Influenza Virus Reassortment Is Enhanced by Semi-infectious Particles but Can Be Suppressed by Defective Interfering Particles

PubMed Central

Tao, Hui; Steel, John; Lowen, Anice C.

2015-01-01

A high particle to infectivity ratio is a feature common to many RNA viruses, with ~90–99% of particles unable to initiate a productive infection under low multiplicity conditions. A recent publication by Brooke et al. revealed that, for influenza A virus (IAV), a proportion of these seemingly non-infectious particles are in fact semi-infectious. Semi-infectious (SI) particles deliver an incomplete set of viral genes to the cell, and therefore cannot support a full cycle of replication unless complemented through co-infection. In addition to SI particles, IAV populations often contain defective-interfering (DI) particles, which actively interfere with production of infectious progeny. With the aim of understanding the significance to viral evolution of these incomplete particles, we tested the hypothesis that SI and DI particles promote diversification through reassortment. Our approach combined computational simulations with experimental determination of infection, co-infection and reassortment levels following co-inoculation of cultured cells with two distinct influenza A/Panama/2007/99 (H3N2)-based viruses. Computational results predicted enhanced reassortment at a given % infection or multiplicity of infection with increasing semi-infectious particle content. Comparison of experimental data to the model indicated that the likelihood that a given segment is missing varies among the segments and that most particles fail to deliver ≥1 segment. To verify the prediction that SI particles augment reassortment, we performed co-infections using viruses exposed to low dose UV. As expected, the introduction of semi-infectious particles with UV-induced lesions enhanced reassortment. In contrast to SI particles, inclusion of DI particles in modeled virus populations could not account for observed reassortment outcomes. DI particles were furthermore found experimentally to suppress detectable reassortment, relative to that seen with standard virus stocks, most likely by

Comparison of Influenza Virus Particle Purification Using Magnetic Sulfated Cellulose Particles with an Established Centrifugation Method for Analytics.

PubMed

Serve, Anja; Pieler, Michael Martin; Benndorf, Dirk; Rapp, Erdmann; Wolff, Michael Werner; Reichl, Udo

2015-11-03

A method for the purification of influenza virus particles using novel magnetic sulfated cellulose particles is presented and compared to an established centrifugation method for analytics. Therefore, purified influenza A virus particles from adherent and suspension MDCK host cell lines were characterized on the protein level with mass spectrometry to compare the viral and residual host cell proteins. Both methods allowed one to identify all 10 influenza A virus proteins, including low-abundance proteins like the matrix protein 2 and nonstructural protein 1, with a similar impurity level of host cell proteins. Compared to the centrifugation method, use of the novel magnetic sulfated cellulose particles reduced the influenza A virus particle purification time from 3.5 h to 30 min before mass spectrometry analysis.

Visualizing interactions between Sindbis virus and cells by single particle tracking

NASA Astrophysics Data System (ADS)

Williard, Mary

2005-03-01

Sindbis virus infects both mammalian and insect cells. Though not pathogenic in humans, Sindbis is a model for many mosquito- borne viruses that cause human disease, such as West Nile virus. We have used real-time single particle fluorescence microscopy to observe individual Sindbis virus particles as they infect living cells. Fluorescent labels were incorporated into both the viral coat proteins and the lipid envelope of the virus. Kinetics characteristic of free diffusion in solution, slower diffusion inside cells, attachment to spots on the cell surface, and motor protein transport inside cells have been observed. Dequenching of the membrane label is used to report membrane fusion events during the infection process. Tracking individual viral particles allows multiple pathways to be determined without the requirement of synchronicity.

Cellular Antiviral Factors that Target Particle Infectivity of HIV-1.

PubMed

Goffinet, Christine

2016-01-01

In the past decade, the identification and characterization of antiviral genes with the ability to interfere with virus replication has established cell-intrinsic innate immunity as a third line of antiviral defense in addition to adaptive and classical innate immunity. Understanding how cellular factors have evolved to inhibit HIV-1 reveals particularly vulnerable points of the viral replication cycle. Many, but not all, antiviral proteins share type I interferon-upregulated expression and sensitivity to viral counteraction or evasion measures. Whereas well-established restriction factors interfere with early post-entry steps and release of HIV-1, recent research has revealed a diverse set of proteins that reduce the infectious quality of released particles using individual, to date poorly understood modes of action. These include induction of paucity of mature glycoproteins in nascent virions or self-incorporation into the virus particle, resulting in poor infectiousness of the virion and impaired spread of the infection. A better understanding of these newly discovered antiviral factors may open new avenues towards the design of drugs that repress the spread of viruses whose genomes have already integrated.

Virus Particle Detection by Convolutional Neural Network in Transmission Electron Microscopy Images.

PubMed

Ito, Eisuke; Sato, Takaaki; Sano, Daisuke; Utagawa, Etsuko; Kato, Tsuyoshi

2018-06-01

A new computational method for the detection of virus particles in transmission electron microscopy (TEM) images is presented. Our approach is to use a convolutional neural network that transforms a TEM image to a probabilistic map that indicates where virus particles exist in the image. Our proposed approach automatically and simultaneously learns both discriminative features and classifier for virus particle detection by machine learning, in contrast to existing methods that are based on handcrafted features that yield many false positives and require several postprocessing steps. The detection performance of the proposed method was assessed against a dataset of TEM images containing feline calicivirus particles and compared with several existing detection methods, and the state-of-the-art performance of the developed method for detecting virus was demonstrated. Since our method is based on supervised learning that requires both the input images and their corresponding annotations, it is basically used for detection of already-known viruses. However, the method is highly flexible, and the convolutional networks can adapt themselves to any virus particles by learning automatically from an annotated dataset.

Zika Virus Baculovirus-Expressed Virus-Like Particles Induce Neutralizing Antibodies in Mice.

PubMed

Dai, Shiyu; Zhang, Tao; Zhang, Yanfang; Wang, Hualin; Deng, Fei

2018-06-01

The newly emerged mosquito-borne Zika virus (ZIKV) strains pose a global challenge owing to its ability to cause microcephaly and neurological disorders. Several ZIKV vaccine candidates have been proposed, including inactivated and live attenuated virus vaccines, vector-based vaccines, DNA and RNA vaccines. These have been shown to be efficacious in preclinical studies in mice and nonhuman primates, but their use will potentially be a threat to immunocompromised individuals and pregnant women. Virus-like particles (VLPs) are empty particles composed merely of viral proteins, which can serve as a safe and valuable tool for clinical prevention and treatment strategies. In this study, we used a new strategy to produce ZIKV VLPs based on the baculovirus expression system and demonstrated the feasibility of their use as a vaccine candidate. The pre-membrane (prM) and envelope (E) proteins were co-expressed in insect cells and self-assembled into particles similar to ZIKV. We found that the ZIKV VLPs could be quickly and easily prepared in large quantities using this system. The VLPs were shown to have good immunogenicity in immunized mice, as they stimulated high levels of virus neutralizing antibody titers, ZIKV-specific IgG titers and potent memory T cell responses. Thus, the baculovirus-based ZIKV VLP vaccine is a safe, effective and economical vaccine candidate for use against ZIKV.

Nascent Phosphorus Oxide

NASA Astrophysics Data System (ADS)

Sumida, David Shuji

PO(X('2)(PI)) is produced via the collision-free infrared multiple photon dissociation (IRMPD) of volatile organophosphorus molecules, and is detected by 2-frequency 2-photon ionization, using the B('2)(SIGMA)('+) state to provide a spectral signature from which X('2)(PI) populations are obtained. Sequential dissociations occur during the IR laser photolysis, in which nascent fragments continue to undergo IRMPD, and PO(X('2)(PI)) accrues from a series of bond fission reactions. Nascent vibrational, rotational, and translational excitations are in sensible accord with this mechanism, except for a few rotational states near J = 19.5. Unlike the nuclear degrees of freedom, the PO(X('2)(PI)) spin-orbit states are populated quite selectively. The ('2)(PI)(,3/2) state, lying only 224 cm('-1) above the ('2)(PI)(,1/2) ground state, contains only (TURN)11% of the population, compared to 34% for a 300K sample. This result is unambiguous; it persists with all precursors, laser fluences, etc., and is verified by comparisons to spectra obtained using a microwave discharge, a flame, and when thermalizing nascent excitations with an inert diluent. This result underscores the sanctity of the separate potential surfaces which correlate to the product spin -orbit states, and the small amount of ('2)(PI)(,3/2) population can be accounted for by non-adiabatic coupling during dissociation, and/or 'freezing' the amount of S(,1) character in an excited precursor in which S(,0) and S(,1) are coupled non-radiatively. We note that such electronic specificity should be dealt with in the analogous recombination reactions. (Copies available exclusively from Micrographics Department, Doheny Library, USC, Los Angeles, CA 90089.).

Evidence that Hsc70 Is Associated with Cucumber Necrosis Virus Particles and Plays a Role in Particle Disassembly

PubMed Central

Alam, Syed Benazir

2016-01-01

ABSTRACT Uncoating of a virus particle to expose its nucleic acid is a critical aspect of the viral multiplication cycle, as it is essential for the establishment of infection. In the present study, we investigated the role of plant HSP70 homologs in the uncoating process of Cucumber necrosis virus (CNV), a nonenveloped positive-sense single-stranded RNA [(+)ssRNA] virus having a T=3 icosahedral capsid. We have found through Western blot analysis and mass spectrometry that the HSP70 homolog Hsc70-2 copurifies with CNV particles. Virus overlay and immunogold labeling assays suggest that Hsc70-2 is physically bound to virions. Furthermore, trypsin digestion profiles suggest that the bound Hsc70-2 is partially protected by the virus, indicating an intimate association with particles. In investigating a possible role of Hsc70-2 in particle disassembly, we showed that particles incubated with Hsp70/Hsc70 antibody produce fewer local lesions than those incubated with prebleed control antibody on Chenopodium quinoa. In conjunction, CNV virions purified using CsCl and having undetectable amounts of Hsc70-2 produce fewer local lesions. We also have found that plants with elevated levels of HSP70/Hsc70 produce higher numbers of local lesions following CNV inoculation. Finally, incubation of recombinant Nicotiana benthamiana Hsc70-2 with virus particles in vitro leads to conformational changes or partial disassembly of capsids as determined by transmission electron microscopy, and particles are more sensitive to chymotrypsin digestion. This is the first report suggesting that a cellular Hsc70 chaperone is involved in disassembly of a plant virus. IMPORTANCE Virus particles must disassemble and release their nucleic acid in order to establish infection in a cell. Despite the importance of disassembly in the ability of a virus to infect its host, little is known about this process, especially in the case of nonenveloped spherical RNA viruses. Previous work has shown that host

Immunogenicity and efficacy of immunodeficiency virus-like particles pseudotyped with the G protein of vesicular stomatitis virus

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

Kuate, Seraphin; Stahl-Hennig, Christiane; Stoiber, Heribert

2006-07-20

Vaccination with exogenous antigens such as recombinant viral proteins, immunodeficiency virus-derived whole inactivated virus particles, or virus-like particles (VLP) has generally failed to provide sufficient protection in animal models for AIDS. Pseudotyping VLPs with the vesicular stomatitis virus G protein (VSV-G), which is known to mediate entry into dendritic cells, might allow more efficient stimulation of immune responses. Therefore, we pseudotyped noninfectious immunodeficiency virus-like particles with VSV-G and carried out a preliminary screen of their immunogenicity and vaccination efficacy. Incorporation of VSV-G into HIV-1 VLPs led to hundred-fold higher antibody titers to HIV-1 Gag and enhancement of T cell responsesmore » in mice. Repeated vaccination of rhesus monkeys for 65 weeks with VSV-G pseudotyped simian immunodeficiency virus (SIV)-like particles (VLP[G]) provided initial evidence for efficient suppression of viral load after mucosal challenge with the SIVmac239 virus. Challenge of monkeys after a 28 week vaccination regimen with VLP[G] led to a reduction in peak viremia, but persistent suppression of viral load was not achieved. Due to limitations in the number of animals available for this study, improved efficacy of VSV-G pseudotyped VLPs in nonhuman primates could not be demonstrated. However, mouse experiments revealed that pseudotyping of VLPs with fusion-competent VSV-G clearly improves their immunogenicity. Additional strategies, particularly adjuvants, should be considered to provide greater protection against a challenge with pathogenic immunodeficiency virus.« less

Characterization of the Lassa virus matrix protein Z: electron microscopic study of virus-like particles and interaction with the nucleoprotein (NP).

PubMed

Eichler, Robert; Strecker, Thomas; Kolesnikova, Larissa; ter Meulen, Jan; Weissenhorn, Winfried; Becker, Stephan; Klenk, Hans Dieter; Garten, Wolfgang; Lenz, Oliver

2004-03-15

Lassa virus is the causative agent of a hemorrhagic fever endemic in west Africa. The RNA genome of Lassa virus encodes the glycoprotein precursor GP-C, a nucleoprotein (NP), the viral polymerase L and a small protein Z (11 kDa). Here, we analyze the role of Z protein for virus maturation. We have recently shown that expression of Z protein in the absence of other viral proteins is sufficient for the release of enveloped Z-containing particles. In this study, we examined particles secreted into the supernatant of a stably Z protein-expressing CHO cell line by electron microscopy. The observed Z-induced virus-like particles did not significantly differ in their morphology and size from Lassa virus particles. Mutation of two proline-rich domains within Z which are known to drastically reduce the release of virus-like particles, had no effect on the cellular localization of the protein nor on its membrane-association. Furthermore, we present evidence that Z interacts with the NP. We assume that Z recruits NP to cellular membranes where virus assembly takes place. We conclude from our data that Lassa virus Z protein plays an essential role in Lassa virus maturation.

Oligonucleotide Length-Dependent Formation of Virus-Like Particles.

PubMed

Maassen, Stan J; de Ruiter, Mark V; Lindhoud, Saskia; Cornelissen, Jeroen J L M

2018-05-23

Understanding the assembly pathway of viruses can contribute to creating monodisperse virus-based materials. In this study, the cowpea chlorotic mottle virus (CCMV) is used to determine the interactions between the capsid proteins of viruses and their cargo. The assembly of the capsid proteins in the presence of different lengths of short, single-stranded (ss) DNA is studied at neutral pH, at which the protein-protein interactions are weak. Chromatography, electrophoresis, microscopy, and light scattering data show that the assembly efficiency and speed of the particles increase with increasing length of oligonucleotides. The minimal length required for assembly under the conditions used herein is 14 nucleotides. Assembly of particles containing such short strands of ssDNA can take almost a month. This slow assembly process enabled the study of intermediate states, which confirmed a low cooperative assembly for CCMV and allowed for further expansion of current assembly theories. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Localization and force analysis at the single virus particle level using atomic force microscopy

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

Liu, Chih-Hao; Horng, Jim-Tong; Chang, Jeng-Shian

2012-01-06

Highlights: Black-Right-Pointing-Pointer Localization of single virus particle. Black-Right-Pointing-Pointer Force measurements. Black-Right-Pointing-Pointer Force mapping. -- Abstract: Atomic force microscopy (AFM) is a vital instrument in nanobiotechnology. In this study, we developed a method that enables AFM to simultaneously measure specific unbinding force and map the viral glycoprotein at the single virus particle level. The average diameter of virus particles from AFM images and the specificity between the viral surface antigen and antibody probe were integrated to design a three-stage method that sets the measuring area to a single virus particle before obtaining the force measurements, where the influenza virus was usedmore » as the object of measurements. Based on the purposed method and performed analysis, several findings can be derived from the results. The mean unbinding force of a single virus particle can be quantified, and no significant difference exists in this value among virus particles. Furthermore, the repeatability of the proposed method is demonstrated. The force mapping images reveal that the distributions of surface viral antigens recognized by antibody probe were dispersed on the whole surface of individual virus particles under the proposed method and experimental criteria; meanwhile, the binding probabilities are similar among particles. This approach can be easily applied to most AFM systems without specific components or configurations. These results help understand the force-based analysis at the single virus particle level, and therefore, can reinforce the capability of AFM to investigate a specific type of viral surface protein and its distributions.« less

Recombinant Modified Vaccinia Virus Ankara Generating Ebola Virus-Like Particles.

