Genetics coupled to quantitative intact proteomics links heritable aphid and endosymbiont protein isoform expression to polerovirus transmission

USDA-ARS?s Scientific Manuscript database

Yellow dwarf viruses in the family Luteoviridae, such as Cereal yellow dwarf virus-RPV (CYDV-RPV), are vectored by aphids and cause the most economically important virus disease of cereal crops worldwide. The identification of aphid proteins mediating virus transmission will better define transmiss...

Non-structural protein P6 encoded by rice black-streaked dwarf virus is recruited to viral inclusion bodies by binding to the viroplasm matrix protein P9-1.

PubMed

Sun, Liying; Xie, Li; Andika, Ida Bagus; Tan, Zilong; Chen, Jianping

2013-08-01

Like other members of the family Reoviridae, rice black-streaked dwarf virus (RBSDV, genus Fijivirus) is thought to replicate and assemble within cytoplasmic viral inclusion bodies, commonly called viroplasms. RBSDV P9-1 is the key protein for the formation of viroplasms, but little is known about the other proteins of the viroplasm or the molecular interactions amongst its components. RBSDV non-structural proteins were screened for their association with P9-1 using a co-immunoprecipitation assay. Only P6 was found to directly interact with P9-1, an interaction that was confirmed by bimolecular fluorescence complementation assay in Spodoptera frugiperda (Sf9) cells. Immunoelectron microscopy showed that P6 and P9-1 co-localized in electron-dense inclusion bodies, indicating that P6 is a constituent of the viroplasm. In addition, non-structural protein P5 also localized to viroplasms and interacted with P6. In Sf9 cells, P6 was diffusely distributed throughout the cytoplasm when expressed alone, but localized to inclusions when co-expressed with P9-1, suggesting that P6 is recruited to viral inclusion bodies by binding to P9-1. P5 localized to the inclusions formed by P9-1 when co-expressed with P6 but did not when P6 was absent, suggesting that P5 is recruited to viroplasms by binding to P6. This study provides a model by which viral non-structural proteins are recruited to RBSDV viroplasms.

Virus incidence in orchardgrass (Dactylis glomerata L.) seed production fields in the Willamette Valley

USDA-ARS?s Scientific Manuscript database

A survey was conducted over the course of three years (2014-2016) for the presence of Barley yellow dwarf virus (BYDV-MAV and BYDV-PAV), Cereal yellow dwarf virus (CYDV-RPV), and Cocksfoot mottle virus (CfMV) in orchardgrass (Dactylis glomerata) fields in the Willamette Valley, Oregon. There was an ...

Dispersed gold nanoparticles potentially ruin gold barley yellow dwarf virus and eliminate virus infectivity hazards

NASA Astrophysics Data System (ADS)

Alkubaisi, Noorah A.; Aref, Nagwa M. A.

2017-02-01

Gold nanoparticles (AuNPs) application melted barley yellow dwarf virus-PAV (BYDV-PAV) spherical nanoparticle capsids. Synergistic therapeutic effects for plant virus resistance were induced by interaction with binding units of prepared AuNPs in a water solution which was characterized and evaluated by zeta sizer, zeta potential and transmission electron microscopy (TEM). The yield of purified nanoparticles of BYDV-PAV was obtained from Hordeum vulgare (Barley) cultivars, local and Giza 121/Justo. It was 0.62 mg/ml from 27.30 g of infected leaves at an A260/A280 ratio. Virus nanoparticle has a spherical shape 30 nm in size by TEM. BYDV-PAV combined with AuNPs to challenge virus function in vivo and in vitro. Dual AuNPs existence in vivo and in vitro affected compacted configuration of viral capsid protein in the interior surface of capsomers, the outer surface, or between the interface of coat protein subunits for 24 and 48 h incubation period in vitro at room temperature. The sizes of AuNPs that had a potentially dramatic deteriorated effect are 3.151 and 31.67 nm with a different intensity of 75.3% for the former and 24.7% for the latter, which enhances optical sensing applications to eliminate virus infectivity. Damages of capsid protein due to AuNPs on the surface of virus subunits caused variable performance in four different types of TEM named puffed, deteriorated and decorated, ruined and vanished. Viral yield showed remarkably high-intensity degree of particle symmetry and uniformity in the local cultivar greater than in Giza 121/Justo cultivar. A high yield of ruined VLPs in the local cultivar than Justo cultivar was noticed. AuNPs indicated complete lysed VLPs and some deteriorated VLPs at 48 h.

Genome-wide association mapping of barley yellow dwarf virus tolerance in spring oat (Avena sativa L.)

USDA-ARS?s Scientific Manuscript database

Barley yellow dwarf (BYD) is one of the most destructive diseases of cereal crops worldwide. Barley yellow dwarf viruses (BYDVs) are responsible for BYD and affect many cereals including oat (Avena sativa L.). Until recently, the molecular marker technology in oat has not allowed for many marker-t...

Incidence of viruses in fescue (Festuca sp.) seed production fields in the Willamette Valley in 2016

USDA-ARS?s Scientific Manuscript database

Tall Fescue seed production fields of Western Oregon were sampled and tested for the presence or absence of three viruses, Barley yellow dwarf virus (BYDV) -MAV and -PAV, and Cereal yellow dwarf virus (CYDV). There was no BYDV-MAV detected in any of the Fescue seed fields. The BYDV-PAV occurred in ...

In vitro synthesis of minus-strand RNA by an isolated cereal yellow dwarf virus RNA-dependent RNA polymerase requires VPg and a stem-loop structure at the 3' end of the virus RNA.

PubMed

Osman, Toba A M; Coutts, Robert H A; Buck, Kenneth W

2006-11-01

Cereal yellow dwarf virus (CYDV) RNA has a 5'-terminal genome-linked protein (VPg). We have expressed the VPg region of the CYDV genome in bacteria and used the purified protein (bVPg) to raise an antiserum which was able to detect free VPg in extracts of CYDV-infected oat plants. A template-dependent RNA-dependent RNA polymerase (RdRp) has been produced from a CYDV membrane-bound RNA polymerase by treatment with BAL 31 nuclease. The RdRp was template specific, being able to utilize templates from CYDV plus- and minus-strand RNAs but not those of three unrelated viruses, Red clover necrotic mosaic virus, Cucumber mosaic virus, and Tobacco mosaic virus. RNA synthesis catalyzed by the RdRp required a 3'-terminal GU sequence and the presence of bVPg. Additionally, synthesis of minus-strand RNA on a plus-strand RNA template required the presence of a putative stem-loop structure near the 3' terminus of CYDV RNA. The base-paired stem, a single-nucleotide (A) bulge in the stem, and the sequence of a tetraloop were all required for the template activity. Evidence was produced showing that minus-strand synthesis in vitro was initiated by priming by bVPg at the 3' end of the template. The data are consistent with a model in which the RdRp binds to the stem-loop structure which positions the active site to recognize the 3'-terminal GU sequence for initiation of RNA synthesis by the addition of an A residue to VPg.

In Vitro Synthesis of Minus-Strand RNA by an Isolated Cereal Yellow Dwarf Virus RNA-Dependent RNA Polymerase Requires VPg and a Stem-Loop Structure at the 3′ End of the Virus RNA▿

PubMed Central

Osman, Toba A. M.; Coutts, Robert H. A.; Buck, Kenneth W.

2006-01-01

Cereal yellow dwarf virus (CYDV) RNA has a 5′-terminal genome-linked protein (VPg). We have expressed the VPg region of the CYDV genome in bacteria and used the purified protein (bVPg) to raise an antiserum which was able to detect free VPg in extracts of CYDV-infected oat plants. A template-dependent RNA-dependent RNA polymerase (RdRp) has been produced from a CYDV membrane-bound RNA polymerase by treatment with BAL 31 nuclease. The RdRp was template specific, being able to utilize templates from CYDV plus- and minus-strand RNAs but not those of three unrelated viruses, Red clover necrotic mosaic virus, Cucumber mosaic virus, and Tobacco mosaic virus. RNA synthesis catalyzed by the RdRp required a 3′-terminal GU sequence and the presence of bVPg. Additionally, synthesis of minus-strand RNA on a plus-strand RNA template required the presence of a putative stem-loop structure near the 3′ terminus of CYDV RNA. The base-paired stem, a single-nucleotide (A) bulge in the stem, and the sequence of a tetraloop were all required for the template activity. Evidence was produced showing that minus-strand synthesis in vitro was initiated by priming by bVPg at the 3′ end of the template. The data are consistent with a model in which the RdRp binds to the stem-loop structure which positions the active site to recognize the 3′-terminal GU sequence for initiation of RNA synthesis by the addition of an A residue to VPg. PMID:16928757

The complete nucleotide sequence of the barley yellow dwarf GPV isolate from China shows that it is a new member of the genus Polerovirus.

PubMed

Zhang, Wenwei; Cheng, Zhuomin; Xu, Lei; Wu, Maosen; Waterhouse, Peter; Zhou, Guanghe; Li, Shifang

2009-01-01

The complete nucleotide sequence of the ssRNA genome of a Chinese GPV isolate of barley yellow dwarf virus (BYDV) was determined. It comprised 5673 nucleotides, and the deduced genome organization resembled that of members of the genus Polerovirus. It was most closely related to cereal yellow dwarf virus-RPV (77% nt identity over the entire genome; coat protein amino acid identity 79%). The GPV isolate also differs in vector specificity from other BYDV strains. Biological properties, phylogenetic analyses and detailed sequence comparisons suggest that GPV should be considered a member of a new species within the genus, and the name Wheat yellow dwarf virus-GPV is proposed.

First report of a resistance-breaking strain of Raspberry bushy dwarf virus in red raspberry (Rubus idaeus) in North America

USDA-ARS?s Scientific Manuscript database

Raspberry bushy dwarf virus (RBDV) is pollen-transmitted and the most important virus of Rubus worldwide. Infection of RBDV is associated with drupelet abortion, resulting in crumbly fruit. Multiple RBDV strains have been reported, with the Scottish-type (D200) strains being the most prevalent, and...

Pollen Transmitted Diseases, Raspberry bushy dwarf virus

USDA-ARS?s Scientific Manuscript database

Raspberry bushy dwarf virus (RBDV) occurs naturally worldwide in many Rubus species and cultivars. In North America, it naturally infects many red raspberry, black raspberry, blackberry and blackberry-raspberry hybrid cultivars. RBDV also occurs in wild R. idaeus L. var. strigosus, R. occidentali., ...

The complete genome sequence of a virus associated with cotton blue disease, cotton leafroll dwarf virus, confirms that it is a new member of the genus Polerovirus.

PubMed

Distéfano, Ana J; Bonacic Kresic, Ivan; Hopp, H Esteban

2010-11-01

Cotton blue disease is the most important virus disease of cotton in the southern part of America. The complete nucleotide sequence of the ssRNA genome of the cotton blue disease-associated virus was determined for the first time. It comprised 5,866 nucleotides, and the deduced genomic organization resembled that of members of the genus Polerovirus. Sequence homology comparison and phylogenetic analysis confirm that this virus (previous proposed name cotton leafroll dwarf virus) is a member of a new species within the genus Polerovirus.

Identification of a maize chlorotic dwarf virus silencing suppressor protein

USDA-ARS?s Scientific Manuscript database

Maize chlorotic dwarf virus (MCDV), a member of the genus Waikavirus, family Secoviridae, has a 11784 nt (+)ssRNA genome that encodes a 389 kDa proteolytically processed polyprotein. We show that an N-terminal 78kDa polyprotein (R78) has silencing suppressor activity, that it is cleaved by the viral...

Coupling Genetics and Proteomics To Identify Aphid Proteins Associated with Vector-Specific Transmission of Polerovirus (Luteoviridae)▿

PubMed Central

Yang, Xiaolong; Thannhauser, T. W.; Burrows, Mary; Cox-Foster, Diana; Gildow, Fred E.; Gray, Stewart M.

2008-01-01

Cereal yellow dwarf virus-RPV (CYDV-RPV) is transmitted specifically by the aphids Rhopalosiphum padi and Schizaphis graminum in a circulative nonpropagative manner. The high level of vector specificity results from the vector aphids having the functional components of the receptor-mediated endocytotic pathways to allow virus to transverse the gut and salivary tissues. Studies of F2 progeny from crosses of vector and nonvector genotypes of S. graminum showed that virus transmission efficiency is a heritable trait regulated by multiple genes acting in an additive fashion and that gut- and salivary gland-associated factors are not genetically linked. Utilizing two-dimensional difference gel electrophoresis to compare the proteomes of vector and nonvector parental and F2 genotypes, four aphid proteins (S4, S8, S29, and S405) were specifically associated with the ability of S. graminum to transmit CYDV-RPV. The four proteins were coimmunoprecipitated with purified RPV, indicating that the aphid proteins are capable of binding to virus. Analysis by mass spectrometry identified S4 as a luciferase and S29 as a cyclophilin, both of which have been implicated in macromolecular transport. Proteins S8 and S405 were not identified from available databases. Study of this unique genetic system coupled with proteomic analysis indicated that these four virus-binding aphid proteins were specifically inherited and conserved in different generations of vector genotypes and suggests that they play a major role in regulating polerovirus transmission. PMID:17959668

Coupling genetics and proteomics to identify aphid proteins associated with vector-specific transmission of polerovirus (luteoviridae).

PubMed

Yang, Xiaolong; Thannhauser, T W; Burrows, Mary; Cox-Foster, Diana; Gildow, Fred E; Gray, Stewart M

2008-01-01

Cereal yellow dwarf virus-RPV (CYDV-RPV) is transmitted specifically by the aphids Rhopalosiphum padi and Schizaphis graminum in a circulative nonpropagative manner. The high level of vector specificity results from the vector aphids having the functional components of the receptor-mediated endocytotic pathways to allow virus to transverse the gut and salivary tissues. Studies of F(2) progeny from crosses of vector and nonvector genotypes of S. graminum showed that virus transmission efficiency is a heritable trait regulated by multiple genes acting in an additive fashion and that gut- and salivary gland-associated factors are not genetically linked. Utilizing two-dimensional difference gel electrophoresis to compare the proteomes of vector and nonvector parental and F(2) genotypes, four aphid proteins (S4, S8, S29, and S405) were specifically associated with the ability of S. graminum to transmit CYDV-RPV. The four proteins were coimmunoprecipitated with purified RPV, indicating that the aphid proteins are capable of binding to virus. Analysis by mass spectrometry identified S4 as a luciferase and S29 as a cyclophilin, both of which have been implicated in macromolecular transport. Proteins S8 and S405 were not identified from available databases. Study of this unique genetic system coupled with proteomic analysis indicated that these four virus-binding aphid proteins were specifically inherited and conserved in different generations of vector genotypes and suggests that they play a major role in regulating polerovirus transmission.

Barley yellow dwarf virus infection and elevated CO2 alter the antioxidants ascorbate and glutathione in wheat.

PubMed

Vandegeer, Rebecca K; Powell, Kevin S; Tausz, Michael

2016-07-20

Plant antioxidants ascorbate and glutathione play an important role in regulating potentially harmful reactive oxygen species produced in response to virus infection. Barley yellow dwarf virus is a widespread viral pathogen that systemically infects cereal crops including wheat, barley and oats. In addition, rising atmospheric CO 2 will alter plant growth and metabolism, including many potential but not well understood effects on plant-virus interactions. In order to better understand the wheat-BYDV interaction and any potential changes under elevated CO 2 , the total concentration and oxidised fraction of ascorbate and glutathione was measured in leaves of a susceptible wheat cultivar (Triticum aestivum L. 'Yitpi') infected with Barley yellow dwarf virus-PAV (Padi Avenae virus) and grown under elevated CO 2 in controlled environment chambers. Virus infection decreased total leaf ascorbate and glutathione concentrations and increased the fraction of oxidised ascorbate (dehydroascorbate). Elevated CO 2 decreased the fraction of oxidised ascorbate. In this work, we demonstrate that systemic infection by a phloem-restricted virus weakens the antioxidant pools of ascorbate and glutathione. In addition, elevated CO 2 may decrease oxidative stress, for example, from virus infection, but there was no direct evidence for an interactive effect between treatments. Copyright © 2016 Elsevier GmbH. All rights reserved.

Identification of a New Cotton Disease Caused by an Atypical Cotton Leafroll Dwarf Virus in Argentina.

PubMed

Agrofoglio, Yamila C; Delfosse, Verónica C; Casse, María F; Hopp, Horacio E; Kresic, Iván Bonacic; Distéfano, Ana J

2017-03-01

An outbreak of a new disease occurred in cotton (Gossypium hirsutum) fields in northwest Argentina starting in the 2009-10 growing season and is still spreading steadily. The characteristic symptoms of the disease included slight leaf rolling and a bushy phenotype in the upper part of the plant. In this study, we determined the complete nucleotide sequences of two independent virus genomes isolated from cotton blue disease (CBD)-resistant and -susceptible cotton varieties. This virus genome comprised 5,866 nucleotides with an organization similar to that of the genus Polerovirus and was closely related to cotton leafroll dwarf virus, with protein identity ranging from 88 to 98%. The virus was subsequently transmitted to a CBD-resistant cotton variety using Aphis gossypii and symptoms were successfully reproduced. To study the persistence of the virus, we analyzed symptomatic plants from CBD-resistant varieties from different cotton-growing fields between 2013 and 2015 and showed the presence of the same virus strain. In addition, a constructed full-length infectious cDNA clone from the virus caused disease symptoms in systemic leaves of CBD-resistant cotton plants. Altogether, the new leafroll disease in CBD-resistant cotton plants is caused by an atypical cotton leafroll dwarf virus.

Barley yellow dwarf virus: Luteoviridae or Tombusviridae?

PubMed

Miller, W Allen; Liu, Sijun; Beckett, Randy

2002-07-01

Summary Barley yellow dwarf virus (BYDV), the most economically important virus of small grains, features highly specialised relationships with its aphid vectors, a plethora of novel translation mechanisms mediated by long-distance RNA interactions, and an ambiguous taxonomic status. The structural and movement proteins of BYDV that confer aphid transmission and phloem-limitation properties resemble those of the Luteoviridae, the family in which BYDV is classified. In contrast, many genes and cis-acting signals involved in replication and gene expression most closely resemble those of the Tombusviridae. BYDV is in genus Luteovirus, family Luteoviridae. BYDV includes at least two serotypes or viruses: BYDV-PAV and BYDV-MAV. The former BYDV-RPV is now Cereal yellow dwarf virus-RPV (CYDV-RPV). CYDV is in genus Polerovirus, family Luteoviridae. Genus Luteovirus shares many features with family Tombusviridae. Physical properties: approximately 25 nm icosahedral (T = 3) virions. One major (22 kDa) and one minor (50-55 kDa) coat protein. 5.6-5.8 kb positive sense RNA genome with no 5'-cap and no poly(A) tail. Most grasses. Most important in oats, barley and wheat. Also infects maize and rice. Yellowing and dwarfing in barley, stunting in wheat; reddening, yellowing and blasting in oats. Some isolates cause leaf notching and curling. Key attractions: Model for the study of circulative transmission of aphid-transmitted viruses. Plethora of unusual translation mechanisms. Evidence of recombination in recent evolutionary history creates taxonomic ambiguity. Economically important virus of wheat, barley and oats, worldwide. Useful websites/meetings: International symposium: 'Barley Yellow Dwarf Disease: Recent Advances and Future Strategies', CIMMYT, El Batan, Mexico, 1-5 September 2002, http://www.cimmyt.cgiar.org/Research/wheat/Conf_BYD_02/invitation.htm http://www.cimmyt.org/Research/wheat/BYDVNEWS/htm/BYDVNEWS.htm Aphid transmission animation: http://www.ppws.vt.edu/~sforza/tmv/bydv_aph.html.

Genetic analysis of a novel Alaska barley yellow dwarf virus in the family Luteoviridae.

PubMed

Robertson, N L; French, R

2007-02-01

A new plant virus belonging to the family Luteoviridae and isolated from diseased oat (Avena sativa L.) plants was discovered in Alaska in 2003. Even though plants with red/orange leaves were indicative of barley yellow dwarf disease, they were not reactive to specific antibodies corresponding to barley yellow dwarf virus (BYDV)-MAV, -PAV, -SGV, and cereal yellow dwarf virus-RPV from enzyme-linked immunosorbent assays (ELISA). An alternative RT-PCR assay that incorporated Shu-F/Yan-R primers for detection of BYDV-MAV, -PAS, -PAV, and SGV was effective in producing approximately 830-nt fragments that contained genomic sequences to the 3'-terminus of the polymerase gene (ORF 2), the intergenic region ( approximately 113 nt), the coat protein gene (ORF 3), and the putative movement gene (ORF 4). The Alaskan isolates were most similar to BYDV-MAV with only about 77 and 80% amino acid identity in the CP and ORF 4, respectively. The Alaska isolates coat protein gene sequences differed in several regions that otherwise are conserved among BYDV-MAV isolates, and may be important in serological variations, accounting for the negative ELISA results. Based upon sequence and serological differences, we concluded that the Alaskan BYDV-MAV-like isolates formed a novel species tentatively in the genus Luteovirus, and propose the name BYDV-ORV (oat red-leaf virus).

A Raspberry bushy dwarf virus isolate from Ecuadorean Rubus glaucus contains an additional RNA that is a rearrangement of RNA 2

USDA-ARS?s Scientific Manuscript database

A new Raspberry bushy dwarf virus isolate was found in commercial blackberry (Rubus glaucus) in Azuay, province of Ecuador and named RBDV-Ec-Az. The complete bipartite genome was sequenced using dsRNA as initial template. RNA 1 was 5449 nucleotides (nt) long and the normal RBDV RNA 2 was 2231 nt lon...

Genome sequence variation in the constricta strain dramatically alters the protein interaction and localization map of Potato yellow dwarf virus

USDA-ARS?s Scientific Manuscript database

The genome sequence of the constricta strain of Potato yellow dwarf virus (CYDV) was determined to be 12,792 nucleotides long and organized into seven open reading frames with the gene order 3’-N-X-P-Y-M-G-L-5’, which encodes the nucleocapsid, phosphoprotein, movement, matrix, glycoprotein and RNA-d...

A novel strategy for the determination of a rhabdovirus genome and its application to sequencing of Eggplant mottled dwarf virus.

PubMed

Pappi, Polyxeni G; Dovas, Chrysostomos I; Efthimiou, Konstantinos E; Maliogka, Varvara I; Katis, Nikolaos I

2013-08-01

A novel strategy employing the rhabdovirus untranslated conserved intergenic regions was developed and applied successfully for the determination of the complete nucleotide sequence of Eggplant mottled dwarf virus (EMDV). The EMDV genome contains seven open reading frames with the same organization as Potato yellow dwarf virus (PYDV), the type species of the genus Nucleorhabdovirus. These two species encode five core genes [nucleocapsid (N), phosphoprotein (P), matrix (M), glycoprotein (G), and the polymerase (L)] like other viruses of the genus and an additional one (X), located between N and P, giving rise to a protein with currently unknown function. Furthermore, both EMDV and PYDV contain a gene (Y), inserted between P and M, which probably encodes the virus movement protein, in concordance with the rest of the plant-infecting rhabdoviruses. Phylogenetic analysis of the polymerase gene confirmed the classification of EMDV within the genus Nucleorhabdovirus and showed a close evolutionary relationship to PYDV. The novel sequencing strategy developed is a useful tool for the genome determination of yet uncharacterized rhabdoviruses.

Epidemiology and integrated management of persistently transmitted aphid-borne viruses of legume and cereal crops in West Asia and North Africa.

PubMed

Makkouk, Khaled M; Kumari, Safaa G

2009-05-01

Cool-season food legumes (faba bean, lentil, chickpea and pea) and cereals (bread and durum wheat and barley) are the most important and widely cultivated crops in West Asia and North Africa (WANA), where they are the main source of carbohydrates and protein for the majority of the population. Persistently transmitted aphid-borne viruses pose a significant limitation to legume and cereal production worldwide. Surveys conducted in many countries in WANA during the last three decades established that the most important of these viruses are: Faba bean necrotic yellows virus (FBNYV: genus Nanovirus; family Nanoviridae), Bean leafroll virus (BLRV: genus Luteovirus; family Luteoviridae), Beet western yellows virus (BWYV: genus Polerovirus; family Luteoviridae), Soybean dwarf virus (SbDV: genus Luteovirus; family Luteoviridae) and Chickpea chlorotic stunt virus (CpCSV: genus Polerovirus; family Luteoviridae) which affect legume crops, and Barley yellow dwarf virus-PAV (BYDV-PAV: genus Luteovirus; family Luteoviridae), Barley yellow dwarf virus-MAV (BYDV-MAV: genus Luteovirus; family Luteoviridae) and Cereal yellow dwarf virus-RPV (CYDV-RPV: genus Polerovirus; family Luteoviridae) which affect cereal crops. Loss in yield caused by these viruses is usually high when infection occurs early in the growing season. Many aphid vector species for the above-mentioned viruses are reported to be prevalent in the WANA region. In addition, in this region many wild species (annual or perennial) were found infected with these viruses and may play an important role in their ecology and spread. Fast spread of these diseases was always associated with high aphid vector populations and activity. Although virus disease management can be achieved by combining several control measures, development of resistant genotypes is undoubtedly one of the most appropriate control methods. Over the last three decades barley and wheat genotypes resistant to BYDV, faba bean genotypes resistant to BLRV, and lentil genotypes resistant to BLRV, FBNYV and SbDV have been successfully identified. Moreover, progress has been made in disease management of some of these viruses using a combination of management options. Experience gathered over the last few decades clearly showed that no single method of virus disease control suffices to reduce yield losses in legume and cereal crops.

Potential Military Chemical/Biological Agents and Compounds

DTIC Science & Technology

2005-01-01

synonym: fowl plague) Bluetongue virus Fungi Foot and mouth disease virus Colletorichum coffeanum var Goat pox virus Cochiliobolus...Swine vesicular disease virus) Virus Rinderpest virus (synonym: Cattle plague Barley Yellow Dwarf Virus Sheep pox virus Teschen disease virus...2) Occurrence. Infection has been documented throughout the US, in Canada, western and central Mexico , Panama, Costa Rica, Colombia, Argentina

Simultaneous Detection of Mixed Infection of Onion yellow dwarf virus and an Allexivirus in RT-PCR for Ensuring Virus Free Onion Bulbs.

PubMed

Kumar, Sandeep; Baranwal, V K; Joshi, Subodh; Arya, Meenakshi; Majumder, S

2010-06-01

Reduced seed production in onion is associated with Onion yellow dwarf virus (OYDV), a filamentous Potyvirus. Onion is also infected with other filamentous virus particles suspected to be Allexivirus. RT-PCR was used to detect mixed infection of both the viruses in leaves and bulbs. A duplex RT-PCR was developed, which simultaneously detected the presence of these two viruses in winter (Rabi) onion bulb. In summer (Kharif) onion bulbs only Allexivirus was detected. The absence of OYDV in summer crop is discussed. The sequencing of RT-PCR amplified products confirmed the identity of OYDV and Allexivirus, the latter showing closer identity to Garlic virus C (GVC)/Garlic mite-borne mosaic virus. This makes the first detection of an Allexivirus in onion crop in India. The duplex RT-PCR to detect these viruses (OYDV and Allexivirus) would be an improvement for indexing of viruses in onion bulbs for seed production.

Rose spring dwarf-associated virus has RNA structural and gene-expression features like those of Barley yellow dwarf virus

PubMed Central

Salem, Nida’ M.; Miller, W. Allen; Rowhani, Adib; Golino, Deborah A.; Moyne, Anne-Laure; Falk, Bryce W.

2015-01-01

We determined the complete nucleotide sequence of the Rose spring dwarf-associated virus (RSDaV) genomic RNA (GenBank accession no. EU024678) and compared its predicted RNA structural characteristics affecting gene expression. A cDNA library was derived from RSDaV double-stranded RNAs (dsRNAs) purified from infected tissue. Nucleotide sequence analysis of the cloned cDNAs, plus for clones generated by 5′- and 3′-RACE showed the RSDaV genomic RNA to be 5,808 nucleotides. The genomic RNA contains five major open reading frames (ORFs), and three small ORFs in the 3′-terminal 800 nucleotides, typical for viruses of genus Luteovirus in the family Luteoviridae. Northern blot hybridization analysis revealed the genomic RNA and two prominent subgenomic RNAs of approximately 3 kb and 1 kb. Putative 5′ ends of the sgRNAs were predicted by identification of conserved sequences and secondary structures which resembled the Barley yellow dwarf virus (BYDV) genomic RNA 5′ end and subgenomic RNA promoter sequences. Secondary structures of the BYDV-like ribosomal frameshift elements and cap-independent translation elements, including long-distance base pairing spanning four kb were identified. These contain similarities but also informative differences with the BYDV structures, including a strikingly different structure predicted for the 3′ cap-independent translation element. These analyses of the RSDaV genomic RNA show more complexity for the RNA structural elements for members of the Luteoviridae. PMID:18329064

Rose spring dwarf-associated virus has RNA structural and gene-expression features like those of Barley yellow dwarf virus.

PubMed

Salem, Nida' M; Miller, W Allen; Rowhani, Adib; Golino, Deborah A; Moyne, Anne-Laure; Falk, Bryce W

2008-06-05

We determined the complete nucleotide sequence of the Rose spring dwarf-associated virus (RSDaV) genomic RNA (GenBank accession no. EU024678) and compared its predicted RNA structural characteristics affecting gene expression. A cDNA library was derived from RSDaV double-stranded RNAs (dsRNAs) purified from infected tissue. Nucleotide sequence analysis of the cloned cDNAs, plus for clones generated by 5'- and 3'-RACE showed the RSDaV genomic RNA to be 5808 nucleotides. The genomic RNA contains five major open reading frames (ORFs), and three small ORFs in the 3'-terminal 800 nucleotides, typical for viruses of genus Luteovirus in the family Luteoviridae. Northern blot hybridization analysis revealed the genomic RNA and two prominent subgenomic RNAs of approximately 3 kb and 1 kb. Putative 5' ends of the sgRNAs were predicted by identification of conserved sequences and secondary structures which resembled the Barley yellow dwarf virus (BYDV) genomic RNA 5' end and subgenomic RNA promoter sequences. Secondary structures of the BYDV-like ribosomal frameshift elements and cap-independent translation elements, including long-distance base pairing spanning four kb were identified. These contain similarities but also informative differences with the BYDV structures, including a strikingly different structure predicted for the 3' cap-independent translation element. These analyses of the RSDaV genomic RNA show more complexity for the RNA structural elements for members of the Luteoviridae.

Let-7b Regulates Myoblast Proliferation by Inhibiting IGF2BP3 Expression in Dwarf and Normal Chicken

PubMed Central

Lin, Shumao; Luo, Wen; Ye, Yaqiong; Bekele, Endashaw J.; Nie, Qinghua; Li, Yugu; Zhang, Xiquan

2017-01-01

The sex-linked dwarf chicken is caused by the mutation of growth hormone receptor (GHR) gene and characterized by shorter shanks, lower body weight, smaller muscle fiber diameter and fewer muscle fiber number. However, the precise regulatory pathways that lead to the inhibition of skeletal muscle growth in dwarf chickens still remain unclear. Here we found a let-7b mediated pathway might play important role in the regulation of dwarf chicken skeletal muscle growth. Let-7b has higher expression in the skeletal muscle of dwarf chicken than in normal chicken, and the expression of insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3), which is a translational activator of IGF2, showed opposite expression trend to let-7b. In vitro cellular assays validated that let-7b directly inhibits IGF2BP3 expression through binding to its 3′UTR region, and the protein level but not mRNA level of IGF2 would be reduced in let-7b overexpressed chicken myoblast. Let-7b can inhibit cell proliferation and induce cell cycle arrest in chicken myoblast through let-7b-IGF2BP3-IGF2 signaling pathway. Additionally, let-7b can also regulate skeletal muscle growth through let-7b-GHR-GHR downstream genes pathway, but this pathway is non-existent in dwarf chicken because of the deletion mutation of GHR 3′UTR. Notably, as the loss binding site of GHR for let-7b, let-7b has enhanced its binding and inhibition on IGF2BP3 in dwarf myoblast, suggesting that the miRNA can balance its inhibiting effect through dynamic regulate its binding to target genes. Collectively, these results not only indicate that let-7b can inhibit skeletal muscle growth through let-7b-IGF2BP3-IGF2 signaling pathway, but also show that let-7b regulates myoblast proliferation by inhibiting IGF2BP3 expression in dwarf and normal chickens. PMID:28736533

Phloem-limited reoviruses universally induce sieve element hyperplasia and more flexible gateways, providing more channels for their movement in plants.

PubMed

Lv, Ming-Fang; Xie, Li; Song, Xi-Jiao; Hong, Jian; Mao, Qian-Zhuo; Wei, Tai-Yun; Chen, Jian-Ping; Zhang, Heng-Mu

2017-11-28

Virion distribution and ultrastructural changes induced by the infection of maize or rice with four different reoviruses were examined. Rice black streaked dwarf virus (RBSDV, genus Fijivirus), Rice ragged stunt virus (RRSV, genus Oryzavirus), and Rice gall dwarf virus (RGDV, genus Phytoreovirus) were all phloem-limited and caused cellular hyperplasia in the phloem resulting in tumors or vein swelling and modifying the cellular arrangement of sieve elements (SEs). In contrast, virions of Rice dwarf virus (RDV, genus Phytoreovirus) were observed in both phloem and mesophyll and the virus did not cause hyperplasia of SEs. The three phloem-limited reoviruses (but not RDV) all induced more flexible gateways at the SE-SE interfaces, especially the non-sieve plate interfaces. These flexible gateways were also observed for the first time at the cellular interfaces between SE and phloem parenchyma (PP). In plants infected with any of the reoviruses, virus-like particles could be seen within the flexible gateways, suggesting that these gateways may serve as channels for the movement of plant reoviruses with their large virions between SEs or between SEs and PP. SE hyperplasia and the increase in flexible gateways may be a universal strategy for the movement of phloem-limited reoviruses.

Comparison of ELISA and RT-PCR for the detection of Prunus necrotic ring spot virus and prune dwarf virus in almond (Prunus dulcis).

PubMed

Mekuria, Genet; Ramesh, Sunita A; Alberts, Evita; Bertozzi, Terry; Wirthensohn, Michelle; Collins, Graham; Sedgley, Margaret

2003-12-01

A technique based on the reverse transcriptase-polymerase chain reaction (RT-PCR) has been developed to detect the presence of Prunus necrotic ringspot virus (PNRSV) and prune dwarf virus (PDV) simultaneously in almond. This paper presents the results of a 3-year study comparing both enzyme-linked immunosorbent assay (ELISA) and RT-PCR for the detection of PNRSV and PDV using 175 almond leaf samples. Multiplex RT-PCR was found to be more sensitive than ELISA, especially when followed by nested PCR for the detection of PDV. The RT-PCR technique has the added advantage that plant material can be tested at any time throughout the growing season.

Discovery and targeted LC-MS/MS of purified polerovirus reveals differences in the virus-host interactome associated with altered aphid transmission

USDA-ARS?s Scientific Manuscript database

The transmission of viruses in the Luteoviridae, such as Cereal yellow dwarf virus (CYDV), requires a series of precisely orchestrated interactions between virus proteins, plant proteins, and aphid proteins. These viruses are retained in the phloem for aphid acquisition and are transmitted by aphids...

Genomic and Proteomic Analysis of Schizaphis graminum Reveals Cyclophilin Proteins Are Involved in the Transmission of Cereal Yellow Dwarf Virus

PubMed Central

Tamborindeguy, Cecilia; Bereman, Michael S.; DeBlasio, Stacy; Igwe, David; Smith, Dawn M.; White, Frank; MacCoss, Michael J.; Gray, Stewart M.; Cilia, Michelle

2013-01-01

Yellow dwarf viruses cause the most economically important virus diseases of cereal crops worldwide and are transmitted by aphid vectors. The identification of aphid genes and proteins mediating virus transmission is critical to develop agriculturally sustainable virus management practices and to understand viral strategies for circulative movement in all insect vectors. Two cyclophilin B proteins, S28 and S29, were identified previously in populations of Schizaphisgraminum that differed in their ability to transmit the RPV strain of Cereal yellow dwarf virus (CYDV-RPV). The presence of S29 was correlated with F2 genotypes that were efficient virus transmitters. The present study revealed the two proteins were isoforms, and a single amino acid change distinguished S28 and S29. The distribution of the two alleles was determined in 12 F2 genotypes segregating for CYDV-RPV transmission capacity and in 11 genetically independent, field-collected S . graminum biotypes. Transmission efficiency for CYDV-RPV was determined in all genotypes and biotypes. The S29 isoform was present in all genotypes or biotypes that efficiently transmit CYDV-RPV and more specifically in genotypes that efficiently transport virus across the hindgut. We confirmed a direct interaction between CYDV-RPV and both S28 and S29 using purified virus and bacterially expressed, his-tagged S28 and S29 proteins. Importantly, S29 failed to interact with a closely related virus that is transported across the aphid midgut. We tested for in vivo interactions using an aphid-virus co-immunoprecipitation strategy coupled with a bottom-up LC-MS/MS analysis using a Q Exactive mass spectrometer. This analysis enabled us to identify a third cyclophilin protein, cyclophilin A, interacting directly or in complex with purified CYDV-RPV. Taken together, these data provide evidence that both cyclophilin A and B interact with CYDV-RPV, and these interactions may be important but not sufficient to mediate virus transport from the hindgut lumen into the hemocoel. PMID:23951206

The rice dwarf virus P2 protein interacts with ent-kaurene oxidases in vivo, leading to reduced biosynthesis of gibberellins and rice dwarf symptoms.

PubMed

Zhu, Shifeng; Gao, Feng; Cao, Xuesong; Chen, Mao; Ye, Gongyin; Wei, Chunhong; Li, Yi

2005-12-01

The mechanisms of viral diseases are a major focus of biology. Despite intensive investigations, how a plant virus interacts with host factors to cause diseases remains poorly understood. The Rice dwarf virus (RDV), a member of the genus Phytoreovirus, causes dwarfed growth phenotypes in infected rice (Oryza sativa) plants. The outer capsid protein P2 is essential during RDV infection of insects and thus influences transmission of RDV by the insect vector. However, its role during RDV infection within the rice host is unknown. By yeast two-hybrid and coimmunoprecipitation assays, we report that P2 of RDV interacts with ent-kaurene oxidases, which play a key role in the biosynthesis of plant growth hormones gibberellins, in infected plants. Furthermore, the expression of ent-kaurene oxidases was reduced in the infected plants. The level of endogenous GA1 (a major active gibberellin in rice vegetative tissues) in the RDV-infected plants was lower than that in healthy plants. Exogenous application of GA3 to RDV-infected rice plants restored the normal growth phenotypes. These results provide evidence that the P2 protein of RDV interferes with the function of a cellular factor, through direct physical interactions, that is important for the biosynthesis of a growth hormone leading to symptom expression. In addition, the interaction between P2 and rice ent-kaurene oxidase-like proteins may decrease phytoalexin biosynthesis and make plants more competent for virus replication. Moreover, P2 may provide a novel tool to investigate the regulation of GA metabolism for plant growth and development.

The Rice Dwarf Virus P2 Protein Interacts with ent-Kaurene Oxidases in Vivo, Leading to Reduced Biosynthesis of Gibberellins and Rice Dwarf Symptoms1

PubMed Central

Zhu, Shifeng; Gao, Feng; Cao, Xuesong; Chen, Mao; Ye, Gongyin; Wei, Chunhong; Li, Yi

2005-01-01

The mechanisms of viral diseases are a major focus of biology. Despite intensive investigations, how a plant virus interacts with host factors to cause diseases remains poorly understood. The Rice dwarf virus (RDV), a member of the genus Phytoreovirus, causes dwarfed growth phenotypes in infected rice (Oryza sativa) plants. The outer capsid protein P2 is essential during RDV infection of insects and thus influences transmission of RDV by the insect vector. However, its role during RDV infection within the rice host is unknown. By yeast two-hybrid and coimmunoprecipitation assays, we report that P2 of RDV interacts with ent-kaurene oxidases, which play a key role in the biosynthesis of plant growth hormones gibberellins, in infected plants. Furthermore, the expression of ent-kaurene oxidases was reduced in the infected plants. The level of endogenous GA1 (a major active gibberellin in rice vegetative tissues) in the RDV-infected plants was lower than that in healthy plants. Exogenous application of GA3 to RDV-infected rice plants restored the normal growth phenotypes. These results provide evidence that the P2 protein of RDV interferes with the function of a cellular factor, through direct physical interactions, that is important for the biosynthesis of a growth hormone leading to symptom expression. In addition, the interaction between P2 and rice ent-kaurene oxidase-like proteins may decrease phytoalexin biosynthesis and make plants more competent for virus replication. Moreover, P2 may provide a novel tool to investigate the regulation of GA metabolism for plant growth and development. PMID:16299167

Complete genome sequence of a divergent strain of lettuce chlorosis virus from Periwinkle in China

USDA-ARS?s Scientific Manuscript database

A novel strain of Lettuce chlorosis virus (LCV) was identified from periwinkle in China (PW) with foliar interveinal chlorosis and plant dwarfing. Complete nucleotide (nt) sequences of genomic RNA1 and RNA2 of the virus are 8,602 nt and 8,456 nt, respectively. The genomic organization of LCV-PW rese...

Low Temperature Storage of Southern Rice Black-Streaked Dwarf Virus-Infected Rice Plants Cannot Sustain Virus Transmission by the Vector.

PubMed

Liu, Danfeng; Li, Pei; Han, Yongqiang; Lei, Wenbin; Hou, Maolin

2016-02-01

Southern rice black-streaked dwarf virus (SRBSDV) is a novel virus transmitted by white-backed planthopper Sogatella furcifera (Hováth) (Hemiptera: Delphacidae). Due to low virus transmission efficiency by the planthopper, researchers are frequently confronted with shortage of viruliferous vectors or infected rice plants, especially in winter and the following spring. To find new ways to maintain virus-infected materials, viral rice plants were stored at -80°C for 45 or 140 d and evaluated as virus sources in virus transmission by the vector. SRBSDV virions were not degraded during storage at -80°C as indicated by reverse transcription-polymerase chain reaction and reverse transcription real-time PCR detection. The planthopper nymphs fed on the infected thawed plants for 48 h survived at about 40% and showed positive detection of SRBSDV, but they lost the virus after feeding for another 20 d (the circulative transmission period) on noninfected plants. Transmission electron microscope images indicated broken capsid of virions in infected thawed leaves in contrast to integrity capsid of virions in infected fresh leaves. These results show that low temperature storage of SRBSDV-infected rice plants cannot sustain virus transmission by white-backed planthopper. © The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Detection and discrimination of members of the family Luteoviridae by real-time PCR and SYBR® GreenER™ melting curve analysis.

PubMed

Chomic, Anastasija; Winder, Louise; Armstrong, Karen F; Pearson, Michael N; Hampton, John G

2011-01-01

This study investigated the suitability of a two step real-time RT-PCR melting curve analysis as a tool for the detection and discrimination of nine species in the plant virus family Luteoviridae, being Soybean dwarf virus [SbDV], Bean leafroll virus [BLRV], Beet chlorosis virus [BChV], Beet mild yellowing virus [BMYV], Beet western yellows virus [BWYV], Cereal yellow dwarf virus-RPV [CYDV-RPV], Cucurbit aphid-borne yellows virus [CABYV], Potato leafroll virus [PLRV] and Turnip yellows virus [TuYV]. Melting temperature and shape of the melting peak were analysed for 68 bp and 148 bp coat protein gene amplicons using SYBR® GreenER™ fluorescent dye. Specific melting peaks with unique melting temperature were observed for the various species of the family Luteoviridae using the 68 bp amplicon, but not with the 148 bp amplicon. Due to the high variability of sequences for some members of this family, different melting temperatures were also observed between different isolates of the species CYDV-RPV and TuYV. Nevertheless, discrimination between species was achieved for SbDV, BLRV, BChV, BMYV, CABYV and either PLRV or BWYV. Melting curve analysis, in this study, is a faster and more discriminatory alternative to gel electrophoresis of end-point PCR products for the detection of Luteoviridae infection. Copyright © 2010 Elsevier B.V. All rights reserved.

Complete sequence and diversity of a maize-associated Polerovirus in East Africa.

PubMed

Massawe, Deogracious P; Stewart, Lucy R; Kamatenesi, Jovia; Asiimwe, Theodore; Redinbaugh, Margaret G

2018-06-01

Since 2011-2012, Maize lethal necrosis (MLN) has emerged in East Africa, causing massive yield loss and propelling research to identify viruses and virus populations present in maize. As expected, next generation sequencing (NGS) has revealed diverse and abundant viruses from the family Potyviridae, primarily sugarcane mosaic virus (SCMV), and maize chlorotic mottle virus (MCMV) (Tombusviridae), which are known to cause MLN by synergistic co-infection. In addition to these expected viruses, we identified a virus in the genus Polerovirus (family Luteoviridae) in 104/172 samples selected for MLN or other potential virus symptoms from Kenya, Uganda, Rwanda, and Tanzania. This polerovirus (MF974579) nucleotide sequence is 97% identical to maize-associated viruses recently reported in China, termed 'maize yellow mosaic virus' (MaYMV) and maize yellow dwarf virus (MaYMV; KU291101, KU291107, MYDV-RMV2; KT992824); and 99% identical to MaYMV (KY684356) infecting sugarcane and itch grass in Nigeria; 83% identical to a barley-associated polerovirus recently identified in Korea (BVG; KT962089); and 79% identical to the U.S. maize-infecting polerovirus maize yellow dwarf virus (MYDV-RMV; KT992824). Nucleotide sequences from ORF0 of 20 individual East African isolates collected from Kenya, Uganda, Rwanda, and Tanzania shared 98% or higher identity, and were detected in 104/172 (60.5%) of samples collected for virus-like symptoms, indicating extensive prevalence but limited diversity of this virus in East Africa. We refer to this virus as "MYDV-like polerovirus" until symptoms of the virus in maize are known.

Coherent diffraction of single Rice Dwarf virus particles using hard X-rays at the Linac Coherent Light Source

PubMed Central

Munke, Anna; Andreasson, Jakob; Aquila, Andrew; Awel, Salah; Ayyer, Kartik; Barty, Anton; Bean, Richard J.; Berntsen, Peter; Bielecki, Johan; Boutet, Sébastien; Bucher, Maximilian; Chapman, Henry N.; Daurer, Benedikt J.; DeMirci, Hasan; Elser, Veit; Fromme, Petra; Hajdu, Janos; Hantke, Max F.; Higashiura, Akifumi; Hogue, Brenda G.; Hosseinizadeh, Ahmad; Kim, Yoonhee; Kirian, Richard A.; Reddy, Hemanth K.N.; Lan, Ti-Yen; Larsson, Daniel S.D.; Liu, Haiguang; Loh, N. Duane; Maia, Filipe R.N.C.; Mancuso, Adrian P.; Mühlig, Kerstin; Nakagawa, Atsushi; Nam, Daewoong; Nelson, Garrett; Nettelblad, Carl; Okamoto, Kenta; Ourmazd, Abbas; Rose, Max; van der Schot, Gijs; Schwander, Peter; Seibert, M. Marvin; Sellberg, Jonas A.; Sierra, Raymond G.; Song, Changyong; Svenda, Martin; Timneanu, Nicusor; Vartanyants, Ivan A.; Westphal, Daniel; Wiedorn, Max O.; Williams, Garth J.; Xavier, Paulraj Lourdu; Yoon, Chun Hong; Zook, James

2016-01-01

Single particle diffractive imaging data from Rice Dwarf Virus (RDV) were recorded using the Coherent X-ray Imaging (CXI) instrument at the Linac Coherent Light Source (LCLS). RDV was chosen as it is a well-characterized model system, useful for proof-of-principle experiments, system optimization and algorithm development. RDV, an icosahedral virus of about 70 nm in diameter, was aerosolized and injected into the approximately 0.1 μm diameter focused hard X-ray beam at the CXI instrument of LCLS. Diffraction patterns from RDV with signal to 5.9 Ångström were recorded. The diffraction data are available through the Coherent X-ray Imaging Data Bank (CXIDB) as a resource for algorithm development, the contents of which are described here. PMID:27478984

Coherent diffraction of single Rice Dwarf virus particles using hard X-rays at the Linac Coherent Light Source

DOE PAGES

Munke, Anna; Andreasson, Jakob; Aquila, Andrew; ...

2016-08-01

Single particle diffractive imaging data from Rice Dwarf Virus (RDV) were recorded using the Coherent X-ray Imaging (CXI) instrument at the Linac Coherent Light Source (LCLS). RDV was chosen as it is a well-characterized model system, useful for proof-of-principle experiments, system optimization and algorithm development. RDV, an icosahedral virus of about 70 nm in diameter, was aerosolized and injected into the approximately 0.1 μm diameter focused hard X-ray beam at the CXI instrument of LCLS. Diffraction patterns from RDV with signal to 5.9 Ångström were recorded. Here, the diffraction data are available through the Coherent X-ray Imaging Data Bank (CXIDB)more » as a resource for algorithm development, the contents of which are described here.« less

Coherent diffraction of single Rice Dwarf virus particles using hard X-rays at the Linac Coherent Light Source.

PubMed

Munke, Anna; Andreasson, Jakob; Aquila, Andrew; Awel, Salah; Ayyer, Kartik; Barty, Anton; Bean, Richard J; Berntsen, Peter; Bielecki, Johan; Boutet, Sébastien; Bucher, Maximilian; Chapman, Henry N; Daurer, Benedikt J; DeMirci, Hasan; Elser, Veit; Fromme, Petra; Hajdu, Janos; Hantke, Max F; Higashiura, Akifumi; Hogue, Brenda G; Hosseinizadeh, Ahmad; Kim, Yoonhee; Kirian, Richard A; Reddy, Hemanth K N; Lan, Ti-Yen; Larsson, Daniel S D; Liu, Haiguang; Loh, N Duane; Maia, Filipe R N C; Mancuso, Adrian P; Mühlig, Kerstin; Nakagawa, Atsushi; Nam, Daewoong; Nelson, Garrett; Nettelblad, Carl; Okamoto, Kenta; Ourmazd, Abbas; Rose, Max; van der Schot, Gijs; Schwander, Peter; Seibert, M Marvin; Sellberg, Jonas A; Sierra, Raymond G; Song, Changyong; Svenda, Martin; Timneanu, Nicusor; Vartanyants, Ivan A; Westphal, Daniel; Wiedorn, Max O; Williams, Garth J; Xavier, Paulraj Lourdu; Yoon, Chun Hong; Zook, James

2016-08-01

Single particle diffractive imaging data from Rice Dwarf Virus (RDV) were recorded using the Coherent X-ray Imaging (CXI) instrument at the Linac Coherent Light Source (LCLS). RDV was chosen as it is a well-characterized model system, useful for proof-of-principle experiments, system optimization and algorithm development. RDV, an icosahedral virus of about 70 nm in diameter, was aerosolized and injected into the approximately 0.1 μm diameter focused hard X-ray beam at the CXI instrument of LCLS. Diffraction patterns from RDV with signal to 5.9 Ångström were recorded. The diffraction data are available through the Coherent X-ray Imaging Data Bank (CXIDB) as a resource for algorithm development, the contents of which are described here.

Host Adaptation of Soybean Dwarf Virus Following Serial Passages on Pea (Pisum sativum) and Soybean (Glycine max)

PubMed Central

Tian, Bin; Gildow, Frederick E.; Stone, Andrew L.; Sherman, Diana J.; Damsteegt, Vernon D.; Schneider, William L.

2017-01-01

Soybean Dwarf Virus (SbDV) is an important plant pathogen, causing economic losses in soybean. In North America, indigenous strains of SbDV mainly infect clover, with occasional outbreaks in soybean. To evaluate the risk of a US clover strain of SbDV adapting to other plant hosts, the clover isolate SbDV-MD6 was serially transmitted to pea and soybean by aphid vectors. Sequence analysis of SbDV-MD6 from pea and soybean passages identified 11 non-synonymous mutations in soybean, and six mutations in pea. Increasing virus titers with each sequential transmission indicated that SbDV-MD6 was able to adapt to the plant host. However, aphid transmission efficiency on soybean decreased until the virus was no longer transmissible. Our results clearly demonstrated that the clover strain of SbDV-MD6 is able to adapt to soybean crops. However, mutations that improve replication and/or movement may have trade-off effects resulting in decreased vector transmission. PMID:28635666

A peptide derived from enzymatic digestion of globulins from amaranth shows strong affinity binding to the replication origin of Tomato yellow leaf curl virus reducing viral replication in Nicotiana benthamiana.

PubMed

Mendoza-Figueroa, J S; Kvarnheden, A; Méndez-Lozano, J; Rodríguez-Negrete, E-A; Arreguín-Espinosa de Los Monteros, R; Soriano-García, M

2018-02-01

Tomato yellow leaf curl virus (TYLCV; genus Begomovirus; family Geminiviridae) infects mainly plants of the family Solanaceae, and the infection induces curling and chlorosis of leaves, dwarfing of the whole plant, and reduced fruit production. Alternatives for direct control of TYLCV and other geminiviruses have been reported, for example, the use of esterified whey proteins, peptide aptamer libraries or artificial zinc finger proteins. The two latter alternatives affect directly the replication of TYLCV as well as of other geminiviruses because the replication structures and sequences are highly conserved within this virus family. Because peptides and proteins offer a potential solution for virus replication control, in this study we show the isolation, biochemical characterization and antiviral activity of a peptide derived from globulins of amaranth seeds (Amaranthus hypochondriacus) that binds to the replication origin sequence (OriRep) of TYLCV and affects viral replication with a consequent reduction of disease symptoms in Nicotiana benthamiana. Aromatic peptides obtained from papain digests of extracted globulins and albumins of amaranth were tested by intrinsic fluorescent titration and localized surface resonance plasmon to analyze their binding affinity to OriRep of TYLCV. The peptide AmPep1 (molecular weight 2.076 KDa) showed the highest affinity value (Kd = 1.8 nM) for OriRep. This peptide shares a high amino acid similarity with a part of an amaranth 11S globulin, and the strong affinity of AmPep1 could be explained by the presence of tryptophan and lysine facilitating interaction with the secondary structure of OriRep. In order to evaluate the effect of the peptide on in vitro DNA synthesis, rolling circle amplification (RCA) was performed using as template DNA from plants infected with TYLCV or another begomovirus, pepper huasteco yellow vein virus (PHYVV), and adding AmPep1 peptide at different concentrations. The results showed a decrease in DNA synthesis of both viruses at increasing concentrations of AmPep1. To further confirm the antiviral activity of the peptide in vivo, AmPep1 was infiltrated into leaves of N. benthamiana plants previously infected with TYLCV. Plants treated with AmPep1 showed a significant decrease in virus titer compared with untreated N. benthamiana plants as well as reduced symptom progression due to the effect of AmPep1 curtailing TYLCV replication in the plant. The peptide also showed antiviral activity in plants infected with PHYVV. This is the first report, in which a peptide is directly used for DNA virus control in plants, supplied as exogenous application and without generation of transgenic lines. Copyright © 2018 Elsevier Inc. All rights reserved.

Characterization of an RNA silencing suppressor encoded by maize yellow dwarf virus-RMV2.

PubMed

Wang, Fang; Zhao, Xia; Dong, Qing; Zhou, Benguo; Gao, Zhengliang

2018-05-11

Maize yellow dwarf virus-RMV2 (MYDV-RMV2) causes dwarfing and yellowing symptoms on leaves in field-grown maize plants in Anhui province in China. Herein, we evaluated the RNA silencing suppressor (RSS) activity of the P0 protein from MYDV-RMV2 by co-infiltration assays using wild-type and GFP-transgenic Nicotiana benthamiana (line 16C). The P0 of MYDV-RMV2 exhibited RSS activity and inhibited RNA silencing both locally and systemically. MYDV-RMV2 P0 acts as an F-box-like motif, and mutations to Ala at positions 67, 68, and 81 in the F-box-like motif (67LPxx81P) abolished the RSS activity of P0. However, MYDV-RMV2 P0 failed to interact with AGO1 from Arabidopsis thaliana. Expressing P0 induced developmental defects. P0 was targeted to both the nuclei and cytoplasm of plant cells. These findings expand our knowledge of the role of polerovirus P0 proteins in RNA silencing.

Sequencing and Validation of Reference Genes to Analyze Endogenous Gene Expression and Quantify Yellow Dwarf Viruses Using RT-qPCR in Viruliferous Rhopalosiphum padi

PubMed Central

Wu, Keke; Liu, Wenwen; Mar, Thithi; Liu, Yan; Wu, Yunfeng; Wang, Xifeng

2014-01-01

The bird cherry-oat aphid (Rhopalosiphum padi), an important pest of cereal crops, not only directly sucks sap from plants, but also transmits a number of plant viruses, collectively the yellow dwarf viruses (YDVs). For quantifying changes in gene expression in vector aphids, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is a touchstone method, but the selection and validation of housekeeping genes (HKGs) as reference genes to normalize the expression level of endogenous genes of the vector and for exogenous genes of the virus in the aphids is critical to obtaining valid results. Such an assessment has not been done, however, for R. padi and YDVs. Here, we tested three algorithms (GeNorm, NormFinder and BestKeeper) to assess the suitability of candidate reference genes (EF-1α, ACT1, GAPDH, 18S rRNA) in 6 combinations of YDV and vector aphid morph. EF-1α and ACT1 together or in combination with GAPDH or with GAPDH and 18S rRNA could confidently be used to normalize virus titre and expression levels of endogenous genes in winged or wingless R. padi infected with Barley yellow dwarf virus isolates (BYDV)-PAV and BYDV-GAV. The use of only one reference gene, whether the most stably expressed (EF-1α) or the least stably expressed (18S rRNA), was not adequate for obtaining valid relative expression data from the RT-qPCR. Because of discrepancies among values for changes in relative expression obtained using 3 regions of the same gene, different regions of an endogenous aphid gene, including each terminus and the middle, should be analyzed at the same time with RT-qPCR. Our results highlight the necessity of choosing the best reference genes to obtain valid experimental data and provide several HKGs for relative quantification of virus titre in YDV-viruliferous aphids. PMID:24810421

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.

First report of Raspberry bushy dwarf virus in blackberry in Ecuador

USDA-ARS?s Scientific Manuscript database

During the past two decades, several viruses have been identified from Rubus (blackberry and raspberry) in wild and commercial plantings around the world (1) In Ecuador; approximately 14 tons of blackberries (Rubus glaucus) are produced each year in an estimated area of 5,500 hectares. This crop pro...

Complete genome sequence and integrated protein localization and interaction map for alfalfa dwarf virus, which combines properties of both cytoplasmic and nuclear plant rhabdoviruses

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

Bejerman, Nicolás, E-mail: n.bejerman@uq.edu.au; Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St Lucia, QLD 4072; Giolitti, Fabián

Summary: We have determined the full-length 14,491-nucleotide genome sequence of a new plant rhabdovirus, alfalfa dwarf virus (ADV). Seven open reading frames (ORFs) were identified in the antigenomic orientation of the negative-sense, single-stranded viral RNA, in the order 3′-N-P-P3-M-G-P6-L-5′. The ORFs are separated by conserved intergenic regions and the genome coding region is flanked by complementary 3′ leader and 5′ trailer sequences. Phylogenetic analysis of the nucleoprotein amino acid sequence indicated that this alfalfa-infecting rhabdovirus is related to viruses in the genus Cytorhabdovirus. When transiently expressed as GFP fusions in Nicotiana benthamiana leaves, most ADV proteins accumulated in the cellmore » periphery, but unexpectedly P protein was localized exclusively in the nucleus. ADV P protein was shown to have a homotypic, and heterotypic nuclear interactions with N, P3 and M proteins by bimolecular fluorescence complementation. ADV appears unique in that it combines properties of both cytoplasmic and nuclear plant rhabdoviruses. - Highlights: • The complete genome of alfalfa dwarf virus is obtained. • An integrated localization and interaction map for ADV is determined. • ADV has a genome sequence similarity and evolutionary links with cytorhabdoviruses. • ADV protein localization and interaction data show an association with the nucleus. • ADV combines properties of both cytoplasmic and nuclear plant rhabdoviruses.« less

Spatio-temporal distribution and environmental drivers of Barley yellow dwarf virus and vector abundance in Kansas.

PubMed

Enders, Laramy; Hefley, Trevor; Girvin, John; Whitworth, Robert; Smith, Charles

2018-05-11

Several aphid species transmit barley yellow dwarf, a globally destructive disease caused by viruses that infect cereal grain crops. Data from >400 samples collected across Kansas wheat fields in 2014 and 2015 were used to develop spatio-temporal models predicting the extent to which landcover, temperature and precipitation affect spring aphid vector abundance and presence of individuals carrying Barley yellow dwarf virus (BYDV). The distribution of Rhopalosiphum padi abundance was not correlated with climate or landcover, but Sitobion avenae abundance was positively correlated to fall temperature and negatively correlated to spring temperature and precipitation. The abundance of Schizaphis graminum was negatively correlated with fall precipitation and winter temperature. The incidence of viruliferous (+BYDV) R. padi was positively correlated with fall precipitation but negatively correlated with winter precipitation. In contrast, the probability of +BYDV S. avenae was unaffected by precipitation but was positively correlated with average fall temperatures and distance to nearest forest or shrubland. R. padi and S. avenae were more prevalent at Eastern sample sites where ground cover is more grassland than cropland, suggesting that grassland may provide over-summering sites for vectors and pose a risk as potential BYDV reservoirs. Nevertheless, land cover patterns were not strongly associated with differences in abundance or probability that viruliferous aphids were present.

Polycistronic artificial miRNA-mediated resistance to Wheat dwarf virus in barley is highly efficient at low temperature.

PubMed

Kis, András; Tholt, Gergely; Ivanics, Milán; Várallyay, Éva; Jenes, Barnabás; Havelda, Zoltán

2016-04-01

Infection of Wheat dwarf virus (WDV) strains on barley results in dwarf disease, imposing severe economic losses on crop production. As the natural resistance resources against this virus are limited, it is imperative to elaborate a biotechnological approach that will provide effective and safe immunity to a wide range of WDV strains. Because vector insect-mediated WDV infection occurs during cool periods in nature, it is important to identify a technology which is effective at lower temperature. In this study, we designed artificial microRNAs (amiRNAs) using a barley miRNA precursor backbone, which target different conservative sequence elements of the WDV strains. Potential amiRNA sequences were selected to minimize the off-target effects and were tested in a transient sensor system in order to select the most effective constructs at low temperature. On the basis of the data obtained, a polycistronic amiRNA precursor construct (VirusBuster171) was built expressing three amiRNAs simultaneously. The construct was transformed into barley under the control of a constitutive promoter. The transgenic lines were kept at 12-15 °C to mimic autumn and spring conditions in which major WDV infection and accumulation take place. We were able to establish a stable barley transgenic line displaying resistance to insect-mediated WDV infection. Our study demonstrates that amiRNA technology can be an efficient tool for the introduction of highly efficient resistance in barley against a DNA virus belonging to the Geminiviridae family, and this resistance is effective at low temperature where the natural insect vector mediates the infection process. © 2015 BSPP and John Wiley & Sons Ltd.

Evidence for lysine acetylation in the coat protein of a Polerovirus

USDA-ARS?s Scientific Manuscript database

Virions of the RPV strain of Cereal yellow dwarf virus (CYDV-RPV) were purified from infected oat tissue and analyzed by mass spectrometry. Two conserved residues, K147 and K181, residing in the virus coat protein, were confidently identified to contain epsilon-N-acetyl groups. While no functional ...

Development and Practical Use of RT-PCR for Seed-transmitted Prune dwarf virus in Quarantine

PubMed Central

Lee, Siwon; Shin, Yong-Gil

2014-01-01

Among imported plants, seeds are the items that have many latent pathogens and are difficult to inspect. Also, they are the import and export items whose market is expected to expand. The biggest problem with seeds is viruses. Prune dwarf virus (PDV) is the virus that is commonly inspected in Prunus cerasifera, P. persica, P. armeniaca, P. mandshurica, P. cerasus, P. avium or P. serotina seeds. In this study, two RT-PCR primer sets, which can promptly and specifically diagnose plant quarantine seed-transmitted PDV, were developed; and nested PCR primers, where products amplify 739 and 673 nucleotides (nt), and an nested PCR-product, 305 nt, can be obtained as these products are amplified again, were developed. Also, a modified-positive control plasmid was developed, where the restriction enzyme XhoI, which can identify the contamination of samples from the control, was inserted. The method developed in this study has detected PDV in 18 cases since 2007, and is expected to continuously contribute to the plant quarantine in Korea. PMID:25289000

Complete genomic characterization of milk vetch dwarf virus isolates from cowpea and broad bean in Anhui province, China.

PubMed

Zhang, Chenhua; Zheng, Hongying; Yan, Dankan; Han, Kelei; Song, Xijiao; Liu, Yong; Zhang, Dongfang; Chen, Jianping; Yan, Fei

2017-08-01

Cowpea and broad bean plants showing severe stunting and leaf rolling symptoms were observed in Hefei city, Anhui province, China, in 2014. Symptomatic plants from both species were shown to be infected with milk vetch dwarf virus (MDV) by PCR. The complete genomes of MDV isolates from cowpea and broad bean were sequenced. Each of them had eight genomic DNAs that differed between the two isolates by 10.7% in their overall nucleotide sequences. In addition, the MDV genomes from cowpea and broad bean were associated with two and three alphasatellite DNAs, respectively. This is the first report of MDV on cowpea in China and the first complete genome sequences of Chinese MDV isolates.

Agricultural Bioterrorism: Why It Is A Concern And What We Must Do

DTIC Science & Technology

2003-04-07

that causes smallpox. • Fungus are any of a major group (Fungi) of saprophytic and parasitic lower plants that lack chlorophyll and include molds, rusts ...fever* • Sheep and goat pox* • Swine vesicular disease* • Vesicular stomatitis* Plant Pathogens • Rice blast (Magnaporthe griesea) • Wheat stem rust ...Puccinia graminis) • Wheat smut (Fusarium graminearum) Wheat Pathogens • Wheat dwarf geminivirus • Barley yellow dwarf virus • Pseudomonas fascovaginaei

Efficacy of anise oil, dwarf-pine oil and chamomile oil against thymidine-kinase-positive and thymidine-kinase-negative herpesviruses.

PubMed

Koch, Christine; Reichling, Jürgen; Kehm, Roland; Sharaf, Mona M; Zentgraf, Hanswalter; Schneele, Jürgen; Schnitzler, Paul

2008-11-01

The effect of anise oil, dwarf-pine oil and chamomile oil against different thymidine-kinase-positive (aciclovir-sensitive) and thymidine-kinase-negative (aciclovir-resistant) herpes simplex virus type 1 (HSV-1) strains was examined. Clinical HSV-1 isolates containing frameshift mutations in the thymidine kinase (TK) gene, an insertion or a deletion, yield a non-functional thymidine kinase enzyme resulting in phenotypical resistance against aciclovir. The inhibitory activity of three different essential oils against herpes simplex virus isolates was tested in-vitro using a plaque reduction assay. All essential oils exhibited high levels of antiviral activity against aciclovir-sensitive HSV strain KOS and aciclovir-resistant clinical HSV isolates as well as aciclovir-resistant strain Angelotti. At maximum noncytotoxic concentrations of the plant oils, plaque formation was significantly reduced by 96.6-99.9%, when herpesviruses were preincubated with drugs before attachment to host cells. No significant effect on viral infectivity could be achieved by adding these compounds during the replication phase. These results indicate that anise oil, dwarf-pine oil and chamomile oil affected the virus by interrupting adsorption of herpesviruses and in a different manner than aciclovir, which is effective after attachment inside the infected cells. Thus the investigated essential oils are capable of exerting a direct effect on HSV and might be useful in the treatment of drug-resistant viruses. Chamomile oil did not reveal any irritating potential on hen's egg chorioallantoic membrane, demonstrated the highest selectivity index among the oils tested and was highly active against clinically relevant aciclovir-resistant HSV-1 strains.

Effects of egg-adaptation on receptor-binding and antigenic properties of recent influenza A (H3N2) vaccine viruses.

PubMed

Parker, Lauren; Wharton, Stephen A; Martin, Stephen R; Cross, Karen; Lin, Yipu; Liu, Yan; Feizi, Ten; Daniels, Rodney S; McCauley, John W

2016-06-01

Influenza A virus (subtype H3N2) causes seasonal human influenza and is included as a component of influenza vaccines. The majority of vaccine viruses are isolated and propagated in eggs, which commonly results in amino acid substitutions in the haemagglutinin (HA) glycoprotein. These substitutions can affect virus receptor-binding and alter virus antigenicity, thereby, obfuscating the choice of egg-propagated viruses for development into candidate vaccine viruses. To evaluate the effects of egg-adaptive substitutions seen in H3N2 vaccine viruses on sialic acid receptor-binding, we carried out quantitative measurement of virus receptor-binding using surface biolayer interferometry with haemagglutination inhibition (HI) assays to correlate changes in receptor avidity with antigenic properties. Included in these studies was a panel of H3N2 viruses generated by reverse genetics containing substitutions seen in recent egg-propagated vaccine viruses and corresponding cell culture-propagated wild-type viruses. These assays provide a quantitative approach to investigating the importance of individual amino acid substitutions in influenza receptor-binding. Results show that viruses with egg-adaptive HA substitutions R156Q, S219Y, and I226N, have increased binding avidity to α2,3-linked receptor-analogues and decreased binding avidity to α2,6-linked receptor-analogues. No measurable binding was detected for the viruses with amino acid substitution combination 156Q+219Y and receptor-binding increased in viruses where egg-adaptation mutations were introduced into cell culture-propagated virus. Substitutions at positions 156 and 190 appeared to be primarily responsible for low reactivity in HI assays with post-infection ferret antisera raised against 2012-2013 season H3N2 viruses. Egg-adaptive substitutions at position 186 caused substantial differences in binding avidity with an insignificant effect on antigenicity.

Genome-Wide Association Mapping of Barley Yellow Dwarf Virus Tolerance in Spring Oat (Avena sativa L.)

PubMed Central

Foresman, Bradley J.; Oliver, Rebekah E.; Jackson, Eric W.; Chao, Shiaoman; Arruda, Marcio P.; Kolb, Frederic L.

2016-01-01

Barley yellow dwarf viruses (BYDVs) are responsible for the disease barley yellow dwarf (BYD) and affect many cereals including oat (Avena sativa L.). Until recently, the molecular marker technology in oat has not allowed for many marker-trait association studies to determine the genetic mechanisms for tolerance. A genome-wide association study (GWAS) was performed on 428 spring oat lines using a recently developed high-density oat single nucleotide polymorphism (SNP) array as well as a SNP-based consensus map. Marker-trait associations were performed using a Q-K mixed model approach to control for population structure and relatedness. Six significant SNP-trait associations representing two QTL were found on chromosomes 3C (Mrg17) and 18D (Mrg04). This is the first report of BYDV tolerance QTL on chromosome 3C (Mrg17) and 18D (Mrg04). Haplotypes using the two QTL were evaluated and distinct classes for tolerance were identified based on the number of favorable alleles. A large number of lines carrying both favorable alleles were observed in the panel. PMID:27175781

Minimized virus binding for tests of barrier materials.

PubMed Central

Lytle, C D; Routson, L B

1995-01-01

Viruses are used to test the barrier properties of materials. Binding of virus particles during passage through holes in the material may yield misleading test results. The choices of challenge virus and suspending medium may be important for minimizing confounding effects that might arise from such binding. In this study, different surrogate viruses, as well as different support media, were evaluated to determine optimal test parameters. Two membranes with high-binding properties (nitrocellulose and cationic polysulfone) were used as filters to compare binding activities of different surrogate challenge viruses (MS2, phi X174, T7, PRD1, and phi 6) in different media. The media consisted of buffered saline with surfactants, serum, or culture broth as additives. In addition, elution rates of viruses that bound to the membranes were determined. The results suggest that viruses can bind by hydrophobic and electrostatic interactions, with phi X174 displaying the lowest level of binding by either process. The nonionic detergents Triton X-100 and Tween 80 (0.1%) equally minimized hydrophobic interactions. Neither anionic nor cationic surfactants were as effective at nontoxic levels. Serum was effective at reducing both hydrophobic and electrostatic binding, with 2% being sufficient for eliminating binding under our test conditions. Thus, phi X174 remains the best choice as a surrogate virus to test barrier materials, and Triton X-100 (0.1%) remains a good choice for reducing hydrophobic binding. In addition, binding of viruses by barrier materials is unlikely to prevent passage of blood-borne pathogens. PMID:7574603

Effect of receptor binding specificity on the immunogenicity and protective efficacy of influenza virus A H1 vaccines

PubMed Central

Sun, Xiangjie; Cao, Weiping; Pappas, Claudia; Liu, Feng; Katz, Jacqueline M.; Tumpey, Terrence M.

2018-01-01

The biological basis for the poor immunogenicity of unadjuvanted avian influenza A virus vaccines in mammals is not well understood. Here, we mutated the hemagglutinin (HA) of two H1N1 virus vaccines to determine whether virus receptor binding specificity contributes to the low immunogenicity of avian influenza virus vaccines. Mutations were introduced into the HA of an avian influenza virus, A/Duck/New York/15024–21/96 (Dk/96) which switched the binding preference from α2,3- to α2,6-linked sialic acid (SA). A switch in receptor specificity of the human A/South Carolina/1/18 (SC/18) virus generated a mutant virus with α2,3 SA (avian) binding preference. Inactivated vaccines were generated and administered to mice and ferrets intramuscularly. We found that the vaccines with human receptor binding preference induced slightly higher antibody titers and cell-mediated immune responses compared to their isogenic viruses with avian receptor binding specificity. Upon challenge with DK/96 or SC18 virus, differences in lung virus titers between the vaccine groups with different receptor-binding specificities were minimal. Overall, our data suggest that receptor binding specificity contributes only marginally to the immunogenicity of avian influenza vaccines and that other factors may also be involved. PMID:25078114

Human H3N2 Influenza Viruses Isolated from 1968 To 2012 Show Varying Preference for Receptor Substructures with No Apparent Consequences for Disease or Spread

PubMed Central

Gulati, Shelly; Smith, David F.; Cummings, Richard D.; Couch, Robert B.; Griesemer, Sara B.; St. George, Kirsten; Webster, Robert G.; Air, Gillian M.

2013-01-01

It is generally accepted that human influenza viruses bind glycans containing sialic acid linked α2–6 to the next sugar, that avian influenza viruses bind glycans containing the α2–3 linkage, and that mutations that change the binding specificity might change the host tropism. We noted that human H3N2 viruses showed dramatic differences in their binding specificity, and so we embarked on a study of representative human H3N2 influenza viruses, isolated from 1968 to 2012, that had been isolated and minimally passaged only in mammalian cells, never in eggs. The 45 viruses were grown in MDCK cells, purified, fluorescently labeled and screened on the Consortium for Functional Glycomics Glycan Array. Viruses isolated in the same season have similar binding specificity profiles but the profiles show marked year-to-year variation. None of the 610 glycans on the array (166 sialylated glycans) bound to all viruses; the closest was Neu5Acα2–6(Galβ1–4GlcNAc)3 in either a linear or biantennary form, that bound 42 of the 45 viruses. The earliest human H3N2 viruses preferentially bound short, branched sialylated glycans while recent viruses bind better to long polylactosamine chains terminating in sialic acid. Viruses isolated in 1996, 2006, 2010 and 2012 bind glycans with α2–3 linked sialic acid; for 2006, 2010 and 2012 viruses this binding was inhibited by oseltamivir, indicating binding of α2–3 sialylated glycans by neuraminidase. More significantly, oseltamivir inhibited virus entry of 2010 and 2012 viruses into MDCK cells. All of these viruses were representative of epidemic strains that spread around the world, so all could infect and transmit between humans with high efficiency. We conclude that the year-to-year variation in receptor binding specificity is a consequence of amino acid sequence changes driven by antigenic drift, and that viruses with quite different binding specificity and avidity are equally fit to infect and transmit in the human population. PMID:23805213

Diminished hepatic growth hormone receptor binding in sex-linked dwarf broiler and leghorn chickens.

PubMed

Leung, F C; Styles, W J; Rosenblum, C I; Lilburn, M S; Marsh, J A

1987-02-01

Hepatic growth hormone (GH) receptor binding was compared in normal and sex-linked dwarfs (SLD) from both Hubbard and Cornell strain chickens. At 6, 8, and 20 weeks of age, hepatic GH receptor binding in the Hubbard SLD chickens was significantly lower than that of normal fast-growing birds. At 20 weeks of age, only 2 of 22 SLD chickens in the Hubbard broiler strain showed positive binding at a high enough level to allow for Scatchard analysis. The affinity constants and binding capacities of these two SLD chickens were numerically (but not significantly) lower than those of the normal fast-growing birds. We further examined hepatic GH receptor binding in two closely related White Leghorn strains of chickens that have been maintained as closed breeding populations for many years. We observed no detectable hepatic GH binding in the Cornell SLD chickens (N = 20), as compared to the normal-growing control strain (K strain). In both SLD strains, pretreatment with 4 M MgCl2 did not enhance GH binding, suggesting that there was no endogenous GH binding to the receptor. Based on these data, we suggest that the lack, or greatly reduced number, of GH receptors may be a major contributing factor to the dwarfism observed in these strains.

The Incidence and Genetic Diversity of Apple Mosaic Virus (ApMV) and Prune Dwarf Virus (PDV) in Prunus Species in Australia

PubMed Central

Constable, Fiona E.; Nancarrow, Narelle; Rodoni, Brendan

2018-01-01

Apple mosaic virus (ApMV) and prune dwarf virus (PDV) are amongst the most common viruses infecting Prunus species worldwide but their incidence and genetic diversity in Australia is not known. In a survey of 127 Prunus tree samples collected from five states in Australia, ApMV and PDV occurred in 4 (3%) and 13 (10%) of the trees respectively. High-throughput sequencing (HTS) of amplicons from partial conserved regions of RNA1, RNA2, and RNA3, encoding the methyltransferase (MT), RNA-dependent RNA polymerase (RdRp), and the coat protein (CP) genes respectively, of ApMV and PDV was used to determine the genetic diversity of the Australian isolates of each virus. Phylogenetic comparison of Australian ApMV and PDV amplicon HTS variants and full length genomes of both viruses with isolates occurring in other countries identified genetic strains of each virus occurring in Australia. A single Australian Prunus infecting ApMV genetic strain was identified as all ApMV isolates sequence variants formed a single phylogenetic group in each of RNA1, RNA2, and RNA3. Two Australian PDV genetic strains were identified based on the combination of observed phylogenetic groups in each of RNA1, RNA2, and RNA3 and one Prunus tree had both strains. The accuracy of amplicon sequence variants phylogenetic analysis based on segments of each virus RNA were confirmed by phylogenetic analysis of full length genome sequences of Australian ApMV and PDV isolates and all published ApMV and PDV genomes from other countries. PMID:29562672

Coat protein expression strategy of oat blue dwarf virus.

PubMed

Edwards, Michael C; Weiland, John J

2014-02-01

Oat blue dwarf virus (OBDV) is a member of the genus Marafivirus whose genome encodes a 227 kDa polyprotein (p227) ostensibly processed post-translationally into its functional components. Encoded near the 3' terminus and coterminal with the p227 ORF are ORFs specifying major and minor capsid proteins (CP). Since the CP expression strategy of marafiviruses has not been thoroughly investigated, we produced a series of point mutants in the OBDV CP encoding gene and examined expression in protoplasts. Results support a model in which the 21 kDa major CP is the product of direct translation of a sgRNA, while the 24 kDa minor CP is a cleavage product derived from both the polyprotein and a larger ~26 kDa precursor translated directly from the sgRNA. Cleavage occurs at an LXG[G/A] motif conserved in many viruses that use papain-like proteases for polyprotein processing and protection against degradation via the ubiquitin-proteasome system. Published by Elsevier Inc.

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

Genetics coupled to quantitative intact proteomics links heritable aphid and endosymbiont protein expression to circulative polerovirus transmission.

PubMed

Cilia, M; Tamborindeguy, C; Fish, T; Howe, K; Thannhauser, T W; Gray, S

2011-03-01

Yellow dwarf viruses in the family Luteoviridae, which are the causal agents of yellow dwarf disease in cereal crops, are each transmitted most efficiently by different species of aphids in a circulative manner that requires the virus to interact with a multitude of aphid proteins. Aphid proteins differentially expressed in F2 Schizaphis graminum genotypes segregating for the ability to transmit Cereal yellow dwarf virus-RPV (CYDV-RPV) were identified using two-dimensional difference gel electrophoresis (DIGE) coupled to either matrix-assisted laser desorption ionization-tandem mass spectrometry or online nanoscale liquid chromatography coupled to electrospray tandem mass spectrometry. A total of 50 protein spots, containing aphid proteins and proteins from the aphid's obligate and maternally inherited bacterial endosymbiont, Buchnera, were identified as differentially expressed between transmission-competent and refractive aphids. Surprisingly, in virus transmission-competent F2 genotypes, the isoelectric points of the Buchnera proteins did not match those in the maternal Buchnera proteome as expected, but instead they aligned with the Buchnera proteome of the transmission-competent paternal parent. Among the aphid proteins identified, many were involved in energy metabolism, membrane trafficking, lipid signaling, and the cytoskeleton. At least eight aphid proteins were expressed as heritable, isoelectric point isoform pairs, one derived from each parental lineage. In the F2 genotypes, the expression of aphid protein isoforms derived from the competent parental lineage aligned with the virus transmission phenotype with high precision. Thus, these isoforms are candidate biomarkers for CYDV-RPV transmission in S. graminum. Our combined genetic and DIGE approach also made it possible to predict where several of the proteins may be expressed in refractive aphids with different barriers to transmission. Twelve proteins were predicted to act in the hindgut of the aphid, while six proteins were predicted to be associated with the accessory salivary glands or hemolymph. Knowledge of the proteins that regulate virus transmission and their predicted locations will aid in understanding the biochemical mechanisms regulating circulative virus transmission in aphids, as well as in identifying new targets to block transmission.

Genetics Coupled to Quantitative Intact Proteomics Links Heritable Aphid and Endosymbiont Protein Expression to Circulative Polerovirus Transmission▿ †

PubMed Central

Cilia, M.; Tamborindeguy, C.; Fish, T.; Howe, K.; Thannhauser, T. W.; Gray, S.

2011-01-01

Yellow dwarf viruses in the family Luteoviridae, which are the causal agents of yellow dwarf disease in cereal crops, are each transmitted most efficiently by different species of aphids in a circulative manner that requires the virus to interact with a multitude of aphid proteins. Aphid proteins differentially expressed in F2 Schizaphis graminum genotypes segregating for the ability to transmit Cereal yellow dwarf virus-RPV (CYDV-RPV) were identified using two-dimensional difference gel electrophoresis (DIGE) coupled to either matrix-assisted laser desorption ionization-tandem mass spectrometry or online nanoscale liquid chromatography coupled to electrospray tandem mass spectrometry. A total of 50 protein spots, containing aphid proteins and proteins from the aphid's obligate and maternally inherited bacterial endosymbiont, Buchnera, were identified as differentially expressed between transmission-competent and refractive aphids. Surprisingly, in virus transmission-competent F2 genotypes, the isoelectric points of the Buchnera proteins did not match those in the maternal Buchnera proteome as expected, but instead they aligned with the Buchnera proteome of the transmission-competent paternal parent. Among the aphid proteins identified, many were involved in energy metabolism, membrane trafficking, lipid signaling, and the cytoskeleton. At least eight aphid proteins were expressed as heritable, isoelectric point isoform pairs, one derived from each parental lineage. In the F2 genotypes, the expression of aphid protein isoforms derived from the competent parental lineage aligned with the virus transmission phenotype with high precision. Thus, these isoforms are candidate biomarkers for CYDV-RPV transmission in S. graminum. Our combined genetic and DIGE approach also made it possible to predict where several of the proteins may be expressed in refractive aphids with different barriers to transmission. Twelve proteins were predicted to act in the hindgut of the aphid, while six proteins were predicted to be associated with the accessory salivary glands or hemolymph. Knowledge of the proteins that regulate virus transmission and their predicted locations will aid in understanding the biochemical mechanisms regulating circulative virus transmission in aphids, as well as in identifying new targets to block transmission. PMID:21159868

Evaluation of Rice Resistance to Southern Rice Black-Streaked Dwarf Virus and Rice Ragged Stunt Virus through Combined Field Tests, Quantitative Real-Time PCR, and Proteome Analysis.

PubMed

Wang, Zhenchao; Yu, Lu; Jin, Linhong; Wang, Wenli; Zhao, Qi; Ran, Longlu; Li, Xiangyang; Chen, Zhuo; Guo, Rong; Wei, Yongtian; Yang, Zhongcheng; Liu, Enlong; Hu, Deyu; Song, Baoan

2017-02-22

Diseases caused by southern rice black-streaked dwarf virus (SRBSDV) and rice ragged stunt virus (RRSV) considerably decrease grain yield. Therefore, determining rice cultivars with high resistance to SRBSDV and RRSV is necessary. In this study, rice cultivars with high resistance to SRBSDV and RRSV were evaluated through field trials in Shidian and Mangshi county, Yunnan province, China. SYBR Green I-based quantitative real-time polymerase chain reaction (qRT-PCR) analysis was used to quantitatively detect virus gene expression levels in different rice varieties. The following parameters were applied to evaluate rice resistance: acre yield (A.Y.), incidence of infected plants (I.I.P.), virus load (V.L.), disease index (D.I.), and insect quantity (I.Q.) per 100 clusters. Zhongzheyou1 (Z1) and Liangyou2186 (L2186) were considered the most suitable varieties with integrated higher A.Y., lower I.I.P., V.L., D.I. and I.Q. In order to investigate the mechanism of rice resistance, comparative label-free shotgun liquid chromatography tandem-mass spectrometry (LC-MS/MS) proteomic approaches were applied to comprehensively describe the proteomics of rice varieties' SRBSDV tolerance. Systemic acquired resistance (SAR)-related proteins in Z1 and L2186 may result in the superior resistance of these varieties compared with Fengyouxiangzhan (FYXZ).

