mutations and can establish persistent infection. Identification of the immune mechanisms required for clearance of MeV RNA from multiple sites will enlighten our understanding of the development of disease due to persistent infection. PMID:22316382

Vector competence of Peruvian mosquitoes (Diptera: Culicidae) for a subtype IIIC virus in the Venezuelan equine encephalomyelitis complex isolated from mosquitoes captured in Peru.

PubMed

Turell, M J; Dohm, D J; Fernandez, R; Calampa, C; O'Guinn, M L

2006-03-01

We evaluated mosquitoes collected in the Amazon Basin, near Iquitos, Peru, for their susceptibility to a subtype IIIC strain of the Venezuelan equine encephalomyelitis complex. This virus had been previously isolated from a pool of mixed Culex vomerifer and Cx. gnomatos captured near Iquitos, Peru, in 1997. After feeding on hamsters with viremias of about 10(8) plaque-forming units of virus per ml, Cx. gnomatos was the most efficient vector. Other species, such as Ochlerotatus fulvus and Psorophora cingulata, although highly susceptible to infection, were not efficient laboratory vectors of this virus due to a significant salivary gland barrier. The Cx. (Culex) species, consisting mostly of Cx. (Cux.) coronator, were nearly refractory to subtype IIIC virus and exhibited both midgut infection as well as salivary gland barriers. Additional studies on biting behavior, mosquito population densities, and vertebrate reservoir hosts of subtype IIIC virus are needed to determine the role that these species play in the maintenance and spread of this virus in the Amazon Basin region.

Domain Interaction Studies of Herpes Simplex Virus 1 Tegument Protein UL16 Reveal Its Interaction with Mitochondria

PubMed Central

Chadha, Pooja; Sarfo, Akua; Zhang, Dan; Abraham, Thomas; Carmichael, Jillian

2016-01-01

ABSTRACT The UL16 tegument protein of herpes simplex virus 1 (HSV-1) is conserved among all herpesviruses and plays many roles during replication. This protein has an N-terminal domain (NTD) that has been shown to bind to several viral proteins, including UL11, VP22, and glycoprotein E, and these interactions are negatively regulated by a C-terminal domain (CTD). Thus, in pairwise transfections, UL16 binding is enabled only when the CTD is absent or altered. Based on these results, we hypothesized that direct interactions occur between the NTD and the CTD. Here we report that the separated and coexpressed functional domains of UL16 are mutually responsive to each other in transfected cells and form complexes that are stable enough to be captured in coimmunoprecipitation assays. Moreover, we found that the CTD can associate with itself. To our surprise, the CTD was also found to contain a novel and intrinsic ability to localize to specific spots on mitochondria in transfected cells. Subsequent analyses of HSV-infected cells by immunogold electron microscopy and live-cell confocal imaging revealed a population of UL16 that does not merely accumulate on mitochondria but in fact makes dynamic contacts with these organelles in a time-dependent manner. These findings suggest that the domain interactions of UL16 serve to regulate not just the interaction of this tegument protein with its viral binding partners but also its interactions with mitochondria. The purpose of this novel interaction remains to be determined. IMPORTANCE The HSV-1-encoded tegument protein UL16 is involved in multiple events of the virus replication cycle, ranging from virus assembly to cell-cell spread of the virus, and hence it can serve as an important drug target. Unfortunately, a lack of both structural and functional information limits our understanding of this protein. The discovery of domain interactions within UL16 and the novel ability of UL16 to interact with mitochondria in HSV-infected cells lays a foundational framework for future investigations aimed at deciphering the structure and function of not just UL16 of HSV-1 but also its homologs in other herpesviruses. PMID:27847362

Interaction with Tsg101 is necessary for the efficient transport and release of nucleocapsids in marburg virus-infected cells.

PubMed

Dolnik, Olga; Kolesnikova, Larissa; Welsch, Sonja; Strecker, Thomas; Schudt, Gordian; Becker, Stephan

2014-10-01

Endosomal sorting complex required for transport (ESCRT) machinery supports the efficient budding of Marburg virus (MARV) and many other enveloped viruses. Interaction between components of the ESCRT machinery and viral proteins is predominantly mediated by short tetrapeptide motifs, known as late domains. MARV contains late domain motifs in the matrix protein VP40 and in the genome-encapsidating nucleoprotein (NP). The PSAP late domain motif of NP recruits the ESCRT-I protein tumor susceptibility gene 101 (Tsg101). Here, we generated a recombinant MARV encoding NP with a mutated PSAP late domain (rMARV(PSAPmut)). rMARV(PSAPmut) was attenuated by up to one log compared with recombinant wild-type MARV (rMARV(wt)), formed smaller plaques and exhibited delayed virus release. Nucleocapsids in rMARV(PSAPmut)-infected cells were more densely packed inside viral inclusions and more abundant in the cytoplasm than in rMARV(wt)-infected cells. A similar phenotype was detected when MARV-infected cells were depleted of Tsg101. Live-cell imaging analyses revealed that Tsg101 accumulated in inclusions of rMARV(wt)-infected cells and was co-transported together with nucleocapsids. In contrast, rMARV(PSAPmut) nucleocapsids did not display co-localization with Tsg101, had significantly shorter transport trajectories, and migration close to the plasma membrane was severely impaired, resulting in reduced recruitment into filopodia, the major budding sites of MARV. We further show that the Tsg101 interacting protein IQGAP1, an actin cytoskeleton regulator, was recruited into inclusions and to individual nucleocapsids together with Tsg101. Moreover, IQGAP1 was detected in a contrail-like structure at the rear end of migrating nucleocapsids. Down regulation of IQGAP1 impaired release of MARV. These results indicate that the PSAP motif in NP, which enables binding to Tsg101, is important for the efficient actin-dependent transport of nucleocapsids to the sites of budding. Thus, the interaction between NP and Tsg101 supports several steps of MARV assembly before virus fission.

Regulation of host translational machinery by African swine fever virus.

PubMed

Castelló, Alfredo; Quintas, Ana; Sánchez, Elena G; Sabina, Prado; Nogal, Marisa; Carrasco, Luis; Revilla, Yolanda

2009-08-01

African swine fever virus (ASFV), like other complex DNA viruses, deploys a variety of strategies to evade the host's defence systems, such as inflammatory and immune responses and cell death. Here, we analyse the modifications in the translational machinery induced by ASFV. During ASFV infection, eIF4G and eIF4E are phosphorylated (Ser1108 and Ser209, respectively), whereas 4E-BP1 is hyperphosphorylated at early times post infection and hypophosphorylated after 18 h. Indeed, a potent increase in eIF4F assembly is observed in ASFV-infected cells, which is prevented by rapamycin treatment. Phosphorylation of eIF4E, eIF4GI and 4E-BP1 is important to enhance viral protein production, but is not essential for ASFV infection as observed in rapamycin- or CGP57380-treated cells. Nevertheless, eIF4F components are indispensable for ASFV protein synthesis and virus spread, since eIF4E or eIF4G depletion in COS-7 or Vero cells strongly prevents accumulation of viral proteins and decreases virus titre. In addition, eIF4F is not only activated but also redistributed within the viral factories at early times of infection, while eIF4G and eIF4E are surrounding these areas at late times. In fact, other components of translational machinery such as eIF2alpha, eIF3b, eIF4E, eEF2 and ribosomal P protein are enriched in areas surrounding ASFV factories. Notably, the mitochondrial network is polarized in ASFV-infected cells co-localizing with ribosomes. Thus, translation and ATP synthesis seem to be coupled and compartmentalized at the periphery of viral factories. At later times after ASFV infection, polyadenylated mRNAs disappear from the cytoplasm of Vero cells, except within the viral factories. The distribution of these pools of mRNAs is similar to the localization of viral late mRNAs. Therefore, degradation of cellular polyadenylated mRNAs and recruitment of the translation machinery to viral factories may contribute to the inhibition of host protein synthesis, facilitating ASFV protein production in infected cells.

Regulation of Host Translational Machinery by African Swine Fever Virus

PubMed Central

Castelló, Alfredo; Quintas, Ana; Sánchez, Elena G.; Sabina, Prado; Nogal, Marisa; Carrasco, Luis; Revilla, Yolanda

2009-01-01

African swine fever virus (ASFV), like other complex DNA viruses, deploys a variety of strategies to evade the host's defence systems, such as inflammatory and immune responses and cell death. Here, we analyse the modifications in the translational machinery induced by ASFV. During ASFV infection, eIF4G and eIF4E are phosphorylated (Ser1108 and Ser209, respectively), whereas 4E-BP1 is hyperphosphorylated at early times post infection and hypophosphorylated after 18 h. Indeed, a potent increase in eIF4F assembly is observed in ASFV-infected cells, which is prevented by rapamycin treatment. Phosphorylation of eIF4E, eIF4GI and 4E-BP1 is important to enhance viral protein production, but is not essential for ASFV infection as observed in rapamycin- or CGP57380-treated cells. Nevertheless, eIF4F components are indispensable for ASFV protein synthesis and virus spread, since eIF4E or eIF4G depletion in COS-7 or Vero cells strongly prevents accumulation of viral proteins and decreases virus titre. In addition, eIF4F is not only activated but also redistributed within the viral factories at early times of infection, while eIF4G and eIF4E are surrounding these areas at late times. In fact, other components of translational machinery such as eIF2α, eIF3b, eIF4E, eEF2 and ribosomal P protein are enriched in areas surrounding ASFV factories. Notably, the mitochondrial network is polarized in ASFV-infected cells co-localizing with ribosomes. Thus, translation and ATP synthesis seem to be coupled and compartmentalized at the periphery of viral factories. At later times after ASFV infection, polyadenylated mRNAs disappear from the cytoplasm of Vero cells, except within the viral factories. The distribution of these pools of mRNAs is similar to the localization of viral late mRNAs. Therefore, degradation of cellular polyadenylated mRNAs and recruitment of the translation machinery to viral factories may contribute to the inhibition of host protein synthesis, facilitating ASFV protein production in infected cells. PMID:19714237

Paleogeographic controls of coal accumulation, Cretaceous Blackhawk Formation and Star Point Sandstone, Wasatch Plateau, Utah.

USGS Publications Warehouse

Flores, R.M.; Blanchard, L.F.; Sanchez, J.D.; Marley, W.E.; Muldoon, W.J.

1984-01-01

Considers the paleogeographic controls affecting the accumulation of coals in delta-barrier-island complexes. Progradation, lateral shifting, and abandonment of these complexes created four major landward-thinning tongues.-from Authors

Global Reprogramming of Host SUMOylation during Influenza Virus Infection

PubMed Central

Domingues, Patricia; Golebiowski, Filip; Tatham, Michael H.; Lopes, Antonio M.; Taggart, Aislynn; Hay, Ronald T.; Hale, Benjamin G.