PubMed

Schweneker, Marc; Laimbacher, Andrea S; Zimmer, Gert; Wagner, Susanne; Schraner, Elisabeth M; Wolferstätter, Michael; Klingenberg, Marieken; Dirmeier, Ulrike; Steigerwald, Robin; Lauterbach, Henning; Hochrein, Hubertus; Chaplin, Paul; Suter, Mark; Hausmann, Jürgen

2017-06-01

There are currently no approved therapeutics or vaccines to treat or protect against the severe hemorrhagic fever and death caused by Ebola virus (EBOV). Ebola virus-like particles (EBOV VLPs) consisting of the matrix protein VP40, the glycoprotein (GP), and the nucleoprotein (NP) are highly immunogenic and protective in nonhuman primates against Ebola virus disease (EVD). We have constructed a modified vaccinia virus Ankara-Bavarian Nordic (MVA-BN) recombinant coexpressing VP40 and GP of EBOV Mayinga and the NP of Taï Forest virus (TAFV) (MVA-BN-EBOV-VLP) to launch noninfectious EBOV VLPs as a second vaccine modality in the MVA-BN-EBOV-VLP-vaccinated organism. Human cells infected with either MVA-BN-EBOV-VLP or MVA-BN-EBOV-GP showed comparable GP expression levels and transport of complex N-glycosylated GP to the cell surface. Human cells infected with MVA-BN-EBOV-VLP produced large amounts of EBOV VLPs that were decorated with GP spikes but excluded the poxviral membrane protein B5, thus resembling authentic EBOV particles. The heterologous TAFV NP enhanced EBOV VP40-driven VLP formation with efficiency similar to that of the homologous EBOV NP in a transient-expression assay, and both NPs were incorporated into EBOV VLPs. EBOV GP-specific CD8 T cell responses were comparable between MVA-BN-EBOV-VLP- and MVA-BN-EBOV-GP-immunized mice. The levels of EBOV GP-specific neutralizing and binding antibodies, as well as GP-specific IgG1/IgG2a ratios induced by the two constructs, in mice were also similar, raising the question whether the quality rather than the quantity of the GP-specific antibody response might be altered by an EBOV VLP-generating MVA recombinant. IMPORTANCE The recent outbreak of Ebola virus (EBOV), claiming more than 11,000 lives, has underscored the need to advance the development of safe and effective filovirus vaccines. Virus-like particles (VLPs), as well as recombinant viral vectors, have proved to be promising vaccine candidates. Modified

Prophylaxis and Immunization in Mice by Use of Virus-Free Defective T Particles to Protect Against Intracerebral Infection by Vesicular Stomatitis Virus

PubMed Central

Doyle, Michael; Holland, John J.

1973-01-01

Defective interfering T particles of vesicular stomatitis virus provide remarkable protection against viral disease and death when introduced intracerebrally in large numbers along with an otherwise rapidly fatal low dose of standard infectious virus. This profound prophylactic effect of defective T particles is due to homologous autointerference since it is serotype-specific and interferon is not induced. This protective effect can be demonstrated only with preparations of T particles that have been purified completely free of infectious virions. When pure T particles are injected intracerebrally along with large doses of infectious virus, they convert an otherwise rapidly fatal disease process to a slowly progressing virus infection that generally terminates in death after many days of wasting disease and paralysis. Intracerebral injection of virus-free T particles alone is apparently innocuous to mice and stimulates immunity to massive doses of homologous infectious virus. In vitro, virus-free T particles at extremely high multiplicities depress cellular RNA and protein synthesis and kill BHK21 cells in culture, but do not exhibit such effects at moderately high multiplicities. PMID:4352972

Selective counting and sizing of single virus particles using fluorescent aptamer-based nanoparticle tracking analysis.

PubMed

Szakács, Zoltán; Mészáros, Tamás; de Jonge, Marien I; Gyurcsányi, Róbert E

2018-05-30

Detection and counting of single virus particles in liquid samples are largely limited to narrow size distribution of viruses and purified formulations. To address these limitations, here we propose a calibration-free method that enables concurrently the selective recognition, counting and sizing of virus particles as demonstrated through the detection of human respiratory syncytial virus (RSV), an enveloped virus with a broad size distribution, in throat swab samples. RSV viruses were selectively labeled through their attachment glycoproteins (G) with fluorescent aptamers, which further enabled their identification, sizing and counting at the single particle level by fluorescent nanoparticle tracking analysis. The proposed approach seems to be generally applicable to virus detection and quantification. Moreover, it could be successfully applied to detect single RSV particles in swab samples of diagnostic relevance. Since the selective recognition is associated with the sizing of each detected particle, this method enables to discriminate viral elements linked to the virus as well as various virus forms and associations.

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

PubMed

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

2014-11-01

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

Cytoplasmic Motifs in the Nipah Virus Fusion Protein Modulate Virus Particle Assembly and Egress.

PubMed

Johnston, Gunner P; Contreras, Erik M; Dabundo, Jeffrey; Henderson, Bryce A; Matz, Keesha M; Ortega, Victoria; Ramirez, Alfredo; Park, Arnold; Aguilar, Hector C

2017-05-15

Nipah virus (NiV), a paramyxovirus in the genus Henipavirus , has a mortality rate in humans of approximately 75%. While several studies have begun our understanding of NiV particle formation, the mechanism of this process remains to be fully elucidated. For many paramyxoviruses, M proteins drive viral assembly and egress; however, some paramyxoviral glycoproteins have been reported as important or essential in budding. For NiV the matrix protein (M), the fusion glycoprotein (F) and, to a much lesser extent, the attachment glycoprotein (G) autonomously induce the formation of virus-like particles (VLPs). However, functional interactions between these proteins during assembly and egress remain to be fully understood. Moreover, if the F-driven formation of VLPs occurs through interactions with host cell machinery, the cytoplasmic tail (CT) of F is a likely interactive domain. Therefore, we analyzed NiV F CT deletion and alanine mutants and report that several but not all regions of the F CT are necessary for efficient VLP formation. Two of these regions contain YXXØ or dityrosine motifs previously shown to interact with cellular machinery involved in F endocytosis and transport. Importantly, our results showed that F-driven, M-driven, and M/F-driven viral particle formation enhanced the recruitment of G into VLPs. By identifying key motifs, specific residues, and functional viral protein interactions important for VLP formation, we improve our understanding of the viral assembly/egress process and point to potential interactions with host cell machinery. IMPORTANCE Henipaviruses can cause deadly infections of medical, veterinary, and agricultural importance. With recent discoveries of new henipa-like viruses, understanding the mechanisms by which these viruses reproduce is paramount. We have focused this study on identifying the functional interactions of three Nipah virus proteins during viral assembly and particularly on the role of one of these proteins, the

Remarkable morphological diversity of viruses and virus-like particles in hot terrestrial environments.

PubMed

Rachel, R; Bettstetter, M; Hedlund, B P; Häring, M; Kessler, A; Stetter, K O; Prangishvili, D

2002-12-01

Electron microscopic studies of the viruses in two hot springs (85 degrees C, pH 1.5-2.0, and 75-93 degrees C, pH 6.5) in Yellowstone National Park revealed particles with twelve different morphotypes. This diversity encompassed known viruses of hyperthermophilic archaea, filamentous Lipothrixviridae, rod-shaped Rudiviridae, and spindle-shaped Fuselloviridae, and novel morphotypes previously not observed in nature. Two virus types resembled head-and-tail bacteriophages from the families Siphoviridae and Podoviridae, and constituted the first observation of these viruses in a hydrothermal environment. Viral hosts in the acidic spring were members of the hyperthermophilic archaeal genus Acidianus.

Viruses and viruslike particles of eukaryotic algae.

PubMed Central

Van Etten, J L; Lane, L C; Meints, R H

1991-01-01

Until recently there was little interest or information on viruses and viruslike particles of eukaryotic algae. However, this situation is changing. In the past decade many large double-stranded DNA-containing viruses that infect two culturable, unicellular, eukaryotic green algae have been discovered. These viruses can be produced in large quantities, assayed by plaque formation, and analyzed by standard bacteriophage techniques. The viruses are structurally similar to animal iridoviruses, their genomes are similar to but larger (greater than 300 kbp) than that of poxviruses, and their infection process resembles that of bacteriophages. Some of the viruses have DNAs with low levels of methylated bases, whereas others have DNAs with high concentrations of 5-methylcytosine and N6-methyladenine. Virus-encoded DNA methyltransferases are associated with the methylation and are accompanied by virus-encoded DNA site-specific (restriction) endonucleases. Some of these enzymes have sequence specificities identical to those of known bacterial enzymes, and others have previously unrecognized specificities. A separate rod-shaped RNA-containing algal virus has structural and nucleotide sequence affinities to higher plant viruses. Quite recently, viruses have been associated with rapid changes in marine algal populations. In the next decade we envision the discovery of new algal viruses, clarification of their role in various ecosystems, discovery of commercially useful genes in these viruses, and exploitation of algal virus genetic elements in plant and algal biotechnology. Images PMID:1779928

Tomato Spotted Wilt Virus Particle Morphogenesis in Plant Cells

PubMed Central

Kikkert, Marjolein; Van Lent, Jan; Storms, Marc; Bodegom, Pentcho; Kormelink, Richard; Goldbach, Rob

1999-01-01

A model for the maturation of tomato spotted wilt virus (TSWV) particles is proposed, mainly based on results with a protoplast infection system, in which the chronology of different maturation events could be determined. By using specific monoclonal and polyclonal antisera in immunofluorescence and electron microscopy, the site of TSWV particle morphogenesis was determined to be the Golgi system. The viral glycoproteins G1 and G2 accumulate in the Golgi prior to a process of wrapping, by which the viral nucleocapsids obtain a double membrane. In a later stage of the maturation, these doubly enveloped particles fuse to each other and to the endoplasmic reticulum to form singly enveloped particles clustered in membranes. Similarities and differences between the maturation of animal-infecting (bunya)viruses and plant-infecting tospoviruses are discussed. PMID:9971812

Virus-like particles as nanovaccine candidates

NASA Astrophysics Data System (ADS)

Guillen, G.; Aguilar, J. C.; Dueñas, S.; Hermida, L.; Iglesias, E.; Penton, E.; Lobaina, Y.; Lopez, M.; Mussachio, A.; Falcon, V.; Alvarez, L.; Martinez, G.; Gil, L.; Valdes, I.; Izquierdo, A.; Lazo, L.; Marcos, E.; Guzman, G.; Muzio, V.; Herrera, L.

2013-03-01

The existing vaccines are mainly limited to the microorganisms we are able to culture and produce and/or to those whose killing is mediated by humoral response (antibody mediated). It has been more difficult to develop vaccines capable of inducing a functional cellular response needed to prevent or cure chronic diseases. New strategies should be taken into account in the improvement of cell-based immune responses in order to prevent and control the infections and eventually clear the virus. Preclinical and clinical results with vaccine candidates developed as a vaccine platform based on virus-like particles (VLPs) evidenced their ability to stimulate mucosal as well as systemic immunity. Particles based on envelope, membrane or nucleocapsid microbial proteins induce a strong immune response after nasal or parenteral administration in mice, non-human primates and humans. In addition, the immune response obtained was modulated in a Th1 sense. The VLPs were also able to immunoenhance the humoral and cellular immune responses against several viral pathogens. Studies in animals and humans with nasal and systemic formulations evidenced that it is possible to induce functional immune response against HBV, HCV, HIV and dengue virus. Invited talk at the 6th International Workshop on Advanced Materials Science and Nanotechnology, 30 October - 2 November 2012, Ha Long, Vietnam.

Seipin is required for converting nascent to mature lipid droplets

PubMed Central

Wang, Huajin; Becuwe, Michel; Housden, Benjamin E; Chitraju, Chandramohan; Porras, Ashley J; Graham, Morven M; Liu, Xinran N; Thiam, Abdou Rachid; Savage, David B; Agarwal, Anil K; Garg, Abhimanyu; Olarte, Maria-Jesus; Lin, Qingqing; Fröhlich, Florian; Hannibal-Bach, Hans Kristian; Upadhyayula, Srigokul; Perrimon, Norbert; Kirchhausen, Tomas; Ejsing, Christer S; Walther, Tobias C; Farese, Robert V

2016-01-01

How proteins control the biogenesis of cellular lipid droplets (LDs) is poorly understood. Using Drosophila and human cells, we show here that seipin, an ER protein implicated in LD biology, mediates a discrete step in LD formation—the conversion of small, nascent LDs to larger, mature LDs. Seipin forms discrete and dynamic foci in the ER that interact with nascent LDs to enable their growth. In the absence of seipin, numerous small, nascent LDs accumulate near the ER and most often fail to grow. Those that do grow prematurely acquire lipid synthesis enzymes and undergo expansion, eventually leading to the giant LDs characteristic of seipin deficiency. Our studies identify a discrete step of LD formation, namely the conversion of nascent LDs to mature LDs, and define a molecular role for seipin in this process, most likely by acting at ER-LD contact sites to enable lipid transfer to nascent LDs. DOI: http://dx.doi.org/10.7554/eLife.16582.001 PMID:27564575

Zika virus-like particle (VLP) based vaccine

PubMed Central

Boigard, Hélène; Alimova, Alexandra; Martin, George R.; Katz, Al; Gottlieb, Paul

2017-01-01

The newly emerged mosquito-borne Zika virus poses a major public challenge due to its ability to cause significant birth defects and neurological disorders. The impact of sexual transmission is unclear but raises further concerns about virus dissemination. No specific treatment or vaccine is currently available, thus the development of a safe and effective vaccine is paramount. Here we describe a novel strategy to assemble Zika virus-like particles (VLPs) by co-expressing the structural (CprME) and non-structural (NS2B/NS3) proteins, and demonstrate their effectiveness as vaccines. VLPs are produced in a suspension culture of mammalian cells and self-assembled into particles closely resembling Zika viruses as shown by electron microscopy studies. We tested various VLP vaccines and compared them to analogous compositions of an inactivated Zika virus (In-ZIKV) used as a reference. VLP immunizations elicited high titers of antibodies, as did the In-ZIKV controls. However, in mice the VLP vaccine stimulated significantly higher virus neutralizing antibody titers than comparable formulations of the In-ZIKV vaccine. The serum neutralizing activity elicited by the VLP vaccine was enhanced using a higher VLP dose and with the addition of an adjuvant, reaching neutralizing titers greater than those detected in the serum of a patient who recovered from a Zika infection in Brazil in 2015. Discrepancies in neutralization levels between the VLP vaccine and the In-ZIKV suggest that chemical inactivation has deleterious effects on neutralizing epitopes within the E protein. This along with the inability of a VLP vaccine to cause infection makes it a preferable candidate for vaccine development. PMID:28481898

How Messenger RNA and Nascent Chain Sequences Regulate Translation Elongation.