Assessment of reference gene stability in Rice stripe virus and Rice black streaked dwarf virus infection rice by quantitative Real-time PCR.

PubMed

Fang, Peng; Lu, Rongfei; Sun, Feng; Lan, Ying; Shen, Wenbiao; Du, Linlin; Zhou, Yijun; Zhou, Tong

2015-10-24

Stably expressed reference gene(s) normalization is important for the understanding of gene expression patterns by quantitative Real-time PCR (RT-qPCR), particularly for Rice stripe virus (RSV) and Rice black streaked dwarf virus (RBSDV) that caused seriously damage on rice plants in China and Southeast Asia. The expression of fourteen common used reference genes of Oryza sativa L. were evaluated by RT-qPCR in RSV and RBSDV infected rice plants. Suitable normalization reference gene(s) were identified by geNorm and NormFinder algorithms. UBQ 10 + GAPDH and UBC + Actin1 were identified as suitable reference genes for RT-qPCR normalization under RSV and RBSDV infection, respectively. When using multiple reference genes, the expression patterns of OsPRIb and OsWRKY, two virus resistance genes, were approximately similar with that reported previously. Comparatively, by using single reference gene (TIP41-Like), a weaker inducible response was observed. We proposed that the combination of two reference genes could obtain more accurate and reliable normalization of RT-qPCR results in RSV- and RBSDV-infected plants. This work therefore sheds light on establishing a standardized RT-qPCR procedure in RSV- and RBSDV-infected rice plants, and might serve as an important point for discovering complex regulatory networks and identifying genes relevant to biological processes or implicated in virus.

Mannose-binding lectin binds to Ebola and Marburg envelope glycoproteins, resulting in blocking of virus interaction with DC-SIGN and complement-mediated virus neutralization.

PubMed

Ji, Xin; Olinger, Gene G; Aris, Sheena; Chen, Ying; Gewurz, Henry; Spear, Gregory T

2005-09-01

Mannose-binding lectin (MBL), a serum lectin that mediates innate immune functions including activation of the lectin complement pathway, binds to carbohydrates expressed on some viral glycoproteins. In this study, the ability of MBL to bind to virus particles pseudotyped with Ebola and Marburg envelope glycoproteins was evaluated. Virus particles bearing either Ebola (Zaire strain) or Marburg (Musoke strain) envelope glycoproteins bound at significantly higher levels to immobilized MBL compared with virus particles pseudotyped with vesicular stomatitis virus glycoprotein or with no virus glycoprotein. As observed in previous studies, Ebola-pseudotyped virus bound to cells expressing the lectin DC-SIGN (dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin). However, pre-incubation of virus with MBL blocked DC-SIGN-mediated binding to cells, suggesting that the two lectins bind at the same or overlapping sites on the Ebola glycoprotein. Neutralization experiments showed that virus pseudotyped with Ebola or Marburg (Musoke) glycoprotein was neutralized by complement, while the Marburg (Ravn strain) glycoprotein-pseudotyped virus was less sensitive to neutralization. Neutralization was partially mediated through the lectin complement pathway, since a complement source deficient in MBL was significantly less effective at neutralizing viruses pseudotyped with filovirus glycoproteins and addition of purified MBL to the MBL-deficient complement increased neutralization. These experiments demonstrated that MBL binds to filovirus envelope glycoproteins resulting in important biological effects and suggest that MBL can interact with filoviruses during infection in humans.

Evolutionary relationships in the ilarviruses: nucleotide sequence of prunus necrotic ringspot virus RNA 3.

PubMed

Sánchez-Navarro, J A; Pallás, V

1997-01-01

The complete nucleotide sequence of an isolate of prunus necrotic ringspot virus (PNRSV) RNA 3 has been determined. Elucidation of the amino acid sequence of the proteins encoded by the two large open reading frames (ORFs) allowed us to carry out comparative and phylogenetic studies on the movement (MP) and coat (CP) proteins in the ilarvirus group. Amino acid sequence comparison of the MP revealed a highly conserved basic sequence motif with an amphipathic alpha-helical structure preceding the conserved motif of the '30K superfamily' proposed by Mushegian and Koonin [26] for MP's. Within this '30K' motif a strictly conserved transmembrane domain is present in all ilarviruses sequenced so far. At the amino-terminal end, prune dwarf virus (PDV) has an extension not present in other ilarviruses but which is observed in all bromo- and cucumoviruses, suggesting a common ancestor or a recombinational event in the Bromoviridae family. Examination of the N-terminus of the CP's of all ilarviruses revealed a highly basic region, part of which resembles the Arg-rich motif that has been characterized in the RNA-binding protein family. This motif has also been found in the other members of the Bromoviridae family, suggesting its involvement in a structural function. Furthermore this region is required for infectivity in ilarviruses. The similarities found in this Arg-rich motif are discussed in terms of this process known as genome activation. Finally, phylogenetic analysis of both the MP and CP proteins revealed a higher relationship of A1MV to PNRSV, apple mosaic virus (ApMV) and PDV than any other member of the ilarvirus group. In that sense, A1MV should be considered as a true ilarvirus instead of forming a distinct group of viruses.

Adaptation of avian influenza A (H6N1) virus from avian to human receptor-binding preference

PubMed Central

Wang, Fei; Qi, Jianxun; Bi, Yuhai; Zhang, Wei; Wang, Min; Zhang, Baorong; Wang, Ming; Liu, Jinhua; Yan, Jinghua; Shi, Yi; Gao, George F

2015-01-01

The receptor-binding specificity of influenza A viruses is a major determinant for the host tropism of the virus, which enables interspecies transmission. In 2013, the first human case of infection with avian influenza A (H6N1) virus was reported in Taiwan. To gather evidence concerning the epidemic potential of H6 subtype viruses, we performed comprehensive analysis of receptor-binding properties of Taiwan-isolated H6 HAs from 1972 to 2013. We propose that the receptor-binding properties of Taiwan-isolated H6 HAs have undergone three major stages: initially avian receptor-binding preference, secondarily obtaining human receptor-binding capacity, and recently human receptor-binding preference, which has been confirmed by receptor-binding assessment of three representative virus isolates. Mutagenesis work revealed that E190V and G228S substitutions are important to acquire the human receptor-binding capacity, and the P186L substitution could reduce the binding to avian receptor. Further structural analysis revealed how the P186L substitution in the receptor-binding site of HA determines the receptor-binding preference change. We conclude that the human-infecting H6N1 evolved into a human receptor preference. PMID:25940072

The Roles of Hemagglutinin Phe-95 in Receptor Binding and Pathogenicity of Influenza B Virus

PubMed Central

Ni, Fengyun; Mbawuike, Innocent Nnadi; Kondrashkina, Elena; Wang, Qinghua

2014-01-01

Diverged ~4,000 years ago, influenza B virus has several important differences from influenza A virus, including lower receptor-binding affinity and highly restricted host range. Based on our prior structural studies, we hypothesized that a single-residue difference in the receptor-binding site of hemagglutinin (HA), Phe-95 in influenza B virus versus Tyr-98 in influenza A/H1~H15, is possibly a key determinant for the low receptor-binding affinity. Here we demonstrate that the mutation Phe95→Tyr in influenza B virus HA restores all three hydrogen bonds made by Tyr-98 in influenza A/H3 HA and has the potential to enhance receptor binding. However, the full realization of this potential is influenced by the local environment into which the mutation is introduced. The binding and replication of the recombinant viruses correlate well with the receptor-binding capabilities of HA. These results are discussed in relation to the roles of Phe-95 in receptor binding and pathogenicity of influenza B virus. PMID:24503069

A Small Molecule Inhibits Virion Attachment to Heparan Sulfate- or Sialic Acid-Containing Glycans

PubMed Central

Colpitts, Che C.

2014-01-01

ABSTRACT Primary attachment to cellular glycans is a critical entry step for most human viruses. Some viruses, such as herpes simplex virus type 1 (HSV-1) and hepatitis C virus (HCV), bind to heparan sulfate, whereas others, such as influenza A virus (IAV), bind to sialic acid. Receptor mimetics that interfere with these interactions are active against viruses that bind to either heparan sulfate or to sialic acid. However, no molecule that inhibits the attachment of viruses in both groups has yet been identified. Epigallocatechin gallate (EGCG), a green tea catechin, is active against many unrelated viruses, including several that bind to heparan sulfate or to sialic acid. We sought to identify the basis for the broad-spectrum activity of EGCG. Here, we show that EGCG inhibits the infectivity of a diverse group of enveloped and nonenveloped human viruses. EGCG acts directly on the virions, without affecting the fluidity or integrity of the virion envelopes. Instead, EGCG interacts with virion surface proteins to inhibit the attachment of HSV-1, HCV, IAV, vaccinia virus, adenovirus, reovirus, and vesicular stomatitis virus (VSV) virions. We further show that EGCG competes with heparan sulfate for binding of HSV-1 and HCV virions and with sialic acid for binding of IAV virions. Therefore, EGCG inhibits unrelated viruses by a common mechanism. Most importantly, we have identified EGCG as the first broad-spectrum attachment inhibitor. Our results open the possibility for the development of small molecule broad-spectrum antivirals targeting virion attachment. IMPORTANCE This study shows that it is possible to develop a small molecule antiviral or microbicide active against the two largest groups of human viruses: those that bind to glycosaminoglycans and those that bind to sialoglycans. This group includes the vast majority of human viruses, including herpes simplex viruses, cytomegalovirus, influenza virus, poxvirus, hepatitis C virus, HIV, and many others. PMID:24789779

Highly Pathogenic Influenza A(H5Nx) Viruses with Altered H5 Receptor-Binding Specificity

PubMed Central

Guo, Hongbo; de Vries, Erik; McBride, Ryan; Dekkers, Jojanneke; Peng, Wenjie; Bouwman, Kim M.; Nycholat, Corwin; Verheije, M. Helene; Paulson, James C.; van Kuppeveld, Frank J.M.

2017-01-01

Emergence and intercontinental spread of highly pathogenic avian influenza A(H5Nx) virus clade 2.3.4.4 is unprecedented. H5N8 and H5N2 viruses have caused major economic losses in the poultry industry in Europe and North America, and lethal human infections with H5N6 virus have occurred in Asia. Knowledge of the evolution of receptor-binding specificity of these viruses, which might affect host range, is urgently needed. We report that emergence of these viruses is accompanied by a change in receptor-binding specificity. In contrast to ancestral clade 2.3.4 H5 proteins, novel clade 2.3.4.4 H5 proteins bind to fucosylated sialosides because of substitutions K222Q and S227R, which are unique for highly pathogenic influenza virus H5 proteins. North American clade 2.3.4.4 virus isolates have retained only the K222Q substitution but still bind fucosylated sialosides. Altered receptor-binding specificity of virus clade 2.3.4.4 H5 proteins might have contributed to emergence and spread of H5Nx viruses. PMID:27869615

Receptor-Binding Profiles of H7 Subtype Influenza Viruses in Different Host Species

PubMed Central

Gambaryan, Alexandra S.; Matrosovich, Tatyana Y.; Philipp, Jennifer; Munster, Vincent J.; Fouchier, Ron A. M.; Cattoli, Giovanni; Capua, Ilaria; Krauss, Scott L.; Webster, Robert G.; Banks, Jill; Bovin, Nicolai V.; Klenk, Hans-Dieter

2012-01-01

Influenza viruses of gallinaceous poultry and wild aquatic birds usually have distinguishable receptor-binding properties. Here we used a panel of synthetic sialylglycopolymers and solid-phase receptor-binding assays to characterize receptor-binding profiles of about 70 H7 influenza viruses isolated from aquatic birds, land-based poultry, and horses in Eurasia and America. Unlike typical duck influenza viruses with non-H7 hemagglutinin (HA), all avian H7 influenza viruses, irrespective of the host species, displayed a poultry-virus-like binding specificity, i.e., preferential binding to sulfated oligosaccharides Neu5Acα2-3Galβ1-4(6-O-HSO3)GlcNAc and Neu5Acα2-3Galβ1-4(Fucα1-3)(6-O-HSO3)GlcNAc. This phenotype correlated with the unique amino acid sequence of the amino acid 185 to 189 loop of H7 HA and seemed to be dependent on ionic interactions between the sulfate group of the receptor and Lys193 and on the lack of sterical clashes between the fucose residue and Gln222. Many North American and Eurasian H7 influenza viruses displayed weak but detectable binding to the human-type receptor moiety Neu5Acα2-6Galβ1-4GlcNAc, highlighting the potential of H7 influenza viruses for avian-to-human transmission. Equine H7 influenza viruses differed from other viruses by preferential binding to the N-glycolyl form of sialic acid. Our data suggest that the receptor-binding site of contemporary H7 influenza viruses in aquatic and terrestrial birds was formed after the introduction of their common precursor from ducks to a new host, presumably, gallinaceous poultry. The uniformity of the receptor-binding profile of H7 influenza viruses in various wild and domestic birds indicates that there is no strong receptor-mediated host range restriction in birds on viruses with this HA subtype. This notion agrees with repeated interspecies transmission of H7 influenza viruses from aquatic birds to poultry. PMID:22345462

Nuclear factors that bind to the enhancer region of nondefective Friend murine leukemia virus.

PubMed Central

Manley, N R; O'Connell, M A; Sharp, P A; Hopkins, N

1989-01-01

Nondefective Friend murine leukemia virus (MuLV) causes erythroleukemia when injected into newborn NFS mice, while Moloney MuLV causes T-cell lymphoma. Exchange of the Friend virus enhancer region, a sequence of about 180 nucleotides including the direct repeat and a short 3'-adjacent segment, for the corresponding region in Moloney MuLV confers the ability to cause erythroid disease on Moloney MuLV. We have used the electrophoretic mobility shift assay and methylation interference analysis to identify cellular factors which bind to the Friend virus enhancer region and compared these with factors, previously identified, that bind to the Moloney virus direct repeat (N. A. Speck and D. Baltimore, Mol. Cell. Biol. 7:1101-1110, 1987). We identified five binding sites for sequence-specific DNA-binding proteins in the Friend virus enhancer region. While some binding sites are present in both the Moloney and Friend virus enhancers, both viruses contain unique sites not present in the other. Although none of the factors identified in this report which bind to these unique sites are present exclusively in T cells or erythroid cells, they bind to three regions of the enhancer shown by genetic analysis to encode disease specificity and thus are candidates to mediate the tissue-specific expression and distinct disease specificities encoded by these virus enhancer elements. Images PMID:2778872

The role of receptor binding specificity in interspecies transmission of influenza viruses

PubMed Central

Imai, Masaki; Kawaoka, Yoshihiro

2017-01-01

Influenza A virus infection begins with the binding of the hemagglutinin (HA) glycoprotein to sialic acid-containing receptors on the surface of the target cell. Avian influenza viruses, including avian H5N1, H7, and H9N2 viruses, can occasionally cross the species barrier and infect humans; however, these viruses do not spread efficiently from person to person, perhaps, in part, due to differences in the receptor-binding specificities of human and avian influenza viruses. The HAs of avian influenza viruses must adapt to receptors in humans to acquire efficient human-to-human transmissibility. In this review, we discuss the receptor binding specificity of influenza A viruses and its role in interspecies transmission. PMID:22445963

Enhanced Human-Type Receptor Binding by Ferret-Transmissible H5N1 with a K193T Mutation.

PubMed

Peng, Wenjie; Bouwman, Kim M; McBride, Ryan; Grant, Oliver C; Woods, Robert J; Verheije, Monique H; Paulson, James C; de Vries, Robert P

2018-05-15

All human influenza pandemics have originated from avian influenza viruses. Although multiple changes are needed for an avian virus to be able to transmit between humans, binding to human-type receptors is essential. Several research groups have reported mutations in H5N1 viruses that exhibit specificity for human-type receptors and promote respiratory droplet transmission between ferrets. Upon detailed analysis, we have found that these mutants exhibit significant differences in fine receptor specificity compared to human H1N1 and H3N2 and retain avian-type receptor binding. We have recently shown that human influenza viruses preferentially bind to α2-6-sialylated branched N-linked glycans, where the sialic acids on each branch can bind to receptor sites on two protomers of the same hemagglutinin (HA) trimer. In this binding mode, the glycan projects over the 190 helix at the top of the receptor-binding pocket, which in H5N1 would create a stearic clash with lysine at position 193. Thus, we hypothesized that a K193T mutation would improve binding to branched N-linked receptors. Indeed, the addition of the K193T mutation to the H5 HA of a respiratory-droplet-transmissible virus dramatically improves both binding to human trachea epithelial cells and specificity for extended α2-6-sialylated N-linked glycans recognized by human influenza viruses. IMPORTANCE Infections by avian H5N1 viruses are associated with a high mortality rate in several species, including humans. Fortunately, H5N1 viruses do not transmit between humans because they do not bind to human-type receptors. In 2012, three seminal papers have shown how these viruses can be engineered to transmit between ferrets, the human model for influenza virus infection. Receptor binding, among others, was changed, and the viruses now bind to human-type receptors. Receptor specificity was still markedly different compared to that of human influenza viruses. Here we report an additional mutation in ferret-transmissible H5N1 that increases human-type receptor binding. K193T seems to be a common receptor specificity determinant, as it increases human-type receptor binding in multiple subtypes. The K193T mutation can now be used as a marker during surveillance of emerging viruses to assess potential pandemic risk. Copyright © 2018 American Society for Microbiology.

Localization of sweet potato chlorotic stunt virus (SPCSV) in synergic infection with potyviruses in sweet potato.

PubMed

Nome, Claudia Fernanda; Nome, Sergio Fernando; Guzmán, Fabiana; Conci, Luis; Laguna, Irma Graciela

2007-01-01

Among diseases reported worldwidely for sweet potato (Ipomoea batatas (L) Lam) crop, one of the most frequent is the Sweet potato virus disease (SPVD), caused by sweet potato chlorotic stunt virus (SPCSV) and sweet potato feathery mottle virus (SPFMV) co-infection. In Argentina, there exists the sweet potato chlorotic dwarf (SPCD), a sweet potato disease caused by triple co-infection with SPCSV, SPFMV and sweet potato mild speckling virus (SPMSV). Both diseases cause a synergism between the potyviruses (SPFMV and SPMSV) and the crinivirus (SPCSV). Up to date, studies carried out on the interaction among these three viruses have not described their localization in the infected tissues. In single infections, virions of the crinivirus genus are limited to the phloem while potyviral virions are found in most tissues of the infected plant. The purpose of this work was to localize the heat shock protein 70 homolog (HSP70h), a movement protein for genus crinivirus, of an Argentinean SPCSV isolate in its single infection and in its double and triple co-infection with SPFMV and SPMSV. The localization was made by in situ hybridization (ISH) for electron microscopy (EM) on ultrathin sections of sweet potato cv. Morada INTA infected tissues. The results demonstrated that viral RNA coding HSP70h is restricted to phloem cells during crinivirus single infection, while it was detected outside the phloem in infections combined with the potyviruses involved in chlorotic dwarf disease.

SEARCHING FOR BINARY Y DWARFS WITH THE GEMINI MULTI-CONJUGATE ADAPTIVE OPTICS SYSTEM (GeMS)

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

Opitz, Daniela; Tinney, C. G.; Faherty, Jacqueline K.

The NASA Wide-field Infrared Survey Explorer (WISE) has discovered almost all the known members of the new class of Y-type brown dwarfs. Most of these Y dwarfs have been identified as isolated objects in the field. It is known that binaries with L- and T-type brown dwarf primaries are less prevalent than either M-dwarf or solar-type primaries, they tend to have smaller separations and are more frequently detected in near-equal mass configurations. The binary statistics for Y-type brown dwarfs, however, are sparse, and so it is unclear if the same trends that hold for L- and T-type brown dwarfs alsomore » hold for Y-type ones. In addition, the detection of binary companions to very cool Y dwarfs may well be the best means available for discovering even colder objects. We present results for binary properties of a sample of five WISE Y dwarfs with the Gemini Multi-Conjugate Adaptive Optics System. We find no evidence for binary companions in these data, which suggests these systems are not equal-luminosity (or equal-mass) binaries with separations larger than ∼0.5–1.9 AU. For equal-mass binaries at an age of 5 Gyr, we find that the binary binding energies ruled out by our observations (i.e., 10{sup 42} erg) are consistent with those observed in previous studies of hotter ultra-cool dwarfs.« less

Analyses of pea necrotic yellow dwarf virus-encoded proteins.

PubMed

Krenz, Björn; Schießl, Ingrid; Greiner, Eva; Krapp, Susanna

2017-06-01

Pea necrotic yellow dwarf virus (PNYDV) is a multipartite, circular, single-stranded DNA plant virus. PNYDV encodes eight proteins and the function of three of which remains unknown-U1, U2, and U4. PNYDV proteins cellular localization was analyzed by GFP tagging and bimolecular fluorescence complementation (BiFC) studies. The interactions of all eight PNYDV proteins were tested pairwise in planta (36 combinations in total). Seven interactions were identified and two (M-Rep with CP and MP with U4) were characterized further. MP and U4 complexes appeared as vesicle-like spots and were localized at the nuclear envelope and cell periphery. These vesicle-like spots were associated with the endoplasmatic reticulum. In addition, a nuclear localization signal (NLS) was mapped for U1, and a mutated U1 with NLS disrupted localized at plasmodesmata and therefore might also have a role in movement. Taken together, this study provides evidence for previously undescribed nanovirus protein-protein interactions and their cellular localization with novel findings not only for those proteins with unknown function, but also for characterized proteins such as the CP.

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.

Passion Fruit Chlorotic Mottle Virus: Molecular Characterization of a New Divergent Geminivirus in Brazil.

PubMed

Fontenele, Rafaela S; Abreu, Rayane A; Lamas, Natalia S; Alves-Freitas, Dione M T; Vidal, Andreza H; Poppiel, Raul R; Melo, Fernando L; Lacorte, Cristiano; Martin, Darren P; Campos, Magnolia A; Varsani, Arvind; Ribeiro, Simone G

2018-04-02

Brazil is one of the major passion fruit producers worldwide. Viral diseases are among the most important constraints for passion fruit production. Here we identify and characterize a new passion fruit infecting-virus belonging to the family Geminiviridae : passion fruit chlorotic mottle virus (PCMoV). PCMoV is a divergent geminivirus unlike previously characterized passion fruit-infecting geminiviruses that belonged to the genus Begomovirus . Among the presently known geminiviruses, it is most closely related to, and shares ~62% genome-wide identity with citrus chlorotic dwarf associated virus (CCDaV) and camelia chlorotic dwarf associated virus (CaCDaV). The 3743 nt PCMoV genome encodes a capsid protein (CP) and replication-associated protein (Rep) that respectively share 56 and 60% amino acid identity with those encoded by CaCDaV. The CPs of PCMoV, CCDaV, and CaCDaV cluster with those of begomovirus whereas their Reps with those of becurtoviruses. Hence, these viruses likely represent a lineage of recombinant begomo-like and becurto-like ancestral viruses. Furthermore, PCMoV, CCDaV, and CaCDaV genomes are ~12-30% larger than monopartite geminiviruses and this is primarily due to the encoded movement protein (MP; 891-921 nt) and this MP is most closely related to that encoded by the DNA-B component of bipartite begomoviruses. Hence, PCMoV, CCDaV, and CaCDaV lineage of viruses may represent molecules in an intermediary step in the evolution of bipartite begomoviruses (~5.3 kb) from monopartite geminiviruses (~2.7-3 kb). An infectious clone of PCMoV systemically infected Nicotiana benthamina , Arabidopsis thaliana , and Passiflora edulis .

Passion Fruit Chlorotic Mottle Virus: Molecular Characterization of a New Divergent Geminivirus in Brazil

PubMed Central

Fontenele, Rafaela S.; Abreu, Rayane A.; Lamas, Natalia S.; Alves-Freitas, Dione M. T.; Vidal, Andreza H.; Melo, Fernando L.; Lacorte, Cristiano; Martin, Darren P.; Campos, Magnolia A.; Ribeiro, Simone G.

2018-01-01

Brazil is one of the major passion fruit producers worldwide. Viral diseases are among the most important constraints for passion fruit production. Here we identify and characterize a new passion fruit infecting-virus belonging to the family Geminiviridae: passion fruit chlorotic mottle virus (PCMoV). PCMoV is a divergent geminivirus unlike previously characterized passion fruit-infecting geminiviruses that belonged to the genus Begomovirus. Among the presently known geminiviruses, it is most closely related to, and shares ~62% genome-wide identity with citrus chlorotic dwarf associated virus (CCDaV) and camelia chlorotic dwarf associated virus (CaCDaV). The 3743 nt PCMoV genome encodes a capsid protein (CP) and replication-associated protein (Rep) that respectively share 56 and 60% amino acid identity with those encoded by CaCDaV. The CPs of PCMoV, CCDaV, and CaCDaV cluster with those of begomovirus whereas their Reps with those of becurtoviruses. Hence, these viruses likely represent a lineage of recombinant begomo-like and becurto-like ancestral viruses. Furthermore, PCMoV, CCDaV, and CaCDaV genomes are ~12–30% larger than monopartite geminiviruses and this is primarily due to the encoded movement protein (MP; 891–921 nt) and this MP is most closely related to that encoded by the DNA-B component of bipartite begomoviruses. Hence, PCMoV, CCDaV, and CaCDaV lineage of viruses may represent molecules in an intermediary step in the evolution of bipartite begomoviruses (~5.3 kb) from monopartite geminiviruses (~2.7–3 kb). An infectious clone of PCMoV systemically infected Nicotiana benthamina, Arabidopsis thaliana, and Passiflora edulis. PMID:29614801

Crop-associated virus reduces the rooting depth of non-crop perennial native grass more than non-crop-associated virus with known viral suppressor of RNA silencing (VSR).

PubMed

Malmstrom, Carolyn M; Bigelow, Patrick; Trębicki, Piotr; Busch, Anna K; Friel, Colleen; Cole, Ellen; Abdel-Azim, Heba; Phillippo, Colin; Alexander, Helen M

2017-09-15

As agricultural acreage expanded and came to dominate landscapes across the world, viruses gained opportunities to move between crop and wild native plants. In the Midwestern USA, virus exchange currently occurs between widespread annual Poaceae crops and remnant native perennial prairie grasses now under consideration as bioenergy feedstocks. In this region, the common aphid species Rhopalosiphum padi L. (the bird cherry-oat aphid) transmits several virus species in the family Luteoviridae, including Barley yellow dwarf virus (BYDV-PAV, genus Luteovirus) and Cereal yellow dwarf virus (CYDV-RPV and -RPS, genus Polerovirus). The yellow dwarf virus (YDV) species in these two genera share genetic similarities in their 3'-ends, but diverge in the 5'-regions. Most notably, CYDVs encode a P0 viral suppressor of RNA silencing (VSR) absent in BYDV-PAV. Because BYDV-PAV has been reported more frequently in annual cereals and CYDVs in perennial non-crop grasses, we examine the hypothesis that the viruses' genetic differences reflect different affinities for crop and non-crop hosts. Specifically, we ask (i) whether CYDVs might persist within and affect a native non-crop grass more strongly than BYDV-PAV, on the grounds that the polerovirus VSR could better moderate the defenses of a well-defended perennial, and (ii) whether the opposite pattern of effects might occur in a less defended annual crop. Because previous work found that the VSR of CYDV-RPS possessed greater silencing suppressor efficiency than that of CYDV-RPV, we further explored (iii) whether a novel grass-associated CYDV-RPS isolate would influence a native non-crop grass more strongly than a comparable CYDV-RPV isolate. In growth chamber studies, we found support for this hypothesis: only grass-associated CYDV-RPS stunted the shoots and crowns of Panicum virgatum L. (switchgrass), a perennial native North American prairie grass, whereas crop-associated BYDV-PAV (and coinfection with BYDV-PAV and CYDV-RPS) most stunted annual Avena sativa L. (oats). These findings suggest that some of the diversity in grass-infecting Luteoviridae reflects viral capacity to modulate defenses in different host types. Intriguingly, while all virus treatments also reduced root production in both host species, only crop-associated BYDV-PAV (or co-infection) reduced rooting depths. Such root effects may increase host susceptibility to drought, and indicate that BYDV-PAV pathogenicity is determined by something other than a P0 VSR. These findings contribute to growing evidence that pathogenic crop-associated viruses may harm native species as well as crops. Critical next questions include the extent to which crop-associated selection pressures drive viral pathogenesis. Copyright © 2017 Elsevier B.V. All rights reserved.

Simultaneous detection of four garlic viruses by multiplex reverse transcription PCR and their distribution in Indian garlic accessions.

PubMed

Majumder, S; Baranwal, V K

2014-06-01

Indian garlic is infected with Onion yellow dwarf virus (OYDV), Shallot latent virus (SLV), Garlic common latent virus (GarCLV) and allexiviruses. Identity and distribution of garlic viruses in various garlic accessions from different geographical regions of India were investigated. OYDV and allexiviruses were observed in all the garlic accessions, while SLV and GarCLV were observed only in a few accessions. A multiplex reverse transcription (RT)-PCR method was developed for the simultaneous detection and identification of OYDV, SLV, GarCLV and Allexivirus infecting garlic accessions in India. This multiplex protocol standardized in this study will be useful in indexing of garlic viruses and production of virus free seed material. Copyright © 2014 Elsevier B.V. All rights reserved.

The P0 protein encoded by cotton leafroll dwarf virus (CLRDV) inhibits local but not systemic RNA silencing.

PubMed

Delfosse, Verónica C; Agrofoglio, Yamila C; Casse, María F; Kresic, Iván Bonacic; Hopp, H Esteban; Ziegler-Graff, Véronique; Distéfano, Ana J

2014-02-13

Plants employ RNA silencing as a natural defense mechanism against viruses. As a counter-defense, viruses encode silencing suppressor proteins (SSPs) that suppress RNA silencing. Most, but not all, the P0 proteins encoded by poleroviruses have been identified as SSP. In this study, we demonstrated that cotton leafroll dwarf virus (CLRDV, genus Polerovirus) P0 protein suppressed local silencing that was induced by sense or inverted repeat transgenes in Agrobacterium co-infiltration assay in Nicotiana benthamiana plants. A CLRDV full-length infectious cDNA clone that is able to infect N. benthamiana through Agrobacterium-mediated inoculation also inhibited local silencing in co-infiltration assays, suggesting that the P0 protein exhibits similar RNA silencing suppression activity when expressed from the full-length viral genome. On the other hand, the P0 protein did not efficiently inhibit the spread of systemic silencing signals. Moreover, Northern blotting indicated that the P0 protein inhibits the generation of secondary but not primary small interfering RNAs. The study of CLRDV P0 suppression activity may contribute to understanding the molecular mechanisms involved in the induction of cotton blue disease by CLRDV infection. Copyright © 2013 Elsevier B.V. All rights reserved.

Two suppressors of RNA silencing encoded by cereal-infecting members of the family Luteoviridae.

PubMed

Liu, Yan; Zhai, Hao; Zhao, Kun; Wu, Beilei; Wang, Xifeng

2012-08-01

Several members of the family Luteoviridae are important pathogens of cultivated plant species of the family Gramineae. In this study, we explored RNA-silencing suppressors (RSSs) encoded by two cereal-infecting luteoviruses: barley yellow dwarf virus and wheat yellow dwarf virus (BYDV and WYDV, respectively). The P0 protein of WYDV-GPV (P0(GPV)) and the P6 protein of BYDV-GAV (P6(GAV)) displayed RSS activities when expressed in agro-infiltrated leaves of Nicotiana benthamiana, by their local ability to inhibit post-transcriptional gene silencing of GFP. Analysis of GFP, mRNA and GFP-specific small interfering RNA indicated that both P0(GPV) and P6(GAV) are suppressors of silencing that can restrain not only local but also systemic gene silencing. This is the first report of RSS activity of the P6 protein in a member of the genus Luteovirus.

A Panoptic Uncovering of the Dynamical Evolution of the Zika Virus NS5 Methyltransferase Binding Site Loops- Zeroing in on the Molecular Landscape.

PubMed

Devnarain, Nikita; Soliman, Mahmoud E S

2018-06-20

The global threat of the Zika virus to humanity is real. Innovative and potent anti-Zika virus drugs are still at large, due to the lack of anti-Zika virus drugs that have passed phase 1 trials. Experimental research has revealed novel inhibitors of Zika virus NS5 methyltransferase enzyme. This study has taken a step further to provide insight into the molecular dynamics of Zika virus and inhibitor binding, which have not been established experimentally. Movements of the methyltransferase binding site loops have a large role to play in the methylation of the viral mRNA cap, which is essential for Zika virus replication. Here we pinpoint the binding interactions between each potential inhibitor and the methyltransferase, residues that are responsible for binding, as well as which inhibitor-bound complex renders the methyltransferase more stable. We also highlight the conformational changes that occur within the methyltransferase to accommodate binding of inhibitors and consequences of those changes upon the RNA- and cap-binding sites in the methyltransferase. This research will improve the understanding of the Zika virus NS5 methyltransferase enzyme, and will be beneficial in driving the development of anti-Zika virus drugs. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Monoclonal antibody-based serological assays and immunocapture-RT-PCR for detecting Rice dwarf virus in field rice plants and leafhopper vectors.

PubMed

Wu, Jianxiang; Ni, Yuequn; Liu, Huan; Ding, Ming; Zhou, Xueping

2014-01-01

Rice dwarf virus (RDV) causes Rice dwarf disease, which leads to considerable losses in rice production in Asia. Purified RDV virions were used as the immunogen to prepare monoclonal antibodies (mAbs). Three murine mAbs against RDV were prepared. Plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA), dot enzyme-linked immunosorbent assay (dot-ELISA) and immunocapture-RT-PCR (IC-RT-PCR) were then developed for sensitive, specific, and rapid detection of RDV in rice and leafhopper samples obtained in the field using the mAbs. The PTA-ELISA, dot-ELISA and IC-RT-PCR detected the virus in infected tissue crude extracts diluted at 1:81,920, 1:10,240 and 1:655,360 (w/v, g mL(-1)), in individual viruliferous rice green leafhopper crude extracts diluted at 1:25,600, 1:6400 and 1:3,276,800 (individual leafhopper/μL), respectively. 878 rice field samples and 531 leafhopper field samples from ten provinces of China were screened for the presence of RDV using the two serological assays and the IC-RT-PCR and the results indicated that 37 of the 878 rice samples and 22 of the 531 leafhopper samples were infected by RDV. All positive samples were from Yunnan Province, indicating that RDV is prevalent in this province, but not in the other nine provinces. The dot-ELISA is suitable for routine detection of large-scale rice and leafhopper samples in field surveys. Copyright © 2013 Elsevier B.V. All rights reserved.

Changes in the hemagglutinin of H5N1 viruses during human infection – Influence on receptor binding☆

PubMed Central

Crusat, Martin; Liu, Junfeng; Palma, Angelina S.; Childs, Robert A.; Liu, Yan; Wharton, Stephen A.; Lin, Yi Pu; Coombs, Peter J.; Martin, Stephen R.; Matrosovich, Mikhail; Chen, Zi; Stevens, David J.; Hien, Vo Minh; Thanh, Tran Tan; Nhu, Le Nguyen Truc; Nguyet, Lam Anh; Ha, Do Quang; van Doorn, H.Rogier; Hien, Tran Tinh; Conradt, Harald S.; Kiso, Makoto; Gamblin, Steve J.; Chai, Wengang; Skehel, John J.; Hay, Alan J.; Farrar, Jeremy; de Jong, Menno D.; Feizi, Ten

2013-01-01

As avian influenza A(H5N1) viruses continue to circulate in Asia and Africa, global concerns of an imminent pandemic persist. Recent experimental studies suggest that efficient transmission between humans of current H5N1 viruses only requires a few genetic changes. An essential step is alteration of the virus hemagglutinin from preferential binding to avian receptors for the recognition of human receptors present in the upper airway. We have identified receptor-binding changes which emerged during H5N1 infection of humans, due to single amino acid substitutions, Ala134Val and Ile151Phe, in the hemagglutinin. Detailed biological, receptor-binding, and structural analyses revealed reduced binding of the mutated viruses to avian-like receptors, but without commensurate increased binding to the human-like receptors investigated, possibly reflecting a receptor-binding phenotype intermediate in adaptation to more human-like characteristics. These observations emphasize that evolution in nature of avian H5N1 viruses to efficient binding of human receptors is a complex multistep process. PMID:24050651

Characterization of Receptor Binding Profiles of Influenza A Viruses Using An Ellipsometry-Based Label-Free Glycan Microarray Assay Platform

PubMed Central

Fei, Yiyan; Sun, Yung-Shin; Li, Yanhong; Yu, Hai; Lau, Kam; Landry, James P.; Luo, Zeng; Baumgarth, Nicole; Chen, Xi; Zhu, Xiangdong

2015-01-01

A key step leading to influenza viral infection is the highly specific binding of a viral spike protein, hemagglutinin (HA), with an extracellular glycan receptor of a host cell. Detailed and timely characterization of virus-receptor binding profiles may be used to evaluate and track the pandemic potential of an influenza virus strain. We demonstrate a label-free glycan microarray assay platform for acquiring influenza virus binding profiles against a wide variety of glycan receptors. By immobilizing biotinylated receptors on a streptavidin-functionalized solid surface, we measured binding curves of five influenza A virus strains with 24 glycans of diverse structures and used the apparent equilibrium dissociation constants (avidity constants, 10–100 pM) as characterizing parameters of viral receptor profiles. Furthermore by measuring binding kinetic constants of solution-phase glycans to immobilized viruses, we confirmed that the glycan-HA affinity constant is in the range of 10 mM and the reaction is enthalpy-driven. PMID:26193329

Characterization of Receptor Binding Profiles of Influenza A Viruses Using An Ellipsometry-Based Label-Free Glycan Microarray Assay Platform.

PubMed

Fei, Yiyan; Sun, Yung-Shin; Li, Yanhong; Yu, Hai; Lau, Kam; Landry, James P; Luo, Zeng; Baumgarth, Nicole; Chen, Xi; Zhu, Xiangdong

2015-07-16

A key step leading to influenza viral infection is the highly specific binding of a viral spike protein, hemagglutinin (HA), with an extracellular glycan receptor of a host cell. Detailed and timely characterization of virus-receptor binding profiles may be used to evaluate and track the pandemic potential of an influenza virus strain. We demonstrate a label-free glycan microarray assay platform for acquiring influenza virus binding profiles against a wide variety of glycan receptors. By immobilizing biotinylated receptors on a streptavidin-functionalized solid surface, we measured binding curves of five influenza A virus strains with 24 glycans of diverse structures and used the apparent equilibrium dissociation constants (avidity constants, 10-100 pM) as characterizing parameters of viral receptor profiles. Furthermore by measuring binding kinetic constants of solution-phase glycans to immobilized viruses, we confirmed that the glycan-HA affinity constant is in the range of 10 mM and the reaction is enthalpy-driven.

Destabilization of strigolactone receptor DWARF14 by binding of ligand and E3-ligase signaling effector DWARF3

PubMed Central

Zhao, Li-Hua; Zhou, X Edward; Yi, Wei; Wu, Zhongshan; Liu, Yue; Kang, Yanyong; Hou, Li; de Waal, Parker W; Li, Suling; Jiang, Yi; Scaffidi, Adrian; Flematti, Gavin R; Smith, Steven M; Lam, Vinh Q; Griffin, Patrick R; Wang, Yonghong; Li, Jiayang; Melcher, Karsten; Xu, H Eric

2015-01-01

Strigolactones (SLs) are endogenous hormones and exuded signaling molecules in plant responses to low levels of mineral nutrients. Key mediators of the SL signaling pathway in rice include the α/β-fold hydrolase DWARF 14 (D14) and the F-box component DWARF 3 (D3) of the ubiquitin ligase SCFD3 that mediate ligand-dependent degradation of downstream signaling repressors. One perplexing feature is that D14 not only functions as the SL receptor but is also an active enzyme that slowly hydrolyzes diverse natural and synthetic SLs including GR24, preventing the crystallization of a binary complex of D14 with an intact SL as well as the ternary D14/SL/D3 complex. Here we overcome these barriers to derive a structural model of D14 bound to intact GR24 and identify the interface that is required for GR24-mediated D14-D3 interaction. The mode of GR24-mediated signaling, including ligand recognition, hydrolysis by D14, and ligand-mediated D14-D3 interaction, is conserved in structurally diverse SLs. More importantly, D14 is destabilized upon the binding of ligands and D3, thus revealing an unusual mechanism of SL recognition and signaling, in which the hormone, the receptor, and the downstream effectors are systematically destabilized during the signal transduction process. PMID:26470846

Mechanism of human antibody-mediated neutralization of Marburg virus.

PubMed

Flyak, Andrew I; Ilinykh, Philipp A; Murin, Charles D; Garron, Tania; Shen, Xiaoli; Fusco, Marnie L; Hashiguchi, Takao; Bornholdt, Zachary A; Slaughter, James C; Sapparapu, Gopal; Klages, Curtis; Ksiazek, Thomas G; Ward, Andrew B; Saphire, Erica Ollmann; Bukreyev, Alexander; Crowe, James E

2015-02-26

The mechanisms by which neutralizing antibodies inhibit Marburg virus (MARV) are not known. We isolated a panel of neutralizing antibodies from a human MARV survivor that bind to MARV glycoprotein (GP) and compete for binding to a single major antigenic site. Remarkably, several of the antibodies also bind to Ebola virus (EBOV) GP. Single-particle EM structures of antibody-GP complexes reveal that all of the neutralizing antibodies bind to MARV GP at or near the predicted region of the receptor-binding site. The presence of the glycan cap or mucin-like domain blocks binding of neutralizing antibodies to EBOV GP, but not to MARV GP. The data suggest that MARV-neutralizing antibodies inhibit virus by binding to infectious virions at the exposed MARV receptor-binding site, revealing a mechanism of filovirus inhibition. Copyright © 2015 Elsevier Inc. All rights reserved.

Interactive Effects of Southern Rice Black-Streaked Dwarf Virus Infection of Host Plant and Vector on Performance of the Vector, Sogatella furcifera (Homoptera: Delphacidae).

PubMed

Lei, Wenbin; Liu, Danfeng; Li, Pei; Hou, Maolin

2014-10-01

Performance of insect vectors can be influenced by the viruses they transmit, either directly by infection of the vectors or indirectly via infection of the host plants. Southern rice black-streaked dwarf virus (SRBSDV) is a propagative virus transmitted by the white-backed planthopper, Sogatella furcifera (Hovath). To elucidate the influence of SRBSDV on the performance of white-backed planthopper, life parameters of viruliferous and nonviruliferous white-backed planthopper fed rice seedlings infected or noninfected with SRBSDV were measured using a factorial design. Regardless of the infection status of the rice plant host, viruliferous white-backed planthopper nymphs took longer to develop from nymph to adult than did nonviruliferous nymphs. Viruliferous white-backed planthopper females deposited fewer eggs than nonviruliferous females and both viruliferous and nonviruliferous white-backed planthopper females laid fewer eggs on infected than on noninfected plants. Longevity of white-backed planthopper females was also affected by the infection status of the rice plant and white-backed planthopper. Nonviruliferous white-backed planthopper females that fed on infected rice plants lived longer than the other three treatment groups. These results indicate that the performance of white-backed planthopper is affected by SRBSDV either directly (by infection of white-backed planthopper) or indirectly (by infection of rice plant). The extended development of viruliferous nymphs and the prolonged life span of nonviruliferous adults on infected plants may increase their likelihood of transmitting virus, which would increase virus spread. © 2014 Entomological Society of America.

Profile of small interfering RNAs from cotton plants infected with the polerovirus Cotton leafroll dwarf virus.

PubMed

Silva, Tatiane F; Romanel, Elisson A C; Andrade, Roberto R S; Farinelli, Laurent; Østerås, Magne; Deluen, Cécile; Corrêa, Régis L; Schrago, Carlos E G; Vaslin, Maite F S

2011-08-24

In response to infection, viral genomes are processed by Dicer-like (DCL) ribonuclease proteins into viral small RNAs (vsRNAs) of discrete sizes. vsRNAs are then used as guides for silencing the viral genome. The profile of vsRNAs produced during the infection process has been extensively studied for some groups of viruses. However, nothing is known about the vsRNAs produced during infections of members of the economically important family Luteoviridae, a group of phloem-restricted viruses. Here, we report the characterization of a population of vsRNAs from cotton plants infected with Cotton leafroll dwarf virus (CLRDV), a member of the genus Polerovirus, family Luteoviridae. Deep sequencing of small RNAs (sRNAs) from leaves of CLRDV-infected cotton plants revealed that the vsRNAs were 21- to 24-nucleotides (nt) long and that their sequences matched the viral genome, with higher frequencies of matches in the 3- region. There were equivalent amounts of sense and antisense vsRNAs, and the 22-nt class of small RNAs was predominant. During infection, cotton Dcl transcripts appeared to be up-regulated, while Dcl2 appeared to be down-regulated. This is the first report on the profile of sRNAs in a plant infected with a virus from the family Luteoviridae. Our sequence data strongly suggest that virus-derived double-stranded RNA functions as one of the main precursors of vsRNAs. Judging by the profiled size classes, all cotton DCLs might be working to silence the virus. The possible causes for the unexpectedly high accumulation of 22-nt vsRNAs are discussed. CLRDV is the causal agent of Cotton blue disease, which occurs worldwide. Our results are an important contribution for understanding the molecular mechanisms involved in this and related diseases.

First Discovery of Acetone Extract from Cottonseed Oil Sludge as a Novel Antiviral Agent against Plant Viruses

PubMed Central

Zhao, Lei; Feng, Chaohong; Hou, Caiting; Hu, Lingyun; Wang, Qiaochun; Wu, Yunfeng

2015-01-01

A novel acetone extract from cottonseed oil sludge was firstly discovered against plant viruses including Tobacco mosaic virus (TMV), Rice stripe virus (RSV) and Southern rice black streaked dwarf virus (SRBSDV). Gossypol and β-sitosterol separated from the acetone extract were tested for their effects on anti-TMV and analysed by nuclear magnetic resonance (NMR) assay. In vivo and field trials in different geographic distributions and different host varieties declared that this extract mixture was more efficient than the commercial agent Ningnanmycin with a broad spectrum of anti-plant-viruses activity. No phytotoxic activity was observed in the treated plants and environmental toxicology showed that this new acetone extract was environmentally friendly, indicating that this acetone extract has potential application in the control of plant virus in the future. PMID:25705894

First discovery of acetone extract from cottonseed oil sludge as a novel antiviral agent against plant viruses.

PubMed

Zhao, Lei; Feng, Chaohong; Hou, Caiting; Hu, Lingyun; Wang, Qiaochun; Wu, Yunfeng

2015-01-01

A novel acetone extract from cottonseed oil sludge was firstly discovered against plant viruses including Tobacco mosaic virus (TMV), Rice stripe virus (RSV) and Southern rice black streaked dwarf virus (SRBSDV). Gossypol and β-sitosterol separated from the acetone extract were tested for their effects on anti-TMV and analysed by nuclear magnetic resonance (NMR) assay. In vivo and field trials in different geographic distributions and different host varieties declared that this extract mixture was more efficient than the commercial agent Ningnanmycin with a broad spectrum of anti-plant-viruses activity. No phytotoxic activity was observed in the treated plants and environmental toxicology showed that this new acetone extract was environmentally friendly, indicating that this acetone extract has potential application in the control of plant virus in the future.

Hairpin RNA Targeting Multiple Viral Genes Confers Strong Resistance to Rice Black-Streaked Dwarf Virus.

PubMed

Wang, Fangquan; Li, Wenqi; Zhu, Jinyan; Fan, Fangjun; Wang, Jun; Zhong, Weigong; Wang, Ming-Bo; Liu, Qing; Zhu, Qian-Hao; Zhou, Tong; Lan, Ying; Zhou, Yijun; Yang, Jie

2016-05-11

Rice black-streaked dwarf virus (RBSDV) belongs to the genus Fijivirus in the family of Reoviridae and causes severe yield loss in rice-producing areas in Asia. RNA silencing, as a natural defence mechanism against plant viruses, has been successfully exploited for engineering virus resistance in plants, including rice. In this study, we generated transgenic rice lines harbouring a hairpin RNA (hpRNA) construct targeting four RBSDV genes, S1, S2, S6 and S10, encoding the RNA-dependent RNA polymerase, the putative core protein, the RNA silencing suppressor and the outer capsid protein, respectively. Both field nursery and artificial inoculation assays of three generations of the transgenic lines showed that they had strong resistance to RBSDV infection. The RBSDV resistance in the segregating transgenic populations correlated perfectly with the presence of the hpRNA transgene. Furthermore, the hpRNA transgene was expressed in the highly resistant transgenic lines, giving rise to abundant levels of 21-24 nt small interfering RNA (siRNA). By small RNA deep sequencing, the RBSDV-resistant transgenic lines detected siRNAs from all four viral gene sequences in the hpRNA transgene, indicating that the whole chimeric fusion sequence can be efficiently processed by Dicer into siRNAs. Taken together, our results suggest that long hpRNA targeting multiple viral genes can be used to generate stable and durable virus resistance in rice, as well as other plant species.

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

Analysis of RNA binding by the dengue virus NS5 RNA capping enzyme.

PubMed

Henderson, Brittney R; Saeedi, Bejan J; Campagnola, Grace; Geiss, Brian J

2011-01-01

Flaviviruses are small, capped positive sense RNA viruses that replicate in the cytoplasm of infected cells. Dengue virus and other related flaviviruses have evolved RNA capping enzymes to form the viral RNA cap structure that protects the viral genome and directs efficient viral polyprotein translation. The N-terminal domain of NS5 possesses the methyltransferase and guanylyltransferase activities necessary for forming mature RNA cap structures. The mechanism for flavivirus guanylyltransferase activity is currently unknown, and how the capping enzyme binds its diphosphorylated RNA substrate is important for deciphering how the flavivirus guanylyltransferase functions. In this report we examine how flavivirus NS5 N-terminal capping enzymes bind to the 5' end of the viral RNA using a fluorescence polarization-based RNA binding assay. We observed that the K(D) for RNA binding is approximately 200 nM Dengue, Yellow Fever, and West Nile virus capping enzymes. Removal of one or both of the 5' phosphates reduces binding affinity, indicating that the terminal phosphates contribute significantly to binding. RNA binding affinity is negatively affected by the presence of GTP or ATP and positively affected by S-adensyl methoninine (SAM). Structural superpositioning of the dengue virus capping enzyme with the Vaccinia virus VP39 protein bound to RNA suggests how the flavivirus capping enzyme may bind RNA, and mutagenesis analysis of residues in the putative RNA binding site demonstrate that several basic residues are critical for RNA binding. Several mutants show differential binding to 5' di-, mono-, and un-phosphorylated RNAs. The mode of RNA binding appears similar to that found with other methyltransferase enzymes, and a discussion of diphosphorylated RNA binding is presented.

Genome activation by raspberry bushy dwarf virus coat protein.

PubMed

Macfarlane, Stuart A; McGavin, Wendy J

2009-03-01

Two sets of infectious cDNA clones of raspberry bushy dwarf virus (RBDV) have been constructed, enabling either the synthesis of infectious RNA transcripts or the delivery of infectious binary plasmid DNA by infiltration of Agrobacterium tumefaciens. In whole plants and in protoplasts, inoculation of RBDV RNA1 and RNA2 transcripts led to a low level of infection, which was greatly increased by the addition of RNA3, a subgenomic RNA coding for the RBDV coat protein (CP). Agroinfiltration of RNA1 and RNA2 constructs did not produce a detectable infection but, again, inclusion of a construct encoding the CP led to high levels of infection. Thus, RBDV replication is greatly stimulated by the presence of the CP, a mechanism that also operates with ilarviruses and alfalfa mosaic virus, where it is referred to as genome activation. Mutation to remove amino acids from the N terminus of the CP showed that the first 15 RBDV CP residues are not required for genome activation. Other experiments, in which overlapping regions at the CP N terminus were fused to the monomeric red fluorescent protein, showed that sequences downstream of the first 48 aa are not absolutely required for genome activation.

Genetics of resistance to the geminivirus, Bean dwarf mosaic virus, and the role of the hypersensitive response in common bean.

PubMed

Seo, Y-S; Gepts, P; Gilbertson, R L

2004-03-01

Bean dwarf mosaic virus (BDMV) is a single-stranded DNA virus (genus: Begomovirus, family: Geminiviridae) that infects common bean ( Phaseolus vulgaris L.) and causes stunted plant growth, and mosaic and mottle symptoms in leaves. BDMV shows differential pathogenicity in common bean, infecting germplasm of the Andean gene pool (e.g., the snap bean cultivar Topcrop), but not that of the Middle American gene pool (e.g., the pinto bean cultivar Othello). Resistance to BDMV in Othello is associated with development of a hypersensitive response (HR) in vascular (phloem) tissues. In this study, Middle American germplasm representing the four recognized races (i.e., Durango, Guatemala, Jalisco, and Mesoamerica) and the parents of Othello were inoculated with BDMV and a BDMV-green fluorescent protein (GFP) reporter. All genotypes showed partial or complete resistance to BDMV and BDMV-GFP, indicating the widespread distribution of resistance in the Middle American gene pool. A number of BDMV-resistant germplasm did not show the HR, indicating it is not correlated with resistance. In the F(1), F(2), and F(3) of reciprocal crosses between Othello and Topcrop, a single dominant allele, Bdm, conferred BDMV resistance.

Response of chickpea genotypes to Agrobacterium-mediated delivery of Chickpea chlorotic dwarf virus (CpCDV) genome and identification of resistance source.

PubMed

Kanakala, S; Verma, H N; Vijay, P; Saxena, D R; Malathi, V G

2013-11-01

Chickpea stunt disease caused by Chickpea chlorotic dwarf virus (CpCDV) (genus Mastrevirus, family Geminiviridae) is the most important biotic stress affecting chickpea crops worldwide. A survey conducted on the incidence of stunt disease clearly revealed high incidence of the disease with severe symptom expression in both indigenous and imported genotypes. To manage the disease in a sustainable way, resistant genotypes need to be bred by adopting objective and precise assessment of the disease response of chickpea genotypes. At present, evaluation of CpCDV resistance is conducted on the basis of natural infection in the field, which is bound to be erroneous due to vagaries in vector population. To circumvent the above problems, we devised an agroinoculation technique that involves the delivery of viral genomic DNA through Agrobacterium tumefaciens. An objective scoring system assigning quantitative value to different symptoms has been evolved to assess the response of chickpea genotypes to CpCDV inoculation. Using the inoculation and scoring techniques, we screened 70 genotypes, which helped in differentiating field resistance that is more due to resistance to vector feeding than resistance to the virus.

Slow-binding inhibition of sialidase from influenza virus.

PubMed

Pegg, M S; von Itzstein, M

1994-04-01

Sialidase from influenza virus A (Tokyo/3/67, N2) is inhibited in slow-binding fashion by 2,3-didehydro-2,4-dideoxy-4-guanidino-N-acetyl-D-neuraminic acid. The Ki observed for the tightly-bound form at steady-state is 3 x 10(-11) M. Slow-binding, which is a consequence of the guanidinyl moiety of the inhibitor, is observed only for influenza virus A sialidase and not for influenza virus B or any other viral, bacterial, or mammalian sialidase investigated. The different results obtained for sialidases from influenza virus A and B, whose active sites are conserved, point to the involvement of the expulsion of a structural water molecule in the slow-binding mechanism.

Mapping of the Lassa virus LAMP1 binding site reveals unique determinants not shared by other old world arenaviruses.

PubMed

Israeli, Hadar; Cohen-Dvashi, Hadas; Shulman, Anastasiya; Shimon, Amir; Diskin, Ron

2017-04-01

Cell entry of many enveloped viruses occurs by engagement with cellular receptors, followed by internalization into endocytic compartments and pH-induced membrane fusion. A previously unnoticed step of receptor switching was found to be critical during cell entry of two devastating human pathogens: Ebola and Lassa viruses. Our recent studies revealed the functional role of receptor switching to LAMP1 for triggering membrane fusion by Lassa virus and showed the involvement of conserved histidines in this switching, suggesting that other viruses from this family may also switch to LAMP1. However, when we investigated viruses that are genetically close to Lassa virus, we discovered that they cannot bind LAMP1. A crystal structure of the receptor-binding module from Morogoro virus revealed structural differences that allowed mapping of the LAMP1 binding site to a unique set of Lassa residues not shared by other viruses in its family, illustrating a key difference in the cell-entry mechanism of Lassa virus that may contribute to its pathogenicity.

Complete genome sequence of a new enamovirus from Argentina infecting alfalfa plants showing dwarfism symptoms.

PubMed

Bejerman, Nicolás; Giolitti, Fabián; Trucco, Verónica; de Breuil, Soledad; Dietzgen, Ralf G; Lenardon, Sergio

2016-07-01

Alfalfa dwarf disease, probably caused by synergistic interactions of mixed virus infections, is a major and emergent disease that threatens alfalfa production in Argentina. Deep sequencing of diseased alfalfa plant samples from the central region of Argentina resulted in the identification of a new virus genome resembling enamoviruses in sequence and genome structure. Phylogenetic analysis suggests that it is a new member of the genus Enamovirus, family Luteoviridae. The virus is tentatively named "alfalfa enamovirus 1" (AEV-1). The availability of the AEV-1 genome sequence will make it possible to assess the genetic variability of this virus and to construct an infectious clone to investigate its role in alfalfa dwarfism disease.

The brown dwarf kinematics project

NASA Astrophysics Data System (ADS)

Faherty, Jackie K.

2010-10-01

Brown dwarfs are a recent addition to the plethora of objects studied in Astronomy. With theoretical masses between 13 and 75 MJupiter , they lack sustained stable Hydrogen burning so they never join the stellar main sequence. They have physical properties similar to both planets and low-mass stars so studies of their population inform on both. The distances and kinematics of brown dwarfs provide key statistical constraints on their ages, moving group membership, absolute brightnesses, evolutionary trends, and multiplicity. Yet, until my thesis, fundamental measurements of parallax and proper motion were made for only a relatively small fraction of the known population. To address this deficiency, I initiated the Brown Dwarf Kinematics (BDKP). Over the past four years I have re-imaged the majority of spectroscopically confirmed field brown dwarfs (or ultracool dwarfs---UCDs) and created the largest proper motion catalog for ultracool dwarfs to date. Using new astrometric information I examined population characteristics such as ages calculated from velocity dispersions and correlations between kinematics and colors. Using proper motions, I identified several new wide co-moving companions and investigated binding energy (and hence formation) limitations as well as the frequency of hierarchical companions. Concurrently over the past four years I have been conducting a parallax survey of 84 UCDs including those showing spectral signatures of youth, metal-poor brown dwarfs, and those within 20 pc of the Sun. Using absolute magnitude relations in J,H, and K, I identified overluminous binary candidates and investigated known flux-reversal binaries. Using current evolutionary models, I compared the MK vs J-K color magnitude diagram to model predictions and found that the low-surface gravity dwarfs are significantly red-ward and underluminous of predictions and a handful of late-type T dwarfs may require thicker clouds to account for their scatter.

Measles virus fusion machinery activated by sialic acid binding globular domain.

PubMed

Talekar, Aparna; Moscona, Anne; Porotto, Matteo

2013-12-01

Paramyxoviruses, including the human pathogen measles virus (MV) and the avian Newcastle disease virus (NDV), enter host cells through fusion of the viral envelope with the target cell membrane. This fusion is driven by the concerted action of two viral envelope glycoproteins: the receptor binding protein and the fusion protein (F). The MV receptor binding protein (hemagglutinin [H]) attaches to proteinaceous receptors on host cells, while the receptor binding protein of NDV (hemagglutinin-neuraminidase [HN]) interacts with sialic acid-containing receptors. The receptor-bound HN/H triggers F to undergo conformational changes that render it competent to mediate fusion of the viral and cellular membranes. The mechanism of fusion activation has been proposed to be different for sialic acid-binding viruses and proteinaceous receptor-binding viruses. We report that a chimeric protein containing the NDV HN receptor binding region and the MV H stalk domain can activate MV F to fuse, suggesting that the signal to the stalk of a protein-binding receptor binding molecule can be transmitted from a sialic acid binding domain. By engineering the NDV HN globular domain to interact with a proteinaceous receptor, the fusion activation signal was preserved. Our findings are consistent with a unified mechanism of fusion activation, at least for the Paramyxovirinae subfamily, in which the receptor binding domains of the receptor binding proteins are interchangeable and the stalk determines the specificity of F activation.

Virions at the gates: receptors and the host-virus arms race.

PubMed

Coffin, John M

2013-01-01

All viruses need to bind to specific receptor molecules on the surface of target cells to initiate infection. Virus-receptor binding is highly specific, and this specificity determines both the species and the cell type that can be infected by a given virus. In some well-studied cases, the virus-binding region on the receptor has been found to be unrelated to the receptor's normal cellular function. Resistance to virus infection can thus evolve by selection of mutations that alter amino acids in the binding region with minimal effect on normal function. This sort of positive selection can be used to infer the history of the host-virus "arms race" during their coevolution. In a new study, Demogines et al. use a combination of phylogenetic, structural, and virological analysis to infer the history and significance of positive selection on the transferrin receptor TfR1, a housekeeping protein required for iron uptake and the cell surface receptor for at least three different types of virus. The authors show that only two parts of the rodent TfR1 molecule have been subject to positive selection and that these correspond to the binding sites for two of these viruses-the mouse mammary tumor virus (a retrovirus) and Machupo virus (an arenavirus). They confirmed this result by introducing the inferred binding site mutations into the wild-type protein and testing for receptor function. Related arenaviruses are beginning to spread in human populations in South America as the cause of often fatal hemorrhagic fevers, and, although Demogines et al. could find no evidence of TfR1 mutations in this region that might have been selected as a consequence of human infection, the authors identified one such mutation in Asian populations that affects infection with these viruses.

Dwarfism and Altered Craniofacial Development in Rabbits Is Caused by a 12.1 kb Deletion at the HMGA2 Locus.

PubMed

Carneiro, Miguel; Hu, Dou; Archer, John; Feng, Chungang; Afonso, Sandra; Chen, Congying; Blanco-Aguiar, José A; Garreau, Hervé; Boucher, Samuel; Ferreira, Paula G; Ferrand, Nuno; Rubin, Carl-Johan; Andersson, Leif

2017-02-01

The dwarf phenotype characterizes the smallest of rabbit breeds and is governed largely by the effects of a single dwarfing allele with an incompletely dominant effect on growth. Dwarf rabbits typically weigh under 1 kg and have altered craniofacial morphology. The dwarf allele is recessive lethal and dwarf homozygotes die within a few days of birth. The dwarf phenotype is expressed in heterozygous individuals and rabbits from dwarf breeds homozygous for the wild-type allele are normal, although smaller when compared to other breeds. Here, we show that the dwarf allele constitutes a ∼12.1 kb deletion overlapping the promoter region and first three exons of the HMGA2 gene leading to inactivation of this gene. HMGA2 has been frequently associated with variation in body size across species. Homozygotes for null alleles are viable in mice but not in rabbits and probably not in humans. RNA-sequencing analysis of rabbit embryos showed that very few genes (4-29 genes) were differentially expressed among the three HMGA2/dwarf genotypes, suggesting that dwarfism and inviability in rabbits are caused by modest changes in gene expression. Our results show that HMGA2 is critical for normal expression of IGF2BP2, which encodes an RNA-binding protein. Finally, we report a catalog of regions of elevated genetic differentiation between dwarf and normal-size rabbits, including LCORL-NCAPG, STC2, HOXD cluster, and IGF2BP2 Levels and patterns of genetic diversity at the LCORL-NCAPG locus further suggest that small size in dwarf breeds was enhanced by crosses with wild rabbits. Overall, our results imply that small size in dwarf rabbits results from a large effect, loss-of-function (LOF) mutation in HMGA2 combined with polygenic selection. Copyright © 2017 by the Genetics Society of America.

Dwarfism and Altered Craniofacial Development in Rabbits Is Caused by a 12.1 kb Deletion at the HMGA2 Locus

PubMed Central

Hu, Dou; Archer, John; Feng, Chungang; Afonso, Sandra; Chen, Congying; Blanco-Aguiar, José A.; Garreau, Hervé; Boucher, Samuel; Ferreira, Paula G.; Ferrand, Nuno; Rubin, Carl-Johan

2017-01-01

The dwarf phenotype characterizes the smallest of rabbit breeds and is governed largely by the effects of a single dwarfing allele with an incompletely dominant effect on growth. Dwarf rabbits typically weigh under 1 kg and have altered craniofacial morphology. The dwarf allele is recessive lethal and dwarf homozygotes die within a few days of birth. The dwarf phenotype is expressed in heterozygous individuals and rabbits from dwarf breeds homozygous for the wild-type allele are normal, although smaller when compared to other breeds. Here, we show that the dwarf allele constitutes a ∼12.1 kb deletion overlapping the promoter region and first three exons of the HMGA2 gene leading to inactivation of this gene. HMGA2 has been frequently associated with variation in body size across species. Homozygotes for null alleles are viable in mice but not in rabbits and probably not in humans. RNA-sequencing analysis of rabbit embryos showed that very few genes (4–29 genes) were differentially expressed among the three HMGA2/dwarf genotypes, suggesting that dwarfism and inviability in rabbits are caused by modest changes in gene expression. Our results show that HMGA2 is critical for normal expression of IGF2BP2, which encodes an RNA-binding protein. Finally, we report a catalog of regions of elevated genetic differentiation between dwarf and normal-size rabbits, including LCORL-NCAPG, STC2, HOXD cluster, and IGF2BP2. Levels and patterns of genetic diversity at the LCORL-NCAPG locus further suggest that small size in dwarf breeds was enhanced by crosses with wild rabbits. Overall, our results imply that small size in dwarf rabbits results from a large effect, loss-of-function (LOF) mutation in HMGA2 combined with polygenic selection. PMID:27986804

Simultaneous detection of six stone fruit viruses by non-isotopic molecular hybridization using a unique riboprobe or 'polyprobe'.

PubMed

Herranz, M Carmen; Sanchez-Navarro, Jesus A; Aparicio, Frederic; Pallás, Vicente

2005-03-01

A new strategy for the simultaneous detection of plant viruses by molecular hybridization has been developed. Two, four or six viral sequences were fused in tandem and transcribed to render unique riboprobes and designated as 'polyprobes'. The 'polyprobe four' (poly 4) covered the four ilarviruses affecting stone fruit trees including apple mosaic virus (ApMV), prunus necrotic ringspot virus (PNRSV), prune dwarf virus (PDV), and American plum line pattern virus (APLPV) whereas the 'polyprobe two' (poly 2) was designed to detect simultaneously, plum pox virus (PPV) and apple chlorotic leaf spot virus (ACLSV), the two more important viruses affecting these trees. Finally, a 'polyprobe six' (poly 6) was generated to detect any of the six viruses. The three polyprobes were comparable to the individual riboprobes in terms of end-point dilution limit and specificity. The validation of the new simultaneous detection strategy was confirmed by the analysis of 46 field samples from up to seven different hosts collected from 10 different geographical areas.

Structural basis of efficient contagion: measles variations on a theme by parainfluenza viruses.

PubMed

Mateo, Mathieu; Navaratnarajah, Chanakha K; Cattaneo, Roberto

2014-04-01

A quartet of attachment proteins and a trio of fusion protein subunits play the cell entry concert of parainfluenza viruses. While many of these viruses bind sialic acid to enter cells, wild type measles binds exclusively two tissue-specific proteins, the lymphatic receptor signaling lymphocytic activation molecule (SLAM), and the epithelial receptor nectin-4. SLAM binds near the stalk-head junction of the hemagglutinin. Nectin-4 binds a hydrophobic groove located between blades 4 and 5 of the hemagglutinin β-propeller head. The mutated vaccine strain hemagglutinin binds in addition the ubiquitous protein CD46, which explains attenuation. The measles virus entry concert has four movements. Andante misterioso: the virus takes over the immune system. Allegro con brio: it rapidly spreads in the upper airway's epithelia. 'Targeting' fugue: the versatile orchestra takes off. Presto furioso: the virus exits the host with thunder. Be careful: music is contagious. Copyright © 2014 Elsevier B.V. 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

Nanomechanical mapping of first binding steps of a virus to animal cells

NASA Astrophysics Data System (ADS)

Alsteens, David; Newton, Richard; Schubert, Rajib; Martinez-Martin, David; Delguste, Martin; Roska, Botond; Müller, Daniel J.

2017-02-01

Viral infection is initiated when a virus binds to cell surface receptors. Because the cell membrane is dynamic and heterogeneous, imaging living cells and simultaneously quantifying the first viral binding events is difficult. Here, we show an atomic force and confocal microscopy set-up that allows the surface receptor landscape of cells to be imaged and the virus binding events within the first millisecond of contact with the cell to be mapped at high resolution (<50 nm). We present theoretical approaches to contour the free-energy landscape of early binding events between an engineered virus and cell surface receptors. We find that the first bond formed between the viral glycoprotein and its cognate cell surface receptor has relatively low lifetime and free energy, but this increases as additional bonds form rapidly (≤1 ms). The formation of additional bonds occurs with positive allosteric modulation and the three binding sites of the viral glycoprotein are quickly occupied. Our quantitative approach can be readily applied to study the binding of other viruses to animal cells.

Transcriptional changes of rice in response to rice black-streaked dwarf virus.

PubMed

Ahmed, Mohamed M S; Ji, Wen; Wang, Muyue; Bian, Shiquan; Xu, Meng; Wang, Weiyun; Zhang, Jiangxiang; Xu, Zhihao; Yu, Meimei; Liu, Qiaoquan; Zhang, Changquan; Zhang, Honggen; Tang, Shuzhu; Gu, Minghong; Yu, Hengxiu

2017-09-10

Rice black-streaked dwarf virus (RBSDV), a member of the genus Fijivirus in the family Reoviridae, causes significant economic losses in rice production in China and many other Asian countries. Although a great deal of effort has been made to elucidate the interactions among the virus, insect vectors, host and environmental conditions, few RBSDV proteins involved in pathogenesis have been identified, and the biological basis of disease development in rice remains largely unknown. Transcriptomic information associated with the disease development in rice would be helpful to unravel the biological mechanism. To determine how the rice transcriptome changes in response to RBSDV infection, we carried out RNA-Seq to perform a genome-wide gene expression analysis of a susceptible rice cultivar KTWYJ3. The transcriptomes of RBSDV-infected samples were compared to those of RBSDV-free (healthy) at two time points (time points are represented by group I and II). The results derived from the differential expression analysis in RBSDV-infected libraries vs. healthy ones in group I revealed that 102 out of a total of 281 significant differentially expressed genes (DEGs) were up-regulated and 179 DEGs were down-regulated. Of the 2592 identified DEGs in group II, 1588 DEGs were up-regulated and 1004 DEGs were down-regulated. A total of 66 DEGs were commonly identified in both groups. Of these 66 DEGs, expression patterns for 36 DEGs were similar in both groups. Our analysis demonstrated that some genes related to disease defense and stress resistance were up-regulated while genes associated with chloroplast were down-regulated in response to RBSDV infection. In addition, some genes associated with plant-height were differentially expressed. This result indicates those genes might be involved in dwarf symptoms caused by RBSDV. Taken together, our results provide a genome-wide transcriptome analysis for rice plants in response to RBSDV infection which may contribute to the understanding of the regulatory mechanisms involved in rice-RBSDV interaction and the biological basis of rice black-streaked dwarf disease development in rice. Copyright © 2017 Elsevier B.V. All rights reserved.

Variability in H9N2 haemagglutinin receptor-binding preference and the pH of fusion.

PubMed

Peacock, Thomas P; Benton, Donald J; Sadeyen, Jean-Remy; Chang, Pengxiang; Sealy, Joshua E; Bryant, Juliet E; Martin, Stephen R; Shelton, Holly; McCauley, John W; Barclay, Wendy S; Iqbal, Munir

2017-03-22

H9N2 avian influenza viruses are primarily a disease of poultry; however, they occasionally infect humans and are considered a potential pandemic threat. Little work has been performed to assess the intrinsic biochemical properties related to zoonotic potential of H9N2 viruses. The objective of this study, therefore, was to investigate H9N2 haemagglutinins (HAs) using two well-known correlates for human adaption: receptor-binding avidity and pH of fusion. Receptor binding was characterized using bio-layer interferometry to measure virus binding to human and avian-like receptor analogues and the pH of fusion was assayed by syncytium formation in virus-infected cells at different pHs. We characterized contemporary H9N2 viruses of the zoonotic G1 lineage, as well as representative viruses of the zoonotic BJ94 lineage. We found that most contemporary H9N2 viruses show a preference for sulphated avian-like receptor analogues. However, the 'Eastern' G1 H9N2 viruses displayed a consistent preference in binding to a human-like receptor analogue. We demonstrate that the presence of leucine at position 226 of the HA receptor-binding site correlated poorly with the ability to bind a human-like sialic acid receptor. H9N2 HAs also display variability in their pH of fusion, ranging between pH 5.4 and 5.85 which is similar to that of the first wave of human H1N1pdm09 viruses but lower than the pH of fusion seen in zoonotic H5N1 and H7N9 viruses. Our results suggest possible molecular mechanisms that may underlie the relatively high prevalence of human zoonotic infection by particular H9N2 virus lineages.

Variability in H9N2 haemagglutinin receptor-binding preference and the pH of fusion

PubMed Central

Peacock, Thomas P; Benton, Donald J; Sadeyen, Jean-Remy; Chang, Pengxiang; Sealy, Joshua E; Bryant, Juliet E; Martin, Stephen R; Shelton, Holly; McCauley, John W; Barclay, Wendy S; Iqbal, Munir

2017-01-01

H9N2 avian influenza viruses are primarily a disease of poultry; however, they occasionally infect humans and are considered a potential pandemic threat. Little work has been performed to assess the intrinsic biochemical properties related to zoonotic potential of H9N2 viruses. The objective of this study, therefore, was to investigate H9N2 haemagglutinins (HAs) using two well-known correlates for human adaption: receptor-binding avidity and pH of fusion. Receptor binding was characterized using bio-layer interferometry to measure virus binding to human and avian-like receptor analogues and the pH of fusion was assayed by syncytium formation in virus-infected cells at different pHs. We characterized contemporary H9N2 viruses of the zoonotic G1 lineage, as well as representative viruses of the zoonotic BJ94 lineage. We found that most contemporary H9N2 viruses show a preference for sulphated avian-like receptor analogues. However, the ‘Eastern' G1 H9N2 viruses displayed a consistent preference in binding to a human-like receptor analogue. We demonstrate that the presence of leucine at position 226 of the HA receptor-binding site correlated poorly with the ability to bind a human-like sialic acid receptor. H9N2 HAs also display variability in their pH of fusion, ranging between pH 5.4 and 5.85 which is similar to that of the first wave of human H1N1pdm09 viruses but lower than the pH of fusion seen in zoonotic H5N1 and H7N9 viruses. Our results suggest possible molecular mechanisms that may underlie the relatively high prevalence of human zoonotic infection by particular H9N2 virus lineages. PMID:28325922

Antigenic Properties of the HIV Envelope on Virions in Solution

PubMed Central

Mengistu, Meron; Lewis, George K.; Lakowicz, Joseph R.

2014-01-01

The structural flexibility found in human immunodeficiency virus (HIV) envelope glycoproteins creates a complex relationship between antigenicity and sensitivity to antiviral antibodies. The study of this issue in the context of viral particles is particularly problematic as conventional virus capture approaches can perturb antigenicity profiles. Here, we employed a unique analytical system based on fluorescence correlation spectroscopy (FCS), which measures antibody-virion binding with all reactants continuously in solution. Panels of nine anti-envelope monoclonal antibodies (MAbs) and five virus types were used to connect antibody binding profiles with neutralizing activities. Anti-gp120 MAbs against the 2G12 or b12 epitope, which marks functional envelope structures, neutralized viruses expressing CCR5-tropic envelopes and exhibited efficient virion binding in solution. MAbs against CD4-induced (CD4i) epitopes considered hidden on functional envelope structures poorly bound these viruses and were not neutralizing. Anti-gp41 MAb 2F5 was neutralizing despite limited virion binding. Similar antigenicity patterns occurred on CXCR4-tropic viruses, except that anti-CD4i MAbs 17b and 19e were neutralizing despite little or no virion binding. Notably, anti-gp120 MAb PG9 and anti-gp41 MAb F240 bound to both CCR5-tropic and CXCR4-tropic viruses without exerting neutralizing activity. Differences in the virus production system altered the binding efficiencies of some antibodies but did not enhance antigenicity of aberrant gp120 structures. Of all viruses tested, only JRFL pseudoviruses showed a direct relationship between MAb binding efficiency and neutralizing potency. Collectively, these data indicate that the antigenic profiles of free HIV particles generally favor the exposure of functional over aberrant gp120 structures. However, the efficiency of virion-antibody interactions in solution inconsistently predicts neutralizing activity in vitro. PMID:24284318

Label-Free Quantitative Proteomic Analysis of Chitosan Oligosaccharide-Treated Rice Infected with Southern Rice Black-Streaked Dwarf Virus.

PubMed

Yang, Anming; Yu, Lu; Chen, Zhuo; Zhang, Shanxue; Shi, Jing; Zhao, Xiaozhen; Yang, Yuanyou; Hu, Deyu; Song, Baoan

2017-05-18

Southern rice black-streaked dwarf virus (SRBSDV) has spread from thesouth of China to the north of Vietnam in the past few years and severelyinfluenced rice production. Its long incubation period and early symptoms are not evident; thus, controlling it is difficult. Chitosan oligosaccharide (COS) is a green plant immunomodulator. Early studies showed that preventing and controlling SRBSDV have a certain effect and reduce disease infection rate, but its underlying controlling and preventing mechanism is unclear. In this study, label-free proteomics was used to analyze differentially expressed proteins in rice after COS treatment. The results showed that COS can up-regulate the plant defense-related proteins and down-regulate the protein expression levels of SRBSDV. Meanwhile, quantitative real-time PCR test results showed that COS can improve defense gene expression in rice. Moreover, COS can enhance the defense enzymatic activities of peroxidase, superoxide dismutase and catalase through mitogen-activated protein kinase signaling cascade pathway, and enhance the rice disease resistance.

Bean Yellow Dwarf Virus replicons for high-level transgene expression in transgenic plants and cell cultures.

PubMed

Zhang, Xiuren; Mason, Hugh

2006-02-05

A novel stable transgenic plant expression system was developed using elements of the replication machinery of Bean Yellow Dwarf Virus (BeYDV). The system contains two transgenes: 1) The BeYDV replicon vector with an expression cassette flanked by cis-acting DNA elements of BeYDV, and 2) The viral replication initiator protein (Rep) controlled by an alcohol-inducible promoter. When Rep expression was triggered by treatment with ethanol, it induced release of the BeYDV replicon from stably integrated T-DNA and episomal replication to high copy number. Replicon amplification resulted in substantially increased transgene mRNA levels (up to 80-fold) and translation products (up to 10-fold) after induction of Rep expression by ethanol treatment in tobacco NT1 cells and leaves of whole potato plants. Thus, the BeYDV stable transformant replicon system is a powerful tool for plant-based production of recombinant proteins. (c) 2005 Wiley Periodicals, Inc.

HMGB1 Protein Binds to Influenza Virus Nucleoprotein and Promotes Viral Replication

PubMed Central

Moisy, Dorothée; Avilov, Sergiy V.; Jacob, Yves; Laoide, Brid M.; Ge, Xingyi; Baudin, Florence; Jestin, Jean-Luc

2012-01-01

Influenza virus has evolved replication strategies that hijack host cell pathways. To uncover interactions between viral macromolecules and host proteins, we applied a phage display strategy. A library of human cDNA expression products displayed on filamentous phages was submitted to affinity selection for influenza viral ribonucleoproteins (vRNPs). High-mobility-group box (HMGB) proteins were found to bind to the nucleoprotein (NP) component of vRNPs. HMGB1 and HMGB2 bind directly to the purified NP in the absence of viral RNA, and the HMG box A domain is sufficient to bind the NP. We show that HMGB1 associates with the viral NP in the nuclei of infected cells, promotes viral growth, and enhances the activity of the viral polymerase. The presence of a functional HMGB1 DNA-binding site is required to enhance influenza virus replication. Glycyrrhizin, which reduces HMGB1 binding to DNA, inhibits influenza virus polymerase activity. Our data show that the HMGB1 protein can play a significant role in intranuclear replication of influenza viruses, thus extending previous findings on the bornavirus and on a number of DNA viruses. PMID:22696656

Functional assignment to JEV proteins using SVM.

PubMed

Sahoo, Ganesh Chandra; Dikhit, Manas Ranjan; Das, Pradeep

2008-01-01

Identification of different protein functions facilitates a mechanistic understanding of Japanese encephalitis virus (JEV) infection and opens novel means for drug development. Support vector machines (SVM), useful for predicting the functional class of distantly related proteins, is employed to ascribe a possible functional class to Japanese encephalitis virus protein. Our study from SVMProt and available JE virus sequences suggests that structural and nonstructural proteins of JEV genome possibly belong to diverse protein functions, are expected to occur in the life cycle of JE virus. Protein functions common to both structural and non-structural proteins are iron-binding, metal-binding, lipid-binding, copper-binding, transmembrane, outer membrane, channels/Pores - Pore-forming toxins (proteins and peptides) group of proteins. Non-structural proteins perform functions like actin binding, zinc-binding, calcium-binding, hydrolases, Carbon-Oxygen Lyases, P-type ATPase, proteins belonging to major facilitator family (MFS), secreting main terminal branch (MTB) family, phosphotransfer-driven group translocators and ATP-binding cassette (ABC) family group of proteins. Whereas structural proteins besides belonging to same structural group of proteins (capsid, structural, envelope), they also perform functions like nuclear receptor, antibiotic resistance, RNA-binding, DNA-binding, magnesium-binding, isomerase (intra-molecular), oxidoreductase and participate in type II (general) secretory pathway (IISP).

Functional assignment to JEV proteins using SVM

PubMed Central

Sahoo, Ganesh Chandra; Dikhit, Manas Ranjan; Das, Pradeep

2008-01-01

Identification of different protein functions facilitates a mechanistic understanding of Japanese encephalitis virus (JEV) infection and opens novel means for drug development. Support vector machines (SVM), useful for predicting the functional class of distantly related proteins, is employed to ascribe a possible functional class to Japanese encephalitis virus protein. Our study from SVMProt and available JE virus sequences suggests that structural and nonstructural proteins of JEV genome possibly belong to diverse protein functions, are expected to occur in the life cycle of JE virus. Protein functions common to both structural and non-structural proteins are iron-binding, metal-binding, lipid-binding, copper-binding, transmembrane, outer membrane, channels/Pores - Pore-forming toxins (proteins and peptides) group of proteins. Non-structural proteins perform functions like actin binding, zinc-binding, calcium-binding, hydrolases, Carbon-Oxygen Lyases, P-type ATPase, proteins belonging to major facilitator family (MFS), secreting main terminal branch (MTB) family, phosphotransfer-driven group translocators and ATP-binding cassette (ABC) family group of proteins. Whereas structural proteins besides belonging to same structural group of proteins (capsid, structural, envelope), they also perform functions like nuclear receptor, antibiotic resistance, RNA-binding, DNA-binding, magnesium-binding, isomerase (intra-molecular), oxidoreductase and participate in type II (general) secretory pathway (IISP). PMID:19052658

Barley yellow dwarf virus Infection Leads to Higher Chemical Defense Signals and Lower Electrophysiological Reactions in Susceptible Compared to Tolerant Barley Genotypes.

PubMed

Paulmann, Maria K; Kunert, Grit; Zimmermann, Matthias R; Theis, Nina; Ludwig, Anatoli; Meichsner, Doreen; Oelmüller, Ralf; Gershenzon, Jonathan; Habekuss, Antje; Ordon, Frank; Furch, Alexandra C U; Will, Torsten

2018-01-01

Barley yellow dwarf virus (BYDV) is a phloem limited virus that is persistently transmitted by aphids. Due to huge yield losses in agriculture, the virus is of high economic relevance. Since the control of the virus itself is not possible, tolerant barley genotypes are considered as the most effective approach to avoid yield losses. Although several genes and quantitative trait loci are known and used in barley breeding for virus tolerance, little is known about molecular and physiological backgrounds of this trait. Therefore, we compared the anatomy and early defense responses of a virus susceptible to those of a virus-tolerant cultivar. One of the very early defense responses is the transmission of electrophysiological reactions. Electrophysiological reactions to BYDV infection might differ between susceptible and tolerant cultivars, since BYDV causes disintegration of sieve elements in susceptible cultivars. The structure of vascular bundles, xylem vessels and sieve elements was examined using microscopy. All three were significantly decreased in size in infected susceptible plants where the virus causes disintegration of sieve elements. This could be associated with an uncontrolled ion exchange between the sieve-element lumen and apoplast. Further, a reduced electrophysiological isolation would negatively affect the propagation of electrophysiological reactions. To test the influence of BYDV infection on electrophysiological reactions, electropotential waves (EPWs) induced by leaf-tip burning were recorded using aphids as bioelectrodes. EPWs in infected susceptible plants disappeared already after 10 cm in contrast to those in healthy susceptible or infected tolerant or healthy tolerant plants. Another early plant defense reaction is an increase in reactive oxygen species (ROS). Using a fluorescent dye, we found a significant increase in ROS content in infected susceptible plants but not in infected tolerant plants. Similar results were found for the phytohormones abscisic acid and three jasmonates. Salicylic acid levels were generally higher after BYDV infection compared to uninfected plants. Heat stimulation caused an increase in jasmonates. By shedding light on the plant defense mechanisms against BYDV, this study, provides further knowledge for breeding virus tolerant plants.