2015-01-01

Summary Dynamic nuclear SUMO modifications play essential roles in orchestrating cellular responses to proteotoxic stress, DNA damage, and DNA virus infection. Here, we describe a non-canonical host SUMOylation response to the nuclear-replicating RNA pathogen, influenza virus, and identify viral RNA polymerase activity as a major contributor to SUMO proteome remodeling. Using quantitative proteomics to compare stress-induced SUMOylation responses, we reveal that influenza virus infection triggers unique re-targeting of SUMO to 63 host proteins involved in transcription, mRNA processing, RNA quality control, and DNA damage repair. This is paralleled by widespread host deSUMOylation. Depletion screening identified ten virus-induced SUMO targets as potential antiviral factors, including C18orf25 and the SMC5/6 and PAF1 complexes. Mechanistic studies further uncovered a role for SUMOylation of the PAF1 complex component, parafibromin (CDC73), in potentiating antiviral gene expression. Our global characterization of influenza virus-triggered SUMO redistribution provides a proteomic resource to understand host nuclear SUMOylation responses to infection. PMID:26549460

Structure-Based Drug Design Targeting a Subunit Interaction of Influenza Virus RNA Polymerase

NASA Astrophysics Data System (ADS)

Sugiyama, Kanako; Obayashi, Eiji; Yoshida, Hisashi; Park, Sam-Yong

Influenza A virus is a major human and animal pathogen with the potential to cause catastrophic loss of life. Influenza virus reproduces rapidly, mutates frequently, and occasionally crosses species barriers. The recent emergence of swine-origin influenza H1N1 and avian influenza related to highly pathogenic forms of the human virus has highlighted the urgent need for new effective treatments. Here, we describe two crystal structures of complexes made by fragments of PA and PB1, and PB1 and PB2. These novel interfaces are surprisingly small, yet they play a crucial role in regulating the 250 kDa polymerase complex, and are completely conserved among swine, avian and human influenza viruses. Given their importance to viral replication and strict conservation, the PA/PB1 and PB1/PB2 interfaces appear to be promising targets for novel anti-influenza drugs of use against all strains of influenza A virus. It is hoped that the structures presented here will assist the search for such compounds.

Purification and crystallization of dengue and West Nile virus NS2B-NS3 complexes.

PubMed

D'Arcy, Allan; Chaillet, Maxime; Schiering, Nikolaus; Villard, Frederic; Lim, Siew Pheng; Lefeuvre, Peggy; Erbel, Paul

2006-02-01

Both dengue and West Nile virus infections are an increasing risk to humans, not only in tropical and subtropical areas, but also in North America and parts of Europe. These viral infections are generally transmitted by mosquitoes, but may also be tick-borne. Infection usually results in mild flu-like symptoms, but can also cause encephalitis and fatalities. Approximately 2799 severe West Nile virus cases were reported this year in the United States, resulting in 102 fatalities. With this alarming increase in the number of West Nile virus infections in western countries and the fact that dengue virus already affects millions of people per year in tropical and subtropical climates, there is a real need for effective medicines. A possible therapeutic target to combat these viruses is the protease, which is essential for virus replication. In order to provide structural information to help to guide a lead identification and optimization program, crystallizations of the NS2B-NS3 protease complexes from both dengue and West Nile viruses have been initiated. Crystals that diffract to high resolution, suitable for three-dimensional structure determinations, have been obtained.

The Human Immunodeficiency Virus 1 ASP RNA promotes viral latency by recruiting the Polycomb Repressor Complex 2 and promoting nucleosome assembly

PubMed Central

Zapata, Juan C.; Campilongo, Federica; Barclay, Robert A.; DeMarino, Catherine; Iglesias-Ussel, Maria D.; Kashanchi, Fatah; Romerio, Fabio

2017-01-01

Various epigenetic marks at the HIV-1 5′LTR suppress proviral expression and promote latency. Cellular antisense transcripts known as long noncoding RNAs (lncRNAs) recruit the polycomb repressor complex 2 (PRC2) to gene promoters, which catalyzes trimethylation of lysine 27 on histone H3 (H3K27me3), thus promoting nucleosome assembly and suppressing gene expression. We found that an HIV-1 antisense transcript expressed from the 3′LTR and encoding the antisense protein ASP promotes proviral latency. Expression of ASP RNA reduced HIV-1 replication in Jurkat cells. Moreover, ASP RNA expression promoted the establishment and maintenance of HIV-1 latency in Jurkat E4 cells. We show that this transcript interacts with and recruits PRC2 to the HIV-1 5′LTR, increasing accumulation of the suppressive epigenetic mark H3K27me3, while reducing RNA Polymerase II and thus proviral transcription. Altogether, our results suggest that the HIV-1 ASP transcript promotes epigenetic silencing of the HIV-1 5′LTR and proviral latency through the PRC2 pathway. PMID:28340355

The Human Immunodeficiency Virus 1 ASP RNA promotes viral latency by recruiting the Polycomb Repressor Complex 2 and promoting nucleosome assembly.

PubMed

Zapata, Juan C; Campilongo, Federica; Barclay, Robert A; DeMarino, Catherine; Iglesias-Ussel, Maria D; Kashanchi, Fatah; Romerio, Fabio

2017-06-01

Various epigenetic marks at the HIV-1 5'LTR suppress proviral expression and promote latency. Cellular antisense transcripts known as long noncoding RNAs (lncRNAs) recruit the polycomb repressor complex 2 (PRC2) to gene promoters, which catalyzes trimethylation of lysine 27 on histone H3 (H3K27me3), thus promoting nucleosome assembly and suppressing gene expression. We found that an HIV-1 antisense transcript expressed from the 3'LTR and encoding the antisense protein ASP promotes proviral latency. Expression of ASP RNA reduced HIV-1 replication in Jurkat cells. Moreover, ASP RNA expression promoted the establishment and maintenance of HIV-1 latency in Jurkat E4 cells. We show that this transcript interacts with and recruits PRC2 to the HIV-1 5'LTR, increasing accumulation of the suppressive epigenetic mark H3K27me3, while reducing RNA Polymerase II and thus proviral transcription. Altogether, our results suggest that the HIV-1 ASP transcript promotes epigenetic silencing of the HIV-1 5'LTR and proviral latency through the PRC2 pathway. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Global circulation patterns of seasonal influenza viruses vary with antigenic drift

PubMed Central

Bedford, Trevor; Riley, Steven; Barr, Ian G.; Broor, Shobha; Chadha, Mandeep; Cox, Nancy J.; Daniels, Rodney S.; Gunasekaran, C. Palani; Hurt, Aeron C.; Kelso, Anne; Lewis, Nicola S.; Li, Xiyan; McCauley, John W.; Odagiri, Takato; Potdar, Varsha; Rambaut, Andrew; Shu, Yuelong; Skepner, Eugene; Smith, Derek J.; Suchard, Marc A.; Tashiro, Masato; Wang, Dayan; Xu, Xiyan; Lemey, Philippe; Russell, Colin A.

2015-01-01

Understanding the spatio-temporal patterns of emergence and circulation of new human seasonal influenza virus variants is a key scientific and public health challenge. The global circulation patterns of influenza A/H3N2 viruses are well-characterized1-7 but the patterns of A/H1N1 and B viruses have remained largely unexplored. Here, based on analyses of 9,604 hemagglutinin sequences of human seasonal influenza viruses from 2000–2012, we show that the global circulation patterns of A/H1N1 (up to 2009), B/Victoria, and B/Yamagata viruses differ substantially from those of A/H3N2 viruses. While genetic variants of A/H3N2 viruses did not persist locally between epidemics and were reseeded from East and Southeast (E-SE) Asia, genetic variants of A/H1N1 and B viruses persisted across multiple seasons and exhibited complex global dynamics with E-SE Asia playing a limited role in disseminating new variants. The less frequent global movement of influenza A/H1N1 and B viruses coincided with slower rates of antigenic evolution, lower ages of infection, and smaller less frequent epidemics compared to A/H3N2 viruses. Detailed epidemic models support differences in age of infection, combined with the less frequent travel of children, as likely drivers of the differences in the patterns of global circulation, suggesting a complex interaction between virus evolution, epidemiology and human behavior. PMID:26053121

Global circulation patterns of seasonal influenza viruses vary with antigenic drift

NASA Astrophysics Data System (ADS)

Bedford, Trevor; Riley, Steven; Barr, Ian G.; Broor, Shobha; Chadha, Mandeep; Cox, Nancy J.; Daniels, Rodney S.; Gunasekaran, C. Palani; Hurt, Aeron C.; Kelso, Anne; Klimov, Alexander; Lewis, Nicola S.; Li, Xiyan; McCauley, John W.; Odagiri, Takato; Potdar, Varsha; Rambaut, Andrew; Shu, Yuelong; Skepner, Eugene; Smith, Derek J.; Suchard, Marc A.; Tashiro, Masato; Wang, Dayan; Xu, Xiyan; Lemey, Philippe; Russell, Colin A.

2015-07-01

Understanding the spatiotemporal patterns of emergence and circulation of new human seasonal influenza virus variants is a key scientific and public health challenge. The global circulation patterns of influenza A/H3N2 viruses are well characterized, but the patterns of A/H1N1 and B viruses have remained largely unexplored. Here we show that the global circulation patterns of A/H1N1 (up to 2009), B/Victoria, and B/Yamagata viruses differ substantially from those of A/H3N2 viruses, on the basis of analyses of 9,604 haemagglutinin sequences of human seasonal influenza viruses from 2000 to 2012. Whereas genetic variants of A/H3N2 viruses did not persist locally between epidemics and were reseeded from East and Southeast Asia, genetic variants of A/H1N1 and B viruses persisted across several seasons and exhibited complex global dynamics with East and Southeast Asia playing a limited role in disseminating new variants. The less frequent global movement of influenza A/H1N1 and B viruses coincided with slower rates of antigenic evolution, lower ages of infection, and smaller, less frequent epidemics compared to A/H3N2 viruses. Detailed epidemic models support differences in age of infection, combined with the less frequent travel of children, as probable drivers of the differences in the patterns of global circulation, suggesting a complex interaction between virus evolution, epidemiology, and human behaviour.

The RSV F and G glycoproteins interact to form a complex on the surface of infected cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Low, Kit-Wei; Tan, Timothy; Ng, Ken

2008-02-08

In this study, the interaction between the respiratory syncytial virus (RSV) fusion (F) protein, attachment (G) protein, and small hydrophobic (SH) proteins was examined. Immunoprecipitation analysis suggested that the F and G proteins exist as a protein complex on the surface of RSV-infected cells, and this conclusion was supported by ultracentrifugation analysis that demonstrated co-migration of surface-expressed F and G proteins. Although our analysis provided evidence for an interaction between the G and SH proteins, no evidence was obtained for a single protein complex involving all three of the virus proteins. These data suggest the existence of multiple virus glycoproteinmore » complexes within the RSV envelope. Although the stimulus that drives RSV-mediated membrane fusion is unknown, the association between the G and F proteins suggest an indirect role for the G protein in this process.« less

A plant virus movement protein forms ringlike complexes with the major nucleolar protein, fibrillarin, in vitro.