PubMed

Choi, Junhong; Grosely, Rosslyn; Prabhakar, Arjun; Lapointe, Christopher P; Wang, Jinfan; Puglisi, Joseph D

2018-06-20

Translation elongation is a highly coordinated, multistep, multifactor process that ensures accurate and efficient addition of amino acids to a growing nascent-peptide chain encoded in the sequence of translated messenger RNA (mRNA). Although translation elongation is heavily regulated by external factors, there is clear evidence that mRNA and nascent-peptide sequences control elongation dynamics, determining both the sequence and structure of synthesized proteins. Advances in methods have driven experiments that revealed the basic mechanisms of elongation as well as the mechanisms of regulation by mRNA and nascent-peptide sequences. In this review, we highlight how mRNA and nascent-peptide elements manipulate the translation machinery to alter the dynamics and pathway of elongation.

Presence of Two Virus-Like Particles in Penicillium citrinum

PubMed Central

Volterra, L.; Cassone, A.; Tonolo, A.; Bruzzone, M. L.

1975-01-01

Two icosahedral virus-like particles (28 and 19 nm in diameter, respectively) have been detected in sporogenic and asporogenic segregants of a strain of Penicillium citrinum. The distribution of the two particles differed among the two segregants. Images PMID:50049

Self-assembled virus-like particles with magnetic cores.

PubMed

Huang, Xinlei; Bronstein, Lyudmila M; Retrum, John; Dufort, Chris; Tsvetkova, Irina; Aniagyei, Stella; Stein, Barry; Stucky, Galen; McKenna, Brandon; Remmes, Nicholas; Baxter, David; Kao, C Cheng; Dragnea, Bogdan

2007-08-01

Efficient encapsulation of functionalized spherical nanoparticles by viral protein cages was found to occur even if the nanoparticle is larger than the inner cavity of the native capsid. This result raises the intriguing possibility of reprogramming the self-assembly of viral structural proteins. The iron oxide nanotemplates used in this work are superparamagnetic, with a blocking temperature of about 250 K, making these virus-like particles interesting for applications such as magnetic resonance imaging and biomagnetic materials. Another novel feature of the virus-like particle assembly described in this work is the use of an anionic lipid micelle coat instead of a molecular layer covalently bound to the inorganic nanotemplate. Differences between the two functionalization strategies are discussed.

Virus-like particles as universal influenza vaccines

PubMed Central

Kang, Sang-Moo; Kim, Min-Chul; Compans, Richard W

2012-01-01

Current influenza vaccines are primarily targeted to induce immunity to the influenza virus strain-specific hemagglutinin antigen and are not effective in controlling outbreaks of new pandemic viruses. An approach for developing universal vaccines is to present highly conserved antigenic epitopes in an immunogenic conformation such as virus-like particles (VLPs) together with an adjuvant to enhance the vaccine immunogenicity. In this review, the authors focus on conserved antigenic targets and molecular adjuvants that were presented in VLPs. Conserved antigenic targets that include the hemagglutinin stalk domain, the external domain of influenza M2 and neuraminidase are discussed in addition to molecular adjuvants that are engineered to be incorporated into VLPs in a membrane-anchored form. PMID:23002980

A novel compound inhibits rHDL assembly and blocks nascent HDL biogenesis downstream of apoAI binding to ABCA1 expressing cells

PubMed Central

Lyssenko, Nicholas N.; Brubaker, Gregory; Smith, Bradley D.; Smith, Jonathan D.

2011-01-01

Objective Nascent high-density lipoprotein (HDL) particles form from cellular lipids and extracellular lipid-free apolipoprotein AI (apoAI) in a process mediated by ATP-binding cassette transporter A1 (ABCA1). We have sought out compounds that inhibit nascent HDL biogenesis without affecting ABCA1 activity. Methods and Results Reconstituted HDL (rHDL) formation and cellular cholesterol efflux assays were used to show that two compounds that bond via hydrogen with phospholipids inhibit rHDL and nascent HDL production. In rHDL formation assays, the inhibitory effect of compound 1 (methyl 3α-acetoxy-7α,12α-di[(phenylaminocarbonyl)amino]-5β-cholan-24-oate), the more active of the two, depended on its ability to associate with phospholipids. In cell assays, compound 1 suppressed ABCA1-mediated cholesterol efflux to apoAI, the 18A peptide, and taurocholate with high specificity, without affecting ABCA1-independent cellular cholesterol efflux to HDL and endocytosis of acetylated low-density lipoprotein (AcLDL) and transferrin. Furthermore, compound 1 did not affect ABCA1 activity adversely, as ABCA1-mediated shedding of microparticles proceeded unabated and apoAI binding to ABCA1-expressing cells increased in its presence. Conclusions The inhibitory effects of compound 1 support a three-step model of nascent HDL biogenesis: plasma membrane remodeling by ABCA1, apoAI binding to ABCA1, and lipoprotein particle assembly. The compound inhibits the final step, causing accumulation of apoAI in ABCA1-expressing cells. PMID:21836073

Virus-like particles as a vaccine delivery system: myths and facts.

PubMed

Roy, Polly; Noad, Rob

2009-01-01

Vaccines against viral disease have traditionally relied on attenuated virus strains or inactivation of infectious virus. Subunit vaccines based on viral proteins expressed in heterologous systems have been effective for some pathogens, but have often suffered from poor immunogenicity due to incorrect protein folding or modification. In this chapter we focus on a specific class of viral subunit vaccine that mimics the overall structure of virus particles and thus preserves the native antigenic conformation of the immunogenic proteins. These virus-like particles (VLPs) have been produced for a wide range of taxonomically and structurally distinct viruses, and have unique advantages in terms of safety and immunogenicity over previous approaches. With new VLP vaccines for papillomavirus beginning to reach the market place we argue that this technology has now 'come-of-age' and must be considered a viable vaccine strategy.

Single-Particle Detection of Transcription following Rotavirus Entry

PubMed Central

Salgado, Eric N.; Upadhyayula, Srigokul

2017-01-01

ABSTRACT Infectious rotavirus particles are triple-layered, icosahedral assemblies. The outer layer proteins, VP4 (cleaved to VP8* and VP5*) and VP7, surround a transcriptionally competent, double-layer particle (DLP), which they deliver into the cytosol. During entry of rhesus rotavirus, VP8* interacts with cell surface gangliosides, allowing engulfment into a membrane vesicle by a clathrin-independent process. Escape into the cytosol and outer-layer shedding depend on interaction of a hydrophobic surface on VP5* with the membrane bilayer and on a large-scale conformational change. We report here experiments that detect the fate of released DLPs and their efficiency in initiating RNA synthesis. By replacing the outer layer with fluorescently tagged, recombinant proteins and also tagging the DLP, we distinguished particles that have lost their outer layer and entered the cytosol (uncoated) from those still within membrane vesicles. We used fluorescent in situ hybridization with probes for nascent transcripts to determine how soon after uncoating transcription began and what fraction of the uncoated particles were active in initiating RNA synthesis. We detected RNA synthesis by uncoated particles as early as 15 min after adding virus. The uncoating efficiency was 20 to 50%; of the uncoated particles, about 10 to 15% synthesized detectable RNA. In the format of our experiments, about 10% of the added particles attached to the cell surface, giving an overall ratio of added particles to RNA-synthesizing particles of between 250:1 and 500:1, in good agreement with the ratio of particles to focus-forming units determined by infectivity assays. Thus, RNA synthesis by even a single, uncoated particle can initiate infection in a cell. IMPORTANCE The pathways by which a virus enters a cell transform its packaged genome into an active one. Contemporary fluorescence microscopy can detect individual virus particles as they enter cells, allowing us to map their multistep entry

ALIX/AIP1 is required for NP incorporation into Mopeia virus Z-induced virus-like particles.

PubMed

Shtanko, Olena; Watanabe, Shinji; Jasenosky, Luke D; Watanabe, Tokiko; Kawaoka, Yoshihiro

2011-04-01

During virus particle assembly, the arenavirus nucleoprotein (NP) associates with the viral genome to form nucleocapsids, which ultimately become incorporated into new virions at the cell membrane. Virion release is facilitated by the viral matrix Z protein through its interaction with the cellular endosomal sorting complex required for transport (ESCRT) machinery. However, the mechanism of nucleocapsid incorporation into virions is not well understood. Here, we demonstrate that ALIX/AIP1, an ESCRT-associated host protein, is required for the incorporation of the NP of Mopeia virus, a close relative of Lassa virus, into Z-induced virus-like particles (VLPs). Furthermore, we show that the Bro1 domain of ALIX/AIP1 interacts with the NP and Z proteins simultaneously, facilitating their interaction, and we identify residues 342 to 399 of NP as being necessary for its interaction with ALIX/AIP1. Our observations suggest a potential role for ALIX/AIP1 in linking Mopeia virus NP to Z and the budding apparatus, thereby promoting NP incorporation into virions.

Pervasive Targeting of Nascent Transcripts by Hfq.

PubMed

Kambara, Tracy K; Ramsey, Kathryn M; Dove, Simon L

2018-05-01

Hfq is an RNA chaperone and an important post-transcriptional regulator in bacteria. Using chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-seq), we show that Hfq associates with hundreds of different regions of the Pseudomonas aeruginosa chromosome. These associations are abolished when transcription is inhibited, indicating that they reflect Hfq binding to transcripts during their synthesis. Analogous ChIP-seq analyses with the post-transcriptional regulator Crc reveal that it associates with many of the same nascent transcripts as Hfq, an activity we show is Hfq dependent. Our findings indicate that Hfq binds many transcripts co-transcriptionally in P. aeruginosa, often in concert with Crc, and uncover direct regulatory targets of these proteins. They also highlight a general approach for studying the interactions of RNA-binding proteins with nascent transcripts in bacteria. The binding of post-transcriptional regulators to nascent mRNAs may represent a prevalent means of controlling translation in bacteria where transcription and translation are coupled. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Photoionization mass spectrometry for the investigation of combustion generated nascent nanoparticles and their relation to laser induced incandescence

NASA Astrophysics Data System (ADS)

Grotheer, H.-H.; Wolf, K.; Hoffmann, K.

2011-08-01

Premixed laminar flat ethylene flames were investigated for nascent nanoparticles through photoionization mass spectrometry (PIMS). Using an atmospheric McKenna burner and ethylene air flames coupled to an atmospheric sampling system, within a relatively narrow C/O range two modes of these particles were found, which can be clearly distinguished with regard to their temperature dependence, their reactivity, and their ionization behaviour. Behind a diesel engine the same particles were observed. These results were corroborated using a low pressure ethylene-O2 flame coupled to a high resolution mass spectrometer. In this case, due to a special inlet system, it was possible to operate the flame in a fairly wide C/O range without clogging of the inlet nozzles. This allowed pursuing the development of particle size distribution functions (PSDF) well into the regime of mature soot. In addition, on the low mass side of the particle spectra measurements with unity resolution were possible and this allowed gaining information concerning their growth mechanism and structure. Finally, in an attempt to mimic Laser Induced Incandescence (LII) experiments the soot-laden molecular beam was exposed to IR irradiation. This resulted in a near complete destruction of nascent particles under LII typical fluences. Small C clusters between 3 and 17 C atoms were found. In addition and with much higher intensities, clusters comprising several hundreds of C atoms were also detected, the latter even at very low fluences when small clusters were totally absent.

Mosquito cell-derived West Nile virus replicon particles mimic arbovirus inoculum and have reduced spread in mice.

PubMed

Boylan, Brendan T; Moreira, Fernando R; Carlson, Tim W; Bernard, Kristen A

2017-02-01

Half of the human population is at risk of infection by an arthropod-borne virus. Many of these arboviruses, such as West Nile, dengue, and Zika viruses, infect humans by way of a bite from an infected mosquito. This infectious inoculum is insect cell-derived giving the virus particles distinct qualities not present in secondary infectious virus particles produced by infected vertebrate host cells. The insect cell-derived particles differ in the glycosylation of virus structural proteins and the lipid content of the envelope, as well as their induction of cytokines. Thus, in order to accurately mimic the inoculum delivered by arthropods, arboviruses should be derived from arthropod cells. Previous studies have packaged replicon genome in mammalian cells to produce replicon particles, which undergo only one round of infection, but no studies exist packaging replicon particles in mosquito cells. Here we optimized the packaging of West Nile virus replicon genome in mosquito cells and produced replicon particles at high concentration, allowing us to mimic mosquito cell-derived viral inoculum. These particles were mature with similar genome equivalents-to-infectious units as full-length West Nile virus. We then compared the mosquito cell-derived particles to mammalian cell-derived particles in mice. Both replicon particles infected skin at the inoculation site and the draining lymph node by 3 hours post-inoculation. The mammalian cell-derived replicon particles spread from the site of inoculation to the spleen and contralateral lymph nodes significantly more than the particles derived from mosquito cells. This in vivo difference in spread of West Nile replicons in the inoculum demonstrates the importance of using arthropod cell-derived particles to model early events in arboviral infection and highlights the value of these novel arthropod cell-derived replicon particles for studying the earliest virus-host interactions for arboviruses.

Comparative analysis of rabbit hemorrhagic disease virus (RHDV) and new RHDV2 virus antigenicity, using specific virus-like particles.

PubMed

Bárcena, Juan; Guerra, Beatriz; Angulo, Iván; González, Julia; Valcárcel, Félix; Mata, Carlos P; Castón, José R; Blanco, Esther; Alejo, Alí

2015-09-24

In 2010 a new Lagovirus related to rabbit haemorrhagic disease virus (RHDV) emerged in France and has since rapidly spread throughout domestic and wild rabbit populations of several European countries. The new virus, termed RHDV2, exhibits distinctive genetic, antigenic and pathogenic features. Notably, RHDV2 kills rabbits previously vaccinated with RHDV vaccines. Here we report for the first time the generation and characterization of RHDV2-specific virus-like particles (VLPs). Our results further confirmed the differential antigenic properties exhibited by RHDV and RHDV2, highlighting the need of using RHDV2-specific diagnostic assays to monitor the spread of this new virus.

Diagnostic aptitude of West Nile virus-like particles expressed in insect cells.