Barley yellow dwarf virus Infection Leads to Higher Chemical Defense Signals and Lower Electrophysiological Reactions in Susceptible Compared to Tolerant Barley Genotypes

PubMed Central

Paulmann, Maria K.; Kunert, Grit; Zimmermann, Matthias R.; Theis, Nina; Ludwig, Anatoli; Meichsner, Doreen; Oelmüller, Ralf; Gershenzon, Jonathan; Habekuss, Antje; Ordon, Frank; Furch, Alexandra C. U.; Will, Torsten

2018-01-01

Barley yellow dwarf virus (BYDV) is a phloem limited virus that is persistently transmitted by aphids. Due to huge yield losses in agriculture, the virus is of high economic relevance. Since the control of the virus itself is not possible, tolerant barley genotypes are considered as the most effective approach to avoid yield losses. Although several genes and quantitative trait loci are known and used in barley breeding for virus tolerance, little is known about molecular and physiological backgrounds of this trait. Therefore, we compared the anatomy and early defense responses of a virus susceptible to those of a virus-tolerant cultivar. One of the very early defense responses is the transmission of electrophysiological reactions. Electrophysiological reactions to BYDV infection might differ between susceptible and tolerant cultivars, since BYDV causes disintegration of sieve elements in susceptible cultivars. The structure of vascular bundles, xylem vessels and sieve elements was examined using microscopy. All three were significantly decreased in size in infected susceptible plants where the virus causes disintegration of sieve elements. This could be associated with an uncontrolled ion exchange between the sieve-element lumen and apoplast. Further, a reduced electrophysiological isolation would negatively affect the propagation of electrophysiological reactions. To test the influence of BYDV infection on electrophysiological reactions, electropotential waves (EPWs) induced by leaf-tip burning were recorded using aphids as bioelectrodes. EPWs in infected susceptible plants disappeared already after 10 cm in contrast to those in healthy susceptible or infected tolerant or healthy tolerant plants. Another early plant defense reaction is an increase in reactive oxygen species (ROS). Using a fluorescent dye, we found a significant increase in ROS content in infected susceptible plants but not in infected tolerant plants. Similar results were found for the phytohormones abscisic acid and three jasmonates. Salicylic acid levels were generally higher after BYDV infection compared to uninfected plants. Heat stimulation caused an increase in jasmonates. By shedding light on the plant defense mechanisms against BYDV, this study, provides further knowledge for breeding virus tolerant plants. PMID:29563918

Targeted entry of enveloped viruses: measles and herpes simplex virus I.

PubMed

Navaratnarajah, Chanakha K; Miest, Tanner S; Carfi, Andrea; Cattaneo, Roberto

2012-02-01

We compare the receptor-based mechanisms that a small RNA virus and a larger DNA virus have evolved to drive the fusion of viral and cellular membranes. Both systems rely on tight control over triggering the concerted refolding of a trimeric fusion protein. While measles virus entry depends on a receptor-binding protein and a fusion protein only, the herpes simplex virus (HSV) is more complex and requires four viral proteins. Nevertheless, in both viruses a receptor-binding protein is required for triggering the membrane fusion process. Moreover, specificity domains can be appended to these receptor-binding proteins to target virus entry to cells expressing a designated receptor. We discuss how principles established with measles and HSV can be applied to targeting other enveloped viruses, and alternatively how retargeted envelopes can be fitted on foreign capsids. Copyright © 2011 Elsevier B.V. All rights reserved.

A new virus in Luteoviridae is associated with raspberry leaf curl disease

USDA-ARS?s Scientific Manuscript database

To determine the etiology of Raspberry Leaf Curl Disease (RLCD), which causes leaf curling, leaf distortion, leaf chlorosis, shoot dwarfing, shoot proliferation in raspberries and can kill plants within three years, a next generation sequences approach was applied. Two red raspberry plants collected...

Characterizing Functional Domains for TIM-Mediated Enveloped Virus Entry

PubMed Central

Moller-Tank, Sven; Albritton, Lorraine M.; Rennert, Paul D.

2014-01-01

ABSTRACT T-cell immunoglobulin and mucin domain 1 (TIM-1) and other TIM family members were recently identified as phosphatidylserine (PtdSer)-mediated virus entry-enhancing receptors (PVEERs). These proteins enhance entry of Ebola virus (EBOV) and other viruses by binding PtdSer on the viral envelope, concentrating virus on the cell surface, and promoting subsequent internalization. The PtdSer-binding activity of the immunoglobulin-like variable (IgV) domain is essential for both virus binding and internalization by TIM-1. However, TIM-3, whose IgV domain also binds PtdSer, does not effectively enhance virus entry, indicating that other domains of TIM proteins are functionally important. Here, we investigate the domains supporting enhancement of enveloped virus entry, thereby defining the features necessary for a functional PVEER. Using a variety of chimeras and deletion mutants, we found that in addition to a functional PtdSer-binding domain PVEERs require a stalk domain of sufficient length, containing sequences that promote an extended structure. Neither the cytoplasmic nor the transmembrane domain of TIM-1 is essential for enhancing virus entry, provided the protein is still plasma membrane bound. Based on these defined characteristics, we generated a mimic lacking TIM sequences and composed of annexin V, the mucin-like domain of α-dystroglycan, and a glycophosphatidylinositol anchor that functioned as a PVEER to enhance transduction of virions displaying Ebola, Chikungunya, Ross River, or Sindbis virus glycoproteins. This identification of the key features necessary for PtdSer-mediated enhancement of virus entry provides a basis for more effective recognition of unknown PVEERs. IMPORTANCE T-cell immunoglobulin and mucin domain 1 (TIM-1) and other TIM family members are recently identified phosphatidylserine (PtdSer)-mediated virus entry-enhancing receptors (PVEERs). These proteins enhance virus entry by binding the phospholipid, PtdSer, present on the viral membrane. While it is known that the PtdSer binding is essential for the PVEER function of TIM-1, TIM-3 shares this binding activity but does not enhance virus entry. No comprehensive studies have been done to characterize the other domains of TIM-1. In this study, using a variety of chimeric proteins and deletion mutants, we define the features necessary for a functional PVEER. With these features in mind, we generated a TIM-1 mimic using functionally similar domains from other proteins. This mimic, like TIM-1, effectively enhanced transduction. These studies provide insight into the key features necessary for PVEERs and will allow for more effective identification of unknown PVEERs. PMID:24696470

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

Influence of rice black streaked dwarf virus on the ecological fitness of non-vector planthopper Nilaparvata lugens (Hemiptera: Delphacidae).

PubMed

Xu, Hong-Xing; He, Xiao-Chan; Zheng, Xu-Song; Yang, Ya-Jun; Lu, Zhong-Xian

2014-08-01

Rice black streak dwarf virus (RBSDV) is transmitted by the small brown planthopper (SBPH), Laodelphax striatellus (Fallen). Non-vector rice brown planthopper (BPH), Nilaparvata lugens (Stål), shares the same host rice plants with SBPH in paddy fields. The changes in nutritional composition of rice plants infected by RBSDV and the ecological fitness of BPH feeding on the infected plants were studied under both artificial climate chamber and field conditions. Contents of 16 detected amino acids and soluble sugar in RBSDV infected rice plants were higher than those in the healthy ones. On the diseased plants BPH had significantly higher nymphal survival rates, nymphal duration of the males, weight of the female adults, as well as egg hatchability compared to BPH being fed on healthy plants. However, there was no obvious difference in female nymph duration, longevity and fecundity. Defense enzymes (superoxidase dismutase, SOD and catalase, CAT) and detoxifying enzymes (carboxylesterase, CAE and glutathione S-transferase, GST) in BPH adults fed on diseased plants had markedly higher activities. The results indicate rice plants infected by RBSDV improved the ecological fitness of the brown planthopper, a serious pest but not a transmitter of the RBSDV virus. © 2013 Institute of Zoology, Chinese Academy of Sciences.

Function and diversity of P0 proteins among cotton leafroll dwarf virus isolates.

PubMed

Cascardo, Renan S; Arantes, Ighor L G; Silva, Tatiane F; Sachetto-Martins, Gilberto; Vaslin, Maité F S; Corrêa, Régis L

2015-08-12

The RNA silencing pathway is an important anti-viral defense mechanism in plants. As a counter defense, some members of the viral family Luteoviridae are able to evade host immunity by encoding the P0 RNA silencing suppressor protein. Here we explored the functional diversity of P0 proteins among eight cotton leafroll dwarf virus (CLRDV) isolates, a virus associated with a worldwide cotton disease known as cotton blue disease (CBD). CLRDV-infected cotton plants of different varieties were collected from five growing fields in Brazil and their P0 sequences compared to three previously obtained isolates. P0's silencing suppression activities were scored based on transient expression experiments in Nicotiana benthamiana leaves. High sequence diversity was observed among CLRDV P0 proteins, indicating that some isolates found in cotton varieties formerly resistant to CLRDV should be regarded as new genotypes within the species. All tested proteins were able to suppress local and systemic silencing, but with significantly variable degrees. All P0 proteins were able to mediate the decay of ARGONAUTE proteins, a key component of the RNA silencing machinery. The sequence diversity observed in CLRDV P0s is also reflected in their silencing suppression capabilities. However, the strength of local and systemic silencing suppression was not correlated for some proteins.

Mutation of mapped TIA-1/TIAR binding sites in the 3' terminal stem-loop of West Nile virus minus-strand RNA in an infectious clone negatively affects genomic RNA amplification.

PubMed

Emara, Mohamed M; Liu, Hsuan; Davis, William G; Brinton, Margo A

2008-11-01

Previous data showed that the cellular proteins TIA-1 and TIAR bound specifically to the West Nile virus 3' minus-strand stem-loop [WNV3'(-)SL] RNA (37) and colocalized with flavivirus replication complexes in WNV- and dengue virus-infected cells (21). In the present study, the sites on the WNV3'(-)SL RNA required for efficient in vitro T-cell intracellular antigen-related (TIAR) and T-cell intracellular antigen-1 (TIA-1) protein binding were mapped to short AU sequences (UAAUU) located in two internal loops of the WNV3'(-)SL RNA structure. Infectious clone RNAs with all or most of the binding site nucleotides in one of the 3' (-)SL loops deleted or substituted did not produce detectable virus after transfection or subsequent passage. With one exception, deletion/mutation of a single terminal nucleotide in one of the binding sequences had little effect on the efficiency of protein binding or virus production, but mutation of a nucleotide in the middle of a binding sequence reduced both the in vitro protein binding efficiency and virus production. Plaque size, intracellular genomic RNA levels, and virus production progressively decreased with decreasing in vitro TIAR/TIA-1 binding activity, but the translation efficiency of the various mutant RNAs was similar to that of the parental RNA. Several of the mutant RNAs that inefficiently interacted with TIAR/TIA-1 in vitro rapidly reverted in vivo, indicating that they could replicate at a low level and suggesting that an interaction between TIAR/TIA-1 and the viral 3'(-)SL RNA is not required for initial low-level symmetric RNA replication but instead facilitates the subsequent asymmetric amplification of genome RNA from the minus-strand template.

Virus Neutralisation: New Insights from Kinetic Neutralisation Curves

PubMed Central

Magnus, Carsten

2013-01-01

Antibodies binding to the surface of virions can lead to virus neutralisation. Different theories have been proposed to determine the number of antibodies that must bind to a virion for neutralisation. Early models are based on chemical binding kinetics. Applying these models lead to very low estimates of the number of antibodies needed for neutralisation. In contrast, according to the more conceptual approach of stoichiometries in virology a much higher number of antibodies is required for virus neutralisation by antibodies. Here, we combine chemical binding kinetics with (virological) stoichiometries to better explain virus neutralisation by antibody binding. This framework is in agreement with published data on the neutralisation of the human immunodeficiency virus. Knowing antibody reaction constants, our model allows us to estimate stoichiometrical parameters from kinetic neutralisation curves. In addition, we can identify important parameters that will make further analysis of kinetic neutralisation curves more valuable in the context of estimating stoichiometries. Our model gives a more subtle explanation of kinetic neutralisation curves in terms of single-hit and multi-hit kinetics. PMID:23468602

Bacterial lipopolysaccharide binding enhances virion stability and promotes environmental fitness of an enteric virus

PubMed Central

Robinson, Christopher M.; Jesudhasan, Palmy R.; Pfeiffer, Julie K.

2014-01-01

Summary Enteric viruses, including poliovirus and reovirus, encounter a vast microbial community in the mammalian gastrointestinal tract, which has been shown to promote virus replication and pathogenesis. Investigating the underlying mechanisms, we find that poliovirus binds bacterial surface polysaccharides, which enhances virion stability and cell attachment by increasing binding to the viral receptor. Additionally, we identified a poliovirus mutant, VP1-T99K, with reduced lipopolysaccharide (LPS) binding. Although T99K and WT poliovirus cell attachment, replication and pathogenesis in mice are equivalent, following peroral inoculation of mice, VP1-T99K poliovirus was unstable in feces. Consequently, the ratio of mutant virus in feces is reduced following additional cycles of infection in mice. Thus, the mutant virus incurs a fitness cost when environmental stability is a factor. These data suggest that poliovirus binds bacterial surface polysaccharides, enhancing cell attachment and environmental stability, potentially promoting transmission to a new host. PMID:24439896

Global alteration of microRNAs and transposon-derived small RNAs in cotton (Gossypium hirsutum) during Cotton leafroll dwarf polerovirus (CLRDV) infection.

PubMed

Romanel, Elisson; Silva, Tatiane F; Corrêa, Régis L; Farinelli, Laurent; Hawkins, Jennifer S; Schrago, Carlos E G; Vaslin, Maite F S

2012-11-01

Small RNAs (sRNAs) are a class of non-coding RNAs ranging from 20- to 40-nucleotides (nts) that are present in most eukaryotic organisms. In plants, sRNAs are involved in the regulation of development, the maintenance of genome stability and the antiviral response. Viruses, however, can interfere with and exploit the silencing-based regulatory networks, causing the deregulation of sRNAs, including small interfering RNAs (siRNAs) and microRNAs (miRNAs). To understand the impact of viral infection on the plant sRNA pathway, we deep sequenced the sRNAs in cotton leaves infected with Cotton leafroll dwarf virus (CLRDV), which is a member of the economically important virus family Luteoviridae. A total of 60 putative conserved cotton miRNAs were identified, including 19 new miRNA families that had not been previously described in cotton. Some of these miRNAs were clearly misregulated during viral infection, and their possible role in symptom development and disease progression is discussed. Furthermore, we found that the 24-nt heterochromatin-associated siRNAs were quantitatively and qualitatively altered in the infected plant, leading to the reactivation of at least one cotton transposable element. This is the first study to explore the global alterations of sRNAs in virus-infected cotton plants. Our results indicate that some CLRDV-induced symptoms may be correlated with the deregulation of miRNA and/or epigenetic networks.

Profile of small interfering RNAs from cotton plants infected with the polerovirus Cotton leafroll dwarf virus

PubMed Central

2011-01-01

Background In response to infection, viral genomes are processed by Dicer-like (DCL) ribonuclease proteins into viral small RNAs (vsRNAs) of discrete sizes. vsRNAs are then used as guides for silencing the viral genome. The profile of vsRNAs produced during the infection process has been extensively studied for some groups of viruses. However, nothing is known about the vsRNAs produced during infections of members of the economically important family Luteoviridae, a group of phloem-restricted viruses. Here, we report the characterization of a population of vsRNAs from cotton plants infected with Cotton leafroll dwarf virus (CLRDV), a member of the genus Polerovirus, family Luteoviridae. Results Deep sequencing of small RNAs (sRNAs) from leaves of CLRDV-infected cotton plants revealed that the vsRNAs were 21- to 24-nucleotides (nt) long and that their sequences matched the viral genome, with higher frequencies of matches in the 3- region. There were equivalent amounts of sense and antisense vsRNAs, and the 22-nt class of small RNAs was predominant. During infection, cotton Dcl transcripts appeared to be up-regulated, while Dcl2 appeared to be down-regulated. Conclusions This is the first report on the profile of sRNAs in a plant infected with a virus from the family Luteoviridae. Our sequence data strongly suggest that virus-derived double-stranded RNA functions as one of the main precursors of vsRNAs. Judging by the profiled size classes, all cotton DCLs might be working to silence the virus. The possible causes for the unexpectedly high accumulation of 22-nt vsRNAs are discussed. CLRDV is the causal agent of Cotton blue disease, which occurs worldwide. Our results are an important contribution for understanding the molecular mechanisms involved in this and related diseases. PMID:21864377

SECRET domain of variola virus CrmB protein can be a member of poxviral type II chemokine-binding proteins family

PubMed Central

2010-01-01

Background Variola virus (VARV) the causative agent of smallpox, eradicated in 1980, have wide spectrum of immunomodulatory proteins to evade host immunity. Recently additional biological activity was discovered for VARV CrmB protein, known to bind and inhibit tumour necrosis factor (TNF) through its N-terminal domain homologous to cellular TNF receptors. Besides binding TNF, this protein was also shown to bind with high affinity several chemokines which recruit B- and T-lymphocytes and dendritic cells to sites of viral entry and replication. Ability to bind chemokines was shown to be associated with unique C-terminal domain of CrmB protein. This domain named SECRET (Smallpox virus-Encoded Chemokine Receptor) is unrelated to the host proteins and lacks significant homology with other known viral chemokine-binding proteins or any other known protein. Findings De novo modelling of VARV-CrmB SECRET domain spatial structure revealed its apparent structural homology with cowpox virus CC-chemokine binding protein (vCCI) and vaccinia virus A41 protein, despite low sequence identity between these three proteins. Potential ligand-binding surface of modelled VARV-CrmB SECRET domain was also predicted to bear prominent electronegative charge which is characteristic to known orthopoxviral chemokine-binding proteins. Conclusions Our results suggest that SECRET should be included into the family of poxviral type II chemokine-binding proteins and that it might have been evolved from the vCCI-like predecessor protein. PMID:20979600

SECRET domain of variola virus CrmB protein can be a member of poxviral type II chemokine-binding proteins family.

PubMed

Antonets, Denis V; Nepomnyashchikh, Tatyana S; Shchelkunov, Sergei N

2010-10-27

Variola virus (VARV) the causative agent of smallpox, eradicated in 1980, have wide spectrum of immunomodulatory proteins to evade host immunity. Recently additional biological activity was discovered for VARV CrmB protein, known to bind and inhibit tumour necrosis factor (TNF) through its N-terminal domain homologous to cellular TNF receptors. Besides binding TNF, this protein was also shown to bind with high affinity several chemokines which recruit B- and T-lymphocytes and dendritic cells to sites of viral entry and replication. Ability to bind chemokines was shown to be associated with unique C-terminal domain of CrmB protein. This domain named SECRET (Smallpox virus-Encoded Chemokine Receptor) is unrelated to the host proteins and lacks significant homology with other known viral chemokine-binding proteins or any other known protein. De novo modelling of VARV-CrmB SECRET domain spatial structure revealed its apparent structural homology with cowpox virus CC-chemokine binding protein (vCCI) and vaccinia virus A41 protein, despite low sequence identity between these three proteins. Potential ligand-binding surface of modelled VARV-CrmB SECRET domain was also predicted to bear prominent electronegative charge which is characteristic to known orthopoxviral chemokine-binding proteins. Our results suggest that SECRET should be included into the family of poxviral type II chemokine-binding proteins and that it might have been evolved from the vCCI-like predecessor protein.

Nuclear proteins hijacked by mammalian cytoplasmic plus strand RNA viruses

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

Lloyd, Richard E., E-mail: rlloyd@bcm.edu

Plus strand RNA viruses that replicate in the cytoplasm face challenges in supporting the numerous biosynthetic functions required for replication and propagation. Most of these viruses are genetically simple and rely heavily on co-opting cellular proteins, particularly cellular RNA-binding proteins, into new roles for support of virus infection at the level of virus-specific translation, and building RNA replication complexes. In the course of infectious cycles many nuclear-cytoplasmic shuttling proteins of mostly nuclear distribution are detained in the cytoplasm by viruses and re-purposed for their own gain. Many mammalian viruses hijack a common group of the same factors. This review summarizesmore » recent gains in our knowledge of how cytoplasmic RNA viruses use these co-opted host nuclear factors in new functional roles supporting virus translation and virus RNA replication and common themes employed between different virus groups. - Highlights: • Nuclear shuttling host proteins are commonly hijacked by RNA viruses to support replication. • A limited group of ubiquitous RNA binding proteins are commonly hijacked by a broad range of viruses. • Key virus proteins alter roles of RNA binding proteins in different stages of virus replication.« less

A novel method for transmitting southern rice black-streaked dwarf virus to rice without insect vector.

PubMed

Yu, Lu; Shi, Jing; Cao, Lianlian; Zhang, Guoping; Wang, Wenli; Hu, Deyu; Song, Baoan

2017-08-15

Southern rice black-streaked dwarf virus (SRBSDV) has spread from the south of China to the north of Vietnam in the past few years, and has severely influenced rice production. However, previous study of traditional SRBSDV transmission method by the natural virus vector, the white-backed planthopper (WBPH, Sogatella furcifera), in the laboratory, researchers are frequently confronted with lack of enough viral samples due to the limited life span of infected vectors and rice plants and low virus acquisition and inoculation efficiency by the vector. Meanwhile, traditional mechanical inoculation of virus only apply to dicotyledon because of the higher content of lignin in the leaves of the monocot. Therefore, establishing an efficient and persistent-transmitting model, with a shorter virus transmission time and a higher virus transmission efficiency, for screening novel anti-SRBSDV drugs is an urgent need. In this study, we firstly reported a novel method for transmitting SRBSDV in rice using the bud-cutting method. The transmission efficiency of SRBSDV in rice was investigated via the polymerase chain reaction (PCR) method and the replication of SRBSDV in rice was also investigated via the proteomics analysis. Rice infected with SRBSDV using the bud-cutting method exhibited similar symptoms to those infected by the WBPH, and the transmission efficiency (>80.00%), which was determined using the PCR method, and the virus transmission time (30 min) were superior to those achieved that transmitted by the WBPH. Proteomics analysis confirmed that SRBSDV P1, P2, P3, P4, P5-1, P5-2, P6, P8, P9-1, P9-2, and P10 proteins were present in infected rice seedlings infected via the bud-cutting method. The results showed that SRBSDV could be successfully transmitted via the bud-cutting method and plants infected SRBSDV exhibited the symptoms were similar to those transmitted by the WBPH. Therefore, the use of the bud-cutting method to generate a cheap, efficient, reliable supply of SRBSDV-infected rice seedlings should aid the development of disease control strategies. Meanwhile, this method also could provide a new idea for the other virus transmission in monocot.

Role of the Phosphatidylserine Receptor TIM-1 in Enveloped-Virus Entry

PubMed Central

Moller-Tank, Sven; Kondratowicz, Andrew S.; Davey, Robert A.; Rennert, Paul D.

2013-01-01

The cell surface receptor T cell immunoglobulin mucin domain 1 (TIM-1) dramatically enhances filovirus infection of epithelial cells. Here, we showed that key phosphatidylserine (PtdSer) binding residues of the TIM-1 IgV domain are critical for Ebola virus (EBOV) entry through direct interaction with PtdSer on the viral envelope. PtdSer liposomes but not phosphatidylcholine liposomes competed with TIM-1 for EBOV pseudovirion binding and transduction. Further, annexin V (AnxV) substituted for the TIM-1 IgV domain, supporting a PtdSer-dependent mechanism. Our findings suggest that TIM-1-dependent uptake of EBOV occurs by apoptotic mimicry. Additionally, TIM-1 enhanced infection of a wide range of enveloped viruses, including alphaviruses and a baculovirus. As further evidence of the critical role of enveloped-virion-associated PtdSer in TIM-1-mediated uptake, TIM-1 enhanced internalization of pseudovirions and virus-like proteins (VLPs) lacking a glycoprotein, providing evidence that TIM-1 and PtdSer-binding receptors can mediate virus uptake independent of a glycoprotein. These results provide evidence for a broad role of TIM-1 as a PtdSer-binding receptor that mediates enveloped-virus uptake. Utilization of PtdSer-binding receptors may explain the wide tropism of many of these viruses and provide new avenues for controlling their virulence. PMID:23698310

Nanophotonic detection of freely interacting molecules on a single influenza virus

NASA Astrophysics Data System (ADS)

Kang, Pilgyu; Schein, Perry; Serey, Xavier; O'Dell, Dakota; Erickson, David

2015-07-01

Biomolecular interactions, such as antibody-antigen binding, are fundamental to many biological processes. At present, most techniques for analyzing these interactions require immobilizing one or both of the interacting molecules on an assay plate or a sensor surface. This is convenient experimentally but can constrain the natural binding affinity and capacity of the molecules, resulting in data that can deviate from the natural free-solution behavior. Here we demonstrate a label-free method for analyzing free-solution interactions between a single influenza virus and specific antibodies at the single particle level using near-field optical trapping and light-scattering techniques. We determine the number of specific antibodies binding to an optically trapped influenza virus by analyzing the change of the Brownian fluctuations of the virus. We develop an analytical model that determines the increased size of the virus resulting from antibodies binding to the virus membrane with uncertainty of ±1-2 nm. We present stoichiometric results of 26 ± 4 (6.8 ± 1.1 attogram) anti-influenza antibodies binding to an H1N1 influenza virus. Our technique can be applied to a wide range of molecular interactions because the nanophotonic tweezer can handle molecules from tens to thousands of nanometers in diameter.

Viral Epitopes and Monoclonal Antibodies: Isolation of Blocking Antibodies that Inhibit Virus Neutralization

NASA Astrophysics Data System (ADS)

Massey, Richard J.; Schochetman, Gerald

1981-07-01

The inability of pathogenic animal viruses to be completely neutralized by antibodies can lead to chronic viral infections in which infectious virus persists even in the presence of excess neutralizing antibody. A mechanism that results in this nonneutralized fraction of virus was defined by the topographical relationships of viral epitopes identified with monoclonal antibodies wherein monoclonal antibodies bind to virus and sterically block the binding of neutralizing antibodies.

The effect of interferon on the receptor sites to rabies virus on mouse neuroblastoma cells

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

Briggs, D.J.

1989-01-01

The binding of rabies virus to mouse neuroblastoma cells (MNA) primed with alpha interferon (IFN-{alpha}), beta interferon (IFN-{beta}), or alpha bungarotoxin (BTX) was examined. A saturable number of receptor sites to rabies virus was calculated by increasing the amount of {sup 3}H-CVS added to a constant number of untreated MNA cells. MNA cells were then exposed to 20 I.U. of IFN-{alpha}, IFN-{beta}, or 1 {mu}g of BTX and assayed to determine if these treatments had an effect on the number of receptor sites to rabies virus. Total amount of {sup 3}H-CVS bound to MNA cells was determined during a threemore » hour incubation period. Cold competition assays using 1,000 fold excess unlabeled CVS were used to determine non-specific binding for each treatment. Specific binding was then calculated by subtracting non-specific binding from the total amount of CVS bound to MNA cells. A similar amount of total viral protein bound to untreated and IFN-{beta}, and BTX treated cells after 180 minutes of incubation. The bound protein varied by only 0.07 {mu}g. However, the amount of specific and non-specific binding varied a great deal between treatments. BTX caused an increase in non-specific and a decrease in specific binding of rabies virus. IFN-{beta} produced variable results in non-specific and specific binding while IFN-{alpha} caused mainly specific binding to occur. The most significant change brought about by IFN-{alpha} was an increase in the rate of viral attachment. At 30 minutes post-infection, IFN-{alpha} treated cells had bound 90% of the total amount of virus bound to untreated cells after 180 minutes. The increased binding rate did not cause a productive infection of rabies virus. No viral production was evident after an incubation period of 48 hours in either IFN-{alpha} or IFN-{beta} treated cells.« less

Mutation of the C/EBP binding sites in the Rous sarcoma virus long terminal repeat and gag enhancers.

PubMed Central

Ryden, T A; de Mars, M; Beemon, K

1993-01-01

Several C/EBP binding sites within the Rous sarcoma virus (RSV) long terminal repeat (LTR) and gag enhancers were mutated, and the effect of these mutations on viral gene expression was assessed. Minimal site-specific mutations in each of three adjacent C/EBP binding sites in the LTR reduced steady-state viral RNA levels. Double mutation of the two 5' proximal LTR binding sites resulted in production of 30% of wild-type levels of virus. DNase I footprinting analysis of mutant DNAs indicated that the mutations blocked C/EBP binding at the affected sites. Additional C/EBP binding sites were identified upstream of the 3' LTR and within the 5' end of the LTRs. Point mutations in the RSV gag intragenic enhancer region, which blocked binding of C/EBP at two of three adjacent C/EBP sites, also reduced virus production significantly. Nuclear extracts prepared from both chicken embryo fibroblasts (CEFs) and chicken muscle contained proteins binding to the same RSV DNA sites as did C/EBP, and mutations that prevented C/EBP binding also blocked binding of these chicken proteins. It appears that CEFs and chicken muscle contain distinct proteins binding to these RSV DNA sites; the CEF binding protein was heat stable, as is C/EBP, while the chicken muscle protein was heat sensitive. Images PMID:8386280

Binding affinity of the L-742,001 inhibitor to the endonuclease domain of A/H1N1/PA influenza virus variants: Molecular simulation approaches

NASA Astrophysics Data System (ADS)

Nguyen, Hung; Nguyen, Hoang Linh; Linh, Huynh Quang; Nguyen, Minh Tho

2018-01-01

The steered molecular dynamics (SMD), molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) and free energy perturbation (FEP) methods were used to determine the binding affinity of the L-742,001 inhibitor to the endonuclease domain of the A/H1N1/PA influenza viruses (including wild type (WT) and three mutations I79L, E119D and F105S for both pH1N1 PA and PR8 PA viruses). Calculated results showed that the L-742,001 inhibitor not only binds to the PR8 PAs (1934 A influenza virus) better than to the pH1N1 PAs (2009 A influenza virus) but also more strongly interacts with the WT endonuclease domain than with three mutant variants for both pH1N1 PA and PR8 PA viruses. The binding affinities obtained by the SMD, MM-PBSA and FEP methods attain high correlation with available experimental data. Here the FEP method appears to provide a more accurate determination of the binding affinity than the SMD and MM-PBSA counterparts.

Heparin octasaccharide decoy liposomes inhibit replication of multiple viruses

PubMed Central

Hendricks, Gabriel L.; Velazquez, Lourdes; Pham, Serena; Qaisar, Natasha; Delaney, James C.; Viswanathan, Karthik; Albers, Leila; Comolli, James C.; Shriver, Zachary; Knipe, David M.; Kurt-Jones, Evelyn A.; Fygenson, Deborah K.; Trevejo, Jose M.

2016-01-01

Heparan sulfate (HS) is a ubiquitous glycosaminoglycan that serves as a cellular attachment site for a number of significant human pathogens, including respiratory syncytial virus (RSV), human parainfluenza virus 3 (hPIV3), and herpes simplex virus (HSV). Decoy receptors can target pathogens by binding to the receptor pocket on viral attachment proteins, acting as ‘molecular sinks’ and preventing the pathogen from binding to susceptible host cells. Decoy receptors functionalized with HS could bind to pathogens and prevent infection, so we generated decoy liposomes displaying HS-octasaccharide (HS-octa). These decoy liposomes significantly inhibited RSV, hPIV3, and HSV infectivity in vitro to a greater degree than the original HS-octa building block. The degree of inhibition correlated with the density of HS-octa displayed on the liposome surface. Decoy liposomes with HS-octa inhibited infection of viruses to a greater extent than either full-length heparin or HS-octa alone. Decoy liposomes were effective when added prior to infection or following the initial infection of cells in vitro. By targeting the well-conserved receptor-binding sites of HS-binding viruses, decoy liposomes functionalized with HS-octa are a promising therapeutic antiviral agent and illustrate the utility of the liposome delivery platform. PMID:25637710

A Single Mutation at Position 190 in Hemagglutinin Enhances Binding Affinity for Human Type Sialic Acid Receptor and Replication of H9N2 Avian Influenza Virus in Mice

PubMed Central

Teng, Qiaoyang; Xu, Dawei; Shen, Weixia; Liu, Qinfang; Rong, Guangyu; Li, Xuesong; Yan, Liping; Yang, Jianmei; Chen, Hongjun; Yu, Hai

2016-01-01

ABSTRACT H9N2 avian influenza virus (AIV) has an extended host range, but the molecular basis underlying H9N2 AIV transmission to mammals remains unclear. We isolated more than 900 H9N2 AIVs in our 3-year surveillance in live bird markets in China from 2009 to 2012. Thirty-seven representative isolates were selected for further detailed characterization. These isolates were categorized into 8 genotypes (B64 to B71) and formed a distinct antigenic subgroup. Three isolates belonging to genotype B69, which is a predominant genotype circulating in China, replicated efficiently in mice, while the viruses tested in parallel in other genotypes replicated poorly, although they, like the three B69 isolates, have a leucine at position 226 in the hemagglutinin (HA) receptor binding site, which is critical for binding human type sialic acid receptors. Further molecular and single mutation analysis revealed that a valine (V) residue at position 190 in HA is responsible for efficient replication of these H9N2 viruses in mice. The 190V in HA does not affect virus receptor binding specificity but enhances binding affinity to human cells and lung tissues from mouse and humans. All these data indicate that the 190V in HA is one of the important determinants for H9N2 AIVs to cross the species barrier to infect mammals despite multiple genes conferring adaptation and replication of H9N2 viruses in mammals. Our findings provide novel insights on understanding host range expansion of H9N2 AIVs. IMPORTANCE Influenza virus hemagglutinin (HA) is responsible for binding to host cell receptors and therefore influences the viral host range and pathogenicity in different species. We showed that the H9N2 avian influenza viruses harboring 190V in the HA exhibit enhanced virus replication in mice. Further studies demonstrate that 190V in the HA does not change virus receptor binding specificity but enhances virus binding affinity of the H9N2 virus to human cells and attachment to lung tissues from humans and mouse. Our findings suggest that more attention should be given to the H9N2 AIVs with HA-190V during surveillance due to their potential threat to mammals, including humans. PMID:27558420

A Single Mutation at Position 190 in Hemagglutinin Enhances Binding Affinity for Human Type Sialic Acid Receptor and Replication of H9N2 Avian Influenza Virus in Mice.

PubMed

Teng, Qiaoyang; Xu, Dawei; Shen, Weixia; Liu, Qinfang; Rong, Guangyu; Li, Xuesong; Yan, Liping; Yang, Jianmei; Chen, Hongjun; Yu, Hai; Ma, Wenjun; Li, Zejun

2016-11-01

H9N2 avian influenza virus (AIV) has an extended host range, but the molecular basis underlying H9N2 AIV transmission to mammals remains unclear. We isolated more than 900 H9N2 AIVs in our 3-year surveillance in live bird markets in China from 2009 to 2012. Thirty-seven representative isolates were selected for further detailed characterization. These isolates were categorized into 8 genotypes (B64 to B71) and formed a distinct antigenic subgroup. Three isolates belonging to genotype B69, which is a predominant genotype circulating in China, replicated efficiently in mice, while the viruses tested in parallel in other genotypes replicated poorly, although they, like the three B69 isolates, have a leucine at position 226 in the hemagglutinin (HA) receptor binding site, which is critical for binding human type sialic acid receptors. Further molecular and single mutation analysis revealed that a valine (V) residue at position 190 in HA is responsible for efficient replication of these H9N2 viruses in mice. The 190V in HA does not affect virus receptor binding specificity but enhances binding affinity to human cells and lung tissues from mouse and humans. All these data indicate that the 190V in HA is one of the important determinants for H9N2 AIVs to cross the species barrier to infect mammals despite multiple genes conferring adaptation and replication of H9N2 viruses in mammals. Our findings provide novel insights on understanding host range expansion of H9N2 AIVs. Influenza virus hemagglutinin (HA) is responsible for binding to host cell receptors and therefore influences the viral host range and pathogenicity in different species. We showed that the H9N2 avian influenza viruses harboring 190V in the HA exhibit enhanced virus replication in mice. Further studies demonstrate that 190V in the HA does not change virus receptor binding specificity but enhances virus binding affinity of the H9N2 virus to human cells and attachment to lung tissues from humans and mouse. Our findings suggest that more attention should be given to the H9N2 AIVs with HA-190V during surveillance due to their potential threat to mammals, including humans. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Structure of unliganded HSV gD reveals a mechanism for receptor-mediated activation of virus entry

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

Krummenacher, Claude; Supekar, Vinit M.; Whitbeck, J. Charles

2010-07-19

Herpes simplex virus (HSV) entry into cells requires binding of the envelope glycoprotein D (gD) to one of several cell surface receptors. The 50 C-terminal residues of the gD ectodomain are essential for virus entry, but not for receptor binding. We have determined the structure of an unliganded gD molecule that includes these C-terminal residues. The structure reveals that the C-terminus is anchored near the N-terminal region and masks receptor-binding sites. Locking the C-terminus in the position observed in the crystals by an intramolecular disulfide bond abolished receptor binding and virus entry, demonstrating that this region of gD moves uponmore » receptor binding. Similarly, a point mutant that would destabilize the C-terminus structure was nonfunctional for entry, despite increased affinity for receptors. We propose that a controlled displacement of the gD C-terminus upon receptor binding is an essential feature of HSV entry, ensuring the timely activation of membrane fusion.« less

Detection of receptor-specific murine leukemia virus binding to cells by immunofluorescence analysis.

PubMed Central

Kadan, M J; Sturm, S; Anderson, W F; Eglitis, M A

1992-01-01

Four classes of murine leukemia virus (MuLV) which display distinct cellular tropisms and bind to different retrovirus receptors to initiate virus infection have been described. In the present study, we describe a rapid, sensitive immunofluorescence assay useful for characterizing the initial binding of MuLV to cells. By using the rat monoclonal antibody 83A25 (L. H. Evans, R. P. Morrison, F. G. Malik, J. Portis, and W. J. Britt, J. Virol. 64:6176-6183, 1990), which recognizes an epitope of the envelope gp70 molecule common to the different classes of MuLV, it is possible to analyse the binding of ecotropic, amphotropic, or xenotropic MuLV by using only a single combination of primary and secondary antibodies. The MuLV binding detected by this assay is envelope receptor specific and matches the susceptibility to infection determined for cells from a variety of species. The binding of amphotropic MuLV to NIH 3T3 cells was shown to be rapid, saturable, and temperature dependent. Chinese hamster ovary (CHO-K1) cells normally lack the ability to bind ecotropic virus and are not infectible by ecotropic vectors. Expression of the cloned ecotropic retrovirus receptor gene (Rec) in CHO-K1 cells confers high levels of ecotropic virus-specific binding and confers susceptibility to infection. Characterization of MuLV binding to primary cells may provide insight into the infectibility of cells by retroviruses and aid in the selection of appropriate vectors for gene transfer experiments. PMID:1312632

Matrix proteins of Nipah and Hendra viruses interact with beta subunits of AP-3 complexes.

PubMed

Sun, Weina; McCrory, Thomas S; Khaw, Wei Young; Petzing, Stephanie; Myers, Terrell; Schmitt, Anthony P

2014-11-01

Paramyxoviruses and other negative-strand RNA viruses encode matrix proteins that coordinate the virus assembly process. The matrix proteins link the viral glycoproteins and the viral ribonucleoproteins at virus assembly sites and often recruit host machinery that facilitates the budding process. Using a co-affinity purification strategy, we have identified the beta subunit of the AP-3 adapter protein complex, AP3B1, as a binding partner for the M proteins of the zoonotic paramyxoviruses Nipah virus and Hendra virus. Binding function was localized to the serine-rich and acidic Hinge domain of AP3B1, and a 29-amino-acid Hinge-derived polypeptide was sufficient for M protein binding in coimmunoprecipitation assays. Virus-like particle (VLP) production assays were used to assess the relationship between AP3B1 binding and M protein function. We found that for both Nipah virus and Hendra virus, M protein expression in the absence of any other viral proteins led to the efficient production of VLPs in transfected cells, and this VLP production was potently inhibited upon overexpression of short M-binding polypeptides derived from the Hinge region of AP3B1. Both human and bat (Pteropus alecto) AP3B1-derived polypeptides were highly effective at inhibiting the production of VLPs. VLP production was also impaired through small interfering RNA (siRNA)-mediated depletion of AP3B1 from cells. These findings suggest that AP-3-directed trafficking processes are important for henipavirus particle production and identify a new host protein-virus protein binding interface that could become a useful target in future efforts to develop small molecule inhibitors to combat paramyxoviral infections. Henipaviruses cause deadly infections in humans, with a mortality rate of about 40%. Hendra virus outbreaks in Australia, all involving horses and some involving transmission to humans, have been a continuing problem. Nipah virus caused a large outbreak in Malaysia in 1998, killing 109 people, and smaller outbreaks have since occurred in Bangladesh and India. In this study, we have defined, for the first time, host factors that interact with henipavirus M proteins and contribute to viral particle assembly. We have also defined a new host protein-viral protein binding interface that can potentially be targeted for the inhibition of paramyxovirus infections. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Matrix Proteins of Nipah and Hendra Viruses Interact with Beta Subunits of AP-3 Complexes

PubMed Central

Sun, Weina; McCrory, Thomas S.; Khaw, Wei Young; Petzing, Stephanie; Myers, Terrell

2014-01-01

ABSTRACT Paramyxoviruses and other negative-strand RNA viruses encode matrix proteins that coordinate the virus assembly process. The matrix proteins link the viral glycoproteins and the viral ribonucleoproteins at virus assembly sites and often recruit host machinery that facilitates the budding process. Using a co-affinity purification strategy, we have identified the beta subunit of the AP-3 adapter protein complex, AP3B1, as a binding partner for the M proteins of the zoonotic paramyxoviruses Nipah virus and Hendra virus. Binding function was localized to the serine-rich and acidic Hinge domain of AP3B1, and a 29-amino-acid Hinge-derived polypeptide was sufficient for M protein binding in coimmunoprecipitation assays. Virus-like particle (VLP) production assays were used to assess the relationship between AP3B1 binding and M protein function. We found that for both Nipah virus and Hendra virus, M protein expression in the absence of any other viral proteins led to the efficient production of VLPs in transfected cells, and this VLP production was potently inhibited upon overexpression of short M-binding polypeptides derived from the Hinge region of AP3B1. Both human and bat (Pteropus alecto) AP3B1-derived polypeptides were highly effective at inhibiting the production of VLPs. VLP production was also impaired through small interfering RNA (siRNA)-mediated depletion of AP3B1 from cells. These findings suggest that AP-3-directed trafficking processes are important for henipavirus particle production and identify a new host protein-virus protein binding interface that could become a useful target in future efforts to develop small molecule inhibitors to combat paramyxoviral infections. IMPORTANCE Henipaviruses cause deadly infections in humans, with a mortality rate of about 40%. Hendra virus outbreaks in Australia, all involving horses and some involving transmission to humans, have been a continuing problem. Nipah virus caused a large outbreak in Malaysia in 1998, killing 109 people, and smaller outbreaks have since occurred in Bangladesh and India. In this study, we have defined, for the first time, host factors that interact with henipavirus M proteins and contribute to viral particle assembly. We have also defined a new host protein-viral protein binding interface that can potentially be targeted for the inhibition of paramyxovirus infections. PMID:25210190

In vitro evidence for RNA binding properties of the coat protein of prunus necrotic ringspot ilarvirus and their comparison to related and unrelated viruses.

PubMed

Pallás, V; Sánchez-Navarro, J A; Díez, J

1999-01-01

The RNA binding properties of the prunus necrotic ringspot virus (PNRSV) coat protein (CP) were demonstrated by northwestern and dot-blot analyses. The capability to bind PNRSV RNA 4 was compared with viruses representing three different interactions prevailing in the assembly and architecture of virions. The results showed that cucumber mosaic virus (CMV) and PNRSV CPs, which stabilise their virions mainly through RNA-protein interactions bound PNRSV RNA 4 even at very high salt concentrations. The CP of cherry leaf roll nepovirus, whose virions are predominantly stabilised by protein-protein interactions did not bind even at the lowest salt concentration tested. Finally the CP of carnation mottle carmovirus, that has an intermediate position in which both RNA-protein and protein-protein interactions are equally important showed a salt-dependent RNA binding.

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

Conserved Receptor-Binding Domains of Lake Victoria Marburgvirus and Zaire Ebolavirus Bind a Shared Receptor

DTIC Science & Technology

2006-04-14

virion, because of the functional importance of and limited variation in this region (44, 45). In some cases, such as murine and feline leukemia viruses ...murine leukemia virus ; PBS, phos- phate-buffered saline; RBD, receptor-binding domain; SARS, severe acute respiratory syndrome; VSV, vesicular stomatitis...entryofpseudotypedret- roviruses. A Moloney murine leukemia virus vector expressing GFP was pseudotyped with the GP1,2 of MARV-Mus (MARV/MLV), a mucin-like

Molecular characterization of a virus from the family Luteoviridae associated with cotton blue disease.

PubMed

Corrêa, R L; Silva, T F; Simões-Araújo, J L; Barroso, P A V; Vidal, M S; Vaslin, M F S

2005-07-01

Cotton blue disease is an aphid-transmitted cotton disease described in Brazil in 1962 as Vein Mosaic "var. Ribeirão Bonito". At present it causes economically important losses in cotton crops if control measures are not implemented. The observed symptoms and mode of transmission have prompted researchers to speculate that cotton blue disease could be attributed to a member of the family Luteoviridae, but there was no molecular evidence supporting this hypothesis. We have amplified part of the genome of a virus associated with this disease using degenerate primers for members of the family Luteoviridae. Sequence analysis of the entire capsid and a partial RdRp revealed a virus probably belonging to the genus Polerovirus. Based on our results we propose that cotton blue disease is associated with a virus with the putative name Cotton leafroll dwarf virus (CLRDV).

Putative recombination events and evolutionary history of five economically important viruses of fruit trees based on coat protein-encoding gene sequence analysis.

PubMed

Boulila, Moncef

2010-06-01

To enhance the knowledge of recombination as an evolutionary process, 267 accessions retrieved from GenBank were investigated, all belonging to five economically important viruses infecting fruit crops (Plum pox, Apple chlorotic leaf spot, Apple mosaic, Prune dwarf, and Prunus necrotic ringspot viruses). Putative recombinational events were detected in the coat protein (CP)-encoding gene using RECCO and RDP version 3.31beta algorithms. Based on RECCO results, all five viruses were shown to contain potential recombination signals in the CP gene. Reconstructed trees with modified topologies were proposed. Furthermore, RECCO performed better than the RDP package in detecting recombination events and exhibiting their evolution rate along the sequences of the five viruses. RDP, however, provided the possible major and minor parents of the recombinants. Thus, the two methods should be considered complementary.

Expanding the scope of a single pathogen survey to solve the mystery of the ailing red raspberries

USDA-ARS?s Scientific Manuscript database

Washington state produces almost 60% of the processed raspberries ($79 M value) in the U.S. Production is severely limited by Raspberry Bushy Dwarf Virus (RBDV) and the soilborne pathogens Phytophthora rubi and Pratylenchus penetrans. However, in 2012, growers began noticing plants with unusual symp...

Thinopyrum 7Ai-1-derived small chromatin with Barley Yellow Dwarf Virus (BYDV) resistance gene integrated into the wheat genome with retrotransposon.

PubMed

Ma, Y-Z; Tomita, M

2013-01-01

Thinopyrum intermedium is a useful source of resistance genes for Barley Yellow Dwarf Virus (BYDV), one of the most damaging wheat diseases. In this study, wheat/Th. intermedium translocation lines with a BYDV resistance gene were developed using the Th. intermedium 7Ai- 1 chromosome. Genomic in situ hybridization (GISH), using a Th. intermedium total genomic DNA probe, enabled detection of 7Ai-1-derived small chromatins containing a BYDV resistance gene, which were translocated onto the end of wheat chromosomes in the lines Y95011 and Y960843. Random amplified polymorphic DNA (RAPD) analyses using 120 random 10-mer primers were conducted to compare the BYDV-resistant translocation lines with susceptible lines. Two primers amplified the DNA fragments specific to the resistant line that would be useful as molecular markers to identify 7Ai-1-derived BYDV resistance chromatin in the wheat genome. Additionally, the isolated Th. intermedium-specific retrotransposon-like sequence pTi28 can be used to identify Th. intermedium chromatin transferred to the wheat genome.

Characterization of the N-terminal segment used by the barley yellow dwarf virus movement protein to promote interaction with the nuclear membrane of host plant cells.

PubMed

Dennison, Sarah Rachel; Harris, Frederick; Brandenburg, Klaus; Phoenix, David Andrew

2007-11-01

The barley yellow dwarf virus movement protein (BYDV-MP) requires its N-terminal sequence to promote the transport of viral RNA into the nuclear compartment of host plant cells. Here, graphical analysis predicts that this sequence would form a membrane interactive amphiphilic alpha-helix. Confirming this prediction, NT1, a peptide homologue of the BYDV-MP N-terminal sequence, was found to be alpha-helical (65%) in the presence of vesicles mimics of the nuclear membrane. The peptide increased the fluidity of these nuclear membrane mimics (rise in wavenumber of circa 0.5-1.0 cm(-1)) and induced surface pressure changes of 2 mN m(-1) in lipid monolayers with corresponding compositions. Taken with isotherm analysis these results suggest that BYDV-MP forms an N-terminal amphiphilic alpha-helix, which partitions into the nuclear membrane primarily through thermodynamically stable associations with the membrane lipid headgroup region. We speculate that these associations may play a role in targeting of the nuclear membrane by BYDM-MP.

Lack of conventional oxygen-linked proton and anion binding sites does not impair allosteric regulation of oxygen binding in dwarf caiman hemoglobin

PubMed Central

Fago, Angela; Malte, Hans; Storz, Jay F.; Gorr, Thomas A.

2013-01-01

In contrast to other vertebrate hemoglobins (Hbs) whose high intrinsic O2 affinities are reduced by red cell allosteric effectors (mainly protons, CO2, organic phosphates, and chloride ions), crocodilian Hbs exhibit low sensitivity to organic phosphates and high sensitivity to bicarbonate (HCO3−), which is believed to augment Hb-O2 unloading during diving and postprandial alkaline tides when blood HCO3− levels and metabolic rates increase. Examination of α- and β-globin amino acid sequences of dwarf caiman (Paleosuchus palpebrosus) revealed a unique combination of substitutions at key effector binding sites compared with other vertebrate and crocodilian Hbs: β82Lys→Gln, β143His→Val, and β146His→Tyr. These substitutions delete positive charges and, along with other distinctive changes in residue charge and polarity, may be expected to disrupt allosteric regulation of Hb-O2 affinity. Strikingly, however, P. palpebrosus Hb shows a strong Bohr effect, and marked deoxygenation-linked binding of organic phosphates (ATP and DPG) and CO2 as carbamate (contrasting with HCO3− binding in other crocodilians). Unlike other Hbs, it polymerizes to large complexes in the oxygenated state. The highly unusual properties of P. palpebrosus Hb align with a high content of His residues (potential sites for oxygenation-linked proton binding) and distinctive surface Cys residues that may form intermolecular disulfide bridges upon polymerization. On the basis of its singular properties, P. palpebrosus Hb provides a unique opportunity for studies on structure-function coupling and the evolution of compensatory mechanisms for maintaining tissue O2 delivery in Hbs that lack conventional effector-binding residues. PMID:23720132

Insulin-like growth factor type-1 receptor down-regulation associated with dwarfism in Holstein calves.

PubMed

Blum, J W; Elsasser, T H; Greger, D L; Wittenberg, S; de Vries, F; Distl, O

2007-10-01

Perturbations in endocrine functions can impact normal growth. Endocrine traits were studied in three dwarf calves exhibiting retarded but proportionate growth and four phenotypically normal half-siblings, sired by the same bull, and four unrelated control calves. Plasma 3,5,3'-triiodothyronine and thyroxine concentrations in dwarfs and half-siblings were in the physiological range and responded normally to injected thyroid-releasing hormone. Plasma glucagon concentrations were different (dwarfs, controls>half-siblings; P<0.05). Plasma growth hormone (GH), insulin-like growth factor-1 (IGF-1) and insulin concentrations in the three groups during an 8-h period were similar, but integrated GH concentrations (areas under concentration curves) were different (dwarfs>controls, P<0.02; half-siblings>controls, P=0.08). Responses of GH to xylazine and to a GH-releasing-factor analogue were similar in dwarfs and half-siblings. Relative gene expression of IGF-1, IGF-2, GH receptor (GHR), insulin receptor, IGF-1 type-1 and -2 receptors (IGF-1R, IGF-2R), and IGF binding proteins were measured in liver and anconeus muscle. GHR mRNA levels were different in liver (dwarfs

Propagation of classical swine fever virus in vitro circumventing heparan sulfate-adaptation.

PubMed

Eymann-Häni, Rita; Leifer, Immanuel; McCullough, Kenneth C; Summerfield, Artur; Ruggli, Nicolas

2011-09-01

Amplification of natural virus isolates in permanent cell lines can result in adaptation, in particular enhanced binding to heparan sulfate (HS)-containing glycosaminoglycans present on most vertebrate cells. This has been reported for several viruses, including the pestivirus classical swine fever virus (CSFV), the causative agent of a highly contagious hemorrhagic disease in pigs. Propagation of CSFV in cell culture is essential in virus diagnostics and research. Adaptation of CSFV to HS-binding has been related to amino acid changes in the viral E(rns) glycoprotein, resulting in viruses with altered replication characteristics in vitro and in vivo. Consequently, a compound blocking the HS-containing structures on cell surfaces was employed to monitor conversion from HS-independency to HS-dependency. It was shown that the porcine PEDSV.15 cell line permitted propagation of CSFV within a limited number of passages without adaptation to HS-binding. The selection of HS-dependent CSFV mutants was also prevented by propagation of the virus in the presence of DSTP 27. The importance of these findings can be seen from the altered ratio of cell-associated to secreted virus upon acquisition of enhanced HS-binding affinity, a phenotype proposed previously to be related to virulence in the natural host. Copyright © 2011 Elsevier B.V. All rights reserved.

Rapid detection of fifteen known soybean viruses by dot-immunobinding assay.

PubMed

Ali, Akhtar

2017-11-01

A dot-immunobinding assay (DIBA) was optimized and used successfully for the rapid detection of 15 known viruses [Alfalfa mosaic virus (AMV), Bean pod mottle virus (BPMV), Bean yellow mosaic virus (BYMV), Cowpea mild mottle virus (CPMMV), Cowpea severe mosaic virus (CPSMV), Cucumber mosaic virus (CMV), Peanut mottle virus (PeMoV), Peanut stunt virus (PSV), Southern bean mosaic virus (SBMV), Soybean dwarf virus (SbDV), Soybean mosaic virus (SMV), Soybean vein necrosis virus (SVNV), Tobacco ringspot virus (TRSV), Tomato ringspot virus (ToRSV), and Tobacco streak virus (TSV)] infecting soybean plants in Oklahoma. More than 1000 leaf samples were collected in approximately 100 commercial soybean fields in 24 counties of Oklahoma, during the 2012-2013 growing seasons. All samples were tested by DIBA using polyclonal antibodies of the above 15 plant viruses. Thirteen viruses were detected, and 8 of them were reported for the first time in soybean crops of Oklahoma. The highest average incidence was recorded for PeMoV (13.5%) followed by SVNV (6.9%), TSV (6.4%), BYMV, (4.5%), and TRSV (3.9%), while the remaining seven viruses were detected in less than 2% of the samples tested. The DIBA was quick, and economical to screen more than 1000 samples against 15 known plant viruses in a very short time. Copyright © 2017 Elsevier B.V. All rights reserved.

Sialylneolacto-N-tetraose c (LSTc)-bearing Liposomal Decoys Capture Influenza A Virus*

PubMed Central

Hendricks, Gabriel L.; Weirich, Kim L.; Viswanathan, Karthik; Li, Jing; Shriver, Zachary H.; Ashour, Joseph; Ploegh, Hidde L.; Kurt-Jones, Evelyn A.; Fygenson, Deborah K.; Finberg, Robert W.; Comolli, James C.; Wang, Jennifer P.

2013-01-01

Influenza is a severe disease in humans and animals with few effective therapies available. All strains of influenza virus are prone to developing drug resistance due to the high mutation rate in the viral genome. A therapeutic agent that targets a highly conserved region of the virus could bypass resistance and also be effective against multiple strains of influenza. Influenza uses many individually weak ligand binding interactions for a high avidity multivalent attachment to sialic acid-bearing cells. Polymerized sialic acid analogs can form multivalent interactions with influenza but are not ideal therapeutics due to solubility and toxicity issues. We used liposomes as a novel means for delivery of the glycan sialylneolacto-N-tetraose c (LSTc). LSTc-bearing decoy liposomes form multivalent, polymer-like interactions with influenza virus. Decoy liposomes competitively bind influenza virus in hemagglutination inhibition assays and inhibit infection of target cells in a dose-dependent manner. Inhibition is specific for influenza virus, as inhibition of Sendai virus and respiratory syncytial virus is not observed. In contrast, monovalent LSTc does not bind influenza virus or inhibit infectivity. LSTc decoy liposomes prevent the spread of influenza virus during multiple rounds of replication in vitro and extend survival of mice challenged with a lethal dose of virus. LSTc decoy liposomes co-localize with fluorescently tagged influenza virus, whereas control liposomes do not. Considering the conservation of the hemagglutinin binding pocket and the ability of decoy liposomes to form high avidity interactions with influenza hemagglutinin, our decoy liposomes have potential as a new therapeutic agent against emerging influenza strains. PMID:23362274

RNA binding properties of the US11 protein from four primate simplexviruses.

PubMed

Tohme, Sarah; Cukier, Cyprian D; Severini, Alberto

2011-11-03

The protein encoded by the Us11 gene of herpes simplex viruses is a dsRNA binding protein which inhibits protein kinase R activity, thereby preventing the interferon-induced shut down of protein synthesis following viral infection. Us11 protein is not essential for infectivity in vitro and in mice in herpes simplex virus type 1 (HSV1), however this virus has a second, and apparently more important, inhibitor of PKR activity, the γ134.5 protein. Recently sequenced simian simplexviruses SA8, HVP2 and B virus do not have an ORF corresponding to the γ134.5 protein, yet they have similar, or greater, infectivity as HSV1 and HSV2. We have expressed the US11 proteins of the simplexviruses HSV1, HSV2, HVP2 and B virus and measured their abilities to bind dsRNA, in order to investigate possible differences that could complement the absence of the γ134.5 protein. We employed a filter binding technique that allows binding of the Us11 protein under condition of excess dsRNA substrate and therefore a measurement of the true Kd value of Us11-dsRNA binding. The results show a Kd of binding in the range of 0.89 nM to 1.82 nM, with no significant difference among the four Us11 proteins.

RNA binding properties of the US11 protein from four primate simplexviruses

PubMed Central

2011-01-01

Background The protein encoded by the Us11 gene of herpes simplex viruses is a dsRNA binding protein which inhibits protein kinase R activity, thereby preventing the interferon-induced shut down of protein synthesis following viral infection. Us11 protein is not essential for infectivity in vitro and in mice in herpes simplex virus type 1 (HSV1), however this virus has a second, and apparently more important, inhibitor of PKR activity, the γ134.5 protein. Recently sequenced simian simplexviruses SA8, HVP2 and B virus do not have an ORF corresponding to the γ134.5 protein, yet they have similar, or greater, infectivity as HSV1 and HSV2. Methods We have expressed the US11 proteins of the simplexviruses HSV1, HSV2, HVP2 and B virus and measured their abilities to bind dsRNA, in order to investigate possible differences that could complement the absence of the γ134.5 protein. We employed a filter binding technique that allows binding of the Us11 protein under condition of excess dsRNA substrate and therefore a measurement of the true Kd value of Us11-dsRNA binding. Results and Conclusions The results show a Kd of binding in the range of 0.89 nM to 1.82 nM, with no significant difference among the four Us11 proteins. PMID:22054255

Monomeric ephrinB2 binding induces allosteric changes in Nipah virus G that precede its full activation.

PubMed

Wong, Joyce J W; Young, Tracy A; Zhang, Jiayan; Liu, Shiheng; Leser, George P; Komives, Elizabeth A; Lamb, Robert A; Zhou, Z Hong; Salafsky, Joshua; Jardetzky, Theodore S

2017-10-03

Nipah virus is an emergent paramyxovirus that causes deadly encephalitis and respiratory infections in humans. Two glycoproteins coordinate the infection of host cells, an attachment protein (G), which binds to cell surface receptors, and a fusion (F) protein, which carries out the process of virus-cell membrane fusion. The G protein binds to ephrin B2/3 receptors, inducing G conformational changes that trigger F protein refolding. Using an optical approach based on second harmonic generation, we show that monomeric and dimeric receptors activate distinct conformational changes in G. The monomeric receptor-induced changes are not detected by conformation-sensitive monoclonal antibodies or through electron microscopy analysis of G:ephrinB2 complexes. However, hydrogen/deuterium exchange experiments confirm the second harmonic generation observations and reveal allosteric changes in the G receptor binding and F-activating stalk domains, providing insights into the pathway of receptor-activated virus entry.Nipah virus causes encephalitis in humans. Here the authors use a multidisciplinary approach to study the binding of the viral attachment protein G to its host receptor ephrinB2 and show that monomeric and dimeric receptors activate distinct conformational changes in G and discuss implications for receptor-activated virus entry.

Heparin octasaccharide decoy liposomes inhibit replication of multiple viruses.

PubMed

Hendricks, Gabriel L; Velazquez, Lourdes; Pham, Serena; Qaisar, Natasha; Delaney, James C; Viswanathan, Karthik; Albers, Leila; Comolli, James C; Shriver, Zachary; Knipe, David M; Kurt-Jones, Evelyn A; Fygenson, Deborah K; Trevejo, Jose M; Wang, Jennifer P; Finberg, Robert W

2015-04-01

Heparan sulfate (HS) is a ubiquitous glycosaminoglycan that serves as a cellular attachment site for a number of significant human pathogens, including respiratory syncytial virus (RSV), human parainfluenza virus 3 (hPIV3), and herpes simplex virus (HSV). Decoy receptors can target pathogens by binding to the receptor pocket on viral attachment proteins, acting as 'molecular sinks' and preventing the pathogen from binding to susceptible host cells. Decoy receptors functionalized with HS could bind to pathogens and prevent infection, so we generated decoy liposomes displaying HS-octasaccharide (HS-octa). These decoy liposomes significantly inhibited RSV, hPIV3, and HSV infectivity in vitro to a greater degree than the original HS-octa building block. The degree of inhibition correlated with the density of HS-octa displayed on the liposome surface. Decoy liposomes with HS-octa inhibited infection of viruses to a greater extent than either full-length heparin or HS-octa alone. Decoy liposomes were effective when added prior to infection or following the initial infection of cells in vitro. By targeting the well-conserved receptor-binding sites of HS-binding viruses, decoy liposomes functionalized with HS-octa are a promising therapeutic antiviral agent and illustrate the utility of the liposome delivery platform. Copyright © 2015 Elsevier B.V. All rights reserved.

A Single Mutation in the E2 Glycoprotein Important for Neurovirulence Influences Binding of Sindbis Virus to Neuroblastoma Cells

PubMed Central

Lee, Peiyu; Knight, Ronald; Smit, Jolanda M.; Wilschut, Jan; Griffin, Diane E.

2002-01-01

The amino acid at position 55 of the E2 glycoprotein (E255) of Sindbis virus (SV) is a critical determinant of SV neurovirulence in mice. Recombinant virus strain TE (E255 = histidine) differs only at this position from virus strain 633 (E255= glutamine), yet TE is considerably more neurovirulent than 633. TE replicates better than 633 in a neuroblastoma cell line (N18), but similarly in BHK cells. Immunofluorescence staining showed that most N18 cells were infected by TE at a multiplicity of infection (MOI) of 50 to 500 and by 633 only at an MOI of 5,000, while both viruses infected essentially 100% of BHK cells at an MOI of 5. When exposed to pH 5, TE and 633 viruses fused to similar extents with liposomes derived from BHK or N18 cell lipids, but fusion with N18-derived liposomes was less extensive (15 to 20%) than fusion with BHK-derived liposomes (∼50%). Binding of TE and 633 to N18, but not BHK, cells was dependent on the medium used for virus binding. Differences between TE and 633 binding to N18 cells were evident in Dulbecco's modified Eagle medium (DMEM), but not in RPMI. In DMEM, the binding efficiency of 633 decreased significantly as the pH was raised from 6.5 to 8.0, while that of TE did not change. The same pattern was observed with RPMI when the ionic strength of RPMI was increased to that of DMEM. TE bound better to heparin-Sepharose than 633, but this difference was not pH dependent. Growth of N18 and BHK cells in sodium chlorate to eliminate all sulfation decreased virus-cell binding, suggesting the involvement of sulfated molecules on the cell surface. Taken together, the presence of glutamine at E255 impairs SV binding to neural cells under conditions characteristic of interstitial fluid. We conclude that mutation to histidine participates in or stabilizes the interaction between the virus and the surface of neural cells, contributing to greater neurovirulence. PMID:12021363

Jasmonic acid-mediated defense suppresses brassinosteroid-mediated susceptibility to Rice black streaked dwarf virus infection in rice.

PubMed

He, Yuqing; Zhang, Hehong; Sun, Zongtao; Li, Junmin; Hong, Gaojie; Zhu, Qisong; Zhou, Xuebiao; MacFarlane, Stuart; Yan, Fei; Chen, Jianping

2017-04-01

Plant hormones play a vital role in plant immune responses. However, in contrast to the relative wealth of information on hormone-mediated immunity in dicot plants, little information is available on monocot-virus defense systems. We used a high-throughput-sequencing approach to compare the global gene expression of Rice black-streaked dwarf virus (RBSDV)-infected rice plants with that of healthy plants. Exogenous hormone applications and transgenic rice were used to test RBSDV infectivity and pathogenicity. Our results revealed that the jasmonic acid (JA) pathway was induced while the brassinosteroid (BR) pathway was suppressed in infected plants. Foliar application of methyl jasmonate (MeJA) or brassinazole (BRZ) resulted in a significant reduction in RBSDV incidence, while epibrassinolide (BL) treatment increased RBSDV infection. Infection studies using coi1-13 and Go mutants demonstrated JA-mediated resistance and BR-mediated susceptibility to RBSDV infection. A mixture of MeJA and BL treatment resulted in a significant reduction in RBSDV infection compared with a single BL treatment. MeJA application efficiently suppressed the expression of BR pathway genes, and this inhibition depended on the JA coreceptor OsCOI1. Collectively, our results reveal that JA-mediated defense can suppress the BR-mediated susceptibility to RBSDV infection. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Molecular Biology of Prune Dwarf Virus—A Lesser Known Member of the Bromoviridae but a Vital Component in the Dynamic Virus–Host Cell Interaction Network

PubMed Central

Bujarski, Józef J.

2017-01-01

Prune dwarf virus (PDV) is one of the members of Bromoviridae family, genus Ilarvirus. Host components that participate in the regulation of viral replication or cell-to-cell movement via plasmodesmata are still unknown. In contrast, viral infections caused by some other Bromoviridae members are well characterized. Bromoviridae can be distinguished based on localization of their replication process in infected cells, cell-to-cell movement mechanisms, and plant-specific response reactions. Depending upon the genus, “genome activation” and viral replication are linked to various membranous structures ranging from endoplasmic reticulum, to tonoplast. In the case of PDV, there is still no evidence of natural resistance sources in the host plants susceptible to virus infection. Apparently, PDV has a great ability to overcome the natural defense responses in a wide spectrum of plant hosts. The first manifestations of PDV infection are specific cell membrane alterations, and the formation of replicase complexes that support PDV RNA replication inside the spherules. During each stage of its life cycle, the virus uses cell components to replicate and to spread in whole plants, within the largely suppressed cellular immunity environment. This work presents the above stages of the PDV life cycle in the context of current knowledge about other Bromoviridae members. PMID:29258199

Innate immune response of channel catfish (Ictalurus punctatus) mannose-binding lectin to channel catfish virus

USDA-ARS?s Scientific Manuscript database

The channel catfish virus (CCV) is a pathogenic herpesvirus that infects channel catfish (Ictalurus punctatus) in pond aquaculture in the Southeast USA. The innate immune protein mannose-binding lectin (MBL) could play an important role in the innate response of channel catfish by binding to the CC...

Characterization of the receptor-binding domain of Ebola glycoprotein in viral entry.

PubMed

Wang, Jizhen; Manicassamy, Balaji; Caffrey, Michael; Rong, Lijun

2011-06-01

Ebola virus infection causes severe hemorrhagic fever in human and non-human primates with high mortality. Viral entry/infection is initiated by binding of glycoprotein GP protein on Ebola virion to host cells, followed by fusion of virus-cell membrane also mediated by GP. Using an human immunodeficiency virus (HIV)-based pseudotyping system, the roles of 41 Ebola GP1 residues in the receptor-binding domain in viral entry were studied by alanine scanning substitutions. We identified that four residues appear to be involved in protein folding/structure and four residues are important for viral entry. An improved entry interference assay was developed and used to study the role of these residues that are important for viral entry. It was found that R64 and K95 are involved in receptor binding. In contrast, some residues such as I170 are important for viral entry, but do not play a major role in receptor binding as indicated by entry interference assay and/or protein binding data, suggesting that these residues are involved in post-binding steps of viral entry. Furthermore, our results also suggested that Ebola and Marburg viruses share a common cellular molecule for entry.

The epidermal growth factor receptor (EGFR) promotes uptake of influenza A viruses (IAV) into host cells.

PubMed

Eierhoff, Thorsten; Hrincius, Eike R; Rescher, Ursula; Ludwig, Stephan; Ehrhardt, Christina

2010-09-09

Influenza A viruses (IAV) bind to sialic-acids at cellular surfaces and enter cells by using endocytotic routes. There is evidence that this process does not occur constitutively but requires induction of specific cellular signals, including activation of PI3K that promotes virus internalization. This implies engagement of cellular signaling receptors during viral entry. Here, we present first indications for an interplay of IAV with receptor tyrosine kinases (RTKs). As representative RTK family-members the epidermal growth factor receptor (EGFR) and the c-Met receptor were studied. Modulation of expression or activity of both RTKs resulted in altered uptake of IAV, showing that these receptors transmit entry relevant signals upon virus binding. More detailed studies on EGFR function revealed that virus binding lead to clustering of lipid-rafts, suggesting that multivalent binding of IAV to cells induces a signaling platform leading to activation of EGFR and other RTKs that in turn facilitates IAV uptake.

The Epidermal Growth Factor Receptor (EGFR) Promotes Uptake of Influenza A Viruses (IAV) into Host Cells

PubMed Central

Eierhoff, Thorsten; Hrincius, Eike R.; Rescher, Ursula; Ludwig, Stephan; Ehrhardt, Christina

2010-01-01

Influenza A viruses (IAV) bind to sialic-acids at cellular surfaces and enter cells by using endocytotic routes. There is evidence that this process does not occur constitutively but requires induction of specific cellular signals, including activation of PI3K that promotes virus internalization. This implies engagement of cellular signaling receptors during viral entry. Here, we present first indications for an interplay of IAV with receptor tyrosine kinases (RTKs). As representative RTK family-members the epidermal growth factor receptor (EGFR) and the c-Met receptor were studied. Modulation of expression or activity of both RTKs resulted in altered uptake of IAV, showing that these receptors transmit entry relevant signals upon virus binding. More detailed studies on EGFR function revealed that virus binding lead to clustering of lipid-rafts, suggesting that multivalent binding of IAV to cells induces a signaling platform leading to activation of EGFR and other RTKs that in turn facilitates IAV uptake. PMID:20844577

Host and viral RNA-binding proteins involved in membrane targeting, replication and intercellular movement of plant RNA virus genomes

PubMed Central

Hyodo, Kiwamu; Kaido, Masanori; Okuno, Tetsuro

2014-01-01

Many plant viruses have positive-strand RNA [(+)RNA] as their genome. Therefore, it is not surprising that RNA-binding proteins (RBPs) play important roles during (+)RNA virus infection in host plants. Increasing evidence demonstrates that viral and host RBPs play critical roles in multiple steps of the viral life cycle, including translation and replication of viral genomic RNAs, and their intra- and intercellular movement. Although studies focusing on the RNA-binding activities of viral and host proteins, and their associations with membrane targeting, and intercellular movement of viral genomes have been limited to a few viruses, these studies have provided important insights into the molecular mechanisms underlying the replication and movement of viral genomic RNAs. In this review, we briefly overview the currently defined roles of viral and host RBPs whose RNA-binding activity have been confirmed experimentally in association with their membrane targeting, and intercellular movement of plant RNA virus genomes. PMID:25071804

Dimeric Architecture of the Hendra Virus Attachment Glycoprotein: Evidence for a Conserved Mode of Assembly▿ †

PubMed Central

Bowden, Thomas A.; Crispin, Max; Harvey, David J.; Jones, E. Yvonne; Stuart, David I.

2010-01-01

Hendra virus is a negative-sense single-stranded RNA virus within the Paramyxoviridae family which, together with Nipah virus, forms the Henipavirus genus. Infection with bat-borne Hendra virus leads to a disease with high mortality rates in humans. We determined the crystal structure of the unliganded six-bladed β-propeller domain and compared it to the previously reported structure of Hendra virus attachment glycoprotein (HeV-G) in complex with its cellular receptor, ephrin-B2. As observed for the related unliganded Nipah virus structure, there is plasticity in the Glu579-Pro590 and Lys236-Ala245 ephrin-binding loops prior to receptor engagement. These data reveal that henipaviral attachment glycoproteins undergo common structural transitions upon receptor binding and further define the structural template for antihenipaviral drug design. Our analysis also provides experimental evidence for a dimeric arrangement of HeV-G that exhibits striking similarity to those observed in crystal structures of related paramyxovirus receptor-binding glycoproteins. The biological relevance of this dimer is further supported by the positional analysis of glycosylation sites from across the paramyxoviruses. In HeV-G, the sites lie away from the putative dimer interface and remain accessible to α-mannosidase processing on oligomerization. We therefore propose that the overall mode of dimer assembly is conserved for all paramyxoviruses; however, while the geometry of dimerization is rather closely similar for those viruses that bind flexible glycan receptors, significant (up to 60°) and different reconfigurations of the subunit packing (associated with a significant decrease in the size of the dimer interface) have accompanied the independent switching to high-affinity protein receptor binding in Hendra and measles viruses. PMID:20375167

Structures of Receptor Complexes of a North American H7N2 Influenza Hemagglutinin with a Loop Deletion in the Receptor Binding Site

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

Yang, Hua; Chen, Li-Mei; Carney, Paul J.

2012-02-21

Human infections with subtype H7 avian influenza viruses have been reported as early as 1979. In 1996, a genetically stable 24-nucleotide deletion emerged in North American H7 influenza virus hemagglutinins, resulting in an eight amino acid deletion in the receptor-binding site. The continuous circulation of these viruses in live bird markets, as well as its documented ability to infect humans, raises the question of how these viruses achieve structural stability and functionality. Here we report a detailed molecular analysis of the receptor binding site of the North American lineage subtype H7N2 virus A/New York/107/2003 (NY107), including complexes with an avianmore » receptor analog (3'-sialyl-N-acetyllactosamine, 3'SLN) and two human receptor analogs (6'-sialyl-N-acetyllactosamine, 6'SLN; sialyllacto-N-tetraose b, LSTb). Structural results suggest a novel mechanism by which residues Arg220 and Arg229 (H3 numbering) are used to compensate for the deletion of the 220-loop and form interactions with the receptor analogs. Glycan microarray results reveal that NY107 maintains an avian-type ({alpha}2-3) receptor binding profile, with only moderate binding to human-type ({alpha}2-6) receptor. Thus despite its dramatically altered receptor binding site, this HA maintains functionality and confirms a need for continued influenza virus surveillance of avian and other animal reservoirs to define their zoonotic potential.« less

Humoral markers of active Epstein-Barr virus infection associate with anti-extractable nuclear antigen autoantibodies and plasma galectin-3 binding protein in systemic lupus erythematosus.

PubMed

Rasmussen, N S; Nielsen, C T; Houen, G; Jacobsen, S

2016-12-01

We investigated if signs of active Epstein-Barr virus and cytomegalovirus infections associate with certain autoantibodies and a marker of type I interferon activity in patients with systemic lupus erythematosus. IgM and IgG plasma levels against Epstein-Barr virus early antigen diffuse and cytomegalovirus pp52 were applied as humoral markers of ongoing/recently active Epstein-Barr virus and cytomegalovirus infections, respectively. Plasma galectin-3 binding protein served as a surrogate marker of type I interferon activity. The measurements were conducted in 57 systemic lupus erythematosus patients and 29 healthy controls using ELISAs. Regression analyses and univariate comparisons were performed for associative evaluation between virus serology, plasma galectin-3 binding protein and autoantibodies, along with other clinical and demographic parameters. Plasma galectin-3 binding protein concentrations were significantly higher in systemic lupus erythematosus patients (P = 0.009) and associated positively with Epstein-Barr virus early antigen diffuse-directed antibodies and the presence of autoantibodies against extractable nuclear antigens in adjusted linear regressions (B = 2.02 and 2.02, P = 0.02 and P = 0.002, respectively). Furthermore, systemic lupus erythematosus patients with anti-extractable nuclear antigens had significantly higher antibody levels against Epstein-Barr virus early antigen diffuse (P = 0.02). Our study supports a link between active Epstein-Barr virus infections, positivity for anti-extractable nuclear antigens and increased plasma galectin-3 binding protein concentrations/type I interferon activity in systemic lupus erythematosus patients. © The Author(s) 2016.

Dephosphorylation of HuR Protein during Alphavirus Infection Is Associated with HuR Relocalization to the Cytoplasm*

PubMed Central

Dickson, Alexa M.; Anderson, John R.; Barnhart, Michael D.; Sokoloski, Kevin J.; Oko, Lauren; Opyrchal, Mateusz; Galanis, Evanthia; Wilusz, Carol J.; Morrison, Thomas E.; Wilusz, Jeffrey

2012-01-01

We have demonstrated previously that the cellular HuR protein binds U-rich elements in the 3′ untranslated region (UTR) of Sindbis virus RNA and relocalizes from the nucleus to the cytoplasm upon Sindbis virus infection in 293T cells. In this study, we show that two alphaviruses, Ross River virus and Chikungunya virus, lack the conserved high-affinity U-rich HuR binding element in their 3′ UTRs but still maintain the ability to interact with HuR with nanomolar affinities through alternative binding elements. The relocalization of HuR protein occurs during Sindbis infection of multiple mammalian cell types as well as during infections with three other alphaviruses. Interestingly, the relocalization of HuR is not a general cellular reaction to viral infection, as HuR protein remained largely nuclear during infections with dengue and measles virus. Relocalization of HuR in a Sindbis infection required viral gene expression, was independent of the presence of a high-affinity U-rich HuR binding site in the 3′ UTR of the virus, and was associated with an alteration in the phosphorylation state of HuR. Sindbis virus-induced HuR relocalization was mechanistically distinct from the movement of HuR observed during a cellular stress response, as there was no accumulation of caspase-mediated HuR cleavage products. Collectively, these data indicate that virus-induced HuR relocalization to the cytoplasm is specific to alphavirus infections and is associated with distinct posttranslational modifications of this RNA-binding protein. PMID:22915590

Identification and Structural Characterization of the ALIX-Binding Late Domains of Simian Immunodeficiency Virus SIV mac239 and SIV agmTan-1

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

Q Zhai; M Landesman; H Robinson

2011-12-31

Retroviral Gag proteins contain short late-domain motifs that recruit cellular ESCRT pathway proteins to facilitate virus budding. ALIX-binding late domains often contain the core consensus sequence YPX{sub n}L (where X{sub n} can vary in sequence and length). However, some simian immunodeficiency virus (SIV) Gag proteins lack this consensus sequence, yet still bind ALIX. We mapped divergent, ALIX-binding late domains within the p6{sup Gag} proteins of SIV{sub MAC239} ({sub 40}SREK{und P}YKE{und VT}ED{und L}LHLNSLF{sub 59}) and SIV{sub agmTan-1} ({sub 24}AAG{und A}YDP{und AR}KL{und L}EQYAKK{sub 41}). Crystal structures revealed that anchoring tyrosines (in lightface) and nearby hydrophobic residues (underlined) contact the ALIX V domain,more » revealing how lentiviruses employ a diverse family of late-domain sequences to bind ALIX and promote virus budding.« less

Identification and Structural Characterization of the ALIX-Binding Late Domains of Simian Immunodeficiency Virus SIVmac239 and SIVagmTan-1

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

Zhai, Q.; Robinson, H.; Landesman, M. B.

2011-01-01

Retroviral Gag proteins contain short late-domain motifs that recruit cellular ESCRT pathway proteins to facilitate virus budding. ALIX-binding late domains often contain the core consensus sequence YPX{sub n}L (where X{sub n} can vary in sequence and length). However, some simian immunodeficiency virus (SIV) Gag proteins lack this consensus sequence, yet still bind ALIX. We mapped divergent, ALIX-binding late domains within the p6{sup Gag} proteins of SIV{sub mac239} ({sub 40}SREK{und P}YKE{und VT}ED{und L}LHLNSLF{sub 59}) and SIV{sub agmTan-1} ({sub 24}AAG{und A}YDP{und AR}KL{und L}EQYAKK{sub 41}). Crystal structures revealed that anchoring tyrosines (in lightface) and nearby hydrophobic residues (underlined) contact the ALIX V domain,more » revealing how lentiviruses employ a diverse family of late-domain sequences to bind ALIX and promote virus budding.« less

Use of virus-attached antibodies or insulin molecules to mediate fusion between Sendai virus envelopes and neuraminidase-treated cells.

PubMed

Gitman, A G; Kahane, I; Loyter, A

1985-05-21

Anti-human erythrocyte antibodies or insulin molecules were covalently coupled to the glycoproteins (the hemagglutinin/neuraminidase and the fusion polypeptides) of Sendai virus envelopes with N-succinimidyl 3-(2-pyridyldithio)propionate and succinimidyl 4-(p-maleimidophenyl)butyrate as cross-linking reagents. Reconstituted Sendai virus envelopes, bearing covalently attached anti-human erythrocyte antibodies or insulin molecules, were able to bind to but not fuse with virus receptor depleted human erythrocytes (neuraminidase-treated human erythrocytes). Only coreconstitution of Sendai virus glycoproteins, bearing attached anti-human erythrocyte antibodies or insulin molecules with intact, untreated viral glycoproteins, led to the formation of fusogenic, targeted reconstituted Sendai virus envelopes. Binding and fusion of reconstituted Sendai virus envelopes, bearing anti-human erythrocyte antibodies or insulin molecules, with neuraminidase-treated human erythrocytes were blocked by the monovalent fraction, obtained after papain digestion of immunoglobulins, made of anti-human erythrocyte antibodies or free insulin molecules, respectively. The results of this work demonstrate an active role of the viral binding protein (hemagglutinin/neuraminidase polypeptide) in the virus membrane fusion process and show a novel and efficient method for the construction of targeted, fusogenic Sendai virus envelopes.

The GHR mutations related to individual’s dwarf

USDA-ARS?s Scientific Manuscript database

Growth hormone (GH) promotes body’s growth through binding with two receptors (GHRs) at the cell surface to interact with Janus kinase and signal transducers and activators of transcription, and then to stimulate metabolic effects and insulin-like growth factor (IGF) synthesis. However, the disorder...

Impact of Mutations in the Hemagglutinin of H10N7 Viruses Isolated from Seals on Virus Replication in Avian and Human Cells.

PubMed

Dittrich, Anne; Scheibner, David; Salaheldin, Ahmed H; Veits, Jutta; Gischke, Marcel; Mettenleiter, Thomas C; Abdelwhab, Elsayed M

2018-02-14

Wild birds are the reservoir for low-pathogenic avian influenza viruses, which are frequently transmitted to domestic birds and occasionally to mammals. In 2014, an H10N7 virus caused severe mortality in harbor seals in northeastern Europe. Although the hemagglutinin (HA) of this virus was closely related to H10 of avian H10N4 virus, it possessed unique nonsynonymous mutations, particularly in the HA1 subunit in or adjacent to the receptor binding domain and proteolytic cleavage site. Here, the impact of these mutations on virus replication was studied in vitro. Using reverse genetics, an avian H10N4 virus was cloned, and nine recombinant viruses carrying one of eight unique mutations or the complete HA from the seal virus were rescued. Receptor binding affinity, replication in avian and mammalian cell cultures, cell-to-cell spread, and HA cleavability of these recombinant viruses were studied. Results show that wild-type recombinant H10N4 virus has high affinity to avian-type sialic acid receptors and no affinity to mammalian-type receptors. The H10N7 virus exhibits dual receptor binding affinity. Interestingly, Q220L (H10 numbering) in the rim of the receptor binding pocket increased the affinity of the H10N4 virus to mammal-type receptors and completely abolished the affinity to avian-type receptors. No remarkable differences in cell-to-cell spread or HA cleavability were observed. All viruses, including the wild-type H10N7 virus, replicated at higher levels in chicken cells than in human cells. These results indicate that H10N7 acquired adaptive mutations (e.g., Q220L) to enhance replication in mammals and retained replication efficiency in the original avian host.