PubMed

Canetta, Elisabetta; Kim, Sang Hyon; Kalinina, Natalia O; Shaw, Jane; Adya, Ashok K; Gillespie, Trudi; Brown, John W S; Taliansky, Michael

2008-02-29

Fibrillarin, one of the major proteins of the nucleolus, has methyltransferase activity directing 2'-O-ribose methylation of rRNA and snRNAs and is required for rRNA processing. The ability of the plant umbravirus, groundnut rosette virus, to move long distances through the phloem, the specialized plant vascular system, has been shown to strictly depend on the interaction of one of its proteins, the ORF3 protein (protein encoded by open reading frame 3), with fibrillarin. This interaction is essential for several stages in the groundnut rosette virus life cycle such as nucleolar import of the ORF3 protein via Cajal bodies, relocalization of some fibrillarin from the nucleolus to cytoplasm, and assembly of cytoplasmic umbraviral ribonucleoprotein particles that are themselves required for the long-distance spread of the virus and systemic infection. Here, using atomic force microscopy, we determine the architecture of these complexes as single-layered ringlike structures with a diameter of 18-22 nm and a height of 2.0+/-0.4 nm, which consist of several (n=6-8) distinct protein granules. We also estimate the molar ratio of fibrillarin to ORF3 protein in the complexes as approximately 1:1. Based on these data, we propose a model of the structural organization of fibrillarin-ORF3 protein complexes and discuss potential mechanistic and functional implications that may also apply to other viruses.

Polypeptide synthesis induced in Nicotiana clevelandii protoplasts by infection with raspberry ringspot nepovirus.

PubMed

Acosta, O; Mayo, M A

1993-01-01

Infection of Nicotiana clevelandii protoplasts by raspberry ringspot nepovirus resulted in the accumulation of about 24 polypeptides that differed in M(r) and pI from polypeptides accumulating in mock-inoculated protoplasts. Similar polypeptides accumulated in protoplasts infected with the S and E strains of RRV but different infection-specific polypeptides were detected in protoplasts infected with tobacco ringspot nepovirus. The M(r) of RRV-specific polypeptides ranged from 210,000 to 18,000 and most are presumed to be derived from others by proteolytic cleavage. No evidence was found for marked changes in polypeptide abundance with time after inoculation or for any virus-specific polypeptide becoming disproportionately abundant in the medium during culture.

Genetic Variability and Evolutionary Implications of RNA Silencing Suppressor Genes in RNA1 of Sweet Potato Chlorotic Stunt Virus Isolates Infecting Sweetpotato and Related Wild Species

PubMed Central

Tugume, Arthur K.; Amayo, Robert; Weinheimer, Isabel; Mukasa, Settumba B.; Rubaihayo, Patrick R.; Valkonen, Jari P. T.

2013-01-01

Background The bipartite single-stranded RNA genome of Sweet potato chlorotic stunt virus (SPCSV, genus Crinivirus; Closteroviridae) encodes a Class 1 RNase III (RNase3), a putative hydrophobic protein (p7) and a 22-kDa protein (p22) from genes located in RNA1. RNase3 and p22 suppress RNA silencing, the basal antiviral defence mechanism in plants. RNase3 is sufficient to render sweetpotato (Ipomoea batatas) virus-susceptible and predisposes it to development of severe diseases following infection with unrelated virus. The incidence, strains and gene content of SPCSV infecting wild plant species have not been studied. Methodology/Principal Findings Thirty SPCSV isolates were characterized from 10 wild Ipomoea species, Hewittia sublobata or Lepistemon owariensis (family Convolvulaceae) in Uganda and compared with 34 local SPCSV isolates infecting sweetpotatoes. All isolates belonged to the East African (EA) strain of SPCSV and contained RNase3 and p7, but p22 was not detected in six isolates. The three genes showed only limited genetic variability and the proteins were under purifying selection. SPCSV isolates lacking p22 synergized with Sweet potato feathery mottle virus (SPFMV, genus potyvirus; Potyviridae) and caused severe symptoms in co-infected sweetpotato plants. One SPCSV isolate enhanced accumulation of SPFMV, but no severe symptoms developed. A new whitefly-transmitted virus (KML33b) encoding an RNase3 homolog (<56% identity to SPCSV RNase3) able to suppresses sense-mediated RNA silencing was detected in I. sinensis. Conclusions/Significance SPCSV isolates infecting wild species and sweetpotato in Uganda were genetically undifferentiated, suggesting inter-species transmission of SPCSV. Most isolates in Uganda contained p22, unlike SPCSV isolates characterized from other countries and continents. Enhanced accumulation of SPFMV and increased disease severity were found to be uncoupled phenotypic outcomes of RNase3-mediated viral synergism in sweetpotato. A second virus encoding an RNase3-like RNA silencing suppressor was detected. Overall, results provided many novel and important insights into evolutionary biology of SPCSV. PMID:24278443

ESCRT-II's involvement in HIV-1 genomic RNA trafficking and assembly.

PubMed

Ghoujal, Bashar; Milev, Miroslav P; Ajamian, Lara; Abel, Karen; Mouland, Andrew J

2012-12-01

Several host proteins play crucial roles in the HIV-1 replication cycle. The endosomal sorting complex required for transport (ESCRT) exemplifies a large, multi-component host machinery that is required by HIV-1 for viral budding. ESCRT promotes the inward budding of vesicles from the membranes of late endosomes to generate multi-vesicular bodies. However, HIV-1 co-opts the ESCRT to enable outwards budding of virus particles from the plasma membrane, a phenomenon that is topologically similar to multi-vesicular body biogenesis. A role for ESCRTII in mRNA trafficking has been established in Drosophila in which the ESCRT-II components, Vps22 and Vps36, promote the localisation of the bicoid mRNA in the fertilised egg. This is achieved via specific interactions with the Staufen protein. In this work, we investigated a possible implication of ESCRT-II in the HIV-1 replication cycle. Co-immunoprecipitation analyses and live cell tri-molecular fluorescence complementation assays revealed that interactions between EAP30 and Gag and another between EAP30 and Staufen1 occur in mammalian cells. We then depleted EAP30 (the orthologue for Vps22) by siRNA to target ESCRT-II in HIV-1 expressing cells. This treatment disrupted ESCRT-II function and leads to the degradation of the two other ESCRT-II complex proteins, EAP45 and EAP20, as well as the associated Rab7-interacting lysosomal protein. The depletion of EAP30 led to dramatically reduced viral structural protein Gag and virus production levels, without any effect on viral RNA levels. On the contrary, the overexpression of EAP30 led to a several-fold increase in virus production. Unexpec-tedly, siRNA-mediated depletion of EAP30 led to a block to HIV-1 genomic RNA trafficking and resulted in the accumulation of genomic RNA in the nucleus and juxtanuclear domains. Our data provide the first evidence that the Staufen1-ESCRT-II interaction is evolutionarily conserved from lower to higher eukaryotes and reveal a novel role for EAP30 in the control of HIV-1 RNA trafficking and gene expression. Copyright © 2012 Wiley-Liss, Inc.

Human Papillomavirus 16 Infection Induces VAP-Dependent Endosomal Tubulation.

PubMed

Siddiqa, Abida; Massimi, Paola; Pim, David; Broniarczyk, Justyna; Banks, Lawrence

2018-03-15

Human papillomavirus (HPV) infection involves complex interactions with the endocytic transport machinery, which ultimately facilitates the entry of the incoming viral genomes into the trans -Golgi network (TGN) and their subsequent nuclear entry during mitosis. The endosomal pathway is a highly dynamic intracellular transport system, which consists of vesicular compartments and tubular extensions, although it is currently unclear whether incoming viruses specifically alter the endocytic machinery. In this study, using MICAL-L1 as a marker for tubulating endosomes, we show that incoming HPV-16 virions induce a profound alteration in global levels of endocytic tubulation. In addition, we also show a critical requirement for the endoplasmic reticulum (ER)-anchored protein VAP in this process. VAP plays an essential role in actin nucleation and endosome-to-Golgi transport. Indeed, the loss of VAP results in a dramatic decrease in the level of endosomal tubulation induced by incoming HPV-16 virions. This is also accompanied by a marked reduction in virus infectivity. In VAP knockdown cells, we see that the defect in virus trafficking occurs after capsid disassembly but prior to localization at the trans -Golgi network, with the incoming virion-transduced DNA accumulating in Vps29/TGN46-positive hybrid vesicles. Taken together, these studies demonstrate that infection with HPV-16 virions induces marked alterations of endocytic transport pathways, some of which are VAP dependent and required for the endosome-to-Golgi transport of the incoming viral L2/DNA complex. IMPORTANCE Human papillomavirus infectious entry involves multiple interactions with the endocytic transport machinery. In this study, we show that incoming HPV-16 virions induce a dramatic increase in endocytic tubulation. This tubulation requires ER-associated VAP, which plays a critical role in ensuring the delivery of cargoes from the endocytic compartments to the trans -Golgi network. Indeed, the loss of VAP blocks HPV infectious entry at a step after capsid uncoating but prior to localization at the trans -Golgi network. These results define a critical role for ER-associated VAP in endocytic tubulation and in HPV-16 infectious entry. Copyright © 2018 American Society for Microbiology.

Gene repertoire of amoeba-associated giant viruses.

PubMed

Colson, Philippe; Raoult, Didier

2010-01-01

Acanthamoeba polyphaga mimivirus, Marseillevirus, and Sputnik, a virophage, are intra-amoebal viruses that have been isolated from water collected in cooling towers. They have provided fascinating data and have raised exciting questions about viruses definition and evolution. Mimivirus and Marseillevirus have been classified in the nucleo-cytoplasmic large DNA viruses (NCLDVs) class. Their genomes are the largest and fifth largest viral genomes sequenced so far. The gene repertoire of these amoeba-associated viruses can be divided into four groups: the core genome, genes acquired by lateral gene transfer, duplicated genes, and ORFans. Open reading frames (ORFs) that have homologs in the NCLDVs core gene set represent 2.9 and 6.1% of the Mimivirus and Marseillevirus gene contents, respectively. A substantial proportion of the Mimivirus, Marseillevirus and Sputnik ORFs exhibit sequence similarities to homologs found in bacteria, archaea, eukaryotes or viruses. The large amount of chimeric genes in these viral genomes might have resulted from acquisitions by lateral gene transfers, implicating sympatric bacteria and viruses with an intra-amoebal lifestyle. In addition, lineage-specific gene expansion may have played a major role in the genome shaping. Altogether, the data so far accumulated on amoeba-associated giant viruses are a powerful incentive to isolate and study additional strains to gain better understanding of their pangenome. Copyright 2010 S. Karger AG, Basel.

Samba virus: a novel mimivirus from a giant rain forest, the Brazilian Amazon.

PubMed

Campos, Rafael K; Boratto, Paulo V; Assis, Felipe L; Aguiar, Eric R G R; Silva, Lorena C F; Albarnaz, Jonas D; Dornas, Fabio P; Trindade, Giliane S; Ferreira, Paulo P; Marques, João T; Robert, Catherine; Raoult, Didier; Kroon, Erna G; La Scola, Bernard; Abrahão, Jônatas S

2014-05-14

The identification of novel giant viruses from the nucleocytoplasmic large DNA viruses group and their virophages has increased in the last decade and has helped to shed light on viral evolution. This study describe the discovery, isolation and characterization of Samba virus (SMBV), a novel giant virus belonging to the Mimivirus genus, which was isolated from the Negro River in the Brazilian Amazon. We also report the isolation of an SMBV-associated virophage named Rio Negro (RNV), which is the first Mimivirus virophage to be isolated in the Americas. Based on a phylogenetic analysis, SMBV belongs to group A of the putative Megavirales order, possibly a new virus related to Acanthamoeba polyphaga mimivirus (APMV). SMBV is the largest virus isolated in Brazil, with an average particle diameter about 574 nm. The SMBV genome contains 938 ORFs, of which nine are ORFans. The 1,213.6 kb SMBV genome is one of the largest genome of any group A Mimivirus described to date. Electron microscopy showed RNV particle accumulation near SMBV and APMV factories resulting in the production of defective SMBV and APMV particles and decreasing the infectivity of these two viruses by several logs. This discovery expands our knowledge of Mimiviridae evolution and ecology.