PubMed

Rebollo, Belén; Sarraseca, Javier; Rodríguez, Mª José; Sanz, Antonio; Jiménez-Clavero, Miguel Ángel; Venteo, Ángel

2018-02-10

West Nile virus is a globally spread zoonotic arbovirus. The laboratory diagnosis of WNV infection relies on virus identification by RT-PCR or on specific antibody detection by serological tests, such as ELISA or virus-neutralization. These methods usually require a preparation of the whole virus as antigen, entailing biosafety issues and therefore requiring BSL-3 facilities. For this reason, recombinant antigenic structures enabling effective antibody recognition comparable to that of the native virions, would be advantageous as diagnostic reagents. WNV virions are enveloped spherical particles made up of 3 structural proteins (C, capsid; M, membrane and E, envelope) enclosing the viral RNA. This study describes the co-expression of these 3 proteins yielding non-infectious virus-like particles (VLPs) and the results of the initial assessment of these VLPs, used instead of the whole virus, that were shown to perform correctly in two different ELISAs for WNV diagnosis. Copyright © 2018. Published by Elsevier Inc.

Nipah Virus Matrix Protein Influences Fusogenicity and Is Essential for Particle Infectivity and Stability.

PubMed

Dietzel, Erik; Kolesnikova, Larissa; Sawatsky, Bevan; Heiner, Anja; Weis, Michael; Kobinger, Gary P; Becker, Stephan; von Messling, Veronika; Maisner, Andrea

2015-12-16

Nipah virus (NiV) causes fatal encephalitic infections in humans. To characterize the role of the matrix (M) protein in the viral life cycle, we generated a reverse genetics system based on NiV strain Malaysia. Using an enhanced green fluorescent protein (eGFP)-expressing M protein-deleted NiV, we observed a slightly increased cell-cell fusion, slow replication kinetics, and significantly reduced peak titers compared to the parental virus. While increased amounts of viral proteins were found in the supernatant of cells infected with M-deleted NiV, the infectivity-to-particle ratio was more than 100-fold reduced, and the particles were less thermostable and of more irregular morphology. Taken together, our data demonstrate that the M protein is not absolutely required for the production of cell-free NiV but is necessary for proper assembly and release of stable infectious NiV particles. Henipaviruses cause a severe disease with high mortality in human patients. Therefore, these viruses can be studied only in biosafety level 4 (BSL-4) laboratories, making it more challenging to characterize their life cycle. Here we investigated the role of the Nipah virus matrix protein in virus-mediated cell-cell fusion and in the formation and release of newly produced particles. We found that even though low levels of infectious viruses are produced in the absence of the matrix protein, it is required for the release of highly infectious and stable particles. Fusogenicity of matrixless viruses was slightly enhanced, further demonstrating the critical role of this protein in different steps of Nipah virus spread. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Progress in Developing Virus-like Particle Influenza Vaccines

PubMed Central

Quan, Fu-Shi; Lee, Young-Tae; Kim, Ki-Hye; Kim, Min-Chul; Kang, Sang-Moo

2016-01-01

Summary Recombinant vaccines based on virus-like particles (VLPs) or nanoparticles have been successful in their safety and efficacy in preclinical and clinical studies. The technology of expressing enveloped VLP vaccines has combined with molecular engineering of proteins in membrane-anchor and immunogenic forms mimicking the native conformation of surface proteins on the enveloped viruses. This review summarizes recent developments in influenza VLP vaccines against seasonal, pandemic, and avian influenza viruses from the perspective of use in humans. The immunogenicity and efficacies of influenza VLP vaccine in the homologous and cross-protection were reviewed. Discussions include limitations of current influenza vaccination strategies and future directions to confer broadly cross protective new influenza vaccines as well as vaccination. PMID:27058302

Identification of HNRNPK as Regulator of Hepatitis C Virus Particle Production

PubMed Central

Poenisch, Marion; Metz, Philippe; Blankenburg, Hagen; Ruggieri, Alessia; Lee, Ji-Young; Rupp, Daniel; Rebhan, Ilka; Diederich, Kathrin; Kaderali, Lars; Domingues, Francisco S.; Albrecht, Mario; Lohmann, Volker; Erfle, Holger; Bartenschlager, Ralf

2015-01-01

Hepatitis C virus (HCV) is a major cause of chronic liver disease affecting around 130 million people worldwide. While great progress has been made to define the principle steps of the viral life cycle, detailed knowledge how HCV interacts with its host cells is still limited. To overcome this limitation we conducted a comprehensive whole-virus RNA interference-based screen and identified 40 host dependency and 16 host restriction factors involved in HCV entry/replication or assembly/release. Of these factors, heterogeneous nuclear ribonucleoprotein K (HNRNPK) was found to suppress HCV particle production without affecting viral RNA replication. This suppression of virus production was specific to HCV, independent from assembly competence and genotype, and not found with the related Dengue virus. By using a knock-down rescue approach we identified the domains within HNRNPK required for suppression of HCV particle production. Importantly, HNRNPK was found to interact specifically with HCV RNA and this interaction was impaired by mutations that also reduced the ability to suppress HCV particle production. Finally, we found that in HCV-infected cells, subcellular distribution of HNRNPK was altered; the protein was recruited to sites in close proximity of lipid droplets and colocalized with core protein as well as HCV plus-strand RNA, which was not the case with HNRNPK variants unable to suppress HCV virion formation. These results suggest that HNRNPK might determine efficiency of HCV particle production by limiting the availability of viral RNA for incorporation into virions. This study adds a new function to HNRNPK that acts as central hub in the replication cycle of multiple other viruses. PMID:25569684

Immune responses and expression of the virus-like particle antigen of the porcine encephalomyocarditis virus.

PubMed

Jeoung, Hye-Young; Lee, Won-Ha; Jeong, Wooseog; Ko, Young-Joon; Choi, Cheong-Up; An, Dong-Jun

2010-10-01

Virus-like particles (VLPs) are particles that consist of viral capsid proteins and are structurally similar to authentic virus. To express VLPs of the porcine encephalomyocarditis virus (EMCV) and investigate their efficacy and immuno response in vivo, a plasmid (P12A3C-pCI) containing the P12A and 3C genes of the EMCV-K3 viral strain was constructed. The VLPs of EMCV-K3 were successfully assembled in 293FT cells on 3 days after transfection with P12A3C-pCI and were identified as particles of about 30-40 nm using transmission electron microscopy (TEM). In an in vivo experiment, the murine cytokines induced by VLPs of naked DNA vaccine showed that the Th1 indicators IL-2, TNF-alpha and GM-CSF, and the Th2 indicators IL-4 and IL-10 were increased. The immunization of mice with the P12A3C-pCI plasmid induced high levels of neutralizing antibody from 128- to 256-fold and led to a significant protection ratio (90%) after challenge with EMCV-K3 (wild-type strain). These VLPs may represent a novel vaccine strategy for the control of EMCV infection on pig farms. 2010 Elsevier Ltd. All rights reserved.

Noninfectious virus-like particles produced by Moloney murine leukemia virus-based retrovirus packaging cells deficient in viral envelope become infectious in the presence of lipofection reagents

PubMed Central

Sharma, Sanjai; Murai, Fukashi; Miyanohara, Atsushi; Friedmann, Theodore

1997-01-01

Retrovirus packaging cell lines expressing the Moloney murine leukemia virus gag and pol genes but lacking virus envelope genes produce virus-like particles constitutively, whether or not they express a transcript from an integrated retroviral provirus. In the absence of a proviral transcript, the assembled particles contain processed gag and reverse transcriptase, and particles made by cells expressing an integrated lacZ provirus also contain viral RNA. The virus-like particles from both cell types are enveloped and are secreted/budded into the extracellular space but are noninfectious. Their physicochemical properties are similar to those of mature retroviral particles. The noninfectious gag pol RNA particles can readily be made infectious by the addition of lipofection reagents to produce preparations with titers of up to 105 colony-forming units per ml. PMID:9380714

Separation of HIV-1 gag virus-like particles from vesicular particles impurities by hydroxyl-functionalized monoliths.

PubMed

Steppert, Petra; Burgstaller, Daniel; Klausberger, Miriam; Kramberger, Petra; Tover, Andres; Berger, Eva; Nöbauer, Katharina; Razzazi-Fazeli, Ebrahim; Jungbauer, Alois

2017-02-01

The downstream processing of enveloped virus-like particles is very challenging because of the biophysical and structural similarity between correctly assembled particles and contaminating vesicular particles present in the feedstock. We used hydroxyl-functionalized polymethacrylate monoliths, providing hydrophobic and electrostatic binding contributions, for the purification of HIV-1 gag virus-like particles. The clarified culture supernatant was conditioned with ammonium sulfate and after membrane filtration loaded onto a 1 mL monolith. The binding capacity was 2 × 10 12 /mL monolith and was only limited by the pressure drop. By applying either a linear or a step gradient elution, to decrease the ammonium sulfate concentration, the majority of double-stranded DNA (88-90%) and host cell protein impurities (39-61%) could be removed while the particles could be separated into two fractions. Proteomic analysis and evaluation of the p24 concentration showed that one fraction contained majority of the HIV-1 gag and the other fraction was less contaminated with proteins originated from intracellular compartments. We were able to process up to 92 bed volumes of conditioned loading material within 3 h and eluted in average 7.3 × 10 11 particles per particle fraction, which is equivalent to 730 vaccination doses of 1 × 10 9 particles. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Rab1A is required for assembly of classical swine fever virus particle.

PubMed

Lin, Jihui; Wang, Chengbao; Liang, Wulong; Zhang, Jing; Zhang, Longxiang; Lv, Huifang; Dong, Wang; Zhang, Yanming

2018-01-15

Rab1A belongs to the small Rab GTPase family and is involved in the lifecycle of numerous viruses. Here, knockdown of Rab1A inhibited CSFV growth. Further study revealed that Rab1A depletion decreased intracellular and extracellular CSFV titers, but did not affect intracellular virus genome copies and E2 protein expression within a virus lifecycle, which suggested that Rab1A is required for CSFV particle assembly rather than for genome replication or virion release. This was proofed by blocking the spread of virus using neutralizing antibodies, through which the negative effects of Rab1A knockdown on multi-cycle replication of CSFV were eliminated. Moreover, co-immunoprecipitation and confocal microscopy assays showed that Rab1A bound to CSFV NS5A protein, indicating that Rab1A and viral NS5A proteins may work cooperatively during CSFV particle assembly. In conclusion, this study demonstrated for the first time that Rab1A is required for CSFV particle assembly and binds to viral particle assembly-related NS5A protein. Copyright © 2017 Elsevier Inc. All rights reserved.

Experimental investigation of virus and clay particles cotransport in partially saturated columns packed with glass beads.

PubMed

Syngouna, Vasiliki I; Chrysikopoulos, Constantinos V

2015-02-15

Suspended clay particles in groundwater can play a significant role as carriers of viruses, because, depending on the physicochemical conditions, clay particles may facilitate or hinder the mobility of viruses. This experimental study examines the effects of clay colloids on the transport of viruses in variably saturated porous media. All cotransport experiments were conducted in both saturated and partially saturated columns packed with glass beads, using bacteriophages MS2 and ΦX174 as model viruses, and kaolinite (KGa-1b) and montmorillonite (STx-1b) as model clay colloids. The various experimental collision efficiencies were determined using the classical colloid filtration theory. The experimental data indicated that the mass recovery of viruses and clay colloids decreased as the water saturation decreased. Temporal moments of the various breakthrough concentrations collected, suggested that the presence of clays significantly influenced virus transport and irreversible deposition onto glass beads. The mass recovery of both viruses, based on total effluent virus concentrations, was shown to reduce in the presence of suspended clay particles. Furthermore, the transport of suspended virus and clay-virus particles was retarded, compared to the conservative tracer. Under unsaturated conditions both clay particles facilitated the transport of ΦX174, while hindered the transport of MS2. Moreover, the surface properties of viruses, clays and glass beads were employed for the construction of classical DLVO and capillary potential energy profiles, and the results suggested that capillary forces play a significant role on colloid retention. It was estimated that the capillary potential energy of MS2 is lower than that of ΦX174, and the capillary potential energy of KGa-1b is lower than that of STx-1b, assuming that the protrusion distance through the water film is the same for each pair of particles. Moreover, the capillary potential energy is several orders of

Cotranslational structure acquisition of nascent polypeptides monitored by NMR spectroscopy.

PubMed

Eichmann, Cédric; Preissler, Steffen; Riek, Roland; Deuerling, Elke

2010-05-18

The folding of proteins in living cells may start during their synthesis when the polypeptides emerge gradually at the ribosomal exit tunnel. However, our current understanding of cotranslational folding processes at the atomic level is limited. We employed NMR spectroscopy to monitor the conformation of the SH3 domain from alpha-spectrin at sequential stages of elongation via in vivo ribosome-arrested (15)N,(13)C-labeled nascent polypeptides. These nascent chains exposed either the entire SH3 domain or C-terminally truncated segments thereof, thus providing snapshots of the translation process. We show that nascent SH3 polypeptides remain unstructured during elongation but fold into a compact, native-like beta-sheet assembly when the entire sequence information is available. Moreover, the ribosome neither imposes major conformational constraints nor significantly interacts with exposed unfolded nascent SH3 domain moieties. Our data provide evidence for a domainwise folding of the SH3 domain on ribosomes without significant population of folding intermediates. The domain follows a thermodynamically favorable pathway in which sequential folding units are stabilized, thus avoiding kinetic traps during the process of cotranslational folding.

Distinct Morphology of Human T-Cell Leukemia Virus Type 1-Like Particles

PubMed Central

Maldonado, José O.; Cao, Sheng; Zhang, Wei; Mansky, Louis M.

2016-01-01

The Gag polyprotein is the main retroviral structural protein and is essential for the assembly and release of virus particles. In this study, we have analyzed the morphology and Gag stoichiometry of human T-cell leukemia virus type 1 (HTLV-1)-like particles and authentic, mature HTLV-1 particles by using cryogenic transmission electron microscopy (cryo-TEM) and scanning transmission electron microscopy (STEM). HTLV-1-like particles mimicked the morphology of immature authentic HTLV-1 virions. Importantly, we have observed for the first time that the morphology of these virus-like particles (VLPs) has the unique local feature of a flat Gag lattice that does not follow the curvature of the viral membrane, resulting in an enlarged distance between the Gag lattice and the viral membrane. Other morphological features that have been previously observed with other retroviruses include: (1) a Gag lattice with multiple discontinuities; (2) membrane regions associated with the Gag lattice that exhibited a string of bead-like densities at the inner leaflet; and (3) an arrangement of the Gag lattice resembling a railroad track. Measurement of the average size and mass of VLPs and authentic HTLV-1 particles suggested a consistent range of size and Gag copy numbers in these two groups of particles. The unique local flat Gag lattice morphological feature observed suggests that HTLV-1 Gag could be arranged in a lattice structure that is distinct from that of other retroviruses characterized to date. PMID:27187442

Marburg virus-like particles produced in insect cells induce neutralizing antibodies in rhesus macaques.