Impact of Mutations in the Hemagglutinin of H10N7 Viruses Isolated from Seals on Virus Replication in Avian and Human Cells

PubMed Central

Dittrich, Anne; Scheibner, David; Salaheldin, Ahmed H.; Veits, Jutta; Gischke, Marcel

2018-01-01

Wild birds are the reservoir for low-pathogenic avian influenza viruses, which are frequently transmitted to domestic birds and occasionally to mammals. In 2014, an H10N7 virus caused severe mortality in harbor seals in northeastern Europe. Although the hemagglutinin (HA) of this virus was closely related to H10 of avian H10N4 virus, it possessed unique nonsynonymous mutations, particularly in the HA1 subunit in or adjacent to the receptor binding domain and proteolytic cleavage site. Here, the impact of these mutations on virus replication was studied in vitro. Using reverse genetics, an avian H10N4 virus was cloned, and nine recombinant viruses carrying one of eight unique mutations or the complete HA from the seal virus were rescued. Receptor binding affinity, replication in avian and mammalian cell cultures, cell-to-cell spread, and HA cleavability of these recombinant viruses were studied. Results show that wild-type recombinant H10N4 virus has high affinity to avian-type sialic acid receptors and no affinity to mammalian-type receptors. The H10N7 virus exhibits dual receptor binding affinity. Interestingly, Q220L (H10 numbering) in the rim of the receptor binding pocket increased the affinity of the H10N4 virus to mammal-type receptors and completely abolished the affinity to avian-type receptors. No remarkable differences in cell-to-cell spread or HA cleavability were observed. All viruses, including the wild-type H10N7 virus, replicated at higher levels in chicken cells than in human cells. These results indicate that H10N7 acquired adaptive mutations (e.g., Q220L) to enhance replication in mammals and retained replication efficiency in the original avian host. PMID:29443887

Structural Basis for a Switch in Receptor Binding Specificity of Two H5N1 Hemagglutinin Mutants

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

Zhu, Xueyong; Viswanathan, Karthik; Raman, Rahul

Avian H5N1 influenza viruses continue to spread in wild birds and domestic poultry with sporadic infection in humans. Receptor binding specificity changes are a prerequisite for H5N1 viruses and other zoonotic viruses to be transmitted among humans. Previous reported hemagglutinin (HA) mutants from ferret-transmissible H5N1 viruses of A/Viet Nam/1203/04 and A/Indonesia/5/05 showed slightly increased, but still very weak, binding to human receptors. From mutagenesis and glycan array studies, we previously identified two H5N1 HA mutants that could more effectively switch receptor specificity to human-like α2-6 linked sialosides with avidity comparable to wild-type H5 HA binding to avian-like α2-3 linked sialosides.more » Here, crystal structures of these two H5 HA mutants free and in complex with human and avian glycan receptor analogues reveal the structural basis for their preferential binding to human receptors. These findings suggest continuous surveillance should be maintained to monitor and assess human-to-human transmission potential of H5N1 viruses.« less

Structural Basis for a Switch in Receptor Binding Specificity of Two H5N1 Hemagglutinin Mutants

DOE PAGES

Zhu, Xueyong; Viswanathan, Karthik; Raman, Rahul; ...

2015-11-01

Avian H5N1 influenza viruses continue to spread in wild birds and domestic poultry with sporadic infection in humans. Receptor binding specificity changes are a prerequisite for H5N1 viruses and other zoonotic viruses to be transmitted among humans. Previous reported hemagglutinin (HA) mutants from ferret-transmissible H5N1 viruses of A/Viet Nam/1203/04 and A/Indonesia/5/05 showed slightly increased, but still very weak, binding to human receptors. From mutagenesis and glycan array studies, we previously identified two H5N1 HA mutants that could more effectively switch receptor specificity to human-like α2-6 linked sialosides with avidity comparable to wild-type H5 HA binding to avian-like α2-3 linked sialosides.more » Here, crystal structures of these two H5 HA mutants free and in complex with human and avian glycan receptor analogues reveal the structural basis for their preferential binding to human receptors. These findings suggest continuous surveillance should be maintained to monitor and assess human-to-human transmission potential of H5N1 viruses.« less

Discovery and molecular characterization of a new luteovirus identified by high-throughput sequencing from apple

USDA-ARS?s Scientific Manuscript database

‘Rapid Apple Decline’ (RAD) is a newly emerging problem of young, dwarf apple trees in the northeastern USA. The affected trees show trunk necrosis, bark cracking and canker formation before collapsing in the summer. In this study, a new luteovirus and three common viruses were identified from apple...

Spatiotemporal model of barley and cereal yellow dwarf virus transmission dynamics with seasonality and plant competition

Treesearch

S.M. Moore; C.A. Manore; V.A. Bokil; E.T. Borer; P.R. Hosseini

2011-01-01

Many generalist pathogens are influenced by the spatial distributions and relative abundances of susceptible host species. The spatial structure of host populations can influence patterns of infection incidence (or disease outbreaks), and the effects of a generalist pathogen on host community dynamics in a spatially heterogeneous community may differ from predictions...

Heterogeneity of envelope molecules expressed on primary human immunodeficiency virus type 1 particles as probed by the binding of neutralizing and nonneutralizing antibodies.

PubMed

Poignard, Pascal; Moulard, Maxime; Golez, Edwin; Vivona, Veronique; Franti, Michael; Venturini, Sara; Wang, Meng; Parren, Paul W H I; Burton, Dennis R

2003-01-01

Virion capture assays, in which immobilized antibodies (Abs) capture virus particles, have been used to suggest that nonneutralizing Abs bind effectively to human immunodeficiency virus type 1 (HIV-1) primary viruses. Here, we show that virion capture assays, under conditions commonly reported in the literature, give a poor indication of epitope expression on the surface of infectious primary HIV-1. First, estimation of primary HIV-1 capture by p24 measurements shows a very poor correlation with an estimation based on infectivity measurements. Second, virion capture appears to require relatively low Ab affinity for the virion, as shown by the ability of a monoclonal Ab to capture a wild-type and a neutralization escape variant virus equally well. Nevertheless, in a more interpretable competition format, it is shown that nonneutralizing anti-CD4 binding site (CD4bs) Abs compete with a neutralizing anti-CD4bs Ab (b12) for virus capture, suggesting that the nonneutralizing anti-CD4bs Abs are able to bind to the envelope species that is involved in virion capture in these experiments. However, the nonneutralizing anti-CD4bs Abs do not inhibit neutralization by b12 even at considerable excess. This suggests that the nonneutralizing Abs are unable to bind effectively to the envelope species required for virus infectivity. The results were obtained for three different primary virus envelopes. The explanation that we favor is that infectious HIV-1 primary virions can express two forms of gp120, an accessible nonfunctional form and a functional form with limited access. Binding to the nonfunctional form, which needs only to be present at relatively low density on the virion, permits capture but does not lead to neutralization. The expression of a nonfunctional but accessible form of gp120 on virions may contribute to the general failure of HIV-1 infection to elicit cross-neutralizing Abs and may represent a significant problem for vaccines based on viruses or virus-like particles.

Receptor specificity of the influenza virus hemagglutinin modulates sensitivity to soluble collectins of the innate immune system and virulence in mice.

PubMed

Tate, Michelle D; Brooks, Andrew G; Reading, Patrick C

2011-04-25

The hemagglutinin (HA) glycoprotein of influenza virus binds to cell surface sialic acid (SA) to initiate infection. In this study, a mutant of influenza A virus strain BJx109 (H3N2) was plaque-purified from the lungs of virus-infected mice that had been depleted of airway macrophages. Sequence analysis identified a single amino acid substitution (S186I) in the vicinity of the receptor-binding site of HA. This substitution was associated with enhanced binding to α(2,3)-Gal-linked SA and an increased ability to infect murine airway epithelial cells. Mutant viruses were less sensitive to neutralization by mouse airway fluids and less efficient in their ability to infect murine macrophages. Moreover, infection of mice with viruses bearing the S186I substitution led to severe disease, characterized by enhanced virus replication, lung pathology and pulmonary edema. Together, these studies confirm that residue 186 of H3 subtype viruses is a critical determinant of virulence in a mouse model of influenza infection. Copyright © 2010 Elsevier Inc. All rights reserved.

Proteomic analysis of interaction between P7-1 of Southern rice black-streaked dwarf virus and the insect vector reveals diverse insect proteins involved in successful transmission.

PubMed

Mar, ThiThi; Liu, Wenwen; Wang, Xifeng

2014-05-06

Southern rice black-streaked dwarf virus (SRBSDV), transmitted by the white-backed planthopper (Sogatella furcifera) in a persistent-propagative manner, has caused serious yield losses in Asia. Here in a yeast two-hybrid system, protein interactions between SRBSDV P7-1 as a bait protein and a cDNA library of S. furcifera as prey protein were assessed. Of 153 proteins identified as putative interactors, 24 were selected for further analysis. Of the 24 proteins, 18 were further confirmed in a chemiluminescent coimmunoprecipitation (Co-IP) assay as true positive interactors with different strengths of interactions. Six potential candidate proteins (neuroglian, myosin light chain 2 [MLC2], polyubiquitin, E3 ubiquitin ligase, ribophorin ii, and profilin) were analyzed for gene expression in five organs by qRT-PCR; mRNA levels were highest in the gut for neuroglian, MLC2, polyubiquitin and profilin, in the salivary glands for ribophorin ii, and in the haemolymph for E3 ubiquitin ligase. A virus-host protein interaction network was constructed using SRBSDV P7-1 and 18 prey positive protein homologs of Drosophila melanogaster. Our findings suggest that these proteins are involved in the complex host reaction to infection by SRBSDV and provide new insights into the molecular basis of transmission. Southern rice black-streaked dwarf virus (SRBSDV), transmitted by S. furcifera in a persistent-propagative manner, is a new found virus and a tentative member of the genus Fijivirus in the family Reoviridae. It was widely noted by plant virologist, government officials and the farmers in Asia in recent years because of its epidemic outbreak and causing serious yield losses after 2009. However, the molecular mechanism by which SRBSDV successfully infects and replicates in both plant and insect hosts remains unclear, and much less is known about how the virus spreads from initially infected cells to adjacent cells in the insect vector. In the present study, we examined protein interactions between SRBSDV P7-1 as the bait and cDNA library of WBPH as the prey by using yeast two-hybrid system, 153 proteins were identified as putative interactors and 24 putative proteins were selected for chemiluminescent coimmunoprecipitation (Co-IP) assay, and then constructed a viral protein-host protein interaction network with homologs of D. melanogaster. Six WBPH proteins were confirmed as potential P7-1 partners that take part in a pivotal role for viral movement in insect vector. These findings will greatly facilitate the understanding of the transmission mechanisms of SRBSDV by its insect vector. This is the first to study the molecular interaction between SRBSDV and its insect vector. Copyright © 2014 Elsevier B.V. All rights reserved.

Exploring Strategies for Labeling Viruses with Gold Nanoclusters through Non-equilibrium Molecular Dynamics Simulations.

PubMed

Pohjolainen, Emmi; Malola, Sami; Groenhof, Gerrit; Häkkinen, Hannu

2017-09-20

Biocompatible gold nanoclusters can be utilized as contrast agents in virus imaging. The labeling of viruses can be achieved noncovalently but site-specifically by linking the cluster to the hydrophobic pocket of a virus via a lipid-like pocket factor. We have estimated the binding affinities of three different pocket factors of echovirus 1 (EV1) in molecular dynamics simulations combined with non-equilibrium free-energy calculations. We have also studied the effects on binding affinities with a pocket factor linked to the Au 102 pMBA 44 nanocluster in different protonation states. Although the absolute binding affinities are over-estimated for all the systems, the trend is in agreement with recent experiments.3 Our results suggest that the natural pocket factor (palmitic acid) can be replaced by molecules pleconaril (drug) and its derivative Kirtan1 that have higher estimated binding affinities. Our results also suggest that including the gold nanocluster does not decrease the affinity of the pocket factor to the virus, but the affinity is sensitive to the protonation state of the nanocluster, i.e., to pH conditions. The methodology introduced in this work helps in the design of optimal strategies for gold-virus bioconjugation for virus detection and manipulation.

Glycosaminoglycans mediate retention of the poxvirus type I interferon binding protein at the cell surface to locally block interferon antiviral responses

PubMed Central

Montanuy, Imma; Alejo, Ali; Alcami, Antonio

2011-01-01

Eradication of smallpox was accomplished 30 yr ago, but poxviral infections still represent a public health concern due to the potential release of variola virus or the emergence of zoonotic poxviruses, such as monkeypox virus. A critical determinant of poxvirus virulence is the inhibition of interferons (IFNs) by the virus-encoded type I IFN-binding protein (IFNα/βBP). This immunomodulatory protein is secreted and has the unique property of interacting with the cell surface in order to prevent IFN-mediated antiviral responses. However, the mechanism of its attachment to the cell surface remains unknown. Using surface plasmon resonance and cell-binding assays, we report that the IFNα/βBP from vaccinia virus, the smallpox vaccine, interacts with cell surface glycosaminoglycans (GAGs). Analysis of the contribution of different regions of the protein to cell surface binding demonstrated that clusters of basic residues in the first immunoglobulin domain mediate GAG interactions. Furthermore, mutation of the GAG-interaction motifs does not affect its IFN-binding and -blocking capacity. Functional conservation of GAG-binding sites is demonstrated for the IFNα/βBP from variola and monkeypox viruses, extending our understanding of immune modulation by the most virulent human poxviruses. These results are relevant for the design of improved vaccines and intervention strategies.—Montanuy, I., Alejo, A., Alcami, A. Glycosaminoglycans mediate retention of the poxvirus type I interferon binding protein at the cell surface to locally block interferon antiviral responses. PMID:21372110

Role of phosphatidylserine receptors in enveloped virus infection.

PubMed

Morizono, Kouki; Chen, Irvin S Y

2014-04-01

We recently demonstrated that a soluble protein, Gas6, can facilitate viral entry by bridging viral envelope phosphatidylserine to Axl, a receptor tyrosine kinase expressed on target cells. The interaction between phosphatidylserine, Gas6, and Axl was originally shown to be a molecular mechanism through which phagocytes recognize phosphatidylserine exposed on dead cells. Since our initial report, several groups have confirmed that Axl/Gas6, as well as other phosphatidylserine receptors, facilitate entry of dengue, West Nile, and Ebola viruses. Virus binding by viral envelope phosphatidylserine is now a viral entry mechanism generalized to many families of viruses. In addition to Axl/Gas6, various molecules are known to recognize phosphatidylserine; however, the effects of these molecules on virus binding and entry have not been comprehensively evaluated and compared. In this study, we examined most of the known human phosphatidylserine-recognizing molecules, including MFG-E8, TIM-1, -3, and -4, CD300a, BAI1, and stabilin-1 and -2, for their abilities to facilitate virus binding and infection. Using pseudotyped lentiviral vectors, we found that a soluble phosphatidylserine-binding protein, MFG-E8, enhances transduction. Cell surface receptors TIM-1 and -4 also enhance virus binding/transduction. The extent of enhancement by these molecules varies, depending on the type of pseudotyping envelope proteins. Mutated MFG-E8, which binds viral envelope phosphatidylserine without bridging virus to cells, but, surprisingly, not annexin V, which has been used to block phagocytosis of dead cells by concealing phosphatidylserine, efficiently blocks these phosphatidylserine-dependent viral entry mechanisms. These results provide insight into understanding the role of viral envelope phosphatidylserine in viral infection. Envelope phosphatidylserine has previously been shown to be important for replication of various envelope viruses, but details of this mechanism(s) were unclear. We were the first to report that a bifunctional serum protein, Gas6, bridges envelope phosphatidylserine to a cell surface receptor, Axl. Recent studies demonstrated that many envelope viruses, including vaccinia, dengue, West Nile, and Ebola viruses, utilize Axl/Gas6 to facilitate their entry, suggesting that the phosphatidylserine-mediated viral entry mechanism can be shared by various enveloped viruses. In addition to Axl/Gas6, various molecules are known to recognize phosphatidylserine; however, the effects of these molecules on virus binding and entry have not been comprehensively evaluated and compared. In this study, we examined most human phosphatidylserine-recognizing molecules for their abilities to facilitate viral infection. The results provide insights into the role(s) of envelope phosphatidylserine in viral infection, which can be applicable to the development of novel antiviral reagents that block phosphatidylserine-mediated viral entry.

Host-Primed Ebola Virus GP Exposes a Hydrophobic NPC1 Receptor-Binding Pocket, Revealing a Target for Broadly Neutralizing Antibodies.

PubMed

Bornholdt, Zachary A; Ndungo, Esther; Fusco, Marnie L; Bale, Shridhar; Flyak, Andrew I; Crowe, James E; Chandran, Kartik; Saphire, Erica Ollmann

2016-02-23

The filovirus surface glycoprotein (GP) mediates viral entry into host cells. Following viral internalization into endosomes, GP is cleaved by host cysteine proteases to expose a receptor-binding site (RBS) that is otherwise hidden from immune surveillance. Here, we present the crystal structure of proteolytically cleaved Ebola virus GP to a resolution of 3.3 Å. We use this structure in conjunction with functional analysis of a large panel of pseudotyped viruses bearing mutant GP proteins to map the Ebola virus GP endosomal RBS at molecular resolution. Our studies indicate that binding of GP to its endosomal receptor Niemann-Pick C1 occurs in two distinct stages: the initial electrostatic interactions are followed by specific interactions with a hydrophobic trough that is exposed on the endosomally cleaved GP1 subunit. Finally, we demonstrate that monoclonal antibodies targeting the filovirus RBS neutralize all known filovirus GPs, making this conserved pocket a promising target for the development of panfilovirus therapeutics. Ebola virus uses its glycoprotein (GP) to enter new host cells. During entry, GP must be cleaved by human enzymes in order for receptor binding to occur. Here, we provide the crystal structure of the cleaved form of Ebola virus GP. We demonstrate that cleavage exposes a site at the top of GP and that this site binds the critical domain C of the receptor, termed Niemann-Pick C1 (NPC1). We perform mutagenesis to find parts of the site essential for binding NPC1 and map distinct roles for an upper, charged crest and lower, hydrophobic trough in cleaved GP. We find that this 3-dimensional site is conserved across the filovirus family and that antibody directed against this site is able to bind cleaved GP from every filovirus tested and neutralize viruses bearing those GPs. Copyright © 2016 Bornholdt et al.

Interaction of CSFV E2 Protein with Swine Host Factors as Detected by Yeast Two-Hybrid System

PubMed Central

Gladue, Douglas P.; Baker-Bransetter, Ryan; Holinka, Lauren G.; Fernandez-Sainz, Ignacio J.; O’Donnell, Vivian; Fletcher, Paige; Lu, Zhiqiang; Borca, Manuel V.

2014-01-01

E2 is one of the envelope glycoproteins of pestiviruses, including classical swine fever virus (CSFV) and bovine viral diarrhea virus (BVDV). E2 is involved in several critical functions, including virus entry into target cells, induction of a protective immune response and virulence in swine. However, there is no information regarding any host binding partners for the E2 proteins. Here, we utilized the yeast two-hybrid system and identified fifty-seven host proteins as positive binding partners which bound E2 from both CSFV and BVDV with the exception of two proteins that were found to be positive for binding only to CSFV E2. Alanine scanning of CSFV E2 demonstrated that the binding sites for these cellular proteins on E2 are likely non-linear binding sites. The possible roles of the identified host proteins are discussed as the results presented here will be important for future studies to elucidate mechanisms of host protein-virus interactions during pestivirus infection. However, due to the limitations of the yeast two hybrid system, the proteins identified is not exhaustive and each interaction identified needs to be confirmed by independent experimental approaches in the context of virus-infected cells before any definitive conclusion can be drawn on relevance for the virus life cycle. PMID:24416391

The Neck Region of the C-type Lectin DC-SIGN Regulates Its Surface Spatiotemporal Organization and Virus-binding Capacity on Antigen-presenting Cells*

PubMed Central

Manzo, Carlo; Torreno-Pina, Juan A.; Joosten, Ben; Reinieren-Beeren, Inge; Gualda, Emilio J.; Loza-Alvarez, Pablo; Figdor, Carl G.; Garcia-Parajo, Maria F.; Cambi, Alessandra

2012-01-01

The C-type lectin DC-SIGN expressed on dendritic cells (DCs) facilitates capture and internalization of a plethora of different pathogens. Although it is known that DC-SIGN organizes in nanoclusters at the surface of DCs, the molecular mechanisms responsible for this well defined nanopatterning and role in viral binding remain enigmatic. By combining biochemical and advanced biophysical techniques, including optical superresolution and single particle tracking, we demonstrate that DC-SIGN intrinsic nanoclustering strictly depends on its molecular structure. DC-SIGN nanoclusters exhibited free, Brownian diffusion on the cell membrane. Truncation of the extracellular neck region, known to abrogate tetramerization, significantly reduced nanoclustering and concomitantly increased lateral diffusion. Importantly, DC-SIGN nanocluster dissolution exclusively compromised binding to nanoscale size pathogens. Monte Carlo simulations revealed that heterogeneity on nanocluster density and spatial distribution confers broader binding capabilities to DC-SIGN. As such, our results underscore a direct relationship between spatial nanopatterning, driven by intermolecular interactions between the neck regions, and receptor diffusion to provide DC-SIGN with the exquisite ability to dock pathogens at the virus length scale. Insight into how virus receptors are organized prior to virus binding and how they assemble into functional platforms for virus docking is helpful to develop novel strategies to prevent virus entry and infection. PMID:23019323

Antibody neutralization of retargeted measles viruses

PubMed Central

Lech, Patrycja J.; Pappoe, Roland; Nakamura, Takafumi; Tobin, Gregory J.; Nara, Peter L.; Russell, Stephen J.

2014-01-01

The measles virus (MV) vaccine lineage is a promising oncolytic but prior exposure to the measles vaccine or wild-type MV strains limits treatment utility due to the presence of anti-measles antibodies. MV entry can be redirected by displaying a polypeptide ligand on the Hemagglutinin (H) C-terminus. We hypothesized that retargeted MV would escape neutralization by monoclonal antibodies (mAbs) recognizing the H receptor-binding surface and be less susceptible to neutralization by human antisera. Using chimeric H proteins, with and without mutations that ablate MV receptor binding, we show that retargeted MVs escape mAbs that target the H receptor-binding surface by virtue of mutations that ablate infection via SLAM and CD46. However, C-terminally displayed domains do not mediate virus entry in the presence of human antibodies that bind to the underlying H domain. In conclusion, utility of retargeted oncolytic measles viruses does not extend to evasion of human serum neutralization. PMID:24725950

Five of Five VHHs Neutralizing Poliovirus Bind the Receptor-Binding Site

PubMed Central

Strauss, Mike; Schotte, Lise; Thys, Bert; Filman, David J.

2016-01-01

ABSTRACT Nanobodies, or VHHs, that recognize poliovirus type 1 have previously been selected and characterized as candidates for antiviral agents or reagents for standardization of vaccine quality control. In this study, we present high-resolution cryo-electron microscopy reconstructions of poliovirus with five neutralizing VHHs. All VHHs bind the capsid in the canyon at sites that extensively overlap the poliovirus receptor-binding site. In contrast, the interaction involves a unique (and surprisingly extensive) surface for each of the five VHHs. Five regions of the capsid were found to participate in binding with all five VHHs. Four of these five regions are known to alter during the expansion of the capsid associated with viral entry. Interestingly, binding of one of the VHHs, PVSS21E, resulted in significant changes of the capsid structure and thus seems to trap the virus in an early stage of expansion. IMPORTANCE We describe the cryo-electron microscopy structures of complexes of five neutralizing VHHs with the Mahoney strain of type 1 poliovirus at resolutions ranging from 3.8 to 6.3Å. All five VHHs bind deep in the virus canyon at similar sites that overlap extensively with the binding site for the receptor (CD155). The binding surfaces on the VHHs are surprisingly extensive, but despite the use of similar binding surfaces on the virus, the binding surface on the VHHs is unique for each VHH. In four of the five complexes, the virus remains essentially unchanged, but for the fifth there are significant changes reminiscent of but smaller in magnitude than the changes associated with cell entry, suggesting that this VHH traps the virus in a previously undescribed early intermediate state. The neutralizing mechanisms of the VHHs and their potential use as quality control agents for the end game of poliovirus eradication are discussed. PMID:26764003

Five of Five VHHs Neutralizing Poliovirus Bind the Receptor-Binding Site.

PubMed

Strauss, Mike; Schotte, Lise; Thys, Bert; Filman, David J; Hogle, James M

2016-01-13

Nanobodies, or VHHs, that recognize poliovirus type 1 have previously been selected and characterized as candidates for antiviral agents or reagents for standardization of vaccine quality control. In this study, we present high-resolution cryo-electron microscopy reconstructions of poliovirus with five neutralizing VHHs. All VHHs bind the capsid in the canyon at sites that extensively overlap the poliovirus receptor-binding site. In contrast, the interaction involves a unique (and surprisingly extensive) surface for each of the five VHHs. Five regions of the capsid were found to participate in binding with all five VHHs. Four of these five regions are known to alter during the expansion of the capsid associated with viral entry. Interestingly, binding of one of the VHHs, PVSS21E, resulted in significant changes of the capsid structure and thus seems to trap the virus in an early stage of expansion. We describe the cryo-electron microscopy structures of complexes of five neutralizing VHHs with the Mahoney strain of type 1 poliovirus at resolutions ranging from 3.8 to 6.3Å. All five VHHs bind deep in the virus canyon at similar sites that overlap extensively with the binding site for the receptor (CD155). The binding surfaces on the VHHs are surprisingly extensive, but despite the use of similar binding surfaces on the virus, the binding surface on the VHHs is unique for each VHH. In four of the five complexes, the virus remains essentially unchanged, but for the fifth there are significant changes reminiscent of but smaller in magnitude than the changes associated with cell entry, suggesting that this VHH traps the virus in a previously undescribed early intermediate state. The neutralizing mechanisms of the VHHs and their potential use as quality control agents for the end game of poliovirus eradication are discussed. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

A novel mechanism of RNase L inhibition: Theiler's virus L* protein prevents 2-5A from binding to RNase L

PubMed Central

Drappier, Melissa; Elliott, Ruth; Zhang, Rong; Weiss, Susan R.; Silverman, Robert H.

2018-01-01

The OAS/RNase L pathway is one of the best-characterized effector pathways of the IFN antiviral response. It inhibits the replication of many viruses and ultimately promotes apoptosis of infected cells, contributing to the control of virus spread. However, viruses have evolved a range of escape strategies that act against different steps in the pathway. Here we unraveled a novel escape strategy involving Theiler’s murine encephalomyelitis virus (TMEV) L* protein. Previously we found that L* was the first viral protein binding directly RNase L. Our current data show that L* binds the ankyrin repeats R1 and R2 of RNase L and inhibits 2’-5’ oligoadenylates (2-5A) binding to RNase L. Thereby, L* prevents dimerization and oligomerization of RNase L in response to 2-5A. Using chimeric mouse hepatitis virus (MHV) expressing TMEV L*, we showed that L* efficiently inhibits RNase L in vivo. Interestingly, those data show that L* can functionally substitute for the MHV-encoded phosphodiesterase ns2, which acts upstream of L* in the OAS/RNase L pathway, by degrading 2-5A. PMID:29652922

Rapid Bedside Inactivation of Ebola Virus for Safe Nucleic Acid Tests.

PubMed

Rosenstierne, Maiken Worsøe; Karlberg, Helen; Bragstad, Karoline; Lindegren, Gunnel; Stoltz, Malin Lundahl; Salata, Cristiano; Kran, Anne-Marte Bakken; Dudman, Susanne Gjeruldsen; Mirazimi, Ali; Fomsgaard, Anders

2016-10-01

Rapid bedside inactivation of Ebola virus would be a solution for the safety of medical and technical staff, risk containment, sample transport, and high-throughput or rapid diagnostic testing during an outbreak. We show that the commercially available Magna Pure lysis/binding buffer used for nucleic acid extraction inactivates Ebola virus. A rapid bedside inactivation method for nucleic acid tests is obtained by simply adding Magna Pure lysis/binding buffer directly into vacuum blood collection EDTA tubes using a thin needle and syringe prior to sampling. The ready-to-use inactivation vacuum tubes are stable for more than 4 months, and Ebola virus RNA is preserved in the Magna Pure lysis/binding buffer for at least 5 weeks independent of the storage temperature. We also show that Ebola virus RNA can be manually extracted from Magna Pure lysis/binding buffer-inactivated samples using the QIAamp viral RNA minikit. We present an easy and convenient method for bedside inactivation using available blood collection vacuum tubes and reagents. We propose to use this simple method for fast, safe, and easy bedside inactivation of Ebola virus for safe transport and routine nucleic acid detection. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Genetics, Receptor Binding, Replication, and Mammalian Transmission of H4 Avian Influenza Viruses Isolated from Live Poultry Markets in China

PubMed Central

Liang, Libin; Deng, Guohua; Shi, Jianzhong; Wang, Shuai; Zhang, Qianyi; Kong, Huihui; Gu, Chunyang; Guan, Yuntao; Suzuki, Yasuo; Li, Yanbing; Jiang, Yongping; Tian, Guobin; Liu, Liling

2015-01-01

ABSTRACT H4 avian influenza virus (AIV) is one of the most prevalent influenza virus subtypes in the world. However, whether H4 AIVs pose a threat to public health remains largely unclear. Here, we analyzed the phylogenetic relationships, receptor binding properties, replication, and transmissibility in mammals of H4 AIVs isolated from live poultry markets in China between 2009 and 2012. Genomic sequence analysis of 36 representative H4 viruses revealed 32 different genotypes, indicating that these viruses are undergoing complex and frequent reassortment events. All 32 viruses tested could replicate in the respiratory organs of infected mice without prior adaptation. Receptor binding analysis demonstrated that the H4 AIVs bound to α-2,6-linked glycans, although they retained the binding preference for α-2,3-linked glycans. When we tested the direct-contact transmission of 10 H4 viruses in guinea pigs, we found that three viruses did not transmit to any of the contact animals, one virus transmitted to one of three contact animals, and six viruses transmitted to all three contact animals. When we further tested the respiratory droplet transmissibility of four of the viruses that transmitted efficiently via direct contact, we found that three of them could transmit to one or two of the five exposed animals. Our study demonstrates that the current circulating H4 AIVs can infect, replicate in, and transmit to mammalian hosts, thereby posing a potential threat to human health. These findings emphasize the continual need for enhanced surveillance of H4 AIVs. IMPORTANCE Numerous surveillance studies have documented the wide distribution of H4 AIVs throughout the world, yet the biological properties of H4 viruses have not been well studied. In this study, we found that multiple genotypes of H4 viruses are cocirculating in the live poultry markets of China and that H4 viruses can replicate in mice, possess human-type receptor binding specificity, and transmit between guinea pigs via direct contact. Strikingly, some H4 strains also can transmit via respiratory droplet, albeit with limited efficiency. These results clearly show the potential threat posed by H4 viruses to public health. PMID:26581996

Deposition of steeply infalling debris - pebbles, boulders, snowballs, asteroids, comets - around stars

NASA Astrophysics Data System (ADS)

Brown, J. C.; Veras, D.; Gänsicke, B. T.

2017-09-01

When Comet Lovejoy plunged into the Sun, and survived, questions arose about the physics of infall of small bodies. [1,2] has already described this infall in detail. However, a more general analysis for any type of star has been missing. [3] generalized previous studies, with specific applications to white dwarfs. High-metallicity pollution is common in white dwarf stars hosting remnant planetary systems. However, they rarely have detectable debris accretion discs, possibly because much of the influx is fast steeply infalling debris in star-grazing orbits, producing a more tenuous signature than a slowly accreting disc. Processes governing such deposition between the Roche radius and photosphere have so far received little attention and we model them here analytically by extending recent work on sun-grazing comets to white dwarf systems. We find that the evolution of cm-to-km size infallers most strongly depends on two combinations of parameters, which effectively measure sublimation rate and binding strength. We then provide an algorithm to determine the fate of infallers for any white dwarf, and apply the algorithm to four limiting combinations of hot versus cool (young/old) white dwarfs with snowy (weak, volatile) versus rocky (strong, refractory) infallers. We find: (i) Total sublimation above the photosphere befalls all small infallers across the entire white dwarf temperature range, the threshold size rising with it and 100× larger for rock than snow. (ii) All very large objects fragment tidally regardless of temperature: for rock, a0 ≽ 105 cm; for snow, a0 ≽ 103 - 3 × 104 cm across all white dwarf cooling ages. (iii) A considerable range of infaller sizes avoids fragmentation and total sublimation, yielding impacts or grazes with cold white dwarfs. This range rapidly narrows with increasing temperature, especially for snowy bodies. Finally, we briefly discuss how the various forms of deposited debris may finally reach the photosphere surface itself.

Using mutagenesis to explore conserved residues in the RNA-binding groove of influenza A virus nucleoprotein for antiviral drug development

NASA Astrophysics Data System (ADS)

Liu, Chia-Lin; Hung, Hui-Chen; Lo, Shou-Chen; Chiang, Ching-Hui; Chen, I.-Jung; Hsu, John T.-A.; Hou, Ming-Hon

2016-02-01

Nucleoprotein (NP) is the most abundant type of RNA-binding viral protein in influenza A virus-infected cells and is necessary for viral RNA transcription and replication. Recent studies demonstrated that influenza NP is a valid target for antiviral drug development. The surface of the groove, covered with numerous conserved residues between the head and body domains of influenza A NP, plays a crucial role in RNA binding. To explore the mechanism by which NP binds RNA, we performed a series of site-directed mutagenesis in the RNA-binding groove, followed by surface plasmon resonance (SPR), to characterize the interactions between RNA and NP. Furthermore, a role of Y148 in NP stability and NP-RNA binding was evaluated. The aromatic residue of Y148 was found to stack with a nucleotide base. By interrupting the stacking interaction between Y148 and an RNA base, we identified an influenza virus NP inhibitor, (E, E)-1,7-bis(4-hydroxy-3-methoxyphenyl) -1,6-heptadiene-3,5-dione; this inhibitor reduced the NP’s RNA-binding affinity and hindered viral replication. Our findings will be useful for the development of new drugs that disrupt the interaction between RNA and viral NP in the influenza virus.

An immunoassay for the study of DNA-binding activities of herpes simplex virus protein ICP8.

PubMed

Lee, C K; Knipe, D M

1985-06-01

An immunoassay was used to examine the interaction between a herpes simplex virus protein, ICP8, and various types of DNA. The advantage of this assay is that the protein is not subjected to harsh purification procedures. We characterized the binding of ICP8 to both single-stranded (ss) and double-stranded (ds) DNA. ICP8 bound ss DNA fivefold more efficiently than ds DNA, and both binding activities were most efficient in 150 mM NaCl. Two lines of evidence indicate that the binding activities were not identical: (i) ds DNA failed to complete with ss DNA binding even with a large excess of ds DNA; (ii) Scatchard plots of DNA binding with various amounts of DNA were fundamentally different for ss DNA and ds DNA. However, the two activities were related in that ss DNA efficiently competed with the binding of ds DNA. We conclude that the ds DNA-binding activity of ICP8 is probably distinct from the ss DNA-binding activity. No evidence for sequence-specific ds DNA binding was obtained for either the entire herpes simplex virus genome or cloned viral sequences.

Structure of the hepatitis E virus-like particle suggests mechanisms for virus assembly and receptor binding

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

Guu, Tom S.Y.; Liu, Zheng; Ye, Qiaozhen

Hepatitis E virus (HEV), a small, non-enveloped RNA virus in the family Hepeviridae, is associated with endemic and epidemic acute viral hepatitis in developing countries. Our 3.5-{angstrom} structure of a HEV-like particle (VLP) shows that each capsid protein contains 3 linear domains that form distinct structural elements: S, the continuous capsid; P1, 3-fold protrusions; and P2, 2-fold spikes. The S domain adopts a jelly-roll fold commonly observed in small RNA viruses. The P1 and P2 domains both adopt {beta}-barrel folds. Each domain possesses a potential polysaccharide-binding site that may function in cell-receptor binding. Sugar binding to P1 at the capsidmore » protein interface may lead to capsid disassembly and cell entry. Structural modeling indicates that native T = 3 capsid contains flat dimers, with less curvature than those of T = 1 VLP. Our findings significantly advance the understanding of HEV molecular biology and have application to the development of vaccines and antiviral medications.« less

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

Wu, Kailang; Li, Weikai; Peng, Guiqing

NL63 coronavirus (NL63-CoV), a prevalent human respiratory virus, is the only group I coronavirus known to use angiotensin-converting enzyme 2 (ACE2) as its receptor. Incidentally, ACE2 is also used by group II SARS coronavirus (SARS-CoV). We investigated how different groups of coronaviruses recognize the same receptor, whereas homologous group I coronaviruses recognize different receptors. We determined the crystal structure of NL63-CoV spike protein receptor-binding domain (RBD) complexed with human ACE2. NL63-CoV RBD has a novel {beta}-sandwich core structure consisting of 2 layers of {beta}-sheets, presenting 3 discontinuous receptor-binding motifs (RBMs) to bind ACE2. NL63-CoV and SARS-CoV have no structural homologymore » in RBD cores or RBMs; yet the 2 viruses recognize common ACE2 regions, largely because of a 'virus-binding hotspot' on ACE2. Among group I coronaviruses, RBD cores are conserved but RBMs are variable, explaining how these viruses recognize different receptors. These results provide a structural basis for understanding viral evolution and virus-receptor interactions.« less

Association analyses of large-scale glycan microarray data reveal novel host-specific substructures in influenza A virus binding glycans

NASA Astrophysics Data System (ADS)

Zhao, Nan; Martin, Brigitte E.; Yang, Chun-Kai; Luo, Feng; Wan, Xiu-Feng

2015-10-01

Influenza A viruses can infect a wide variety of animal species and, occasionally, humans. Infection occurs through the binding formed by viral surface glycoprotein hemagglutinin and certain types of glycan receptors on host cell membranes. Studies have shown that the α2,3-linked sialic acid motif (SA2,3Gal) in avian, equine, and canine species; the α2,6-linked sialic acid motif (SA2,6Gal) in humans; and SA2,3Gal and SA2,6Gal in swine are responsible for the corresponding host tropisms. However, more detailed and refined substructures that determine host tropisms are still not clear. Thus, in this study, we applied association mining on a set of glycan microarray data for 211 influenza viruses from five host groups: humans, swine, canine, migratory waterfowl, and terrestrial birds. The results suggest that besides Neu5Acα2-6Galβ, human-origin viruses could bind glycans with Neu5Acα2-8Neu5Acα2-8Neu5Ac and Neu5Gcα2-6Galβ1-4GlcNAc substructures; Galβ and GlcNAcβ terminal substructures, without sialic acid branches, were associated with the binding of human-, swine-, and avian-origin viruses; sulfated Neu5Acα2-3 substructures were associated with the binding of human- and swine-origin viruses. Finally, through three-dimensional structure characterization, we revealed that the role of glycan chain shapes is more important than that of torsion angles or of overall structural similarities in virus host tropisms.

High molecular weight polysaccharide that binds and inhibits virus

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

Konowalchuk, Thomas W.; Konowalchuk, Jack

This invention provides a high molecular weight polysaccharide capable of binding to and inhibiting virus and related pharmaceutical formulations and methods of inhibiting viral infectivity and/or pathogenicity, as well as immunogenic compositions. The invention further includes methods of inhibiting the growth of cancer cells and of ameliorating a symptom of aging. Additionally, the invention provides methods of detecting and/or quantifying and/or isolating viruses.

High molecular weight polysaccharide that binds and inhibits virus

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

Konowalchuk, Thomas W

This invention provides a high molecular weight polysaccharide capable of binding to and inhibiting virus and related pharmaceutical formulations and methods on inhibiting viral infectivity and/or pathogenicity, as well as immunogenic compositions. The invention further methods of inhibiting the growth of cancer cells and of ameliorating a symptom of aging. Additionally, the invention provides methods of detecting and/or quantifying and/or isolating viruses.

The neuraminidases of MDCK grown human influenza A(H3N2) viruses isolated since 1994 can demonstrate receptor binding.

PubMed

Mohr, Peter G; Deng, Yi-Mo; McKimm-Breschkin, Jennifer L

2015-04-22

The neuraminidases (NAs) of MDCK passaged human influenza A(H3N2) strains isolated since 2005 are reported to have dual functions of cleavage of sialic acid and receptor binding. NA agglutination of red blood cells (RBCs) can be inhibited by neuraminidase inhibitors (NAIs), thus distinguishing it from haemagglutinin (HA) binding. We wanted to know if viruses prior to 2005 can demonstrate this property. Pairs of influenza A(H3N2) isolates ranging from 1993-2008 passaged in parallel only in eggs or in MDCK cells were tested for inhibition of haemagglutination by various NAIs. Only viruses isolated since 1994 and cultured in MDCK cells bound chicken RBCs solely through their NA. NAI inhibition of agglutination of turkey RBCs was seen for some, but not all of these same MDCK grown viruses. Efficacy of inhibition of enzyme activity and haemagglutination differed between NAIs. For many viruses lower concentrations of oseltamivir could inhibit agglutination compared to zanamivir, although they could both inhibit enzyme activity at comparable concentrations. An E119V mutation reduced sensitivity to oseltamivir and 4-aminoDANA for both the enzyme assay and inhibition of agglutination. Sequence analysis of the NAs and HAs of some paired viruses revealed mutations in the haemagglutinin of all egg passaged viruses. For many of the paired egg and MDCK cultured viruses we found no differences in their NA sequences by Sanger sequencing. However, deep sequencing of MDCK grown isolates revealed low levels of variant populations with mutations at either D151 or T148 in the NA, suggesting mutations at either site may be able to confer this property. The NA active site of MDCK cultured human influenza A(H3N2) viruses isolated since 1994 can express dual enzyme and receptor binding functions. Binding correlated with either D151 or T148 mutations. The catalytic and receptor binding sites do not appear to be structurally identical since relative concentrations of the NAIs to inhibit enzyme activity and agglutination differ.

Cis- and trans-regulation of luteovirus gene expression by the 3’ end of the viral genome

PubMed Central

Miller, W. Allen; Jackson, Jacquelyn; Feng, Ying

2016-01-01

Translation of the 5.7 kb luteovirus genome is controlled by the 3’ untranslated region (UTR). Base pairing between regions of the 3’ UTR and sequences kilobases upstream is required for cap-independent translation and ribosomal frameshifting needed to synthesize the viral replicase. Luteoviruses produce subgenomic RNAs, which can serve as mRNA, but one sgRNA also regulates translation initiation in trans. As on all viruses, the 3’ and 5’ ends contain structures that are presumed to facilitate RNA synthesis. This review describes the structures and interactions of Barley yellow dwarf virus RNA that facilitate the complex interplay between the above events and result in a successful virus infection. We also present surprising results on the apparent lack of need for some subgenomic RNAs for the virus to infect cells or whole plants. In summary, the UTRs of luteoviruses are highly complex entities that control and fine-tune many key events of the virus replication cycle. PMID:25858272

MicroRNAs responding to southern rice black-streaked dwarf virus infection and their target genes associated with symptom development in rice.

PubMed

Xu, Donglin; Mou, Guiping; Wang, Kang; Zhou, Guohui

2014-09-22

Southern rice black-streaked dwarf virus (SRBSDV) is a recently emerged rice virus that has spread across Asia. This devastating virus causes rice plants to produce a variety of symptoms during different growth stages. MicroRNAs (miRNAs) comprise a large group of 21-24-nt RNA molecules that are important regulators of plant development processes and stress responses. In this study, we used microarray profiling to investigate rice miRNAs responding to SRBSDV infection at 3, 9, 15, and 20 days post-inoculation (dpi). Expression levels of 56 miRNAs were altered in SRBSDV-infected rice plants, with these changes classified into eight different regulation patterns according to their temporal expression dynamics. Fourteen miRNAs belonging to six families (miR164, R396, R530, R1846, R1858, and R2097) were significantly regulated at 20 dpi. We used RT-qPCR to search for expression level correlations between members of these families and their putative targets at 3, 9, and 15 dpi. Some members of the miR164, R396, R530, and R1846 families were found to be positively or negatively correlated with their respective targets during 3-15 days after SRBSDV infection, whereas in more cases the rice miRNAs were not in correlation with their targets along the post-inoculation period, suggesting that some additional factors may be involved in rice miRNA-target interactions. The reported functions of rice genes targeted by the miR164, R396, R530, R1846, and R1858 families indicated that these genes are associated with symptom development. These results provide insights into miRNA-mediated SRBSDV-rice interactions. Copyright © 2014 Elsevier B.V. All rights reserved.

Unity in diversity: Shared mechanism of entry among paramyxoviruses

PubMed Central

Palgen, Jean-Louis; Jurgens, Eric M.; Moscona, Anne; Palermo, Laura M.; Porotto, Matteo

2015-01-01

The Paramyxoviridae family includes many viruses that are pathogenic in humans, including parainfluenza viruses, measles virus, respiratory syncytial virus and the emerging zoonotic Henipaviruses. No effective treatments are currently available for these viruses, and there is a need for efficient antiviral therapies. Paramyxoviruses enter the target cell by binding to a cell surface receptor and then fusing the viral envelope with the target cell membrane, allowing the release of the viral genome into the cytoplasm. Blockage of these crucial steps prevents infection and disease. Binding and fusion are driven by two virus encoded glycoproteins, the receptor-binding protein and the fusion protein, that together form the viral “fusion machinery”. The development of efficient antiviral drugs requires a deeper understanding of the mechanism of action of the Paramyxoviridae fusion machinery, which is still controversial. Here we review recent structural and functional data on these proteins and the current understanding of the mechanism of the paramyxovirus cell entry process. PMID:25595799

Electrical detection of single viruses

NASA Astrophysics Data System (ADS)

Patolsky, Fernando; Zheng, Gengfeng; Hayden, Oliver; Lakadamyali, Melike; Zhuang, Xiaowei; Lieber, Charles M.

2004-09-01

We report direct, real-time electrical detection of single virus particles with high selectivity by using nanowire field effect transistors. Measurements made with nanowire arrays modified with antibodies for influenza A showed discrete conductance changes characteristic of binding and unbinding in the presence of influenza A but not paramyxovirus or adenovirus. Simultaneous electrical and optical measurements using fluorescently labeled influenza A were used to demonstrate conclusively that the conductance changes correspond to binding/unbinding of single viruses at the surface of nanowire devices. pH-dependent studies further show that the detection mechanism is caused by a field effect, and that the nanowire devices can be used to determine rapidly isoelectric points and variations in receptor-virus binding kinetics for different conditions. Lastly, studies of nanowire devices modified with antibodies specific for either influenza or adenovirus show that multiple viruses can be selectively detected in parallel. The possibility of large-scale integration of these nanowire devices suggests potential for simultaneous detection of a large number of distinct viral threats at the single virus level.

Lifting DELLA repression of Arabidopsis seed germination by nonproteolytic gibberellin signaling

USDA-ARS?s Scientific Manuscript database

DELLA repression of Arabidopsis seed germination can be lifted through the ubiquitin-proteasome pathway and proteolysis-independent GA signaling. GA-binding to the GID1 (GIBBERELLIN-INSENSITIVE DWARF1) GA receptors stimulates GID1-GA-DELLA complex formation which in turn triggers DELLA protein ubiq...

Image Restoration and Analysis of Influenza Virions Binding to Membrane Receptors Reveal Adhesion-Strengthening Kinetics

PubMed Central

Lee, Donald W.; Hsu, Hung-Lun; Bacon, Kaitlyn B.; Daniel, Susan

2016-01-01

With the development of single-particle tracking (SPT) microscopy and host membrane mimics called supported lipid bilayers (SLBs), stochastic virus-membrane binding interactions can be studied in depth while maintaining control over host receptor type and concentration. However, several experimental design challenges and quantitative image analysis limitations prevent the widespread use of this approach. One main challenge of SPT studies is the low signal-to-noise ratio of SPT videos, which is sometimes inevitable due to small particle sizes, low quantum yield of fluorescent dyes, and photobleaching. These situations could render current particle tracking software to yield biased binding kinetic data caused by intermittent tracking error. Hence, we developed an effective image restoration algorithm for SPT applications called STAWASP that reveals particles with a signal-to-noise ratio of 2.2 while preserving particle features. We tested our improvements to the SPT binding assay experiment and imaging procedures by monitoring X31 influenza virus binding to α2,3 sialic acid glycolipids. Our interests lie in how slight changes to the peripheral oligosaccharide structures can affect the binding rate and residence times of viruses. We were able to detect viruses binding weakly to a glycolipid called GM3, which was undetected via assays such as surface plasmon resonance. The binding rate was around 28 folds higher when the virus bound to a different glycolipid called GD1a, which has a sialic acid group extending further away from the bilayer surface than GM3. The improved imaging allowed us to obtain binding residence time distributions that reflect an adhesion-strengthening mechanism via multivalent bonds. We empirically fitted these distributions using a time-dependent unbinding rate parameter, koff, which diverges from standard treatment of koff as a constant. We further explain how to convert these models to fit ensemble-averaged binding data obtained by assays such as surface plasmon resonance. PMID:27695072

Glycan Engagement Dictates Hydrocephalus Induction by Serotype 1 Reovirus

PubMed Central

Stencel-Baerenwald, Jennifer; Reiss, Kerstin; Blaum, Bärbel S.; Colvin, Daniel; Li, Xiao-Nan; Abel, Ty; Boyd, Kelli; Stehle, Thilo

2015-01-01

ABSTRACT Receptors expressed on the host cell surface adhere viruses to target cells and serve as determinants of viral tropism. Several viruses bind cell surface glycans to facilitate entry, but the contribution of specific glycan moieties to viral disease is incompletely understood. Reovirus provides a tractable experimental model for studies of viral neuropathogenesis. In newborn mice, serotype 1 (T1) reovirus causes hydrocephalus, whereas serotype 3 (T3) reovirus causes encephalitis. T1 and T3 reoviruses engage distinct glycans, suggesting that glycan-binding capacity contributes to these differences in pathogenesis. Using structure-guided mutagenesis, we engineered a mutant T1 reovirus incapable of binding the T1 reovirus-specific glycan receptor, GM2. The mutant virus induced substantially less hydrocephalus than wild-type virus, an effect phenocopied by wild-type virus infection of GM2-deficient mice. In comparison to wild-type virus, yields of mutant virus were diminished in cultured ependymal cells, the cell type that lines the brain ventricles. These findings suggest that GM2 engagement targets reovirus to ependymal cells in mice and illuminate the function of glycan engagement in reovirus serotype-dependent disease. PMID:25736887

ADENOVIRUS INTERACTION WITH ITS CELLULAR RECEPTOR CAR.

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

HOWITT,J.; ANDERSON,C.W.; FREIMUTH,P.

The mechanism of adenovirus attachment to the host cell plasma membrane has been revealed in detail by research over the past 10 years. It has long been known that receptor binding activity is associated with the viral fibers, trimeric spike proteins that protrude radially from the vertices of the icosahedral capsid (Philipson et al. 1968). In some adenovirus serotypes, fiber and other virus structural proteins are synthesized in excess and accumulate in the cell nucleus during late stages of infection. Fiber protein can be readily purified from lysates of cells infected with subgroup C viruses, for example Ad2 and Ad5more » (Boulanger and Puvion 1973). Addition of purified fiber protein to virus suspensions during adsorption strongly inhibits infection, indicating that fiber and intact virus particles compete for binding sites on host cells (Philipson et al. 1968; Hautala et al. 1998). Cell binding studies using purified radiolabeled fiber demonstrated that fiber binds specifically and with high affinity to the cell plasma membrane, and that cell lines typically used for laboratory propagation of adenovirus have approximately 10{sup 4} high-affinity receptor sites per cell (Persson et al. 1985; Freimuth 1996). Similar numbers of high-affinity binding sites for radiolabeled intact virus particles also were observed (Seth et al. 1994).« less

Characterization of soluble glycoprotein D-mediated herpes simplex virus type 1 infection

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

Tsvitov, Marianna; Frampton, Arthur R.; Shah, Waris A.

2007-04-10

Herpes simplex virus type 1 (HSV-1) entry into permissive cells involves attachment to cell-surface glycosaminoglycans (GAGs) and fusion of the virus envelope with the cell membrane triggered by the binding of glycoprotein D (gD) to cognate receptors. In this study, we characterized the observation that soluble forms of the gD ectodomain (sgD) can mediate entry of gD-deficient HSV-1. We examined the efficiency and receptor specificity of this activity and used sequential incubation protocols to determine the order and stability of the initial interactions required for entry. Surprisingly, virus binding to GAGs did not increase the efficiency of sgD-mediated entry andmore » gD-deficient virus was capable of attaching to GAG-deficient cells in the absence of sgD. These observations suggested a novel binding interaction that may play a role in normal HSV infection.« less

Novel rabies virus-neutralizing epitope recognized by human monoclonal antibody: fine mapping and escape mutant analysis.

PubMed

Marissen, Wilfred E; Kramer, R Arjen; Rice, Amy; Weldon, William C; Niezgoda, Michael; Faber, Milosz; Slootstra, Jerry W; Meloen, Rob H; Clijsters-van der Horst, Marieke; Visser, Therese J; Jongeneelen, Mandy; Thijsse, Sandra; Throsby, Mark; de Kruif, John; Rupprecht, Charles E; Dietzschold, Bernhard; Goudsmit, Jaap; Bakker, Alexander B H

2005-04-01

Anti-rabies virus immunoglobulin combined with rabies vaccine protects humans from lethal rabies infections. For cost and safety reasons, replacement of the human or equine polyclonal immunoglobulin is advocated, and the use of rabies virus-specific monoclonal antibodies (MAbs) is recommended. We produced two previously described potent rabies virus-neutralizing human MAbs, CR57 and CRJB, in human PER.C6 cells. The two MAbs competed for binding to rabies virus glycoprotein. Using CR57 and a set of 15-mer overlapping peptides covering the glycoprotein ectodomain, a neutralization domain was identified between amino acids (aa) 218 and 240. The minimal binding region was identified as KLCGVL (aa 226 to 231), with key residues K-CGV- identified by alanine replacement scanning. The critical binding region of this novel nonconformational rabies virus epitope is highly conserved within rabies viruses of genotype 1. Subsequently, we generated six rabies virus variants escaping neutralization by CR57 and six variants escaping CRJB. The CR57 escape mutants were only partially covered by CRJB, and all CRJB-resistant variants completely escaped neutralization by CR57. Without exception, the CR57-resistant variants showed a mutation at key residues within the defined minimal binding region, while the CRJB escape viruses showed a single mutation distant from the CR57 epitope (N182D) combined with mutations in the CR57 epitope. The competition between CR57 and CRJB, the in vitro escape profile, and the apparent overlap between the recognized epitopes argues against including both CR57 and CRJB in a MAb cocktail aimed at replacing classical immunoglobulin preparations.

Antibody Pressure by a Human Monoclonal Antibody Targeting the 2009 Pandemic H1N1 Virus Hemagglutinin Drives the Emergence of a Virus with Increased Virulence in Mice

PubMed Central

O’Donnell, Christopher D.; Vogel, Leatrice; Wright, Amber; Das, Suman R.; Wrammert, Jens; Li, Gui-Mei; McCausland, Megan; Zheng, Nai-Ying; Yewdell, Jonathan W.; Ahmed, Rafi; Wilson, Patrick C.; Subbarao, Kanta

2012-01-01

ABSTRACT In 2009, a novel H1N1 influenza A virus (2009 pH1N1) emerged and caused a pandemic. A human monoclonal antibody (hMAb; EM4C04), highly specific for the 2009 pH1N1 virus hemagglutinin (HA), was isolated from a severely ill 2009 pH1N1 virus-infected patient. We postulated that under immune pressure with EM4C04, the 2009 pH1N1 virus would undergo antigenic drift and mutate at sites that would identify the antibody binding site. To do so, we infected MDCK cells in the presence of EM4C04 and generated 11 escape mutants, displaying 7 distinct amino acid substitutions in the HA. Six substitutions greatly reduced MAb binding (K123N, D131E, K133T, G134S, K157N, and G158E). Residues 131, 133, and 134 are contiguous with residues 157 and 158 in the globular domain structure and contribute to a novel pH1N1 antibody epitope. One mutation near the receptor binding site, S186P, increased the binding affinity of the HA to the receptor. 186P and 131E are present in the highly virulent 1918 virus HA and were recently identified as virulence determinants in a mouse-passaged pH1N1 virus. We found that pH1N1 escape variants expressing these substitutions enhanced replication and lethality in mice compared to wild-type 2009 pH1N1 virus. The increased virulence of these viruses was associated with an increased affinity for α2,3 sialic acid receptors. Our study demonstrates that antibody pressure by an hMAb targeting a novel epitope in the Sa region of 2009 pH1N1 HA is able to inadvertently drive the development of a more virulent virus with altered receptor binding properties. This broadens our understanding of antigenic drift. PMID:22647789

Tripartite Interactions of Barley Yellow Dwarf Virus, Sitobion avenae and Wheat Varieties

PubMed Central

Liu, Xiao-Feng; Hu, Xiang-Shun; Keller, Mike A.; Zhao, Hui-Yan; Wu, Yun-Feng; Liu, Tong-Xian

2014-01-01

The tripartite interactions in a pathosystem involving wheat (Triticum aestivum L.), the Barley yellow dwarf virus (BYDV), and the BYDV vector aphid Sitobion avenae were studied under field conditions to determine the impact of these interactions on aphid populations, virus pathology and grain yield. Wheat varietal resistance to BYDV and aphids varied among the three wheat varieties studied over two consecutive years. The results demonstrated that (1) aphid peak number (APN) in the aphid + BYDV (viruliferous aphid) treatment was greater and occurred earlier than that in the non-viruliferous aphid treatment. The APN and the area under the curve of population dynamics (AUC) on a S. avenae-resistant variety 98-10-30 was significantly lower than on two aphid-susceptible varieties Tam200(13)G and Xiaoyan6. (2) The production of alatae (PA) was greater on the variety 98-10-30 than on the other varieties, and PA was greater in the aphid + BYDV treatment on 98-10-30 than in the non-viruliferous aphid treatment, but this trend was reversed on Tam200(13)G and Xiaoyan6. (3) The BYDV disease incidence (DIC) on the variety 98-10-30 was greater than that on the other two varieties in 2012, and the disease index (DID) on Tam200(13)G was lower than on the other varieties in the aphid + BYDV and BYDV treatments in 2012, but not in 2011 when aphid vector numbers were generally lower. (4) Yield loss in the aphid + BYDV treatment tended to be greater than that in the aphid or BYDV alone treatments across varieties and years. We suggested that aphid population development and BYDV transmission tend to promote each other under field conditions. The aphids + BYDV treatment caused greater yield reductions than non-viruliferous aphids or virus treatment. Wheat varietal resistance in 98-10-30 affects the aphid dispersal, virus transmission and wheat yield loss though inhibits aphid populations from increasing. PMID:25184214

RNAi-mediated resistance to rice black-streaked dwarf virus in transgenic rice.

PubMed

Ahmed, Mohamed M S; Bian, Shiquan; Wang, Muyue; Zhao, Jing; Zhang, Bingwei; Liu, Qiaoquan; Zhang, Changquan; Tang, Shuzhu; Gu, Minghong; Yu, Hengxiu

2017-04-01

Rice black-streaked dwarf virus (RBSDV), a member of the genus Fijivirus in the family Reoviridae, causes significant economic losses in rice production in China and many other Asian countries. Development of resistant varieties by using conventional breeding methods is limited, as germplasm with high level of resistance to RBSDV have not yet been found. One of the most promising methods to confer resistance against RBSDV is the use of RNA interference (RNAi) technology. RBSDV non-structural protein P7-2, encoded by S7-2 gene, is a potential F-box protein and involved in the plant-virus interaction through the ubiquitination pathway. P8, encoded by S8 gene, is the minor core protein that possesses potent active transcriptional repression activity. In this study, we transformed rice calli using a mini-twin T-DNA vector harboring RNAi constructs of the RBSDV genes S7-2 or S8, and obtained plants harboring the target gene constructs and the selectable marker gene, hygromycin phosphotransferase (HPT). From the offspring of these transgenic plants, we obtained selectable marker (HPT gene)-free plants. Homozygous T 5 transgenic lines which harbored either S7-2-RNAi or S8-RNAi exhibited high level resistance against RBSDV under field infection pressure from indigenous viruliferous small brown planthoppers. Thus, our results showed that RNA interference with the expression of S7-2 or S8 genes seemed an effective way to induce high level resistance in rice against RBSD disease.

Virtual screening of potential inhibitors from TCM for the CPSF30 binding site on the NS1A protein of influenza A virus.

PubMed

Ai, Haixin; Zhang, Li; Chang, Alan K; Wei, Hongyun; Che, Yuchen; Liu, Hongsheng

2014-03-01

Inhibition of CPSF30 function by the effector domain of influenza A virus of non-structural protein 1 (NS1A) protein plays a critical role in the suppression of host key antiviral response. The CPSF30-binding site of NS1A appears to be a very attractive target for the development of new drugs against influenza A virus. In this study, structure-based molecular docking was utilized to screen more than 30,000 compounds from a Traditional Chinese Medicine (TCM) database. Four drug-like compounds were selected as potential inhibitors for the CPSF30-binding site of NS1A. Docking conformation analysis results showed that these potential inhibitors could bind to the CPSF30-binding site with strong hydrophobic interactions and weak hydrogen bonds. Molecular dynamics simulations and MM-PBSA calculations suggested that two of the inhibitors, compounds 32056 and 31674, could stably bind to the CPSF30-binding site with high binding free energy. These two compounds could be modified to achieve higher binding affinity, so that they may be used as potential leads in the development of new anti-influenza drugs.

Entry Inhibition of Influenza Viruses with High Mannose Binding Lectin ESA-2 from the Red Alga Eucheuma serra through the Recognition of Viral Hemagglutinin

PubMed Central

Sato, Yuichiro; Morimoto, Kinjiro; Kubo, Takanori; Sakaguchi, Takemasa; Nishizono, Akira; Hirayama, Makoto; Hori, Kanji

2015-01-01

Lectin sensitivity of the recent pandemic influenza A virus (H1N1-2009) was screened for 12 lectins with various carbohydrate specificity by a neutral red dye uptake assay with MDCK cells. Among them, a high mannose (HM)-binding anti-HIV lectin, ESA-2 from the red alga Eucheuma serra, showed the highest inhibition against infection with an EC50 of 12.4 nM. Moreover, ESA-2 exhibited a wide range of antiviral spectrum against various influenza strains with EC50s of pico molar to low nanomolar levels. Besides ESA-2, HM-binding plant lectin ConA, fucose-binding lectins such as fungal AOL from Aspergillus oryzae and AAL from Aleuria aurantia were active against H1N1-2009, but the potency of inhibition was of less magnitude compared with ESA-2. Direct interaction between ESA-2 and a viral envelope glycoprotein, hemagglutinin (HA), was demonstrated by ELISA assay. This interaction was effectively suppressed by glycoproteins bearing HM-glycans, indicating that ESA-2 binds to the HA of influenza virus through HM-glycans. Upon treatment with ESA-2, no viral antigens were detected in the host cells, indicating that ESA-2 inhibited the initial steps of virus entry into the cells. ESA-2 would thus be useful as a novel microbicide to prevent penetration of viruses such as HIV and influenza viruses to the host cells. PMID:26035023

The Crystal Structure and RNA-Binding of an Orthomyxovirus Nucleoprotein

PubMed Central

Zheng, Wenjie; Olson, John; Vakharia, Vikram; Tao, Yizhi Jane

2013-01-01

Genome packaging for viruses with segmented genomes is often a complex problem. This is particularly true for influenza viruses and other orthomyxoviruses, whose genome consists of multiple negative-sense RNAs encapsidated as ribonucleoprotein (RNP) complexes. To better understand the structural features of orthomyxovirus RNPs that allow them to be packaged, we determined the crystal structure of the nucleoprotein (NP) of a fish orthomyxovirus, the infectious salmon anemia virus (ISAV) (genus Isavirus). As the major protein component of the RNPs, ISAV-NP possesses a bi-lobular structure similar to the influenza virus NP. Because both RNA-free and RNA-bound ISAV NP forms stable dimers in solution, we were able to measure the NP RNA binding affinity as well as the stoichiometry using recombinant proteins and synthetic oligos. Our RNA binding analysis revealed that each ISAV-NP binds ∼12 nts of RNA, shorter than the 24–28 nts originally estimated for the influenza A virus NP based on population average. The 12-nt stoichiometry was further confirmed by results from electron microscopy and dynamic light scattering. Considering that RNPs of ISAV and the influenza viruses have similar morphologies and dimensions, our findings suggest that NP-free RNA may exist on orthomyxovirus RNPs, and selective RNP packaging may be accomplished through direct RNA-RNA interactions. PMID:24068932

Sequence- and Interactome-Based Prediction of Viral Protein Hotspots Targeting Host Proteins: A Case Study for HIV Nef

PubMed Central

Sarmady, Mahdi; Dampier, William; Tozeren, Aydin

2011-01-01

Virus proteins alter protein pathways of the host toward the synthesis of viral particles by breaking and making edges via binding to host proteins. In this study, we developed a computational approach to predict viral sequence hotspots for binding to host proteins based on sequences of viral and host proteins and literature-curated virus-host protein interactome data. We use a motif discovery algorithm repeatedly on collections of sequences of viral proteins and immediate binding partners of their host targets and choose only those motifs that are conserved on viral sequences and highly statistically enriched among binding partners of virus protein targeted host proteins. Our results match experimental data on binding sites of Nef to host proteins such as MAPK1, VAV1, LCK, HCK, HLA-A, CD4, FYN, and GNB2L1 with high statistical significance but is a poor predictor of Nef binding sites on highly flexible, hoop-like regions. Predicted hotspots recapture CD8 cell epitopes of HIV Nef highlighting their importance in modulating virus-host interactions. Host proteins potentially targeted or outcompeted by Nef appear crowding the T cell receptor, natural killer cell mediated cytotoxicity, and neurotrophin signaling pathways. Scanning of HIV Nef motifs on multiple alignments of hepatitis C protein NS5A produces results consistent with literature, indicating the potential value of the hotspot discovery in advancing our understanding of virus-host crosstalk. PMID:21738584

High doses of recombinant mannan-binding lectin inhibit the binding of influenza A(H1N1)pdm09 virus with cells expressing DC-SIGN.

PubMed

Yu, Lei; Shang, Shiqiang; Tao, Ran; Wang, Caiyun; Zhang, Li; Peng, Hao; Chen, Yinghu

2017-07-01

The pandemic influenza A (H1N1)pdm09 virus continues to be a threat to human health. Low doses of mannan-binding lectin (MBL) (<1 μg/mL) were shown not to protect against influenza A(H1N1)pdm09 infection. However, the effect of high doses of MBL has not been investigated. Dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) has been proposed as an alternative receptor for influenza A(H1N1)pdm09 virus. In this study, we examined the expression of DC-SIGN on DCs as well as on acute monocytic leukemia cell line, THP-1. High doses of recombinant or human MBL inhibited binding of influenza A(H1N1)pdm09 to both these cell types in the presence of complement derived from bovine serum. Further, anti-DC-SIGN monoclonal antibody inhibited binding of influenza A(H1N1)pdm09 to both DC-SIGN-expressing DCs and THP-1 cells. This study demonstrates that high doses of MBL can inhibit binding of influenza A(H1N1)pdm09 virus to DC-SIGN-expressing cells in the presence of complement. Our results suggest that DC-SIGN may be an alternative receptor for influenza A(H1N1)pdm09 virus. © 2017 APMIS. Published by John Wiley & Sons Ltd.

Plasticity in Structural and Functional Interactions between the Phosphoprotein and Nucleoprotein of Measles Virus*

PubMed Central

Shu, Yaoling; Habchi, Johnny; Costanzo, Stéphanie; Padilla, André; Brunel, Joanna; Gerlier, Denis; Oglesbee, Michael; Longhi, Sonia

2012-01-01

The measles virus (MeV) phosphoprotein (P) tethers the polymerase to the nucleocapsid template for transcription and genome replication. Binding of P to nucleocapsid is mediated by the X domain of P (XD) and a conserved sequence (Box-2) within the C-terminal domain of the nucleoprotein (NTAIL). XD binding induces NTAIL α-helical folding, which in turn has been proposed to stabilize the polymerase-nucleocapsid complex, with cycles of binding and release required for transcription and genome replication. The current work directly assessed the relationships among XD-induced NTAIL folding, XD-NTAIL binding affinity, and polymerase activity. Amino acid substitutions that abolished XD-induced NTAIL α-helical folding were created within Box-2 of Edmonston MeV NTAIL. Polymerase activity in minireplicons was maintained despite a 35-fold decrease in XD-NTAIL binding affinity or reduction/loss of XD-induced NTAIL alpha-helical folding. Recombinant infectious virus was recovered for all mutants, and transcriptase elongation rates remained within a 1.7-fold range of parent virus. Box-2 mutations did however impose a significant cost to infectivity, reflected in an increase in the amount of input genome required to match the infectivity of parent virus. Diminished infectivity could not be attributed to changes in virion protein composition or production of defective interfering particles, where changes from parent virus were within a 3-fold range. The results indicated that MeV polymerase activity, but not infectivity, tolerates amino acid changes in the XD-binding region of the nucleoprotein. Selectional pressure for conservation of the Box-2 sequence may thus reflect a role in assuring the fidelity of polymerase functions or the assembly of viral particles required for optimal infectivity. PMID:22318731

Plasticity in structural and functional interactions between the phosphoprotein and nucleoprotein of measles virus.

PubMed

Shu, Yaoling; Habchi, Johnny; Costanzo, Stéphanie; Padilla, André; Brunel, Joanna; Gerlier, Denis; Oglesbee, Michael; Longhi, Sonia

2012-04-06

The measles virus (MeV) phosphoprotein (P) tethers the polymerase to the nucleocapsid template for transcription and genome replication. Binding of P to nucleocapsid is mediated by the X domain of P (XD) and a conserved sequence (Box-2) within the C-terminal domain of the nucleoprotein (N(TAIL)). XD binding induces N(TAIL) α-helical folding, which in turn has been proposed to stabilize the polymerase-nucleocapsid complex, with cycles of binding and release required for transcription and genome replication. The current work directly assessed the relationships among XD-induced N(TAIL) folding, XD-N(TAIL) binding affinity, and polymerase activity. Amino acid substitutions that abolished XD-induced N(TAIL) α-helical folding were created within Box-2 of Edmonston MeV N(TAIL). Polymerase activity in minireplicons was maintained despite a 35-fold decrease in XD-N(TAIL) binding affinity or reduction/loss of XD-induced N(TAIL) alpha-helical folding. Recombinant infectious virus was recovered for all mutants, and transcriptase elongation rates remained within a 1.7-fold range of parent virus. Box-2 mutations did however impose a significant cost to infectivity, reflected in an increase in the amount of input genome required to match the infectivity of parent virus. Diminished infectivity could not be attributed to changes in virion protein composition or production of defective interfering particles, where changes from parent virus were within a 3-fold range. The results indicated that MeV polymerase activity, but not infectivity, tolerates amino acid changes in the XD-binding region of the nucleoprotein. Selectional pressure for conservation of the Box-2 sequence may thus reflect a role in assuring the fidelity of polymerase functions or the assembly of viral particles required for optimal infectivity.

Conserved neutralizing epitope at globular head of hemagglutinin in H3N2 influenza viruses.

PubMed

Iba, Yoshitaka; Fujii, Yoshifumi; Ohshima, Nobuko; Sumida, Tomomi; Kubota-Koketsu, Ritsuko; Ikeda, Mariko; Wakiyama, Motoaki; Shirouzu, Mikako; Okada, Jun; Okuno, Yoshinobu; Kurosawa, Yoshikazu; Yokoyama, Shigeyuki

2014-07-01

Neutralizing antibodies that target the hemagglutinin of influenza virus either inhibit binding of hemagglutinin to cellular receptors or prevent the low-pH-induced conformational change in hemagglutinin required for membrane fusion. In general, the former type of antibody binds to the globular head formed by HA1 and has narrow strain specificity, while the latter type binds to the stem mainly formed by HA2 and has broad strain specificity. In the present study, we analyzed the epitope and function of a broadly neutralizing human antibody against H3N2 viruses, F005-126. The crystal structure of F005-126 Fab in complex with hemagglutinin revealed that the antibody binds to the globular head, spans a cleft formed by two hemagglutinin monomers in a hemagglutinin trimer, and cross-links them. It recognizes two peptide portions (sites L and R) and a glycan linked to asparagine at residue 285 using three complementarity-determining regions and framework 3 in the heavy chain. Binding of the antibody to sites L (residues 171 to 173, 239, and 240) and R (residues 91, 92, 270 to 273, 284, and 285) is mediated mainly by van der Waals contacts with the main chains of the peptides in these sites and secondarily by hydrogen bonds with a few side chains of conserved sequences in HA1. Furthermore, the glycan recognized by F005-126 is conserved among H3N2 viruses. F005-126 has the ability to prevent low-pH-induced conformational changes in hemagglutinin. The newly identified conserved epitope, including the glycan, should be immunogenic in humans and may induce production of broadly neutralizing antibodies against H3 viruses. Antibodies play an important role in protection against influenza virus, and hemagglutinin is the major target for virus neutralizing antibodies. It has long been believed that all effective neutralizing antibodies bind to the surrounding regions of the sialic acid-binding pocket and inhibit the binding of hemagglutinin to the cellular receptor. Since mutations are readily introduced into such epitopes, this type of antibody shows narrow strain specificity. Recently, however, broadly neutralizing antibodies have been isolated. Most of these bind either to conserved sites in the stem region or to the sialic acid-binding pocket itself. In the present study, we identified a new neutralizing epitope in the head region recognized by a broadly neutralizing human antibody against H3N2. This epitope may be useful for design of vaccines. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Conserved Neutralizing Epitope at Globular Head of Hemagglutinin in H3N2 Influenza Viruses

PubMed Central

Iba, Yoshitaka; Fujii, Yoshifumi; Ohshima, Nobuko; Sumida, Tomomi; Kubota-Koketsu, Ritsuko; Ikeda, Mariko; Wakiyama, Motoaki; Shirouzu, Mikako; Okada, Jun; Okuno, Yoshinobu; Yokoyama, Shigeyuki

2014-01-01

ABSTRACT Neutralizing antibodies that target the hemagglutinin of influenza virus either inhibit binding of hemagglutinin to cellular receptors or prevent the low-pH-induced conformational change in hemagglutinin required for membrane fusion. In general, the former type of antibody binds to the globular head formed by HA1 and has narrow strain specificity, while the latter type binds to the stem mainly formed by HA2 and has broad strain specificity. In the present study, we analyzed the epitope and function of a broadly neutralizing human antibody against H3N2 viruses, F005-126. The crystal structure of F005-126 Fab in complex with hemagglutinin revealed that the antibody binds to the globular head, spans a cleft formed by two hemagglutinin monomers in a hemagglutinin trimer, and cross-links them. It recognizes two peptide portions (sites L and R) and a glycan linked to asparagine at residue 285 using three complementarity-determining regions and framework 3 in the heavy chain. Binding of the antibody to sites L (residues 171 to 173, 239, and 240) and R (residues 91, 92, 270 to 273, 284, and 285) is mediated mainly by van der Waals contacts with the main chains of the peptides in these sites and secondarily by hydrogen bonds with a few side chains of conserved sequences in HA1. Furthermore, the glycan recognized by F005-126 is conserved among H3N2 viruses. F005-126 has the ability to prevent low-pH-induced conformational changes in hemagglutinin. The newly identified conserved epitope, including the glycan, should be immunogenic in humans and may induce production of broadly neutralizing antibodies against H3 viruses. IMPORTANCE Antibodies play an important role in protection against influenza virus, and hemagglutinin is the major target for virus neutralizing antibodies. It has long been believed that all effective neutralizing antibodies bind to the surrounding regions of the sialic acid-binding pocket and inhibit the binding of hemagglutinin to the cellular receptor. Since mutations are readily introduced into such epitopes, this type of antibody shows narrow strain specificity. Recently, however, broadly neutralizing antibodies have been isolated. Most of these bind either to conserved sites in the stem region or to the sialic acid-binding pocket itself. In the present study, we identified a new neutralizing epitope in the head region recognized by a broadly neutralizing human antibody against H3N2. This epitope may be useful for design of vaccines. PMID:24719430

A pseudoreceptor modelling study of the varicella-zoster virus and human thymidine kinase binding sites

NASA Astrophysics Data System (ADS)

Greenidge, Paulette A.; Merz, Alfred; Folkers, Gerd

1995-12-01

A representative range of pyrimidine nucleoside analogues that are known to inhibit herpes simplex virus (HSV) replication have been used to construct receptor binding site models for the varicella-zoster virus (VZV), thymidine kinase (TK) and human TK1. Given a set of interacting ligands, superimposed in such a manner as to define a pharmacophore, the pseudoreceptor modelling technique Yak provides a means of building binding site models of macromolecules for which no three-dimensional experimental structures are available. Once the models have been evaluated by their ability to reproduce experimental binding data [Vedani et al., J. Am. Chem. Soc., 117 (1995) 4987], they can be used for predictive purposes. Calculated and experimental values of relative binding affinity are compared. Our models suggest that the substitution of one residue may be sufficient to determine ligand subtype affinity.

Molecular mechanism of respiratory syncytial virus fusion inhibitors

PubMed Central

Battles, Michael B; Langedijk, Johannes P; Furmanova-Hollenstein, Polina; Chaiwatpongsakorn, Supranee; Costello, Heather M; Kwanten, Leen; Vranckx, Luc; Vink, Paul; Jaensch, Steffen; Jonckers, Tim H M; Koul, Anil; Arnoult, Eric; Peeples, Mark E; Roymans, Dirk; McLellan, Jason S

2016-01-01

Respiratory syncytial virus (RSV) is a leading cause of pneumonia and bronchiolitis in young children and the elderly. Therapeutic small molecules have been developed that bind the RSV F glycoprotein and inhibit membrane fusion, yet their binding sites and molecular mechanisms of action remain largely unknown. Here we show that these inhibitors bind to a three-fold-symmetric pocket within the central cavity of the metastable prefusion conformation of RSV F. Inhibitor binding stabilizes this conformation by tethering two regions that must undergo a structural rearrangement to facilitate membrane fusion. Inhibitor-escape mutations occur in residues that directly contact the inhibitors or are involved in the conformational rearrangements required to accommodate inhibitor binding. Resistant viruses do not propagate as well as wild-type RSV in vitro, indicating a fitness cost for inhibitor escape. Collectively, these findings provide new insight into class I viral fusion proteins and should facilitate development of optimal RSV fusion inhibitors. PMID:26641933

Identification of the heparin binding site on adeno-associated virus serotype 3B (AAV-3B)

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

Lerch, Thomas F.; Chapman, Michael S., E-mail: chapmami@ohsu.edu

2012-02-05

Adeno-associated virus is a promising vector for gene therapy. In the current study, the binding site on AAV serotype 3B for the heparan sulfate proteoglycan (HSPG) receptor has been characterized. X-ray diffraction identified a disaccharide binding site at the most positively charged region on the virus surface. The contributions of basic amino acids at this and other sites were characterized using site-directed mutagenesis. Both heparin and cell binding are correlated to positive charge at the disaccharide binding site, and transduction is significantly decreased in AAV-3B vectors mutated at this site to reduce heparin binding. While the receptor attachment sites ofmore » AAV-3B and AAV-2 are both in the general vicinity of the viral spikes, the exact amino acids that participate in electrostatic interactions are distinct. Diversity in the mechanisms of cell attachment by AAV serotypes will be an important consideration for the rational design of improved gene therapy vectors.« less

Identification of the heparin binding site on adeno-associated virus serotype 3B (AAV-3B)

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

Lerch, Thomas F.; Chapman, Michael S.

2012-05-24

Adeno-associated virus is a promising vector for gene therapy. In the current study, the binding site on AAV serotype 3B for the heparan sulfate proteoglycan (HSPG) receptor has been characterized. X-ray diffraction identified a disaccharide binding site at the most positively charged region on the virus surface. The contributions of basic amino acids at this and other sites were characterized using site-directed mutagenesis. Both heparin and cell binding are correlated to positive charge at the disaccharide binding site, and transduction is significantly decreased in AAV-3B vectors mutated at this site to reduce heparin binding. While the receptor attachment sites ofmore » AAV-3B and AAV-2 are both in the general vicinity of the viral spikes, the exact amino acids that participate in electrostatic interactions are distinct. Diversity in the mechanisms of cell attachment by AAV serotypes will be an important consideration for the rational design of improved gene therapy vectors.« less

Cationic polymer brush-modified cellulose nanocrystals for high-affinity virus binding

NASA Astrophysics Data System (ADS)

Rosilo, Henna; McKee, Jason R.; Kontturi, Eero; Koho, Tiia; Hytönen, Vesa P.; Ikkala, Olli; Kostiainen, Mauri A.

2014-09-01

Surfaces capable of high-affinity binding of biomolecules are required in several biotechnological applications, such as purification, transfection, and sensing. Therein, the rod-shaped, colloidal cellulose nanocrystals (CNCs) are appealing due to their large surface area available for functionalization. In order to exploit electrostatic binding, their intrinsically anionic surfaces have to be cationized as biological supramolecules are predominantly anionic. Here we present a facile way to prepare cationic CNCs by surface-initiated atom-transfer radical polymerization of poly(N,N-dimethylaminoethyl methacrylate) and subsequent quaternization of the polymer pendant amino groups. The cationic polymer brush-modified CNCs maintained excellent dispersibility and colloidal stability in water and showed a ζ-potential of +38 mV. Dynamic light scattering and electron microscopy showed that the modified CNCs electrostatically bind cowpea chlorotic mottle virus and norovirus-like particles with high affinity. Addition of only a few weight percent of the modified CNCs in water dispersions sufficed to fully bind the virus capsids to form micrometer-sized assemblies. This enabled the concentration and extraction of the virus particles from solution by low-speed centrifugation. These results show the feasibility of the modified CNCs in virus binding and concentrating, and pave the way for their use as transduction enhancers for viral delivery applications.Surfaces capable of high-affinity binding of biomolecules are required in several biotechnological applications, such as purification, transfection, and sensing. Therein, the rod-shaped, colloidal cellulose nanocrystals (CNCs) are appealing due to their large surface area available for functionalization. In order to exploit electrostatic binding, their intrinsically anionic surfaces have to be cationized as biological supramolecules are predominantly anionic. Here we present a facile way to prepare cationic CNCs by surface-initiated atom-transfer radical polymerization of poly(N,N-dimethylaminoethyl methacrylate) and subsequent quaternization of the polymer pendant amino groups. The cationic polymer brush-modified CNCs maintained excellent dispersibility and colloidal stability in water and showed a ζ-potential of +38 mV. Dynamic light scattering and electron microscopy showed that the modified CNCs electrostatically bind cowpea chlorotic mottle virus and norovirus-like particles with high affinity. Addition of only a few weight percent of the modified CNCs in water dispersions sufficed to fully bind the virus capsids to form micrometer-sized assemblies. This enabled the concentration and extraction of the virus particles from solution by low-speed centrifugation. These results show the feasibility of the modified CNCs in virus binding and concentrating, and pave the way for their use as transduction enhancers for viral delivery applications. Electronic supplementary information (ESI) available: CNC surface chain fraction and degree of substitution after BriBBr modification, NMR spectra of the SI-ATRP reaction mixture at 0 and 120 min, conversion of the DMAEMA monomer during SI-ATRP, DLS size distribution profiles of CNCs and CNC-g-P(QDMAEMA), TEM images of NoV-VLPs and their complexes with CNC-g-P(QDMAEMA) at 0 mM NaCl. See DOI: 10.1039/c4nr03584d

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

Clausen, Drew; Wade, Richard A.; Kopparapu, Ravi Kumar

Binaries that contain a hot subdwarf (sdB) star and a main-sequence companion may have interacted in the past. This binary population has historically helped determine our understanding of binary stellar evolution. We have computed a grid of binary population synthesis models using different assumptions about the minimum core mass for helium ignition, the envelope binding energy, the common-envelope ejection efficiency, the amount of mass and angular momentum lost during stable mass transfer, and the criteria for stable mass transfer on the red giant branch and in the Hertzsprung gap. These parameters separately and together can significantly change the entire predictedmore » population of sdBs. Nonetheless, several different parameter sets can reproduce the observed subpopulation of sdB + white dwarf and sdB + M dwarf binaries, which has been used to constrain these parameters in previous studies. The period distribution of sdB + early F dwarf binaries offers a better test of different mass transfer scenarios for stars that fill their Roche lobes on the red giant branch.« less

A universal PCR primer to detect members of the Potyviridae and its use to examine the taxonomic status of several members of the family.