BECN1-dependent CASP2 incomplete autophagy induction by binding to rabies virus phosphoprotein.

PubMed

Liu, Juan; Wang, Hailong; Gu, Jinyan; Deng, Tingjuan; Yuan, Zhuangchuan; Hu, Boli; Xu, Yunbin; Yan, Yan; Zan, Jie; Liao, Min; DiCaprio, Erin; Li, Jianrong; Su, Shuo; Zhou, Jiyong

2017-04-03

Autophagy is an essential component of host immunity and used by viruses for survival. However, the autophagy signaling pathways involved in virus replication are poorly documented. Here, we observed that rabies virus (RABV) infection triggered intracellular autophagosome accumulation and results in incomplete autophagy by inhibiting autophagy flux. Subsequently, we found that RABV infection induced the reduction of CASP2/caspase 2 and the activation of AMP-activated protein kinase (AMPK)-AKT-MTOR (mechanistic target of rapamycin) and AMPK-MAPK (mitogen-activated protein kinase) pathways. Further investigation revealed that BECN1/Beclin 1 binding to viral phosphoprotein (P) induced an incomplete autophagy via activating the pathways CASP2-AMPK-AKT-MTOR and CASP2-AMPK-MAPK by decreasing CASP2. Taken together, our data first reveals a crosstalk of BECN1 and CASP2-dependent autophagy pathways by RABV infection.

Could viruses contribute to the worldwide epidemic of obesity?

PubMed

Atkinson, Richard L

2008-01-01

The prevalence of obesity in children increased rapidly starting about 1980 in both developed and developing countries. Studies of changes in diet and physical activity, television watching, and food advertisements on television suggest that these are not sufficient to explain the epidemic. The pattern of rapid spread is suggestive of an infectious origin. The concept of virus-induced obesity is not new. Eight viruses have been shown to cause obesity in animals and there is evidence for virus-induced obesity in humans. Recent evidence on animal and human adenoviruses suggests that these adenoviruses may infect adipocytes to alter enzymes and transcription factors resulting in accumulation of triglycerides and differentiation of preadipocytes into mature adipocytes. The E4orf1 gene of Ad-36 has been shown to be responsible for the adipogenic effect. It appears that a portion of the worldwide epidemic of obesity since 1980 could be due to infections with human adenoviruses.

Evolution of Cocirculating Varicella-Zoster Virus Genotypes during a Chickenpox Outbreak in Guinea-Bissau

PubMed Central

Gray, Eleanor R.; Kundu, Samit; Cooray, Samantha; Poulsen, Anja; Aaby, Peter; Breuer, Judith

2014-01-01

ABSTRACT Varicella-zoster virus (VZV), a double-stranded DNA alphaherpesvirus, is associated with seasonal outbreaks of varicella in nonimmunized populations. Little is known about whether these outbreaks are associated with a single or multiple viral genotypes and whether new mutations rapidly accumulate during transmission. Here, we take advantage of a well-characterized population cohort in Guinea-Bissau and produce a unique set of 23 full-length genome sequences, collected over 7 months from eight households. Comparative sequence analysis reveals that four distinct genotypes cocirculated among the population, three of which were present during the first week of the outbreak, although no patients were coinfected, which indicates that exposure to infectious virus from multiple sources is common during VZV outbreaks. Transmission of VZV was associated with length polymorphisms in the R1 repeat region and the origin of DNA replication. In two cases, these were associated with the formation of distinct lineages and point to the possible coevolution of these loci, despite the lack of any known functional link in VZV or related herpesviruses. We show that these and all other sequenced clade 5 viruses possess a distinct R1 repeat motif that increases the acidity of an ORF11p protein domain and postulate that this has either arisen or been lost following divergence of the major clades. Thus, sequencing of whole VZV genomes collected during an outbreak has provided novel insights into VZV biology, transmission patterns, and (recent) natural history. IMPORTANCE VZV is a highly infectious virus and the causative agent of chickenpox and shingles, the latter being particularly associated with the risk of painful complications. Seasonal outbreaks of chickenpox are very common among young children, yet little is known about the dynamics of the virus during person-to-person to transmission or whether multiple distinct viruses seed and/or cocirculate during an outbreak. In this study, we have sequenced chickenpox viruses from an outbreak in Guinea-Bissau that are supported by detailed epidemiological data. Our data show that multiple different virus strains seeded and were maintained throughout the 6-month outbreak period and that viruses transmitted between individuals accumulated new mutations in specific genomic regions. Of particular interest is the potential coevolution of two distinct parts of the genomes and our calculations of the rate of viral mutation, both of which increase our understanding of how VZV evolves over short periods of time in human populations. PMID:25275123

Evolution of cocirculating varicella-zoster virus genotypes during a chickenpox outbreak in Guinea-Bissau.

PubMed

Depledge, Daniel P; Gray, Eleanor R; Kundu, Samit; Cooray, Samantha; Poulsen, Anja; Aaby, Peter; Breuer, Judith

2014-12-01

Varicella-zoster virus (VZV), a double-stranded DNA alphaherpesvirus, is associated with seasonal outbreaks of varicella in nonimmunized populations. Little is known about whether these outbreaks are associated with a single or multiple viral genotypes and whether new mutations rapidly accumulate during transmission. Here, we take advantage of a well-characterized population cohort in Guinea-Bissau and produce a unique set of 23 full-length genome sequences, collected over 7 months from eight households. Comparative sequence analysis reveals that four distinct genotypes cocirculated among the population, three of which were present during the first week of the outbreak, although no patients were coinfected, which indicates that exposure to infectious virus from multiple sources is common during VZV outbreaks. Transmission of VZV was associated with length polymorphisms in the R1 repeat region and the origin of DNA replication. In two cases, these were associated with the formation of distinct lineages and point to the possible coevolution of these loci, despite the lack of any known functional link in VZV or related herpesviruses. We show that these and all other sequenced clade 5 viruses possess a distinct R1 repeat motif that increases the acidity of an ORF11p protein domain and postulate that this has either arisen or been lost following divergence of the major clades. Thus, sequencing of whole VZV genomes collected during an outbreak has provided novel insights into VZV biology, transmission patterns, and (recent) natural history. VZV is a highly infectious virus and the causative agent of chickenpox and shingles, the latter being particularly associated with the risk of painful complications. Seasonal outbreaks of chickenpox are very common among young children, yet little is known about the dynamics of the virus during person-to-person to transmission or whether multiple distinct viruses seed and/or cocirculate during an outbreak. In this study, we have sequenced chickenpox viruses from an outbreak in Guinea-Bissau that are supported by detailed epidemiological data. Our data show that multiple different virus strains seeded and were maintained throughout the 6-month outbreak period and that viruses transmitted between individuals accumulated new mutations in specific genomic regions. Of particular interest is the potential coevolution of two distinct parts of the genomes and our calculations of the rate of viral mutation, both of which increase our understanding of how VZV evolves over short periods of time in human populations. Copyright © 2014 Depledge et al.

Zika Virus: An Emergent Neuropathological Agent

PubMed Central

White, Martyn K.; Wollebo, Hassen S.; Beckham, J. David; Tyler, Kenneth L.; Khalili, Kamel

2016-01-01

The emergence of Zika virus in the Americas has followed a pattern that is familiar from earlier epidemics of other viruses, where a new disease is introduced into a human population and then spreads rapidly with important public health consequences. In the case of Zika virus, an accumulating body of recent evidence implicates the virus in the etiology of serious pathologies of the human nervous system, that is, the occurrence of microcephaly in neonates and Guillain–Barré syndrome in adults. Zika virus is an arbovirus (arthropod-borne virus) and a member of the family Flaviviridae, genus Flavivirus. Zika virions are enveloped and icosahedral, and contain a nonsegmented, single-stranded, positive-sense RNA genome, which encodes 3 structural and 7 nonstructural proteins that are expressed as a single polyprotein that undergoes cleavage. Zika genomic RNA replicates in the cytoplasm of infected host cells. Zika virus was first detected in 1947 in the blood of a febrile monkey in Uganda’s Zika Forest and in crushed suspensions of the Aedes mosquito, which is one of the vectors for Zika virus. The virus remained obscure, with a few human cases confined to Africa and Asia. There are two lineages of the Zika virus, African and Asian, with the Asian strain causing outbreaks in Micronesia in 2007 and French Polynesia in 2013–2014. From here, the virus spread to Brazil with the first report of autochthonous Zika transmission in the Americas in March 2015. The rapid advance of the virus in the Americas and its likely association with microcephaly and Guillain–Barré syndrome make Zika an urgent public health concern. PMID:27464346

Hypotensive Effect and Accumulation of Dinitrosyl Iron Complexes in Blood and Tissues after Intravenous and Subcutaneous Injection.

PubMed

Timoshin, A A; Lakomkin, V L; Abramov, A A; Ruuge, E K; Vanin, A F

2016-12-01

Subcutaneous injection of Oxacom with glutathione-bound dinitrosyl iron complex as the active principle produced a slower drop of mean BP and longer accumulation of protein-bound dinitrosyl iron complexes in whole blood and tissues than intravenous injection of this drug, while durations of hypotensive effect in both cases were practically identical. In contrast to intravenous injection of the drug, its subcutaneous administration was not characterized by a high concentration of protein-bound dinitrosyl iron complexes in the blood at the onset of experiment; in addition, accumulation of these NO forms in the lungs was more pronounced after subcutaneous injection than after intravenous one.

Complex genetic structure of the rabies virus in Bangkok and its surrounding provinces, Thailand: implications for canine rabies control.

PubMed

Lumlertdacha, Boonlert; Wacharapluesadee, Supaporn; Denduangboripant, Jessada; Ruankaew, Nipada; Hoonsuwan, Wirongrong; Puanghat, Apirom; Sakarasaeranee, Plyyonk; Briggs, Deborrah; Hemachudha, Thiravat

2006-03-01

Dog vaccination and population management have been suggested as priorities in attempts at disease control in canine rabies-endemic countries. Budget limitations and the complexity of social, cultural and religious variables have complicated progress in the developing world. In Bangkok, Thailand, an intensive canine vaccination and sterilization programme has been in place since November 2002. Our objective was to determine if the rabies virus could be mapped according to its genetic variations and geographical location on the small localized scale of Bangkok and its surrounding provinces. Phylogenetic characterization of 69 samples from Bangkok and five neighbouring and two remote provinces, by limited sequence analysis of the rabies virus nucleoprotein gene, distinguished six different clades. Rabies viruses of four clades were intermixed in Bangkok and in the surrounding highly populated regions whereas the other two clades were confined to rural and less populated provinces. Such a complex pattern of gene flow, particularly in Bangkok, may affect the outcome of canine control programmes.