PubMed

Weiwei, Gai; Xuexing, Zheng; Chong, Wang; Yongkun, Zhao; Qi, Wang; Hualei, Wang; Gary, Wong; Ying, Xie; Haijun, Wang; Zengguo, Cao; Na, Feng; Hang, Chi; Tiecheng, Wang; Yuwei, Gao; Junjie, Shan; Songtao, Yang; Xianzhu, Xia

2017-12-01

Marburg virus (MARV), which is one of the most virulent agents in the world, causes lethal haemorrhagic fever in humans and nonhuman primates (NHPs) with a mortality rate of up to 90%. Currently, there is no effective treatment or approved vaccine for MARV for human use to control disease outbreak and spread. Virus-like particles (VLPs), which are morphologically identical to the native infectious virus particle, are efficacious as vaccines against many viruses, including human papilloma virus (HPV), porcine circovirus (PCV) type 2 and hepatitis B virus (HBV). In this study, we generated MARV virus-like particles (VLPs) by co-expressing a glycoprotein (GP) and matrix protein (VP40) using the baculovirus expression system. Rhesus macaques vaccinated with MARV VLPs mixed with adjuvant Poria cocos polysaccharides (PCP-II) produced a GP-specific IgG titer of up to 1:1280 and virus-neutralizing antibody titers that reached 1:320. MARV VLPs also elicited interferon-γ (IFN-γ) and interleukin-4 (IL-4) secretion associated with T-helper 1 cell (Th1)- and T-helper 2 cell (Th2)-mediated immunity, as detected using enzyme-linked immunospot (ELISpot) assays. These data indicate that MARV VLPs mixed with adjuvant PCP-II have excellent immunogenicity in rhesus macaques and may be a promising candidate vaccine against MARV. © 2017 Wiley Periodicals, Inc.

High sensitivity detection and sorting of infectious human immunodeficiency virus (HIV-1) particles by flow virometry

PubMed Central

Bonar, Micha M.; Tilton, John C.

2017-01-01

Detection of viruses by flow cytometry is complicated by their small size. Here, we characterized the ability of a standard (FACSAria II) and a sub-micron flow cytometer (A50 Micro) to resolve HIV-1 viruses. The A50 was superior at resolving small particles but did not reliably distinguish HIV-1, extracellular vesicles, and laser noise by light scatter properties alone. However, single fluorescent HIV-1 particles could readily be detected by both cytometers. Fluorescent particles were sorted and retained infectivity, permitting further exploration of the functional consequences of HIV-1 heterogeneity. Finally, flow cytometry had a limit of detection of 80 viruses/ml, nearly equal to PCR assays. These studies demonstrate the power of flow cytometry to detect and sort viral particles and provide a critical toolkit to validate methods to label wild-type HIV-1; quantitatively assess integrity and aggregation of viruses and virus-based therapeutics; and efficiently screen drugs inhibiting viral assembly and release. PMID:28235684

High sensitivity detection and sorting of infectious human immunodeficiency virus (HIV-1) particles by flow virometry.

PubMed

Bonar, Michał M; Tilton, John C

2017-05-01

Detection of viruses by flow cytometry is complicated by their small size. Here, we characterized the ability of a standard (FACSAria II) and a sub-micron flow cytometer (A50 Micro) to resolve HIV-1 viruses. The A50 was superior at resolving small particles but did not reliably distinguish HIV-1, extracellular vesicles, and laser noise by light scatter properties alone. However, single fluorescent HIV-1 particles could readily be detected by both cytometers. Fluorescent particles were sorted and retained infectivity, permitting further exploration of the functional consequences of HIV-1 heterogeneity. Finally, flow cytometry had a limit of detection of 80 viruses/ml, nearly equal to PCR assays. These studies demonstrate the power of flow cytometry to detect and sort viral particles and provide a critical toolkit to validate methods to label wild-type HIV-1; quantitatively assess integrity and aggregation of viruses and virus-based therapeutics; and efficiently screen drugs inhibiting viral assembly and release. Copyright © 2017 Elsevier Inc. All rights reserved.

Influenza vaccines based on virus-like particles

PubMed Central

Kang, Sang-Moo; Song, Jae-Min; Quan, Fu-Shi; Compans, Richard W.

2009-01-01

The simultaneous expression of structural proteins of virus can produce virus-like particles (VLPs) by a self-assembly process in a viral life cycle even in the absence of genomic material. Taking an advantage of structural and morphological similarities of VLPs to native virions, VLPs have been suggested as a promising platform for new viral vaccines. In the light of a pandemic threat, influenza VLPs have been recently developed as a new generation of non-egg based cell culture-derived vaccine candidates against influenza infection. Animals vaccinated with VLPs containing hemagglutinin (HA) or HA and neuraminidase (NA) were protected from morbidity and mortality resulting from lethal influenza infections. Influenza VLPs serve as an excellent model system of an enveloped virus for understanding the properties of VLPs in inducing protective immunity. In this review, we briefly describe the characteristics of influenza VLPs assembled with a lipid bilayer containing glycoproteins, and summarize the current progress on influenza VLPs as an alternative vaccine candidate against seasonal as well as pandemic influenza viruses. In addition, the protective immune correlates induced by vaccination with influenza VLPs are discussed. PMID:19374929

DYNAMICS OF NASCENT AND ACTIVE ZONE ULTRASTRUCTURE AS SYNAPSES ENLARGE DURING LTP IN MATURE HIPPOCAMPUS

PubMed Central

Bell, Maria Elizabeth; Bourne, Jennifer N.; Chirillo, Michael A.; Mendenhall, John M.; Kuwajima, Masaaki; Harris, Kristen M.

2014-01-01

Nascent zones and active zones are adjacent synaptic regions that share a postsynaptic density, but nascent zones lack the presynaptic vesicles found at active zones. Here dendritic spine synapses were reconstructed through serial section electron microscopy (3DEM) and EM tomography to investigate nascent zone dynamics during long-term potentiation (LTP) in mature rat hippocampus. LTP was induced with theta-burst stimulation and comparisons were made to control stimulation in the same hippocampal slices at 5 minutes, 30 minutes, and 2 hours post-induction and to perfusion-fixed hippocampus in vivo. Nascent zones were present at the edges of ~35% of synapses in perfusion-fixed hippocampus and as many as ~50% of synapses in some hippocampal slice conditions. By 5 minutes, small dense core vesicles known to transport active zone proteins moved into more presynaptic boutons. By 30 minutes, nascent zone area decreased without significant change in synapse area, suggesting that presynaptic vesicles were recruited to pre-existing nascent zones. By 2 hours, both nascent and active zones were enlarged. Immunogold labeling revealed that glutamate receptors can be found in nascent zones; however, average distances from nascent zones to docked presynaptic vesicles ranged from 170±5 nm in perfusion-fixed hippocampus to 251±4 nm at enlarged synapses by 2 hours during LTP. Prior stochastic modeling suggests that falloff in glutamate concentration reduces the probability of glutamate receptor activation from 0.4 at the center of release to 0.1 just 200 nm away. Thus, conversion of nascent zones to functional active zones likely requires the recruitment of presynaptic vesicles during LTP. PMID:25043676

Virus like particles as a platform for cancer vaccine development.

PubMed

Ong, Hui Kian; Tan, Wen Siang; Ho, Kok Lian

2017-01-01

Cancers have killed millions of people in human history and are still posing a serious health problem worldwide. Therefore, there is an urgent need for developing preventive and therapeutic cancer vaccines. Among various cancer vaccine development platforms, virus-like particles (VLPs) offer several advantages. VLPs are multimeric nanostructures with morphology resembling that of native viruses and are mainly composed of surface structural proteins of viruses but are devoid of viral genetic materials rendering them neither infective nor replicative. In addition, they can be engineered to display multiple, highly ordered heterologous epitopes or peptides in order to optimize the antigenicity and immunogenicity of the displayed entities. Like native viruses, specific epitopes displayed on VLPs can be taken up, processed, and presented by antigen-presenting cells to elicit potent specific humoral and cell-mediated immune responses. Several studies also indicated that VLPs could overcome the immunosuppressive state of the tumor microenvironment and break self-tolerance to elicit strong cytotoxic lymphocyte activity, which is crucial for both virus clearance and destruction of cancerous cells. Collectively, these unique characteristics of VLPs make them optimal cancer vaccine candidates. This review discusses current progress in the development of VLP-based cancer vaccines and some potential drawbacks of VLPs in cancer vaccine development. Extracellular vesicles with close resembling to viral particles are also discussed and compared with VLPs as a platform in cancer vaccine developments.

Virus-like particles in cystic mammary adenoma of a snow leopard.

PubMed

Chandra, S; Laughlin, D C

1975-11-01

Virus-like particles were observed in the giant cells of a mammary adenoma of a snow leopard kept in captivity. Particles that measured 115 to 125 nm in diameter budded from the lamella of endoplasmic reticulum and were studded on their inner surfaces with dense granules (approximately 12 nm) that gave them their unique ultrastructural morphology. Such particles were not observed extracellularly. Type B or type C particles were not seen in the tumor tissue.

Characterization of a novel isoform of alpha-nascent polypeptide-associated complex as IgE-defined autoantigen.

PubMed

Mossabeb, Roschanak; Seiberler, Susanne; Mittermann, Irene; Reininger, Renate; Spitzauer, Susanne; Natter, Susanne; Verdino, Petra; Keller, Walter; Kraft, Dietrich; Valenta, Rudolf

2002-10-01

The nascent polypeptide-associated complex is required for intracellular translocation of newly synthesized polypeptides in eukaryotic cells. It may also act as a transcriptional coactivator in humans and various eukaryotic organisms and binds to nucleic acids. Recently, we provided evidence that a component of nascent polypeptide-associated complex, alpha-nascent polypeptide-associated complex, represents an IgE-reactive autoantigen for atopic dermatitis patients. By oligonucleotide screening we isolated a complete cDNA coding for a so far unknown alpha-nascent polypeptide-associated complex isoform from a human epithelial cDNA library. Southern blot hybridization experiments provided further evidence that alpha-nascent polypeptide-associated complex is encoded by a gene family. Recombinant alpha-nascent polypeptide-associated complex was expressed in Escherichia coli as a soluble, His-tagged protein, and purified via nickel affinity chromatography. By circular dichroism analysis it is demonstrated that purified recombinant alpha-nascent polypeptide-associated complex represents a folded protein of mixed alpha-helical and beta-sheet conformation with unusual high thermal stability and remarkable refolding capacity. Complete recombinant alpha-nascent polypeptide-associated complex (215 amino acids) and its 86 amino acid C-terminal fragment specifically bound IgE autoantibodies. Recombinant alpha-nascent polypeptide-associated complex also inhibited IgE binding to natural alpha-nascent polypeptide-associated complex, demonstrating the presence of common IgE epitopes between the recombinant and natural protein. Furthermore, recombinant alpha-nascent polypeptide-associated complex induced specific lymphoproliferative responses in peripheral blood mononuclear cells of a sensitized atopic dermatitis patient. As has been proposed for environmental allergens it is possible that T cell responses to IgE-defined autoantigens may contribute to the chronic skin manifestations

Sequential protein association with nascent 60S ribosomal particles.

PubMed

Saveanu, Cosmin; Namane, Abdelkader; Gleizes, Pierre-Emmanuel; Lebreton, Alice; Rousselle, Jean-Claude; Noaillac-Depeyre, Jacqueline; Gas, Nicole; Jacquier, Alain; Fromont-Racine, Micheline

2003-07-01

Ribosome biogenesis in eukaryotes depends on the coordinated action of ribosomal and nonribosomal proteins that guide the assembly of preribosomal particles. These intermediate particles follow a maturation pathway in which important changes in their protein composition occur. The mechanisms involved in the coordinated assembly of the ribosomal particles are poorly understood. We show here that the association of preribosomal factors with pre-60S complexes depends on the presence of earlier factors, a phenomenon essential for ribosome biogenesis. The analysis of the composition of purified preribosomal complexes blocked in maturation at specific steps allowed us to propose a model of sequential protein association with, and dissociation from, early pre-60S complexes for several preribosomal factors such as Mak11, Ssf1, Rlp24, Nog1, and Nog2. The presence of either Ssf1 or Nog2 in complexes that contain the 27SB pre-rRNA defines novel, distinct pre-60S particles that contain the same pre-rRNA intermediates and that differ only by the presence or absence of specific proteins. Physical and functional interactions between Rlp24 and Nog1 revealed that the assembly steps are, at least in part, mediated by direct protein-protein interactions.

Generation of Tioman virus nucleocapsid-like particles in yeast Saccharomyces cerevisiae.

PubMed

Petraityte, Rasa; Tamosiunas, Paulius L; Juozapaitis, Mindaugas; Zvirbliene, Aurelija; Sasnauskas, Kestutis; Shiell, Brian; Russell, Gail; Bingham, John; Michalski, Wojtek P

2009-10-01

Tioman virus (TioV) was isolated from a number of pooled urine samples of Tioman Island flying foxes (Pteropus hypomelanus) during the search for the reservoir host of Nipah virus. Studies have established TioV as a new virus in the family Paramyxoviridae. This novel paramyxovirus is antigenically related to Menangle virus that was isolated in Australia in 1997 during disease outbreak in pigs. TioV causes mild disease in pigs and has a predilection for lymphoid tissues. Recent serosurvey showed that 1.8% of Tioman Islanders had neutralizing antibodies against TioV, indicating probable past infection. For the development of convenient serological tests for this virus, recombinant TioV nucleocapsid (N) protein was expressed in the yeast Saccharomyces cerevisiae. High yields of recombinant TioV N protein were obtained. Electron microscopy demonstrated that purified recombinant N protein self-assembled into nucleocapsid-like particles which were identical in density and morphology to authentic nucleocapsids from paramyxovirus-infected cells. Different size nucleocapsid-like particles were stable and readily purified by CsCl gradient ultracentrifugation. Polyclonal sera raised in rabbits after immunization with recombinant TioV N protein reacted reliably with TioV infected tissues in immunohistochemistry tests. It confirmed that the antigenic properties of yeast derived TioV N protein are identical to authentic viral protein.

Crystal Structure and Proteomics Analysis of Empty Virus-like Particles of Cowpea Mosaic Virus

PubMed Central

Huynh, Nhung T.; Hesketh, Emma L.; Saxena, Pooja; Meshcheriakova, Yulia; Ku, You-Chan; Hoang, Linh T.; Johnson, John E.; Ranson, Neil A.; Lomonossoff, George P.; Reddy, Vijay S.