PubMed

Chen, J; Chen, J; Adams, M J

2001-01-01

A universal primer (Sprimer: 5'-GGX AAY AAY AGY GGX CAZ CC-3', X = A, G, C or T; Y = T or C; Z = A or G), designed from the consensus sequences that code for the conserved sequence GNNSGQP in the NIb region of members of the family Potyviridae, was used to amplify by RT-PCR the 3'-terminal genome regions from infected plant samples representing 21 different viruses in the family. Sequencing of some of the fragments (c. 1.7 kb) showed that the type strain (ATTC PV-107) of Oat necrotic mottle virus is not a distinct species in the genus Rymovirus, but is synonymous with Brome streak mosaic virus (genus Tritimovirus) and that Celery mosaic virus is a distinct member of the genus Potyvirus not closely related to any other sequenced species. Potyviruses infecting crops in China were also investigated, showing that viruses on cowpea and maize in Hangzhou, Zhejiang province were respectively Bean common mosaic virus and Sugarcane mosaic virus and that one on garlic in Nanjing, Jiangsu province was Onion yellow dwarf virus. Fragments were also sequenced from Chinese isolates of Lettuce mosaic virus and Soybean mosaic virus (from Hangzhou), Turnip mosaic virus (2 different isolates from Zhejiang province) and RNA1 of Wheat yellow mosaic virus (from Rongcheng, Shandong province).

Antibody neutralization of retargeted measles viruses.

PubMed

Lech, Patrycja J; Pappoe, Roland; Nakamura, Takafumi; Tobin, Gregory J; Nara, Peter L; Russell, Stephen J

2014-04-01

The measles virus (MV) vaccine lineage is a promising oncolytic but prior exposure to the measles vaccine or wild-type MV strains limits treatment utility due to the presence of anti-measles antibodies. MV entry can be redirected by displaying a polypeptide ligand on the Hemagglutinin (H) C-terminus. We hypothesized that retargeted MV would escape neutralization by monoclonal antibodies (mAbs) recognizing the H receptor-binding surface and be less susceptible to neutralization by human antisera. Using chimeric H proteins, with and without mutations that ablate MV receptor binding, we show that retargeted MVs escape mAbs that target the H receptor-binding surface by virtue of mutations that ablate infection via SLAM and CD46. However, C-terminally displayed domains do not mediate virus entry in the presence of human antibodies that bind to the underlying H domain. In conclusion, utility of retargeted oncolytic measles viruses does not extend to evasion of human serum neutralization. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Structure of Epstein-Barr Virus Glycoprotein 42 Suggests a Mechanism for Triggering Receptor-Activated Virus Entry

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

Kirschner, Austin N.; Sorem, Jessica; Longnecker, Richard

Epstein-Barr virus requires glycoproteins gH/gL, gB, and gp42 to fuse its lipid envelope with B cells. Gp42 is a type II membrane protein consisting of a flexible N-terminal region, which binds gH/gL, and a C-terminal lectin-like domain that binds to the B-cell entry receptor human leukocyte antigen (HLA) class II. Gp42 triggers membrane fusion after HLA binding, a process that requires simultaneous binding to gH/gL and a functional hydrophobic pocket in the lectin domain adjacent to the HLA binding site. Here we present the structure of gp42 in its unbound form. Comparisons to the previously determined structure of a gp42:HLAmore » complex reveals additional N-terminal residues forming part of the gH/gL binding site and structural changes in the receptor binding domain. Although the core of the lectin domain remains similar, significant shifts in two loops and an {alpha} helix bordering the essential hydrophobic pocket suggest a structural mechanism for triggering fusion.« less

Mutations in type 3 reovirus that determine binding to sialic acid are contained in the fibrous tail domain of viral attachment protein sigma1.

PubMed

Chappell, J D; Gunn, V L; Wetzel, J D; Baer, G S; Dermody, T S

1997-03-01

The reovirus attachment protein, sigma1, determines numerous aspects of reovirus-induced disease, including viral virulence, pathways of spread, and tropism for certain types of cells in the central nervous system. The sigma1 protein projects from the virion surface and consists of two distinct morphologic domains, a virion-distal globular domain known as the head and an elongated fibrous domain, termed the tail, which is anchored into the virion capsid. To better understand structure-function relationships of sigma1 protein, we conducted experiments to identify sequences in sigma1 important for viral binding to sialic acid, a component of the receptor for type 3 reovirus. Three serotype 3 reovirus strains incapable of binding sialylated receptors were adapted to growth in murine erythroleukemia (MEL) cells, in which sialic acid is essential for reovirus infectivity. MEL-adapted (MA) mutant viruses isolated by serial passage in MEL cells acquired the capacity to bind sialic acid-containing receptors and demonstrated a dependence on sialic acid for infection of MEL cells. Analysis of reassortant viruses isolated from crosses of an MA mutant virus and a reovirus strain that does not bind sialic acid indicated that the sigma1 protein is solely responsible for efficient growth of MA mutant viruses in MEL cells. The deduced sigma1 amino acid sequences of the MA mutant viruses revealed that each strain contains a substitution within a short region of sequence in the sigma1 tail predicted to form beta-sheet. These studies identify specific sequences that determine the capacity of reovirus to bind sialylated receptors and suggest a location for a sialic acid-binding domain. Furthermore, the results support a model in which type 3 sigma1 protein contains discrete receptor binding domains, one in the head and another in the tail that binds sialic acid.

VP24 Is a Chitin-Binding Protein Involved in White Spot Syndrome Virus Infection

PubMed Central

Li, Zaipeng; Han, Yali; Xu, Limei

2015-01-01

ABSTRACT Oral ingestion is the major route of infection for the white spot syndrome virus (WSSV). However, the mechanism by which virus particles in the digestive tract invade host cells is unknown. In the present study, we demonstrate that WSSV virions can bind to chitin through one of the major envelope proteins (VP24). Mutagenesis analysis indicated that amino acids (aa) 186 to 200 in the C terminus of VP24 were required for chitin binding. Moreover, the P-VP24186–200 peptide derived from the VP24 chitin binding region significantly inhibited the VP24-chitin interaction and the WSSV-chitin interaction, implying that VP24 participates in WSSV binding to chitin. Oral inoculation experiments showed that P-VP24186–200 treatment reduced the number of virus particles remaining in the digestive tract during the early stage of infection and greatly hindered WSSV proliferation in shrimp. These data indicate that binding of WSSV to chitin through the viral envelope protein VP24 is essential for WSSV per os infection and provide new ideas for preventing WSSV infection in shrimp farms. IMPORTANCE In this study, we show that WSSV can bind to chitin through the envelope protein VP24. The chitin-binding domain of VP24 maps to amino acids 186 to 200 in the C terminus. Binding of WSSV to chitin through the viral envelope protein VP24 is essential for WSSV per os infection. These findings not only extend our knowledge of WSSV infection but also provide new insights into strategies to prevent WSSV infection in shrimp farms. PMID:26512091

Migration of rice planthoppers and their vectored re-emerging and novel rice viruses in East Asia

PubMed Central

Otuka, Akira

2013-01-01

This review examines recent studies of the migration of three rice planthoppers, Laodelphax striatellus, Sogatella furcifera, and Nilaparvata lugens, in East Asia. Laodelphax striatellus has recently broken out in Jiangsu province, eastern China. The population density in the province started to increase in the early 2000s and peaked in 2004. In 2005, Rice stripe virus (RSV) viruliferous rate of L. striatellus peaked at 31.3%. Since then, rice stripe disease spread severely across the whole province. Due to the migration of the RSV vectors, the rice stripe disease spread to neighboring countries Japan and Korea. An overseas migration of L. striatellus that occurred in 2008 was analyzed, when a slow-moving cold vortex, a type of low pressure system, reached western Japan from Jiangsu, carrying the insects into Japan. Subsequently the rice stripe diseases struck these areas in Japan severely. In Korea, similar situations occurred in 2009, 2011, and 2012. Their migration sources were also estimated to be in Jiangsu by backward trajectory analysis. Rice black-streaked dwarf virus, whose vector is L. striatellus, has recently re-emerged in eastern China, and the evidence for overseas migrations of the virus, just like the RSV’s migrations, has been given. A method of predicting the overseas migration of L. striatellus has been developed by Japanese, Chinese, and Korean institutes. An evaluation of the prediction showed that this method properly predicted migration events that occurred in East Asia from 2008 to 2011. Southern rice black-streaked dwarf virus (SRBSDV) was first found in Guangdong province. Its vector is S. furcifera. An outbreak of SRBSDV occurred in southern China in 2009 and spread to Vietnam the same year. This disease and virus were also found in Japan in 2010. The epidemic triggered many migration studies to investigate concrete spring-summer migration routes in China, and the addition of migration sources for early arrivals in Guangdong and Guangxi have been proposed. Nilaparvata lugens is also an important insect pest of rice. Its migration situations on the Indochina peninsula and return migrations in China are discussed. PMID:24312081

Virus infection mediates the effects of elevated CO2 on plants and vectors.

PubMed

Trębicki, Piotr; Vandegeer, Rebecca K; Bosque-Pérez, Nilsa A; Powell, Kevin S; Dader, Beatriz; Freeman, Angela J; Yen, Alan L; Fitzgerald, Glenn J; Luck, Jo E

2016-03-04

Atmospheric carbon dioxide (CO2) concentration has increased significantly and is projected to double by 2100. To increase current food production levels, understanding how pests and diseases respond to future climate driven by increasing CO2 is imperative. We investigated the effects of elevated CO2 (eCO2) on the interactions among wheat (cv. Yitpi), Barley yellow dwarf virus and an important pest and virus vector, the bird cherry-oat aphid (Rhopalosiphum padi), by examining aphid life history, feeding behavior and plant physiology and biochemistry. Our results showed for the first time that virus infection can mediate effects of eCO2 on plants and pathogen vectors. Changes in plant N concentration influenced aphid life history and behavior, and N concentration was affected by virus infection under eCO2. We observed a reduction in aphid population size and increased feeding damage on noninfected plants under eCO2 but no changes to population and feeding on virus-infected plants irrespective of CO2 treatment. We expect potentially lower future aphid populations on noninfected plants but no change or increased aphid populations on virus-infected plants therefore subsequent virus spread. Our findings underscore the complexity of interactions between plants, insects and viruses under future climate with implications for plant disease epidemiology and crop production.

Virus infection mediates the effects of elevated CO2 on plants and vectors

PubMed Central

Trębicki, Piotr; Vandegeer, Rebecca K.; Bosque-Pérez, Nilsa A.; Powell, Kevin S.; Dader, Beatriz; Freeman, Angela J.; Yen, Alan L.; Fitzgerald, Glenn J.; Luck, Jo E.

2016-01-01

Atmospheric carbon dioxide (CO2) concentration has increased significantly and is projected to double by 2100. To increase current food production levels, understanding how pests and diseases respond to future climate driven by increasing CO2 is imperative. We investigated the effects of elevated CO2 (eCO2) on the interactions among wheat (cv. Yitpi), Barley yellow dwarf virus and an important pest and virus vector, the bird cherry-oat aphid (Rhopalosiphum padi), by examining aphid life history, feeding behavior and plant physiology and biochemistry. Our results showed for the first time that virus infection can mediate effects of eCO2 on plants and pathogen vectors. Changes in plant N concentration influenced aphid life history and behavior, and N concentration was affected by virus infection under eCO2. We observed a reduction in aphid population size and increased feeding damage on noninfected plants under eCO2 but no changes to population and feeding on virus-infected plants irrespective of CO2 treatment. We expect potentially lower future aphid populations on noninfected plants but no change or increased aphid populations on virus-infected plants therefore subsequent virus spread. Our findings underscore the complexity of interactions between plants, insects and viruses under future climate with implications for plant disease epidemiology and crop production. PMID:26941044

Virus infection mediates the effects of elevated CO2 on plants and vectors

NASA Astrophysics Data System (ADS)

Trębicki, Piotr; Vandegeer, Rebecca K.; Bosque-Pérez, Nilsa A.; Powell, Kevin S.; Dader, Beatriz; Freeman, Angela J.; Yen, Alan L.; Fitzgerald, Glenn J.; Luck, Jo E.

2016-03-01

Atmospheric carbon dioxide (CO2) concentration has increased significantly and is projected to double by 2100. To increase current food production levels, understanding how pests and diseases respond to future climate driven by increasing CO2 is imperative. We investigated the effects of elevated CO2 (eCO2) on the interactions among wheat (cv. Yitpi), Barley yellow dwarf virus and an important pest and virus vector, the bird cherry-oat aphid (Rhopalosiphum padi), by examining aphid life history, feeding behavior and plant physiology and biochemistry. Our results showed for the first time that virus infection can mediate effects of eCO2 on plants and pathogen vectors. Changes in plant N concentration influenced aphid life history and behavior, and N concentration was affected by virus infection under eCO2. We observed a reduction in aphid population size and increased feeding damage on noninfected plants under eCO2 but no changes to population and feeding on virus-infected plants irrespective of CO2 treatment. We expect potentially lower future aphid populations on noninfected plants but no change or increased aphid populations on virus-infected plants therefore subsequent virus spread. Our findings underscore the complexity of interactions between plants, insects and viruses under future climate with implications for plant disease epidemiology and crop production.

Biophysical Characterization of Nucleophosmin Interactions with Human Immunodeficiency Virus Rev and Herpes Simplex Virus US11

PubMed Central

Nouri, Kazem; Moll, Jens M.; Milroy, Lech-Gustav; Hain, Anika; Dvorsky, Radovan; Amin, Ehsan; Lenders, Michael; Nagel-Steger, Luitgard; Howe, Sebastian; Smits, Sander H. J.; Hengel, Hartmut; Schmitt, Lutz; Münk, Carsten; Brunsveld, Luc; Ahmadian, Mohammad R.

2015-01-01

Nucleophosmin (NPM1, also known as B23, numatrin or NO38) is a pentameric RNA-binding protein with RNA and protein chaperon functions. NPM1 has increasingly emerged as a potential cellular factor that directly associates with viral proteins; however, the significance of these interactions in each case is still not clear. In this study, we have investigated the physical interaction of NPM1 with both human immunodeficiency virus type 1 (HIV-1) Rev and Herpes Simplex virus type 1 (HSV-1) US11, two functionally homologous proteins. Both viral proteins show, in mechanistically different modes, high affinity for a binding site on the N-terminal oligomerization domain of NPM1. Rev, additionally, exhibits low-affinity for the central histone-binding domain of NPM1. We also showed that the proapoptotic cyclic peptide CIGB-300 specifically binds to NPM1 oligomerization domain and blocks its association with Rev and US11. Moreover, HIV-1 virus production was significantly reduced in the cells treated with CIGB-300. Results of this study suggest that targeting NPM1 may represent a useful approach for antiviral intervention. PMID:26624888

Novel Rabies Virus-Neutralizing Epitope Recognized by Human Monoclonal Antibody: Fine Mapping and Escape Mutant Analysis†

PubMed Central

Marissen, Wilfred E.; Kramer, R. Arjen; Rice, Amy; Weldon, William C.; Niezgoda, Michael; Faber, Milosz; Slootstra, Jerry W.; Meloen, Rob H.; Clijsters-van der Horst, Marieke; Visser, Therese J.; Jongeneelen, Mandy; Thijsse, Sandra; Throsby, Mark; de Kruif, John; Rupprecht, Charles E.; Dietzschold, Bernhard; Goudsmit, Jaap; Bakker, Alexander B. H.

2005-01-01

Anti-rabies virus immunoglobulin combined with rabies vaccine protects humans from lethal rabies infections. For cost and safety reasons, replacement of the human or equine polyclonal immunoglobulin is advocated, and the use of rabies virus-specific monoclonal antibodies (MAbs) is recommended. We produced two previously described potent rabies virus-neutralizing human MAbs, CR57 and CRJB, in human PER.C6 cells. The two MAbs competed for binding to rabies virus glycoprotein. Using CR57 and a set of 15-mer overlapping peptides covering the glycoprotein ectodomain, a neutralization domain was identified between amino acids (aa) 218 and 240. The minimal binding region was identified as KLCGVL (aa 226 to 231), with key residues K-CGV- identified by alanine replacement scanning. The critical binding region of this novel nonconformational rabies virus epitope is highly conserved within rabies viruses of genotype 1. Subsequently, we generated six rabies virus variants escaping neutralization by CR57 and six variants escaping CRJB. The CR57 escape mutants were only partially covered by CRJB, and all CRJB-resistant variants completely escaped neutralization by CR57. Without exception, the CR57-resistant variants showed a mutation at key residues within the defined minimal binding region, while the CRJB escape viruses showed a single mutation distant from the CR57 epitope (N182D) combined with mutations in the CR57 epitope. The competition between CR57 and CRJB, the in vitro escape profile, and the apparent overlap between the recognized epitopes argues against including both CR57 and CRJB in a MAb cocktail aimed at replacing classical immunoglobulin preparations. PMID:15795253

Mucin-like Region of Herpes Simplex Virus Type 1 Attachment Protein Glycoprotein C (gC) Modulates the Virus-Glycosaminoglycan Interaction*

PubMed Central

Altgärde, Noomi; Eriksson, Charlotta; Peerboom, Nadia; Phan-Xuan, Tuan; Moeller, Stephanie; Schnabelrauch, Matthias; Svedhem, Sofia; Trybala, Edward; Bergström, Tomas; Bally, Marta

2015-01-01

Glycoprotein C (gC) mediates the attachment of HSV-1 to susceptible host cells by interacting with glycosaminoglycans (GAGs) on the cell surface. gC contains a mucin-like region located near the GAG-binding site, which may affect the binding activity. Here, we address this issue by studying a HSV-1 mutant lacking the mucin-like domain in gC and the corresponding purified mutant protein (gCΔmuc) in cell culture and GAG-binding assays, respectively. The mutant virus exhibited two functional alterations as compared with native HSV-1 (i.e. decreased sensitivity to GAG-based inhibitors of virus attachment to cells and reduced release of viral particles from the surface of infected cells). Kinetic and equilibrium binding characteristics of purified gC were assessed using surface plasmon resonance-based sensing together with a surface platform consisting of end-on immobilized GAGs. Both native gC and gCΔmuc bound via the expected binding region to chondroitin sulfate and sulfated hyaluronan but not to the non-sulfated hyaluronan, confirming binding specificity. In contrast to native gC, gCΔmuc exhibited a decreased affinity for GAGs and a slower dissociation, indicating that once formed, the gCΔmuc-GAG complex is more stable. It was also found that a larger number of gCΔmuc bound to a single GAG chain, compared with native gC. Taken together, our data suggest that the mucin-like region of HSV-1 gC is involved in the modulation of the GAG-binding activity, a feature of importance both for unrestricted virus entry into the cells and release of newly produced viral particles from infected cells. PMID:26160171

Isolation of a High Affinity Neutralizing Monoclonal Antibody against 2009 Pandemic H1N1 Virus That Binds at the ‘Sa’ Antigenic Site

PubMed Central

Mishra, Arpita; Yeolekar, Leena; Dhere, Rajeev; Kapre, Subhash; Varadarajan, Raghavan; Gupta, Satish Kumar

2013-01-01

Influenza virus evades host immunity through antigenic drift and shift, and continues to circulate in the human population causing periodic outbreaks including the recent 2009 pandemic. A large segment of the population was potentially susceptible to this novel strain of virus. Historically, monoclonal antibodies (MAbs) have been fundamental tools for diagnosis and epitope mapping of influenza viruses and their importance as an alternate treatment option is also being realized. The current study describes isolation of a high affinity (K D = 2.1±0.4 pM) murine MAb, MA2077 that binds specifically to the hemagglutinin (HA) surface glycoprotein of the pandemic virus. The antibody neutralized the 2009 pandemic H1N1 virus in an in vitro microneutralization assay (IC50 = 0.08 µg/ml). MA2077 also showed hemagglutination inhibition activity (HI titre of 0.50 µg/ml) against the pandemic virus. In a competition ELISA, MA2077 competed with the binding site of the human MAb, 2D1 (isolated from a survivor of the 1918 Spanish flu pandemic) on pandemic H1N1 HA. Epitope mapping studies using yeast cell-surface display of a stable HA1 fragment, wherein ‘Sa’ and ‘Sb’ sites were independently mutated, localized the binding site of MA2077 within the ‘Sa’ antigenic site. These studies will facilitate our understanding of antigen antibody interaction in the context of neutralization of the pandemic influenza virus. PMID:23383214

Late summer disease symptoms in western Washington red raspberry fields associated with co-occurrence of Phytophthora rubi, Verticillium dahliae, and Pratylenchus penetrans, but not Raspberry bushy dwarf virus

USDA-ARS?s Scientific Manuscript database

60% of the $109 million processed by the U.S. red raspberry industry is in northern Washington. In 2012, late summer disease symptoms were observed in many raspberry fields. These symptoms were initially attributed to Verticillium dahliae, but other soilborne pathogens (Phytophthora rubi, Pratylench...

Reading in Place

ERIC Educational Resources Information Center

Price, Leah

2012-01-01

The digital age is rendering books more common, not less. It is true that there is nothing new about "furniture books": The trade in reading material has long been dwarfed by the market for coffee-table books, books that steakhouse chains buy by the yard, empty bindings that interior decorators use to accessorize the upholstery. As coffee-table…

Enlargement of interscapular brown adipose tissue in growth hormone antagonist transgenic and in growth hormone receptor gene-disrupted dwarf mice.

PubMed

Li, Yuesheng; Knapp, Joanne R; Kopchick, John J

2003-02-01

Growth hormone (GH) acts on adipose tissue by accelerating fat expenditure, preventing triglyceride accumulation, and facilitating lipid mobilization. To investigate whether GH is involved in the development and metabolism of interscapular brown adipose tissue (BAT), a site of nonshivering thermogenesis, we employed three lines of transgenic mice. Two of the lines are dwarf due to expression of a GH antagonist (GHA) or disruption of the GH receptor/binding-protein gene. A third mouse line is giant due to overexpression of a bovine GH (bGH) transgene. We have found that the body weights of those animals are proportional to their body lengths at 10 weeks of age. However, GHA dwarf mice tend to catch up with the nontransgenic (NT) littermates in body weight but not in body length at 52 weeks of age. The increase of body mass index (BMI) for GHA mice accelerates rapidly relative to controls as a function of age. We have also observed that BAT in both dwarf mouse lines but not in giant mice is enlarged in contrast to nontransgenic littermates. This enlargement occurs as a function of age. Northern analysis suggests that BAT can be a GH-responsive tissue because GHR/BP mRNAs were found there. Finally, the level of uncoupling protein-1 (UCP1) RNA was found to be higher in dwarf mice and lower in giant animals relative to controls, suggesting that GH-mediated signaling may negatively regulate UCP1 gene expression in BAT.

Prediction of the binding mode of N2-phenylguanine derivative inhibitors to herpes simplex virus type 1 thymidine kinase

NASA Astrophysics Data System (ADS)

Gaudio, Anderson Coser; Takahata, Yuji; Richards, William Graham

1998-01-01

The probable binding mode of the herpes simplex virus thymidine kinase (HSV1 TK) N2-[substituted]-phenylguanine inhibitors is proposed. A computational experiment was designed to check some qualitative binding parameters and to calculate the interaction binding energies of alternative binding modes of N2-phenylguanines. The known binding modes of the HSV1 TK natural substrate deoxythymidine and one of its competitive inhibitors ganciclovir were used as templates. Both the qualitative and quantitative parts of the computational experiment indicated that the N2-phenylguanine derivatives bind to the HSV1 TK active site in the deoxythymidine-like binding mode. An experimental observation that N2-phenylguanosine derivatives are not phosphorylated during the interaction with the HSV1 TK gives support to the proposed binding mode.

The Receptor-Binding Site of the Measles Virus Hemagglutinin Protein Itself Constitutes a Conserved Neutralizing Epitope

PubMed Central

Ohno, Shinji; Sakai, Kouji; Ito, Yuri; Fukuhara, Hideo; Komase, Katsuhiro; Brindley, Melinda A.; Rota, Paul A.; Plemper, Richard K.; Maenaka, Katsumi; Takeda, Makoto

2013-01-01

Here, we provide direct evidence that the receptor-binding site of measles virus (MV) hemagglutinin protein itself forms an effective conserved neutralizing epitope (CNE). Several receptor-interacting residues constitute the CNE. Thus, viral escape from neutralization has to be associated with loss of receptor-binding activity. Since interactions with both the signaling lymphocyte activation molecule (SLAM) and nectin4 are critical for MV pathogenesis, its escape, which results from loss of receptor-binding activity, should not occur in nature. PMID:23283964

Early Antibody Response Contributes to the Virus Eradication and Clinical Recovery of H7N9 Influenza Infection.

PubMed

Liu, Xi; Yang, Zheng; Yuan, Jing; Liao, Jian; Duan, Lian; Wang, Wenfei; Zhang, Fuping; Chen, Xinchun; Zhou, Boping

2017-09-01

A new type of highly pathogenic avian influenza virus, H7N9, has been a great threat to public health since its 2013 outbreak. The humoral immune response plays a critical role in protection from the influenza virus, but its role and kinetics in H7N9-infected patients remain to be determined. In this study, we performed a retrospective investigation of the antibody response in plasma samples from 37 cases of hospitalized patients and analysed the relationship between the antibody response and the clinical outcomes. Our results showed that the HA7-binding antibody was generated earlier than the neutralizing antibody. Higher titer of HA7-binding antibody during the first 14 days after disease onset were associated with a shorter virus-positive continuation period, which is an important risk predictor ( P <0.05). Additionally, the titers of HA7-binding antibody were consistently and significantly lower in patients who died than those who recovered from the severe disease. Unexpectedly, no correlation between the titer of neutralizing antibody and the resulting clinical outcomes was found, suggesting that a neutralizing antibody-independent mechanism also contributed to virus control. In summary, our data suggests that an early antibody response against H7N9 influenza virus contributes to the eradication of the virus. A higher, early HA7-binding antibody response is associated with better clinical outcomes in H7N9 patients. © 2017 by the Association of Clinical Scientists, Inc.

Crystal Structure of the Marburg Virus Nucleoprotein Core Domain Chaperoned by a VP35 Peptide Reveals a Conserved Drug Target for Filovirus.

PubMed

Zhu, Tengfei; Song, Hao; Peng, Ruchao; Shi, Yi; Qi, Jianxun; Gao, George F

2017-09-15

Filovirus nucleoprotein (NP), viral protein 35 (VP35), and polymerase L are essential for viral replication and nucleocapsid formation. Here, we identify a 28-residue peptide (NP binding peptide [NPBP]) from Marburg virus (MARV) VP35 through sequence alignment with previously identified Ebola virus (EBOV) NPBP, which bound to the core region (residues 18 to 344) of the N-terminal portion of MARV NP with high affinity. The crystal structure of the MARV NP core/NPBP complex at a resolution of 2.6 Å revealed that NPBP binds to the C-terminal region of the NP core via electrostatic and nonpolar interactions. Further structural analysis revealed that the MARV and EBOV NP cores hold a conserved binding pocket for NPBP, and this pocket could serve as a promising target for the design of universal drugs against filovirus infection. In addition, cross-binding assays confirmed that the NP core of MARV or EBOV can bind the NPBP from the other virus, although with moderately reduced binding affinities that result from termini that are distinct between the MARV and EBOV NPBPs. IMPORTANCE Historically, Marburg virus (MARV) has caused severe disease with up to 90% lethality. Among the viral proteins produced by MARV, NP and VP35 are both multifunctional proteins that are essential for viral replication. In its relative, Ebola virus (EBOV), an N-terminal peptide from VP35 binds to the NP N-terminal region with high affinity. Whether this is a common mechanism among filoviruses is an unsolved question. Here, we present the crystal structure of a complex that consists of the core domain of MARV NP and the NPBP peptide from VP35. As we compared MARV NPBP with EBOV NPBP, several different features at the termini were identified. Although these differences reduce the affinity of the NP core for NPBPs across genera, a conserved pocket in the C-terminal region of the NP core makes cross-species binding possible. Our results expand our knowledge of filovirus NP-VP35 interactions and provide more details for therapeutic intervention. Copyright © 2017 American Society for Microbiology.

Mutation of CD2AP and SH3KBP1 Binding Motif in Alphavirus nsP3 Hypervariable Domain Results in Attenuated Virus.

PubMed

Mutso, Margit; Morro, Ainhoa Moliner; Smedberg, Cecilia; Kasvandik, Sergo; Aquilimeba, Muriel; Teppor, Mona; Tarve, Liisi; Lulla, Aleksei; Lulla, Valeria; Saul, Sirle; Thaa, Bastian; McInerney, Gerald M; Merits, Andres; Varjak, Margus

2018-04-27

Infection by Chikungunya virus (CHIKV) of the Old World alphaviruses (family Togaviridae) in humans can cause arthritis and arthralgia. The virus encodes four non-structural proteins (nsP) (nsP1, nsp2, nsP3 and nsP4) that act as subunits of the virus replicase. These proteins also interact with numerous host proteins and some crucial interactions are mediated by the unstructured C-terminal hypervariable domain (HVD) of nsP3. In this study, a human cell line expressing EGFP tagged with CHIKV nsP3 HVD was established. Using quantitative proteomics, it was found that CHIKV nsP3 HVD can bind cytoskeletal proteins, including CD2AP, SH3KBP1, CAPZA1, CAPZA2 and CAPZB. The interaction with CD2AP was found to be most evident; its binding site was mapped to the second SH3 ligand-like element in nsP3 HVD. Further assessment indicated that CD2AP can bind to nsP3 HVDs of many different New and Old World alphaviruses. Mutation of the short binding element hampered the ability of the virus to establish infection. The mutation also abolished ability of CD2AP to co-localise with nsP3 and replication complexes of CHIKV; the same was observed for Semliki Forest virus (SFV) harbouring a similar mutation. Similar to CD2AP, its homolog SH3KBP1 also bound the identified motif in CHIKV and SFV nsP3.

Use of superparamagnetic beads for the isolation of a peptide with specificity to cymbidium mosaic virus.

PubMed

Ooi, Diana Jia Miin; Dzulkurnain, Adriya; Othman, Rofina Yasmin; Lim, Saw Hoon; Harikrishna, Jennifer Ann

2006-09-01

A modified method for the rapid isolation of specific ligands to whole virus particles is described. Biopanning against cymbidium mosaic virus was carried out with a commercial 12-mer random peptide display library. A solution phase panning method was devised using streptavidin-coated superparamagnetic beads. The solution based panning method was more efficient than conventional immobilized target panning when using whole viral particles of cymbidium mosaic virus as a target. Enzyme-linked immunosorbent assay of cymbidium mosaic virus-binding peptides isolated from the library identified seven peptides with affinity for cymbidium mosaic virus and one peptide which was specific to cymbidium mosaic virus and had no significant binding to odontoglossum ringspot virus. This method should have broad application for the screening of whole viral particles towards the rapid development of diagnostic reagents without the requirement for cloning and expression of single antigens.

Parvovirus B19 VLP recognizes globoside in supported lipid bilayers.

PubMed

Nasir, Waqas; Nilsson, Jonas; Olofsson, Sigvard; Bally, Marta; Rydell, Gustaf E

2014-05-01

Studies have suggested that the glycosphingolipid globoside (Gb4Cer) is a receptor for human parvovirus B19. Virus-like particles bind to Gb4Cer on thin-layer chromatograms, but a direct interaction between the virus and lipid membrane-associated Gb4Cer has been debated. Here, we characterized the binding of parvovirus B19 VP1/VP2 virus-like particles to glycosphingolipids (i) on thin-layer chromatograms (TLCs) and (ii) incorporated into supported lipid bilayers (SLBs) acting as cell-membrane mimics. The binding specificities of parvovirus B19 determined in the two systems were in good agreement; the VLP recognized both Gb4Cer and the Forssman glycosphingolipid on TLCs and in SLBs compatible with the role of Gb4Cer as a receptor for this virus. Copyright © 2014 Elsevier Inc. All rights reserved.

Molecular Docking Studies to Explore Potential Binding Pockets and Inhibitors for Chikungunya Virus Envelope Glycoproteins.

PubMed

Nguyen, Phuong T V; Yu, Haibo; Keller, Paul A

2017-03-11

The chikungunya virus (CHIKV) envelope glycoproteins are considered important potential targets for anti-CHIKV drug discovery due to their crucial roles in virus attachment and virus entry. In this study, using two available crystal structures of the immature and mature forms of envelope glycoproteins, virtual screenings based on blind dockings and focused dockings were carried out to identify potential binding pockets and hit compounds for the virus. The chemical library database of compounds, NCI Diversity Set II, was used in these docking studies. In addition to reproducing previously reported examples, new binding pockets were identified, e.g., Pocket 2 in the 3N40, and Pocket 2 and Pocket 3 in the 3N42. Convergences in conformational sampling in docking using AutoDock Vina were evaluated. An analysis of docking results was carried out to understand interactions of the envelope glycoproteins complexes. Some key residues for interactions, for example Gly91 and His230, are identified as possessing important roles in the fusion process.

Role of Multiple Hosts in the Cross-Species Transmission and Emergence of a Pandemic Parvovirus

PubMed Central

Allison, Andrew B.; Harbison, Carole E.; Pagan, Israel; Stucker, Karla M.; Kaelber, Jason T.; Brown, Justin D.; Ruder, Mark G.; Keel, M. Kevin; Dubovi, Edward J.; Holmes, Edward C.

2012-01-01

Understanding the mechanisms of cross-species virus transmission is critical to anticipating emerging infectious diseases. Canine parvovirus type 2 (CPV-2) emerged as a variant of a feline parvovirus when it acquired mutations that allowed binding to the canine transferrin receptor type 1 (TfR). However, CPV-2 was soon replaced by a variant virus (CPV-2a) that differed in antigenicity and receptor binding. Here we show that the emergence of CPV involved an additional host range variant virus that has circulated undetected in raccoons for at least 24 years, with transfers to and from dogs. Raccoon virus capsids showed little binding to the canine TfR, showed little infection of canine cells, and had altered antigenic structures. Remarkably, in capsid protein (VP2) phylogenies, most raccoon viruses fell as evolutionary intermediates between the CPV-2 and CPV-2a strains, suggesting that passage through raccoons assisted in the evolution of CPV-2a. This highlights the potential role of alternative hosts in viral emergence. PMID:22072763

Assembly-directed antivirals differentially bind quasiequivalent pockets to modify hepatitis B virus capsid tertiary and quaternary structure.

PubMed

Katen, Sarah P; Tan, Zhenning; Chirapu, Srinivas Reddy; Finn, M G; Zlotnick, Adam

2013-08-06

Hepatitis B virus (HBV) is a major cause of liver disease. Assembly of the HBV capsid is a critical step in virus production and an attractive target for new antiviral therapies. We determined the structure of HBV capsid in complex with AT-130, a member of the phenylpropenamide family of assembly effectors. AT-130 causes tertiary and quaternary structural changes but does not disrupt capsid structure. AT-130 binds a hydrophobic pocket that also accommodates the previously characterized heteroaryldihydropyrimidine compounds but favors a unique quasiequivalent location on the capsid surface. Thus, this pocket is a promiscuous drug-binding site and a likely target for different assembly effectors with a broad range of mechanisms of activity. That AT-130 successfully decreases virus production by increasing capsid assembly rate without disrupting capsid structure delineates a paradigm in antiviral design, that disrupting reaction timing is a viable strategy for assembly effectors of HBV and other viruses. Copyright © 2013 Elsevier Ltd. All rights reserved.

Role of multiple hosts in the cross-species transmission and emergence of a pandemic parvovirus.

PubMed

Allison, Andrew B; Harbison, Carole E; Pagan, Israel; Stucker, Karla M; Kaelber, Jason T; Brown, Justin D; Ruder, Mark G; Keel, M Kevin; Dubovi, Edward J; Holmes, Edward C; Parrish, Colin R

2012-01-01

Understanding the mechanisms of cross-species virus transmission is critical to anticipating emerging infectious diseases. Canine parvovirus type 2 (CPV-2) emerged as a variant of a feline parvovirus when it acquired mutations that allowed binding to the canine transferrin receptor type 1 (TfR). However, CPV-2 was soon replaced by a variant virus (CPV-2a) that differed in antigenicity and receptor binding. Here we show that the emergence of CPV involved an additional host range variant virus that has circulated undetected in raccoons for at least 24 years, with transfers to and from dogs. Raccoon virus capsids showed little binding to the canine TfR, showed little infection of canine cells, and had altered antigenic structures. Remarkably, in capsid protein (VP2) phylogenies, most raccoon viruses fell as evolutionary intermediates between the CPV-2 and CPV-2a strains, suggesting that passage through raccoons assisted in the evolution of CPV-2a. This highlights the potential role of alternative hosts in viral emergence.

High-Dose Mannose-Binding Lectin Therapy for Ebola Virus Infection

DTIC Science & Technology

2010-06-01

viruses . N-glycosylation of viral envelopes is an important such target shared between in- fluenza, HIV, HCV, West Nile virus , SARS-CoV, Hendra virus ...host cells. Therefore, MBL preferentially recognizes glycosylated viruses including influenza virus , human immunodeficiency virus , severe acute...respiratory syndrome coronovirus (SARS-CoV), Ebola virus , and Marburg virus . It also recognizes many glycosylated gram- positive and gram-negative bacteria [1

Immune Escape Variants of H9N2 Influenza Viruses Containing Deletions at the Hemagglutinin Receptor Binding Site Retain Fitness In Vivo and Display Enhanced Zoonotic Characteristics.

PubMed

Peacock, Thomas P; Benton, Donald J; James, Joe; Sadeyen, Jean-Remy; Chang, Pengxiang; Sealy, Joshua E; Bryant, Juliet E; Martin, Stephen R; Shelton, Holly; Barclay, Wendy S; Iqbal, Munir

2017-07-15

H9N2 avian influenza viruses are enzootic in poultry across Asia and North Africa, where they pose a threat to human health as both zoonotic agents and potential pandemic candidates. Poultry vaccination against H9N2 viruses has been employed in many regions; however, vaccine effectiveness is frequently compromised due to antigenic drift arising from amino acid substitutions in the major influenza virus antigen hemagglutinin (HA). Using selection with HA-specific monoclonal antibodies, we previously identified H9N2 antibody escape mutants that contained deletions of amino acids in the 220 loop of the HA receptor binding sites (RBSs). Here we analyzed the impact of these deletions on virus zoonotic infection characteristics and fitness. We demonstrated that mutant viruses with RBS deletions are able to escape polyclonal antiserum binding and are able to infect and be transmitted between chickens. We showed that the deletion mutants have increased binding to human-like receptors and greater replication in primary human airway cells; however, the mutant HAs also displayed reduced pH and thermal stability. In summary, we infer that variant influenza viruses with deletions in the 220 loop could arise in the field due to immune selection pressure; however, due to reduced HA stability, we conclude that these viruses are unlikely to be transmitted from human to human by the airborne route, a prerequisite for pandemic emergence. Our findings underscore the complex interplay between antigenic drift and viral fitness for avian influenza viruses as well as the challenges of predicting which viral variants may pose the greatest threats for zoonotic and pandemic emergence. IMPORTANCE Avian influenza viruses, such as H9N2, cause disease in poultry as well as occasionally infecting humans and are therefore considered viruses with pandemic potential. Many countries have introduced vaccination of poultry to try to control the disease burden; however, influenza viruses are able to rapidly evolve to escape immune pressure in a process known as "antigenic drift." Previously, we experimentally generated antigenic-drift variants in the laboratory, and here, we test our "drifted" viruses to assess their zoonotic infection characteristics and transmissibility in chickens. We found that the drifted viruses were able to infect and be transmitted between chickens and showed increased binding to human-like receptors. However, the drift mutant viruses displayed reduced stability, and we predict that they are unlikely to be transmitted from human to human and cause an influenza pandemic. These results demonstrate the complex relationship between antigenic drift and the potential of avian influenza viruses to infect humans. Copyright © 2017 Peacock et al.

Mathematical Modeling of Herpes Simplex Virus Distribution in Solid Tumors: Implications for Cancer Gene Therapy

PubMed Central

Mok, Wilson; Stylianopoulos, Triantafyllos; Boucher, Yves; Jain, Rakesh K.

2010-01-01

Purpose Although oncolytic viral vectors show promise for the treatment of various cancers, ineffective initial distribution and propagation throughout the tumor mass often limit the therapeutic response. A mathematical model is developed to describe the spread of herpes simplex virus from the initial injection site. Experimental Design The tumor is modeled as a sphere of radius R. The model incorporates reversible binding, interstitial diffusion, viral degradation, and internalization and physiologic parameters. Three species are considered as follows: free interstitial virus, virus bound to cell surfaces, and internalized virus. Results This analysis reveals that both rapid binding and internalization as well as hindered diffusion contain the virus to the initial injection volume, with negligible spread to the surrounding tissue. Unfortunately, increasing the dose to saturate receptors and promote diffusion throughout the tumor is not a viable option: the concentration necessary would likely compromise safety. However, targeted modifications to the virus that decrease the binding affinity have the potential to increase the number of infected cells by 1.5-fold or more. An increase in the effective diffusion coefficient can result in similar gains. Conclusions This analysis suggests criteria by which the potential response of a tumor to oncolytic herpes simplex virus therapy can be assessed. Furthermore, it reveals the potential of modifications to the vector delivery method, physicochemical properties of the virus, and tumor extracellular matrix composition to enhance efficacy. PMID:19318482

Comprehensive Analysis of DWARF14-LIKE2 (DLK2) Reveals Its Functional Divergence from Strigolactone-Related Paralogs

PubMed Central

Végh, Attila; Incze, Norbert; Fábián, Attila; Huo, Heqiang; Bradford, Kent J.; Balázs, Ervin; Soós, Vilmos

2017-01-01

Strigolactones (SLs) and related butenolides, originally identified as active seed germination stimulants of parasitic weeds, play important roles in many aspects of plant development. Two members of the D14 α/β hydrolase protein family, DWARF14 (D14) and KARRIKIN INSENSITIVE2 (KAI2) are essential for SL/butenolide signaling. The third member of the family in Arabidopsis, DWARF 14-LIKE2 (DLK2) is structurally very similar to D14 and KAI2, but its function is unknown. We demonstrated that DLK2 does not bind nor hydrolyze natural (+)5-deoxystrigol [(+)5DS], and weakly hydrolyzes non-natural strigolactone (-)5DS. A detailed genetic analysis revealed that DLK2 does not affect SL responses and can regulate seedling photomorphogenesis. DLK2 is upregulated in the dark dependent upon KAI2 and PHYTOCHROME INTERACTING FACTORS (PIFs), indicating that DLK2 might function in light signaling pathways. In addition, unlike its paralog proteins, DLK2 is not subject to rac-GR24-induced degradation, suggesting that DLK2 acts independently of MORE AXILLARY GROWTH2 (MAX2); however, regulation of DLK2 transcription is mostly accomplished through MAX2. In conclusion, these data suggest that DLK2 represents a divergent member of the DWARF14 family. PMID:28970845

Comprehensive Analysis of DWARF14-LIKE2 (DLK2) Reveals Its Functional Divergence from Strigolactone-Related Paralogs.

PubMed

Végh, Attila; Incze, Norbert; Fábián, Attila; Huo, Heqiang; Bradford, Kent J; Balázs, Ervin; Soós, Vilmos

2017-01-01

Strigolactones (SLs) and related butenolides, originally identified as active seed germination stimulants of parasitic weeds, play important roles in many aspects of plant development. Two members of the D14 α/β hydrolase protein family, DWARF14 (D14) and KARRIKIN INSENSITIVE2 (KAI2) are essential for SL/butenolide signaling. The third member of the family in Arabidopsis, DWARF 14-LIKE2 (DLK2) is structurally very similar to D14 and KAI2, but its function is unknown. We demonstrated that DLK2 does not bind nor hydrolyze natural (+)5-deoxystrigol [(+)5DS], and weakly hydrolyzes non-natural strigolactone (-)5DS. A detailed genetic analysis revealed that DLK2 does not affect SL responses and can regulate seedling photomorphogenesis. DLK2 is upregulated in the dark dependent upon KAI2 and PHYTOCHROME INTERACTING FACTORS (PIFs), indicating that DLK2 might function in light signaling pathways. In addition, unlike its paralog proteins, DLK2 is not subject to rac -GR24-induced degradation, suggesting that DLK2 acts independently of MORE AXILLARY GROWTH2 (MAX2); however, regulation of DLK2 transcription is mostly accomplished through MAX2. In conclusion, these data suggest that DLK2 represents a divergent member of the DWARF14 family.

Conquering systematics in the timing of the pulsar triple system J0337+1715: Towards a unique and robust test of the strong equivalence principle

NASA Astrophysics Data System (ADS)

Gusinskaia, N. V.; Archibald, A. M.; Hessels, J. W. T.; Lorimer, D. R.; Ransom, S. M.; Stairs, I. H.; Lynch, R. S.

2017-12-01

PSR J0337+1715 is a millisecond radio pulsar in a hierarchical stellar triple system containing two white dwarfs. The pulsar orbits the inner white dwarf every 1.6 days. In turn, this inner binary system orbits the outer white dwarf every 327 days. The gravitational influence of the outer white dwarf strongly accelerates the inner binary, making this system an excellent laboratory in which to test the strong equivalence principle (SEP) of general relativity - especially because the neutron star has significant gravitational self-binding energy. This system has been intensively monitored using three radio telescopes: Arecibo, Green Bank and Westerbork. Using the more than 25000 pulse times of arrival (TOAs) collected to date, we have modeled the system using direct 3-body numerical integration. Here we present our efforts to quantify the effects of systematics in the TOAs and timing residuals, which can limit the precision to which we can test the SEP in this system. In this work we describe Fourier-based techniques that we apply to the residuals in order to isolate the effects of systematics that could masquerade as an SEP violation. We also demonstrate that tidal effects are insignificant in the modeling.

Mutational analysis of vaccinia virus E3 protein: the biological functions do not correlate with its biochemical capacity to bind double-stranded RNA.

PubMed

Dueck, Kevin J; Hu, YuanShen Sandy; Chen, Peter; Deschambault, Yvon; Lee, Jocelyn; Varga, Jessie; Cao, Jingxin

2015-05-01

Vaccinia E3 protein has the biochemical capacity of binding to double-stranded RNA (dsRNA). The best characterized biological functions of the E3 protein include its host range function, suppression of cytokine expression, and inhibition of interferon (IFN)-induced antiviral activity. Currently, the role of the dsRNA binding capacity in the biological functions of the E3 protein is not clear. To further understand the mechanism of the E3 protein biological functions, we performed alanine scanning of the entire dsRNA binding domain of the E3 protein to examine the link between its biochemical capacity of dsRNA binding and biological functions. Of the 115 mutants examined, 20 were defective in dsRNA binding. Although the majority of the mutants defective in dsRNA binding also showed defective replication in HeLa cells, nine mutants (I105A, Y125A, E138A, F148A, F159A, K171A, L182A, L183A, and I187/188A) retained the host range function to various degrees. Further examination of a set of representative E3L mutants showed that residues essential for dsRNA binding are not essential for the biological functions of E3 protein, such as inhibition of protein kinase R (PKR) activation, suppression of cytokine expression, and apoptosis. Thus, data described in this communication strongly indicate the E3 protein performs its biological functions via a novel mechanism which does not correlate with its dsRNA binding activity. dsRNAs produced during virus replication are important pathogen-associated molecular patterns (PAMPs) for inducing antiviral immune responses. One of the strategies used by many viruses to counteract such antiviral immune responses is achieved by producing dsRNA binding proteins, such as poxvirus E3 family proteins, influenza virus NS1, and Ebola virus V35 proteins. The most widely accepted model for the biological functions of this class of viral dsRNA binding proteins is that they bind to and sequester viral dsRNA PAMPs; thus, they suppress the related antiviral immune responses. However, no direct experimental data confirm such a model. In this study of vaccinia E3 protein, we found that the biological functions of the E3 protein are not necessarily linked to its biochemical capacity of dsRNA binding. Thus, our data strongly point to a new concept of virus modulation of cellular antiviral responses triggered by dsRNA PAMPs. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Synergy of two low-affinity NLSs determines the high avidity of influenza A virus nucleoprotein NP for human importin α isoforms.

PubMed

Wu, Wei; Sankhala, Rajeshwer S; Florio, Tyler J; Zhou, Lixin; Nguyen, Nhan L T; Lokareddy, Ravi K; Cingolani, Gino; Panté, Nelly

2017-09-12

The influenza A virus nucleoprotein (NP) is an essential multifunctional protein that encapsidates the viral genome and functions as an adapter between the virus and the host cell machinery. NPs from all strains of influenza A viruses contain two nuclear localization signals (NLSs): a well-studied monopartite NLS1 and a less-characterized NLS2, thought to be bipartite. Through site-directed mutagenesis and functional analysis, we found that NLS2 is also monopartite and is indispensable for viral infection. Atomic structures of importin α bound to two variants of NLS2 revealed NLS2 primarily binds the major-NLS binding site of importin α, unlike NLS1 that associates with the minor NLS-pocket. Though peptides corresponding to NLS1 and NLS2 bind weakly to importin α, the two NLSs synergize in the context of the full length NP to confer high avidity for importin α7, explaining why the virus efficiently replicates in the respiratory tract that exhibits high levels of this isoform. This study, the first to functionally characterize NLS2, demonstrates NLS2 plays an important and unexpected role in influenza A virus infection. We propose NLS1 and NLS2 form a bipartite NLS in trans, which ensures high avidity for importin α7 while preventing non-specific binding to viral RNA.

Small Interfering RNA Pathway Modulates Initial Viral Infection in Midgut Epithelium of Insect after Ingestion of Virus.

PubMed

Lan, Hanhong; Chen, Hongyan; Liu, Yuyan; Jiang, Chaoyang; Mao, Qianzhuo; Jia, Dongsheng; Chen, Qian; Wei, Taiyun

2016-01-15

Numerous viruses are transmitted in a persistent manner by insect vectors. Persistent viruses establish their initial infection in the midgut epithelium, from where they disseminate to the midgut visceral muscles. Although propagation of viruses in insect vectors can be controlled by the small interfering RNA (siRNA) antiviral pathway, whether the siRNA pathway can control viral dissemination from the midgut epithelium is unknown. Infection by a rice virus (Southern rice black streaked dwarf virus [SRBSDV]) of its incompetent vector (the small brown planthopper [SBPH]) is restricted to the midgut epithelium. Here, we show that the siRNA pathway is triggered by SRBSDV infection in continuously cultured cells derived from the SBPH and in the midgut of the intact insect. Knockdown of the expression of the core component Dicer-2 of the siRNA pathway by RNA interference strongly increased the ability of SRBSDV to propagate in continuously cultured SBPH cells and in the midgut epithelium, allowing viral titers in the midgut epithelium to reach the threshold (1.99 × 10(9) copies of the SRBSDV P10 gene/μg of midgut RNA) needed for viral dissemination into the SBPH midgut muscles. Our results thus represent the first elucidation of the threshold for viral dissemination from the insect midgut epithelium. Silencing of Dicer-2 further facilitated the transmission of SRBSDV into rice plants by SBPHs. Taken together, our results reveal the new finding that the siRNA pathway can control the initial infection of the insect midgut epithelium by a virus, which finally affects the competence of the virus's vector. Many viral pathogens that cause significant global health and agricultural problems are transmitted via insect vectors. The first bottleneck in viral infection, the midgut epithelium, is a principal determinant of the ability of an insect species to transmit a virus. Southern rice black streaked dwarf virus (SRBSDV) is restricted exclusively to the midgut epithelium of an incompetent vector, the small brown planthopper (SBPH). Here, we show that silencing of the core component Dicer-2 of the small interfering RNA (siRNA) pathway increases viral titers in the midgut epithelium past the threshold (1.99 × 10(9) copies of the SRBSDV P10 gene/μg of midgut RNA) for viral dissemination into the midgut muscles and then into the salivary glands, allowing the SBPH to become a competent vector of SRBSDV. This result is the first evidence that the siRNA antiviral pathway has a direct role in the control of viral dissemination from the midgut epithelium and that it affects the competence of the virus's vector. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Small Interfering RNA Pathway Modulates Initial Viral Infection in Midgut Epithelium of Insect after Ingestion of Virus

PubMed Central

Lan, Hanhong; Chen, Hongyan; Liu, Yuyan; Jiang, Chaoyang; Mao, Qianzhuo; Jia, Dongsheng; Chen, Qian

2015-01-01

ABSTRACT Numerous viruses are transmitted in a persistent manner by insect vectors. Persistent viruses establish their initial infection in the midgut epithelium, from where they disseminate to the midgut visceral muscles. Although propagation of viruses in insect vectors can be controlled by the small interfering RNA (siRNA) antiviral pathway, whether the siRNA pathway can control viral dissemination from the midgut epithelium is unknown. Infection by a rice virus (Southern rice black streaked dwarf virus [SRBSDV]) of its incompetent vector (the small brown planthopper [SBPH]) is restricted to the midgut epithelium. Here, we show that the siRNA pathway is triggered by SRBSDV infection in continuously cultured cells derived from the SBPH and in the midgut of the intact insect. Knockdown of the expression of the core component Dicer-2 of the siRNA pathway by RNA interference strongly increased the ability of SRBSDV to propagate in continuously cultured SBPH cells and in the midgut epithelium, allowing viral titers in the midgut epithelium to reach the threshold (1.99 × 109 copies of the SRBSDV P10 gene/μg of midgut RNA) needed for viral dissemination into the SBPH midgut muscles. Our results thus represent the first elucidation of the threshold for viral dissemination from the insect midgut epithelium. Silencing of Dicer-2 further facilitated the transmission of SRBSDV into rice plants by SBPHs. Taken together, our results reveal the new finding that the siRNA pathway can control the initial infection of the insect midgut epithelium by a virus, which finally affects the competence of the virus's vector. IMPORTANCE Many viral pathogens that cause significant global health and agricultural problems are transmitted via insect vectors. The first bottleneck in viral infection, the midgut epithelium, is a principal determinant of the ability of an insect species to transmit a virus. Southern rice black streaked dwarf virus (SRBSDV) is restricted exclusively to the midgut epithelium of an incompetent vector, the small brown planthopper (SBPH). Here, we show that silencing of the core component Dicer-2 of the small interfering RNA (siRNA) pathway increases viral titers in the midgut epithelium past the threshold (1.99 × 109 copies of the SRBSDV P10 gene/μg of midgut RNA) for viral dissemination into the midgut muscles and then into the salivary glands, allowing the SBPH to become a competent vector of SRBSDV. This result is the first evidence that the siRNA antiviral pathway has a direct role in the control of viral dissemination from the midgut epithelium and that it affects the competence of the virus's vector. PMID:26537672

Evaluation of the Biological Properties and Cross-Reactive Antibody Response to H10 Influenza Viruses in Ferrets

PubMed Central

Sutton, Troy C.; Lamirande, Elaine W.; Czako, Rita

2017-01-01

ABSTRACT The recent outbreak of avian origin H10N7 influenza among seals in northern Europe and two fatal human infections with an avian H10N8 virus in China have demonstrated that H10 viruses can spread between mammals and cause severe disease in humans. To gain insight into the potential for H10 viruses to cross the species barrier and to identify a candidate vaccine strain, we evaluated the in vitro and in vivo properties and antibody response in ferrets to 20 diverse H10 viruses. H10 virus infection of ferrets caused variable weight loss, and all 20 viruses replicated throughout the respiratory tract; however, replication in the lungs was highly variable. In glycan-binding assays, the H10 viruses preferentially bound “avian-like” α2,3-linked sialic acids. Importantly, several isolates also displayed strong binding to long-chain “human-like” α2,6-linked sialic acids and exhibited comparable or elevated neuraminidase activity relative to human H1N1, H2N2, and H3N2 viruses. In hemagglutination inhibition assays, 12 antisera cross-reacted with ≥14 of 20 H10 viruses, and 7 viruses induced neutralizing activity against ≥15 of the 20 viruses. By combining data on weight loss, viral replication, and the cross-reactive antibody response, we identified A/mallard/Portugal/79906/2009 (H10N7) as a suitable virus for vaccine development. Collectively, our findings suggest that H10 viruses may continue to sporadically infect humans and other mammals, underscoring the importance of developing an H10 vaccine for pandemic preparedness. IMPORTANCE Avian origin H10 influenza viruses sporadically infect humans and other mammals; however, little is known about viruses of this subtype. Thus, we characterized the biological properties of 20 H10 viruses in vitro and in ferrets. Infection caused mild to moderate weight loss (5 to 15%), with robust viral replication in the nasal tissues and variable replication in the lung. H10 viruses preferentially bind “avian-like” sialic acids, although several isolates also displayed binding to “human-like” sialic acid receptors. This is consistent with the ability of H10 viruses to cross the species barrier and warrants selection of an H10 vaccine strain. By evaluating the cross-reactive antibody response to the H10 viruses and combining this analysis with viral replication and weight loss findings, we identified A/mallard/Portugal/79906/2009 (H10N7) as a suitable H10 vaccine strain. PMID:28701401

Integrin-Using Rotaviruses Bind α2β1 Integrin α2 I Domain via VP4 DGE Sequence and Recognize αXβ2 and αVβ3 by Using VP7 during Cell Entry

PubMed Central

Graham, Kate L.; Halasz, Peter; Tan, Yan; Hewish, Marilyn J.; Takada, Yoshikazu; Mackow, Erich R.; Robinson, Martyn K.; Coulson, Barbara S.

2003-01-01

Integrins α2β1, αXβ2, and αVβ3 have been implicated in rotavirus cell attachment and entry. The virus spike protein VP4 contains the α2β1 ligand sequence DGE at amino acid positions 308 to 310, and the outer capsid protein VP7 contains the αXβ2 ligand sequence GPR. To determine the viral proteins and sequences involved and to define the roles of α2β1, αXβ2, and αVβ3, we analyzed the ability of rotaviruses and their reassortants to use these integrins for cell binding and infection and the effect of peptides DGEA and GPRP on these events. Many laboratory-adapted human, monkey, and bovine viruses used integrins, whereas all porcine viruses were integrin independent. The integrin-using rotavirus strains each interacted with all three integrins. Integrin usage related to VP4 serotype independently of sialic acid usage. Analysis of rotavirus reassortants and assays of virus binding and infectivity in integrin-transfected cells showed that VP4 bound α2β1, and VP7 interacted with αXβ2 and αVβ3 at a postbinding stage. DGEA inhibited rotavirus binding to α2β1 and infectivity, whereas GPRP binding to αXβ2 inhibited infectivity but not binding. The truncated VP5* subunit of VP4, expressed as a glutathione S-transferase fusion protein, bound the expressed α2 I domain. Alanine mutagenesis of D308 and G309 in VP5* eliminated VP5* binding to the α2 I domain. In a novel process, integrin-using viruses bind the α2 I domain of α2β1 via DGE in VP4 and interact with αXβ2 (via GPR) and αVβ3 by using VP7 to facilitate cell entry and infection. PMID:12941907

Footprinting of Chlorella virus DNA ligase bound at a nick in duplex DNA.

PubMed

Odell, M; Shuman, S

1999-05-14

The 298-amino acid ATP-dependent DNA ligase of Chlorella virus PBCV-1 is the smallest eukaryotic DNA ligase known. The enzyme has intrinsic specificity for binding to nicked duplex DNA. To delineate the ligase-DNA interface, we have footprinted the enzyme binding site on DNA and the DNA binding site on ligase. The size of the exonuclease III footprint of ligase bound a single nick in duplex DNA is 19-21 nucleotides. The footprint is asymmetric, extending 8-9 nucleotides on the 3'-OH side of the nick and 11-12 nucleotides on the 5'-phosphate side. The 5'-phosphate moiety is essential for the binding of Chlorella virus ligase to nicked DNA. Here we show that the 3'-OH moiety is not required for nick recognition. The Chlorella virus ligase binds to a nicked ligand containing 2',3'-dideoxy and 5'-phosphate termini, but cannot catalyze adenylation of the 5'-end. Hence, the 3'-OH is important for step 2 chemistry even though it is not itself chemically transformed during DNA-adenylate formation. A 2'-OH cannot substitute for the essential 3'-OH in adenylation at a nick or even in strand closure at a preadenylated nick. The protein side of the ligase-DNA interface was probed by limited proteolysis of ligase with trypsin and chymotrypsin in the presence and absence of nicked DNA. Protease accessible sites are clustered within a short segment from amino acids 210-225 located distal to conserved motif V. The ligase is protected from proteolysis by nicked DNA. Protease cleavage of the native enzyme prior to DNA addition results in loss of DNA binding. These results suggest a bipartite domain structure in which the interdomain segment either comprises part of the DNA binding site or undergoes a conformational change upon DNA binding. The domain structure of Chlorella virus ligase inferred from the solution experiments is consistent with the structure of T7 DNA ligase determined by x-ray crystallography.

In silico analysis of AHJD-like viruses, Staphylococcus aureus phages S24-1 and S13′, and study of phage S24-1 adsorption

PubMed Central

Uchiyama, Jumpei; Takemura-Uchiyama, Iyo; Kato, Shin-ichiro; Sato, Miho; Ujihara, Takako; Matsui, Hidehito; Hanaki, Hideaki; Daibata, Masanori; Matsuzaki, Shigenobu

2014-01-01

Staphylococcus aureus is a clinically important bacterium that is commensal in both humans and animals. Bacteriophage (phage) attachment to the host bacterial surface is an important process during phage infection, which involves interactions between phage receptor-binding proteins and host receptor molecules. However, little information is available on the receptor-binding protein of S. aureus phages. S. aureus virulent phages S24-1 and S13′ (family Podoviridae, genus AHJD-like viruses) were isolated from sewage. In the present study, we investigated the receptor-binding protein of AHJD-like viruses using phage S24-1. First, based on a comparative genomic analysis of phages S24-1 and S13′, open reading frame 16 (ORF16) of phage S24-1 was speculated to be the receptor-binding protein, which possibly determines the host range. Second, we demonstrated that this was the receptor-binding protein of phage S24-1. Third, our study suggested that wall teichoic acids in the cell walls of S. aureus are the main receptor molecules for ORF16 and phage S24-1. Finally, the C-terminal region of ORF16 may be essential for binding to S. aureus. These results strongly suggest that ORF16 of phage S24-1 and its homologs may be the receptor-binding proteins of AHJD-like viruses. PMID:24591378

A mammary cell-specific enhancer in mouse mammary tumor virus DNA is composed of multiple regulatory elements including binding sites for CTF/NFI and a novel transcription factor, mammary cell-activating factor.

PubMed Central

Mink, S; Härtig, E; Jennewein, P; Doppler, W; Cato, A C

1992-01-01

Mouse mammary tumor virus (MMTV) is a milk-transmitted retrovirus involved in the neoplastic transformation of mouse mammary gland cells. The expression of this virus is regulated by mammary cell type-specific factors, steroid hormones, and polypeptide growth factors. Sequences for mammary cell-specific expression are located in an enhancer element in the extreme 5' end of the long terminal repeat region of this virus. This enhancer, when cloned in front of the herpes simplex thymidine kinase promoter, endows the promoter with mammary cell-specific response. Using functional and DNA-protein-binding studies with constructs mutated in the MMTV long terminal repeat enhancer, we have identified two main regulatory elements necessary for the mammary cell-specific response. These elements consist of binding sites for a transcription factor in the family of CTF/NFI proteins and the transcription factor mammary cell-activating factor (MAF) that recognizes the sequence G Pu Pu G C/G A A G G/T. Combinations of CTF/NFI- and MAF-binding sites or multiple copies of either one of these binding sites but not solitary binding sites mediate mammary cell-specific expression. The functional activities of these two regulatory elements are enhanced by another factor that binds to the core sequence ACAAAG. Interdigitated binding sites for CTF/NFI, MAF, and/or the ACAAAG factor are also found in the 5' upstream regions of genes encoding whey milk proteins from different species. These findings suggest that mammary cell-specific regulation is achieved by a concerted action of factors binding to multiple regulatory sites. Images PMID:1328867

HIV-1 matrix domain removal ameliorates virus assembly and processing defects incurred by positive nucleocapsid charge elimination.

PubMed

Ko, Li-Jung; Yu, Fu-Hsien; Huang, Kuo-Jung; Wang, Chin-Tien

2015-01-01

Human immunodeficiency virus type 1 nucleocapsid (NC) basic residues presumably contribute to virus assembly via RNA, which serves as a scaffold for Gag-Gag interaction during particle assembly. To determine whether NC basic residues play a role in Gag cleavage (thereby impacting virus assembly), Gag processing efficiency and virus particle production were analyzed for an HIV-1 mutant NC15A, with alanine serving as a substitute for all NC basic residues. Results indicate that NC15A significantly impaired virus maturation in addition to significantly affecting Gag membrane binding and assembly. Interestingly, removal of the matrix (MA) central globular domain ameliorated the NC15A assembly and processing defects, likely through enhancement of Gag multimerization and membrane binding capacities.

Selection of reference genes for gene expression studies in virus-infected monocots using quantitative real-time PCR.

PubMed

Zhang, Kun; Niu, Shaofang; Di, Dianping; Shi, Lindan; Liu, Deshui; Cao, Xiuling; Miao, Hongqin; Wang, Xianbing; Han, Chenggui; Yu, Jialin; Li, Dawei; Zhang, Yongliang

2013-10-10

Both genome-wide transcriptomic surveys of the mRNA expression profiles and virus-induced gene silencing-based molecular studies of target gene during virus-plant interaction involve the precise estimation of the transcript abundance. Quantitative real-time PCR (qPCR) is the most widely adopted technique for mRNA quantification. In order to obtain reliable quantification of transcripts, identification of the best reference genes forms the basis of the preliminary work. Nevertheless, the stability of internal controls in virus-infected monocots needs to be fully explored. In this work, the suitability of ten housekeeping genes (ACT, EF1α, FBOX, GAPDH, GTPB, PP2A, SAND, TUBβ, UBC18 and UK) for potential use as reference genes in qPCR were investigated in five different monocot plants (Brachypodium, barley, sorghum, wheat and maize) under infection with different viruses including Barley stripe mosaic virus (BSMV), Brome mosaic virus (BMV), Rice black-streaked dwarf virus (RBSDV) and Sugarcane mosaic virus (SCMV). By using three different algorithms, the most appropriate reference genes or their combinations were identified for different experimental sets and their effectiveness for the normalisation of expression studies were further validated by quantitative analysis of a well-studied PR-1 gene. These results facilitate the selection of desirable reference genes for more accurate gene expression studies in virus-infected monocots. Copyright © 2013 Elsevier B.V. All rights reserved.

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

Sprague, E.R.; Wang, C.; Baker, D.

Herpes simplex virus type-1 expresses a heterodimeric Fc receptor, gE-gI, on the surfaces of virions and infected cells that binds the Fc region of host immunoglobulin G and is implicated in the cell-to-cell spread of virus. gE-gI binds immunoglobulin G at the basic pH of the cell surface and releases it at the acidic pH of lysosomes, consistent with a role in facilitating the degradation of antiviral antibodies. Here we identify the C-terminal domain of the gE ectodomain (CgE) as the minimal Fc-binding domain and present a 1.78-{angstrom} CgE structure. A 5-{angstrom} gE-gI/Fc crystal structure, which was independently verified bymore » a theoretical prediction method, reveals that CgE binds Fc at the C{sub H}2-C{sub H}3 interface, the binding site for several mammalian and bacterial Fc-binding proteins. The structure identifies interface histidines that may confer pH-dependent binding and regions of CgE implicated in cell-to-cell spread of virus. The ternary organization of the gE-gI/Fc complex is compatible with antibody bipolar bridging, which can interfere with the antiviral immune response.« less

Molecular mechanism of respiratory syncytial virus fusion inhibitors

DOE PAGES

Battles, Michael B.; Langedijk, Johannes P.; Furmanova-Hollenstein, Polina; ...

2015-12-07

Respiratory syncytial virus (RSV) is a leading cause of pneumonia and bronchiolitis in young children and the elderly. Therapeutic small molecules have been developed that bind the RSV F glycoprotein and inhibit membrane fusion, yet their binding sites and molecular mechanisms of action remain largely unknown. In this paper, we show that these inhibitors bind to a three-fold-symmetric pocket within the central cavity of the metastable prefusion conformation of RSV F. Inhibitor binding stabilizes this conformation by tethering two regions that must undergo a structural rearrangement to facilitate membrane fusion. Inhibitor-escape mutations occur in residues that directly contact the inhibitorsmore » or are involved in the conformational rearrangements required to accommodate inhibitor binding. Resistant viruses do not propagate as well as wild-type RSV in vitro, indicating a fitness cost for inhibitor escape. Finally and collectively, these findings provide new insight into class I viral fusion proteins and should facilitate development of optimal RSV fusion inhibitors.« less

Genetics, Receptor Binding Property, and Transmissibility in Mammals of Naturally Isolated H9N2 Avian Influenza Viruses

PubMed Central

Deng, Guohua; Zhang, Qianyi; Wang, Jinliang; He, Xijun; Wang, Kaicheng; Chen, Jiming; Li, Yuanyuan; Fan, Jun; Kong, Huiui; Gu, Chunyang; Guan, Yuantao; Suzuki, Yasuo; Kawaoka, Yoshihiro; Liu, Liling; Jiang, Yongping; Tian, Guobin; Li, Yanbing; Bu, Zhigao; Chen, Hualan

2014-01-01

H9N2 subtype influenza viruses have been detected in different species of wild birds and domestic poultry in many countries for several decades. Because these viruses are of low pathogenicity in poultry, their eradication is not a priority for animal disease control in many countries, which has allowed them to continue to evolve and spread. Here, we characterized the genetic variation, receptor-binding specificity, replication capability, and transmission in mammals of a series of H9N2 influenza viruses that were detected in live poultry markets in southern China between 2009 and 2013. Thirty-five viruses represented 17 genotypes on the basis of genomic diversity, and one specific “internal-gene-combination” predominated among the H9N2 viruses. This gene combination was also present in the H7N9 and H10N8 viruses that have infected humans in China. All of the 35 viruses preferentially bound to the human-like receptor, although two also retained the ability to bind to the avian-like receptor. Six of nine viruses tested were transmissible in ferrets by respiratory droplet; two were highly transmissible. Some H9N2 viruses readily acquired the 627K or 701N mutation in their PB2 gene upon infection of ferrets, further enhancing their virulence and transmission in mammals. Our study indicates that the widespread dissemination of H9N2 viruses poses a threat to human health not only because of the potential of these viruses to cause an influenza pandemic, but also because they can function as “vehicles” to deliver different subtypes of influenza viruses from avian species to humans. PMID:25411973

Thermal Inactivation of Enteric Viruses and Bioaccumulation of Enteric Foodborne Viruses in Live Oysters (Crassostrea virginica)

PubMed Central

Araud, Elbashir; DiCaprio, Erin; Ma, Yuanmei; Lou, Fangfei; Gao, Yu; Kingsley, David; Hughes, John H.

2016-01-01

Human enteric viruses are among the main causative agents of shellfish-associated outbreaks. In this study, the kinetics of viral bioaccumulation in live oysters and the heat stabilities of the predominant enteric viruses were determined both in tissue culture and in oyster tissues. A human norovirus (HuNoV) GII.4 strain, HuNoV surrogates (murine norovirus [MNV-1], Tulane virus [TV]), hepatitis A virus (HAV), and human rotavirus (RV) bioaccumulated to high titers within oyster tissues, with different patterns of bioaccumulation for the different viruses. We tested the thermal stability of each virus at 62, 72, and 80°C in culture medium. The viruses can be ranked from the most heat resistant to the least stable as follows: HAV, RV, TV, MNV-1. In addition, we found that oyster tissues provided protection to the viruses during heat treatment. To decipher the mechanism underlying viral inactivation by heat, purified TV was treated at 80°C for increasing time intervals. It was found that the integrity of the viral capsid was disrupted, whereas viral genomic RNA remained intact. Interestingly, heat treatment leading to complete loss of TV infectivity was not sufficient to completely disrupt the receptor binding activity of TV, as determined by the porcine gastric mucin–magnetic bead binding assay. Similarly, HuNoV virus-like particles (VLPs) and a HuNoV GII.4 strain retained some receptor binding ability following heat treatment. Although foodborne viruses have variable heat stability, 80°C for >6 min was sufficient to completely inactivate enteric viruses in oysters, with the exception of HAV. PMID:26826225

Identification and expression profiling analysis of calmodulin-binding transcription activator genes in maize (Zea mays L.) under abiotic and biotic stresses

PubMed Central

Yue, Runqing; Lu, Caixia; Sun, Tao; Peng, Tingting; Han, Xiaohua; Qi, Jianshuang; Yan, Shufeng; Tie, Shuanggui

2015-01-01

The calmodulin-binding transcription activators (CAMTA) play critical roles in plant growth and responses to environmental stimuli. However, how CAMTAs function in responses to abiotic and biotic stresses in maize (Zea mays L.) is largely unknown. In this study, we first identified all the CAMTA homologous genes in the whole genome of maize. The results showed that nine ZmCAMTA genes showed highly diversified gene structures and tissue-specific expression patterns. Many ZmCAMTA genes displayed high expression levels in the roots. We then surveyed the distribution of stress-related cis-regulatory elements in the −1.5 kb promoter regions of ZmCAMTA genes. Notably, a large number of stress-related elements present in the promoter regions of some ZmCAMTA genes, indicating a genetic basis of stress expression regulation of these genes. Quantitative real-time PCR was used to test the expression of ZmCAMTA genes under several abiotic stresses (drought, salt, and cold), various stress-related hormones [abscisic acid, auxin, salicylic acid (SA), and jasmonic acid] and biotic stress [rice black-streaked dwarf virus (RBSDV) infection]. Furthermore, the expression pattern of ZmCAMTA genes under RBSDV infection was analyzed to investigate their potential roles in responses of different maize cultivated varieties to RBSDV. The expression of most ZmCAMTA genes responded to both abiotic and biotic stresses. The data will help us to understand the roles of CAMTA-mediated Ca2+ signaling in maize tolerance to environmental stresses. PMID:26284092

T Regulatory Cell Induced Foxp3 Binds the IL2, IFNγ, and TNFα Promoters in Virus-Specific CD8+ T Cells from Feline Immunodeficiency Virus Infected Cats.

PubMed

Wang, Yan; Nag, Mukta; Tuohy, Joanne L; De Paris, Kristina; Fogle, Jonathan E

2018-03-01

Polyfunctional CD8 + T cells play a critical role in controlling viremia during AIDS lentiviral infections. However, for most HIV-infected individuals, virus-specific CD8 + T cells exhibit loss of polyfunctionality, including loss of IL2, TNFα, and IFNγ. Using the feline immunodeficiency virus (FIV) model for AIDS lentiviral persistence, our laboratory has demonstrated that FIV-activated Treg cells target CD8 + T cells, leading to a reduction in IL2 and IFNγ production. Furthermore, we have demonstrated that Treg cells induce expression of the repressive transcription factor, Foxp3, in CD8 + T cells. Based upon these findings, we asked if Treg-induced Foxp3 could bind to the IL2, TNFα, and IFNγ promoter regions in virus-specific CD8 + T cells. Following coculture with autologous Treg cells, we demonstrated decreased mRNA levels of IL2 and IFNγ at weeks 4 and 8 postinfection and decreased TNFα at week 4 postinfection in virus-specific CD8 + T cells. We also clearly demonstrated Treg cell-induced Foxp3 expression in virus-specific CD8 + T cells at weeks 1, 4, and 8 postinfection. Finally, we documented Foxp3 binding to the IL2, TNFα, and IFNγ promoters at 8 weeks and 6 months postinfection in virus-specific CD8 + T cells following Treg cell coculture. In summary, the results here clearly demonstrate that Foxp3 inhibits IL2, TNFα, and IFNγ transcription by binding to their promoter regions in lentivirus-specific CD8 + T cells. We believe this is the first description of this process during the course of AIDS lentiviral infection.

Low pathogenic avian influenza isolates from wild birds replicate and transmit via contact in ferrets without prior adaptation.

PubMed

Driskell, Elizabeth A; Pickens, Jennifer A; Humberd-Smith, Jennifer; Gordy, James T; Bradley, Konrad C; Steinhauer, David A; Berghaus, Roy D; Stallknecht, David E; Howerth, Elizabeth W; Tompkins, Stephen Mark

2012-01-01

Direct transmission of avian influenza viruses to mammals has become an increasingly investigated topic during the past decade; however, isolates that have been primarily investigated are typically ones originating from human or poultry outbreaks. Currently there is minimal comparative information on the behavior of the innumerable viruses that exist in the natural wild bird host. We have previously demonstrated the capacity of numerous North American avian influenza viruses isolated from wild birds to infect and induce lesions in the respiratory tract of mice. In this study, two isolates from shorebirds that were previously examined in mice (H1N9 and H6N1 subtypes) are further examined through experimental inoculations in the ferret with analysis of viral shedding, histopathology, and antigen localization via immunohistochemistry to elucidate pathogenicity and transmission of these viruses. Using sequence analysis and glycan binding analysis, we show that these avian viruses have the typical avian influenza binding pattern, with affinity for cell glycoproteins/glycolipids having terminal sialic acid (SA) residues with α 2,3 linkage [Neu5Ac(α2,3)Gal]. Despite the lack of α2,6 linked SA binding, these AIVs productively infected both the upper and lower respiratory tract of ferrets, resulting in nasal viral shedding and pulmonary lesions with minimal morbidity. Moreover, we show that one of the viruses is able to transmit to ferrets via direct contact, despite its binding affinity for α 2,3 linked SA residues. These results demonstrate that avian influenza viruses, which are endemic in aquatic birds, can potentially infect humans and other mammals without adaptation. Finally this work highlights the need for additional study of the wild bird subset of influenza viruses in regard to surveillance, transmission, and potential for reassortment, as they have zoonotic potential.

Targeting CTCF to Control Virus Gene Expression: A Common Theme amongst Diverse DNA Viruses.

PubMed

Pentland, Ieisha; Parish, Joanna L

2015-07-06

All viruses target host cell factors for successful life cycle completion. Transcriptional control of DNA viruses by host cell factors is important in the temporal and spatial regulation of virus gene expression. Many of these factors are recruited to enhance virus gene expression and thereby increase virus production, but host cell factors can also restrict virus gene expression and productivity of infection. CCCTC binding factor (CTCF) is a host cell DNA binding protein important for the regulation of genomic chromatin boundaries, transcriptional control and enhancer element usage. CTCF also functions in RNA polymerase II regulation and in doing so can influence co-transcriptional splicing events. Several DNA viruses, including Kaposi's sarcoma-associated herpesvirus (KSHV), Epstein-Barr virus (EBV) and human papillomavirus (HPV) utilize CTCF to control virus gene expression and many studies have highlighted a role for CTCF in the persistence of these diverse oncogenic viruses. CTCF can both enhance and repress virus gene expression and in some cases CTCF increases the complexity of alternatively spliced transcripts. This review article will discuss the function of CTCF in the life cycle of DNA viruses in the context of known host cell CTCF functions.

Structures of D14 and D14L in the strigolactone and karrikin signaling pathways.

PubMed

Kagiyama, Megumi; Hirano, Yoshinori; Mori, Tomoyuki; Kim, Sun-Yong; Kyozuka, Junko; Seto, Yoshiya; Yamaguchi, Shinjiro; Hakoshima, Toshio

2013-02-01

Strigolactones (SLs) are plant hormones that inhibit shoot branching. DWARF14 (D14) inhibits rice tillering and is an SL receptor candidate in the branching inhibition pathway, whereas the close homologue DWARF14-LIKE (D14L) participates in the signaling pathway of karrikins (KARs), which are derived from burnt vegetation as smoke stimulants of seed germination. We provide the first evidence for direct binding of the bioactive SL analogue GR24 to D14. Isothermal titration calorimetry measurements show a D14-GR24 binding affinity in the sub-micromolar range. Similarly, bioactive KAR1 directly binds D14L in the micromolar range. The crystal structure of rice D14 shows a compact α-/β-fold hydrolase domain forming a deep ligand-binding pocket capable of accommodating GR24. Insertion of four α-helices between β6 strand and αD helix forms the helical cap of the pocket, although the pocket is open to the solvent. The pocket contains the conserved catalytic triad Ser-His-Asp aligned with the oxyanion hole, suggesting hydrolase activity. Although these structural characteristics are conserved in D14L, the D14L pocket is smaller than that of D14. The KAR-insensitive mutation kai2-1 is located at the prominent long β6-αD1 loop, which is characteristic in D14 and D14L, but not in related α-/β-fold hydrolases. © 2013 The Authors Genes to Cells © 2013 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

Transmissible gastroenteritis virus; identification of M protein-binding peptide ligands with antiviral and diagnostic potential

USDA-ARS?s Scientific Manuscript database

The membrane (M) protein is one of the major structural proteins of coronavirus particles. In this study, the M protein of transmissible gastroenteritis virus (TGEV) was used to biopan a 12-mer phage display random peptide library. Three phages expressing TGEV-M-binding peptides were identified and ...

PhOBF1, a petunia OCS element binding factor, plays an important role in antiviral RNA silencing

USDA-ARS?s Scientific Manuscript database

Virus-induced gene silencing (VIGS) is a common strategy of reverse genetics for characterizing function of genes in plant. The detailed mechanism governing RNA silencing efficiency triggered by virus is largely unclear. Here, we revealed that a petunia (Petunia hybrida) ocs element binding factor, ...

75 FR 48355 - Prospective Grant of Exclusive License: Griffithsin, Glycosylation-Resistant Griffithsin, and...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-08-10

... infections where those viral infections are human immunodeficiency virus (HIV) or hepatitis C virus (HCV... Griffithsin inhibits viral binding, fusion and entry into the host cells by binding to viral envelope gp120... a viral infection (incl. HIV), as well as vaccine development, and screening assays. The second...

Integrative proteomics to understand the transmission mechanism of Barley yellow dwarf virus-GPV by its insect vector Rhopalosiphum padi

PubMed Central

Wang, Hui; Wu, Keke; Liu, Yan; Wu, Yunfeng; Wang, Xifeng

2015-01-01

Barley yellow dwarf virus-GPV (BYDV-GPV) is transmitted by Rhopalosiphum padi and Schizaphis graminum in a persistent nonpropagative manner. To improve our understanding of its transmission mechanism by aphid vectors, we used two approaches, isobaric tags for relative and absolute quantitation (iTRAQ) and yeast two-hybrid (YTH) system, to identify proteins in R. padi that may interact with or direct the spread of BYDV-GPV along the circulative transmission pathway. Thirty-three differential aphid proteins in viruliferous and nonviruliferous insects were identified using iTRAQ coupled to 2DLC-MS/MS. With the yeast two-hybrid system, 25 prey proteins were identified as interacting with the readthrough protein (RTP) and eight with the coat protein (CP), which are encoded by BYDV-GPV. Among the aphid proteins identified, most were involved in primary energy metabolism, synaptic vesicle cycle, the proteasome pathway and the cell cytoskeleton organization pathway. In a systematic comparison of the two methods, we found that the information generated by the two methods was complementary. Taken together, our findings provide useful information on the interactions between BYDV-GPV and its vector R. padi to further our understanding of the mechanisms regulating circulative transmission in aphid vectors. PMID:26161807

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

Liu, Yue; Sheng, Ju; Baggen, Jim

Human enterovirus D68 (EV-D68) is a causative agent of childhood respiratory diseases and has now emerged as a global public health threat. Nevertheless, knowledge of the tissue tropism and pathogenesis of EV-D68 has been hindered by a lack of studies on the receptor-mediated EV-D68 entry into host cells. Here we demonstrate that cell surface sialic acid is essential for EV-D68 to bind to and infect susceptible cells. Crystal structures of EV-D68 in complex with sialylated glycan receptor analogues show that they bind into the ‘canyon’ on the virus surface. The sialic acid receptor induces a cascade of conformational changes inmore » the virus to eject a fatty-acid-like molecule that regulates the stability of the virus. Furthermore, virus binding to a sialic acid receptor and to immunoglobulin-like receptors used by most other enteroviruses share a conserved mechanism for priming viral uncoating and facilitating cell entry.« less

Structure of the measles virus hemagglutinin bound to its cellular receptor SLAM.

PubMed

Hashiguchi, Takao; Ose, Toyoyuki; Kubota, Marie; Maita, Nobuo; Kamishikiryo, Jun; Maenaka, Katsumi; Yanagi, Yusuke

2011-02-01

Measles virus, a major cause of childhood morbidity and mortality worldwide, predominantly infects immune cells using signaling lymphocyte activation molecule (SLAM) as a cellular receptor. Here we present crystal structures of measles virus hemagglutinin (MV-H), the receptor-binding glycoprotein, in complex with SLAM. The MV-H head domain binds to a β-sheet of the membrane-distal ectodomain of SLAM using the side of its β-propeller fold. This is distinct from attachment proteins of other paramyxoviruses that bind receptors using the top of their β-propeller. The structure provides templates for antiviral drug design, an explanation for the effectiveness of the measles virus vaccine, and a model of the homophilic SLAM-SLAM interaction involved in immune modulations. Notably, the crystal structures obtained show two forms of the MV-H-SLAM tetrameric assembly (dimer of dimers), which may have implications for the mechanism of fusion triggering.

One-step selection of Vaccinia virus-binding DNA aptamers by MonoLEX

PubMed Central

Nitsche, Andreas; Kurth, Andreas; Dunkhorst, Anna; Pänke, Oliver; Sielaff, Hendrik; Junge, Wolfgang; Muth, Doreen; Scheller, Frieder; Stöcklein, Walter; Dahmen, Claudia; Pauli, Georg; Kage, Andreas

2007-01-01

Background As a new class of therapeutic and diagnostic reagents, more than fifteen years ago RNA and DNA aptamers were identified as binding molecules to numerous small compounds, proteins and rarely even to complete pathogen particles. Most aptamers were isolated from complex libraries of synthetic nucleic acids by a process termed SELEX based on several selection and amplification steps. Here we report the application of a new one-step selection method (MonoLEX) to acquire high-affinity DNA aptamers binding Vaccinia virus used as a model organism for complex target structures. Results The selection against complete Vaccinia virus particles resulted in a 64-base DNA aptamer specifically binding to orthopoxviruses as validated by dot blot analysis, Surface Plasmon Resonance, Fluorescence Correlation Spectroscopy and real-time PCR, following an aptamer blotting assay. The same oligonucleotide showed the ability to inhibit in vitro infection of Vaccinia virus and other orthopoxviruses in a concentration-dependent manner. Conclusion The MonoLEX method is a straightforward procedure as demonstrated here for the identification of a high-affinity DNA aptamer binding Vaccinia virus. MonoLEX comprises a single affinity chromatography step, followed by subsequent physical segmentation of the affinity resin and a single final PCR amplification step of bound aptamers. Therefore, this procedure improves the selection of high affinity aptamers by reducing the competition between aptamers of different affinities during the PCR step, indicating an advantage for the single-round MonoLEX method. PMID:17697378

Hydrophobic pocket targeting probes for enteroviruses.