Study of rubella candidate vaccine based on a structurally modified plant virus.

PubMed

Trifonova, Ekaterina A; Zenin, Vladimir A; Nikitin, Nikolai A; Yurkova, Maria S; Ryabchevskaya, Ekaterina M; Putlyaev, Egor V; Donchenko, Ekaterina K; Kondakova, Olga A; Fedorov, Alexey N; Atabekov, Joseph G; Karpova, Olga V

2017-08-01

A novel rubella candidate vaccine based on a structurally modified plant virus - spherical particles (SPs) - was developed. SPs generated by the thermal remodelling of the tobacco mosaic virus are promising platforms for the development of vaccines. SPs combine unique properties: biosafety, stability, high immunogenicity and the effective adsorption of antigens. We assembled in vitro and characterised complexes (candidate vaccine) based on SPs and the rubella virus recombinant antigen. The candidate vaccine induced a strong humoral immune response against rubella. The IgG isotypes ratio indicated the predominance of IgG1 which plays a key role in immunity to natural rubella infection. The immune response was generally directed against the rubella antigen within the complexes. We suggest that SPs can act as a platform (depot) for the rubella antigen, enhancing specific immune response. Our results demonstrate that SPs-antigen complexes can be an effective and safe candidate vaccine against rubella. Copyright © 2017 Elsevier B.V. All rights reserved.

The RNA synthesis machinery of negative-stranded RNA viruses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ortín, Juan, E-mail: jortin@cnb.csic.es; Martín-Benito, Jaime, E-mail: jmartinb@cnb.csic.es

The group of Negative-Stranded RNA Viruses (NSVs) includes many human pathogens, like the influenza, measles, mumps, respiratory syncytial or Ebola viruses, which produce frequent epidemics of disease and occasional, high mortality outbreaks by transmission from animal reservoirs. The genome of NSVs consists of one to several single-stranded, negative-polarity RNA molecules that are always assembled into mega Dalton-sized complexes by association to many nucleoprotein monomers. These RNA-protein complexes or ribonucleoproteins function as templates for transcription and replication by action of the viral RNA polymerase and accessory proteins. Here we review our knowledge on these large RNA-synthesis machines, including the structure ofmore » their components, the interactions among them and their enzymatic activities, and we discuss models showing how they perform the virus transcription and replication programmes. - Highlights: • Overall organisation of NSV RNA synthesis machines. • Structure and function of the ribonucleoprotein components: Atomic structure of the RNA polymerase complex. • Commonalities and differences between segmented- and non-segmented NSVs. • Transcription versus replication programmes.« less

The crystal structure of Zika virus NS5 reveals conserved drug targets.

PubMed

Duan, Wenqian; Song, Hao; Wang, Haiyuan; Chai, Yan; Su, Chao; Qi, Jianxun; Shi, Yi; Gao, George F

2017-04-03

Zika virus (ZIKV) has emerged as major health concern, as ZIKV infection has been shown to be associated with microcephaly, severe neurological disease and possibly male sterility. As the largest protein component within the ZIKV replication complex, NS5 plays key roles in the life cycle and survival of the virus through its N-terminal methyltransferase (MTase) and C-terminal RNA-dependent RNA polymerase (RdRp) domains. Here, we present the crystal structures of ZIKV NS5 MTase in complex with an RNA cap analogue ( m7 GpppA) and the free NS5 RdRp. We have identified the conserved features of ZIKV NS5 MTase and RdRp structures that could lead to development of current antiviral inhibitors being used against flaviviruses, including dengue virus and West Nile virus, to treat ZIKV infection. These results should inform and accelerate the structure-based design of antiviral compounds against ZIKV. © 2017 The Authors.

Ebola Virus VP35-VP40 Interaction Is Sufficient for Packaging 3E-5E Minigenome RNA into Virus-Like Particles

PubMed Central

Johnson, Reed F.; McCarthy, Sarah E.; Godlewski, Peter J.; Harty, Ronald N.

2006-01-01

The packaging of viral genomic RNA into nucleocapsids and subsequently into virions is not completely understood. Phosphoprotein (P) and nucleoprotein (NP) interactions link NP-RNA complexes with P-L (polymerase) complexes to form viral nucleocapsids. The nucleocapsid then interacts with the viral matrix protein, leading to specific packaging of the nucleocapsid into the virion. A mammalian two-hybrid assay and confocal microscopy were used to demonstrate that Ebola virus VP35 and VP40 interact and colocalize in transfected cells. VP35 was packaged into budding virus-like particles (VLPs) as observed by protease protection assays. Moreover, VP40 and VP35 were sufficient for packaging an Ebola virus minignome RNA into VLPs. Results from immunoprecipitation-reverse transcriptase PCR experiments suggest that VP35 confers specificity of the nucleocapsid for viral genomic RNA by direct VP35-RNA interactions. PMID:16698994

Serologic evidence of Jamestown Canyon and Keystone virus infection in vertebrates of the DelMarVa Peninsula.

PubMed

Watts, D M; LeDuc, J W; Bailey, C L; Dalrymple, J M; Gargan, T P

1982-11-01

Serological data accumulated during the past decade indicated that a variety of feral and domestic animals of the Delaware-Maryland-Virginia (DelMarVa) Peninsula were infected with Jamestown Canyon (JC) and/or Keystone (KEY) viruses (Bunyaviridae, California serogroup). Neutralizing (N) antibody to JC virus was most prevalent in white-tailed deer, sika deer, cottontail rabbits and horses. KEY virus N antibody was detected most frequently in gray squirrels and domestic goats. N antibody indicative of past infection by one or both viruses also was found in raccoons, horses and humans. JC and/or KEY virus N antibodies were not demonstrable in sera of several other species of small mammals and reptiles. Investigations were extended to evaluate the role of domestic goats as an amplifying host of JC and KEY viruses and to assess their potential as sentinels of virus transmission. Goats maintained in the Pocomoke Cypress Swamp during the summer season of 1978, acquired N antibodies to JC and KEY viruses. Following experimental inoculation with either JC or KEY virus, all goats developed N antibody despite the absence of a demonstrable viremia in most animals. Goats proved to be effective as sentinels for monitoring the transmission of JC and KEY viruses; however, the exceptionally low titers or absence of viremia following inoculation with these viruses would seem to preclude a potential virus-amplifying role for this species. Although findings implicated primarily gray squirrels and white-tailed deer as possible amplifying hosts of KEY and JC virus, respectively, further investigations will be required to clarify their role, particularly since both viruses may be maintained entirely by transovarial transmission.

Clomiphene and Its Isomers Block Ebola Virus Particle Entry and Infection with Similar Potency: Potential Therapeutic Implications

PubMed Central

Nelson, Elizabeth A.; Barnes, Alyson B.; Wiehle, Ronald D.; Fontenot, Gregory K.; Hoenen, Thomas; White, Judith M.

2016-01-01

The 2014 outbreak of Ebola virus (EBOV) in Western Africa highlighted the need for anti-EBOV therapeutics. Clomiphene is a U.S. Food and Drug Administration (FDA)-approved drug that blocks EBOV entry and infection in cells and significantly protects EBOV-challenged mice. As provided, clomiphene is, approximately, a 60:40 mixture of two stereoisomers, enclomiphene and zuclomiphene. The pharmacokinetic properties of the two isomers vary, but both accumulate in the eye and male reproductive tract, tissues in which EBOV can persist. Here we compared the ability of clomiphene and its isomers to inhibit EBOV using viral-like particle (VLP) entry and transcription/replication-competent VLP (trVLP) assays. Clomiphene and its isomers inhibited the entry and infection of VLPs and trVLPs with similar potencies. This was demonstrated with VLPs bearing the glycoproteins from three filoviruses (EBOV Mayinga, EBOV Makona, and Marburg virus) and in two cell lines (293T/17 and Vero E6). Visual problems have been noted in EBOV survivors, and viral RNA has been isolated from semen up to nine months post-infection. Since the clomiphene isomers accumulate in these affected tissues, clomiphene or one of its isomers warrants consideration as an anti-EBOV agent, for example, to potentially help ameliorate symptoms in EBOV survivors. PMID:27490565

Inhibition of PKR Activation by the Proline-Rich RNA Binding Domain of the Herpes Simplex Virus Type 1 Us11 Protein

PubMed Central

Poppers, Jeremy; Mulvey, Matthew; Khoo, David; Mohr, Ian

2000-01-01

Upon activation by double-stranded RNA in virus-infected cells, the cellular PKR kinase phosphorylates the translation initiation factor eukaryotic initiation factor 2 (eIF2) and thereby inhibits protein synthesis. The γ34.5 and Us11 gene products encoded by herpes simplex virus type 1 (HSV-1) are dedicated to preventing the accumulation of phosphorylated eIF2. While the γ34.5 gene specifies a regulatory subunit for protein phosphatase 1α, the Us11 gene encodes an RNA binding protein that also prevents PKR activation. γ34.5 mutants fail to grow on a variety of human cells as phosphorylated eIF2 accumulates and protein synthesis ceases prior to the completion of the viral life cycle. We demonstrate that expression of a 68-amino-acid fragment of Us11 containing a novel proline-rich basic RNA binding domain allows for sustained protein synthesis and enhanced growth of γ34.5 mutants. Furthermore, this fragment is sufficient to inhibit activation of the cellular PKR kinase in a cell-free system, suggesting that the intrinsic activities of this small fragment, notably RNA binding and ribosome association, may be required to prevent PKR activation. PMID:11070019

Inhibition of PKR activation by the proline-rich RNA binding domain of the herpes simplex virus type 1 Us11 protein.

PubMed

Poppers, J; Mulvey, M; Khoo, D; Mohr, I

2000-12-01

Upon activation by double-stranded RNA in virus-infected cells, the cellular PKR kinase phosphorylates the translation initiation factor eukaryotic initiation factor 2 (eIF2) and thereby inhibits protein synthesis. The gamma 34.5 and Us11 gene products encoded by herpes simplex virus type 1 (HSV-1) are dedicated to preventing the accumulation of phosphorylated eIF2. While the gamma 34.5 gene specifies a regulatory subunit for protein phosphatase 1 alpha, the Us11 gene encodes an RNA binding protein that also prevents PKR activation. gamma 34.5 mutants fail to grow on a variety of human cells as phosphorylated eIF2 accumulates and protein synthesis ceases prior to the completion of the viral life cycle. We demonstrate that expression of a 68-amino-acid fragment of Us11 containing a novel proline-rich basic RNA binding domain allows for sustained protein synthesis and enhanced growth of gamma 34.5 mutants. Furthermore, this fragment is sufficient to inhibit activation of the cellular PKR kinase in a cell-free system, suggesting that the intrinsic activities of this small fragment, notably RNA binding and ribosome association, may be required to prevent PKR activation.

Clomiphene and Its Isomers Block Ebola Virus Particle Entry and Infection with Similar Potency: Potential Therapeutic Implications.