2016-01-01

SUMMARY Empty virus-like particles (eVLPs) of Cowpea mosaic virus (CPMV) are currently being utilized as reagents in various biomedical and nanotechnology applications. Here, we report the crystal structure of CPMV eVLPs determined using X-ray crystallography at 2.3 Å resolution and compare it with previously reported cryo-electron microscopy (cryo-EM) of eVLPs and virion crystal structures. Although the X-ray and cryo-EM structures of eVLPs are mostly similar, there exist significant differences at the C terminus of the small (S) subunit. The intact C terminus of the S subunit plays a critical role in enabling the efficient assembly of CPMV virions and eVLPs, but undergoes proteolysis after particle formation. In addition, we report the results of mass spectrometry-based proteomics analysis of coat protein subunits from CPMV eVLPs and virions that identify the C termini of S subunits undergo proteolytic cleavages at multiple sites instead of a single cleavage site as previously observed. PMID:27021160

Genotype I of Japanese Encephalitis Virus Virus-like Particles Elicit Sterilizing Immunity against Genotype I and III Viral Challenge in Swine.

PubMed

Fan, Yi-Chin; Chen, Jo-Mei; Lin, Jen-Wei; Chen, Yi-Ying; Wu, Guan-Hong; Su, Kuan-Hsuan; Chiou, Ming-Tang; Wu, Shang-Rung; Yin, Ji-Hang; Liao, Jiunn-Wang; Chang, Gwong-Jen J; Chiou, Shyan-Song

2018-05-10

Swine are a critical amplifying host involved in human Japanese encephalitis (JE) outbreaks. Cross-genotypic immunogenicity and sterile protection are important for the current genotype III (GIII) virus-derived vaccines in swine, especially now that emerging genotype I (GI) JE virus (JEV) has replaced GIII virus as the dominant strain. Herein, we aimed to develop a system to generate GI JEV virus-like particles (VLPs) and evaluate the immunogenicity and protection of the GI vaccine candidate in mice and specific pathogen-free swine. A CHO-heparan sulfate-deficient (CHO-HS(-)) cell clone, named 51-10 clone, stably expressing GI-JEV VLP was selected and continually secreted GI VLPs without signs of cell fusion. 51-10 VLPs formed a homogeneously empty-particle morphology and exhibited similar antigenic activity as GI virus. GI VLP-immunized mice showed balanced cross-neutralizing antibody titers against GI to GIV viruses (50% focus-reduction micro-neutralization assay titers 71 to 240) as well as potent protection against GI or GIII virus infection. GI VLP-immunized swine challenged with GI or GIII viruses showed no fever, viremia, or viral RNA in tonsils, lymph nodes, and brains as compared with phosphate buffered saline-immunized swine. We thus conclude GI VLPs can provide sterile protection against GI and GIII viruses in swine.

Virus-Like Particles That Can Deliver Proteins and RNA | NCI Technology Transfer Center | TTC

Cancer.gov

The present invention describes novel virus-like particles (VLPs) that are capable of binding to and replicating within a target mammalian cell, including human cells. The claimed VLPs are safer than viral delivery because they are incapable of re-infecting target cells. The National Cancer Institute's Protein Expression Laboratory seeks parties interested in licensing the novel delivery of RNA to mammalian cells using virus-like particles.

Canine distemper virus matrix protein influences particle infectivity, particle composition, and envelope distribution in polarized epithelial cells and modulates virulence.

PubMed

Dietzel, Erik; Anderson, Danielle E; Castan, Alexandre; von Messling, Veronika; Maisner, Andrea

2011-07-01

In paramyxoviruses, the matrix (M) protein mediates the interaction between the envelope and internal proteins during particle assembly and egress. In measles virus (MeV), M mutations, such as those found in subacute sclerosing panencephalitis (SSPE) strains, and differences in vaccine and wild-type M proteins can affect the strength of interaction with the envelope glycoproteins, assembly efficiency, and spread. However, the contribution of the M protein to the replication and pathogenesis of the closely related canine distemper virus (CDV) has not been characterized. To this end this, we generated a recombinant wild-type CDV carrying a vaccine strain M protein. The recombinant virus retained the parental growth phenotype in VerodogSLAMtag cells, but displayed an increased particle-to-infectivity ratio very similar to that of the vaccine strain, likely due to inefficient H protein incorporation. Even though infectious virus was released only from the apical surface, consistent with the release polarity of the wild-type CDV strain, envelope protein distribution in polarized epithelial cells reproduced the bipolar pattern seen in vaccine strain-infected cells. Most notably, the chimeric virus was completely attenuated in ferrets and caused only a mild and transient leukopenia, indicating that the differences in particle infectivity and envelope protein sorting mediated by the vaccine M protein contribute importantly to vaccine strain attenuation.

Production and immunogenicity of chimeric virus-like particles containing the spike glycoprotein of infectious bronchitis virus

PubMed Central

Lv, Lishan; Li, Xiaoming; Liu, Genmei; Li, Ran; Liu, Qiliang; Shen, Huifang; Wang, Wei; Xue, Chunyi

2014-01-01

Infectious bronchitis virus (IBV) poses a severe threat to the poultry industry and causes heavy economic losses worldwide. Vaccination is the most effective method of preventing infection and controlling the spread of IBV, but currently available inactivated and attenuated virus vaccines have some disadvantages. We developed a chimeric virus-like particle (VLP)-based candidate vaccine for IBV protection. The chimeric VLP was composed of matrix 1 protein from avian influenza H5N1 virus and a fusion protein neuraminidase (NA)/spike 1 (S1) that was generated by fusing IBV S1 protein to the cytoplasmic and transmembrane domains of NA protein of avian influenza H5N1 virus. The chimeric VLPs elicited significantly higher S1-specific antibody responses in intramuscularly immunized mice and chickens than inactivated IBV viruses. Furthermore, the chimeric VLPs induced significantly higher neutralization antibody levels than inactivated H120 virus in SPF chickens. Finally, the chimeric VLPs induced significantly higher IL-4 production in mice. These results demonstrate that chimeric VLPs have the potential for use in vaccines against IBV infection. PMID:24378590

Production and immunogenicity of chimeric virus-like particles containing the spike glycoprotein of infectious bronchitis virus.

PubMed

Lv, Lishan; Li, Xiaoming; Liu, Genmei; Li, Ran; Liu, Qiliang; Shen, Huifang; Wang, Wei; Xue, Chunyi; Cao, Yongchang

2014-01-01

Infectious bronchitis virus (IBV) poses a severe threat to the poultry industry and causes heavy economic losses worldwide. Vaccination is the most effective method of preventing infection and controlling the spread of IBV, but currently available inactivated and attenuated virus vaccines have some disadvantages. We developed a chimeric virus-like particle (VLP)-based candidate vaccine for IBV protection. The chimeric VLP was composed of matrix 1 protein from avian influenza H5N1 virus and a fusion protein neuraminidase (NA)/spike 1 (S1) that was generated by fusing IBV S1 protein to the cytoplasmic and transmembrane domains of NA protein of avian influenza H5N1 virus. The chimeric VLPs elicited significantly higher S1-specific antibody responses in intramuscularly immunized mice and chickens than inactivated IBV viruses. Furthermore, the chimeric VLPs induced significantly higher neutralization antibody levels than inactivated H120 virus in SPF chickens. Finally, the chimeric VLPs induced significantly higher IL-4 production in mice. These results demonstrate that chimeric VLPs have the potential for use in vaccines against IBV infection.

MINIGENOMES, TRANSCRIPTION AND REPLICATION COMPETENT VIRUS-LIKE PARTICLES AND BEYOND: REVERSE GENETICS SYSTEMS FOR FILOVIRUSES AND OTHER NEGATIVE STRANDED HEMORRHAGIC FEVER VIRUSES

PubMed Central

Hoenen, Thomas; Groseth, Allison; de Kok-Mercado, Fabian; Kuhn, Jens H.; Wahl-Jensen, Victoria

2012-01-01

Reverse-genetics systems are powerful tools enabling researchers to study the replication cycle of RNA viruses, including filoviruses and other hemorrhagic fever viruses, as well as to discover new antivirals. They include full-length clone systems as well as a number of life cycle modeling systems. Full-length clone systems allow for the generation of infectious, recombinant viruses, and thus are an important tool for studying the virus replication cycle in its entirety. In contrast, life cycle modeling systems such as minigenome and transcription and replication competent virus-like particle systems can be used to simulate and dissect parts of the virus life cycle outside of containment facilities. Minigenome systems are used to model viral genome replication and transcription, whereas transcription and replication competent virus-like particle systems also model morphogenesis and budding as well as infection of target cells. As such, these modeling systems have tremendous potential to further the discovery and screening of new antivirals targeting hemorrhagic fever viruses. This review provides an overview of currently established reverse genetics systems for hemorrhagic fever-causing negative-sense RNA viruses, with a particular emphasis on filoviruses, and the potential application of these systems for antiviral research. PMID:21699921

A cell culture-derived whole virus influenza A vaccine based on magnetic sulfated cellulose particles confers protection in mice against lethal influenza A virus infection.

PubMed

Pieler, Michael M; Frentzel, Sarah; Bruder, Dunja; Wolff, Michael W; Reichl, Udo

2016-12-07

Downstream processing and formulation of viral vaccines employs a large number of different unit operations to achieve the desired product qualities. The complexity of individual process steps involved, the need for time consuming studies towards the optimization of virus yields, and very high requirements regarding potency and safety of vaccines results typically in long lead times for the establishment of new processes. To overcome such obstacles, to enable fast screening of potential vaccine candidates, and to explore options for production of low cost veterinary vaccines a new platform for whole virus particle purification and formulation based on magnetic particles has been established. Proof of concept was carried out with influenza A virus particles produced in suspension Madin Darby canine kidney (MDCK) cells. The clarified, inactivated, concentrated, and diafiltered virus particles were bound to magnetic sulfated cellulose particles (MSCP), and directly injected into mice for immunization including positive and negative controls. We show here, that in contrast to the mock-immunized group, vaccination of mice with antigen-loaded MSCP (aMSCP) resulted in high anti-influenza A antibody responses and full protection against a lethal challenge with replication competent influenza A virus. Antiviral protection correlated with a 400-fold reduced number of influenza nucleoprotein gene copies in the lungs of aMSCP immunized mice compared to mock-treated animals, indicating the efficient induction of antiviral immunity by this novel approach. Thus, our data proved the use of MSCP for purification and formulation of the influenza vaccine to be fast and efficient, and to confer protection of mice against influenza A virus infection. Furthermore, the method proposed has the potential for fast purification of virus particles directly from bioreactor harvests with a minimum number of process steps towards formulation of low-cost veterinary vaccines, and for screening

Encephalomyocarditis Virus Ribonucleic Acid Polymerase Associated with 150S Cytoplasmic Particles

PubMed Central

Bases, Robert; Tarikas, Helgi

1969-01-01

Cytoplasmic particles which sedimented at 150S were the smallest structures containing detectable viral ribonucleic acid polymerase in mouse cells infected with encephalomyocarditis virus. PMID:4307906

Characterization of the Quasi-Enveloped Hepatitis E Virus Particles Released by the Cellular Exosomal Pathway

PubMed Central

Nagashima, Shigeo; Takahashi, Masaharu; Kobayashi, Tominari; Nishizawa, Tsutomu; Nishiyama, Takashi; Primadharsini, Putu Prathiwi

2017-01-01

ABSTRACT Our previous studies demonstrated that membrane-associated hepatitis E virus (HEV) particles—now considered “quasi-enveloped particles”—are present in the multivesicular body with intraluminal vesicles (exosomes) in infected cells and that the release of HEV virions is related to the exosomal pathway. In this study, we characterized exosomes purified from the culture supernatants of HEV-infected PLC/PRF/5 cells. Purified CD63-, CD9-, or CD81-positive exosomes derived from the culture supernatants of HEV-infected cells that had been cultivated in serum-free medium were found to contain HEV RNA and the viral capsid (ORF2) and ORF3 proteins, as determined by reverse transcription-PCR (RT-PCR) and Western blotting, respectively. Furthermore, immunoelectron microscopy, with or without prior detergent and protease treatment, revealed the presence of virus-like particles in the exosome fraction. These particles were 39.6 ± 1.0 nm in diameter and were covered with a lipid membrane. After treatment with detergent and protease, the diameter of these virus-like particles was 26.9 ± 0.9 nm, and the treated particles became accessible with an anti-HEV ORF2 monoclonal antibody (MAb). The HEV particles in the exosome fraction were capable of infecting naive PLC/PRF/5 cells but were not neutralized by an anti-HEV ORF2 MAb which efficiently neutralizes nonenveloped HEV particles in cell culture. These results indicate that the membrane-wrapped HEV particles released by the exosomal pathway are copurified with the exosomes in the exosome fraction and suggest that the capsids of HEV particles are individually covered by lipid membranes resembling those of exosomes, similar to enveloped viruses. IMPORTANCE Hepatitis E, caused by HEV, is an important infectious disease that is spreading worldwide. HEV infection can cause acute or fulminant hepatitis and can become chronic in immunocompromised hosts, including patients after organ transplantation. The HEV particles

From Cells to Virus Particles: Quantitative Methods to Monitor RNA Packaging

PubMed Central

Ferrer, Mireia; Henriet, Simon; Chamontin, Célia; Lainé, Sébastien; Mougel, Marylène

2016-01-01

In cells, positive strand RNA viruses, such as Retroviridae, must selectively recognize their full-length RNA genome among abundant cellular RNAs to assemble and release particles. How viruses coordinate the intracellular trafficking of both RNA and protein components to the assembly sites of infectious particles at the cell surface remains a long-standing question. The mechanisms ensuring packaging of genomic RNA are essential for viral infectivity. Since RNA packaging impacts on several essential functions of retroviral replication such as RNA dimerization, translation and recombination events, there are many studies that require the determination of RNA packaging efficiency and/or RNA packaging ability. Studies of RNA encapsidation rely upon techniques for the identification and quantification of RNA species packaged by the virus. This review focuses on the different approaches available to monitor RNA packaging: Northern blot analysis, ribonuclease protection assay and quantitative reverse transcriptase-coupled polymerase chain reaction as well as the most recent RNA imaging and sequencing technologies. Advantages, disadvantages and limitations of these approaches will be discussed in order to help the investigator to choose the most appropriate technique. Although the review was written with the prototypic simple murine leukemia virus (MLV) and complex human immunodeficiency virus type 1 (HIV-1) in mind, the techniques were described in order to benefit to a larger community. PMID:27556480

Particle-to-PFU ratio of Ebola virus influences disease course and survival in cynomolgus macaques.