PubMed

Martikainen, Mari; Salorinne, Kirsi; Lahtinen, Tanja; Malola, Sami; Permi, Perttu; Häkkinen, Hannu; Marjomäki, Varpu

2015-11-07

Visualization and tracking of viruses without compromising their functionality is crucial in order to understand virus targeting to cells and tissues, and to understand the subsequent subcellular steps leading to virus uncoating and replication. Enteroviruses are important human pathogens causing a vast number of acute infections, and are also suggested to contribute to the development of chronic diseases like type I diabetes. Here, we demonstrate a novel method to target site-specifically the hydrophobic pocket of enteroviruses. A probe, a derivative of Pleconaril, was developed and conjugated to various labels that enabled the visualization of enteroviruses under light and electron microscopes. The probe mildly stabilized the virus particle by increasing the melting temperature by 1-3 degrees, and caused a delay in the uncoating of the virus in the cellular endosomes, but could not however inhibit the receptor binding, cellular entry or infectivity of the virus. The hydrophobic pocket binding moiety of the probe was shown to bind to echovirus 1 particle by STD and tr-NOESY NMR methods. Furthermore, binding to echovirus 1 and Coxsackievirus A9, and to a lesser extent to Coxsackie virus B3 was verified by using a gold nanocluster labeled probe by TEM analysis. Molecular modelling suggested that the probe fits the hydrophobic pockets of EV1 and CVA9, but not of CVB3 as expected, correlating well with the variations in the infectivity and stability of the virus particles. EV1 conjugated to the fluorescent dye labeled probe was efficiently internalized into the cells. The virus-fluorescent probe conjugate accumulated in the cytoplasmic endosomes and caused infection starting from 6 hours onwards. Remarkably, before and during the time of replication, the fluorescent probe was seen to leak from the virus-positive endosomes and thus separate from the capsid proteins that were left in the endosomes. These results suggest that, like the physiological hydrophobic content, the probe may be released upon virus uncoating. Our results collectively thus show that the gold and fluorescently labeled probes may be used to track and visualize the studied enteroviruses during the early phases of infection opening new avenues to follow virus uncoating in cells.

Structural virology. Near-atomic cryo-EM structure of the helical measles virus nucleocapsid.

PubMed

Gutsche, Irina; Desfosses, Ambroise; Effantin, Grégory; Ling, Wai Li; Haupt, Melina; Ruigrok, Rob W H; Sachse, Carsten; Schoehn, Guy

2015-05-08

Measles is a highly contagious human disease. We used cryo-electron microscopy and single particle-based helical image analysis to determine the structure of the helical nucleocapsid formed by the folded domain of the measles virus nucleoprotein encapsidating an RNA at a resolution of 4.3 angstroms. The resulting pseudoatomic model of the measles virus nucleocapsid offers important insights into the mechanism of the helical polymerization of nucleocapsids of negative-strand RNA viruses, in particular via the exchange subdomains of the nucleoprotein. The structure reveals the mode of the nucleoprotein-RNA interaction and explains why each nucleoprotein of measles virus binds six nucleotides, whereas the respiratory syncytial virus nucleoprotein binds seven. It provides a rational basis for further analysis of measles virus replication and transcription, and reveals potential targets for drug design. Copyright © 2015, American Association for the Advancement of Science.

Structure of the Nucleoprotein Binding Domain of Mokola Virus Phosphoprotein▿

PubMed Central

Assenberg, René; Delmas, Olivier; Ren, Jingshan; Vidalain, Pierre-Olivier; Verma, Anil; Larrous, Florence; Graham, Stephen C.; Tangy, Frédéric; Grimes, Jonathan M.; Bourhy, Hervé

2010-01-01

Mokola virus (MOKV) is a nonsegmented, negative-sense RNA virus that belongs to the Lyssavirus genus and Rhabdoviridae family. MOKV phosphoprotein P is an essential component of the replication and transcription complex and acts as a cofactor for the viral RNA-dependent RNA polymerase. P recruits the viral polymerase to the nucleoprotein-bound viral RNA (N-RNA) via an interaction between its C-terminal domain and the N-RNA complex. Here we present a structure for this domain of MOKV P, obtained by expression of full-length P in Escherichia coli, which was subsequently truncated during crystallization. The structure has a high degree of homology with P of rabies virus, another member of Lyssavirus genus, and to a lesser degree with P of vesicular stomatitis virus (VSV), a member of the related Vesiculovirus genus. In addition, analysis of the crystal packing of this domain reveals a potential binding site for the nucleoprotein N. Using both site-directed mutagenesis and yeast two-hybrid experiments to measure P-N interaction, we have determined the relative roles of key amino acids involved in this interaction to map the region of P that binds N. This analysis also reveals a structural relationship between the N-RNA binding domain of the P proteins of the Rhabdoviridae and the Paramyxoviridae. PMID:19906936

A chemokine-binding domain in the tumor necrosis factor receptor from variola (smallpox) virus

PubMed Central

Alejo, Alí; Ruiz-Argüello, M. Begoña; Ho, Yin; Smith, Vincent P.; Saraiva, Margarida; Alcami, Antonio

2006-01-01

Variola virus (VaV) is the causative agent of smallpox, one of the most devastating diseases encountered by man, that was eradicated in 1980. The deliberate release of VaV would have catastrophic consequences on global public health. However, the mechanisms that contribute to smallpox pathogenesis are poorly understood at the molecular level. The ability of viruses to evade the host defense mechanisms is an important determinant of viral pathogenesis. Here we show that the tumor necrosis factor receptor (TNFR) homologue CrmB encoded by VaV functions not only as a soluble decoy TNFR but also as a highly specific binding protein for several chemokines that mediate recruitment of immune cells to mucosal surfaces and the skin, sites of virus entry and viral replication at late stages of smallpox. CrmB binds chemokines through its C-terminal domain, which is unrelated to TNFRs, was named smallpox virus-encoded chemokine receptor (SECRET) domain and uncovers a family of poxvirus chemokine inhibitors. An active SECRET domain was found in another viral TNFR (CrmD) and three secreted proteins encoded by orthopoxviruses. These findings identify a previously undescribed chemokine-binding and inhibitory domain unrelated to host chemokine receptors and a mechanism of immune modulation in VaV that may influence smallpox pathogenesis. PMID:16581912

A chemokine-binding domain in the tumor necrosis factor receptor from variola (smallpox) virus.

PubMed

Alejo, Alí; Ruiz-Argüello, M Begoña; Ho, Yin; Smith, Vincent P; Saraiva, Margarida; Alcami, Antonio

2006-04-11

Variola virus (VaV) is the causative agent of smallpox, one of the most devastating diseases encountered by man, that was eradicated in 1980. The deliberate release of VaV would have catastrophic consequences on global public health. However, the mechanisms that contribute to smallpox pathogenesis are poorly understood at the molecular level. The ability of viruses to evade the host defense mechanisms is an important determinant of viral pathogenesis. Here we show that the tumor necrosis factor receptor (TNFR) homologue CrmB encoded by VaV functions not only as a soluble decoy TNFR but also as a highly specific binding protein for several chemokines that mediate recruitment of immune cells to mucosal surfaces and the skin, sites of virus entry and viral replication at late stages of smallpox. CrmB binds chemokines through its C-terminal domain, which is unrelated to TNFRs, was named smallpox virus-encoded chemokine receptor (SECRET) domain and uncovers a family of poxvirus chemokine inhibitors. An active SECRET domain was found in another viral TNFR (CrmD) and three secreted proteins encoded by orthopoxviruses. These findings identify a previously undescribed chemokine-binding and inhibitory domain unrelated to host chemokine receptors and a mechanism of immune modulation in VaV that may influence smallpox pathogenesis.

The complete genomic sequence of a tentative new polerovirus identified in barley in South Korea.

PubMed

Zhao, Fumei; Lim, Seungmo; Yoo, Ran Hee; Igori, Davaajargal; Kim, Sang-Min; Kwak, Do Yeon; Kim, Sun Lim; Lee, Bong Choon; Moon, Jae Sun

2016-07-01

The complete nucleotide sequence of a new barley polerovirus, tentatively named barley virus G (BVG), which was isolated in Gimje, South Korea, has been determined using an RNA sequencing technique combined with polymerase chain reaction methods. The viral genomic RNA of BVG is 5,620 nucleotides long and contains six typical open reading frames commonly observed in other poleroviruses. Sequence comparisons revealed that BVG is most closely related to maize yellow dwarf virus-RMV, with the highest amino acid identities being less than 90 % for all of the corresponding proteins. These results suggested that BVG is a member of a new species in the genus Polerovirus.

Neuraminidase inhibitor R-125489 - A promising drug for treating influenza virus: Steered molecular dynamics approach

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

Mai, Binh Khanh; Li, Mai Suan, E-mail: masli@ifpan.edu.pl

2011-07-08

Highlights: {yields} We study binding affinity of R-125489 and its prodrug CS-8958 to neuraminidase of pathogenic influenza viruses by molecular dynamics simulations. {yields} It is shown that, in agreement with experiments, R-125489 binds to neuraminidase more tightly than CS-8958. {yields} We predict that R-125489 can be used to treat not only wild-type but also tamiflu-resistant N294S, H274Y variants of A/H5N1 virus. {yields} The high correlation between theoretical and experimental data implies that SMD is a very promising tool for drug design. -- Abstract: Two neuraminidase inhibitors, oseltamivir and zanamivir, are important drug treatments for influenza. Oseltamivir-resistant mutants of the influenzamore » virus A/H1N1 and A/H5N1 have emerged, necessitating the development of new long-acting antiviral agents. One such agent is a new neuraminidase inhibitor R-125489 and its prodrug CS-8958. An atomic level understanding of the nature of this antiviral agents binding is still missing. We address this gap in our knowledge by applying steered molecular dynamics (SMD) simulations to different subtypes of seasonal and highly pathogenic influenza viruses. We show that, in agreement with experiments, R-125489 binds to neuraminidase more tightly than CS-8958. Based on results obtained by SMD and the molecular mechanics-Poisson-Boltzmann surface area method, we predict that R-125489 can be used to treat not only wild-type but also tamiflu-resistant N294S, H274Y variants of A/H5N1 virus as its binding affinity does not vary much across these systems. The high correlation level between theoretically determined rupture forces and experimental data on binding energies for the large number of systems studied here implies that SMD is a promising tool for drug design.« less

Envelope residue 375 substitutions in simian–human immunodeficiency viruses enhance CD4 binding and replication in rhesus macaques

PubMed Central

Li, Hui; Wang, Shuyi; Kong, Rui; Ding, Wenge; Lee, Fang-Hua; Parker, Zahra; Kim, Eunlim; Learn, Gerald H.; Hahn, Paul; Policicchio, Ben; Brocca-Cofano, Egidio; Deleage, Claire; Hao, Xingpei; Chuang, Gwo-Yu; Gorman, Jason; Gardner, Matthew; Lewis, Mark G.; Hatziioannou, Theodora; Santra, Sampa; Apetrei, Cristian; Pandrea, Ivona; Alam, S. Munir; Liao, Hua-Xin; Shen, Xiaoying; Tomaras, Georgia D.; Farzan, Michael; Chertova, Elena; Keele, Brandon F.; Estes, Jacob D.; Lifson, Jeffrey D.; Doms, Robert W.; Montefiori, David C.; Haynes, Barton F.; Sodroski, Joseph G.; Kwong, Peter D.; Hahn, Beatrice H.; Shaw, George M.

2016-01-01

Most simian–human immunodeficiency viruses (SHIVs) bearing envelope (Env) glycoproteins from primary HIV-1 strains fail to infect rhesus macaques (RMs). We hypothesized that inefficient Env binding to rhesus CD4 (rhCD4) limits virus entry and replication and could be enhanced by substituting naturally occurring simian immunodeficiency virus Env residues at position 375, which resides at a critical location in the CD4-binding pocket and is under strong positive evolutionary pressure across the broad spectrum of primate lentiviruses. SHIVs containing primary or transmitted/founder HIV-1 subtype A, B, C, or D Envs with genotypic variants at residue 375 were constructed and analyzed in vitro and in vivo. Bulky hydrophobic or basic amino acids substituted for serine-375 enhanced Env affinity for rhCD4, virus entry into cells bearing rhCD4, and virus replication in primary rhCD4 T cells without appreciably affecting antigenicity or antibody-mediated neutralization sensitivity. Twenty-four RMs inoculated with subtype A, B, C, or D SHIVs all became productively infected with different Env375 variants—S, M, Y, H, W, or F—that were differentially selected in different Env backbones. Notably, SHIVs replicated persistently at titers comparable to HIV-1 in humans and elicited autologous neutralizing antibody responses typical of HIV-1. Seven animals succumbed to AIDS. These findings identify Env–rhCD4 binding as a critical determinant for productive SHIV infection in RMs and validate a novel and generalizable strategy for constructing SHIVs with Env glycoproteins of interest, including those that in humans elicit broadly neutralizing antibodies or bind particular Ig germ-line B-cell receptors. PMID:27247400

Role of LAMP1 Binding and pH Sensing by the Spike Complex of Lassa Virus.

PubMed

Cohen-Dvashi, Hadas; Israeli, Hadar; Shani, Orly; Katz, Aliza; Diskin, Ron

2016-11-15

To effectively infect cells, Lassa virus needs to switch in an endosomal compartment from its primary receptor, α-dystroglycan, to a protein termed LAMP1. A unique histidine triad on the surface of the receptor-binding domain from the glycoprotein spike complex of Lassa virus is important for LAMP1 binding. Here we investigate mutated spikes that have an impaired ability to interact with LAMP1 and show that although LAMP1 is important for efficient infectivity, it is not required for spike-mediated membrane fusion per se Our studies reveal important regulatory roles for histidines from the triad in sensing acidic pH and preventing premature spike triggering. We further show that LAMP1 requires a positively charged His230 residue to engage with the spike complex and that LAMP1 binding promotes membrane fusion. These results elucidate the molecular role of LAMP1 binding during Lassa virus cell entry and provide new insights into how pH is sensed by the spike. Lassa virus is a devastating disease-causing agent in West Africa, with a significant yearly death toll and severe long-term complications associated with its infection in survivors. In recent years, we learned that Lassa virus needs to switch receptors in a pH-dependent manner to efficiently infect cells, but neither the molecular mechanisms that allow switching nor the actual effects of switching were known. Here we investigate the activity of the viral spike complex after abrogation of its ability to switch receptors. These studies inform us about the role of switching receptors and provide new insights into how the spike senses acidic pH. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Structural Basis for Suppression of a Host Antiviral Response by Influenza A Virus

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

Das,K.; Ma, L.; Xiao, R.

2008-01-01

Influenza A viruses are responsible for seasonal epidemics and high mortality pandemics. A major function of the viral NS1A protein, a virulence factor, is the inhibition of the production of IFN-{beta}{beta} mRNA and other antiviral mRNAs. The NS1A protein of the human influenza A/Udorn/72 (Ud) virus inhibits the production of these antiviral mRNAs by binding the cellular 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30), which is required for the 3' end processing of all cellular pre-mRNAs. Here we report the 1.95- Angstroms resolution X-ray crystal structure of the complex formed between the second and third zinc fingermore » domain (F2F3) of CPSF30 and the C-terminal domain of the Ud NS1A protein. The complex is a tetramer, in which each of two F2F3 molecules wraps around two NS1A effector domains that interact with each other head-to-head. This structure identifies a CPSF30 binding pocket on NS1A comprised of amino acid residues that are highly conserved among human influenza A viruses. Single amino acid changes within this binding pocket eliminate CPSF30 binding, and a recombinant Ud virus expressing an NS1A protein with such a substitution is attenuated and does not inhibit IFN-{beta} pre-mRNA processing. This binding pocket is a potential target for antiviral drug development. The crystal structure also reveals that two amino acids outside of this pocket, F103 and M106, which are highly conserved (>99%) among influenza A viruses isolated from humans, participate in key hydrophobic interactions with F2F3 that stabilize the complex.« less

Biological effects of individually synthesized TNF-binding domain of variola virus CrmB protein.

PubMed

Tsyrendorzhiev, D D; Orlovskaya, I A; Sennikov, S V; Tregubchak, T V; Gileva, I P; Tsyrendorzhieva, M D; Shchelkunov, S N

2014-06-01

The biological characteristics of a 17-kDa protein synthesized in bacterial cells, a TNF-binding domain (VARV-TNF-BP) of a 47-kDa variola virus CrmB protein (VARV-CrmB) consisting of TNF-binding and chemokine-binding domains, were studied. Removal of the C-terminal chemokine-binding domain from VARV-CrmB protein was inessential for the efficiency of its inhibition of TNF cytotoxicity towards L929 mouse fibroblast culture and for TNF-induced oxidative metabolic activity of mouse blood leukocytes. The results of this study could form the basis for further studies of VARV-TNF-BP mechanisms of activity for prospective use in practical medicine.

Construction of fusogenic vesicles bearing specific antibodies. Targeting of reconstituted Sendai virus envelopes towards neuraminidase-treated human erythrocytes.

PubMed

Gitman, A G; Loyter, A

1984-08-10

The cross-linking reagents succinimidyl-4-(p-maleimidophenyl)-butyrate and N-succinimidyl-3-(2-pyridyldithio)-propionate were used to covalently attach antibodies against human erythrocytes to the thiol-containing paraffin, dodecanethiol. The complex formed, dodecanethiol-maleimidophenylbutyrate (or pyridyldithiopropionate)-antibody was inserted into the membranes of reconstituted Sendai virus envelopes. This was achieved by addition of the dodecanethiol-maleimidophenylbutyrate-antibody to a detergent solution (Triton X-100) containing the viral envelope phospholipids and glycoproteins. Removal of the detergent led to the formation of vesicles containing the viral glycoprotein and the dodecanethiol-maleimidophenylbutyrate (or pyridyldithiopropionate)-antibody complexes within the same membrane. Reconstituted Sendai virus envelope-bearing antibodies against human erythrocytes were able to fuse with human erythrocytes (as was reflected by reconstituted Sendai virus envelope-induced hemolysis) from which the natural virus receptors were removed by treatment with neuraminidase. Thus, it appears that anti-human erythrocyte antibodies could substitute for the viral binding protein (hemagglutinin/neuraminidase glycoprotein) in mediating functional binding of the virus particles to the cell plasma membranes. Furthermore, from the results of the present work, it may be inferred that in addition to being the viral-binding protein, hemagglutinin/neuraminidase glycoprotein actively participates in the process of virus-cell fusion.

Differences in Glycoprotein Complex Receptor Binding Site Accessibility Prompt Poor Cross-Reactivity of Neutralizing Antibodies between Closely Related Arenaviruses

PubMed Central

Brouillette, Rachel B.; Phillips, Elisabeth K.; Ayithan, Natarajan

2017-01-01

ABSTRACT The glycoprotein complex (GPC) of arenaviruses, composed of stable signal peptide, GP1, and GP2, is the only antigen correlated with antibody-mediated neutralization. However, despite strong cross-reactivity of convalescent antisera between related arenavirus species, weak or no cross-neutralization occurs. Two closely related clade B viruses, Machupo virus (MACV) and Junín virus (JUNV), have nearly identical overall GPC architecture and share a host receptor, transferrin receptor 1 (TfR1). Given structural and functional similarities of the GP1 receptor binding site (RBS) of these viruses and the recent demonstration that the RBS is an important target for neutralizing antibodies, it is not clear how these viruses avoid cross-neutralization. To address this, MACV/JUNV chimeric GPCs were assessed for interaction with a group of α-JUNV GPC monoclonal antibodies (MAbs) and mouse antisera against JUNV or MACV GPC. All six MAbs targeted GP1, with those that neutralized JUNV GPC-pseudovirions competing with each other for RBS binding. However, these MAbs were unable to bind to a chimeric GPC composed of JUNV GP1 containing a small disulfide bonded loop (loop 10) unique to MACV GPC, suggesting that this loop may block MAbs interaction with the GP1 RBS. Consistent with this loop causing interference, mouse anti-JUNV GPC antisera that solely neutralized pseudovirions bearing autologous GP1 provided enhanced neutralization of MACV GPC when this loop was removed. Our studies provide evidence that loop 10, which is unique to MACV GP1, is an important impediment to binding of neutralizing antibodies and contributes to the poor cross-neutralization of α-JUNV antisera against MACV. IMPORTANCE Multiple New World arenaviruses can cause severe disease in humans, and some geographic overlap exists among these viruses. A vaccine that protects against a broad range of New World arenaviruses is desirable for purposes of simplicity, cost, and broad protection against multiple National Institute of Allergy and Infectious Disease-assigned category A priority pathogens. In this study, we sought to better understand how closely related arenaviruses elude cross-species neutralization by investigating the structural bases of antibody binding and avoidance. In our studies, we found that neutralizing antibodies against two New World arenaviruses, Machupo virus (MACV) and Junín virus (JUNV), bound to the envelope glycoprotein 1 (GP1) with JUNV monoclonal antibodies targeting the receptor binding site (RBS). We further show that altered structures surrounding the RBS pocket in MACV GP1 impede access of JUNV-elicited antibodies. PMID:28100617

Differences in Glycoprotein Complex Receptor Binding Site Accessibility Prompt Poor Cross-Reactivity of Neutralizing Antibodies between Closely Related Arenaviruses.

PubMed

Brouillette, Rachel B; Phillips, Elisabeth K; Ayithan, Natarajan; Maury, Wendy

2017-04-01

The glycoprotein complex (GPC) of arenaviruses, composed of stable signal peptide, GP1, and GP2, is the only antigen correlated with antibody-mediated neutralization. However, despite strong cross-reactivity of convalescent antisera between related arenavirus species, weak or no cross-neutralization occurs. Two closely related clade B viruses, Machupo virus (MACV) and Junín virus (JUNV), have nearly identical overall GPC architecture and share a host receptor, transferrin receptor 1 (TfR1). Given structural and functional similarities of the GP1 receptor binding site (RBS) of these viruses and the recent demonstration that the RBS is an important target for neutralizing antibodies, it is not clear how these viruses avoid cross-neutralization. To address this, MACV/JUNV chimeric GPCs were assessed for interaction with a group of α-JUNV GPC monoclonal antibodies (MAbs) and mouse antisera against JUNV or MACV GPC. All six MAbs targeted GP1, with those that neutralized JUNV GPC-pseudovirions competing with each other for RBS binding. However, these MAbs were unable to bind to a chimeric GPC composed of JUNV GP1 containing a small disulfide bonded loop (loop 10) unique to MACV GPC, suggesting that this loop may block MAbs interaction with the GP1 RBS. Consistent with this loop causing interference, mouse anti-JUNV GPC antisera that solely neutralized pseudovirions bearing autologous GP1 provided enhanced neutralization of MACV GPC when this loop was removed. Our studies provide evidence that loop 10, which is unique to MACV GP1, is an important impediment to binding of neutralizing antibodies and contributes to the poor cross-neutralization of α-JUNV antisera against MACV. IMPORTANCE Multiple New World arenaviruses can cause severe disease in humans, and some geographic overlap exists among these viruses. A vaccine that protects against a broad range of New World arenaviruses is desirable for purposes of simplicity, cost, and broad protection against multiple National Institute of Allergy and Infectious Disease-assigned category A priority pathogens. In this study, we sought to better understand how closely related arenaviruses elude cross-species neutralization by investigating the structural bases of antibody binding and avoidance. In our studies, we found that neutralizing antibodies against two New World arenaviruses, Machupo virus (MACV) and Junín virus (JUNV), bound to the envelope glycoprotein 1 (GP1) with JUNV monoclonal antibodies targeting the receptor binding site (RBS). We further show that altered structures surrounding the RBS pocket in MACV GP1 impede access of JUNV-elicited antibodies. Copyright © 2017 American Society for Microbiology.

Targeting of loaded Sendai virus envelopes by covalently attached insulin molecules to virus receptor-depleted cells: fusion-mediated microinjection of ricin A and simian virus 40 DNA.

PubMed

Gitman, A G; Graessmann, A; Loyter, A

1985-11-01

Insulin molecules were covalently attached to detergent-solubilized Sendai virus envelope glycoproteins (HN and F polypeptides) by the use of the crosslinking reagent succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB). Reconstitution of modified viral glycoproteins (carrying covalently attached insulin) together with unmodified viral glycoproteins resulted in the formation of "fusogenic" viral envelopes bearing insulin molecules. Reconstitution of such fusogenic viral envelopes in the presence of ricin A or simian virus 40 (SV40) DNA resulted in the formation of viral envelopes bearing insulin molecules and loaded with ricin A or SV40 DNA. Such viral envelopes were able to bind to hepatoma tissue culture cells (HTCC) from which Sendai virus receptors were removed by treatment with neuraminidase. Incubation of viral envelopes loaded with ricin A with virus receptor-depleted HTCC resulted in fusion-mediated injection of the toxin, as inferred from inhibition of protein synthesis and decrease in cell viability of the microinjected cells. Fusion-mediated injection of SV40 DNA was inferred from the appearance of SV40 tumor antigen in microinjected cells. Binding and fusion of the loaded viral envelopes to neuraminidase-treated HTCC was mediated solely by the virus-associated insulin molecules. Addition of free insulin molecules inhibited binding of the viral envelopes and, consequently, the microinjection of ricin A and SV40 DNA.

Targeting of loaded Sendai virus envelopes by covalently attached insulin molecules to virus receptor-depleted cells: fusion-mediated microinjection of ricin A and simian virus 40 DNA.

PubMed Central

Gitman, A G; Graessmann, A; Loyter, A

1985-01-01

Insulin molecules were covalently attached to detergent-solubilized Sendai virus envelope glycoproteins (HN and F polypeptides) by the use of the crosslinking reagent succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB). Reconstitution of modified viral glycoproteins (carrying covalently attached insulin) together with unmodified viral glycoproteins resulted in the formation of "fusogenic" viral envelopes bearing insulin molecules. Reconstitution of such fusogenic viral envelopes in the presence of ricin A or simian virus 40 (SV40) DNA resulted in the formation of viral envelopes bearing insulin molecules and loaded with ricin A or SV40 DNA. Such viral envelopes were able to bind to hepatoma tissue culture cells (HTCC) from which Sendai virus receptors were removed by treatment with neuraminidase. Incubation of viral envelopes loaded with ricin A with virus receptor-depleted HTCC resulted in fusion-mediated injection of the toxin, as inferred from inhibition of protein synthesis and decrease in cell viability of the microinjected cells. Fusion-mediated injection of SV40 DNA was inferred from the appearance of SV40 tumor antigen in microinjected cells. Binding and fusion of the loaded viral envelopes to neuraminidase-treated HTCC was mediated solely by the virus-associated insulin molecules. Addition of free insulin molecules inhibited binding of the viral envelopes and, consequently, the microinjection of ricin A and SV40 DNA. PMID:2997783

Structure, Receptor Binding, and Antigenicity of Influenza Virus Hemagglutinins from the 1957 H2N2 Pandemic

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

Xu, Rui; McBride, Ryan; Paulson, James C.

2010-03-04

The hemagglutinin (HA) envelope protein of influenza viruses mediates essential viral functions, including receptor binding and membrane fusion, and is the major viral antigen for antibody neutralization. The 1957 H2N2 subtype (Asian flu) was one of the three great influenza pandemics of the last century and caused 1 million deaths globally from 1957 to 1968. Three crystal structures of 1957 H2 HAs have been determined at 1.60 to 1.75 {angstrom} resolutions to investigate the structural basis for their antigenicity and evolution from avian to human binding specificity that contributed to its introduction into the human population. These structures, which representmore » the highest resolutions yet recorded for a complete ectodomain of a glycosylated viral surface antigen, along with the results of glycan microarray binding analysis, suggest that a hydrophobicity switch at residue 226 and elongation of receptor-binding sites were both critical for avian H2 HA to acquire human receptor specificity. H2 influenza viruses continue to circulate in birds and pigs and, therefore, remain a substantial threat for transmission to humans. The H2 HA structure also reveals a highly conserved epitope that could be harnessed in the design of a broader and more universal influenza A virus vaccine.« less

VP40 of the Ebola Virus as a Target for EboV Therapy: Comprehensive Conformational and Inhibitor Binding Landscape from Accelerated Molecular Dynamics.

PubMed

Balmith, Marissa; Soliman, Mahmoud E S

2017-03-01

The first account of the dynamic features of the loop region of VP40 of the Ebola virus was studied using accelerated molecular dynamics simulations and reported herein. Among the proteins of the Ebola virus, the matrix protein (VP40) plays a significant role in the virus lifecycle thereby making it a promising therapeutic target. Of interest is the newly elucidated N-terminal domain loop region of VP40 comprising residues K127, T129, and N130 which when mutated to alanine have demonstrated an unrecognized role for N-terminal domain-plasma membrane interaction for efficient VP40-plasma membrane localization, oligomerization, matrix assembly, and egress. The molecular understanding of the conformational features of VP40 in complex with a known inhibitor still remains elusive. Using accelerated molecular dynamics approaches, we conducted a comparative study on VP40 apo and bound systems to understand the conformational features of VP40 at the molecular level and to determine the effect of inhibitor binding with the aid of a number of post-dynamic analytical tools. Significant features were seen in the presence of an inhibitor as per molecular mechanics/generalized born surface area binding free energy calculations. Results revealed that inhibitor binding to VP40 reduces the flexibility and mobility of the protein as supported by root mean square fluctuation and root mean square deviation calculations. The study revealed a characteristic "twisting" motion and coiling of the loop region of VP40 accompanied by conformational changes in the dimer interface upon inhibitor binding. We believe that results presented in this study will ultimately provide useful insight into the binding landscape of VP40 which could assist researchers in the discovery of potent Ebola virus inhibitors for anti-Ebola therapies.

GRB2 Interaction with the Ecotropic Murine Leukemia Virus Receptor, mCAT-1, Controls Virus Entry and Is Stimulated by Virus Binding

PubMed Central

Chen, Zeming; Kolokoltsov, Andrey A.; Wang, Jia; Adhikary, Shramika; Lorinczi, Marta; Elferink, Lisa A.

2012-01-01

For retroviruses such as HIV-1 and murine leukemia virus (MLV), active receptor recruitment and trafficking occur during viral entry. However, the underlying mechanisms and cellular factors involved in the process are largely uncharacterized. The viral receptor for ecotropic MLV (eMLV), a classical model for retrovirus infection mechanisms and pathogenesis, is mouse cationic amino acid transporter 1 (mCAT-1). Growth factor receptor-bound protein 2 (GRB2) is an adaptor protein that has been shown to couple cell surface receptors, such as epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor, to intracellular signaling events. Here we examined if GRB2 could also play a role in controlling infection by retroviruses by affecting receptor function. The GRB2 RNA interference (RNAi)-mediated suppression of endogenous GRB2 resulted in a consistent and significant reduction of virus binding and membrane fusion. The binding between eMLV and cells promoted increased GRB2–mCAT-1 interactions, as detected by immunoprecipitation. Consistently, the increased colocalization of GRB2 and mCAT-1 signals was detected by confocal microscopy. This association was time dependent and paralleled the kinetics of cell-virus membrane fusion. Interestingly, unlike the canonical binding pattern seen for GRB2 and growth factor receptors, GRB2–mCAT-1 binding does not depend on the GRB2-SH2 domain-mediated recognition of tyrosine phosphorylation on the receptor. The inhibition of endogenous GRB2 led to a reduction in surface levels of mCAT-1, which was detected by immunoprecipitation and by a direct binding assay using a recombinant MLV envelope protein receptor binding domain (RBD). Consistent with this observation, the expression of a dominant negative GRB2 mutant (R86K) resulted in the sequestration of mCAT-1 from the cell surface into intracellular vesicles. Taken together, these findings suggest a novel role for GRB2 in ecotropic MLV entry and infection by facilitating mCAT-1 trafficking. PMID:22090132

GRB2 interaction with the ecotropic murine leukemia virus receptor, mCAT-1, controls virus entry and is stimulated by virus binding.

PubMed

Chen, Zeming; Kolokoltsov, Andrey A; Wang, Jia; Adhikary, Shramika; Lorinczi, Marta; Elferink, Lisa A; Davey, Robert A

2012-02-01

For retroviruses such as HIV-1 and murine leukemia virus (MLV), active receptor recruitment and trafficking occur during viral entry. However, the underlying mechanisms and cellular factors involved in the process are largely uncharacterized. The viral receptor for ecotropic MLV (eMLV), a classical model for retrovirus infection mechanisms and pathogenesis, is mouse cationic amino acid transporter 1 (mCAT-1). Growth factor receptor-bound protein 2 (GRB2) is an adaptor protein that has been shown to couple cell surface receptors, such as epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor, to intracellular signaling events. Here we examined if GRB2 could also play a role in controlling infection by retroviruses by affecting receptor function. The GRB2 RNA interference (RNAi)-mediated suppression of endogenous GRB2 resulted in a consistent and significant reduction of virus binding and membrane fusion. The binding between eMLV and cells promoted increased GRB2-mCAT-1 interactions, as detected by immunoprecipitation. Consistently, the increased colocalization of GRB2 and mCAT-1 signals was detected by confocal microscopy. This association was time dependent and paralleled the kinetics of cell-virus membrane fusion. Interestingly, unlike the canonical binding pattern seen for GRB2 and growth factor receptors, GRB2-mCAT-1 binding does not depend on the GRB2-SH2 domain-mediated recognition of tyrosine phosphorylation on the receptor. The inhibition of endogenous GRB2 led to a reduction in surface levels of mCAT-1, which was detected by immunoprecipitation and by a direct binding assay using a recombinant MLV envelope protein receptor binding domain (RBD). Consistent with this observation, the expression of a dominant negative GRB2 mutant (R86K) resulted in the sequestration of mCAT-1 from the cell surface into intracellular vesicles. Taken together, these findings suggest a novel role for GRB2 in ecotropic MLV entry and infection by facilitating mCAT-1 trafficking.

Evidence for zinc binding by two structural proteins of Plodia interpunctella granulosis virus

NASA Technical Reports Server (NTRS)

Funk, C. J.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

1992-01-01

Workers in our laboratory previously reported the possibility of cation involvement in the in vitro dissociation of the Plodia interpunctella granulosis virus nucleocapsids (K. A. Tweeten, L. A. Bulla, Jr., and R. A. Consigli, J. Virol. 33:866-876, 1980; M. E. Wilson and R. A. Consigli, Virology 143:516-525, 1985). The current study found zinc associated with both granulosis virus nucleocapsids and granulin by atomic absorption analysis. A blotting assay with 65Zn2+ specifically identified the radioactive cation as binding to two viral structural proteins, granulin and VP12. These findings indicate that zinc may have a critical role in maintaining virus stability.

Amiodarone affects Ebola virus binding and entry into target cells.

PubMed

Salata, Cristiano; Munegato, Denis; Martelli, Francesco; Parolin, Cristina; Calistri, Arianna; Baritussio, Aldo; Palù, Giorgio

2018-03-02

Ebola Virus Disease is one of the most lethal transmissible infections characterized by a high fatality rate. Several research studies have aimed to identify effective antiviral agents. Amiodarone, a drug used for the treatment of arrhythmias, has been shown to inhibit filovirus infection in vitro by acting at the early step of the viral replication cycle. Here we demonstrate that amiodarone reduces virus binding to target cells and slows down the progression of the viral particles along the endocytic pathway. Overall our data support the notion that amiodarone interferes with Ebola virus infection by affecting cellular pathways/targets involved in the viral entry process.

Diversity, Replication, Pathogenicity and Cell Biology of Crimean Congo Hemorrhagic Fever Virus

DTIC Science & Technology

2006-10-01

recombinant protein produced and purified in E . coli was able to bind to ssRNA in a dot blot filter binding assay. In order to identify domains in the N...Fig. 1. RNA binding domains of the N protein of CCHFV. The depicted GST-fusion proteins were expressed in E . coli and purified using a...detected and NSm protein produced after cleavage of the glycoprotein precursor in virus infected cells. The NSm is stable and transported to the Golgi

Broad-spectrum non-toxic antiviral nanoparticles with a virucidal inhibition mechanism

NASA Astrophysics Data System (ADS)

Cagno, Valeria; Andreozzi, Patrizia; D'Alicarnasso, Marco; Jacob Silva, Paulo; Mueller, Marie; Galloux, Marie; Le Goffic, Ronan; Jones, Samuel T.; Vallino, Marta; Hodek, Jan; Weber, Jan; Sen, Soumyo; Janeček, Emma-Rose; Bekdemir, Ahmet; Sanavio, Barbara; Martinelli, Chiara; Donalisio, Manuela; Rameix Welti, Marie-Anne; Eleouet, Jean-Francois; Han, Yanxiao; Kaiser, Laurent; Vukovic, Lela; Tapparel, Caroline; Král, Petr; Krol, Silke; Lembo, David; Stellacci, Francesco

2018-02-01

Viral infections kill millions yearly. Available antiviral drugs are virus-specific and active against a limited panel of human pathogens. There are broad-spectrum substances that prevent the first step of virus-cell interaction by mimicking heparan sulfate proteoglycans (HSPG), the highly conserved target of viral attachment ligands (VALs). The reversible binding mechanism prevents their use as a drug, because, upon dilution, the inhibition is lost. Known VALs are made of closely packed repeating units, but the aforementioned substances are able to bind only a few of them. We designed antiviral nanoparticles with long and flexible linkers mimicking HSPG, allowing for effective viral association with a binding that we simulate to be strong and multivalent to the VAL repeating units, generating forces (~190 pN) that eventually lead to irreversible viral deformation. Virucidal assays, electron microscopy images, and molecular dynamics simulations support the proposed mechanism. These particles show no cytotoxicity, and in vitro nanomolar irreversible activity against herpes simplex virus (HSV), human papilloma virus, respiratory syncytial virus (RSV), dengue and lenti virus. They are active ex vivo in human cervicovaginal histocultures infected by HSV-2 and in vivo in mice infected with RSV.

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

Development of an immunochromatographic strip test for rapid detection of citrus yellow vein clearing virus.

PubMed

Bin, Yu; Li, Zhongan; Wu, Jianxiang; Wang, Xuefeng; Zhou, Yan; Li, Taisheng; Yang, Fangyun; Zhou, Changyong; Song, Zhen

2018-02-01

A rapid immunochromatographic strip (ICS) test for detection of citrus yellow vein clearing virus (CYVCV) was developed. The test is based on an antibody sandwich format and uses the monoclonal antibody (MAb) 1E1, which is specific for CYVCV. MAb 1E1 labeled with 30-nm colloidal gold particles was coated on a gold conjugate pad. A secondary goat anti-mouse IgG was coated on the surface of a nitrocellulose filter membrane (NC) as the control (C) line, while 1E1 was coated on the surface of the NC as the test (T) line. The ICS test was evaluated for specificity and sensitivity and then applied for virus detection in field samples. There was no cross-reaction with citrus tristeza virus (CTV), satsuma dwarf virus (SDV), citrus tatter leaf virus (CTLV), citrus exocortis viroid (CEVd), citrus mosaic virus (CiMV), citrus psorosis virus (CPV), citrus ringspot virus (RSV) or 'Candidatus Liberibacter asiaticus' (CLas). The ICS test was still able to detect CYVCV in tissue extracts at a dilution of 1: 320 (w/v), which is as efficient as the dot-ELISA assay. In general, the ICS assay is less expensive, faster and simpler to conduct than conventional CYVCV detection methods, so it may be useful for large-scale detection or monitoring of CYVCV.

On revealing the gene targets of Ebola virus microRNAs involved in the human skin microbiome.

PubMed

Hsu, Pei-Chun; Chiou, Bin-Hao; Huang, Chun-Ming

2018-01-01

Ebola virus, a negative-sense single-stranded RNA virus, causes severe viral hemorrhagic fever and has a high mortality rate. Histopathological and immunopathological analyses of Ebola virus have revealed that histopathological changes in skin tissue are associated with various degrees of endothelial cell swelling and necrosis. The interactions of microbes within or on a host are a crucial for the skin immune shield. The discovery of microRNAs (miRNAs) in Ebola virus implies that immune escape, endothelial cell rupture, and tissue dissolution during Ebola virus infection are a result of the effects of Ebola virus miRNAs. Keratinocytes obtained from normal skin can attach and spread through expression of the thrombospondin family of proteins, playing a role in initiation of cell-mediated immune responses in the skin. Several miRNAs have been shown to bind the 3' untranslated region of thrombospondin mRNA, thereby controlling its stability and translational activity. In this study, we discovered short RNA sequences that may act as miRNAs from Propionibacterium acnes using a practical workflow of bioinformatics methods. Subsequently, we deciphered the common target gene. These RNA sequences tended to bind to the same thrombospondin protein, THSD4, emphasizing the potential importance of the synergistic binding of miRNAs from Ebola virus, Propionibacterium acnes , and humans to the target. These results provide important insights into the molecular mechanisms of thrombospondin proteins and miRNAs in Ebola virus infection.

Structural dissection of Ebola virus and its assembly determinants using cryo-electron tomography.

PubMed

Bharat, Tanmay A M; Noda, Takeshi; Riches, James D; Kraehling, Verena; Kolesnikova, Larissa; Becker, Stephan; Kawaoka, Yoshihiro; Briggs, John A G

2012-03-13

Ebola virus is a highly pathogenic filovirus causing severe hemorrhagic fever with high mortality rates. It assembles heterogenous, filamentous, enveloped virus particles containing a negative-sense, single-stranded RNA genome packaged within a helical nucleocapsid (NC). We have used cryo-electron microscopy and tomography to visualize Ebola virus particles, as well as Ebola virus-like particles, in three dimensions in a near-native state. The NC within the virion forms a left-handed helix with an inner nucleoprotein layer decorated with protruding arms composed of VP24 and VP35. A comparison with the closely related Marburg virus shows that the N-terminal region of nucleoprotein defines the inner diameter of the Ebola virus NC, whereas the RNA genome defines its length. Binding of the nucleoprotein to RNA can assemble a loosely coiled NC-like structure; the loose coil can be condensed by binding of the viral matrix protein VP40 to the C terminus of the nucleoprotein, and rigidified by binding of VP24 and VP35 to alternate copies of the nucleoprotein. Four proteins (NP, VP24, VP35, and VP40) are necessary and sufficient to mediate assembly of an NC with structure, symmetry, variability, and flexibility indistinguishable from that in Ebola virus particles released from infected cells. Together these data provide a structural and architectural description of Ebola virus and define the roles of viral proteins in its structure and assembly.

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.

Mutations Abrogating VP35 Interaction with Double-Stranded RNA Render Ebola Virus Avirulent in Guinea Pigs

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

Prins, Kathleen C.; Delpeut, Sebastien; Leung, Daisy W.

2010-10-11

Ebola virus (EBOV) protein VP35 is a double-stranded RNA (dsRNA) binding inhibitor of host interferon (IFN)-{alpha}/{beta} responses that also functions as a viral polymerase cofactor. Recent structural studies identified key features, including a central basic patch, required for VP35 dsRNA binding activity. To address the functional significance of these VP35 structural features for EBOV replication and pathogenesis, two point mutations, K319A/R322A, that abrogate VP35 dsRNA binding activity and severely impair its suppression of IFN-{alpha}/{beta} production were identified. Solution nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography reveal minimal structural perturbations in the K319A/R322A VP35 double mutant and suggest that lossmore » of basic charge leads to altered function. Recombinant EBOVs encoding the mutant VP35 exhibit, relative to wild-type VP35 viruses, minimal growth attenuation in IFN-defective Vero cells but severe impairment in IFN-competent cells. In guinea pigs, the VP35 mutant virus revealed a complete loss of virulence. Strikingly, the VP35 mutant virus effectively immunized animals against subsequent wild-type EBOV challenge. These in vivo studies, using recombinant EBOV viruses, combined with the accompanying biochemical and structural analyses directly correlate VP35 dsRNA binding and IFN inhibition functions with viral pathogenesis. Moreover, these studies provide a framework for the development of antivirals targeting this critical EBOV virulence factor.« less

Phage-display for identifying peptides that bind the spike protein of transmissible gastroenteritis virus and possess diagnostic potential

USDA-ARS?s Scientific Manuscript database

The spike (S) protein is a key structural protein of coronaviruses including, the porcine transmissible gastroenteritis virus (TGEV). The S protein is a type I membrane glycoprotein located in the viral envelope and is responsible for mediating the binding of viral particles to specific cell recepto...

Measurement of Bluetongue Virus Binding to a Mammalian Cell Surface Receptor by an In Situ Immune Fluorescent Staining Technique

USDA-ARS?s Scientific Manuscript database

A quantifiable in situ immune fluorescent assay (IFA) was developed to measure bluetongue virus (BTV) binding to mammalian cells. The utility of the assay was demonstrated with both Chinese hamster ovary (CHO) and bovine pulmonary artery endothelial (CPAE) cells. Since heparin sulfate (HS) has been ...

Natural resistance-associated macrophage protein is a cellular receptor for sindbis virus in both insect and mammalian hosts.

PubMed

Rose, Patrick P; Hanna, Sheri L; Spiridigliozzi, Anna; Wannissorn, Nattha; Beiting, Daniel P; Ross, Susan R; Hardy, Richard W; Bambina, Shelly A; Heise, Mark T; Cherry, Sara

2011-08-18

Alphaviruses, including several emerging human pathogens, are a large family of mosquito-borne viruses with Sindbis virus being a prototypical member of the genus. The host factor requirements and receptors for entry of this class of viruses remain obscure. Using a Drosophila system, we identified the divalent metal ion transporter natural resistance-associated macrophage protein (NRAMP) as a host cell surface molecule required for Sindbis virus binding and entry into Drosophila cells. Consequently, flies mutant for dNRAMP were protected from virus infection. NRAMP2, the ubiquitously expressed vertebrate homolog, mediated binding and infection of Sindbis virus into mammalian cells, and murine cells deficient for NRAMP2 were nonpermissive to infection. Alphavirus glycoprotein chimeras demonstrated that the requirement for NRAMP2 is at the level of Sindbis virus entry. Given the conserved structure of alphavirus glycoproteins, and the widespread use of transporters for viral entry, other alphaviruses may use conserved multipass membrane proteins for infection. Copyright © 2011 Elsevier Inc. All rights reserved.

Dwarf Galaxies and the Cosmic Web

NASA Astrophysics Data System (ADS)

Benítez-Llambay, Alejandro; Navarro, Julio F.; Abadi, Mario G.; Gottlöber, Stefan; Yepes, Gustavo; Hoffman, Yehuda; Steinmetz, Matthias

2013-02-01

We use a cosmological simulation of the formation of the Local Group of Galaxies to identify a mechanism that enables the removal of baryons from low-mass halos without appealing to feedback or reionization. As the Local Group forms, matter bound to it develops a network of filaments and pancakes. This moving web of gas and dark matter drifts and sweeps a large volume, overtaking many halos in the process. The dark matter content of these halos is unaffected but their gas can be efficiently removed by ram pressure. The loss of gas is especially pronounced in low-mass halos due to their lower binding energy and has a dramatic effect on the star formation history of affected systems. This "cosmic web stripping" may help to explain the scarcity of dwarf galaxies compared with the numerous low-mass halos expected in ΛCDM and the large diversity of star formation histories and morphologies characteristic of faint galaxies. Although our results are based on a single high-resolution simulation, it is likely that the hydrodynamical interaction of dwarf galaxies with the cosmic web is a crucial ingredient so far missing from galaxy formation models.

Collision Tomography: Physical Properties of Possible Progenitors of the Andromeda Stellar Stream

NASA Astrophysics Data System (ADS)

Miki, Yohei; Mori, Masao; Rich, R. Michael

2016-08-01

To unveil a progenitor of the Andromeda Giant Stellar Stream, we investigate the interaction between an accreting satellite galaxy and the Andromeda Galaxy using an N-body simulation. We perform a comprehensive exploration of the properties of the progenitor dwarf galaxy, using 247 models of varying mass, mass distribution, and size. We show that the binding energy of the progenitor is the crucial parameter in reproducing the Andromeda Giant Stellar Stream and the shell-like structures surrounding the Andromeda Galaxy. As a result of the simulations, the progenitor must satisfy a simple scaling relation between the core radius, the total mass and the tidal radius. Using this relation, we successfully constrain the physical properties of the progenitors to have masses ranging from 5× {10}8{M}⊙ to 5× {10}9{M}⊙ and central surface densities around {10}3 {M}⊙ {{pc}}-2. A detailed comparison between our result and the nearby observed galaxies indicates that possible progenitors of the Andromeda Giant Stellar Stream include a dwarf elliptical galaxy, a dwarf irregular galaxy, and a small spiral galaxy.

A pH-sensitive heparin-binding sequence from Baculovirus gp64 protein is important for binding to mammalian cells but not to Sf9 insect cells.

PubMed

Wu, Chunxiao; Wang, Shu

2012-01-01

Binding to heparan sulfate is essential for baculovirus transduction of mammalian cells. Our previous study shows that gp64, the major glycoprotein on the virus surface, binds to heparin in a pH-dependent way, with a stronger binding at pH 6.2 than at 7.4. Using fluorescently labeled peptides, we mapped the pH-dependent heparin-binding sequence of gp64 to a 22-amino-acid region between residues 271 and 292. Binding of this region to the cell surface was also pH dependent, and peptides containing this sequence could efficiently inhibit baculovirus transduction of mammalian cells at pH 6.2. When the heparin-binding peptide was immobilized onto the bead surface to mimic the high local concentration of gp64 on the virus surface, the peptide-coated magnetic beads could efficiently pull down cells expressing heparan sulfate but not cells pretreated with heparinase or cells not expressing heparan sulfate. Interestingly, although this heparin-binding function is essential for baculovirus transduction of mammalian cells, it is dispensable for infection of Sf9 insect cells. Virus infectivity on Sf9 cells was not reduced by the presence of heparin or the identified heparin-binding peptide, even though the peptide could bind to Sf9 cell surface and be efficiently internalized. Thus, our data suggest that, depending on the availability of the target molecules on the cell surface, baculoviruses can use two different methods, electrostatic interaction with heparan sulfate and more specific receptor binding, for cell attachment.

Distinctive receptor binding properties of the surface glycoprotein of a natural feline leukemia virus isolate with unusual disease spectrum.

PubMed

Bolin, Lisa L; Chandhasin, Chandtip; Lobelle-Rich, Patricia A; Albritton, Lorraine M; Levy, Laura S

2011-05-13

Feline leukemia virus (FeLV)-945, a member of the FeLV-A subgroup, was previously isolated from a cohort of naturally infected cats. An unusual multicentric lymphoma of non-T-cell origin was observed in natural and experimental infection with FeLV-945. Previous studies implicated the FeLV-945 surface glycoprotein (SU) as a determinant of disease outcome by an as yet unknown mechanism. The present studies demonstrate that FeLV-945 SU confers distinctive properties of binding to the cell surface receptor. Virions bearing the FeLV-945 Env protein were observed to bind the cell surface receptor with significantly increased efficiency, as was soluble FeLV-945 SU protein, as compared to the corresponding virions or soluble protein from a prototype FeLV-A isolate. SU proteins cloned from other cohort isolates exhibited increased binding efficiency comparable to or greater than FeLV-945 SU. Mutational analysis implicated a domain containing variable region B (VRB) to be the major determinant of increased receptor binding, and identified a single residue, valine 186, to be responsible for the effect. The FeLV-945 SU protein binds its cell surface receptor, feTHTR1, with significantly greater efficiency than does that of prototype FeLV-A (FeLV-A/61E) when present on the surface of virus particles or in soluble form, demonstrating a 2-fold difference in the relative dissociation constant. The results implicate a single residue, valine 186, as the major determinant of increased binding affinity. Computational modeling suggests a molecular mechanism by which residue 186 interacts with the receptor-binding domain through residue glutamine 110 to effect increased binding affinity. Through its increased receptor binding affinity, FeLV-945 SU might function in pathogenesis by increasing the rate of virus entry and spread in vivo, or by facilitating entry into a novel target cell with a low receptor density.

Recognition of the murine coronavirus genomic RNA packaging signal depends on the second RNA-binding domain of the nucleocapsid protein.

PubMed

Kuo, Lili; Koetzner, Cheri A; Hurst, Kelley R; Masters, Paul S

2014-04-01

The coronavirus nucleocapsid (N) protein forms a helical ribonucleoprotein with the viral positive-strand RNA genome and binds to the principal constituent of the virion envelope, the membrane (M) protein, to facilitate assembly and budding. Besides these structural roles, N protein associates with a component of the replicase-transcriptase complex, nonstructural protein 3, at a critical early stage of infection. N protein has also been proposed to participate in the replication and selective packaging of genomic RNA and the transcription and translation of subgenomic mRNA. Coronavirus N proteins contain two structurally distinct RNA-binding domains, an unusual characteristic among RNA viruses. To probe the functions of these domains in the N protein of the model coronavirus mouse hepatitis virus (MHV), we constructed mutants in which each RNA-binding domain was replaced by its counterpart from the N protein of severe acute respiratory syndrome coronavirus (SARS-CoV). Mapping of revertants of the resulting chimeric viruses provided evidence for extensive intramolecular interactions between the two RNA-binding domains. Through analysis of viral RNA that was packaged into virions we identified the second of the two RNA-binding domains as a principal determinant of MHV packaging signal recognition. As expected, the interaction of N protein with M protein was not affected in either of the chimeric viruses. Moreover, the SARS-CoV N substitutions did not alter the fidelity of leader-body junction formation during subgenomic mRNA synthesis. These results more clearly delineate the functions of N protein and establish a basis for further exploration of the mechanism of genomic RNA packaging. This work describes the interactions of the two RNA-binding domains of the nucleocapsid protein of a model coronavirus, mouse hepatitis virus. The main finding is that the second of the two domains plays an essential role in recognizing the RNA structure that allows the selective packaging of genomic RNA into assembled virions.

Computational analysis of the receptor binding specificity of novel influenza A/H7N9 viruses.

PubMed

Zhou, Xinrui; Zheng, Jie; Ivan, Fransiskus Xaverius; Yin, Rui; Ranganathan, Shoba; Chow, Vincent T K; Kwoh, Chee-Keong

2018-05-09

Influenza viruses are undergoing continuous and rapid evolution. The fatal influenza A/H7N9 has drawn attention since the first wave of infections in March 2013, and raised more grave concerns with its increased potential to spread among humans. Experimental studies have revealed several host and virulence markers, indicating differential host binding preferences which can help estimate the potential of causing a pandemic. Here we systematically investigate the sequence pattern and structural characteristics of novel influenza A/H7N9 using computational approaches. The sequence analysis highlighted mutations in protein functional domains of influenza viruses. Molecular docking and molecular dynamics simulation revealed that the hemagglutinin (HA) of A/Taiwan/1/2017(H7N9) strain enhanced the binding with both avian and human receptor analogs, compared with the previous A/Shanghai/02/2013(H7N9) strain. The Molecular Mechanics - Poisson Boltzmann Surface Area (MM-PBSA) calculation revealed the change of residue-ligand interaction energy and detected the residues with conspicuous binding preference. The results are novel and specific to the emerging influenza A/Taiwan/1/2017(H7N9) strain compared with A/Shanghai/02/2013(H7N9). Its enhanced ability to bind human receptor analogs, which are abundant in the human upper respiratory tract, may be responsible for the recent outbreak. Residues showing binding preference were detected, which could facilitate monitoring the circulating influenza viruses.

Southern rice black-streaked dwarf virus (SRBSDV) directly affects the feeding and reproduction behavior of its vector, Sogatella furcifera (Horváth) (Hemiptera: Delphacidae).

PubMed

Xu, Hongxing; He, Xiaochan; Zheng, Xusong; Yang, Yajun; Tian, Junce; Lu, Zhongxian

2014-03-24

Southern rice black-streaked dwarf virus (SRBSDV) is a recently discovered member of the genus Fijivirus and it is transmitted by the rice whitebacked planthopper (WBPH), Sogatella furcifera (Horváth). It was found that SRBSDV infected vectors might contribute negatively to the WBPH population, although the longer nymphal period might benefit viral acquisition, transmission and increase infection rate. The interaction between SRBSDV and its vector need to be further explored to gain better understanding of the dispersal of WBPH and the spread of virus disease, in particular the feeding and reproduction behavior of viruliferous WBPH. Newly hatched nymphs of WBPH were fed on healthy rice plant after feeding on SRBSDV-infected rice plants for 2 h, and newly emerged adults were numbered and tested. Feeding behaviors of WBPH adults were monitored electronically within a Faraday cage using a Giga-4 DC EPG amplifier. The newly emerged adults were paired, and the fecundity and egg hatchability were investigated. WBPH was molecularly identified for SRBSDV when they dead. According to the identification results, data on viruliferous and non-viruliferous WBPH were collected and analyzed. Feeding behavior of viruliferous WBPH was different from those of non-viruliferous WBPH. Frequency of phloem sap ingestion of viruliferous WBPH increased significantly, however the total feeding duration did not increase markedly. When both WBPH parents were infected with SRBSDV, their fecundity and hatchability of the eggs produced were significant lower than those of normal WBPH parents. However, if only one of the parents was viruliferous, fecundity and egg hatchability were only slightly affected. Viruliferous WBPH fed on the phloem more frequently than non-viruliferous WBPH and can thus contribute to virus transmission. When both vector parents are viruliferous fecundity and hatchability of the eggs were significantly reduced. However when only one of the parents WBPH was viruliferous, there were no significant effects.

Southern rice black-streaked dwarf virus (SRBSDV) directly affects the feeding and reproduction behavior of its vector, Sogatella furcifera (Horváth) (Hemiptera: Delphacidae)

PubMed Central

2014-01-01

Background Southern rice black-streaked dwarf virus (SRBSDV) is a recently discovered member of the genus Fijivirus and it is transmitted by the rice whitebacked planthopper (WBPH), Sogatella furcifera (Horváth). It was found that SRBSDV infected vectors might contribute negatively to the WBPH population, although the longer nymphal period might benefit viral acquisition, transmission and increase infection rate. The interaction between SRBSDV and its vector need to be further explored to gain better understanding of the dispersal of WBPH and the spread of virus disease, in particular the feeding and reproduction behavior of viruliferous WBPH. Methods Newly hatched nymphs of WBPH were fed on healthy rice plant after feeding on SRBSDV-infected rice plants for 2 h, and newly emerged adults were numbered and tested. Feeding behaviors of WBPH adults were monitored electronically within a Faraday cage using a Giga-4 DC EPG amplifier. The newly emerged adults were paired, and the fecundity and egg hatchability were investigated. WBPH was molecularly identified for SRBSDV when they dead. According to the identification results, data on viruliferous and non-viruliferous WBPH were collected and analyzed. Results Feeding behavior of viruliferous WBPH was different from those of non-viruliferous WBPH. Frequency of phloem sap ingestion of viruliferous WBPH increased significantly, however the total feeding duration did not increase markedly. When both WBPH parents were infected with SRBSDV, their fecundity and hatchability of the eggs produced were significant lower than those of normal WBPH parents. However, if only one of the parents was viruliferous, fecundity and egg hatchability were only slightly affected. Conclusions Viruliferous WBPH fed on the phloem more frequently than non-viruliferous WBPH and can thus contribute to virus transmission. When both vector parents are viruliferous fecundity and hatchability of the eggs were significantly reduced. However when only one of the parents WBPH was viruliferous, there were no significant effects. PMID:24661747

Investigating Ebola virus pathogenicity using molecular dynamics.

PubMed

Pappalardo, Morena; Collu, Francesca; Macpherson, James; Michaelis, Martin; Fraternali, Franca; Wass, Mark N

2017-08-11

Ebolaviruses have been known to cause deadly disease in humans for 40 years and have recently been demonstrated in West Africa to be able to cause large outbreaks. Four Ebolavirus species cause severe disease associated with high mortality in humans. Reston viruses are the only Ebolaviruses that do not cause disease in humans. Conserved amino acid changes in the Reston virus protein VP24 compared to VP24 of other Ebolaviruses have been suggested to alter VP24 binding to host cell karyopherins resulting in impaired inhibition of interferon signalling, which may explain the difference in human pathogenicity. Here we used protein structural analysis and molecular dynamics to further elucidate the interaction between VP24 and KPNA5. As a control experiment, we compared the interaction of wild-type and R137A-mutant (known to affect KPNA5 binding) Ebola virus VP24 with KPNA5. Results confirmed that the R137A mutation weakens direct VP24-KPNA5 binding and enables water molecules to penetrate at the interface. Similarly, Reston virus VP24 displayed a weaker interaction with KPNA5 than Ebola virus VP24, which is likely to reduce the ability of Reston virus VP24 to prevent host cell interferon signalling. Our results provide novel molecular detail on the interaction of Reston virus VP24 and Ebola virus VP24 with human KPNA5. The results indicate a weaker interaction of Reston virus VP24 with KPNA5 than Ebola virus VP24, which is probably associated with a decreased ability to interfere with the host cell interferon response. Hence, our study provides further evidence that VP24 is a key player in determining Ebolavirus pathogenicity.

TIM-1 Mediates Dystroglycan-Independent Entry of Lassa Virus.

PubMed

Brouillette, Rachel B; Phillips, Elisabeth K; Patel, Radhika; Mahauad-Fernandez, Wadie; Moller-Tank, Sven; Rogers, Kai J; Dillard, Jacob A; Cooney, Ashley L; Martinez-Sobrido, Luis; Okeoma, Chioma; Maury, Wendy

2018-06-06

Lassa virus (LASV) is an Old World arenavirus responsible for hundreds of thousands of infections in West Africa every year. LASV entry into a variety of cell types is mediated by interactions with glycosyltransferase LARGE-modified O-linked glycans present on the ubiquitous receptor, α-dystroglycan (αDG). Yet, cells lacking αDG are permissive to LASV infection, suggesting that alternative receptors exist. Previous studies demonstrate that phosphatidylserine (PtdSer)-binding receptors, Axl and Tyro3 along with C-type lectin receptors, mediate αDG-independent entry. Here, we demonstrate that another PtdSer receptor, TIM-1, mediates LASV glycoprotein (GP) pseudotyped virions entry into αDG knocked out HEK 293T and wild-type (WT) Vero which express αDG lacking appropriate glycosylation. To investigate the mechanism by which TIM-1 mediates enhancement of entry, we demonstrate that mutagenesis of the TIM-1 IgV domain PtdSer-binding pocket abrogated transduction. Further, the human TIM-1 IgV domain binding monoclonal antibody, ARD5, blocked transduction of pseudovirions bearing LASV GP in a dose-dependent manner. Finally, as we showed previously for other viruses that use TIM-1 for entry, a chimeric TIM-1 protein that substitutes the proline rich region (PRR) from murine leukemia virus envelope (Env) for the mucin-like domain served as a competent receptor. These studies provide evidence that, in the absence of a functional αDG, TIM-1 mediates entry of LASV pseudoviral particles through interactions of virions with the IgV PtdSer binding pocket of TIM-1. Importance PtdSer receptors, such as TIM-1, are emerging as critical entry factors for many enveloped viruses. Most recently, Hepatitis C virus and Zika virus have been added to a growing list. PtdSer receptors engage with enveloped viruses through binding of PtdSer embedded in the viral envelope, defining them as GP-independent receptors. This GP-independent entry mechanism should effectively mediate entry of all enveloped viruses, yet LASV GP pseudotyped viruses were previously found to be unresponsive to PtdSer receptor enhancement in HEK 293T cells. Here we demonstrate that LASV pseudovirions can utilize the PtdSer receptor TIM-1, but only in the absence of appropriately glycosylated α-dystroglycan (αDG), the high affinity cell surface receptor for LASV. Our studies shed light on LASV receptor utilization and explain why earlier studies performed in α-DG-expressing cells did not find that LASV pseudovirions utilize PtdSer receptors for virus uptake. Copyright © 2018 American Society for Microbiology.

RNA-binding properties and mapping of the RNA-binding domain from the movement protein of Prunus necrotic ringspot virus.

PubMed

Herranz, M Carmen; Pallás, Vicente

2004-03-01

The movement protein (MP) of Prunus necrotic ringspot virus (PNRSV) is involved in intercellular virus transport. In this study, putative RNA-binding properties of the PNRSV MP were studied. The PNRSV MP was produced in Escherichia coli using an expression vector. Electrophoretic mobility shift assays (EMSAs) using DIG-labelled riboprobes demonstrated that PNRSV MP bound ssRNA cooperatively without sequence specificity. Two different ribonucleoprotein complexes were found to be formed depending on the molar MP : PNRSV RNA ratio. The different responses of the complexes to urea treatment strongly suggested that they have different structural properties. Deletion mutagenesis followed by Northwestern analysis allowed location of a nucleic acid binding domain to aa 56-88. This 33 aa RNA-binding motif is the smallest region delineated among members of the family Bromoviridae for which RNA-binding properties have been demonstrated. This domain is highly conserved within all phylogenetic subgroups previously described for PNRSV isolates. Interestingly, the RNA-binding domain described here and the one described for Alfamovirus are located at the N terminus of their corresponding MPs, whereas similar domains previously characterized in members of the genera Bromovirus and Cucumovirus are present at the C terminus, strongly reflecting their corresponding phylogenetic relationships. The evolutionary implications of this observation are discussed.

Hydrophobic pocket targeting probes for enteroviruses

NASA Astrophysics Data System (ADS)

Martikainen, Mari; Salorinne, Kirsi; Lahtinen, Tanja; Malola, Sami; Permi, Perttu; Häkkinen, Hannu; Marjomäki, Varpu

2015-10-01

Visualization and tracking of viruses without compromising their functionality is crucial in order to understand virus targeting to cells and tissues, and to understand the subsequent subcellular steps leading to virus uncoating and replication. Enteroviruses are important human pathogens causing a vast number of acute infections, and are also suggested to contribute to the development of chronic diseases like type I diabetes. Here, we demonstrate a novel method to target site-specifically the hydrophobic pocket of enteroviruses. A probe, a derivative of Pleconaril, was developed and conjugated to various labels that enabled the visualization of enteroviruses under light and electron microscopes. The probe mildly stabilized the virus particle by increasing the melting temperature by 1-3 degrees, and caused a delay in the uncoating of the virus in the cellular endosomes, but could not however inhibit the receptor binding, cellular entry or infectivity of the virus. The hydrophobic pocket binding moiety of the probe was shown to bind to echovirus 1 particle by STD and tr-NOESY NMR methods. Furthermore, binding to echovirus 1 and Coxsackievirus A9, and to a lesser extent to Coxsackie virus B3 was verified by using a gold nanocluster labeled probe by TEM analysis. Molecular modelling suggested that the probe fits the hydrophobic pockets of EV1 and CVA9, but not of CVB3 as expected, correlating well with the variations in the infectivity and stability of the virus particles. EV1 conjugated to the fluorescent dye labeled probe was efficiently internalized into the cells. The virus-fluorescent probe conjugate accumulated in the cytoplasmic endosomes and caused infection starting from 6 hours onwards. Remarkably, before and during the time of replication, the fluorescent probe was seen to leak from the virus-positive endosomes and thus separate from the capsid proteins that were left in the endosomes. These results suggest that, like the physiological hydrophobic content, the probe may be released upon virus uncoating. Our results collectively thus show that the gold and fluorescently labeled probes may be used to track and visualize the studied enteroviruses during the early phases of infection opening new avenues to follow virus uncoating in cells.Visualization and tracking of viruses without compromising their functionality is crucial in order to understand virus targeting to cells and tissues, and to understand the subsequent subcellular steps leading to virus uncoating and replication. Enteroviruses are important human pathogens causing a vast number of acute infections, and are also suggested to contribute to the development of chronic diseases like type I diabetes. Here, we demonstrate a novel method to target site-specifically the hydrophobic pocket of enteroviruses. A probe, a derivative of Pleconaril, was developed and conjugated to various labels that enabled the visualization of enteroviruses under light and electron microscopes. The probe mildly stabilized the virus particle by increasing the melting temperature by 1-3 degrees, and caused a delay in the uncoating of the virus in the cellular endosomes, but could not however inhibit the receptor binding, cellular entry or infectivity of the virus. The hydrophobic pocket binding moiety of the probe was shown to bind to echovirus 1 particle by STD and tr-NOESY NMR methods. Furthermore, binding to echovirus 1 and Coxsackievirus A9, and to a lesser extent to Coxsackie virus B3 was verified by using a gold nanocluster labeled probe by TEM analysis. Molecular modelling suggested that the probe fits the hydrophobic pockets of EV1 and CVA9, but not of CVB3 as expected, correlating well with the variations in the infectivity and stability of the virus particles. EV1 conjugated to the fluorescent dye labeled probe was efficiently internalized into the cells. The virus-fluorescent probe conjugate accumulated in the cytoplasmic endosomes and caused infection starting from 6 hours onwards. Remarkably, before and during the time of replication, the fluorescent probe was seen to leak from the virus-positive endosomes and thus separate from the capsid proteins that were left in the endosomes. These results suggest that, like the physiological hydrophobic content, the probe may be released upon virus uncoating. Our results collectively thus show that the gold and fluorescently labeled probes may be used to track and visualize the studied enteroviruses during the early phases of infection opening new avenues to follow virus uncoating in cells. Electronic supplementary information (ESI) available: Details of the synthesis of the probes, UV-Vis absorption spectra of the probe (2), PAGE separation and the absorption spectra of the gold labeled probe (3), details of the NMR experiments, determination of the cytotoxicity of the studied molecules, TEM micrographs of the gold labeled probe (3) with enteroviruses, live cell imaging of the fluorescent probe (4) in cells, and additional details of modeling of the hydrophobic pockets. See DOI: 10.1039/c5nr04139b

Ilarviruses of Prunus spp.: a continued concern for fruit trees.

PubMed

Pallas, V; Aparicio, F; Herranz, M C; Amari, K; Sanchez-Pina, M A; Myrta, A; Sanchez-Navarro, J A

2012-12-01

Prunus spp. are affected by a large number of viruses, causing significant economic losses through either direct or indirect damage, which results in reduced yield and fruit quality. Among these viruses, members of the genus Ilarvirus (isometric labile ringspot viruses) occupy a significant position due to their distribution worldwide. Although symptoms caused by these types of viruses were reported early in the last century, their molecular characterization was not achieved until the 1990s, much later than for other agronomically relevant viruses. This was mainly due to the characteristic liability of virus particles in tissue extracts. In addition, ilarviruses, together with Alfalfa mosaic virus, are unique among plant viruses in that they require a few molecules of the coat protein in the inoculum in order to be infectious, a phenomenon known as genome activation. Another factor that has made the study of this group of viruses difficult is that infectious clones have been obtained only for the type member of the genus, Tobacco streak virus. Four ilarviruses, Prunus necrotic ringspot virus, Prune dwarf virus, Apple mosaic virus, and American plum line pattern virus, are pathogens of the main cultivated fruit trees. As stated in the 9th Report of the International Committee on Taxonomy of Viruses, virions of this genus are "unpromising subjects for the raising of good antisera." With the advent of molecular approaches for their detection and characterization, it has been possible to get a more precise view of their prevalence and genome organization. This review updates our knowledge on the incidence, genome organization and expression, genetic diversity, modes of transmission, and diagnosis, as well as control of this peculiar group of viruses affecting fruit trees.

Characterization of Human and Murine T-Cell Immunoglobulin Mucin Domain 4 (TIM-4) IgV Domain Residues Critical for Ebola Virus Entry.

PubMed

Rhein, Bethany A; Brouillette, Rachel B; Schaack, Grace A; Chiorini, John A; Maury, Wendy

2016-07-01

Phosphatidylserine (PtdSer) receptors that are responsible for the clearance of dying cells have recently been found to mediate enveloped virus entry. Ebola virus (EBOV), a member of the Filoviridae family of viruses, utilizes PtdSer receptors for entry into target cells. The PtdSer receptors human and murine T-cell immunoglobulin mucin (TIM) domain proteins TIM-1 and TIM-4 mediate filovirus entry by binding to PtdSer on the virion surface via a conserved PtdSer binding pocket within the amino-terminal IgV domain. While the residues within the TIM-1 IgV domain that are important for EBOV entry are characterized, the molecular details of virion-TIM-4 interactions have yet to be investigated. As sequences and structural alignments of the TIM proteins suggest distinct differences in the TIM-1 and TIM-4 IgV domain structures, we sought to characterize TIM-4 IgV domain residues required for EBOV entry. Using vesicular stomatitis virus pseudovirions bearing EBOV glycoprotein (EBOV GP/VSVΔG), we evaluated virus binding and entry into cells expressing TIM-4 molecules mutated within the IgV domain, allowing us to identify residues important for entry. Similar to TIM-1, residues in the PtdSer binding pocket of murine and human TIM-4 (mTIM-4 and hTIM-4) were found to be important for EBOV entry. However, additional TIM-4-specific residues were also found to impact EBOV entry, with a total of 8 mTIM-4 and 14 hTIM-4 IgV domain residues being critical for virion binding and internalization. Together, these findings provide a greater understanding of the interaction of TIM-4 with EBOV virions. With more than 28,000 cases and over 11,000 deaths during the largest and most recent Ebola virus (EBOV) outbreak, there has been increased emphasis on the development of therapeutics against filoviruses. Many therapies under investigation target EBOV cell entry. T-cell immunoglobulin mucin (TIM) domain proteins are cell surface factors important for the entry of many enveloped viruses, including EBOV. TIM family member TIM-4 is expressed on macrophages and dendritic cells, which are early cellular targets during EBOV infection. Here, we performed a mutagenesis screening of the IgV domain of murine and human TIM-4 to identify residues that are critical for EBOV entry. Surprisingly, we identified more human than murine TIM-4 IgV domain residues that are required for EBOV entry. Defining the TIM IgV residues needed for EBOV entry clarifies the virus-receptor interactions and paves the way for the development of novel therapeutics targeting virus binding to this cell surface receptor. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Characterization of Human and Murine T-Cell Immunoglobulin Mucin Domain 4 (TIM-4) IgV Domain Residues Critical for Ebola Virus Entry

PubMed Central

Rhein, Bethany A.; Brouillette, Rachel B.; Schaack, Grace A.; Chiorini, John A.

2016-01-01

ABSTRACT Phosphatidylserine (PtdSer) receptors that are responsible for the clearance of dying cells have recently been found to mediate enveloped virus entry. Ebola virus (EBOV), a member of the Filoviridae family of viruses, utilizes PtdSer receptors for entry into target cells. The PtdSer receptors human and murine T-cell immunoglobulin mucin (TIM) domain proteins TIM-1 and TIM-4 mediate filovirus entry by binding to PtdSer on the virion surface via a conserved PtdSer binding pocket within the amino-terminal IgV domain. While the residues within the TIM-1 IgV domain that are important for EBOV entry are characterized, the molecular details of virion–TIM-4 interactions have yet to be investigated. As sequences and structural alignments of the TIM proteins suggest distinct differences in the TIM-1 and TIM-4 IgV domain structures, we sought to characterize TIM-4 IgV domain residues required for EBOV entry. Using vesicular stomatitis virus pseudovirions bearing EBOV glycoprotein (EBOV GP/VSVΔG), we evaluated virus binding and entry into cells expressing TIM-4 molecules mutated within the IgV domain, allowing us to identify residues important for entry. Similar to TIM-1, residues in the PtdSer binding pocket of murine and human TIM-4 (mTIM-4 and hTIM-4) were found to be important for EBOV entry. However, additional TIM-4-specific residues were also found to impact EBOV entry, with a total of 8 mTIM-4 and 14 hTIM-4 IgV domain residues being critical for virion binding and internalization. Together, these findings provide a greater understanding of the interaction of TIM-4 with EBOV virions. IMPORTANCE With more than 28,000 cases and over 11,000 deaths during the largest and most recent Ebola virus (EBOV) outbreak, there has been increased emphasis on the development of therapeutics against filoviruses. Many therapies under investigation target EBOV cell entry. T-cell immunoglobulin mucin (TIM) domain proteins are cell surface factors important for the entry of many enveloped viruses, including EBOV. TIM family member TIM-4 is expressed on macrophages and dendritic cells, which are early cellular targets during EBOV infection. Here, we performed a mutagenesis screening of the IgV domain of murine and human TIM-4 to identify residues that are critical for EBOV entry. Surprisingly, we identified more human than murine TIM-4 IgV domain residues that are required for EBOV entry. Defining the TIM IgV residues needed for EBOV entry clarifies the virus-receptor interactions and paves the way for the development of novel therapeutics targeting virus binding to this cell surface receptor. PMID:27122575

Characterization of insulin-like growth factor I receptor on human erythrocytes.

PubMed

Hizuka, N; Takano, K; Tanaka, I; Honda, N; Tsushima, T; Shizume, K

1985-12-01

[125I]Insulin-like growth factor I (IGF-I) specifically bound to erythrocytes; the binding was saturable, and time and temperature dependent. Steady state binding was reached at 16 h at 4 C, and specific binding averaged 14.3 +/- 0.7% (+/- SEM) at a concentration of 3.6 X 10(9) cells/ml in seven normal subjects. [125I]IGF-I binding to the cells was displaced by unlabeled IGF-I in a dose-dependent manner. Scatchard analysis indicated a linear plot, and Ka and number of binding sites/cell were 1.43 +/- 0.07 X 10(9) M-1 and 20.7 +/- 2.2, respectively. Compared to IGF-I, the relative potencies of multiplication-stimulating activity and insulin for displacing [125I]IGF-I binding were 20% and 1%, respectively. [125I]IGF-I binding to erythrocytes from patients with acromegaly was lower than binding to cells from pituitary dwarfs. An inverse correlation between plasma IGF-I level and the number of IGF-I-binding sites per cell was found (r = -0.75; P less than 0.005). These results demonstrate that [125I]IGF-I binding to erythrocytes can be used for clinical measurement of the IGF-I receptor.

Si:P as a laboratory analogue for hydrogen on high magnetic field white dwarf stars.

PubMed

Murdin, B N; Li, Juerong; Pang, M L Y; Bowyer, E T; Litvinenko, K L; Clowes, S K; Engelkamp, H; Pidgeon, C R; Galbraith, I; Abrosimov, N V; Riemann, H; Pavlov, S G; Hübers, H-W; Murdin, P G

2013-01-01

Laboratory spectroscopy of atomic hydrogen in a magnetic flux density of 10(5) T (1 gigagauss), the maximum observed on high-field magnetic white dwarfs, is impossible because practically available fields are about a thousand times less. In this regime, the cyclotron and binding energies become equal. Here we demonstrate Lyman series spectra for phosphorus impurities in silicon up to the equivalent field, which is scaled to 32.8 T by the effective mass and dielectric constant. The spectra reproduce the high-field theory for free hydrogen, with quadratic Zeeman splitting and strong mixing of spherical harmonics. They show the way for experiments on He and H(2) analogues, and for investigation of He(2), a bound molecule predicted under extreme field conditions.

Structure and Receptor Binding of the Hemagglutinin from a Human H6N1 Influenza Virus

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

Tzarum, Netanel; de Vries, Robert P.; Zhu, Xueyong

Avian influenza viruses that cause infection and are transmissible in humans involve changes in the receptor binding site (RBS) of the viral hemagglutinin (HA) that alter receptor preference from α2-3-linked (avian-like) to α2-6-linked (human-like) sialosides. A human case of avian-origin H6N1 influenza virus was recently reported, but the molecular mechanisms contributing to it crossing the species barrier are unknown. We find that, although the H6 HA RBS contains D190V and G228S substitutions that potentially promote human receptor binding, recombinant H6 HA preferentially binds α2-3-linked sialosides, indicating no adaptation to human receptors. Crystal structures of H6 HA with avian and humanmore » receptor analogs reveal that H6 HA preferentially interacts with avian receptor analogs. Lastly, this binding mechanism differs from other HA subtypes due to a unique combination of RBS residues, highlighting additional variation in HA-receptor interactions and the challenges in predicting which influenza strains and subtypes can infect humans and cause pandemics.« less

Human importin alpha and RNA do not compete for binding to influenza A virus nucleoprotein

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

Boulo, Sebastien; UJF-EMBL-CNRS UMI 3265, Unit of Virus Host-Cell Interactions, 6 rue Jules Horowitz, 38042 Grenoble Cedex 9; Akarsu, Hatice

2011-01-05

Influenza virus has a segmented genome composed of eight negative stranded RNA segments. Each segment is covered with NP forming ribonucleoproteins (vRNPs) and carries a copy of the heterotrimeric polymerase complex. As a rare phenomenon among the RNA viruses, the viral replication occurs in the nucleus and therefore implies interactions between host and viral factors, such as between importin alpha and nucleoprotein. In the present study we report that through binding with the human nuclear receptor importin {alpha}5 (Imp{alpha}5), the viral NP is no longer oligomeric but maintained as a monomer inside the complex. In this regard, Imp{alpha}5 acts asmore » a chaperone until NP is delivered in the nucleus for viral RNA encapsidation. Moreover, we show that the association of NP with the host transporter does not impair the binding of NP to RNA. The complex human Imp{alpha}5-NP binds RNA with the same affinity as wt NP alone, whereas engineered monomeric NP through point mutations binds RNA with a strongly reduced affinity.« less

A single mutation in Taiwanese H6N1 influenza hemagglutinin switches binding to human-type receptors

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

de Vries, Robert P.; Tzarum, Netanel; Peng, Wenjie

In June 2013, the first case of human infection with an avian H6N1 virus was reported in a Taiwanese woman. Although this was a single non-fatal case, the virus continues to circulate in Taiwanese poultry. As with any emerging avian virus that infects humans, there is concern that acquisition of human-type receptor specificity could enable transmission in the human population. Despite mutations in the receptor-binding pocket of the human H6N1 isolate, it has retained avian-type (NeuAcα2-3Gal) receptor specificity. However, we show here that a single nucleotide substitution, resulting in a change from Gly to Asp at position 225 (G225D), completelymore » switches specificity to human-type (NeuAcα2-6Gal) receptors. Significantly, G225D H6 loses binding to chicken trachea epithelium and is now able to bind to human tracheal tissue. Structural analysis reveals that Asp225 directly interacts with the penultimate Gal of the human-type receptor, stabilizing human receptor binding.« less

Simian Immunodeficiency Virus and Human Immunodeficiency Virus Type 1 Nef Proteins Show Distinct Patterns and Mechanisms of Src Kinase Activation

PubMed Central

Greenway, Alison L.; Dutartre, Hélène; Allen, Kelly; McPhee, Dale A.; Olive, Daniel; Collette, Yves

1999-01-01

The nef gene from human and simian immunodeficiency viruses (HIV and SIV) regulates cell function and viral replication, possibly through binding of the nef product to cellular proteins, including Src family tyrosine kinases. We show here that the Nef protein encoded by SIVmac239 interacts with and also activates the human Src kinases Lck and Hck. This is in direct contrast to the inhibitory effect of HIV type 1 (HIV-1) Nef on Lck catalytic activity. Unexpectedly, however, the interaction of SIV Nef with human Lck or Hck is not mediated via its consensus proline motif, which is known to mediate HIV-1 Nef binding to Src homology 3 (SH3) domains, and various experimental analyses failed to show significant interaction of SIV Nef with the SH3 domain of either kinase. Instead, SIV Nef can bind Lck and Hck SH2 domains, and its N-terminal 50 amino acid residues are sufficient for Src kinase binding and activation. Our results provide evidence for multiple mechanisms by which Nef binds to and regulates Src kinases. PMID:10364375

Structure and Receptor Binding of the Hemagglutinin from a Human H6N1 Influenza Virus

DOE PAGES

Tzarum, Netanel; de Vries, Robert P.; Zhu, Xueyong; ...

2015-03-11

Avian influenza viruses that cause infection and are transmissible in humans involve changes in the receptor binding site (RBS) of the viral hemagglutinin (HA) that alter receptor preference from α2-3-linked (avian-like) to α2-6-linked (human-like) sialosides. A human case of avian-origin H6N1 influenza virus was recently reported, but the molecular mechanisms contributing to it crossing the species barrier are unknown. We find that, although the H6 HA RBS contains D190V and G228S substitutions that potentially promote human receptor binding, recombinant H6 HA preferentially binds α2-3-linked sialosides, indicating no adaptation to human receptors. Crystal structures of H6 HA with avian and humanmore » receptor analogs reveal that H6 HA preferentially interacts with avian receptor analogs. Lastly, this binding mechanism differs from other HA subtypes due to a unique combination of RBS residues, highlighting additional variation in HA-receptor interactions and the challenges in predicting which influenza strains and subtypes can infect humans and cause pandemics.« less

Identification of novel target sites and an inhibitor of the dengue virus E protein.

PubMed

Yennamalli, Ragothaman; Subbarao, Naidu; Kampmann, Thorsten; McGeary, Ross P; Young, Paul R; Kobe, Bostjan

2009-06-01

Dengue and related flaviviruses represent a significant global health threat. The envelope glycoprotein E mediates virus attachment to a host cell and the subsequent fusion of viral and host cell membranes. The fusion process is driven by conformational changes in the E protein and is an essential step in the virus life cycle. In this study, we analyzed the pre-fusion and post-fusion structures of the dengue virus E protein to identify potential novel sites that could bind small molecules, which could interfere with the conformational transitions that mediate the fusion process. We used an in silico virtual screening approach combining three different docking algorithms (DOCK, GOLD and FlexX) to identify compounds that are likely to bind to these sites. Seven structurally diverse molecules were selected to test experimentally for inhibition of dengue virus propagation. The best compound showed an IC(50) in the micromolar range against dengue virus type 2.