PubMed

Nelson, Elizabeth A; Barnes, Alyson B; Wiehle, Ronald D; Fontenot, Gregory K; Hoenen, Thomas; White, Judith M

2016-08-02

The 2014 outbreak of Ebola virus (EBOV) in Western Africa highlighted the need for anti-EBOV therapeutics. Clomiphene is a U.S. Food and Drug Administration (FDA)-approved drug that blocks EBOV entry and infection in cells and significantly protects EBOV-challenged mice. As provided, clomiphene is, approximately, a 60:40 mixture of two stereoisomers, enclomiphene and zuclomiphene. The pharmacokinetic properties of the two isomers vary, but both accumulate in the eye and male reproductive tract, tissues in which EBOV can persist. Here we compared the ability of clomiphene and its isomers to inhibit EBOV using viral-like particle (VLP) entry and transcription/replication-competent VLP (trVLP) assays. Clomiphene and its isomers inhibited the entry and infection of VLPs and trVLPs with similar potencies. This was demonstrated with VLPs bearing the glycoproteins from three filoviruses (EBOV Mayinga, EBOV Makona, and Marburg virus) and in two cell lines (293T/17 and Vero E6). Visual problems have been noted in EBOV survivors, and viral RNA has been isolated from semen up to nine months post-infection. Since the clomiphene isomers accumulate in these affected tissues, clomiphene or one of its isomers warrants consideration as an anti-EBOV agent, for example, to potentially help ameliorate symptoms in EBOV survivors.

The Antiviral Mechanism of an Influenza A Virus Nucleoprotein-Specific Single-Domain Antibody Fragment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hanke, Leo; Knockenhauer, Kevin E.; Brewer, R. Camille

Alpaca-derived single-domain antibody fragments (VHHs) that target the influenza A virus nucleoprotein (NP) can protect cells from infection when expressed in the cytosol. We found that one such VHH, αNP-VHH1, exhibits antiviral activity similar to that of Mx proteins by blocking nuclear import of incoming viral ribonucleoproteins (vRNPs) and viral transcription and replication in the nucleus. We determined a 3.2-Å crystal structure of αNP-VHH1 in complex with influenza A virus NP. The VHH binds to a nonconserved region on the body domain of NP, which has been associated with binding to host factors and serves as a determinant of hostmore » range. Several of the NP/VHH interface residues determine sensitivity of NP to antiviral Mx GTPases. The structure of the NP/αNP-VHH1 complex affords a plausible explanation for the inhibitory properties of the VHH and suggests a rationale for the antiviral properties of Mx proteins. Such knowledge can be leveraged for much-needed novel antiviral strategies. IMPORTANCEInfluenza virus strains can rapidly escape from protection afforded by seasonal vaccines or acquire resistance to available drugs. Additional ways to interfere with the virus life cycle are therefore urgently needed. The influenza virus nucleoprotein is one promising target for antiviral interventions. We have previously isolated alpaca-derived single-domain antibody fragments (VHHs) that protect cells from influenza virus infection if expressed intracellularly. We show here that one such VHH exhibits antiviral activities similar to those of proteins of the cellular antiviral defense (Mx proteins). We determined the three-dimensional structure of this VHH in complex with the influenza virus nucleoprotein and identified the interaction site, which overlaps regions that determine sensitivity of the virus to Mx proteins. Our data define a new vulnerability of influenza virus, help us to better understand the cellular antiviral mechanisms, and provide a well-characterized tool to further study them.« less

Proteomic Analysis of Virus-Host Interactions in an Infectious Context Using Recombinant Viruses*

PubMed Central

Komarova, Anastassia V.; Combredet, Chantal; Meyniel-Schicklin, Laurène; Chapelle, Manuel; Caignard, Grégory; Camadro, Jean-Michel; Lotteau, Vincent; Vidalain, Pierre-Olivier; Tangy, Frédéric

2011-01-01

RNA viruses exhibit small-sized genomes encoding few proteins, but still establish complex networks of interactions with host cell components to achieve replication and spreading. Ideally, these virus-host protein interactions should be mapped directly in infected cell culture, but such a high standard is often difficult to reach when using conventional approaches. We thus developed a new strategy based on recombinant viruses expressing tagged viral proteins to capture both direct and indirect physical binding partners during infection. As a proof of concept, we engineered a recombinant measles virus (MV) expressing one of its virulence factors, the MV-V protein, with a One-STrEP amino-terminal tag. This allowed virus-host protein complex analysis directly from infected cells by combining modified tandem affinity chromatography and mass spectrometry analysis. Using this approach, we established a prosperous list of 245 cellular proteins interacting either directly or indirectly with MV-V, and including four of the nine already known partners of this viral factor. These interactions were highly specific of MV-V because they were not recovered when the nucleoprotein MV-N, instead of MV-V, was tagged. Besides key components of the antiviral response, cellular proteins from mitochondria, ribosomes, endoplasmic reticulum, protein phosphatase 2A, and histone deacetylase complex were identified for the first time as prominent targets of MV-V and the critical role of the later protein family in MV replication was addressed. Most interestingly, MV-V showed some preferential attachment to essential proteins in the human interactome network, as assessed by centrality and interconnectivity measures. Furthermore, the list of MV-V interactors also showed a massive enrichment for well-known targets of other viruses. Altogether, this clearly supports our approach based on reverse genetics of viruses combined with high-throughput proteomics to probe the interaction network that viruses establish in infected cells. PMID:21911578

Analysis of nuclear accumulation of influenza NP antigen in von Magnus virus-infected cells.

PubMed

Maeno, K; Aoki, H; Hamaguchi, M; Iinuma, M; Nagai, Y; Matsumoto, T; Takeura, S; Shibata, M

1981-01-01

When 1-5C-4 cells were infected with von Magnus virus derived from influenza A/RI/5+ virus by successive undiluted passages in chick embryos, virus-specific proteins were synthesized but production of infectious virus was inhibited. In these cells the synthesis of viral RNA was suppressed and the nucleoprotein (NP) antigen was found predominantly in the nucleus in contrast to standard virus-infected cells in which the antigen was distributed throughout the whole cell. The intracellular location and migration of NP were determined by isotope labeling and sucrose gradient centrifugation of subcellular fractions. In standard virus-infected cell NP polypeptide was present predominantly in the cytoplasm in the form of viral ribonucleoprotein (RNP) and intranuclear RNP was detected in reduced amounts. In contrast, in von Magnus virus-infected cells NP polypeptide was present predominantly in the nucleus in a nonassembled, soluble from and the amount of cytoplasmic RNP was considerably reduced. After short-pulse labeling NP was detected exclusively in the cytoplasm in a soluble form and after a chase a large proportion of such soluble NP was seen in the nucleus. It is suggested that a large proportion of the NP synthesized in von Magnus virus-infected cells in not assembled into cytoplasmic RNP because of the lack of available RNA and the NP migrated into the nucleus and remained there.

Ribonuclease from Bacillus Acts as an Antiviral Agent against Negative- and Positive-Sense Single Stranded Human Respiratory RNA Viruses

PubMed Central

Müller, Christin; Romanova, Yulia; Mostafa, Ahmed; Ulyanova, Vera; Pleschka, Stephan; Ilinskaya, Olga

2017-01-01

Bacillus pumilus ribonuclease (binase) was shown to be a promising antiviral agent in animal models and cell cultures. However, the mode of its antiviral action remains unknown. To assess the binase effect on intracellular viral RNA we have selected single stranded negative- and positive-sense RNA viruses, influenza virus, and rhinovirus, respectively, which annually cause respiratory illnesses and are characterized by high contagious nature, mutation rate, and antigen variability. We have shown that binase exerts an antiviral effect on both viruses at the same concentration, which does not alter the spectrum of A549 cellular proteins and expression of housekeeping genes. The titers of influenza A (H1N1pdm) virus and human rhinovirus serotype 1A were reduced by 40% and 65%, respectively. A preincubation of influenza virus with binase before infection significantly reduced viral titer after single-cycle replication of the virus. Using influenza A virus mini genome system we showed that binase reduced GFP reporter signaling indicating a binase action on the expression of viral mRNA. Binase reduced the level of H1N1pdm viral NP mRNA accumulation in A549 cells by 20%. Since the viral mRNA is a possible target for binase this agent could be potentially applied in the antiviral therapy against both negative- and positive-sense RNA viruses. PMID:28546965

Fighting fire with fire: attacking the complexity of human tumors with armed therapeutic viruses.

PubMed

Hermiston, Terry

2002-08-01

Cancer gene therapies have centered on the use of a single gene, directed against a particular property or single aspect of tumor biology, to treat neoplastic disease. These therapies have met with limited clinical success. This is, perhaps, not surprising given the complex and heterogeneous nature of solid tumors. Treatments targeted at confronting multiple dimensions of human tumors are needed. Armed therapeutic viruses (oncolytic viruses carrying therapeutic genes) represent a system where the concerted action of multiple therapeutics can be joined into a single agent, and represent a promising avenue for developing future cancer therapies.

Preventing Spread of Vine leafroll virus and Vine Mealybug

USDA-ARS?s Scientific Manuscript database

Leafroll disease in grapevines is caused by a complex of 11 species of viruses and occurs throughout the California's grape regions but causes more damage in the cooler wine grape regions. These viruses cause leaf chlorosis and leaf margins to roll downward. Leafroll can reduce berry yields up to ...

Identification of gene biomarkers for respiratory synctial virus infection in a bronchical epithelial cell line

EPA Science Inventory

Abstract: Respiratory syncytial virus (RSV) infection involves complex virus-host interplay. In this study, we analyzed gene expression in RSV-infected BEAS-2B cells to discover novel signaling pathways and biomarkers. We hybridized RNAs from RSV- or vehicle-treated BEAS-2B to ...

HIV Structural Database

National Institute of Standards and Technology Data Gateway

SRD 102 HIV Structural Database (Web, free access)   The HIV Protease Structural Database is an archive of experimentally determined 3-D structures of Human Immunodeficiency Virus 1 (HIV-1), Human Immunodeficiency Virus 2 (HIV-2) and Simian Immunodeficiency Virus (SIV) Proteases and their complexes with inhibitors or products of substrate cleavage.

Viral hemorrhagic fevers of animals caused by DNA viruses

USDA-ARS?s Scientific Manuscript database

Here we outline serious diseases of food and fiber animals that cause damaging economic effects on producers all over the world. The only vector-borne DNA virus is included here (i.e., African swine fever virus), and the herpesviruses discussed have a complex epidemiology characterized by outbreaks ...