PubMed

Alfson, Kendra J; Avena, Laura E; Beadles, Michael W; Staples, Hilary; Nunneley, Jerritt W; Ticer, Anysha; Dick, Edward J; Owston, Michael A; Reed, Christopher; Patterson, Jean L; Carrion, Ricardo; Griffiths, Anthony

2015-07-01

This study addresses the role of Ebola virus (EBOV) specific infectivity in virulence. Filoviruses are highly lethal, enveloped, single-stranded negative-sense RNA viruses that can cause hemorrhagic fever. No approved vaccines or therapies exist for filovirus infections, and infectious virus must be handled in maximum containment. Efficacy testing of countermeasures, in addition to investigations of pathogenicity and immune response, often requires a well-characterized animal model. For EBOV, an obstacle in performing accurate disease modeling is a poor understanding of what constitutes an infectious dose in animal models. One well-recognized consequence of viral passage in cell culture is a change in specific infectivity, often measured as a particle-to-PFU ratio. Here, we report that serial passages of EBOV in cell culture resulted in a decrease in particle-to-PFU ratio. Notably, this correlated with decreased potency in a lethal cynomolgus macaque (Macaca fascicularis) model of infection; animals were infected with the same viral dose as determined by plaque assay, but animals that received more virus particles exhibited increased disease. This suggests that some particles are unable to form a plaque in a cell culture assay but are able to result in lethal disease in vivo. These results have a significant impact on how future studies are designed to model EBOV disease and test countermeasures. Ebola virus (EBOV) can cause severe hemorrhagic disease with a high case-fatality rate, and there are no approved vaccines or therapies. Specific infectivity can be considered the total number of viral particles per PFU, and its impact on disease is poorly understood. In stocks of most mammalian viruses, there are particles that are unable to complete an infectious cycle or unable to cause cell pathology in cultured cells. We asked if these particles cause disease in nonhuman primates by infecting monkeys with equal infectious doses of genetically identical stocks

Nascent-Seq reveals novel features of mouse circadian transcriptional regulation

PubMed Central

Menet, Jerome S; Rodriguez, Joseph; Abruzzi, Katharine C; Rosbash, Michael

2012-01-01

A substantial fraction of the metazoan transcriptome undergoes circadian oscillations in many cells and tissues. Based on the transcription feedback loops important for circadian timekeeping, it is commonly assumed that this mRNA cycling reflects widespread transcriptional regulation. To address this issue, we directly measured the circadian dynamics of mouse liver transcription using Nascent-Seq (genome-wide sequencing of nascent RNA). Although many genes are rhythmically transcribed, many rhythmic mRNAs manifest poor transcriptional rhythms, indicating a prominent contribution of post-transcriptional regulation to circadian mRNA expression. This analysis of rhythmic transcription also showed that the rhythmic DNA binding profile of the transcription factors CLOCK and BMAL1 does not determine the transcriptional phase of most target genes. This likely reflects gene-specific collaborations of CLK:BMAL1 with other transcription factors. These insights from Nascent-Seq indicate that it should have broad applicability to many other gene expression regulatory issues. DOI: http://dx.doi.org/10.7554/eLife.00011.001 PMID:23150795

Visualization of the African swine fever virus infection in living cells by incorporation into the virus particle of green fluorescent protein-p54 membrane protein chimera

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

Hernaez, Bruno; Escribano, Jose M.; Alonso, Covadonga

2006-06-20

Many stages of African swine fever virus infection have not yet been studied in detail. To track the behavior of African swine fever virus (ASFV) in the infected cells in real time, we produced an infectious recombinant ASFV (B54GFP-2) that expresses and incorporates into the virus particle a chimera of the p54 envelope protein fused to the enhanced green fluorescent protein (EGFP). The incorporation of the fusion protein into the virus particle was confirmed immunologically and it was determined that p54-EGFP was fully functional by confirmation that the recombinant virus made normal-sized plaques and presented similar growth curves to themore » wild-type virus. The tagged virus was visualized as individual fluorescent particles during the first stages of infection and allowed to visualize the infection progression in living cells through the viral life cycle by confocal microscopy. In this work, diverse potential applications of B54GFP-2 to study different aspects of ASFV infection are shown. By using this recombinant virus it was possible to determine the trajectory and speed of intracellular virus movement. Additionally, we have been able to visualize for first time the ASFV factory formation dynamics and the cytophatic effect of the virus in live infected cells. Finally, we have analyzed virus progression along the infection cycle and infected cell death as time-lapse animations.« less

The Lymphocytic Choriomeningitis Virus Matrix Protein PPXY Late Domain Drives the Production of Defective Interfering Particles

PubMed Central

Ziegler, Christopher M.; Eisenhauer, Philip; Bruce, Emily A.; Weir, Marion E.; King, Benjamin R.; Klaus, Joseph P.; Krementsov, Dimitry N.; Shirley, David J.; Ballif, Bryan A.; Botten, Jason

2016-01-01

Arenaviruses cause severe diseases in humans but establish asymptomatic, lifelong infections in rodent reservoirs. Persistently-infected rodents harbor high levels of defective interfering (DI) particles, which are thought to be important for establishing persistence and mitigating virus-induced cytopathic effect. Little is known about what drives the production of DI particles. We show that neither the PPXY late domain encoded within the lymphocytic choriomeningitis virus (LCMV) matrix protein nor a functional endosomal sorting complex transport (ESCRT) pathway is absolutely required for the generation of standard infectious virus particles. In contrast, DI particle release critically requires the PPXY late domain and is ESCRT-dependent. Additionally, the terminal tyrosine in the PPXY motif is reversibly phosphorylated and our findings indicate that this posttranslational modification may regulate DI particle formation. Thus we have uncovered a new role for the PPXY late domain and a possible mechanism for its regulation. PMID:27010636

Single-Particle Tracking of Human Immunodeficiency Virus Type 1 Productive Entry into Human Primary Macrophages.

PubMed

Li, Qin; Li, Wei; Yin, Wen; Guo, Jia; Zhang, Zhi-Ping; Zeng, Dejun; Zhang, Xiaowei; Wu, Yuntao; Zhang, Xian-En; Cui, Zongqiang

2017-04-25

Macrophages are one of the major targets of human immunodeficiency virus (HIV-1), but the viral entry pathway remains poorly understood in these cells. Noninvasive virus labeling and single-virus tracking are effective tools for studying virus entry. Here, we constructed a quantum dot (QD)-encapsulated infectious HIV-1 particle to track viral entry at a single-particle level in live human primary macrophages. QDs were encapsulated in HIV-1 virions by incorporating viral accessory protein Vpr-conjugated QDs during virus assembly. With the HIV-1 particles encapsulating QDs, we monitored the early phase of viral infection in real time and observed that, during infection, HIV-1 was endocytosed in a clathrin-mediated manner; the particles were translocated into Rab5A-positive endosomes, and the core was released into the cytoplasm by viral envelope-mediated endosomal fusion. Drug inhibition assays verified that endosome fusion contributes to HIV-1 productive infection in primary macrophages. Additionally, we observed that a dynamic actin cytoskeleton is critical for HIV-1 entry and intracellular migration in primary macrophages. HIV-1 dynamics and infection could be blocked by multiple different actin inhibitors. Our study revealed a productive entry pathway in macrophages that requires both endosomal function and actin dynamics, which may assist in the development of inhibitors to block the HIV entry in macrophages.

Rotavirus Virus-Like Particles as Surrogates in Environmental Persistence and Inactivation Studies

PubMed Central

Caballero, Santiago; Abad, F. Xavier; Loisy, Fabienne; Le Guyader, Françoise S.; Cohen, Jean; Pintó, Rosa M.; Bosch, Albert

2004-01-01

Virus-like particles (VLPs) with the full-length VP2 and VP6 rotavirus capsid proteins, produced in the baculovirus expression system, have been evaluated as surrogates of human rotavirus in different environmental scenarios. Green fluorescent protein-labeled VLPs (GFP-VLPs) and particles enclosing a heterologous RNA (pseudoviruses), whose stability may be monitored by flow cytometry and antigen capture reverse transcription-PCR, respectively, were used. After 1 month in seawater at 20°C, no significant differences were observed between the behaviors of GFP-VLPs and of infectious rotavirus, whereas pseudovirus particles showed a higher decay rate. In the presence of 1 mg of free chlorine (FC)/liter both tracers persisted longer in freshwater at 20°C than infectious viruses, whereas in the presence of 0.2 mg of FC/liter no differences were observed between tracers and infectious rotavirus at short contact times. However, from 30 min of contact with FC onward, the decay of infectious rotavirus was higher than that of recombinant particles. The predicted Ct value for a 90% reduction of GFP-VLPs or pseudoviruses induces a 99.99% inactivation of infectious rotavirus. Both tracers were more resistant to UV light irradiation than infectious rotavirus in fresh and marine water. The effect of UV exposure was more pronounced on pseudovirus than in GFP-VLPs. In all types of water, the UV dose to induce a 90% reduction of pseudovirus ensures a 99.99% inactivation of infectious rotavirus. Recombinant virus surrogates open new possibilities for the systematic validation of virus removal practices in actual field situations where pathogenic agents cannot be introduced. PMID:15240262

Quantitative Evaluation of Protein Heterogeneity within Herpes Simplex Virus 1 Particles.

PubMed

El Bilali, Nabil; Duron, Johanne; Gingras, Diane; Lippé, Roger

2017-05-15

Several virulence genes have been identified thus far in the herpes simplex virus 1 genome. It is also generally accepted that protein heterogeneity among virions further impacts viral fitness. However, linking this variability directly with infectivity has been challenging at the individual viral particle level. To address this issue, we resorted to flow cytometry (flow virometry), a powerful approach we recently employed to analyze individual viral particles, to identify which tegument proteins vary and directly address if such variability is biologically relevant. We found that the stoichiometry of the U L 37, ICP0, and VP11/12 tegument proteins in virions is more stable than the VP16 and VP22 tegument proteins, which varied significantly among viral particles. Most interestingly, viruses sorted for their high VP16 or VP22 content yielded modest but reproducible increases in infectivity compared to their corresponding counterparts containing low VP16 or VP22 content. These findings were corroborated for VP16 in short interfering RNA experiments but proved intriguingly more complex for VP22. An analysis by quantitative Western blotting revealed substantial alterations of virion composition upon manipulation of individual tegument proteins and suggests that VP22 protein levels acted indirectly on viral fitness. These findings reaffirm the interdependence of the virion components and corroborate that viral fitness is influenced not only by the genome of viruses but also by the stoichiometry of proteins within each virion. IMPORTANCE The ability of viruses to spread in animals has been mapped to several viral genes, but other factors are clearly involved, including virion heterogeneity. To directly probe whether the latter influences viral fitness, we analyzed the protein content of individual herpes simplex virus 1 particles using an innovative flow cytometry approach. The data confirm that some viral proteins are incorporated in more controlled amounts, while

Quantitative Evaluation of Protein Heterogeneity within Herpes Simplex Virus 1 Particles

PubMed Central

El Bilali, Nabil; Duron, Johanne; Gingras, Diane

2017-01-01

ABSTRACT Several virulence genes have been identified thus far in the herpes simplex virus 1 genome. It is also generally accepted that protein heterogeneity among virions further impacts viral fitness. However, linking this variability directly with infectivity has been challenging at the individual viral particle level. To address this issue, we resorted to flow cytometry (flow virometry), a powerful approach we recently employed to analyze individual viral particles, to identify which tegument proteins vary and directly address if such variability is biologically relevant. We found that the stoichiometry of the UL37, ICP0, and VP11/12 tegument proteins in virions is more stable than the VP16 and VP22 tegument proteins, which varied significantly among viral particles. Most interestingly, viruses sorted for their high VP16 or VP22 content yielded modest but reproducible increases in infectivity compared to their corresponding counterparts containing low VP16 or VP22 content. These findings were corroborated for VP16 in short interfering RNA experiments but proved intriguingly more complex for VP22. An analysis by quantitative Western blotting revealed substantial alterations of virion composition upon manipulation of individual tegument proteins and suggests that VP22 protein levels acted indirectly on viral fitness. These findings reaffirm the interdependence of the virion components and corroborate that viral fitness is influenced not only by the genome of viruses but also by the stoichiometry of proteins within each virion. IMPORTANCE The ability of viruses to spread in animals has been mapped to several viral genes, but other factors are clearly involved, including virion heterogeneity. To directly probe whether the latter influences viral fitness, we analyzed the protein content of individual herpes simplex virus 1 particles using an innovative flow cytometry approach. The data confirm that some viral proteins are incorporated in more controlled amounts

Crystal Structure and Proteomics Analysis of Empty Virus-like Particles of Cowpea Mosaic Virus.

PubMed

Huynh, Nhung T; Hesketh, Emma L; Saxena, Pooja; Meshcheriakova, Yulia; Ku, You-Chan; Hoang, Linh T; Johnson, John E; Ranson, Neil A; Lomonossoff, George P; Reddy, Vijay S

2016-04-05

Empty virus-like particles (eVLPs) of Cowpea mosaic virus (CPMV) are currently being utilized as reagents in various biomedical and nanotechnology applications. Here, we report the crystal structure of CPMV eVLPs determined using X-ray crystallography at 2.3 Å resolution and compare it with previously reported cryo-electron microscopy (cryo-EM) of eVLPs and virion crystal structures. Although the X-ray and cryo-EM structures of eVLPs are mostly similar, there exist significant differences at the C terminus of the small (S) subunit. The intact C terminus of the S subunit plays a critical role in enabling the efficient assembly of CPMV virions and eVLPs, but undergoes proteolysis after particle formation. In addition, we report the results of mass spectrometry-based proteomics analysis of coat protein subunits from CPMV eVLPs and virions that identify the C termini of S subunits undergo proteolytic cleavages at multiple sites instead of a single cleavage site as previously observed. Copyright © 2016 Elsevier Ltd. All rights reserved.

Nascent RNA kinetics: Transient and steady state behavior of models of transcription

NASA Astrophysics Data System (ADS)

Choubey, Sandeep

2018-02-01

Regulation of transcription is a vital process in cells, but mechanistic details of this regulation still remain elusive. The dominant approach to unravel the dynamics of transcriptional regulation is to first develop mathematical models of transcription and then experimentally test the predictions these models make for the distribution of mRNA and protein molecules at the individual cell level. However, these measurements are affected by a multitude of downstream processes which make it difficult to interpret the measurements. Recent experimental advancements allow for counting the nascent mRNA number of a gene as a function of time at the single-inglr cell level. These measurements closely reflect the dynamics of transcription. In this paper, we consider a general mechanism of transcription with stochastic initiation and deterministic elongation and probe its impact on the temporal behavior of nascent RNA levels. Using techniques from queueing theory, we derive exact analytical expressions for the mean and variance of the nascent RNA distribution as functions of time. We apply these analytical results to obtain the mean and variance of nascent RNA distribution for specific models of transcription. These models of initiation exhibit qualitatively distinct transient behaviors for both the mean and variance which further allows us to discriminate between them. Stochastic simulations confirm these results. Overall the analytical results presented here provide the necessary tools to connect mechanisms of transcription initiation to single-cell measurements of nascent RNA.

Complete and Incomplete Hepatitis B Virus Particles: Formation, Function, and Application.