Identification of novel target sites and an inhibitor of the dengue virus E protein

NASA Astrophysics Data System (ADS)

Yennamalli, Ragothaman; Subbarao, Naidu; Kampmann, Thorsten; McGeary, Ross P.; Young, Paul R.; Kobe, Bostjan

2009-06-01

Dengue and related flaviviruses represent a significant global health threat. The envelope glycoprotein E mediates virus attachment to a host cell and the subsequent fusion of viral and host cell membranes. The fusion process is driven by conformational changes in the E protein and is an essential step in the virus life cycle. In this study, we analyzed the pre-fusion and post-fusion structures of the dengue virus E protein to identify potential novel sites that could bind small molecules, which could interfere with the conformational transitions that mediate the fusion process. We used an in silico virtual screening approach combining three different docking algorithms (DOCK, GOLD and FlexX) to identify compounds that are likely to bind to these sites. Seven structurally diverse molecules were selected to test experimentally for inhibition of dengue virus propagation. The best compound showed an IC50 in the micromolar range against dengue virus type 2.

Identification of Cell Surface Molecules Involved in Dystroglycan-Independent Lassa Virus Cell Entry

PubMed Central

Ströher, Ute; Ebihara, Hideki; Feldmann, Heinz

2012-01-01

Although O-mannosylated dystroglycan is a receptor for Lassa virus, a causative agent of Lassa fever, recent findings suggest the existence of an alternative receptor(s). Here we identified four molecules as receptors for Lassa virus: Axl and Tyro3, from the TAM family, and dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) and liver and lymph node sinusoidal endothelial calcium-dependent lectin (LSECtin), from the C-type lectin family. These molecules enhanced the binding of Lassa virus to cells and mediated infection independently of dystroglycan. Axl- or Tyro3-mediated infection required intracellular signaling via the tyrosine kinase activity of Axl or Tyro3, whereas DC-SIGN- or LSECtin-mediated infection and binding were dependent on a specific carbohydrate and on ions. The identification of these four molecules as Lassa virus receptors advances our understanding of Lassa virus cell entry. PMID:22156524

Molecular docking based screening of compounds against VP40 from Ebola virus.

PubMed

M Alam El-Din, Hanaa; A Loutfy, Samah; Fathy, Nasra; H Elberry, Mostafa; M Mayla, Ahmed; Kassem, Sara; Naqvi, Asif

2016-01-01

Ebola virus causes severe and often fatal hemorrhagic fevers in humans. The 2014 Ebola epidemic affected multiple countries. The virus matrix protein (VP40) plays a central role in virus assembly and budding. Since there is no FDA-approved vaccine or medicine against Ebola viral infection, discovering new compounds with different binding patterns against it is required. Therefore, we aim to identify small molecules that target the Arg 134 RNA binding and active site of VP40 protein. 1800 molecules were retrieved from PubChem compound database based on Structure Similarity and Conformers of pyrimidine-2, 4-dione. Molecular docking approach using Lamarckian Genetic Algorithm was carried out to find the potent inhibitors for VP40 based on calculated ligand-protein pairwise interaction energies. The grid maps representing the protein were calculated using auto grid and grid size was set to 60*60*60 points with grid spacing of 0.375 Ǻ. Ten independent docking runs were carried out for each ligand and results were clustered according to the 1.0 Ǻ RMSD criteria. The post-docking analysis showed that binding energies ranged from -8.87 to 0.6 Kcal/mol. We report 7 molecules, which showed promising ADMET results, LD-50, as well as H-bond interaction in the binding pocket. The small molecules discovered could act as potential inhibitors for VP40 and could interfere with virus assembly and budding process.

Molecular docking based screening of compounds against VP40 from Ebola virus

PubMed Central

M Alam El-Din, Hanaa; A. Loutfy, Samah; Fathy, Nasra; H Elberry, Mostafa; M Mayla, Ahmed; Kassem, Sara; Naqvi, Asif

2016-01-01

Ebola virus causes severe and often fatal hemorrhagic fevers in humans. The 2014 Ebola epidemic affected multiple countries. The virus matrix protein (VP40) plays a central role in virus assembly and budding. Since there is no FDA-approved vaccine or medicine against Ebola viral infection, discovering new compounds with different binding patterns against it is required. Therefore, we aim to identify small molecules that target the Arg 134 RNA binding and active site of VP40 protein. 1800 molecules were retrieved from PubChem compound database based on Structure Similarity and Conformers of pyrimidine-2, 4-dione. Molecular docking approach using Lamarckian Genetic Algorithm was carried out to find the potent inhibitors for VP40 based on calculated ligand-protein pairwise interaction energies. The grid maps representing the protein were calculated using auto grid and grid size was set to 60*60*60 points with grid spacing of 0.375 Ǻ. Ten independent docking runs were carried out for each ligand and results were clustered according to the 1.0 Ǻ RMSD criteria. The post-docking analysis showed that binding energies ranged from -8.87 to 0.6 Kcal/mol. We report 7 molecules, which showed promising ADMET results, LD-50, as well as H-bond interaction in the binding pocket. The small molecules discovered could act as potential inhibitors for VP40 and could interfere with virus assembly and budding process. PMID:28149054

C-type lectins do not act as functional receptors for filovirus entry into cells

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

Matsuno, Keita; Nakayama, Eri; Noyori, Osamu

2010-12-03

Research highlights: {yields} Filovirus glycoprotein (GP) having a deficient receptor binding region were generated. {yields} Mutant GPs mediated virus entry less efficiently than wild-type GP. {yields} Mutant GPs bound to C-type lectins but not mediated entire steps of cellular entry. {yields} C-type lectins do not independently mediate filovirus entry into cells. {yields} Other molecule(s) are required for C-type lectin-mediated entry of filoviruses. -- Abstract: Cellular C-type lectins have been reported to facilitate filovirus infection by binding to glycans on filovirus glycoprotein (GP). However, it is not clearly known whether interaction between C-type lectins and GP mediates all the steps ofmore » virus entry (i.e., attachment, internalization, and membrane fusion). In this study, we generated vesicular stomatitis viruses pseudotyped with mutant GPs that have impaired structures of the putative receptor binding regions and thus reduced ability to infect the monkey kidney cells that are routinely used for virus propagation. We found that infectivities of viruses with the mutant GPs dropped in C-type lectin-expressing cells, parallel with those in the monkey kidney cells, whereas binding activities of these GPs to the C-type lectins were not correlated with the reduced infectivities. These results suggest that C-type lectin-mediated entry of filoviruses requires other cellular molecule(s) that may be involved in virion internalization or membrane fusion.« less

Virus altered rice attractiveness to planthoppers is mediated by volatiles and related to virus titre and expression of defence and volatile-biosynthesis genes.

PubMed

Lu, Guanghua; Zhang, Tong; He, Yuange; Zhou, Guohui

2016-12-07

Viruses may induce changes in plant hosts and vectors to enhance their transmission. The white-backed planthopper (WBPH) and brown planthopper (BPH) are vectors of Southern rice black-streaked dwarf virus (SRBSDV) and Rice ragged stunt virus (RRSV), respectively, which cause serious rice diseases. We herein describe the effects of SRBSDV and RRSV infections on host-selection behaviour of vector and non-vector planthoppers at different disease stages. The Y-tube olfactometer choice and free-choice tests indicated that SRBSDV and RRSV infections altered the attractiveness of rice plants to vector and non-vector planthoppers. The attractiveness was mainly mediated by rice volatiles, and varied with disease progression. The attractiveness of the SRBSDV- or RRSV-infected rice plants to the virus-free WBPHs or BPHs initially decreased, then increased, and finally decreased again. For the viruliferous WBPHs and BPHs, SRBSDV or RRSV infection increased the attractiveness of plants more for the non-vector than for the vector planthoppers. Furthermore, we observed that the attractiveness of infected plants to planthoppers was positively correlated with the virus titres. The titre effects were greater for virus-free than for viruliferous planthoppers. Down-regulated defence genes OsAOS1, OsICS, and OsACS2 and up-regulated volatile-biosynthesis genes OsLIS, OsCAS, and OsHPL3 expression in infected plants may influence their attractiveness.

A Sequence in the loop domain of hepatitis C virus E2 protein identified in silico as crucial for the selective binding to human CD81

PubMed Central

Chang, Chun-Chun; Hsu, Hao-Jen; Yen, Jui-Hung; Lo, Shih-Yen

2017-01-01

Hepatitis C virus (HCV) is a species-specific pathogenic virus that infects only humans and chimpanzees. Previous studies have indicated that interactions between the HCV E2 protein and CD81 on host cells are required for HCV infection. To determine the crucial factors for species-specific interactions at the molecular level, this study employed in silico molecular docking involving molecular dynamic simulations of the binding of HCV E2 onto human and rat CD81s. In vitro experiments including surface plasmon resonance measurements and cellular binding assays were applied for simple validations of the in silico results. The in silico studies identified two binding regions on the HCV E2 loop domain, namely E2-site1 and E2-site2, as being crucial for the interactions with CD81s, with the E2-site2 as the determinant factor for human-specific binding. Free energy calculations indicated that the E2/CD81 binding process might follow a two-step model involving (i) the electrostatic interaction-driven initial binding of human-specific E2-site2, followed by (ii) changes in the E2 orientation to facilitate the hydrophobic and van der Waals interaction-driven binding of E2-site1. The sequence of the human-specific, stronger-binding E2-site2 could serve as a candidate template for the future development of HCV-inhibiting peptide drugs. PMID:28481946

Crimean-Congo hemorrhagic fever virus nucleocapsid protein has dual RNA binding modes.

PubMed

Jeeva, Subbiah; Pador, Sean; Voss, Brittany; Ganaie, Safder Saieed; Mir, Mohammad Ayoub

2017-01-01

Crimean Congo hemorrhagic fever, a zoonotic viral disease, has high mortality rate in humans. There is currently no vaccine for Crimean Congo hemorrhagic fever virus (CCHFV) and chemical interventions are limited. The three negative sense genomic RNA segments of CCHFV are specifically encapsidated by the nucleocapsid protein into three ribonucleocapsids, which serve as templates for the viral RNA dependent RNA polymerase. Here we demonstrate that CCHFV nucleocapsid protein has two distinct binding modes for double and single strand RNA. In the double strand RNA binding mode, the nucleocapsid protein preferentially binds to the vRNA panhandle formed by the base pairing of complementary nucleotides at the 5' and 3' termini of viral genome. The CCHFV nucleocapsid protein does not have RNA helix unwinding activity and hence does not melt the duplex vRNA panhandle after binding. In the single strand RNA binding mode, the nucleocapsid protein does not discriminate between viral and non-viral RNA molecules. Binding of both vRNA panhandle and single strand RNA induce a conformational change in the nucleocapsid protein. Nucleocapsid protein remains in a unique conformational state due to simultaneously binding of structurally distinct vRNA panhandle and single strand RNA substrates. Although the role of dual RNA binding modes in the virus replication cycle is unknown, their involvement in the packaging of viral genome and regulation of CCHFV replication in conjunction with RdRp and host derived RNA regulators is highly likely.

Switching on RNA Silencing Suppressor Activity by Restoring Argonaute Binding to a Viral Protein

PubMed Central

Szabó, Edit Z.; Manczinger, Máté; Göblös, Anikó; Kemény, Lajos

2012-01-01

We found that Sweet potato feathery mottle virus (SPFMV) P1, a close homologue of Sweet potato mild mottle virus P1, did not have any silencing suppressor activity. Remodeling the Argonaute (AGO) binding domain of SPFMV P1 by the introduction of two additional WG/GW motifs converted it to a silencing suppressor with AGO binding capacity. To our knowledge, this is the first instance of the transformation of a viral protein of unknown function to a functional silencing suppressor. PMID:22623784

Sialic acid-dependent cell entry of human enterovirus D68

DOE PAGES

Liu, Yue; Sheng, Ju; Baggen, Jim; ...

2015-11-13

Human enterovirus D68 (EV-D68) is a causative agent of childhood respiratory diseases and has now emerged as a global public health threat. Nevertheless, knowledge of the tissue tropism and pathogenesis of EV-D68 has been hindered by a lack of studies on the receptor-mediated EV-D68 entry into host cells. Here we demonstrate that cell surface sialic acid is essential for EV-D68 to bind to and infect susceptible cells. Crystal structures of EV-D68 in complex with sialylated glycan receptor analogues show that they bind into the ‘canyon’ on the virus surface. The sialic acid receptor induces a cascade of conformational changes inmore » the virus to eject a fatty-acid-like molecule that regulates the stability of the virus. Furthermore, virus binding to a sialic acid receptor and to immunoglobulin-like receptors used by most other enteroviruses share a conserved mechanism for priming viral uncoating and facilitating cell entry.« less

The Ebola Virus Nucleoprotein Recruits the Host PP2A-B56 Phosphatase to Activate Transcriptional Support Activity of VP30.

PubMed

Kruse, Thomas; Biedenkopf, Nadine; Hertz, Emil Peter Thrane; Dietzel, Erik; Stalmann, Gertrud; López-Méndez, Blanca; Davey, Norman E; Nilsson, Jakob; Becker, Stephan

2018-01-04

Transcription of the Ebola virus genome depends on the viral transcription factor VP30 in its unphosphorylated form, but the underlying molecular mechanism of VP30 dephosphorylation is unknown. Here we show that the Ebola virus nucleoprotein (NP) recruits the host PP2A-B56 protein phosphatase through a B56-binding LxxIxE motif and that this motif is essential for VP30 dephosphorylation and viral transcription. The LxxIxE motif and the binding site of VP30 in NP are in close proximity, and both binding sites are required for the dephosphorylation of VP30. We generate a specific inhibitor of PP2A-B56 and show that it suppresses Ebola virus transcription and infection. This work dissects the molecular mechanism of VP30 dephosphorylation by PP2A-B56, and it pinpoints this phosphatase as a potential target for therapeutic intervention. Copyright © 2017 Elsevier Inc. All rights reserved.

Sialic acid-dependent cell entry of human enterovirus D68

PubMed Central

Liu, Yue; Sheng, Ju; Baggen, Jim; Meng, Geng; Xiao, Chuan; Thibaut, Hendrik J.; van Kuppeveld, Frank J. M.; Rossmann, Michael G.

2015-01-01

Human enterovirus D68 (EV-D68) is a causative agent of childhood respiratory diseases and has now emerged as a global public health threat. Nevertheless, knowledge of the tissue tropism and pathogenesis of EV-D68 has been hindered by a lack of studies on the receptor-mediated EV-D68 entry into host cells. Here we demonstrate that cell surface sialic acid is essential for EV-D68 to bind to and infect susceptible cells. Crystal structures of EV-D68 in complex with sialylated glycan receptor analogues show that they bind into the ‘canyon' on the virus surface. The sialic acid receptor induces a cascade of conformational changes in the virus to eject a fatty-acid-like molecule that regulates the stability of the virus. Thus, virus binding to a sialic acid receptor and to immunoglobulin-like receptors used by most other enteroviruses share a conserved mechanism for priming viral uncoating and facilitating cell entry. PMID:26563423

Binding of Free and Immune Complex-Associated Hepatitis C Virus to Erythrocytes Is Mediated by the Complement System.

PubMed

Salam, Kazi Abdus; Wang, Richard Y; Grandinetti, Teresa; De Giorgi, Valeria; Alter, Harvey J; Allison, Robert D

2018-05-09

Erythrocytes bind circulating immune complexes (IC) and facilitate IC clearance from the circulation. Chronic hepatitis C virus (HCV) infection is associated with IC-related disorders. In this study we investigated the kinetics and mechanism of HCV and HCV-IC binding to and dissociation from erythrocytes. Cell culture-produced HCV was mixed with erythrocytes from healthy blood donors and erythrocyte-associated virus particles were quantified. Purified complement proteins, complement-depleted serum, and complement receptor antibodies were used to investigate complement-mediated HCV-erythrocyte binding. Purified HCV-specific immunoglobulin G from a chronic HCV-infected patient was used to study complement-mediated HCV-IC-erythrocyte binding. Binding of HCV to erythrocytes increased 200 to 1,000 fold after adding complement active human serum in the absence of antibody. Opsonization of free HCV occurred within 10 minutes and peak binding to erythrocytes was observed at 20-30 minutes. Complement protein C1 was required for binding, while C2, C3 and C4 significantly enhanced binding. Complement receptor 1 (CR1, CD35) antibodies blocked the binding of HCV to erythrocytes isolated from chronically infected HCV patients and healthy blood donors. HCV-ICs significantly enhanced complement-mediated binding to erythrocytes compared to unbound HCV. Dissociation of complement-opsonized HCV from erythrocytes depended on the presence of Factor I. HCV released by Factor I bound preferentially to CD19+ B cells compared to other leukocytes. These results demonstrate that complement mediates the binding of free and IC-associated HCV to CR1 on erythrocytes, and provide a mechanistic rationale for investigating the differential phenotypic expression of HCV-IC-related disease. This article is protected by copyright. All rights reserved. © 2018 by the American Association for the Study of Liver Diseases.

In silico screening of small molecule libraries using the dengue virus envelope E protein has identified compounds with antiviral activity against multiple flaviviruses.

PubMed

Kampmann, Thorsten; Yennamalli, Ragothaman; Campbell, Phillipa; Stoermer, Martin J; Fairlie, David P; Kobe, Bostjan; Young, Paul R

2009-12-01

The flaviviruses comprise a large group of related viruses, many of which pose a significant global human health threat, most notably the dengue viruses (DENV), West Nile virus (WNV) and yellow fever virus (YFV). Flaviviruses enter host cells via fusion of the viral and cellular membranes, a process mediated by the major viral envelope protein E as it undergoes a low pH induced conformational change in the endosomal compartment of the host cell. This essential entry stage in the flavivirus life cycle provides an attractive target for the development of antiviral agents. We performed an in silico docking screen of the Maybridge chemical database within a previously described ligand binding pocket in the dengue E protein structure that is thought to play a key role in the conformational transitions that lead to membrane fusion. The biological activity of selected compounds identified from this screen revealed low micromolar antiviral potency against dengue virus for two of the compounds. Our results also provide the first evidence that compounds selected to bind to this ligand binding site on the flavivirus E protein abrogate fusion activity. Interestingly, one of these compounds also has antiviral activity against both WNV (kunjin strain) and YFV.

Inefficient transmission of H5N1 influenza viruses in a ferret contact model.

PubMed

Yen, Hui-Ling; Lipatov, Aleksandr S; Ilyushina, Natalia A; Govorkova, Elena A; Franks, John; Yilmaz, Neziha; Douglas, Alan; Hay, Alan; Krauss, Scott; Rehg, Jerold E; Hoffmann, Erich; Webster, Robert G

2007-07-01

The abilities to infect and transmit efficiently among humans are essential for a novel influenza A virus to cause a pandemic. To evaluate the pandemic potential of widely disseminated H5N1 influenza viruses, a ferret contact model using experimental groups comprised of one inoculated ferret and two contact ferrets was used to study the transmissibility of four human H5N1 viruses isolated from 2003 to 2006. The effects of viral pathogenicity and receptor binding specificity (affinity to synthetic sialosaccharides with alpha2,3 or alpha2,6 linkages) on transmissibility were assessed. A/Vietnam/1203/04 and A/Vietnam/JP36-2/05 viruses, which possess "avian-like" alpha2,3-linked sialic acid (SA) receptor specificity, caused neurological symptoms and death in ferrets inoculated with 10(3) 50% tissue culture infectious doses. A/Hong Kong/213/03 and A/Turkey/65-596/06 viruses, which show binding affinity for "human-like" alpha2,6-linked SA receptors in addition to their affinity for alpha2,3-linked SA receptors, caused mild clinical symptoms and were not lethal to the ferrets. No transmission of A/Vietnam/1203/04 or A/Turkey/65-596/06 virus was detected. One contact ferret developed neutralizing antibodies to A/Hong Kong/213/03 but did not exhibit any clinical signs or detectable virus shedding. In two groups, one of two naïve contact ferrets had detectable virus after 6 to 8 days when housed together with the A/Vietnam/JP36-2/05 virus-inoculated ferrets. Infected contact ferrets showed severe clinical signs, although little or no virus was detected in nasal washes. This limited virus shedding explained the absence of secondary transmission from the infected contact ferret to the other naïve ferret that were housed together. Our results suggest that despite their receptor binding affinity, circulating H5N1 viruses retain molecular determinants that restrict their spread among mammalian species.

Screening for the Location of RNA using the Chloride Ion Distribution in Simulations of Virus Capsids.

PubMed

Larsson, Daniel S D; van der Spoel, David

2012-07-10

The complete structure of the genomic material inside a virus capsid remains elusive, although a limited amount of symmetric nucleic acid can be resolved in the crystal structure of 17 icosahedral viruses. The negatively charged sugar-phosphate backbone of RNA and DNA as well as the large positive charge of the interior surface of the virus capsids suggest that electrostatic complementarity is an important factor in the packaging of the genomes in these viruses. To test how much packing information is encoded by the electrostatic and steric envelope of the capsid interior, we performed extensive all-atom molecular dynamics (MD) simulations of virus capsids with explicit water molecules and solvent ions. The model systems were two small plant viruses in which significant amounts of RNA has been observed by X-ray crystallography: satellite tobacco mosaic virus (STMV, 62% RNA visible) and satellite tobacco necrosis virus (STNV, 34% RNA visible). Simulations of half-capsids of these viruses with no RNA present revealed that the binding sites of RNA correlated well with regions populated by chloride ions, suggesting that it is possible to screen for the binding sites of nucleic acids by determining the equilibrium distribution of negative ions. By including the crystallographically resolved RNA in addition to ions, we predicted the localization of the unresolved RNA in the viruses. Both viruses showed a hot-spot for RNA binding at the 5-fold symmetry axis. The MD simulations were compared to predictions of the chloride density based on nonlinear Poisson-Boltzmann equation (PBE) calculations with mobile ions. Although the predictions are superficially similar, the PBE calculations overestimate the ion concentration close to the capsid surface and underestimate it far away, mainly because protein dynamics is not taken into account. Density maps from chloride screening can be used to aid in building atomic models of packaged virus genomes. Knowledge of the principles of genome packaging might be exploited for both antiviral therapy and technological applications.

Crystal structure of reovirus attachment protein σ1 in complex with sialylated oligosaccharides.

PubMed

Reiter, Dirk M; Frierson, Johnna M; Halvorson, Elizabeth E; Kobayashi, Takeshi; Dermody, Terence S; Stehle, Thilo

2011-08-01

Many viruses attach to target cells by binding to cell-surface glycans. To gain a better understanding of strategies used by viruses to engage carbohydrate receptors, we determined the crystal structures of reovirus attachment protein σ1 in complex with α-2,3-sialyllactose, α-2,6-sialyllactose, and α-2,8-di-siallylactose. All three oligosaccharides terminate in sialic acid, which serves as a receptor for the reovirus serotype studied here. The overall structure of σ1 resembles an elongated, filamentous trimer. It contains a globular head featuring a compact β-barrel, and a fibrous extension formed by seven repeating units of a triple β-spiral that is interrupted near its midpoint by a short α-helical coiled coil. The carbohydrate-binding site is located between β-spiral repeats two and three, distal from the head. In all three complexes, the terminal sialic acid forms almost all of the contacts with σ1 in an identical manner, while the remaining components of the oligosaccharides make little or no contacts. We used this structural information to guide mutagenesis studies to identify residues in σ1 that functionally engage sialic acid by assessing hemagglutination capacity and growth in murine erythroleukemia cells, which require sialic acid binding for productive infection. Our studies using σ1 mutant viruses reveal that residues 198, 202, 203, 204, and 205 are required for functional binding to sialic acid by reovirus. These findings provide insight into mechanisms of reovirus attachment to cell-surface glycans and contribute to an understanding of carbohydrate binding by viruses. They also establish a filamentous, trimeric carbohydrate-binding module that could potentially be used to endow other trimeric proteins with carbohydrate-binding properties.

Surface IgM λ light chain is involved in the binding and infection of infectious bursal disease virus (IBDV) to DT40 cells.

PubMed

Chi, Jiaqi; You, Leiming; Li, Peipei; Teng, Man; Zhang, Gaiping; Luo, Jun; Wang, Aiping

2018-04-01

Infectious bursal disease virus (IBDV) is an important immunosuppressive virus in chickens. Surface immunoglobulin M (sIgM)-bearing B lymphocytes act as the major targets of IBDV in the bursa of Fabricius, and sIgM may function as one of the membrane binding sites responsible for IBDV infection. Recently, using the virus overlay protein binding assay, the chicken λ light chain of sIgM was identified to specifically interact with IBDV in a virulence-independent manner in vitro. To further investigate sIgM λ light chain-mediated IBDV binding and infection in pre-B cells, the cell line DT40, which is susceptible to both pathogenic and attenuated IBDV, was used. Based on the RNA interference strategy, the DT40 cell line whose λ light chain of sIgM was stably knocked down, herein termed DT40LKD, was generated by the genomic integration of a specific small hairpin RNA and a green fluorescence protein co-expression construct. Flow cytometry analysis indicated that the binding of IBDV to DT40LKD cells was significantly reduced due to the loss of sIgM λ light chain. In particular, reduced viral replication was observed in IBDV-incubated DT40LKD cells, and no viral release into cell culture medium was detected by the IBDV rapid diagnostic strips. In addition, the rescue of sIgM λ light chain expression restored viral binding and replication in DT40LKD cells. These results show that sIgM λ light chain appears to be beneficial for IBDV attachment and infection, suggesting that sIgM acts as a binding site involved in IBDV infection.

Structure-Based Discovery of the Novel Antiviral Properties of Naproxen against the Nucleoprotein of Influenza A Virus

PubMed Central

Lejal, Nathalie; Tarus, Bogdan; Bouguyon, Edwige; Chenavas, Sylvie; Bertho, Nicolas; Delmas, Bernard; Ruigrok, Rob W. H.; Di Primo, Carmelo

2013-01-01

The nucleoprotein (NP) binds the viral RNA genome and associates with the polymerase in a ribonucleoprotein complex (RNP) required for transcription and replication of influenza A virus. NP has no cellular counterpart, and the NP sequence is highly conserved, which led to considering NP a hot target in the search for antivirals. We report here that monomeric nucleoprotein can be inhibited by a small molecule binding in its RNA binding groove, resulting in a novel antiviral against influenza A virus. We identified naproxen, an anti-inflammatory drug that targeted the nucleoprotein to inhibit NP-RNA association required for NP function, by virtual screening. Further docking and molecular dynamics (MD) simulations identified in the RNA groove two NP-naproxen complexes of similar levels of interaction energy. The predicted naproxen binding sites were tested using the Y148A, R152A, R355A, and R361A proteins carrying single-point mutations. Surface plasmon resonance, fluorescence, and other in vitro experiments supported the notion that naproxen binds at a site identified by MD simulations and showed that naproxen competed with RNA binding to wild-type (WT) NP and protected active monomers of the nucleoprotein against proteolytic cleavage. Naproxen protected Madin-Darby canine kidney (MDCK) cells against viral challenges with the H1N1 and H3N2 viral strains and was much more effective than other cyclooxygenase inhibitors in decreasing viral titers of MDCK cells. In a mouse model of intranasal infection, naproxen treatment decreased the viral titers in mice lungs. In conclusion, naproxen is a promising lead compound for novel antivirals against influenza A virus that targets the nucleoprotein in its RNA binding groove. PMID:23459490

Emergence of viruses resistant to neutralization by V3-specific antibodies in experimental human immunodeficiency virus type 1 IIIB infection of chimpanzees.

PubMed Central

Nara, P L; Smit, L; Dunlop, N; Hatch, W; Merges, M; Waters, D; Kelliher, J; Gallo, R C; Fischinger, P J; Goudsmit, J

1990-01-01

Emergence in two chimpanzees of human immunodeficiency virus type 1 (HIV-1) IIIB variants resistant to neutralization by the preexisting antibody is described. Viruses isolated from the HIV-1 IIIB gp120-vaccinated and -challenged animal were more resistant to neutralization by the chimpanzee's own serum than viruses isolated from the naive infected animal, indicating immune pressure as the selective mechanism. However, all reisolated viruses were 16- to 256-fold more neutralization resistant than the inoculum virus to antibodies binding to the third variable domain (V3) of the HIV-1 external envelope. Early chimpanzee serum samples that neutralized the inoculum strain but not the reisolated viruses were found to bind an HIV-1 IIIB common nonapeptide (IQRGPGRAF) derived from the gp120 isolate-specific V3 domain shown to induce isolate-specific neutralization in other animals. Amplification of the V3 coding sequence by polymerase chain reaction and subsequent sequence analysis of the neutralization-resistant variants obtained from in vivo-infected animals indicated that early resistance to neutralization by an HIV-1 IIIB monoclonal antibody (0.5 beta) was conferred by changes outside the direct binding site for the selective neutralizing antibody. The reisolated neutralization-resistant isolates consisted of the lower-replication-competent virus subpopulations of the HIV-1 IIIB stock, as confirmed by biological and sequence analyses. In vitro passage of the HIV-1 IIIB stock through chimpanzee and human peripheral blood mononuclear cell cultures void of HIV-specific antibody resulted in homogenic amplification of the more-replication-competent subpopulation preexisting in the original viral stock, suggesting a role for the immune system in suppressing the more-replication-competent viruses. Images PMID:2370681

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

Munke, Anna; Andreasson, Jakob; Aquila, Andrew

Single particle diffractive imaging data from Rice Dwarf Virus (RDV) were recorded using the Coherent X-ray Imaging (CXI) instrument at the Linac Coherent Light Source (LCLS). RDV was chosen as it is a well-characterized model system, useful for proof-of-principle experiments, system optimization and algorithm development. RDV, an icosahedral virus of about 70 nm in diameter, was aerosolized and injected into the approximately 0.1 μm diameter focused hard X-ray beam at the CXI instrument of LCLS. Diffraction patterns from RDV with signal to 5.9 Ångström were recorded. Here, the diffraction data are available through the Coherent X-ray Imaging Data Bank (CXIDB)more » as a resource for algorithm development, the contents of which are described here.« less

Evidence for lysine acetylation in the coat protein of a polerovirus.

PubMed

Cilia, Michelle; Johnson, Richard; Sweeney, Michelle; DeBlasio, Stacy L; Bruce, James E; MacCoss, Michael J; Gray, Stewart M

2014-10-01

Virions of the RPV strain of Cereal yellow dwarf virus-RPV were purified from infected oat tissue and analysed by MS. Two conserved residues, K147 and K181, in the virus coat protein, were confidently identified to contain epsilon-N-acetyl groups. While no functional data are available for K147, K181 lies within an interfacial region critical for virion assembly and stability. The signature immonium ion at m/z 126.0919 demonstrated the presence of N-acetyllysine, and the sequence fragment ions enabled an unambiguous assignment of the epsilon-N-acetyl modification on K181. We hypothesize that selection favours acetylation of K181 in a fraction of coat protein monomers to stabilize the capsid by promoting intermonomer salt bridge formation.

Low Pathogenic Avian Influenza Isolates from Wild Birds Replicate and Transmit via Contact in Ferrets without Prior Adaptation

PubMed Central

Humberd-Smith, Jennifer; Gordy, James T.; Bradley, Konrad C.; Steinhauer, David A.; Berghaus, Roy D.; Stallknecht, David E.; Howerth, Elizabeth W.; Tompkins, Stephen Mark

2012-01-01

Direct transmission of avian influenza viruses to mammals has become an increasingly investigated topic during the past decade; however, isolates that have been primarily investigated are typically ones originating from human or poultry outbreaks. Currently there is minimal comparative information on the behavior of the innumerable viruses that exist in the natural wild bird host. We have previously demonstrated the capacity of numerous North American avian influenza viruses isolated from wild birds to infect and induce lesions in the respiratory tract of mice. In this study, two isolates from shorebirds that were previously examined in mice (H1N9 and H6N1 subtypes) are further examined through experimental inoculations in the ferret with analysis of viral shedding, histopathology, and antigen localization via immunohistochemistry to elucidate pathogenicity and transmission of these viruses. Using sequence analysis and glycan binding analysis, we show that these avian viruses have the typical avian influenza binding pattern, with affinity for cell glycoproteins/glycolipids having terminal sialic acid (SA) residues with α 2,3 linkage [Neu5Ac(α2,3)Gal]. Despite the lack of α2,6 linked SA binding, these AIVs productively infected both the upper and lower respiratory tract of ferrets, resulting in nasal viral shedding and pulmonary lesions with minimal morbidity. Moreover, we show that one of the viruses is able to transmit to ferrets via direct contact, despite its binding affinity for α 2,3 linked SA residues. These results demonstrate that avian influenza viruses, which are endemic in aquatic birds, can potentially infect humans and other mammals without adaptation. Finally this work highlights the need for additional study of the wild bird subset of influenza viruses in regard to surveillance, transmission, and potential for reassortment, as they have zoonotic potential. PMID:22675507

Next-Generation Sequencing and Genome Editing in Plant Virology

PubMed Central

Hadidi, Ahmed; Flores, Ricardo; Candresse, Thierry; Barba, Marina

2016-01-01

Next-generation sequencing (NGS) has been applied to plant virology since 2009. NGS provides highly efficient, rapid, low cost DNA, or RNA high-throughput sequencing of the genomes of plant viruses and viroids and of the specific small RNAs generated during the infection process. These small RNAs, which cover frequently the whole genome of the infectious agent, are 21–24 nt long and are known as vsRNAs for viruses and vd-sRNAs for viroids. NGS has been used in a number of studies in plant virology including, but not limited to, discovery of novel viruses and viroids as well as detection and identification of those pathogens already known, analysis of genome diversity and evolution, and study of pathogen epidemiology. The genome engineering editing method, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has been successfully used recently to engineer resistance to DNA geminiviruses (family, Geminiviridae) by targeting different viral genome sequences in infected Nicotiana benthamiana or Arabidopsis plants. The DNA viruses targeted include tomato yellow leaf curl virus and merremia mosaic virus (begomovirus); beet curly top virus and beet severe curly top virus (curtovirus); and bean yellow dwarf virus (mastrevirus). The technique has also been used against the RNA viruses zucchini yellow mosaic virus, papaya ringspot virus and turnip mosaic virus (potyvirus) and cucumber vein yellowing virus (ipomovirus, family, Potyviridae) by targeting the translation initiation genes eIF4E in cucumber or Arabidopsis plants. From these recent advances of major importance, it is expected that NGS and CRISPR-Cas technologies will play a significant role in the very near future in advancing the field of plant virology and connecting it with other related fields of biology. PMID:27617007

Gibberellin Receptor GID1: Gibberellin Recognition and Molecular Evolution

NASA Astrophysics Data System (ADS)

Kato, Hiroaki; Sato, Tomomi; Ueguchi-Tanaka, Miyako

Gibberellins (GAs) are phytohormones essential for many developmental processes in plants. We analyzed the crystal structure of a nuclear GA receptor, GIBBERELLIN INSENSITIVE DWARF 1 (GID1) from Oryza sativa. As it was proposed from the sequence similarity, the overall structure of GID1 shows an α/β-hydrolase fold similar to that of the hormone-sensitive lipases (HSLs) except for an amino-terminal lid. The GA-binding site corresponds to the substrate-binding site of HSLs. Almost residues assigned for GA binding showed very little or no activity when they were replaced with Ala. The substitution of the residues corresponding to those of the lycophyte GID1s caused an increase in the binding affinity for GA34, a 2β-hydroxylated GA4. These findings indicate that GID1 originated from HSL and was tinkered to have the specificity for bioactive GAs in the course of plant evolution.

Human immunodeficiency virus type 1 Tat binds to Candida albicans, inducing hyphae but augmenting phagocytosis in vitro

PubMed Central

Gruber, Andreas; Lell, Claudia P; Speth, Cornelia; Stoiber, Heribert; Lass-Flörl, Cornelia; Sonneborn, Anja; Ernst, Joachim F; Dierich, Manfred P; Würzner, Reinhard

2001-01-01

Tat, the human immunodeficiency virus type 1 (HIV-1) transactivating protein, binds through its RGD-motif to human integrin receptors. Candida albicans, the commonest cause of mucosal candidiasis in subjects infected with HIV-1, also possesses RGD-binding capacity. The present study reveals that Tat binds to C. albicans but not to C. tropicalis. Tat binding was markedly reduced by laminin and to a lesser extent by a complement C3 peptide containing the RGD motif, but not by a control peptide. The outgrowth of C. albicans was accelerated following binding of Tat, but phagocytosis of opsonized C. albicans was also increased after Tat binding. Thus, Tat binding promotes fungal virulence by inducing hyphae but may also reduce it by augmenting phagocytosis. The net effect of Tat in vivo is difficult to judge but in view of the many disease-promoting effects of Tat we propose that accelerating the formation of hyphae dominates over the augmentation of phagocytosis. PMID:11899432

Molecular Genetic Analysis and Evolution of Segment 7 in Rice Black-Streaked Dwarf Virus in China

PubMed Central

Chen, Yanping; Wu, Jirong; Meng, Qingchang; Han, Xiaohua; Hao, Zhuanfang; Li, Mingshun; Yong, Hongjun; Zhang, Degui; Zhang, Shihuang; Li, Xinhai

2015-01-01

Rice black-streaked dwarf virus (RBSDV) causes maize rough dwarf disease or rice black-streaked dwarf disease and can lead to severe yield losses in maize and rice. To analyse RBSDV evolution, codon usage bias and genetic structure were investigated in 111 maize and rice RBSDV isolates from eight geographic locations in 2013 and 2014. The linear dsRNA S7 is A+U rich, with overall codon usage biased toward codons ending with A (A3s, S7-1: 32.64%, S7-2: 29.95%) or U (U3s, S7-1: 44.18%, S7-2: 46.06%). Effective number of codons (Nc) values of 45.63 in S7-1 (the first open reading frame of S7) and 39.96 in S7-2 (the second open reading frame of S7) indicate low degrees of RBSDV-S7 codon usage bias, likely driven by mutational bias regardless of year, host, or geographical origin. Twelve optimal codons were detected in S7. The nucleotide diversity (π) of S7 sequences in 2013 isolates (0.0307) was significantly higher than in 2014 isolates (0.0244, P = 0.0226). The nucleotide diversity (π) of S7 sequences in isolates from Jinan (0.0391) was higher than that from the other seven locations (P < 0.01). Only one S7 recombinant was detected in Baoding. RBSDV isolates could be phylogenetically classified into two groups according to S7 sequences, and further classified into two subgroups. S7-1 and S7-2 were under negative and purifying selection, with respective Ka/Ks ratios of 0.0179 and 0.0537. These RBSDV populations were expanding (P < 0.01) as indicated by negative values for Tajima's D, Fu and Li's D, and Fu and Li's F. Genetic differentiation was detected in six RBSDV subpopulations (P < 0.05). Absolute Fst (0.0790) and Nm (65.12) between 2013 and 2014, absolute Fst (0.1720) and Nm (38.49) between maize and rice, and absolute Fst values of 0.0085-0.3069 and Nm values of 0.56-29.61 among these eight geographic locations revealed frequent gene flow between subpopulations. Gene flow between 2013 and 2014 was the most frequent. PMID:26121638

Elution of viruses by ionic and nonionic surfactants.

PubMed Central

Fujito, B T; Lytle, C D

1996-01-01

The ionic and nonionic surfactants sodium dodecyl sulfate and Triton X-100, respectively, eluted two viruses, phi X174 and PRD1, which were adsorbed to the ionic and nonionic binding membranes cationic polysulfone and nitrocellulose, respectively. Results indicated that complete elution was readily achieved only when combinations of surfactants and binding membranes were matched (i.e., ionic-ionic or nonionic-nonionic). PMID:8795240

HIV envelope glycoprotein imaged at high resolution | Center for Cancer Research

Cancer.gov

The outer surface of the human immunodeficiency virus (HIV) is surrounded by an envelope studded with spike-shaped glycoproteins called Env that help the deadly virus identify, bind, and infect cells. When unbound, Env exists in a “closed” conformational state. Upon binding with target cells, such as CD4+ T cells, the protein transitions to an “open” configuration. Given that

Identifying geographic hot spots of reassortment in a multipartite plant virus

PubMed Central

Savory, Fiona R; Varma, Varun; Ramakrishnan, Uma

2014-01-01

Reassortment between different species or strains plays a key role in the evolution of multipartite plant viruses and can have important epidemiological implications. Identifying geographic locations where reassortant lineages are most likely to emerge could be a valuable strategy for informing disease management and surveillance efforts. We developed a predictive framework to identify potential geographic hot spots of reassortment based upon spatially explicit analyses of genome constellation diversity. To demonstrate the utility of this approach, we examined spatial variation in the potential for reassortment among Cardamom bushy dwarf virus (CBDV; Nanoviridae, Babuvirus) isolates in Northeast India. Using sequence data corresponding to six discrete genome components for 163 CBDV isolates, a quantitative measure of genome constellation diversity was obtained for locations across the sampling region. Two key areas were identified where viruses with highly distinct genome constellations cocirculate, and these locations were designated as possible geographic hot spots of reassortment, where novel reassortant lineages could emerge. Our study demonstrates that the potential for reassortment can be spatially dependent in multipartite plant viruses and highlights the use of evolutionary analyses to identify locations which could be actively managed to facilitate the prevention of outbreaks involving novel reassortant strains. PMID:24944570

Recovery of West Nile Virus Envelope Protein Domain III Chimeras with Altered Antigenicity and Mouse Virulence

PubMed Central

McAuley, Alexander J.; Torres, Maricela; Plante, Jessica A.; Huang, Claire Y.-H.; Bente, Dennis A.

2016-01-01

ABSTRACT Flaviviruses are positive-sense, single-stranded RNA viruses responsible for millions of human infections annually. The envelope (E) protein of flaviviruses comprises three structural domains, of which domain III (EIII) represents a discrete subunit. The EIII gene sequence typically encodes epitopes recognized by virus-specific, potently neutralizing antibodies, and EIII is believed to play a major role in receptor binding. In order to assess potential interactions between EIII and the remainder of the E protein and to assess the effects of EIII sequence substitutions on the antigenicity, growth, and virulence of a representative flavivirus, chimeric viruses were generated using the West Nile virus (WNV) infectious clone, into which EIIIs from nine flaviviruses with various levels of genetic diversity from WNV were substituted. Of the constructs tested, chimeras containing EIIIs from Koutango virus (KOUV), Japanese encephalitis virus (JEV), St. Louis encephalitis virus (SLEV), and Bagaza virus (BAGV) were successfully recovered. Characterization of the chimeras in vitro and in vivo revealed differences in growth and virulence between the viruses, with in vivo pathogenesis often not being correlated with in vitro growth. Taken together, the data demonstrate that substitutions of EIII can allow the generation of viable chimeric viruses with significantly altered antigenicity and virulence. IMPORTANCE The envelope (E) glycoprotein is the major protein present on the surface of flavivirus virions and is responsible for mediating virus binding and entry into target cells. Several viable West Nile virus (WNV) variants with chimeric E proteins in which the putative receptor-binding domain (EIII) sequences of other mosquito-borne flaviviruses were substituted in place of the WNV EIII were recovered, although the substitution of several more divergent EIII sequences was not tolerated. The differences in virulence and tissue tropism observed with the chimeric viruses indicate a significant role for this sequence in determining the pathogenesis of the virus within the mammalian host. Our studies demonstrate that these chimeras are viable and suggest that such recombinant viruses may be useful for investigation of domain-specific antibody responses and the more extensive definition of the contributions of EIII to the tropism and pathogenesis of WNV or other flaviviruses. PMID:26912625

Recovery of West Nile Virus Envelope Protein Domain III Chimeras with Altered Antigenicity and Mouse Virulence.

PubMed

McAuley, Alexander J; Torres, Maricela; Plante, Jessica A; Huang, Claire Y-H; Bente, Dennis A; Beasley, David W C

2016-05-01

Flaviviruses are positive-sense, single-stranded RNA viruses responsible for millions of human infections annually. The envelope (E) protein of flaviviruses comprises three structural domains, of which domain III (EIII) represents a discrete subunit. The EIII gene sequence typically encodes epitopes recognized by virus-specific, potently neutralizing antibodies, and EIII is believed to play a major role in receptor binding. In order to assess potential interactions between EIII and the remainder of the E protein and to assess the effects of EIII sequence substitutions on the antigenicity, growth, and virulence of a representative flavivirus, chimeric viruses were generated using the West Nile virus (WNV) infectious clone, into which EIIIs from nine flaviviruses with various levels of genetic diversity from WNV were substituted. Of the constructs tested, chimeras containing EIIIs from Koutango virus (KOUV), Japanese encephalitis virus (JEV), St. Louis encephalitis virus (SLEV), and Bagaza virus (BAGV) were successfully recovered. Characterization of the chimeras in vitro and in vivo revealed differences in growth and virulence between the viruses, within vivo pathogenesis often not being correlated within vitro growth. Taken together, the data demonstrate that substitutions of EIII can allow the generation of viable chimeric viruses with significantly altered antigenicity and virulence. The envelope (E) glycoprotein is the major protein present on the surface of flavivirus virions and is responsible for mediating virus binding and entry into target cells. Several viable West Nile virus (WNV) variants with chimeric E proteins in which the putative receptor-binding domain (EIII) sequences of other mosquito-borne flaviviruses were substituted in place of the WNV EIII were recovered, although the substitution of several more divergent EIII sequences was not tolerated. The differences in virulence and tissue tropism observed with the chimeric viruses indicate a significant role for this sequence in determining the pathogenesis of the virus within the mammalian host. Our studies demonstrate that these chimeras are viable and suggest that such recombinant viruses may be useful for investigation of domain-specific antibody responses and the more extensive definition of the contributions of EIII to the tropism and pathogenesis of WNV or other flaviviruses. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Crystal Structure of West Nile Virus Envelope Glycoprotein Reveals Viral Surface Epitopes

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

Kanai,R.; Kar, K.; Anthony, K.

2006-01-01

West Nile virus, a member of the Flavivirus genus, causes fever that can progress to life-threatening encephalitis. The major envelope glycoprotein, E, of these viruses mediates viral attachment and entry by membrane fusion. We have determined the crystal structure of a soluble fragment of West Nile virus E. The structure adopts the same overall fold as that of the E proteins from dengue and tick-borne encephalitis viruses. The conformation of domain II is different from that in other prefusion E structures, however, and resembles the conformation of domain II in postfusion E structures. The epitopes of neutralizing West Nile virus-specificmore » antibodies map to a region of domain III that is exposed on the viral surface and has been implicated in receptor binding. In contrast, we show that certain recombinant therapeutic antibodies, which cross-neutralize West Nile and dengue viruses, bind a peptide from domain I that is exposed only during the membrane fusion transition. By revealing the details of the molecular landscape of the West Nile virus surface, our structure will assist the design of antiviral vaccines and therapeutics.« less

Ternary Complex Factors and Cofactors Are Essential for Human T-Cell Leukemia Virus Type 1 Tax Transactivation of the Serum Response Element

PubMed Central

Shuh, Maureen; Derse, David

2000-01-01

The human T-cell leukemia virus type 1 Tax protein activates the expression of cellular immediate early genes controlled by the serum response element (SRE), which contains both the serum response factor (SRF) binding element (CArG box) and the ternary complex factor (TCF) binding element (Ets box). We show that TCF binding is necessary for Tax activation of the SRE and that Tax directly interacts with TCFs in vitro. In addition, Tax interactions with CREB binding protein (CBP) and p300- and CBP-associated factor were found to be essential for Tax activation of SRF-mediated transcription. PMID:11070040

Characterization of Interactions between Heparin/Glycosaminoglycan and Adeno-associated Virus

PubMed Central

Zhang, Fuming; Aguilera, Javier; Beaudet, Julie M.; Xie, Qing; Lerch, Thomas F.; Davulcu, Omar; Colón, Wilfredo; Chapman, Michael S.; Linhardt, Robert J.

2013-01-01

Adeno-associated virus (AAV) is a key candidate in the development of gene therapy. In this report, we used surface plasmon resonance spectroscopy to study the interaction between AAV and heparin and other glycosaminoglycans. Surface plasmon resonance results revealed that heparin binds to AAV with extremely high affinity. Solution competition studies shows that AAV binding to heparin is chain length dependent. AAV prefers to bind full chain heparin. All sulfo groups (especially N-sulfo and 6-O-sulfo groups) on heparin are important for the AAV- heparin interaction. Higher levels of sulfo group substitution in GAGs enhance their binding affinities. Atomic force microscopy was also performed to image AAV-2 complexed with heparin. PMID:23952613

An Aptamer That Neutralizes R5 Strains of Human Immunodeficiency Virus Type 1 Blocks gp120-CCR5 Interaction

PubMed Central

Dey, Antu K.; Khati, Makobetsa; Tang, Min; Wyatt, Richard; Lea, Susan M.; James, William

2005-01-01

We recently described the isolation and structural characterization of 2′-fluoropyrimidine-substituted RNA aptamers that bind to gp120 of R5 strains of human immunodeficiency virus type 1 and thereby potently neutralize the infectivity of phylogenetically diverse R5 strains. Here we investigate the physical basis of their antiviral action. We show that both N-linked oligosaccharides and the variable loops V1/V2 and V3 are not required for binding of one aptamer, B40, to gp120. Using surface plasmon resonance binding analyses, we show that the aptamer binds to the CCR5-binding site on gp120 in a relatively CD4-independent manner, providing a mechanistic explanation for its neutralizing potency. PMID:16227301

Using high throughput screening to define virus clearance by chromatography resins.

PubMed

Connell-Crowley, Lisa; Larimore, Elizabeth A; Gillespie, Ron

2013-07-01

High throughput screening (HTS) of chromatography resins can accelerate downstream process development by rapidly providing information on product and impurity partitioning over a wide range of experimental conditions. In addition to the removal of typical product and process-related impurities, chromatography steps are also used to remove potential adventitious viral contaminants and non-infectious retrovirus-like particles expressed by rodent cell lines used for production. This article evaluates the feasibility of using HTS in a 96-well batch-binding format to study removal of the model retrovirus xenotropic murine leukemia virus (xMuLV) from product streams. Two resins were examined: the anion exchange resin Q Sepharose Fast Flow™ (QSFF) and Capto adhere™, a mixed mode resin. QSFF batch-binding HTS data was generated using two mAbs at various pHs, NaCl concentrations, and levels of impurities. Comparison of HTS data to that generated using the column format showed good agreement with respect to virus retentation at different pHs, NaCl concentrations and impurity levels. Results indicate that NaCl concentration and impurity level, but not pH, are key parameters that can impact xMuLV binding to both resins. Binding of xMuLV to Capto adhere appeared to tolerate higher levels of NaCl and impurity than QSFF, and showed some product-specific impact on binding that was not observed with QSFF. Overall, the results demonstrate that the 96-well batch-binding HTS technique can be an effective tool for rapidly defining conditions for robust virus clearance on chromatographic resins. Copyright © 2013 Wiley Periodicals, Inc.

Identification of differentially regulated maize proteins conditioning Sugarcane mosaic virus systemic infection.

PubMed

Chen, Hui; Cao, Yanyong; Li, Yiqing; Xia, Zihao; Xie, Jipeng; Carr, John P; Wu, Boming; Fan, Zaifeng; Zhou, Tao

2017-08-01

Sugarcane mosaic virus (SCMV) is the most important cause of maize dwarf mosaic disease. To identify maize genes responsive to SCMV infection and that may be involved in pathogenesis, a comparative proteomic analysis was performed using the first and second systemically infected leaves (termed 1 SL and 2 SL, respectively). Seventy-one differentially expressed proteins were identified in 1 SL and 2 SL upon SCMV infection. Among them, eight proteins showed the same changing patterns in both 1 SL and 2 SL. Functional annotations of regulated proteins and measurement of photosynthetic activity revealed that photosynthesis was more inhibited and defensive gene expression more pronounced in 1 SL than in 2 SL. Knockdown of regulated proteins in both 1 SL and 2 SL by a brome mosaic virus-based gene silencing vector in maize indicated that protein disulfide isomerase-like and phosphoglycerate kinase were required for optimal SCMV replication. By contrast, knockdown of polyamine oxidase (ZmPAO) significantly increased SCMV accumulation, implying that ZmPAO activity might contribute to resistance or tolerance. The results suggest that combining comparative proteomic analyses of different tissues and virus-induced gene silencing is an efficient way to identify host proteins supporting virus replication or enhancing resistance to virus infection. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Characterization of a new apple luteovirus identified by high-throughput sequencing.

PubMed

Liu, Huawei; Wu, Liping; Nikolaeva, Ekaterina; Peter, Kari; Liu, Zongrang; Mollov, Dimitre; Cao, Mengji; Li, Ruhui

2018-05-15

'Rapid Apple Decline' (RAD) is a newly emerging problem of young, dwarf apple trees in the Northeastern USA. The affected trees show trunk necrosis, cracking and canker before collapse in summer. In this study, we discovered and characterized a new luteovirus from apple trees in RAD-affected orchards using high-throughput sequencing (HTS) technology and subsequent Sanger sequencing. Illumina NextSeq sequencing was applied to total RNAs prepared from three diseased apple trees. Sequence reads were de novo assembled, and contigs were annotated by BLASTx. RT-PCR and 5'/3' RACE sequencing were used to obtain the complete genome of a new virus. RT-PCR was used to detect the virus. Three common apple viruses and a new luteovirus were identified from the diseased trees by HTS and RT-PCR. Sequence analyses of the complete genome of the new virus show that it is a new species of the genus Luteovirus in the family Luteoviridae. The virus is graft transmissible and detected by RT-PCR in apple trees in a couple of orchards. A new luteovirus and/or three known viruses were found to be associated with RAD. Molecular characterization of the new luteovirus provides important information for further investigation of its distribution and etiological role.

Understanding the immune system architecture and transcriptome responses to southern rice black-streaked dwarf virus in Sogatella furcifera.

PubMed

Wang, Lin; Tang, Nan; Gao, Xinlei; Guo, Dongyang; Chang, Zhaoxia; Fu, Yating; Akinyemi, Ibukun A; Wu, Qingfa

2016-11-02

Sogatella furcifera, the white-backed planthopper (WBPH), has become one of the most destructive pests in rice production owing to its plant sap-sucking behavior and efficient transmission of Southern rice black-streaked dwarf virus (SRBSDV) in a circulative, propagative and persistent manner. The dynamic and complex SRBSDV-WBPH-rice plant interaction is still poorly understood. In this study, based on a homology-based genome-wide analysis, 348 immune-related genes belonging to 28 families were identified in WBPH. A transcriptome analysis of non-viruliferous (NVF) and viruliferous groups with high viral titers (HVT) and median viral titers (MVT) revealed that feeding on SRBSDV-infected rice plants has a significant impact on gene expression, regardless of viral titers in insects. We identified 278 up-regulated and 406 down-regulated genes shared among the NVF, MVT, and HVT groups and detected significant down-regulation of primary metabolism-related genes and oxidoreductase. In viruliferous WBPH with viral titer-specific transcriptome changes, 1,906 and 1,467 genes exhibited strict monotonically increasing and decreasing expression, respectively. The RNAi pathway was the major antiviral response to increasing viral titers among diverse immune responses. These results clarify the responses of immune genes and the transcriptome of WBPH to SRBSDV and improve our knowledge of the functional relationship between pathogen, vector, and host.

Mild and severe cereal yellow dwarf viruses differ in silencing suppressor efficiency of the P0 protein.

PubMed

Almasi, Reza; Miller, W Allen; Ziegler-Graff, Véronique

2015-10-02

Viral pathogenicity has often been correlated to the expression of the viral encoded-RNA silencing suppressor protein (SSP). The silencing suppressor activity of the P0 protein encoded by cereal yellow dwarf virus-RPV (CYDV-RPV) and -RPS (CYDV-RPS), two poleroviruses differing in their symptomatology was investigated. CYDV-RPV displays milder symptoms in oat and wheat whereas CYDV-RPS is responsible for more severe disease. We showed that both P0 proteins (P0(CY-RPV) and P0(CY-RPS)) were able to suppress local RNA silencing induced by either sense or inverted repeat transgenes in an Agrobacterium tumefaciens-mediated expression assay in Nicotiana benthamiana. P0(CY-RPS) displayed slightly higher activity. Systemic spread of the silencing signal was not impaired. Analysis of short-interfering RNA (siRNA) abundance revealed that accumulation of primary siRNA was not affected, but secondary siRNA levels were reduced by both CYDV P0 proteins, suggesting that they act downstream of siRNA production. Correlated with this finding we showed that both P0 proteins partially destabilized ARGONAUTE1. Finally both P0(CY-RPV) and P0(CY-RPS) interacted in yeast cells with ASK2, a component of an E3-ubiquitin ligase, with distinct affinities. Copyright © 2015 Elsevier B.V. All rights reserved.

H1PVAT is a novel and potent early-stage inhibitor of poliovirus replication that targets VP1.

PubMed

Tijsma, Aloys; Thibaut, Hendrik Jan; Spieser, Stéphane A H; De Palma, Armando; Koukni, Mohamed; Rhoden, Eric; Oberste, Steve; Pürstinger, Gerhard; Volny-Luraghi, Antonia; Martin, Javier; Marchand, Arnaud; Chaltin, Patrick; Neyts, Johan; Leyssen, Pieter

2014-10-01

A novel small molecule, H1PVAT, was identified as a potent and selective inhibitor of the in vitro replication of all three poliovirus serotypes, whereas no activity was observed against other enteroviruses. Time-of-drug-addition studies revealed that the compound interfered with an early stage of virus replication. Four independently-selected H1PVAT-resistant virus variants uniformly carried the single amino acid substitution I194F in the VP1 capsid protein. Poliovirus type 1 strain Sabin, reverse-engineered to contain this substitution, proved to be completely insensitive to the antiviral effect of H1PVAT and was cross-resistant to the capsid-binding inhibitors V-073 and pirodavir. The VP1 I194F mutant had a smaller plaque phenotype than wild-type virus, and the amino acid substitution rendered the virus more susceptible to heat inactivation. Both for the wild-type and VP1 I194F mutant virus, the presence of H1PVAT increased the temperature at which the virus was inactivated, providing evidence that the compound interacts with the viral capsid, and that capsid stabilization and antiviral activity are not necessarily correlated. Molecular modeling suggested that H1PVAT binds with high affinity in the pocket underneath the floor of the canyon that is involved in receptor binding. Introduction of the I194F substitution in the model of VP1 induced a slight concerted rearrangement of the core β-barrel in this pocket, which disfavors binding of the compound. Taken together, the compound scaffold, to which H1PVAT belongs, may represent another promising class of poliovirus capsid-binding inhibitors next to V-073 and pirodavir. Potent antivirals against poliovirus will be essential in the poliovirus eradication end-game. Copyright © 2014. Published by Elsevier B.V.

Bile Salt-Stimulated Lipase from Human Milk Binds DC-SIGN and Inhibits Human Immunodeficiency Virus Type 1 Transfer to CD4+ T Cells

PubMed Central

Naarding, Marloes A.; Dirac, Annette M.; Ludwig, Irene S.; Speijer, Dave; Lindquist, Susanne; Vestman, Eva-Lotta; Stax, Martijn J.; Geijtenbeek, Teunis B. H.; Pollakis, Georgios; Hernell, Olle; Paxton, William A.

2006-01-01

A wide range of pathogens, including human immunodeficiency virus type 1 (HIV-1), hepatitis C virus, Ebola virus, cytomegalovirus, dengue virus, Mycobacterium, Leishmania, and Helicobacter pylori, can interact with dendritic cell (DC)-specific ICAM3-grabbing nonintegrin (DC-SIGN), expressed on DCs and a subset of B cells. More specifically, the interaction of the gp120 envelope protein of HIV-1 with DC-SIGN can facilitate the transfer of virus to CD4+ T lymphocytes in trans and enhance infection. We have previously demonstrated that a multimeric LeX component in human milk binds to DC-SIGN, preventing HIV-1 from interacting with this receptor. Biochemical analysis reveals that the compound is heat resistant, trypsin sensitive, and larger than 100 kDa, indicating a specific glycoprotein as the inhibitory compound. By testing human milk from three different mothers, we found the levels of DC-SIGN binding and viral inhibition to vary between samples. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and matrix-assisted laser desorption ionization analysis, we identified bile salt-stimulated lipase (BSSL), a Lewis X (LeX)-containing glycoprotein found in human milk, to be the major variant protein between the samples. BSSL isolated from human milk bound to DC-SIGN and inhibited the transfer of HIV-1 to CD4+ T lymphocytes. Two BSSL isoforms isolated from the same human milk sample showed differences in DC-SIGN binding, illustrating that alterations in the BSSL forms explain the differences observed. These results indicate that variations in BSSL lead to alterations in LeX expression by the protein, which subsequently alters the DC-SIGN binding capacity and the inhibitory effect on HIV-1 transfer. Identifying the specific molecular interaction between the different forms may aid in the future design of antimicrobial agents. PMID:17005819

Which Plant Proteins Are Involved in Antiviral Defense? Review on In Vivo and In Vitro Activities of Selected Plant Proteins against Viruses.

PubMed

Musidlak, Oskar; Nawrot, Robert; Goździcka-Józefiak, Anna

2017-11-01

Plants have evolved a variety of defense mechanisms to tackle virus attack. Endogenous plant proteins can function as virus suppressors. Different types of proteins mediate defense responses against plant viruses. Pathogenesis-related (PR) proteins are activated upon pathogen infections or in different stress situations and their production is one of many components in plant defense. Ribosome-inactivating proteins (RIPs) suppress translation by enzymatically damaging ribosomes and they have been found to have antiviral activity. RNA-binding proteins (RBPs) bind to target RNAs via specialized RNA-binding domain and can directly or indirectly function in plant defense system against RNA viruses. Proteins involved in silencing machinery, namely Dicer-like (DCL) proteins, Argonaute (AGO) proteins, and RNA-dependent RNA polymerases (RDRs) confer innate antiviral defense in plants as they are able to degrade foreign RNA of viral origin. This review aims to provide a comprehensive and up-to-date picture of plant proteins participating in antiviral defense. As a result we discuss proteins conferring plant antiviral resistance and their potential future applications in different fields of life including agriculture and medicine.

Which Plant Proteins Are Involved in Antiviral Defense? Review on In Vivo and In Vitro Activities of Selected Plant Proteins against Viruses

PubMed Central

Goździcka-Józefiak, Anna

2017-01-01

Plants have evolved a variety of defense mechanisms to tackle virus attack. Endogenous plant proteins can function as virus suppressors. Different types of proteins mediate defense responses against plant viruses. Pathogenesis-related (PR) proteins are activated upon pathogen infections or in different stress situations and their production is one of many components in plant defense. Ribosome-inactivating proteins (RIPs) suppress translation by enzymatically damaging ribosomes and they have been found to have antiviral activity. RNA-binding proteins (RBPs) bind to target RNAs via specialized RNA-binding domain and can directly or indirectly function in plant defense system against RNA viruses. Proteins involved in silencing machinery, namely Dicer-like (DCL) proteins, Argonaute (AGO) proteins, and RNA-dependent RNA polymerases (RDRs) confer innate antiviral defense in plants as they are able to degrade foreign RNA of viral origin. This review aims to provide a comprehensive and up-to-date picture of plant proteins participating in antiviral defense. As a result we discuss proteins conferring plant antiviral resistance and their potential future applications in different fields of life including agriculture and medicine. PMID:29104238

Shotgun glycomics of pig lung identifies natural endogenous receptors for influenza viruses.

PubMed

Byrd-Leotis, Lauren; Liu, Renpeng; Bradley, Konrad C; Lasanajak, Yi; Cummings, Sandra F; Song, Xuezheng; Heimburg-Molinaro, Jamie; Galloway, Summer E; Culhane, Marie R; Smith, David F; Steinhauer, David A; Cummings, Richard D

2014-06-03

Influenza viruses bind to host cell surface glycans containing terminal sialic acids, but as studies on influenza binding become more sophisticated, it is becoming evident that although sialic acid may be necessary, it is not sufficient for productive binding. To better define endogenous glycans that serve as viral receptors, we have explored glycan recognition in the pig lung, because influenza is broadly disseminated in swine, and swine have been postulated as an intermediary host for the emergence of pandemic strains. For these studies, we used the technology of "shotgun glycomics" to identify natural receptor glycans. The total released N- and O-glycans from pig lung glycoproteins and glycolipid-derived glycans were fluorescently tagged and separated by multidimensional HPLC, and individual glycans were covalently printed to generate pig lung shotgun glycan microarrays. All viruses tested interacted with one or more sialylated N-glycans but not O-glycans or glycolipid-derived glycans, and each virus demonstrated novel and unexpected differences in endogenous N-glycan recognition. The results illustrate the repertoire of specific, endogenous N-glycans of pig lung glycoproteins for virus recognition and offer a new direction for studying endogenous glycan functions in viral pathogenesis.

Identification and Characterization of the UL37 Protein of Herpes Simplex Virus Type 1 and Demonstration that it Interacts with ICP8, the Major DNA Binding Protein of Herpes Simplex Virus

DTIC Science & Technology

1992-10-20

Identification of ORFs HSV DNA binding proteins • 1 3 3 5 7 7 11 17 18 22 reps and its role in HSV replication 23 Biochemical properties . . 23...Figure 1 . 2. 3 • 4. 5. 6. 7. 8. Structural model of the herpesvirus virion Schematic diagram of HSV pathogenesis . Diagram of the main...vaccinia virus- 13. Autoradiogram of an immunoblot of HSV - 1 -infected cell proteins harvested at various times postinfec- 85 tioD probed with anti-UL42

The RNA- and TRIM25-Binding Domains of Influenza Virus NS1 Protein Are Essential for Suppression of NLRP3 Inflammasome-Mediated Interleukin-1β Secretion.

PubMed

Moriyama, Miyu; Chen, I-Yin; Kawaguchi, Atsushi; Koshiba, Takumi; Nagata, Kyosuke; Takeyama, Haruko; Hasegawa, Hideki; Ichinohe, Takeshi

2016-04-01

Inflammasomes are cytosolic multimolecular protein complexes that stimulate the activation of caspase-1 and the release of mature forms of interleukin-1β (IL-1β) and IL-18. We previously demonstrated that the influenza A virus M2 protein stimulates IL-1β secretion following activation of the nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome. The nonstructural protein 1 (NS1) of influenza virus inhibits caspase-1 activation and IL-1β secretion. However, the precise mechanism by which NS1 inhibits IL-1β secretion remains unknown. Here, we showed that J774A.1 macrophages stably expressing the NS1 protein inhibited IL-1β secretion after infection with recombinant influenza virus lacking the NS1 gene. Coimmunoprecipitation assay revealed that the NS1 protein interacts with NLRP3. Importantly, the NS1 protein inhibited the NLRP3/ASC-induced single-speck formation required for full activation of inflammasomes. The NS1 protein of other influenza virus strains, including a recent pandemic strain, also inhibited inflammasome-mediated IL-1β secretion. The NS1 RNA-binding domain (basic residues 38 and 41) and TRIM25-binding domain (acidic residues 96 and 97) were required for suppression of NLRP3 inflammasome-mediated IL-1β secretion. These results shed light on a mechanism by which the NS1 protein of influenza virus suppresses NLRP3 inflammasome-mediated IL-1β secretion. Innate immune sensing of influenza virus via pattern recognition receptors not only plays a key role in generating type I interferons but also triggers inflammatory responses. We previously demonstrated that the influenza A virus M2 protein activates the NLRP3 inflammasome, leading to the secretion of interleukin-1β (IL-1β) and IL-18 following the activation of caspase-1. Although the nonstructural protein 1 (NS1) of influenza virus inhibits IL-1β secretion, the precise mechanism by which it achieves this remains to be defined. Here, we demonstrate that the NS1 protein interacts with NLRP3 to suppress NLRP3 inflammasome activation. J774A.1 macrophages stably expressing the NS1 protein suppressed NLRP3-mediated IL-1β secretion. The NS1 RNA-binding domain (basic residues 38 and 41) and TRIM25-binding domain (acidic residues 96 and 97) are important for suppression of NLRP3 inflammasome-mediated IL-1β secretion. These results will facilitate the development of new anti-inflammatory drugs. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

The RNA- and TRIM25-Binding Domains of Influenza Virus NS1 Protein Are Essential for Suppression of NLRP3 Inflammasome-Mediated Interleukin-1β Secretion

PubMed Central

Moriyama, Miyu; Chen, I-Yin; Kawaguchi, Atsushi; Koshiba, Takumi; Nagata, Kyosuke; Takeyama, Haruko; Hasegawa, Hideki

2016-01-01

ABSTRACT Inflammasomes are cytosolic multimolecular protein complexes that stimulate the activation of caspase-1 and the release of mature forms of interleukin-1β (IL-1β) and IL-18. We previously demonstrated that the influenza A virus M2 protein stimulates IL-1β secretion following activation of the nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome. The nonstructural protein 1 (NS1) of influenza virus inhibits caspase-1 activation and IL-1β secretion. However, the precise mechanism by which NS1 inhibits IL-1β secretion remains unknown. Here, we showed that J774A.1 macrophages stably expressing the NS1 protein inhibited IL-1β secretion after infection with recombinant influenza virus lacking the NS1 gene. Coimmunoprecipitation assay revealed that the NS1 protein interacts with NLRP3. Importantly, the NS1 protein inhibited the NLRP3/ASC-induced single-speck formation required for full activation of inflammasomes. The NS1 protein of other influenza virus strains, including a recent pandemic strain, also inhibited inflammasome-mediated IL-1β secretion. The NS1 RNA-binding domain (basic residues 38 and 41) and TRIM25-binding domain (acidic residues 96 and 97) were required for suppression of NLRP3 inflammasome-mediated IL-1β secretion. These results shed light on a mechanism by which the NS1 protein of influenza virus suppresses NLRP3 inflammasome-mediated IL-1β secretion. IMPORTANCE Innate immune sensing of influenza virus via pattern recognition receptors not only plays a key role in generating type I interferons but also triggers inflammatory responses. We previously demonstrated that the influenza A virus M2 protein activates the NLRP3 inflammasome, leading to the secretion of interleukin-1β (IL-1β) and IL-18 following the activation of caspase-1. Although the nonstructural protein 1 (NS1) of influenza virus inhibits IL-1β secretion, the precise mechanism by which it achieves this remains to be defined. Here, we demonstrate that the NS1 protein interacts with NLRP3 to suppress NLRP3 inflammasome activation. J774A.1 macrophages stably expressing the NS1 protein suppressed NLRP3-mediated IL-1β secretion. The NS1 RNA-binding domain (basic residues 38 and 41) and TRIM25-binding domain (acidic residues 96 and 97) are important for suppression of NLRP3 inflammasome-mediated IL-1β secretion. These results will facilitate the development of new anti-inflammatory drugs. PMID:26865721

Envelope conformational changes induced by human immunodeficiency virus type 1 attachment inhibitors prevent CD4 binding and downstream entry events.

PubMed

Ho, Hsu-Tso; Fan, Li; Nowicka-Sans, Beata; McAuliffe, Brian; Li, Chang-Ben; Yamanaka, Gregory; Zhou, Nannan; Fang, Hua; Dicker, Ira; Dalterio, Richard; Gong, Yi-Fei; Wang, Tao; Yin, Zhiwei; Ueda, Yasutsugu; Matiskella, John; Kadow, John; Clapham, Paul; Robinson, James; Colonno, Richard; Lin, Pin-Fang

2006-04-01

BMS-488043 is a small-molecule human immunodeficiency virus type 1 (HIV-1) CD4 attachment inhibitor with demonstrated clinical efficacy. The compound inhibits soluble CD4 (sCD4) binding to the 11 distinct HIV envelope gp120 proteins surveyed. Binding of BMS-488043 and that of sCD4 to gp120 are mutually exclusive, since increased concentrations of one can completely block the binding of the other without affecting the maximal gp120 binding capacity. Similarly, BMS-488043 inhibited virion envelope trimers from binding to sCD4-immunoglobulin G (IgG), with decreasing inhibition as the sCD4-IgG concentration increased, and BMS-488043 blocked the sCD4-induced exposure of the gp41 groove in virions. In both virion binding assays, BMS-488043 was active only when added prior to sCD4. Collectively, these results indicate that obstruction of gp120-sCD4 interactions is the primary inhibition mechanism of this compound and that compound interaction with envelope must precede CD4 binding. By three independent approaches, BMS-488043 was further shown to induce conformational changes within gp120 in both the CD4 and CCR5 binding regions. These changes likely prevent gp120-CD4 interactions and downstream entry events. However, BMS-488043 could only partially inhibit CD4 binding to an HIV variant containing a specific envelope truncation and altered gp120 conformation, despite effectively inhibiting the pseudotyped virus infection. Taken together, BMS-488043 inhibits viral entry primarily through altering the envelope conformation and preventing CD4 binding, and other downstream entry events could also be inhibited as a result of these induced conformational changes.

The Globoside Receptor Triggers Structural Changes in the B19 Virus Capsid That Facilitate Virus Internalization▿

PubMed Central

Bönsch, Claudia; Zuercher, Christoph; Lieby, Patricia; Kempf, Christoph; Ros, Carlos

2010-01-01

Globoside (Gb4Cer), Ku80 autoantigen, and α5β1 integrin have been identified as cell receptors/coreceptors for human parvovirus B19 (B19V), but their role and mechanism of interaction with the virus are largely unknown. In UT7/Epo cells, expression of Gb4Cer and CD49e (integrin alpha-5) was high, but expression of Ku80 was insignificant. B19V colocalized with Gb4Cer and, to a lesser extent, with CD49e. However, only anti-Gb4Cer antibodies could disturb virus attachment. Only a small proportion of cell-bound viruses were internalized, while the majority became detached from the receptor. When added to uninfected cells, the receptor-detached virus showed superior cell binding capacity and infectivity. Attachment of B19V to cells triggered conformational changes in the capsid leading to the accessibility of the N terminus of VP1 (VP1u) to antibodies, which was maintained in the receptor-detached virus. VP1u became similarly accessible to antibodies following incubation of B19V particles with increasing concentrations of purified Gb4Cer. The receptor-mediated exposure of VP1u is critical for virus internalization, since capsids lacking VP1 could bind to cells but were not internalized. Moreover, an antibody against the N terminus of VP1u disturbed virus internalization, but only when present during and not after virus attachment, indicating the involvement of this region in binding events required for internalization. These results suggest that Gb4Cer is not only the primary receptor for B19V attachment but also the mediator of capsid rearrangements required for subsequent interactions leading to virus internalization. The capacity of the virus to detach and reattach again would enhance the probability of productive infections. PMID:20826697

Cleavage of influenza RNA by using a human PUF-based artificial RNA-binding protein–staphylococcal nuclease hybrid

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

Mori, Tomoaki; Nakamura, Kento; Masaoka, Keisuke

Various viruses infect animals and humans and cause a variety of diseases, including cancer. However, effective methodologies to prevent virus infection have not yet been established. Therefore, development of technologies to inactivate viruses is highly desired. We have already demonstrated that cleavage of a DNA virus genome was effective to prevent its replication. Here, we expanded this methodology to RNA viruses. In the present study, we used staphylococcal nuclease (SNase) instead of the PIN domain (PilT N-terminus) of human SMG6 as an RNA-cleavage domain and fused the SNase to a human Pumilio/fem-3 binding factor (PUF)-based artificial RNA-binding protein to constructmore » an artificial RNA restriction enzyme with enhanced RNA-cleavage rates for influenzavirus. The resulting SNase-fusion nuclease cleaved influenza RNA at rates 120-fold greater than the corresponding PIN-fusion nuclease. The cleaving ability of the PIN-fusion nuclease was not improved even though the linker moiety between the PUF and RNA-cleavage domain was changed. Gel shift assays revealed that the RNA-binding properties of the PUF derivative used was not as good as wild type PUF. Improvement of the binding properties or the design method will allow the SNase-fusion nuclease to cleave an RNA target in mammalian animal cells and/or organisms. - Highlights: • A novel RNA restriction enzyme using SNase was developed tor cleave viral RNA. • Our enzyme cleaved influenza RNA with rates >120-fold higher rates a PIN-fusion one. • Our artificial enzyme with the L5 linker showed the highest RNA cleavage rate. • Our artificial enzyme site-selectively cleaved influenza RNA in vitro.« less

The Enamovirus P0 protein is a silencing suppressor which inhibits local and systemic RNA silencing through AGO1 degradation

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

Fusaro, Adriana F.; CSIRO Plant Industry, Canberra, P.O. Box 1600, ACT 2601; Correa, Regis L.

The P0 protein of poleroviruses and P1 protein of sobemoviruses suppress the plant's RNA silencing machinery. Here we identified a silencing suppressor protein (SSP), P0{sup PE}, in the Enamovirus Pea enation mosaic virus-1 (PEMV-1) and showed that it and the P0s of poleroviruses Potato leaf roll virus and Cereal yellow dwarf virus have strong local and systemic SSP activity, while the P1 of Sobemovirus Southern bean mosaic virus supresses systemic silencing. The nuclear localized P0{sup PE} has no discernable sequence conservation with known SSPs, but proved to be a strong suppressor of local silencing and a moderate suppressor of systemicmore » silencing. Like the P0s from poleroviruses, P0{sup PE} destabilizes AGO1 and this action is mediated by an F-box-like domain. Therefore, despite the lack of any sequence similarity, the poleroviral and enamoviral SSPs have a conserved mode of action upon the RNA silencing machinery.« less

Structures of minute virus of mice replication initiator protein N-terminal domain: Insights into DNA nicking and origin binding

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

Tewary, Sunil K.; Liang, Lingfei; Lin, Zihan

Members of the Parvoviridae family all encode a non-structural protein 1 (NS1) that directs replication of single-stranded viral DNA, packages viral DNA into capsid, and serves as a potent transcriptional activator. Here we report the X-ray structure of the minute virus of mice (MVM) NS1 N-terminal domain at 1.45 Å resolution, showing that sites for dsDNA binding, ssDNA binding and cleavage, nuclear localization, and other functions are integrated on a canonical fold of the histidine-hydrophobic-histidine superfamily of nucleases, including elements specific for this Protoparvovirus but distinct from its Bocaparvovirus or Dependoparvovirus orthologs. High resolution structural analysis reveals a nickase activemore » site with an architecture that allows highly versatile metal ligand binding. The structures support a unified mechanism of replication origin recognition for homotelomeric and heterotelomeric parvoviruses, mediated by a basic-residue-rich hairpin and an adjacent helix in the initiator proteins and by tandem tetranucleotide motifs in the replication origins. - Highlights: • The structure of a parvovirus replication initiator protein has been determined; • The structure sheds light on mechanisms of ssDNA binding and cleavage; • The nickase active site is preconfigured for versatile metal ligand binding; • The binding site for the double-stranded replication origin DNA is identified; • A single domain integrates multiple functions in virus replication.« less

Quantitative Comparison of Human Parainfluenza Virus Hemagglutinin-Neuraminidase Receptor Binding and Receptor Cleavage

PubMed Central

Tappert, Mary M.; Porterfield, J. Zachary; Mehta-D'Souza, Padmaja; Gulati, Shelly

2013-01-01

The human parainfluenza virus (hPIV) hemagglutinin-neuraminidase (HN) protein binds (H) oligosaccharide receptors that contain N-acetylneuraminic acid (Neu5Ac) and cleaves (N) Neu5Ac from these oligosaccharides. In order to determine if one of HN′s two functions is predominant, we measured the affinity of H for its ligands by a solid-phase binding assay with two glycoprotein substrates and by surface plasmon resonance with three monovalent glycans. We compared the dissociation constant (Kd) values from these experiments with previously determined Michaelis-Menten constants (Kms) for the enzyme activity. We found that glycoprotein substrates and monovalent glycans containing Neu5Acα2-3Galβ1-4GlcNAc bind HN with Kd values in the 10 to 100 μM range. Km values for HN were previously determined to be on the order of 1 mM (M. M. Tappert, D. F. Smith, and G. M. Air, J. Virol. 85:12146–12159, 2011). A Km value greater than the Kd value indicates that cleavage occurs faster than the dissociation of binding and will dominate under N-permissive conditions. We propose, therefore, that HN is a neuraminidase that can hold its substrate long enough to act as a binding protein. The N activity can therefore regulate binding by reducing virus-receptor interactions when the concentration of receptor is high. PMID:23740997

Replacement of the V3 domain in the surface subunit of the feline immunodeficiency virus envelope glycoprotein with the equivalent region of a T cell-tropic human immunodeficiency virus type 1 results in a chimeric surface protein that efficiently binds to CXCR4.

PubMed

González, Silvia A; Falcón, Juan I; Affranchino, José L

2014-03-01

Feline immunodeficiency virus (FIV) and the T cell-tropic strains of human immunodeficiency virus type 1 (HIV-1) share the use of the chemokine receptor CXCR4 for cell entry. To study this process further we developed a cell surface binding assay based on the expression of a soluble version of the FIV SU C-terminally tagged with the influenza virus hemagglutinin epitope (HA). The specificity of the assay was demonstrated by the following evidence: (1) the SU-HA protein bound to HeLa cells that express CXCR4 but not to MDCK cells that lack this chemokine receptor; and (2) binding of the SU-HA to HeLa cells was blocked by incubation with the CXCR4 antagonist AMD3100 as well as with the anti-CXCR4 monoclonal antibody (MAb) 12G5. Deletion of the V3 region from the FIV SU glycoprotein abolished its ability to bind CXCR4-expressing cells. Remarkably, substitution of the V3 domain of the FIV SU by the equivalent region of the HIV-1 NL4-3 isolate resulted in efficient cell surface binding of the chimeric SU protein to CXCR4. Moreover, transfection of MDCK cells with a plasmid encoding human CXCR4 allowed the association of the chimeric SU-HA glycoprotein to the transfected cells. Interestingly, while cell binding of the chimeric FIV-HIV SU was inhibited by an anti-HIV-1 V3 MAb, its association with CXCR4 was found to be resistant to AMD3100. Of note, the chimeric FIV-HIV Env glycoprotein was capable of promoting CXCR4-dependent cell-to-cell fusion.

Free energy simulations reveal a double mutant avian H5N1 virus hemagglutinin with altered receptor binding specificity.

PubMed

Das, Payel; Li, Jingyuan; Royyuru, Ajay K; Zhou, Ruhong

2009-08-01

Historically, influenza pandemics have been triggered when an avian influenza virus or a human/avian reassorted virus acquires the ability to replicate efficiently and become transmissible in the human population. Most critically, the major surface glycoprotein hemagglutinin (HA) must adapt to the usage of human-like (alpha-2,6-linked) sialylated glycan receptors. Therefore, identification of mutations that can switch the currently circulating H5N1 HA receptor binding specificity from avian to human might provide leads to the emergence of pandemic H5N1 viruses. To define such mutations in the H5 subtype, here we provide a computational framework that combines molecular modeling with extensive free energy simulations. Our results show that the simulated binding affinities are in good agreement with currently available experimental data. Moreover, we predict that one double mutation (V135S and A138S) in HA significantly enhances alpha-2,6-linked receptor recognition by the H5 subtype. Our simulations indicate that this double mutation in H5N1 HA increases the binding affinity to alpha-2,6-linked sialic acid receptors by 2.6 +/- 0.7 kcal/mol per HA monomer that primarily arises from the electrostatic interactions. Further analyses reveal that introduction of this double mutation results in a conformational change in the receptor binding pocket of H5N1 HA. As a result, a major rearrangement occurs in the hydrogen-bonding network of HA with the human receptor, making the human receptor binding pattern of double mutant H5N1 HA surprisingly similar to that observed in human H1N1 HA. These large scale molecular simulations on single and double mutants thus provide new insights into our understanding toward human adaptation of the avian H5N1 virus. 2009 Wiley Periodicals, Inc.

Characterization of two mosquito STATs, AaSTAT and CtSTAT. Differential regulation of tyrosine phosphorylation and DNA binding activity by lipopolysaccharide treatment and by Japanese encephalitis virus infection.

PubMed

Lin, Chang-Chi; Chou, Chih-Ming; Hsu, Ya-Li; Lien, Jih-Ching; Wang, Yu-Ming; Chen, Shui-Tsung; Tsai, Shu-Chuan; Hsiao, Pei-Wen; Huang, Chang-Jen

2004-01-30

Two mosquito STATs, AaSTAT and CtSTAT, have been cloned from Aedes albopictus and Culex tritaeniorhynchus mosquitoes, respectively. These two STATs are more similar to those of Drosophila, Anopheles, and mammalian STAT5 in the DNA binding and Src homology 2 domains. The mRNA transcripts are expressed at all developmental stages, and the proteins are present predominantly at the pupal and adult stages in both mosquitoes. Stimulation with lipopolysaccharide resulted in an increase of tyrosine phosphorylation and DNA binding activity of AaSTAT and CtSTAT as well as an increase of luciferase activity of a reporter gene containing Drosophila STAT binding motif in mosquito C6/36 cells. After being infected with Japanese encephalitis virus, nuclear extracts of C6/36 cells revealed a decrease of tyrosine phosphorylation and DNA binding activity of AaSTAT which could be restored by sodium orthovanadate treatment. Taking all of the data together, this is the first report to clone and characterize two mosquito STATs with 81% identity and to demonstrate a different response of tyrosine phosphorylation and DNA binding of these two STATs by lipopolysaccharide treatment and by Japanese encephalitis virus infection.

Mutations in CypA Binding Region of HIV-1 Capsid Affect Capsid Stability and Viral Replication in Primary Macrophages.