Exosome-mediated miR-146a transfer suppresses type I interferon response and facilitates EV71 infection

PubMed Central

Fu, Yuxuan; Zhang, Li; Zhang, Fang; Tang, Ting; Zhou, Qi; Feng, Chunhong; Jin, Yu

2017-01-01

Exosomes can transfer genetic materials between cells. Their roles in viral infections are beginning to be appreciated. Researches have shown that exosomes released from virus-infected cells contain a variety of viral and host cellular factors that are able to modulate recipient’s cellular response and result in productive infection of the recipient host. Here, we showed that EV71 infection resulted in upregulated exosome secretion and differential packaging of the viral genomic RNA and miR-146a into exosomes. We provided evidence showing that miR-146a was preferentially enriched in exosomes while the viral RNA was not in infected cells. Moreover, the exosomes contained replication-competent EV71 RNA in complex with miR-146a, Ago2, and GW182 and could mediate EV71 transmission independent of virus-specific receptor. The exosomal viral RNA could be transferred to and replicate in a new target cell while the exosomal miR-146a suppressed type I interferon response in the target cell, thus facilitating the viral replication. Additionally, we found that the IFN-stimulated gene factors (ISGs), BST-2/tetherin, were involved in regulating EV71-induced upregulation of exosome secretion. Importantly, in vivo study showed that exosomal viral RNA exhibited differential tissue accumulation as compared to the free virus particles. Together, our findings provide evidence that exosomes secreted by EV71-infected cells selectively packaged high level miR-146a that can be functionally transferred to and facilitate exosomal EV71 RNA to replicate in the recipient cells by suppressing type I interferon response. PMID:28910400

Phosphorylation of the Antiviral Protein Interferon-inducible Transmembrane Protein 3 (IFITM3) Dually Regulates Its Endocytosis and Ubiquitination*

PubMed Central

Chesarino, Nicholas M.; McMichael, Temet M.; Hach, Jocelyn C.; Yount, Jacob S.

2014-01-01

Interferon-inducible transmembrane protein 3 (IFITM3) is essential for innate defense against influenza virus in mice and humans. IFITM3 localizes to endolysosomes where it prevents virus fusion, although mechanisms controlling its trafficking to this cellular compartment are not fully understood. We determined that both mouse and human IFITM3 are phosphorylated by the protein-tyrosine kinase FYN on tyrosine 20 (Tyr20) and that mouse IFITM3 is also phosphorylated on the non-conserved Tyr27. Phosphorylation led to a cellular redistribution of IFITM3, including plasma membrane accumulation. Mutation of Tyr20 caused a similar redistribution of IFITM3 and resulted in decreased antiviral activity against influenza virus, whereas Tyr27 mutation of mouse IFITM3 showed minimal effects on localization or activity. Using FYN knockout cells, we also found that IFITM3 phosphorylation is not a requirement for its antiviral activity. Together, these results indicate that Tyr20 is part of an endocytosis signal that can be blocked by phosphorylation or by mutation of this residue. Further mutagenesis narrowed this endocytosis-controlling region to four residues conforming to a YXXΦ (where X is any amino acid and Φ is Val, Leu, or Ile) endocytic motif that, when transferred to CD4, resulted in its internalization from the cell surface. Additionally, we found that phosphorylation of IFITM3 by FYN and mutagenesis of Tyr20 both resulted in decreased IFITM3 ubiquitination. Overall, these results suggest that modification of Tyr20 may serve in a cellular checkpoint controlling IFITM3 trafficking and degradation and demonstrate the complexity of posttranslational regulation of IFITM3. PMID:24627473

Selective Degradation of Host RNA Polymerase II Transcripts by Influenza A Virus PA-X Host Shutoff Protein

PubMed Central

Larkins-Ford, Jonah; McCormick, Craig; Gaglia, Marta M.

2016-01-01

Influenza A viruses (IAVs) inhibit host gene expression by a process known as host shutoff. Host shutoff limits host innate immune responses and may also redirect the translation apparatus to the production of viral proteins. Multiple IAV proteins regulate host shutoff, including PA-X, a ribonuclease that remains incompletely characterized. We report that PA-X selectively targets host RNA polymerase II (Pol II) transcribed mRNAs, while sparing products of Pol I and Pol III. Interestingly, we show that PA-X can also target Pol II-transcribed RNAs in the nucleus, including non-coding RNAs that are not destined to be translated, and reporter transcripts with RNA hairpin structures that block ribosome loading. Transcript degradation likely occurs in the nucleus, as PA-X is enriched in the nucleus and its nuclear localization correlates with reduction in target RNA levels. Complete degradation of host mRNAs following PA-X-mediated endonucleolytic cleavage is dependent on the host 5’->3’-exonuclease Xrn1. IAV mRNAs are structurally similar to host mRNAs, but are synthesized and modified at the 3’ end by the action of the viral RNA-dependent RNA polymerase complex. Infection of cells with wild-type IAV or a recombinant PA-X-deficient virus revealed that IAV mRNAs resist PA-X-mediated degradation during infection. At the same time, loss of PA-X resulted in changes in the synthesis of select viral mRNAs and a decrease in viral protein accumulation. Collectively, these results significantly advance our understanding of IAV host shutoff, and suggest that the PA-X causes selective degradation of host mRNAs by discriminating some aspect of Pol II-dependent RNA biogenesis in the nucleus. PMID:26849127

Differential pH-dependent cellular uptake pathways among foamy viruses elucidated using dual-colored fluorescent particles

PubMed Central

2012-01-01

Background It is thought that foamy viruses (FVs) enter host cells via endocytosis because all FV glycoproteins examined display pH-dependent fusion activities. Only the prototype FV (PFV) glycoprotein has also significant fusion activity at neutral pH, suggesting that its uptake mechanism may deviate from other FVs. To gain new insights into the uptake processes of FV in individual live host cells, we developed fluorescently labeled infectious FVs. Results N-terminal tagging of the FV envelope leader peptide domain with a fluorescent protein resulted in efficient incorporation of the fluorescently labeled glycoprotein into secreted virions without interfering with their infectivity. Double-tagged viruses consisting of an eGFP-tagged PFV capsid (Gag-eGFP) and mCherry-tagged Env (Ch-Env) from either PFV or macaque simian FV (SFVmac) were observed during early stages of the infection pathway. PFV Env, but not SFVmac Env, containing particles induced strong syncytia formation on target cells. Both virus types showed trafficking of double-tagged virions towards the cell center. Upon fusion and subsequent capsid release into the cytosol, accumulation of naked capsid proteins was observed within four hours in the perinuclear region, presumably representing the centrosomes. Interestingly, virions harboring fusion-defective glycoproteins still promoted virus attachment and uptake, but failed to show syncytia formation and perinuclear capsid accumulation. Biochemical and initial imaging analysis indicated that productive fusion events occur predominantly within 4–6 h after virus attachment. Non-fused or non-fusogenic viruses are rapidly cleared from the cells by putative lysosomal degradation. Quantitative monitoring of the fraction of individual viruses containing both Env and capsid signals as a function of time demonstrated that PFV virions fused within the first few minutes, whereas fusion of SFVmac virions was less pronounced and observed over the entire 90 minutes measured. Conclusions The characterized double-labeled FVs described here provide new mechanistic insights into FV early entry steps, demonstrating that productive viral fusion occurs early after target cell attachment and uptake. The analysis highlights apparent differences in the uptake pathways of individual FV species. Furthermore, the infectious double-labeled FVs promise to provide important tools for future detailed analyses on individual FV fusion events in real time using advanced imaging techniques. PMID:22935135

Accumulation of p53 in infectious mononucleosis tissues.

PubMed

Ehsan, A; Fan, H; Eagan, P A; Siddiqui, H A; Gulley, M L

2000-11-01

Epstein-Barr virus (EBV) infects lymphocytes, where it persists indefinitely for the life of the host; whether the virus interacts with p53 to maintain itself in these cells is unknown. Lymphoid biopsy samples from 10 patients with infectious mononucleosis (IM) were examined for expression of p53 by immunohistochemistry. Accumulation of p53 was detected in all 10 cases, primarily in large lymphocytes of the expanded paracortex. The presence of EBV was confirmed in all 10 cases by EBER1 (EBV-encoded RNA) in situ hybridization, whereas 11 non-IM control samples lacked significant EBER1 and did not express p53 in paracortical lymphocytes. Interestingly, EBV infection alone does not cause accumulation of intracellular p53, because many more cells expressed EBER1 than p53 in the IM tissues. To determine whether p53 was confined to the subset of infected cells in which viral replication was occurring, BZLF1 immunostains were performed. Viral BZLF1 was detected in 8 of 10 IM tissues; however, the paucity and small size of the BZLF1-expressing lymphocytes suggests that they are not the same cells overexpressing p53. To further examine the relationship between p53 and EBV gene expression, the tissues were studied for latent membrane protein 1 (LMP1) expression by immunohistochemistry. Viral LMP1 was observed in the large paracortical lymphocytes of all 10 cases of IM, indicating co-localization of p53 and LMP1 in these cells. Our findings confirm that p53 overexpression is not specific for nodal malignancy and that p53 accumulation is characteristic of IM. Because p53 was not coexpressed in the same cells as BZLF1, it appears that BZLF1 is not directly responsible for p53 accumulation. Nevertheless, co-localization of p53 and LMP1 in activated-appearing lymphocytes suggests that EBV infection is responsible for p53 accumulation. HUM PATHOL 31:1397-1403. Copyright 2000 by W.B. Saunders Company

Accumulation of a soluble form of human nectin-2 is required for exerting the resistance against herpes simplex virus type 2 infection in transfected cells.

PubMed

Fujimoto, Y; Ozaki, K; Iwamori, N; Takakuwa, H; Ono, E

2016-03-01

Cell entry of herpes simplex virus type 2 (HSV-2) requires the interaction of viral glycoprotein D (gD) with the receptor nectin-1 and herpesvirus entry mediator (HVEM). In addition, it is known that nectin-2 is also functional as a receptor for HSV-2, although the binding to the gD is weak. To examine an antiviral potential of a soluble form of human nectin-2 (hNectin-2Ig), transfected Vero cells expressing the entire ectodomain of nectin-2 fused to the Fc portion of human IgG were established. Specific binding of hNectin-2Ig to HSV-2 gD was confirmed by ELISA. Competitive ELISA demonstrated that accumulation of hNectin-2Ig in transfected cells increased significantly in a cell culture time dependent manner. Viral growth of several HSV-2 strains was significantly inhibited in the transfected cells that were cultured for 72 hr compared with control Vero cells, but not in cells that were cultured for 24 hr. These results indicate that accumulation of a soluble form of nectin-2 is required for exerting the resistance against HSV-2 infection.

Fluoxetine a novel anti-hepatitis C virus agent via ROS-, JNK-, and PPARβ/γ-dependent pathways.

PubMed

Young, Kung-Chia; Bai, Chyi-Huey; Su, Hui-Chen; Tsai, Pei-Ju; Pu, Chien-Yu; Liao, Chao-Sheng; Lin, Yu-Min; Lai, Hsin-Wen; Chong, Lee-Won; Tsai, Yau-Sheng; Tsao, Chiung-Wen

2014-10-01

More than 20% of chronic hepatitis C (CHC) patients receiving interferon-alpha (IFN-α)-based anti-hepatitis C virus (HCV) therapy experienced significant depression, which was relieved by treatment with fluoxetine. However, whether and how fluoxetine affected directly the anti-HCV therapy remained unclear. Here, we demonstrated that fluoxetine inhibited HCV infection and blocked the production of reactive oxygen species (ROS) and lipid accumulation in Huh7.5 cells. Fluoxetine facilitated the IFN-α-mediated antiviral actions via activations of signal transducer and activator of transcription (STAT)-1 and c-Jun amino-terminal kinases (JNK). Alternatively, fluoxetine elevated peroxisome proliferator-activated receptor (PPAR) response element activity under HCV infection. The inhibitory effects of fluoxetine on HCV infection and lipid accumulation, but not production of ROS, were partially reversed by the PPAR-β, -γ, and JNK antagonists. Furthermore, fluoxetine intervention to the IFN-α-2b regimen facilitated to reduce HCV titer and alanine transaminase level for CHC patients. Therefore, fluoxetine intervention to the IFN-α-2b regimen improved the efficacy of anti-HCV treatment, which might be related to blockades of ROS generation and lipid accumulation and activation of host antiviral JNK/STAT-1 and PPARβ/γ signals. Copyright © 2014 Elsevier B.V. All rights reserved.