PubMed

Setiawan, Laurentia C; van Dort, Karel A; Rits, Maarten A N; Kootstra, Neeltje A

2016-04-01

Mutations in the cyclophilin A (CypA) binding region in the HIV-1 capsid affect their dependency on the known HIV-1 cofactor CypA and allow escape from the HIV-1 restriction factor Trim5α in human and simian cells. Here we study the effect of these mutations in the CypA binding region of capsid on cofactor binding, capsid destabilization, and viral replication in primary cells. We showed that the viral capsid with mutations in the CypA binding region (CypA-BR) interacted efficiently with CypA, but had an increased stability upon infection as compared to the wild-type capsid. Interestingly, the wild-type virus was able to infect monocyte-derived macrophages (MDM) more efficiently as compared to the CypA-BR mutant variant. The lower infectivity of the CypA-BR mutant virus in MDM was associated with lower levels of reverse transcription products. Similar to the wild-type virus, the CypA-BR mutant variant was unable to induce a strong innate response in primary macrophages. These data demonstrate that mutations in the CypA binding site of the capsid resulted in higher capsid stability and hampered infectivity in macrophages.

NMR study of complexes between low molecular mass inhibitors and the West Nile virus NS2B-NS3 protease.

PubMed

Su, Xun-Cheng; Ozawa, Kiyoshi; Yagi, Hiromasa; Lim, Siew P; Wen, Daying; Ekonomiuk, Dariusz; Huang, Danzhi; Keller, Thomas H; Sonntag, Sebastian; Caflisch, Amedeo; Vasudevan, Subhash G; Otting, Gottfried

2009-08-01

The two-component NS2B-NS3 protease of West Nile virus is essential for its replication and presents an attractive target for drug development. Here, we describe protocols for the high-yield expression of stable isotope-labelled samples in vivo and in vitro. We also describe the use of NMR spectroscopy to determine the binding mode of new low molecular mass inhibitors of the West Nile virus NS2B-NS3 protease which were discovered using high-throughput in vitro screening. Binding to the substrate-binding sites S1 and S3 is confirmed by intermolecular NOEs and comparison with the binding mode of a previously identified low molecular mass inhibitor. Our results show that all these inhibitors act by occupying the substrate-binding site of the protease rather than by an allosteric mechanism. In addition, the NS2B polypeptide chain was found to be positioned near the substrate-binding site, as observed previously in crystal structures of the protease in complex with peptide inhibitors or bovine pancreatic trypsin inhibitor. This indicates that the new low molecular mass compounds, although inhibiting the protease, also promote the proteolytically active conformation of NS2B, which is very different from the crystal structure of the protein without inhibitor.

Structure-based design of novel naproxen derivatives targeting monomeric nucleoprotein of Influenza A virus

PubMed Central

Tarus, Bogdan; Bertrand, Hélène; Zedda, Gloria; Di Primo, Carmelo; Quideau, Stéphane; Slama-Schwok, Anny

2015-01-01

The nucleoprotein (NP) binds the viral RNA genome as oligomers assembled with the polymerase in a ribonucleoprotein complex required for transcription and replication of influenza A virus. Novel antiviral candidates targeting the nucleoprotein either induced higher order oligomers or reduced NP oligomerization by targeting the oligomerization loop and blocking its insertion into adjacent nucleoprotein subunit. In this study, we used a different structure-based approach to stabilize monomers of the nucleoprotein by drugs binding in its RNA-binding groove. We recently identified naproxen as a drug competing with RNA binding to NP with antiinflammatory and antiviral effects against influenza A virus. Here, we designed novel derivatives of naproxen by fragment extension for improved binding to NP. Molecular dynamics simulations suggested that among these derivatives, naproxen A and C0 were most promising. Their chemical synthesis is described. Both derivatives markedly stabilized NP monomer against thermal denaturation. Naproxen C0 bound tighter to NP than naproxen at a binding site predicted by MD simulations and shown by competition experiments using wt NP or single-point mutants as determined by surface plasmon resonance. MD simulations suggested that impeded oligomerization and stabilization of monomeric NP is likely to be achieved by drugs binding in the RNA grove and inducing close to their binding site conformational changes of key residues hosting the oligomerization loop as observed for the naproxen derivatives. Naproxen C0 is a potential antiviral candidate blocking influenza nucleoprotein function. PMID:25333630

Structure-based design of novel naproxen derivatives targeting monomeric nucleoprotein of Influenza A virus.

PubMed

Tarus, Bogdan; Bertrand, Hélène; Zedda, Gloria; Di Primo, Carmelo; Quideau, Stéphane; Slama-Schwok, Anny

2015-09-01

The nucleoprotein (NP) binds the viral RNA genome as oligomers assembled with the polymerase in a ribonucleoprotein complex required for transcription and replication of influenza A virus. Novel antiviral candidates targeting the nucleoprotein either induced higher order oligomers or reduced NP oligomerization by targeting the oligomerization loop and blocking its insertion into adjacent nucleoprotein subunit. In this study, we used a different structure-based approach to stabilize monomers of the nucleoprotein by drugs binding in its RNA-binding groove. We recently identified naproxen as a drug competing with RNA binding to NP with antiinflammatory and antiviral effects against influenza A virus. Here, we designed novel derivatives of naproxen by fragment extension for improved binding to NP. Molecular dynamics simulations suggested that among these derivatives, naproxen A and C0 were most promising. Their chemical synthesis is described. Both derivatives markedly stabilized NP monomer against thermal denaturation. Naproxen C0 bound tighter to NP than naproxen at a binding site predicted by MD simulations and shown by competition experiments using wt NP or single-point mutants as determined by surface plasmon resonance. MD simulations suggested that impeded oligomerization and stabilization of monomeric NP is likely to be achieved by drugs binding in the RNA grove and inducing close to their binding site conformational changes of key residues hosting the oligomerization loop as observed for the naproxen derivatives. Naproxen C0 is a potential antiviral candidate blocking influenza nucleoprotein function.

Identification of Novel Fusion Inhibitors of Influenza A Virus by Chemical Genetics

PubMed Central

Lai, Kin Kui; Cheung, Nam Nam; Yang, Fang; Dai, Jun; Liu, Li; Chen, Zhiwei; Sze, Kong Hung; Chen, Honglin

2015-01-01

ABSTRACT A previous screening of more than 50,000 compounds led to the identification of a pool of bioactive small molecules with inhibitory effect on the influenza A virus. One of these compounds, now widely known as nucleozin, is a small molecule that targets the influenza A virus nucleoprotein. Here we identify and characterize two structurally different novel fusion inhibitors of the influenza A virus group 1 hemagglutinin (HA), FA-583 and FA-617, with low nanomolar activities. Escape mutants that are highly resistant to each of these compounds were generated, and both were found to carry mutations localized in close proximity to the B-loop of the hemagglutinin 2 protein, which plays a crucial role in the virion-host cell fusion process. Recombinant virus, generated through reverse genetics, confirmed the resistance phenotype. In addition, the proposed binding pockets predicted by molecular docking studies are in accordance with the resistance-bearing mutation sites. We show through mechanistic studies that FA-583 and FA-617 act as fusion inhibitors by prohibiting the low-pH-induced conformational change of hemagglutinin. Our study has offered concrete biological and mechanistic explorations for the strategic development of novel fusion inhibitors of influenza A viruses. IMPORTANCE Here we report two structurally distinctive novel fusion inhibitors of influenza A virus that act by interfering with the structural change of HA at acidic pH, a process necessary for successful entry of the virus. Mutational and molecular docking studies have identified their binding pockets situated in close proximity to the B-loop region of hemagglutinin 2. The reduced sensitivity of FA-583- or FA-617-associated mutants to another compound suggests a close proximity and even partial overlap of their binding sites on hemagglutinin. Amino acid sequence alignments and crystal structure analyses of group 1 and group 2 hemagglutinins have shed light on the possible binding mode of these two compounds. This report offers new lead compounds for the design of fusion inhibitors for influenza A viruses and further shows that analysis by forward chemical genetics is a highly effective approach for the identification of novel compounds that can perturb the infectivity of viruses and to probe new druggable targets or druggable domains in various viruses. PMID:26676787

Sequential Infection in Ferrets with Antigenically Distinct Seasonal H1N1 Influenza Viruses Boosts Hemagglutinin Stalk-Specific Antibodies

PubMed Central

Kirchenbaum, Greg A.; Carter, Donald M.

2015-01-01

ABSTRACT Broadly reactive antibodies targeting the conserved hemagglutinin (HA) stalk region are elicited following sequential infection or vaccination with influenza viruses belonging to divergent subtypes and/or expressing antigenically distinct HA globular head domains. Here, we demonstrate, through the use of novel chimeric HA proteins and competitive binding assays, that sequential infection of ferrets with antigenically distinct seasonal H1N1 (sH1N1) influenza virus isolates induced an HA stalk-specific antibody response. Additionally, stalk-specific antibody titers were boosted following sequential infection with antigenically distinct sH1N1 isolates in spite of preexisting, cross-reactive, HA-specific antibody titers. Despite a decline in stalk-specific serum antibody titers, sequential sH1N1 influenza virus-infected ferrets were protected from challenge with a novel H1N1 influenza virus (A/California/07/2009), and these ferrets poorly transmitted the virus to naive contacts. Collectively, these findings indicate that HA stalk-specific antibodies are commonly elicited in ferrets following sequential infection with antigenically distinct sH1N1 influenza virus isolates lacking HA receptor-binding site cross-reactivity and can protect ferrets against a pathogenic novel H1N1 virus. IMPORTANCE The influenza virus hemagglutinin (HA) is a major target of the humoral immune response following infection and/or seasonal vaccination. While antibodies targeting the receptor-binding pocket of HA possess strong neutralization capacities, these antibodies are largely strain specific and do not confer protection against antigenic drift variant or novel HA subtype-expressing viruses. In contrast, antibodies targeting the conserved stalk region of HA exhibit broader reactivity among viruses within and among influenza virus subtypes. Here, we show that sequential infection of ferrets with antigenically distinct seasonal H1N1 influenza viruses boosts the antibody responses directed at the HA stalk region. Moreover, ferrets possessing HA stalk-specific antibody were protected against novel H1N1 virus infection and did not transmit the virus to naive contacts. PMID:26559834

Viral unmasking of cellular 5S rRNA pseudogene transcripts induces RIG-I-mediated immunity.

PubMed

Chiang, Jessica J; Sparrer, Konstantin M J; van Gent, Michiel; Lässig, Charlotte; Huang, Teng; Osterrieder, Nikolaus; Hopfner, Karl-Peter; Gack, Michaela U

2018-01-01

The sensor RIG-I detects double-stranded RNA derived from RNA viruses. Although RIG-I is also known to have a role in the antiviral response to DNA viruses, physiological RNA species recognized by RIG-I during infection with a DNA virus are largely unknown. Using next-generation RNA sequencing (RNAseq), we found that host-derived RNAs, most prominently 5S ribosomal RNA pseudogene 141 (RNA5SP141), bound to RIG-I during infection with herpes simplex virus 1 (HSV-1). Infection with HSV-1 induced relocalization of RNA5SP141 from the nucleus to the cytoplasm, and virus-induced shutoff of host protein synthesis downregulated the abundance of RNA5SP141-interacting proteins, which allowed RNA5SP141 to bind RIG-I and induce the expression of type I interferons. Silencing of RNA5SP141 strongly dampened the antiviral response to HSV-1 and the related virus Epstein-Barr virus (EBV), as well as influenza A virus (IAV). Our findings reveal that antiviral immunity can be triggered by host RNAs that are unshielded following depletion of their respective binding proteins by the virus.

Autophagy in Measles Virus Infection.

PubMed

Rozières, Aurore; Viret, Christophe; Faure, Mathias

2017-11-24

Autophagy is a biological process that helps cells to recycle obsolete cellular components and which greatly contributes to maintaining cellular integrity in response to environmental stress factors. Autophagy is also among the first lines of cellular defense against invading microorganisms, including viruses. The autophagic destruction of invading pathogens, a process referred to as xenophagy, involves cytosolic autophagy receptors, such as p62/SQSTM1 (Sequestosome 1) or NDP52/CALCOCO2 (Nuclear Dot 52 KDa Protein/Calcium Binding And Coiled-Coil Domain 2), which bind to microbial components and target them towards growing autophagosomes for degradation. However, most, if not all, infectious viruses have evolved molecular tricks to escape from xenophagy. Many viruses even use autophagy, part of the autophagy pathway or some autophagy-associated proteins, to improve their infectious potential. In this regard, the measles virus, responsible for epidemic measles, has a unique interface with autophagy as the virus can induce multiple rounds of autophagy in the course of infection. These successive waves of autophagy result from distinct molecular pathways and seem associated with anti- and/or pro-measles virus consequences. In this review, we describe what the autophagy-measles virus interplay has taught us about both the biology of the virus and the mechanistic orchestration of autophagy.

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

Mannose-binding lectin contributes to deleterious inflammatory response in pandemic H1N1 and avian H9N2 infection.

PubMed

Ling, Man To; Tu, Wenwei; Han, Yan; Mao, Huawei; Chong, Wai Po; Guan, Jing; Liu, Ming; Lam, Kwok Tai; Law, Helen K W; Peiris, J S Malik; Takahashi, K; Lau, Yu Lung

2012-01-01

Mannose-binding lectin (MBL) is a pattern-recognition molecule, which functions as a first line of host defense. Pandemic H1N1 (pdmH1N1) influenza A virus caused massive infection in 2009 and currently circulates worldwide. Avian influenza A H9N2 (H9N2/G1) virus has infected humans and has the potential to be the next pandemic virus. Antiviral function and immunomodulatory role of MBL in pdmH1N1 and H9N2/G1 virus infection have not been investigated. In this study, MBL wild-type (WT) and MBL knockout (KO) murine models were used to examine the role of MBL in pdmH1N1 and H9N2/G1 virus infection. Our study demonstrated that in vitro, MBL binds to pdmH1N1 and H9N2/G1 viruses, likely via the carbohydrate recognition domain of MBL. Wild-type mice developed more severe disease, as evidenced by a greater weight loss than MBL KO mice during influenza virus infection. Furthermore, MBL WT mice had enhanced production of proinflammatory cytokines and chemokines compared with MBL KO mice, suggesting that MBL could upregulate inflammatory responses that may potentially worsen pdmH1N1 and H9N2/G1 virus infections. Our study provided the first in vivo evidence that MBL may be a risk factor during pdmH1N1 and H9N2/G1 infection by upregulating proinflammatory response.

Antigenic Drift of the Pandemic 2009 A(H1N1) Influenza Virus in a Ferret Model

PubMed Central

Guarnaccia, Teagan; Carolan, Louise A.; Maurer-Stroh, Sebastian; Lee, Raphael T. C.; Job, Emma; Reading, Patrick C.; Petrie, Stephen; McCaw, James M.; McVernon, Jodie; Hurt, Aeron C.; Kelso, Anne; Mosse, Jennifer; Barr, Ian G.; Laurie, Karen L.

2013-01-01

Surveillance data indicate that most circulating A(H1N1)pdm09 influenza viruses have remained antigenically similar since they emerged in humans in 2009. However, antigenic drift is likely to occur in the future in response to increasing population immunity induced by infection or vaccination. In this study, sequential passaging of A(H1N1)pdm09 virus by contact transmission through two independent series of suboptimally vaccinated ferrets resulted in selection of variant viruses with an amino acid substitution (N156K, H1 numbering without signal peptide; N159K, H3 numbering without signal peptide; N173K, H1 numbering from first methionine) in a known antigenic site of the viral HA. The N156K HA variant replicated and transmitted efficiently between naïve ferrets and outgrew wildtype virus in vivo in ferrets in the presence and absence of immune pressure. In vitro, in a range of cell culture systems, the N156K variant rapidly adapted, acquiring additional mutations in the viral HA that also potentially affected antigenic properties. The N156K escape mutant was antigenically distinct from wildtype virus as shown by binding of HA-specific antibodies. Glycan binding assays demonstrated the N156K escape mutant had altered receptor binding preferences compared to wildtype virus, which was supported by computational modeling predictions. The N156K substitution, and culture adaptations, have been detected in human A(H1N1)pdm09 viruses with N156K preferentially reported in sequences from original clinical samples rather than cultured isolates. This study demonstrates the ability of the A(H1N1)pdm09 virus to undergo rapid antigenic change to evade a low level vaccine response, while remaining fit in a ferret transmission model of immunization and infection. Furthermore, the potential changes in receptor binding properties that accompany antigenic changes highlight the importance of routine characterization of clinical samples in human A(H1N1)pdm09 influenza surveillance. PMID:23671418

Foamy Virus Vector Carries a Strong Insulator in Its Long Terminal Repeat Which Reduces Its Genotoxic Potential

PubMed Central

2017-01-01

ABSTRACT Strong viral enhancers in gammaretrovirus vectors have caused cellular proto-oncogene activation and leukemia, necessitating the use of cellular promoters in “enhancerless” self-inactivating integrating vectors. However, cellular promoters result in relatively low transgene expression, often leading to inadequate disease phenotype correction. Vectors derived from foamy virus, a nonpathogenic retrovirus, show higher preference for nongenic integrations than gammaretroviruses/lentiviruses and preferential integration near transcriptional start sites, like gammaretroviruses. We found that strong viral enhancers/promoters placed in foamy viral vectors caused extremely low immortalization of primary mouse hematopoietic stem/progenitor cells compared to analogous gammaretrovirus/lentivirus vectors carrying the same enhancers/promoters, an effect not explained solely by foamy virus' modest insertional site preference for nongenic regions compared to gammaretrovirus/lentivirus vectors. Using CRISPR/Cas9-mediated targeted insertion of analogous proviral sequences into the LMO2 gene and then measuring LMO2 expression, we demonstrate a sequence-specific effect of foamy virus, independent of insertional bias, contributing to reduced genotoxicity. We show that this effect is mediated by a 36-bp insulator located in the foamy virus long terminal repeat (LTR) that has high-affinity binding to the CCCTC-binding factor. Using our LMO2 activation assay, LMO2 expression was significantly increased when this insulator was removed from foamy virus and significantly reduced when the insulator was inserted into the lentiviral LTR. Our results elucidate a mechanism underlying the low genotoxicity of foamy virus, identify a novel insulator, and support the use of foamy virus as a vector for gene therapy, especially when strong enhancers/promoters are required. IMPORTANCE Understanding the genotoxic potential of viral vectors is important in designing safe and efficacious vectors for gene therapy. Self-inactivating vectors devoid of viral long-terminal-repeat enhancers have proven safe; however, transgene expression from cellular promoters is often insufficient for full phenotypic correction. Foamy virus is an attractive vector for gene therapy. We found foamy virus vectors to be remarkably less genotoxic, well below what was expected from their integration site preferences. We demonstrate that the foamy virus long terminal repeats contain an insulator element that binds CCCTC-binding factor and reduces its insertional genotoxicity. Our study elucidates a mechanism behind the low genotoxic potential of foamy virus, identifies a unique insulator, and supports the use of foamy virus as a vector for gene therapy. PMID:29046446

Feline leukemia virus infection requires a post-receptor binding envelope-dependent cellular component.

PubMed

Hussain, Naveen; Thickett, Kelly R; Na, Hong; Leung, Cherry; Tailor, Chetankumar S

2011-12-01

Gammaretrovirus receptors have been suggested to contain the necessary determinants to mediate virus binding and entry. Here, we show that murine NIH 3T3 and baby hamster kidney (BHK) cells overexpressing receptors for subgroup A, B, and C feline leukemia viruses (FeLVs) are weakly susceptible (10(1) to 10(2) CFU/ml) to FeLV pseudotype viruses containing murine leukemia virus (MLV) core (Gag-Pol) proteins, whereas FeLV receptor-expressing murine Mus dunni tail fibroblast (MDTF) cells are highly susceptible (10(4) to 10(6) CFU/ml). However, NIH 3T3 cells expressing the FeLV subgroup B receptor PiT1 are highly susceptible to gibbon ape leukemia virus pseudotype virus, which differs from the FeLV pseudotype viruses only in the envelope protein. FeLV resistance is not caused by a defect in envelope binding, low receptor expression levels, or N-linked glycosylation. Resistance is not alleviated by substitution of the MLV core in the FeLV pseudotype virus with FeLV core proteins. Interestingly, FeLV resistance is alleviated by fusion of receptor-expressing NIH 3T3 and BHK cells with MDTF or human TE671 cells, suggesting the absence of an additional cellular component in NIH 3T3 and BHK cells that is required for FeLV infection. The putative FeLV-specific cellular component is not a secreted factor, as MDTF conditioned medium does not alleviate the block to FeLV infection. Together, our findings suggest that FeLV infection requires an additional envelope-dependent cellular component that is absent in NIH 3T3 and BHK cells but that is present in MDTF and TE671 cells.

Use of tissue-specific microRNA to control pathology of wild-type adenovirus without attenuation of its ability to kill cancer cells.

PubMed

Cawood, Ryan; Chen, Hannah H; Carroll, Fionnadh; Bazan-Peregrino, Miriam; van Rooijen, Nico; Seymour, Leonard W

2009-05-01

Replicating viruses have broad applications in biomedicine, notably in cancer virotherapy and in the design of attenuated vaccines; however, uncontrolled virus replication in vulnerable tissues can give pathology and often restricts the use of potent strains. Increased knowledge of tissue-selective microRNA expression now affords the possibility of engineering replicating viruses that are attenuated at the RNA level in sites of potential pathology, but retain wild-type replication activity at sites not expressing the relevant microRNA. To assess the usefulness of this approach for the DNA virus adenovirus, we have engineered a hepatocyte-safe wild-type adenovirus 5 (Ad5), which normally mediates significant toxicity and is potentially lethal in mice. To do this, we have included binding sites for hepatocyte-selective microRNA mir-122 within the 3' UTR of the E1A transcription cassette. Imaging versions of these viruses, produced by fusing E1A with luciferase, showed that inclusion of mir-122 binding sites caused up to 80-fold decreased hepatic expression of E1A following intravenous delivery to mice. Animals administered a ten-times lethal dose of wild-type Ad5 (5x10(10) viral particles/mouse) showed substantial hepatic genome replication and extensive liver pathology, while inclusion of 4 microRNA binding sites decreased replication 50-fold and virtually abrogated liver toxicity. This modified wild-type virus retained full activity within cancer cells and provided a potent, liver-safe oncolytic virus. In addition to providing many potent new viruses for cancer virotherapy, microRNA control of virus replication should provide a new strategy for designing safe attenuated vaccines applied across a broad range of viral diseases.

The RNA-Binding Site of Poliovirus 3C Protein Doubles as a Phosphoinositide-Binding Domain.

PubMed

Shengjuler, Djoshkun; Chan, Yan Mei; Sun, Simou; Moustafa, Ibrahim M; Li, Zhen-Lu; Gohara, David W; Buck, Matthias; Cremer, Paul S; Boehr, David D; Cameron, Craig E

2017-12-05

Some viruses use phosphatidylinositol phosphate (PIP) to mark membranes used for genome replication or virion assembly. PIP-binding motifs of cellular proteins do not exist in viral proteins. Molecular-docking simulations revealed a putative site of PIP binding to poliovirus (PV) 3C protein that was validated using nuclear magnetic resonance spectroscopy. The PIP-binding site was located on a highly dynamic α helix, which also functions in RNA binding. Broad PIP-binding activity was observed in solution using a fluorescence polarization assay or in the context of a lipid bilayer using an on-chip, fluorescence assay. All-atom molecular dynamics simulations of the 3C protein-membrane interface revealed PIP clustering and perhaps PIP-dependent conformations. PIP clustering was mediated by interaction with residues that interact with the RNA phosphodiester backbone. We conclude that 3C binding to membranes will be determined by PIP abundance. We suggest that the duality of function observed for 3C may extend to RNA-binding proteins of other viruses. Copyright © 2017 Elsevier Ltd. All rights reserved.

Lineage-Specific Differences between the gp120 Inner Domain Layer 3 of Human Immunodeficiency Virus and That of Simian Immunodeficiency Virus

PubMed Central

Ding, Shilei; Medjahed, Halima; Prévost, Jérémie; Coutu, Mathieu; Xiang, Shi-Hua

2016-01-01

ABSTRACT Binding of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) gp120 exterior envelope glycoprotein to CD4 triggers conformational changes in gp120 that promote its interaction with one of the chemokine receptors, usually CCR5, ultimately leading to gp41-mediated virus-cell membrane fusion and entry. We previously described that topological layers (layer 1, layer 2, and layer 3) in the gp120 inner domain contribute to gp120-trimer association in the unliganded state but also help secure CD4 binding. Relative to layer 1 of HIV-1 gp120, the SIVmac239 gp120 layer 1 plays a more prominent role in maintaining gp120-trimer association but is minimally involved in promoting CD4 binding, which could be explained by the existence of a well-conserved tryptophan at position 375 (Trp 375) in HIV-2/SIVsmm. In this study, we investigated the role of SIV layer 3 in viral entry, cell-to-cell fusion, and CD4 binding. We observed that a network of interactions involving some residues of the β8-α5 region in SIVmac239 layer 3 may contribute to CD4 binding by helping shape the nearby Phe 43 cavity, which directly contacts CD4. In summary, our results suggest that layer 3 in SIV has a greater impact on CD4 binding than in HIV-1. This work defines lineage-specific differences in layer 3 from HIV-1 and that from SIV. IMPORTANCE CD4-induced conformational changes in the gp120 inner domain involve rearrangements between three topological layers. While the role of layers 1 to 3 for HIV-1 and layers 1 and 2 for SIV on gp120 transition to the CD4-bound conformation has been reported, the role of SIV layer 3 remains unknown. Here we report that SIV layer 3 has a greater impact on CD4 binding than does layer 3 in HIV-1 gp120. This work defines lineage-specific differences in layer 3 from HIV-1 and SIV. PMID:27535053

Bovine Leukemia Virus SU Protein Interacts with Zinc, and Mutations within Two Interacting Regions Differently Affect Viral Fusion and Infectivity In Vivo

PubMed Central

Gatot, Jean-Stéphane; Callebaut, Isabelle; Van Lint, Carine; Demonté, Dominique; Kerkhofs, Pierre; Portetelle, Daniel; Burny, Arsène; Willems, Luc; Kettmann, Richard

2002-01-01

Bovine leukemia virus (BLV) and human T-cell lymphotropic virus type 1 (HTLV-1) belong to the genus of deltaretroviruses. Their entry into the host cell is supposed to be mediated by interactions of the extracellular (SU) envelope glycoproteins with cellular receptors. To gain insight into the mechanisms governing this process, we investigated the ability of SU proteins to interact with specific ligands. In particular, by affinity chromatography, we have shown that BLV SU protein specifically interacted with zinc ions. To identify the protein domains involved in binding, 16 peptides distributed along the sequence were tested. Two of them appeared to be able to interact with zinc. To unravel the role of these SU regions in the biology of the virus, mutations were introduced into the env gene of a BLV molecular clone in order to modify residues potentially interacting with zinc. The fusogenic capacity of envelope mutated within the first zinc-binding region (104 to 123) was completely abolished. Furthermore, the integrity of this domain was also required for in vivo infectivity. In contrast, mutations within the second zinc-binding region (218 to 237) did not hamper the fusogenic capacity; indeed, the syncytia were even larger. In sheep, mutations in region 218 to 237 did not alter infectivity or viral spread. Finally, we demonstrated that the envelope of the related HTLV-1 was also able to bind zinc. Interestingly, zinc ions were found to be associated with the receptor-binding domain (RBD) of Friend murine leukemia virus (Fr-MLV) SU glycoprotein, further supporting their relevance in SU structure. Based on the sequence similarities shared with the Fr-MLV RBD, whose three-dimensional structure has been experimentally determined, we located the BLV zinc-binding peptide 104-123 on the opposite side of the potential receptor-binding surface. This observation supports the hypothesis that zinc ions could mediate interactions of the SU RBD either with the C-terminal part of SU, thereby contributing to the SU structural integrity, or with a partner(s) different from the receptor. PMID:12134000

NKp44 receptor mediates interaction of the envelope glycoproteins from the West-Nile and dengue viruses with Natural Killer cells

PubMed Central

Hershkovitz, Oren; Rosental, Benyamin; Rosenberg, Lior Ann; Navarro-Sanchez, Martha Erika; Jivov, Sergey; Zilka, Alon; Gershoni-Yahalom, Orly; Brient-Litzler, Elodie; Bedouelle, Hugues; Ho, Joanna W.; Campbell, Kerry S.; Rager-Zisman, Bracha; Despres, Philippe; Porgador, Angel

2009-01-01

Dengue virus (DV) and West Nile virus (WNV) have become a global concern due to their widespread distribution and their ability to cause a variety of human diseases. Antiviral immune defenses involve natural killer (NK) cells. In the present study, we investigated the interaction between NK cells and these two flaviviruses. We show that the NK-activating receptor NKp44 is involved in virally-mediated NK activation through direct interaction with the flavivirus envelope protein. Recombinant NKp44 directly binds to purified DV and WNV envelope proteins and specifically to domain III of WNV envelope protein (EIII); it also binds to WNV virus-like particles (VLPs). These WNV-VLPs and WNV-EIII directly bind NK cells expressing high levels of NKp44. Functionally, interaction of NK cells with infective and inactivated WNV results in NKp44-mediated NK de-granulation. Finally, WNV infection of cells results in increased binding of recombinant NKp44 that is specifically inhibited by anti-WNV serum. WNV-infected target cells induce IFNγ secretion and augmented lysis by NKp44-expressing primary NK cells that are blocked by anti-NKp44 antibodies. Our findings show that triggering of NK cells by flavivirus is mediated by interaction of NKp44 with the flavivirus envelope protein. PMID:19635919

Removal of either N-glycan site from the envelope receptor binding domain of Moloney and Friend but not AKV mouse ecotropic gammaretroviruses alters receptor usage

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

Knoper, Ryan C.; Ferrarone, John; Yan Yuhe

2009-09-01

Three N-linked glycosylation sites were removed from the envelope glycoproteins of Friend, Moloney, and AKV mouse ecotropic gammaretroviruses: gs1 and gs2, in the receptor binding domain; and gs8, in a region implicated in post-binding cell fusion. Mutants were tested for their ability to infect rodent cells expressing 4 CAT-1 receptor variants. Three mutants (Mo-gs1, Mo-gs2, and Fr-gs1) infect NIH 3T3 and rat XC cells, but are severely restricted in Mus dunni cells and Lec8, a Chinese hamster cell line susceptible to ecotropic virus. This restriction is reproduced in ferret cells expressing M. dunni dCAT-1, but not in cells expressing NIHmore » 3T3 mCAT-1. Virus binding assays, pseudotype assays, and the use of glycosylation inhibitors further suggest that restriction is primarily due to receptor polymorphism and, in M. dunni cells, to glycosylation of cellular proteins. Virus envelope glycan size or type does not affect infectivity. Thus, host range variation due to N-glycan deletion is receptor variant-specific, cell-specific, virus type-specific, and glycan site-specific.« less

Interaction of zanamivir with DNA and RNA: Models for drug DNA and drug RNA bindings

NASA Astrophysics Data System (ADS)

Nafisi, Shohreh; Kahangi, Fatemeh Ghoreyshi; Azizi, Ebrahim; Zebarjad, Nader; Tajmir-Riahi, Heidar-Ali

2007-03-01

Zanamivir (ZAN) is the first of a new generation of influenza virus-specific drugs known as neuraminidase inhibitors, which acts by interfering with life cycles of influenza viruses A and B. It prevents the virus spreading infection to other cells by blocking the neuraminidase enzyme present on the surface of the virus. The aim of this study was to examine the stability and structural features of calf thymus DNA and yeast RNA complexes with zanamivir in aqueous solution, using constant DNA or RNA concentration (12.5 mM) and various zanamivir/polynucleotide ( P) ratios of 1/20, 1/10, 1/4, and 1/2. FTIR and UV-visible spectroscopy are used to determine the drug external binding modes, the binding constant and the stability of zanamivir-DNA and RNA complexes in aqueous solution. Structural analysis showed major interaction of zanamivir with G-C (major groove) and A-T (minor groove) base pairs and minor perturbations of the backbone PO 2 group with overall binding constants of Kzanamivir-DNA = 1.30 × 10 4 M -1 and Kzanamivir-RNA = 1.38 × 10 4 M -1. The drug interaction induces a partial B to A-DNA transition, while RNA remains in A-conformation.

Novel Chemical Ligands to Ebola Virus and Marburg Virus Nucleoproteins Identified by Combining Affinity Mass Spectrometry and Metabolomics Approaches

PubMed Central

Fu, Xu; Wang, Zhihua; Li, Lixin; Dong, Shishang; Li, Zhucui; Jiang, Zhenzuo; Wang, Yuefei; Shui, Wenqing

2016-01-01

The nucleoprotein (NP) of Ebola virus (EBOV) and Marburg virus (MARV) is an essential component of the viral ribonucleoprotein complex and significantly impacts replication and transcription of the viral RNA genome. Although NP is regarded as a promising antiviral druggable target, no chemical ligands have been reported to interact with EBOV NP or MARV NP. We identified two compounds from a traditional Chinese medicine Gancao (licorice root) that can bind both NPs by combining affinity mass spectrometry and metabolomics approaches. These two ligands, 18β-glycyrrhetinic acid and licochalcone A, were verified by defined compound mixture screens and further characterized with individual ligand binding assays. Accompanying biophysical analyses demonstrate that binding of 18β-glycyrrhetinic acid to EBOV NP significantly reduces protein thermal stability, induces formation of large NP oligomers, and disrupts the critical association of viral ssRNA with NP complexes whereas the compound showed no such activity on MARV NP. Our study has revealed the substantial potential of new analytical techniques in ligand discovery from natural herb resources. In addition, identification of a chemical ligand that influences the oligomeric state and RNA-binding function of EBOV NP sheds new light on antiviral drug development. PMID:27403722

Nucleoprotein of influenza B virus binds to its type A counterpart and disrupts influenza A viral polymerase complex formation

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

Jaru-ampornpan, Peera, E-mail: peera.jar@biotec.or.th; Narkpuk, Jaraspim; Wanitchang, Asawin

Highlights: •FluB nucleoprotein (BNP) can bind to FluA nucleoprotein (ANP). •BNP–ANP interaction inhibits FluA polymerase activity. •BNP binding prevents ANP from forming a functional FluA polymerase complex. •Nuclear localization of BNP is necessary for FluA polymerase inhibition. •Viral RNA is not required for the BNP–ANP interaction. -- Abstract: Upon co-infection with influenza B virus (FluB), influenza A virus (FluA) replication is substantially impaired. Previously, we have shown that the nucleoprotein of FluB (BNP) can inhibit FluA polymerase machinery, retarding the growth of FluA. However, the molecular mechanism underlying this inhibitory action awaited further investigation. Here, we provide evidence that BNPmore » hinders the proper formation of FluA polymerase complex by competitively binding to the nucleoprotein of FluA. To exert this inhibitory effect, BNP must be localized in the nucleus. The interaction does not require the presence of the viral RNA but needs an intact BNP RNA-binding motif. The results highlight the novel role of BNP as an anti-influenza A viral agent and provide insights into the mechanism of intertypic interference.« less

A kinesin-1 binding motif in vaccinia virus that is widespread throughout the human genome

PubMed Central

Dodding, Mark P; Mitter, Richard; Humphries, Ashley C; Way, Michael

2011-01-01

Transport of cargoes by kinesin-1 is essential for many cellular processes. Nevertheless, the number of proteins known to recruit kinesin-1 via its cargo binding light chain (KLC) is still quite small. We also know relatively little about the molecular features that define kinesin-1 binding. We now show that a bipartite tryptophan-based kinesin-1 binding motif, originally identified in Calsyntenin is present in A36, a vaccinia integral membrane protein. This bipartite motif in A36 is required for kinesin-1-dependent transport of the virus to the cell periphery. Bioinformatic analysis reveals that related bipartite tryptophan-based motifs are present in over 450 human proteins. Using vaccinia as a surrogate cargo, we show that regions of proteins containing this motif can function to recruit KLC and promote virus transport in the absence of A36. These proteins interact with the kinesin light chain outside the context of infection and have distinct preferences for KLC1 and KLC2. Our observations demonstrate that KLC binding can be conferred by a common set of features that are found in a wide range of proteins associated with diverse cellular functions and human diseases. PMID:21915095

A single mutation in Taiwanese H6N1 influenza hemagglutinin switches binding to human-type receptors.

PubMed

de Vries, Robert P; Tzarum, Netanel; Peng, Wenjie; Thompson, Andrew J; Ambepitiya Wickramasinghe, Iresha N; de la Pena, Alba T Torrents; van Breemen, Marielle J; Bouwman, Kim M; Zhu, Xueyong; McBride, Ryan; Yu, Wenli; Sanders, Rogier W; Verheije, Monique H; Wilson, Ian A; Paulson, James C

2017-09-01

In June 2013, the first case of human infection with an avian H6N1 virus was reported in a Taiwanese woman. Although this was a single non-fatal case, the virus continues to circulate in Taiwanese poultry. As with any emerging avian virus that infects humans, there is concern that acquisition of human-type receptor specificity could enable transmission in the human population. Despite mutations in the receptor-binding pocket of the human H6N1 isolate, it has retained avian-type (NeuAcα2-3Gal) receptor specificity. However, we show here that a single nucleotide substitution, resulting in a change from Gly to Asp at position 225 (G225D), completely switches specificity to human-type (NeuAcα2-6Gal) receptors. Significantly, G225D H6 loses binding to chicken trachea epithelium and is now able to bind to human tracheal tissue. Structural analysis reveals that Asp225 directly interacts with the penultimate Gal of the human-type receptor, stabilizing human receptor binding. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

Sialic acid content in human saliva and anti-influenza activity against human and avian influenza viruses.

PubMed

Limsuwat, Nattavatchara; Suptawiwat, Ornpreya; Boonarkart, Chompunuch; Puthavathana, Pilaipan; Wiriyarat, Witthawat; Auewarakul, Prasert

2016-03-01

It was shown previously that human saliva has higher antiviral activity against human influenza viruses than against H5N1 highly pathogenic avian influenza viruses, and that the major anti-influenza activity was associated with sialic-acid-containing molecules. To further characterize the differential susceptibility to saliva among influenza viruses, seasonal influenza A and B virus, pandemic H1N1 virus, and 15 subtypes of avian influenza virus were tested for their susceptibility to human and chicken saliva. Human saliva showed higher hemagglutination inhibition (HI) and neutralization (NT) titers against seasonal influenza A virus and the pandemic H1N1 viruses than against influenza B virus and most avian influenza viruses, except for H9N2 and H12N9 avian influenza viruses, which showed high HI and NT titers. To understand the nature of sialic-acid-containing anti-influenza factors in human saliva, α2,3- and α2,6-linked sialic acid was measured in human saliva samples using a lectin binding and dot blot assay. α2,6-linked sialic acid was found to be more abundant than α2,3-linked sialic acid, and a seasonal H1N1 influenza virus bound more efficiently to human saliva than an H5N1 virus in a dot blot analysis. These data indicated that human saliva contains the sialic acid type corresponding to the binding preference of seasonal influenza viruses.

DWARF GALAXIES AND THE COSMIC WEB

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

Benitez-Llambay, Alejandro; Abadi, Mario G.; Navarro, Julio F.

2013-02-01

We use a cosmological simulation of the formation of the Local Group of Galaxies to identify a mechanism that enables the removal of baryons from low-mass halos without appealing to feedback or reionization. As the Local Group forms, matter bound to it develops a network of filaments and pancakes. This moving web of gas and dark matter drifts and sweeps a large volume, overtaking many halos in the process. The dark matter content of these halos is unaffected but their gas can be efficiently removed by ram pressure. The loss of gas is especially pronounced in low-mass halos due tomore » their lower binding energy and has a dramatic effect on the star formation history of affected systems. This 'cosmic web stripping' may help to explain the scarcity of dwarf galaxies compared with the numerous low-mass halos expected in {Lambda}CDM and the large diversity of star formation histories and morphologies characteristic of faint galaxies. Although our results are based on a single high-resolution simulation, it is likely that the hydrodynamical interaction of dwarf galaxies with the cosmic web is a crucial ingredient so far missing from galaxy formation models.« less

Evolution of canine parvovirus--a need for new vaccines?

PubMed

Truyen, Uwe

2006-10-05

Canine parvovirus (CPV) is a new virus, which is continuing to evolve, giving rise to new antigenic types and virus mutants that spread through the dog population. The most successful mutants, from an evolutionary perspective, appear to be selected by improved binding to the CPV receptor, the canine transferrin receptor, and by an extended host range, which for the newer antigenic types now includes both the dog and the cat. The new viruses also show antigenic differences that can be defined by binding of certain monoclonal antibodies; they also differ in their reactivity in virus neutralisation tests, using immune sera raised against the various antigenic types. These differences may influence the susceptibility of young animals to infection at the time when the level of maternally derived antibody decreases to the minimum protective titre. This minimum protective titre may vary depending on the infecting virus type. There is, however, a high degree of cross-protection between the virus types and the true relevance of the differences in neutralisation titer is currently not known.

Tomato yellow leaf curl virus infection of a resistant tomato line with a silenced sucrose transporter gene LeHT1 results in inhibition of growth, enhanced virus spread, and necrosis.

PubMed

Eybishtz, Assaf; Peretz, Yuval; Sade, Dagan; Gorovits, Rena; Czosnek, Henryk

2010-02-01

To identify genes involved in resistance of tomato to Tomato yellow leaf curl virus (TYLCV), cDNA libraries from lines resistant (R) and susceptible (S) to the virus were compared. The hexose transporter LeHT1 was found to be expressed preferentially in R tomato plants. The role of LeHT1 in the establishment of TYLCV resistance was studied in R plants where LeHT1 has been silenced using Tobacco rattle virus-induced gene silencing (TRV VIGS). Following TYLCV inoculation, LeHT1-silenced R plants showed inhibition of growth and enhanced virus accumulation and spread. In addition, a necrotic response was observed along the stem and petioles of infected LeHT1-silenced R plants, but not on infected not-silenced R plants. This response was specific of R plants since it was absent in infected LeHT1-silenced S plants. Necrosis had several characteristics of programmed cell death (PCD): DNA from necrotic tissues presented a PCD-characteristic ladder pattern, the amount of a JNK analogue increased, and production of reactive oxygen was identified by DAB staining. A similar necrotic reaction along stem and petioles was observed in LeHT1-silenced R plants infected with the DNA virus Bean dwarf mosaic virus and the RNA viruses Cucumber mosaic virus and Tobacco mosaic virus. These results constitute the first evidence for a necrotic response backing natural resistance to TYLCV in tomato, confirming that plant defense is organized in multiple layers. They demonstrate that the hexose transporter LeHT1 is essential for the expression of natural resistance against TYLCV and its expression correlates with inhibition of virus replication and movement.

Enhancement of Ebola Virus Infection via Ficolin-1 Interaction with the Mucin Domain of GP Glycoprotein.

PubMed

Favier, Anne-Laure; Gout, Evelyne; Reynard, Olivier; Ferraris, Olivier; Kleman, Jean-Philippe; Volchkov, Viktor; Peyrefitte, Christophe; Thielens, Nicole M

2016-06-01

Ebola virus infection requires the surface viral glycoprotein to initiate entry into the target cells. The trimeric glycoprotein is a highly glycosylated viral protein which has been shown to interact with host C-type lectin receptors and the soluble complement recognition protein mannose-binding lectin, thereby enhancing viral infection. Similarly to mannose-binding lectin, ficolins are soluble effectors of the innate immune system that recognize particular glycans at the pathogen surface. In this study, we demonstrate that ficolin-1 interacts with the Zaire Ebola virus (EBOV) glycoprotein, and we characterized this interaction by surface plasmon resonance spectroscopy. Ficolin-1 was shown to bind to the viral glycoprotein with a high affinity. This interaction was mediated by the fibrinogen-like recognition domain of ficolin-1 and the mucin-like domain of the viral glycoprotein. Using a ficolin-1 control mutant devoid of sialic acid-binding capacity, we identified sialylated moieties of the mucin domain to be potential ligands on the glycoprotein. In cell culture, using both pseudotyped viruses and EBOV, ficolin-1 was shown to enhance EBOV infection independently of the serum complement. We also observed that ficolin-1 enhanced EBOV infection on human monocyte-derived macrophages, described to be major viral target cells,. Competition experiments suggested that although ficolin-1 and mannose-binding lectin recognized different carbohydrate moieties on the EBOV glycoprotein, the observed enhancement of the infection likely depended on a common cellular receptor/partner. In conclusion, ficolin-1 could provide an alternative receptor-mediated mechanism for enhancing EBOV infection, thereby contributing to viral subversion of the host innate immune system. A specific interaction involving ficolin-1 (M-ficolin), a soluble effector of the innate immune response, and the glycoprotein (GP) of EBOV was identified. Ficolin-1 enhanced virus infection instead of tipping the balance toward its elimination. An interaction between the fibrinogen-like recognition domain of ficolin-1 and the mucin-like domain of Ebola virus GP occurred. In this model, the enhancement of infection was shown to be independent of the serum complement. The facilitation of EBOV entry into target host cells by the interaction with ficolin-1 and other host lectins shunts virus elimination, which likely facilitates the survival of the virus in infected host cells and contributes to the virus strategy to subvert the innate immune response. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Virological and pathological characterization of an avian H1N1 influenza A virus.

PubMed

Koo, Bon-Sang; Kim, Hye Kwon; Song, Daesub; Na, Woonsung; Song, Min-Suk; Kwon, Jin Jung; Wong, Sook-San; Noh, Ji Yeong; Ahn, Min-Ju; Kim, Doo-Jin; Webby, Richard J; Yoon, Sun-Woo; Jeong, Dae Gwin

2018-05-01

Gene segments from avian H1N1 influenza A viruses have reassorted with other influenza viruses to generate pandemic strains over the past century. Nevertheless, little effort has been invested in understanding the characteristics of avian H1N1 influenza viruses. Here, we present the genome sequence and a molecular and virological characterization of an avian influenza A virus, A/wild bird/Korea/SK14/2014 (A/SK14, H1N1), isolated from migratory birds in South Korea during the winter season of 2014-2015. Full-genome sequencing and phylogenetic analysis revealed that the virus belongs to the Eurasian avian lineage. Although it retained avian-receptor binding preference, A/SK14 virus also exhibited detectable human-like receptor binding and was able to replicate in differentiated primary normal human bronchial epithelial cells. In animal models, A/SK14 virus was moderately pathogenic in mice, and virus was detected in nasal washes from inoculated guinea pigs, but not in direct-contact guinea pigs. Although A/SK14 showed moderate pathogenicity and no evidence of transmission in a mammalian model, our results suggest that the dual receptor specificity of A/SK14-like virus might allow for a more rapid adaptation to mammals, emphasizing the importance of further continuous surveillance and risk-assessment activities.

Methodological Guidelines for Accurate Detection of Viruses in Wild Plant Species

PubMed Central

Renner, Kurra; Cole, Ellen; Seabloom, Eric W.; Borer, Elizabeth T.; Malmstrom, Carolyn M.

2016-01-01

Ecological understanding of disease risk, emergence, and dynamics and of the efficacy of control strategies relies heavily on efficient tools for microorganism identification and characterization. Misdetection, such as the misclassification of infected hosts as healthy, can strongly bias estimates of disease prevalence and lead to inaccurate conclusions. In natural plant ecosystems, interest in assessing microbial dynamics is increasing exponentially, but guidelines for detection of microorganisms in wild plants remain limited, particularly so for plant viruses. To address this gap, we explored issues and solutions associated with virus detection by serological and molecular methods in noncrop plant species as applied to the globally important Barley yellow dwarf virus PAV (Luteoviridae), which infects wild native plants as well as crops. With enzyme-linked immunosorbent assays (ELISA), we demonstrate how virus detection in a perennial wild plant species may be much greater in stems than in leaves, although leaves are most commonly sampled, and may also vary among tillers within an individual, thereby highlighting the importance of designing effective sampling strategies. With reverse transcription-PCR (RT-PCR), we demonstrate how inhibitors in tissues of perennial wild hosts can suppress virus detection but can be overcome with methods and products that improve isolation and amplification of nucleic acids. These examples demonstrate the paramount importance of testing and validating survey designs and virus detection methods for noncrop plant communities to ensure accurate ecological surveys and reliable assumptions about virus dynamics in wild hosts. PMID:26773088

Methodological Guidelines for Accurate Detection of Viruses in Wild Plant Species.

PubMed

Lacroix, Christelle; Renner, Kurra; Cole, Ellen; Seabloom, Eric W; Borer, Elizabeth T; Malmstrom, Carolyn M

2016-01-15

Ecological understanding of disease risk, emergence, and dynamics and of the efficacy of control strategies relies heavily on efficient tools for microorganism identification and characterization. Misdetection, such as the misclassification of infected hosts as healthy, can strongly bias estimates of disease prevalence and lead to inaccurate conclusions. In natural plant ecosystems, interest in assessing microbial dynamics is increasing exponentially, but guidelines for detection of microorganisms in wild plants remain limited, particularly so for plant viruses. To address this gap, we explored issues and solutions associated with virus detection by serological and molecular methods in noncrop plant species as applied to the globally important Barley yellow dwarf virus PAV (Luteoviridae), which infects wild native plants as well as crops. With enzyme-linked immunosorbent assays (ELISA), we demonstrate how virus detection in a perennial wild plant species may be much greater in stems than in leaves, although leaves are most commonly sampled, and may also vary among tillers within an individual, thereby highlighting the importance of designing effective sampling strategies. With reverse transcription-PCR (RT-PCR), we demonstrate how inhibitors in tissues of perennial wild hosts can suppress virus detection but can be overcome with methods and products that improve isolation and amplification of nucleic acids. These examples demonstrate the paramount importance of testing and validating survey designs and virus detection methods for noncrop plant communities to ensure accurate ecological surveys and reliable assumptions about virus dynamics in wild hosts. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Autophagy pathway induced by a plant virus facilitates viral spread and transmission by its insect vector.

PubMed

Chen, Yong; Chen, Qian; Li, Manman; Mao, Qianzhuo; Chen, Hongyan; Wu, Wei; Jia, Dongsheng; Wei, Taiyun

2017-11-01

Many viral pathogens are persistently transmitted by insect vectors and cause agricultural or health problems. Generally, an insect vector can use autophagy as an intrinsic antiviral defense mechanism against viral infection. Whether viruses can evolve to exploit autophagy to promote their transmission by insect vectors is still unknown. Here, we show that the autophagic process is triggered by the persistent replication of a plant reovirus, rice gall dwarf virus (RGDV) in cultured leafhopper vector cells and in intact insects, as demonstrated by the appearance of obvious virus-containing double-membrane autophagosomes, conversion of ATG8-I to ATG8-II and increased level of autophagic flux. Such virus-containing autophagosomes seem able to mediate nonlytic viral release from cultured cells or facilitate viral spread in the leafhopper intestine. Applying the autophagy inhibitor 3-methyladenine or silencing the expression of Atg5 significantly decrease viral spread in vitro and in vivo, whereas applying the autophagy inducer rapamycin or silencing the expression of Torc1 facilitate such viral spread. Furthermore, we find that activation of autophagy facilitates efficient viral transmission, whereas inhibiting autophagy blocks viral transmission by its insect vector. Together, these results indicate a plant virus can induce the formation of autophagosomes for carrying virions, thus facilitating viral spread and transmission by its insect vector. We believe that such a role for virus-induced autophagy is common for vector-borne persistent viruses during their transmission by insect vectors.

Autophagy pathway induced by a plant virus facilitates viral spread and transmission by its insect vector

PubMed Central

Mao, Qianzhuo; Chen, Hongyan; Wu, Wei

2017-01-01

Many viral pathogens are persistently transmitted by insect vectors and cause agricultural or health problems. Generally, an insect vector can use autophagy as an intrinsic antiviral defense mechanism against viral infection. Whether viruses can evolve to exploit autophagy to promote their transmission by insect vectors is still unknown. Here, we show that the autophagic process is triggered by the persistent replication of a plant reovirus, rice gall dwarf virus (RGDV) in cultured leafhopper vector cells and in intact insects, as demonstrated by the appearance of obvious virus-containing double-membrane autophagosomes, conversion of ATG8-I to ATG8-II and increased level of autophagic flux. Such virus-containing autophagosomes seem able to mediate nonlytic viral release from cultured cells or facilitate viral spread in the leafhopper intestine. Applying the autophagy inhibitor 3-methyladenine or silencing the expression of Atg5 significantly decrease viral spread in vitro and in vivo, whereas applying the autophagy inducer rapamycin or silencing the expression of Torc1 facilitate such viral spread. Furthermore, we find that activation of autophagy facilitates efficient viral transmission, whereas inhibiting autophagy blocks viral transmission by its insect vector. Together, these results indicate a plant virus can induce the formation of autophagosomes for carrying virions, thus facilitating viral spread and transmission by its insect vector. We believe that such a role for virus-induced autophagy is common for vector-borne persistent viruses during their transmission by insect vectors. PMID:29125860

Strain-Specific V3 and CD4 Binding Site Autologous HIV-1 Neutralizing Antibodies Select Neutralization-Resistant Viruses

DOE PAGES

Moody, M.  Anthony; Gao, Feng; Gurley, Thaddeus  C.; ...

2015-09-09

The third variable (V3) loop and the CD4 binding site (CD4bs) of the viral envelope are frequently targeted by neutralizing antibodies (nAbs) in HIV-1-infected individuals. In chronic infection, virus escape mutants repopulate the plasma and V3 and CD4bs nAbs emerge that can neutralize heterologous tier 1 easy-to-neutralize, but not tier 2 difficult-to-neutralize HIV-1 isolates. However, neutralization sensitivity of autologous plasma viruses to this type of nAb response has not been studied. We describe the development and evolution in vivo of antibodies distinguished by their target specificity for V3and CD4bs epitopes on autologous tier 2 viruses but not on heterologous tiermore » 2 viruses. A surprisingly high fraction of autologous circulating viruses was sensitive to these antibodies. These findings demonstrate a role for V3 and CD4bs antibodies in constraining the native envelope trimer in vivo to a neutralization-resistant phenotype, explaining why HIV-1 transmission generally occurs by tier 2 neutralization-resistant viruses.« less

RNA viruses can hijack vertebrate microRNAs to suppress innate immunity

NASA Astrophysics Data System (ADS)

Trobaugh, Derek W.; Gardner, Christina L.; Sun, Chengqun; Haddow, Andrew D.; Wang, Eryu; Chapnik, Elik; Mildner, Alexander; Weaver, Scott C.; Ryman, Kate D.; Klimstra, William B.

2014-02-01

Currently, there is little evidence for a notable role of the vertebrate microRNA (miRNA) system in the pathogenesis of RNA viruses. This is primarily attributed to the ease with which these viruses mutate to disrupt recognition and growth suppression by host miRNAs. Here we report that the haematopoietic-cell-specific miRNA miR-142-3p potently restricts the replication of the mosquito-borne North American eastern equine encephalitis virus in myeloid-lineage cells by binding to sites in the 3' non-translated region of its RNA genome. However, by limiting myeloid cell tropism and consequent innate immunity induction, this restriction directly promotes neurologic disease manifestations characteristic of eastern equine encephalitis virus infection in humans. Furthermore, the region containing the miR-142-3p binding sites is essential for efficient virus infection of mosquito vectors. We propose that RNA viruses can adapt to use antiviral properties of vertebrate miRNAs to limit replication in particular cell types and that this restriction can lead to exacerbation of disease severity.

Strain-Specific V3 and CD4 Binding Site Autologous HIV-1 Neutralizing Antibodies Select Neutralization-Resistant Viruses

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

Moody, M.  Anthony; Gao, Feng; Gurley, Thaddeus  C.

The third variable (V3) loop and the CD4 binding site (CD4bs) of the viral envelope are frequently targeted by neutralizing antibodies (nAbs) in HIV-1-infected individuals. In chronic infection, virus escape mutants repopulate the plasma and V3 and CD4bs nAbs emerge that can neutralize heterologous tier 1 easy-to-neutralize, but not tier 2 difficult-to-neutralize HIV-1 isolates. However, neutralization sensitivity of autologous plasma viruses to this type of nAb response has not been studied. We describe the development and evolution in vivo of antibodies distinguished by their target specificity for V3and CD4bs epitopes on autologous tier 2 viruses but not on heterologous tiermore » 2 viruses. A surprisingly high fraction of autologous circulating viruses was sensitive to these antibodies. These findings demonstrate a role for V3 and CD4bs antibodies in constraining the native envelope trimer in vivo to a neutralization-resistant phenotype, explaining why HIV-1 transmission generally occurs by tier 2 neutralization-resistant viruses.« less

Alterations in Hemagglutinin Receptor-Binding Specificity Accompany the Emergence of Highly Pathogenic Avian Influenza Viruses

PubMed Central

Mochalova, Larisa; Harder, Timm; Tuzikov, Alexander; Bovin, Nicolai; Wolff, Thorsten; Matrosovich, Mikhail; Schweiger, Brunhilde

2015-01-01

ABSTRACT Highly pathogenic avian influenza viruses (HPAIVs) of hemagglutinin H5 and H7 subtypes emerge after introduction of low-pathogenic avian influenza viruses (LPAIVs) from wild birds into poultry flocks, followed by subsequent circulation and evolution. The acquisition of multiple basic amino acids at the endoproteolytical cleavage site of the hemagglutinin (HA) is a molecular indicator for high pathogenicity, at least for infections of gallinaceous poultry. Apart from the well-studied significance of the multibasic HA cleavage site, there is only limited knowledge on other alterations in the HA and neuraminidase (NA) molecules associated with changes in tropism during the emergence of HPAIVs from LPAIVs. We hypothesized that changes in tropism may require alterations of the sialyloligosaccharide specificities of HA and NA. To test this hypothesis, we compared a number of LPAIVs and HPAIVs for their HA-mediated binding and NA-mediated desialylation of a set of synthetic receptor analogs, namely, α2-3-sialylated oligosaccharides. NA substrate specificity correlated with structural groups of NAs and did not correlate with pathogenic potential of the virus. In contrast, all HPAIVs differed from LPAIVs by a higher HA receptor-binding affinity toward the trisaccharides Neu5Acα2-3Galβ1-4GlcNAcβ (3′SLN) and Neu5Acα2-3Galβ1-3GlcNAcβ (SiaLec) and by the ability to discriminate between the nonfucosylated and fucosylated sialyloligosaccharides 3′SLN and Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ (SiaLex), respectively. These results suggest that alteration of the receptor-binding specificity accompanies emergence of the HPAIVs from their low-pathogenic precursors. IMPORTANCE Here, we have found for the first time correlations of receptor-binding properties of the HA with a highly pathogenic phenotype of poultry viruses. Our study suggests that enhanced receptor-binding affinity of HPAIVs for a typical “poultry-like” receptor, 3′SLN, is provided by substitutions in the receptor-binding site of HA which appeared in HA of LPAIVs in the course of transmission of LPAIVs from wild waterfowl into poultry flocks, with subsequent adaptation in poultry. The identification of LPAIVs with receptor characteristics of HPAIVs argues that the sialic acid-binding specificity of the HA may be used as a novel phenotypic marker of HPAIVs. PMID:25741006

Mutagenesis of Paramyxovirus Hemagglutinin-Neuraminidase Membrane-Proximal Stalk Region Influences Stability, Receptor Binding, and Neuraminidase Activity.

PubMed

Adu-Gyamfi, Emmanuel; Kim, Lori S; Jardetzky, Theodore S; Lamb, Robert A

2016-09-01

Paramyxoviridae consist of a large family of enveloped, negative-sense, nonsegmented single-stranded RNA viruses that account for a significant number of human and animal diseases. The fusion process for nearly all paramyxoviruses involves the mixing of the host cell plasma membrane and the virus envelope in a pH-independent fashion. Fusion is orchestrated via the concerted action of two surface glycoproteins: an attachment protein called hemagglutinin-neuraminidase (HN [also called H or G depending on virus type and substrate]), which acts as a receptor binding protein, and a fusion (F) protein, which undergoes a major irreversible refolding process to merge the two membranes. Recent biochemical evidence suggests that receptor binding by HN is dispensable for cell-cell fusion. However, factors that influence the stability and/or conformation of the HN 4-helix bundle (4HB) stalk have not been studied. Here, we used oxidative cross-linking as well as functional assays to investigate the role of the structurally unresolved membrane-proximal stalk region (MPSR) (residues 37 to 58) of HN in the context of headless and full-length HN membrane fusion promotion. Our data suggest that the receptor binding head serves to stabilize the stalk to regulate fusion. Moreover, we found that the MPSR of HN modulates receptor binding and neuraminidase activity without a corresponding regulation of F triggering. Paramyxoviruses require two viral membrane glycoproteins, the attachment protein variously called HN, H, or G and the fusion protein (F), to couple host receptor recognition to virus-cell fusion. The HN protein has a globular head that is attached to a membrane-anchored flexible stalk of ∼80 residues and has three activities: receptor binding, neuraminidase, and fusion activation. In this report, we have identified the functional significance of the membrane-proximal stalk region (MPSR) (HN, residues 37 to 56) of the paramyxovirus parainfluenza virus (PIV5), a region of the HN stalk that has not had its structure determined by X-ray crystallography. Our data suggest that the MPSR influences receptor binding and neuraminidase activity via an indirect mechanism. Moreover, the receptor binding head group stabilizes the 4HB stalk as part of the general mechanism to fine-tune F-activation. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Nuclear TRIM25 Specifically Targets Influenza Virus Ribonucleoproteins to Block the Onset of RNA Chain Elongation.

PubMed

Meyerson, Nicholas R; Zhou, Ligang; Guo, Yusong R; Zhao, Chen; Tao, Yizhi J; Krug, Robert M; Sawyer, Sara L

2017-11-08

TRIM25 is an E3 ubiquitin ligase that activates RIG-I to promote the antiviral interferon response. The NS1 protein from all strains of influenza A virus binds TRIM25, although not all virus strains block the interferon response, suggesting alternative mechanisms for TRIM25 action. Here we present a nuclear role for TRIM25 in specifically restricting influenza A virus replication. TRIM25 inhibits viral RNA synthesis through a direct mechanism that is independent of its ubiquitin ligase activity and the interferon pathway. This activity can be inhibited by the viral NS1 protein. TRIM25 inhibition of viral RNA synthesis results from its binding to viral ribonucleoproteins (vRNPs), the structures containing individual viral RNA segments, the viral polymerase, and multiple viral nucleoproteins. TRIM25 binding does not inhibit initiation of capped-RNA-primed viral mRNA synthesis by the viral polymerase. Rather, the onset of RNA chain elongation is inhibited because TRIM25 prohibits the movement of RNA into the polymerase complex. Copyright © 2017 Elsevier Inc. All rights reserved.

Glycomic characterization of respiratory tract tissues of ferrets: implications for its use in influenza virus infection studies.

PubMed

Jia, Nan; Barclay, Wendy S; Roberts, Kim; Yen, Hui-Ling; Chan, Renee W Y; Lam, Alfred K Y; Air, Gillian; Peiris, J S Malik; Dell, Anne; Nicholls, John M; Haslam, Stuart M

2014-10-10

The initial recognition between influenza virus and the host cell is mediated by interactions between the viral surface protein hemagglutinin and sialic acid-terminated glycoconjugates on the host cell surface. The sialic acid residues can be linked to the adjacent monosaccharide by α2-3- or α2-6-type glycosidic bonds. It is this linkage difference that primarily defines the species barrier of the influenza virus infection with α2-3 binding being associated with avian influenza viruses and α2-6 binding being associated with human strains. The ferret has been extensively used as an animal model to study the transmission of influenza. To better understand the validity of this model system, we undertook glycomic characterization of respiratory tissues of ferret, which allows a comparison of potential viral receptors to be made between humans and ferrets. To complement the structural analysis, lectin staining experiments were performed to characterize the regional distributions of glycans along the respiratory tract of ferrets. Finally, the binding between the glycans identified and the hemagglutinins of different strains of influenza viruses was assessed by glycan array experiments. Our data indicated that the respiratory tissues of ferret heterogeneously express both α2-3- and α2-6-linked sialic acids. However, the respiratory tissues of ferret also expressed the Sda epitope (NeuAcα2-3(GalNAcβ1-4)Galβ1-4GlcNAc) and sialylated N,N'-diacetyllactosamine (NeuAcα2-6GalNAcβ1-4GlcNAc), which have not been observed in the human respiratory tract surface epithelium. The presence of the Sda epitope reduces potential binding sites for avian viruses and thus may have implications for the usefulness of the ferret in the study of influenza virus infection. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Exploration of nucleoprotein α-MoRE and XD interactions of Nipah and Hendra viruses.

PubMed

Shang, Xu; Chu, Wenting; Chu, Xiakun; Xu, Liufang; Longhi, Sonia; Wang, Jin

2018-04-24

Henipavirus, including Hendra virus (HeV) and Nipah virus (NiV), is a newly discovered human pathogen genus. The nucleoprotein of Henipavirus contains an α-helical molecular recognition element (α-MoRE) that folds upon binding to the X domain (XD) of the phosphoprotein (P). In order to explore the conformational dynamics of free α-MoREs and the underlying binding-folding mechanism with XD, atomic force field-based and hybrid structure-based MD simulations were carried out. In our empirical force field-based simulations, characteristic structures and helicities of α-MoREs reveal the co-existence of partially structured and disordered conformations, as in the case of the well characterized cognate measles virus (MeV) α-MoRE. In spite of their overall similarity, the two α-MoREs display subtle helicity differences in their C-terminal region, but much different from that of MeV. For the α-MoRE/XD complexes, the results of our hybrid structure-based simulations provide the coupled binding-folding landscapes, and unveil a wide conformational selection mechanism at early binding stages, followed by a final induce-fit mechanism selection process. However, the HeV and NiV complexes have a lower binding barrier compared to that of MeV. Moreover, the HeV α-MoRE/XD complex shows much less coupling effects between binding and folding compared to that from both NiV and MeV. Our analysis revealed that contrary to NiV and MeV, the N- and C-terminal regions of the HeV α-MoRE maintains a low helicity also in the bound form.

Biopanning of polypeptides binding to bovine ephemeral fever virus G1 protein from phage display peptide library.

PubMed

Hou, Peili; Zhao, Guimin; He, Chengqiang; Wang, Hongmei; He, Hongbin

2018-01-04

The bovine ephemeral fever virus (BEFV) glycoprotein neutralization site 1 (also referred as G 1 protein), is a critical protein responsible for virus infectivity and eliciting immune-protection, however, binding peptides of BEFV G 1 protein are still unclear. Thus, the aim of the present study was to screen specific polypeptides, which bind BEFV G 1 protein with high-affinity and inhibit BEFV replication. The purified BEFV G 1 was coated and then reacted with the M13-based Ph.D.-7 phage random display library. The peptides for target binding were automated sequenced after four rounds of enrichment biopanning. The amino acid sequences of polypeptide displayed on positive clones were deduced and the affinity of positive polypeptides with BEFV G 1 was assayed by ELISA. Then the roles of specific G 1 -binding peptides in the context of BEFV infection were analyzed. The results showed that 27 specific peptide ligands displaying 11 different amino acid sequences were obtained, and the T18 and T25 clone had a higher affinity to G 1 protein than the other clones. Then their antiviral roles of two phage clones (T25 and T18) showed that both phage polypeptide T25 and T18 exerted inhibition on BEFV replication compared to control group. Moreover, synthetic peptide based on T18 (HSIRYDF) and T25 (YSLRSDY) alone or combined use on BEFV replication showed that the synthetic peptides could effectively inhibit the formation of cytopathic plaque and significantly inhibit BEFV RNA replication in a dose-dependent manner. Two antiviral peptide ligands binding to bovine ephemeral fever virus G 1 protein from phage display peptide library were identified, which may provide a potential research tool for diagnostic reagents and novel antiviral agents.

Murine Polyomavirus Cell Surface Receptors Activate Distinct Signaling Pathways Required for Infection.

PubMed

O'Hara, Samantha D; Garcea, Robert L

2016-11-01

Virus binding to the cell surface triggers an array of host responses, including activation of specific signaling pathways that facilitate steps in virus entry. Using mouse polyomavirus (MuPyV), we identified host signaling pathways activated upon virus binding to mouse embryonic fibroblasts (MEFs). Pathways activated by MuPyV included the phosphatidylinositol 3-kinase (PI3K), FAK/SRC, and mitogen-activated protein kinase (MAPK) pathways. Gangliosides and α4-integrin are required receptors for MuPyV infection. MuPyV binding to both gangliosides and the α4-integrin receptors was required for activation of the PI3K pathway; however, either receptor interaction alone was sufficient for activation of the MAPK pathway. Using small-molecule inhibitors, we confirmed that the PI3K and FAK/SRC pathways were required for MuPyV infection, while the MAPK pathway was dispensable. Mechanistically, the PI3K pathway was required for MuPyV endocytosis, while the FAK/SRC pathway enabled trafficking of MuPyV along microtubules. Thus, MuPyV interactions with specific cell surface receptors facilitate activation of signaling pathways required for virus entry and trafficking. Understanding how different viruses manipulate cell signaling pathways through interactions with host receptors could lead to the identification of new therapeutic targets for viral infection. Virus binding to cell surface receptors initiates outside-in signaling that leads to virus endocytosis and subsequent virus trafficking. How different viruses manipulate cell signaling through interactions with host receptors remains unclear, and elucidation of the specific receptors and signaling pathways required for virus infection may lead to new therapeutic targets. In this study, we determined that gangliosides and α4-integrin mediate mouse polyomavirus (MuPyV) activation of host signaling pathways. Of these pathways, the PI3K and FAK/SRC pathways were required for MuPyV infection. Both the PI3K and FAK/SRC pathways have been implicated in human diseases, such as heart disease and cancer, and inhibitors directed against these pathways are currently being investigated as therapies. It is possible that these pathways play a role in human PyV infections and could be targeted to inhibit PyV infection in immunosuppressed patients. Copyright © 2016 O’Hara and Garcea.

Calcium-Dependent Rubella Virus Fusion Occurs in Early Endosomes.

PubMed

Dubé, Mathieu; Etienne, Loïc; Fels, Maximilian; Kielian, Margaret

2016-07-15

The E1 membrane protein of rubella virus (RuV) is a class II membrane fusion protein structurally related to the fusion proteins of the alphaviruses, flaviviruses, and phleboviruses. Virus entry is mediated by a low pH-dependent fusion reaction through E1's insertion into the cell membrane and refolding to a stable homotrimer. Unlike the other described class II proteins, RuV E1 contains 2 fusion loops, which complex a metal ion between them by interactions with residues N88 and D136. Insertion of the E1 protein into the target membrane, fusion, and infection require calcium and are blocked by alanine substitution of N88 or D136. Here we addressed the requirements of E1 for calcium binding and the intracellular location of the calcium requirement during virus entry. Our results demonstrated that N88 and D136 are optimally configured to support RuV fusion and are strongly selected for during the virus life cycle. While E1 has some similarities with cellular proteins that bind calcium and anionic lipids, RuV binding to the membrane was independent of anionic lipids. Virus fusion occurred within early endosomes, and chelation of intracellular calcium showed that calcium within the early endosome was required for virus fusion and infection. Calcium triggered the reversible insertion of E1 into the target membrane at neutral pH, but E1 homotrimer formation and fusion required a low pH. Thus, RuV E1, unlike other known class II fusion proteins, has distinct triggers for membrane insertion and fusion protein refolding mediated, respectively, by endosomal calcium and low pH. Rubella virus causes a mild disease of childhood, but infection of pregnant women frequently results in miscarriage or severe birth defects. In spite of an effective vaccine, RuV disease remains a serious problem in many developing countries. RuV infection of host cells involves endocytic uptake and low pH-triggered membrane fusion and is unusual in its requirement for calcium binding by the membrane fusion protein. Here we addressed the mechanism of the calcium requirement and the required location of calcium during virus entry. Both calcium and low pH were essential during the virus fusion reaction, which was shown to occur in the early endosome compartment. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Calcium-Dependent Rubella Virus Fusion Occurs in Early Endosomes

PubMed Central

Dubé, Mathieu; Etienne, Loïc; Fels, Maximilian

2016-01-01

ABSTRACT The E1 membrane protein of rubella virus (RuV) is a class II membrane fusion protein structurally related to the fusion proteins of the alphaviruses, flaviviruses, and phleboviruses. Virus entry is mediated by a low pH-dependent fusion reaction through E1's insertion into the cell membrane and refolding to a stable homotrimer. Unlike the other described class II proteins, RuV E1 contains 2 fusion loops, which complex a metal ion between them by interactions with residues N88 and D136. Insertion of the E1 protein into the target membrane, fusion, and infection require calcium and are blocked by alanine substitution of N88 or D136. Here we addressed the requirements of E1 for calcium binding and the intracellular location of the calcium requirement during virus entry. Our results demonstrated that N88 and D136 are optimally configured to support RuV fusion and are strongly selected for during the virus life cycle. While E1 has some similarities with cellular proteins that bind calcium and anionic lipids, RuV binding to the membrane was independent of anionic lipids. Virus fusion occurred within early endosomes, and chelation of intracellular calcium showed that calcium within the early endosome was required for virus fusion and infection. Calcium triggered the reversible insertion of E1 into the target membrane at neutral pH, but E1 homotrimer formation and fusion required a low pH. Thus, RuV E1, unlike other known class II fusion proteins, has distinct triggers for membrane insertion and fusion protein refolding mediated, respectively, by endosomal calcium and low pH. IMPORTANCE Rubella virus causes a mild disease of childhood, but infection of pregnant women frequently results in miscarriage or severe birth defects. In spite of an effective vaccine, RuV disease remains a serious problem in many developing countries. RuV infection of host cells involves endocytic uptake and low pH-triggered membrane fusion and is unusual in its requirement for calcium binding by the membrane fusion protein. Here we addressed the mechanism of the calcium requirement and the required location of calcium during virus entry. Both calcium and low pH were essential during the virus fusion reaction, which was shown to occur in the early endosome compartment. PMID:27122589

Myxoma Virus Protein M029 Is a Dual Function Immunomodulator that Inhibits PKR and Also Conscripts RHA/DHX9 to Promote Expanded Host Tropism and Viral Replication

PubMed Central

Rahman, Masmudur M.; Liu, Jia; Chan, Winnie M.; Rothenburg, Stefan; McFadden, Grant

2013-01-01

Myxoma virus (MYXV)-encoded protein M029 is a member of the poxvirus E3 family of dsRNA-binding proteins that antagonize the cellular interferon signaling pathways. In order to investigate additional functions of M029, we have constructed a series of targeted M029-minus (vMyx-M029KO and vMyx-M029ID) and V5-tagged M029 MYXV. We found that M029 plays a pivotal role in determining the cellular tropism of MYXV in all mammalian cells tested. The M029-minus viruses were able to replicate only in engineered cell lines that stably express a complementing protein, such as vaccinia E3, but underwent abortive or abated infection in all other tested mammalian cell lines. The M029-minus viruses were dramatically attenuated in susceptible host European rabbits and caused no observable signs of myxomatosis. Using V5-tagged M029 virus, we observed that M029 expressed as an early viral protein is localized in both the nuclear and cytosolic compartments in virus-infected cells, and is also incorporated into virions. Using proteomic approaches, we have identified Protein Kinase R (PKR) and RNA helicase A (RHA)/DHX9 as two cellular binding partners of M029 protein. In virus-infected cells, M029 interacts with PKR in a dsRNA-dependent manner, while binding with DHX9 was not dependent on dsRNA. Significantly, PKR knockdown in human cells rescued the replication defect of the M029-knockout viruses. Unexpectedly, this rescue of M029-minus virus replication by PKR depletion could then be reversed by RHA/DHX9 knockdown in human monocytic THP1 cells. This indicates that M029 not only inhibits generic PKR anti-viral pathways, but also binds and conscripts RHA/DHX9 as a pro-viral effector to promote virus replication in THP1 cells. Thus, M029 is a critical host range and virulence factor for MYXV that is required for replication in all mammalian cells by antagonizing PKR-mediated anti-viral functions, and also conscripts pro-viral RHA/DHX9 to promote viral replication specifically in myeloid cells. PMID:23853588

Biological Nanoplatforms for Self-Assembled Electronics

DTIC Science & Technology

2015-03-24

as M13 , a virus that infects Escherichia coli. Approximately one billion different amino acid sequences are displayed on different viruses in the...sequence when contained within a phage M13 coat protein sequence, not chemically linked to the surface of phage MS2 VLPs. Thus, binding properties may...gallium arsenide in a bacteriophage M13 phage display library, MS2 VLPs modified with the metal binding peptides do not display the same activity

Glycosylation at Asn91 of H1N1 haemagglutinin affects binding to glycan receptors

PubMed Central

Jayaraman, Akila; Koh, Xiaoying; Li, Jing; Raman, Rahul; Viswanathan, Karthik; Shriver, Zachary; Sasisekharan, Ram

2012-01-01

The glycoprotein HA (haemagglutinin) on the surface of influenza A virus plays a central role in recognition and binding to specific host cell-surface glycan receptors and in fusion of viral membrane to the host nuclear membrane during viral replication. Given the abundance of HA on the viral surface, this protein is also the primary target for host innate and adaptive immune responses. Although addition of glycosylation sites on HA are a part of viral evolution to evade the host immune responses, there are specific glycosylation sites that are conserved during most of the evolution of the virus. In the present study, it was demonstrated that one such conserved glycosylation site at Asn91 in H1N1 HA critically governs the glycan receptor-binding specificity and hence would potentially impinge on the host adaptation of the virus. PMID:22642577

Glycosylation at Asn91 of H1N1 haemagglutinin affects binding to glycan receptors.

PubMed

Jayaraman, Akila; Koh, Xiaoying; Li, Jing; Raman, Rahul; Viswanathan, Karthik; Shriver, Zachary; Sasisekharan, Ram

2012-06-15

The glycoprotein HA (haemagglutinin) on the surface of influenza A virus plays a central role in recognition and binding to specific host cell-surface glycan receptors and in fusion of viral membrane to the host nuclear membrane during viral replication. Given the abundance of HA on the viral surface, this protein is also the primary target for host innate and adaptive immune responses. Although addition of glycosylation sites on HA are a part of viral evolution to evade the host immune responses, there are specific glycosylation sites that are conserved during most of the evolution of the virus. In the present study, it was demonstrated that one such conserved glycosylation site at Asn(91) in H1N1 HA critically governs the glycan receptor-binding specificity and hence would potentially impinge on the host adaptation of the virus.

Vaccine-elicited receptor-binding site antibodies neutralize two New World hemorrhagic fever arenaviruses.

PubMed

Clark, Lars E; Mahmutovic, Selma; Raymond, Donald D; Dilanyan, Taleen; Koma, Takaaki; Manning, John T; Shankar, Sundaresh; Levis, Silvana C; Briggiler, Ana M; Enria, Delia A; Wucherpfennig, Kai W; Paessler, Slobodan; Abraham, Jonathan

2018-05-14

While five arenaviruses cause human hemorrhagic fevers in the Western Hemisphere, only Junin virus (JUNV) has a vaccine. The GP1 subunit of their envelope glycoprotein binds transferrin receptor 1 (TfR1) using a surface that substantially varies in sequence among the viruses. As such, receptor-mimicking antibodies described to date are type-specific and lack the usual breadth associated with this mode of neutralization. Here we isolate, from the blood of a recipient of the live attenuated JUNV vaccine, two antibodies that cross-neutralize Machupo virus with varying efficiency. Structures of GP1-Fab complexes explain the basis for efficient cross-neutralization, which involves avoiding receptor mimicry and targeting a conserved epitope within the receptor-binding site (RBS). The viral RBS, despite its extensive sequence diversity, is therefore a target for cross-reactive antibodies with activity against New World arenaviruses of public health concern.

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.

Detection and Characterization of Viral Species/Subspecies Using Isothermal Recombinase Polymerase Amplification (RPA) Assays.

PubMed

Glais, Laurent; Jacquot, Emmanuel

2015-01-01

Numerous molecular-based detection protocols include an amplification step of the targeted nucleic acids. This step is important to reach the expected sensitive detection of pathogens in diagnostic procedures. Amplifications of nucleic acid sequences are generally performed, in the presence of appropriate primers, using thermocyclers. However, the time requested to amplify molecular targets and the cost of the thermocycler machines could impair the use of these methods in routine diagnostics. Recombinase polymerase amplification (RPA) technique allows rapid (short-term incubation of sample and primers in an enzymatic mixture) and simple (isothermal) amplification of molecular targets. RPA protocol requires only basic molecular steps such as extraction procedures and agarose gel electrophoresis. Thus, RPA can be considered as an interesting alternative to standard molecular-based diagnostic tools. In this paper, the complete procedures to set up an RPA assay, applied to detection of RNA (Potato virus Y, Potyvirus) and DNA (Wheat dwarf virus, Mastrevirus) viruses, are described. The proposed procedure allows developing species- or subspecies-specific detection assay.

The nucleolar phosphoprotein B23 targets Newcastle disease virus matrix protein to the nucleoli and facilitates viral replication.

PubMed

Duan, Zhiqiang; Chen, Jian; Xu, Haixu; Zhu, Jie; Li, Qunhui; He, Liang; Liu, Huimou; Hu, Shunlin; Liu, Xiufan

2014-03-01

The cellular nucleolar proteins are reported to facilitate the replication cycles of some human and animal viruses by interaction with viral proteins. In this study, a nucleolar phosphoprotein B23 was identified to interact with Newcastle disease virus (NDV) matrix (M) protein. We found that NDV M protein accumulated in the nucleolus by binding B23 early in infection, but resulted in the redistribution of B23 from the nucleoli to the nucleoplasm later in infection. In vitro binding studies utilizing deletion mutants indicated that amino acids 30-60 of M and amino acids 188-245 of B23 were required for binding. Furthermore, knockdown of B23 by siRNA or overexpression of B23 or M-binding B23-derived polypeptides remarkably reduced cytopathic effect and inhibited NDV replication. Collectively, we show that B23 facilitates NDV replication by targeting M to the nucleolus, demonstrating for the first time a direct role for nucleolar protein B23 in a paramyxovirus replication process. Copyright © 2014 Elsevier Inc. All rights reserved.

Role for a region of helically unstable DNA within the Epstein-Barr virus latent cycle origin of DNA replication oriP in origin function

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

Polonskaya, Zhanna; Benham, Craig J.; Hearing, Janet

The minimal replicator of the Epstein-Barr virus (EBV) latent cycle origin of DNA replication oriP is composed of two binding sites for the Epstein-Barr virus nuclear antigen-1 (EBNA-1) and flanking inverted repeats that bind the telomere repeat binding factor TRF2. Although not required for minimal replicator activity, additional binding sites for EBNA-1 and TRF2 and one or more auxiliary elements located to the right of the EBNA-1/TRF2 sites are required for the efficient replication of oriP plasmids. Another region of oriP that is predicted to be destabilized by DNA supercoiling is shown here to be an important functional component ofmore » oriP. The ability of DNA fragments of unrelated sequence and possessing supercoiled-induced DNA duplex destabilized (SIDD) structures, but not fragments characterized by helically stable DNA, to substitute for this component of oriP demonstrates a role for the SIDD region in the initiation of oriP-plasmid DNA replication.« less

Receptor mimicry by antibody F045–092 facilitates universal binding to the H3 subtype of influenza virus

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

Lee, Peter S.; Ohshima, Nobuko; Stanfield, Robyn L.

Influenza viruses present a significant health challenge each year, as in the H3N2 epidemic of 2012–2013. Here we describe an antibody, F045–092, that possesses broadly neutralizing activity against the entire H3 subtype and accommodates the natural variation and additional glycosylation in all strains tested from 1963 to 2011. Crystal structures of F045–092 in complex with HAs from 1975 and 2011 H3N2 viruses reveal the structural basis for its neutralization breadth through insertion of its 23-residue HCDR3 into the receptor-binding site that involves striking receptor mimicry. F045–092 extends its recognition to divergent subtypes, including H1, H2 and H13, using the enhancedmore » avidity of its IgG to overcome lower-affinity Fab binding, as observed with other antibodies that target the receptor-binding site. This unprecedented level of antibody cross-reactivity against the H3 subtype can potentially inform on development of a pan-H3 vaccine or small-molecule therapeutics.« less

Mutational analysis reveals a noncontractile but interactive role of actin and profilin in viral RNA-dependent RNA synthesis.

PubMed

Harpen, Mary; Barik, Tiasha; Musiyenko, Alla; Barik, Sailen

2009-11-01

As obligatory parasites, viruses co-opt a variety of cellular functions for robust replication. The expression of the nonsegmented negative-strand RNA genome of respiratory syncytial virus (RSV), a significant pediatric pathogen, absolutely requires actin and is stimulated by the actin-regulatory protein profilin. As actin is a major contractile protein, it was important to determine whether the known functional domains of actin and profilin were important for their ability to activate RSV transcription. Analyses of recombinant mutants in a reconstituted RSV transcription system suggested that the divalent-cation-binding domain of actin is critically needed for binding to the RSV genome template and for the activation of viral RNA synthesis. In contrast, the nucleotide-binding domain and the N-terminal acidic domain were needed neither for template binding nor for transcription. Specific surface residues of actin, required for actin-actin contact during filamentation, were also nonessential for viral transcription. Unlike actin, profilin did not directly bind to the viral template but was recruited by actin. Mutation of the interactive residues of actin or profilin, resulting in the loss of actin-profilin binding, also abolished profilin's ability to stimulate viral transcription. Together, these results suggest that actin acts as a classical transcription factor for the virus by divalent-cation-dependent binding to the viral template and that profilin acts as a transcriptional cofactor, in part by associating with actin. This essential viral role of actin is independent of its contractile cellular role.