Host-Pathogen Interactions : XXXII. A Fungal Glucan Preparation Protects Nicotianae against Infection by Viruses.

PubMed

Kopp, M; Rouster, J; Fritig, B; Darvill, A; Albersheim, P

1989-05-01

A glucan preparation obtained from the mycelial walls of the fungus Phytophthora megasperma f.sp. glycinea and known as an elicitor of phytoalexins in soybean was shown to be a very efficient inducer of resistance against viruses in tobacco. The glucan preparation protected against mechanically transmitted viral infections on the upper and lower leaf surfaces. Whether the glucan preparation was applied by injection, inoculation, or spraying, it protected the plants if applied before, at the same time as, or not later than 8 hours after virus inoculation. At concentrations ranging from 0.1 to 10 micrograms per milliliter, the glucan preparation induced protection ranging from 50 to 100% against both symptom production (necrotic local lesions, necrotic rings, or systemic mosaic) and virus accumulation in all Nicotiana-virus combinations examined. However, no significant protection against some of the same viruses was observed in bean or turnip. The host plants successfully protected included N. tabacum (9 different cultivars), N. sylvestris, N. glutinosa, and N. clevelandii. The viruses belonged to several taxonomic groups including tobacco mosaic virus, alfalfa mosaic virus, and tomato black ring virus. The glucan preparation did not act directly on the virus and did not interfere with virus disassembly; rather, it appeared to induce changes in the host plant that prevented infections from being initiated or recently established infections from enlarging. The induced resistance does not depend on induction of pathogenesis-related proteins, the phenylpropanoid pathway, lignin-like substances, or callose-like materials. We believe the induced resistance results from a mechanism that has yet to be described.

The C Terminus of the Herpes Simplex Virus UL25 Protein Is Required for Release of Viral Genomes from Capsids Bound to Nuclear Pores

PubMed Central

Huffman, Jamie B.; Daniel, Gina R.; Falck-Pedersen, Erik; Huet, Alexis

2017-01-01

ABSTRACT The herpes simplex virus (HSV) capsid is released into the cytoplasm after fusion of viral and host membranes, whereupon dynein-dependent trafficking along microtubules targets it to the nuclear envelope. Binding of the capsid to the nuclear pore complex (NPC) is mediated by the capsid protein pUL25 and the capsid-tethered tegument protein pUL36. Temperature-sensitive mutants in both pUL25 and pUL36 dock at the NPC but fail to release DNA. The uncoating reaction has been difficult to study due to the rapid release of the genome once the capsid interacts with the nuclear pore. In this study, we describe the isolation and characterization of a truncation mutant of pUL25. Live-cell imaging and immunofluorescence studies demonstrated that the mutant was not impaired in penetration of the host cell or in trafficking of the capsid to the nuclear membrane. However, expression of viral proteins was absent or significantly delayed in cells infected with the pUL25 mutant virus. Transmission electron microscopy revealed capsids accumulated at nuclear pores that retained the viral genome for at least 4 h postinfection. In addition, cryoelectron microscopy (cryo-EM) reconstructions of virion capsids did not detect any obvious differences in the location or structural organization for the pUL25 or pUL36 proteins on the pUL25 mutant capsids. Further, in contrast to wild-type virus, the antiviral response mediated by the viral DNA-sensing cyclic guanine adenine synthase (cGAS) was severely compromised for the pUL25 mutant. These results demonstrate that the pUL25 capsid protein has a critical role in releasing viral DNA from NPC-bound capsids. IMPORTANCE Herpes simplex virus 1 (HSV-1) is the causative agent of several pathologies ranging in severity from the common cold sore to life-threatening encephalitic infection. Early steps in infection include release of the capsid into the cytoplasm, docking of the capsid at a nuclear pore, and release of the viral genome into the nucleus. A key knowledge gap is how the capsid engages the NPC and what triggers release of the viral genome into the nucleus. Here we show that the C-terminal region of the HSV-1 pUL25 protein is required for releasing the viral genome from capsids docked at nuclear pores. The significance of our research is in identifying pUL25 as a key viral factor for genome uncoating. pUL25 is found at each of the capsid vertices as part of the capsid vertex-specific component and implicates the importance of this complex for NPC binding and genome release. PMID:28490590

Novel triple reassortant H1N2 influenza viruses bearing six internal genes of the pandemic 2009/H1N1 influenza virus were detected in pigs in China.

PubMed

Qiao, Chuanling; Liu, Liping; Yang, Huanliang; Chen, Yan; Xu, Huiyang; Chen, Hualan

2014-12-01

The pandemic A/H1N1 influenza viruses emerged in both Mexico and the United States in March 2009, and were transmitted efficiently in the human population. Transmissions of the pandemic 2009/H1N1 virus from humans to poultry and other species of mammals were reported from several continents during the course of the 2009 H1N1 pandemic. Reassortant H1N1, H1N2, and H3N2 viruses containing genes of the pandemic 2009/H1N1 viruses appeared in pigs in some countries. In winter of 2012, a total of 2600 nasal swabs were collected from healthy pigs in slaughterhouses located throughout 10 provinces in China. The isolated viruses were subjected to genetic and antigenic analysis. Two novel triple-reassortant H1N2 influenza viruses were isolated from swine in China in 2012, with the HA gene derived from Eurasian avian-like swine H1N1, the NA gene from North American swine H1N2, and the six internal genes from the pandemic 2009/H1N1 viruses. The two viruses had similar antigenic features and some significant changes in antigenic characteristics emerged when compared to the previously identified isolates. We inferred that the novel reassortant viruses in China may have arisen from the accumulation of the three types of influenza viruses, which further indicates that swine herds serve as "mixing vessels" for influenza viruses. Influenza virus reassortment is an ongoing process, and our findings highlight the urgent need for continued influenza surveillance among swine herds. Copyright © 2014 Elsevier B.V. All rights reserved.

Complete genome analysis of 33 ecologically and biologically diverse Rift Valley fever virus strains reveals widespread virus movement and low genetic diversity due to recent common ancestry.

PubMed

Bird, Brian H; Khristova, Marina L; Rollin, Pierre E; Ksiazek, Thomas G; Nichol, Stuart T

2007-03-01

Rift Valley fever (RVF) virus is a mosquito-borne RNA virus responsible for large explosive outbreaks of acute febrile disease in humans and livestock in Africa with significant mortality and economic impact. The successful high-throughput generation of the complete genome sequence was achieved for 33 diverse RVF virus strains collected from throughout Africa and Saudi Arabia from 1944 to 2000, including strains differing in pathogenicity in disease models. While several distinct virus genetic lineages were determined, which approximately correlate with geographic origin, multiple exceptions indicative of long-distance virus movement have been found. Virus strains isolated within an epidemic (e.g., Mauritania, 1987, or Egypt, 1977 to 1978) exhibit little diversity, while those in enzootic settings (e.g., 1970s Zimbabwe) can be highly diverse. In addition, the large Saudi Arabian RVF outbreak in 2000 appears to have involved virus introduction from East Africa, based on the close ancestral relationship of a 1998 East African virus. Virus genetic diversity was low (approximately 5%) and primarily involved accumulation of mutations at an average of 2.9 x 10(-4) substitutions/site/year, although some evidence of RNA segment reassortment was found. Bayesian analysis of current RVF virus genetic diversity places the most recent common ancestor of these viruses in the late 1800s, the colonial period in Africa, a time of dramatic changes in agricultural practices and introduction of nonindigenous livestock breeds. In addition to insights into the evolution and ecology of RVF virus, these genomic data also provide a foundation for the design of molecular detection assays and prototype vaccines useful in combating this important disease.

Manganese-Enhanced Magnetic Resonance Imaging Reflects Brain Pathology During Progressive HIV-1 Infection of Humanized Mice.

PubMed

Bade, Aditya N; Gorantla, Santhi; Dash, Prasanta K; Makarov, Edward; Sajja, Balasrinivasa R; Poluektova, Larisa Y; Luo, Jiangtao; Gendelman, Howard E; Boska, Michael D; Liu, Yutong

2016-07-01

Progressive human immunodeficiency viral (HIV) infection commonly leads to a constellation of cognitive, motor, and behavioral impairments. These are collectively termed HIV-associated neurocognitive disorders (HAND). While antiretroviral therapy (ART) reduces HAND severity, it does not affect disease prevalence. Despite decades of research, there remain no biomarkers for HAND and all potential comorbid conditions must first be excluded for a diagnosis to be made. To this end, we now report that manganese (Mn(2+))-enhanced magnetic resonance imaging (MEMRI) can reflect brain region-specific HIV-1-induced neuropathology in chronically virus-infected NOD/scid-IL-2Rγc(null) humanized mice. MEMRI diagnostics mirrors the abilities of Mn(2+) to enter and accumulate in affected neurons during disease. T1 relaxivity and its weighted signal intensity are proportional to Mn(2+) activities in neurons. In 16-week virus-infected humanized mice, altered MEMRI signal enhancement was easily observed in affected brain regions. These included, but were not limited to, the hippocampus, amygdala, thalamus, globus pallidus, caudoputamen, substantia nigra, and cerebellum. MEMRI signal was coordinated with levels of HIV-1 infection, neuroinflammation (astro- and micro-gliosis), and neuronal injury. MEMRI accurately demonstrates the complexities of HIV-1-associated neuropathology in rodents that reflects, in measure, the clinical manifestations of neuroAIDS as it is seen in a human host.

Editing of HIV-1 RNA by the double-stranded RNA deaminase ADAR1 stimulates viral infection

PubMed Central

Doria, Margherita; Neri, Francesca; Gallo, Angela; Farace, Maria Giulia; Michienzi, Alessandro

2009-01-01

Adenosine deaminases that act on dsRNA (ADARs) are enzymes that target double-stranded regions of RNA converting adenosines into inosines (A-to-I editing) thus contributing to genome complexity and fine regulation of gene expression. It has been described that a member of the ADAR family, ADAR1, can target viruses and affect their replication process. Here we report evidence showing that ADAR1 stimulates human immuno deficiency virus type 1 (HIV-1) replication by using both editing-dependent and editing-independent mechanisms. We show that over-expression of ADAR1 in HIV-1 producer cells increases viral protein accumulation in an editing-independent manner. Moreover, HIV-1 virions generated in the presence of over-expressed ADAR1 but not an editing-inactive ADAR1 mutant are released more efficiently and display enhanced infectivity, as demonstrated by challenge assays performed with T cell lines and primary CD4+ T lymphocytes. Finally, we report that ADAR1 associates with HIV-1 RNAs and edits adenosines in the 5′ untranslated region (UTR) and the Rev and Tat coding sequence. Overall these results suggest that HIV-1 has evolved mechanisms to take advantage of specific RNA editing activity of the host cell and disclose a stimulatory function of ADAR1 in the spread of HIV-1. PMID:19651874