PubMed

Hu, Jianming; Liu, Kuancheng

2017-03-21

Hepatitis B virus (HBV) is a para-retrovirus or retroid virus that contains a double-stranded DNA genome and replicates this DNA via reverse transcription of a RNA pregenome. Viral reverse transcription takes place within a capsid upon packaging of the RNA and the viral reverse transcriptase. A major characteristic of HBV replication is the selection of capsids containing the double-stranded DNA, but not those containing the RNA or the single-stranded DNA replication intermediate, for envelopment during virion secretion. The complete HBV virion particles thus contain an outer envelope, studded with viral envelope proteins, that encloses the capsid, which, in turn, encapsidates the double-stranded DNA genome. Furthermore, HBV morphogenesis is characterized by the release of subviral particles that are several orders of magnitude more abundant than the complete virions. One class of subviral particles are the classical surface antigen particles (Australian antigen) that contain only the viral envelope proteins, whereas the more recently discovered genome-free (empty) virions contain both the envelope and capsid but no genome. In addition, recent evidence suggests that low levels of RNA-containing particles may be released, after all. We will summarize what is currently known about how the complete and incomplete HBV particles are assembled. We will discuss briefly the functions of the subviral particles, which remain largely unknown. Finally, we will explore the utility of the subviral particles, particularly, the potential of empty virions and putative RNA virions as diagnostic markers and the potential of empty virons as a vaccine candidate.

Influenza Virus Aerosols in Human Exhaled Breath: Particle Size, Culturability, and Effect of Surgical Masks

PubMed Central

Milton, Donald K.; Cowling, Benjamin J.; Grantham, Michael L.

2013-01-01

The CDC recommends that healthcare settings provide influenza patients with facemasks as a means of reducing transmission to staff and other patients, and a recent report suggested that surgical masks can capture influenza virus in large droplet spray. However, there is minimal data on influenza virus aerosol shedding, the infectiousness of exhaled aerosols, and none on the impact of facemasks on viral aerosol shedding from patients with seasonal influenza. We collected samples of exhaled particles (one with and one without a facemask) in two size fractions (“coarse”>5 µm, “fine”≤5 µm) from 37 volunteers within 5 days of seasonal influenza onset, measured viral copy number using quantitative RT-PCR, and tested the fine-particle fraction for culturable virus. Fine particles contained 8.8 (95% CI 4.1 to 19) fold more viral copies than did coarse particles. Surgical masks reduced viral copy numbers in the fine fraction by 2.8 fold (95% CI 1.5 to 5.2) and in the coarse fraction by 25 fold (95% CI 3.5 to 180). Overall, masks produced a 3.4 fold (95% CI 1.8 to 6.3) reduction in viral aerosol shedding. Correlations between nasopharyngeal swab and the aerosol fraction copy numbers were weak (r = 0.17, coarse; r = 0.29, fine fraction). Copy numbers in exhaled breath declined rapidly with day after onset of illness. Two subjects with the highest copy numbers gave culture positive fine particle samples. Surgical masks worn by patients reduce aerosols shedding of virus. The abundance of viral copies in fine particle aerosols and evidence for their infectiousness suggests an important role in seasonal influenza transmission. Monitoring exhaled virus aerosols will be important for validation of experimental transmission studies in humans. PMID:23505369

Virus-Like Particle Secretion and Genotype-Dependent Immunogenicity of Dengue Virus Serotype 2 DNA Vaccine

PubMed Central

Galula, Jedhan U.; Shen, Wen-Fan; Chuang, Shih-Te

2014-01-01

ABSTRACT Dengue virus (DENV), composed of four distinct serotypes, is the most important and rapidly emerging arthropod-borne pathogen and imposes substantial economic and public health burdens. We constructed candidate vaccines containing the DNA of five of the genotypes of dengue virus serotype 2 (DENV-2) and evaluated the immunogenicity, the neutralizing (Nt) activity of the elicited antibodies, and the protective efficacy elicited in mice immunized with the vaccine candidates. We observed a significant correlation between the level of in vitro virus-like particle secretion, the elicited antibody response, and the protective efficacy of the vaccines containing the DNA of the different DENV genotypes in immunized mice. However, higher total IgG antibody levels did not always translate into higher Nt antibodies against homologous and heterologous viruses. We also found that, in contrast to previous reports, more than 50% of total IgG targeted ectodomain III (EDIII) of the E protein, and a substantial fraction of this population was interdomain highly neutralizing flavivirus subgroup-cross-reactive antibodies, such as monoclonal antibody 1B7-5. In addition, the lack of a critical epitope(s) in the Sylvatic genotype virus recognized by interdomain antibodies could be the major cause of the poor protection of mice vaccinated with the Asian 1 genotype vaccine (pVD2-Asian 1) from lethal challenge with virus of the Sylvatic genotype. In conclusion, although the pVD2-Asian 1 vaccine was immunogenic, elicited sufficient titers of Nt antibodies against all DENV-2 genotypes, and provided 100% protection against challenge with virus of the homologous Asian 1 genotype and virus of the heterologous Cosmopolitan genotype, it is critical to monitor the potential emergence of Sylvatic genotype viruses, since vaccine candidates under development may not protect vaccinated humans from these viruses. IMPORTANCE Five genotype-specific dengue virus serotype 2 (DENV-2) DNA vaccine

Clustering and cellular distribution characteristics of virus particles of Tomato spotted wilt virus and Tomato zonate spot virus in different plant hosts.

PubMed

Zhang, Zhongkai; Zheng, Kuanyu; Dong, Jiahong; Fang, Qi; Hong, Jian; Wang, Xifeng

2016-01-19

Tomato spotted wilt virus (TSWV) and Tomato zonate spot virus (TZSV) are the two dominant species of thrip-transmitted tospoviruses, cause significant losses in crop yield in Yunnan and its neighboring provinces in China. TSWV and TZSV belong to different serogroup of tospoviruses but induce similar symptoms in the same host plant species, which makes diagnostic difficult. We used different electron microscopy preparing methods to investigate clustering and cellular distribution of TSWV and TZSV in the host plant species. Negative staining of samples infected with TSWV and TZSV revealed that particles usually clustered in the vesicles, including single particle (SP), double particles clustering (DPC), triple particles clustering (TPC). In the immunogold labeling negative staining against proteins of TZSV, the antibodies against Gn protein were stained more strongly than the N protein. Ultrathin section and high pressure freeze (HPF)-electron microscopy preparations revealed that TSWV particles were distributed in the cisternae of endoplasmic reticulum (ER), filamentous inclusions (FI) and Golgi bodies in the mesophyll cells. The TSWV particles clustered as multiple particles clustering (MPC) and distributed in globular viroplasm or cisternae of ER in the top leaf cell. TZSV particles were distributed more abundantly in the swollen membrane of ER in the mesophyll cell than those in the phloem parenchyma cells and were not observed in the top leaf cell. However, TZSV virions were mainly present as single particle in the cytoplasm, with few clustering as MPC. In this study, we identified TSWV and TZSV particles had the distinct cellular distribution patterns in the cytoplasm from different tissues and host plants. This is the first report of specific clustering characteristics of tospoviruses particles as well as the cellular distribution of TSWV particles in the FI and globular viroplasm where as TZSV particles inside the membrane of ER. These results indicated that

Annexin V Incorporated into Influenza Virus Particles Inhibits Gamma Interferon Signaling and Promotes Viral Replication

PubMed Central

Berri, Fatma; Haffar, Ghina; Lê, Vuong Ba; Sadewasser, Anne; Paki, Katharina; Lina, Bruno; Wolff, Thorsten

2014-01-01

ABSTRACT During the budding process, influenza A viruses (IAVs) incorporate multiple host cell membrane proteins. However, for most of them, their significance in viral morphogenesis and infectivity remains unknown. We demonstrate here that the expression of annexin V (A5) is upregulated at the cell surface upon IAV infection and that a substantial proportion of the protein is present in lipid rafts, the site of virus budding. Western blotting and immunogold analysis of highly purified IAV particles showed the presence of A5 in the virion. Significantly, gamma interferon (IFN-γ)-induced Stat phosphorylation and IFN-γ-induced 10-kDa protein (IP-10) production in macrophage-derived THP-1 cells was inhibited by purified IAV particles. Disruption of the IFN-γ signaling pathway was A5 dependent since downregulation of its expression or its blockage reversed the inhibition and resulted in decreased viral replication in vitro. The functional significance of these results was also observed in vivo. Thus, IAVs can subvert the IFN-γ antiviral immune response by incorporating A5 into their envelope during the budding process. IMPORTANCE Many enveloped viruses, including influenza A viruses, bud from the plasma membrane of their host cells and incorporate cellular surface proteins into viral particles. However, for the vast majority of these proteins, only the observation of their incorporation has been reported. We demonstrate here that the host protein annexin V is specifically incorporated into influenza virus particles during the budding process. Importantly, we showed that packaged annexin V counteracted the antiviral activity of gamma interferon in vitro and in vivo. Thus, these results showed that annexin V incorporated in the viral envelope of influenza viruses allow viral escape from immune surveillance. Understanding the role of host incorporated protein into virions may reveal how enveloped RNA viruses hijack the host cell machinery for their own purposes. PMID

Expanding the genetic code for site-specific labelling of tobacco mosaic virus coat protein and building biotin-functionalized virus-like particles.

PubMed

Wu, F C; Zhang, H; Zhou, Q; Wu, M; Ballard, Z; Tian, Y; Wang, J Y; Niu, Z W; Huang, Y

2014-04-18

A method for site-specific and high yield modification of tobacco mosaic virus coat protein (TMVCP) utilizing a genetic code expanding technology and copper free cycloaddition reaction has been established, and biotin-functionalized virus-like particles were built by the self-assembly of the protein monomers.

Virus-Like Particle Vaccine Protects against 2009 H1N1 Pandemic Influenza Virus in Mice

PubMed Central

Quan, Fu-Shi; Vunnava, Aswani; Compans, Richard W.; Kang, Sang-Moo

2010-01-01

Background The 2009 influenza pandemic and shortages in vaccine supplies worldwide underscore the need for new approaches to develop more effective vaccines. Methodology/Principal Findings We generated influenza virus-like particles (VLPs) containing proteins derived from the A/California/04/2009 virus, and tested their efficacy as a vaccine in mice. A single intramuscular vaccination with VLPs provided complete protection against lethal challenge with the A/California/04/2009 virus and partial protection against A/PR/8/1934 virus, an antigenically distant human isolate. VLP vaccination induced predominant IgG2a antibody responses, high hemagglutination inhibition (HAI) titers, and recall IgG and IgA antibody responses. HAI titers after VLP vaccination were equivalent to those observed after live virus infection. VLP immune sera also showed HAI responses against diverse geographic pandemic isolates. Notably, a low dose of VLPs could provide protection against lethal infection. Conclusion/Significance This study demonstrates that VLP vaccination provides highly effective protection against the 2009 pandemic influenza virus. The results indicate that VLPs can be developed into an effective vaccine, which can be rapidly produced and avoid the need to isolate high growth reassortants for egg-based production. PMID:20161790

Topographic control on the nascent Mediterranean outflow

NASA Astrophysics Data System (ADS)

Gasser, M.; Pelegrí, J. L.; Nash, J. D.; Peters, H.; García-Lafuente, J.

2011-12-01

Data collected during a 12-day cruise in July 2009 served to examine the structure of the nascent Mediterranean Outflow Water (MOW) immediately west of the Espartel Sill, the westernmost sill in the Strait of Gibraltar. The MOW is characterized by high salinities (>37.0 and reaching 38.3) and high velocities (exceeding 1 m s-1 at 100 m above the seafloor), and follows a submerged valley along a 30 km stretch, the natural western extension of the strait. It is approx. 150 m thick and 10 km wide, and experiences a substantial drop from 420 to 530 m over a distance of some 3 km between two relatively flat regions. Measurements indicate that the nascent MOW behaves as a gravity current with nearly maximal traveling speed; if this condition is maintained, then the maximum MOW velocity would decrease slowly with distance from the Espartel Sill, remaining significantly high until the gravity current excess density is only a small fraction of its original value. The sharp pycnocline between the Mediterranean and the overlying North Atlantic Central waters is dynamically unstable, particularly where the flow interacts with the 100 m decrease in bottom depth. Here, subcritical gradient Richardson numbers coincide with the development of large interfacial undulations and billows. The very energetic downslope flow is likely responsible for the development of a narrow V-shaped channel downstream of the seafloor drop along the axis of the submerged valley, this probably being the very first erosional scour produced by the nascent MOW. The coincidence of subcritical gradient Richardson numbers with relatively high turbidity values above the channel flanks suggests it may be undergoing upstream erosion.

Plant-Derived Chimeric Virus Particles for the Diagnosis of Primary Sjögren Syndrome.

PubMed

Tinazzi, Elisa; Merlin, Matilde; Bason, Caterina; Beri, Ruggero; Zampieri, Roberta; Lico, Chiara; Bartoloni, Elena; Puccetti, Antonio; Lunardi, Claudio; Pezzotti, Mario; Avesani, Linda

2015-01-01

Plants are ideal for the production of protein-based nanomaterials because they synthesize and assemble complex multimeric proteins that cannot be expressed efficiently using other platforms. Plant viruses can be thought of as self-replicating proteinaceous nanomaterials generally stable and easily produced in high titers. We used Potato virus X (PVX), chimeric virus particles, and Cowpea mosaic virus, empty virus-like particles to display a linear peptide (lipo) derived from human lipocalin, which is immunodominant in Sjögren's syndrome (SjS) and is thus recognized by autoantibodies in SjS patient serum. These virus-derived nanoparticles were thus used to develop a diagnostic assay for SjS based on a direct enzyme linked immunosorbent assay format. We found that PVX-lipo formulations were more sensitive than the chemically synthesized immunodominant peptide and equally specific when used to distinguish between healthy individuals and SjS patients. Our novel assay therefore allows the diagnosis of SjS using a simple, low-invasive serum test, contrasting with the invasive labial biopsy required for current tests. Our results demonstrate that nanomaterials based on plant viruses can be used as diagnostic reagents for SjS, and could also be developed for the diagnosis of other diseases.

Microstructure of atmospheric particles revealed by TXM and a new mode of influenza virus transmission

NASA Astrophysics Data System (ADS)

Bao, L. M.; Zhang, G. L.; Lei, Q. T.; Li, Y.; Li, X. L.; Hwu, Y. K.; Yi, J. M.

2015-09-01

For control of influenza, firstly it is important to find the real virus transmission media. Atmospheric aerosol particles are presumably one of the media. In this study, three typical atmospheric inhaled particles in Shanghai were studied by the synchrotron based transmission X-ray microscopes (TXM). Three dimensional microstructure of the particles reveals that there are many pores contained in, particularly the coal combustion fly particles which may be possible virus carrier. The particles can transport over long distance and cause long-range infections due to its light weight. We suggest a mode which is droplet combining with aerosol mode. By this mode the transmission of global and pandemic influenzas and infection between inland avian far from population and poultry or human living in cities along coast may be explained.

Isolated Potato Virus A coat protein possesses unusual properties and forms different short virus-like particles.

PubMed

Ksenofontov, Alexander L; Dobrov, Eugeny N; Fedorova, Natalia V; Serebryakova, Marina V; Prusov, Andrei N; Baratova, Ludmila A; Paalme, Viiu; Järvekülg, Lilian; Shtykova, Eleonora V

2018-05-01

In our previous study, we have observed that the isolated coat proteins (CP) of the Potyvirus Potato Virus A (PVA) virions exhibit an intrinsic tendency to self-associate into various multimeric forms containing some fractions of cross-β-structure. In this report, we studied the effect of solution conditions on the structure and dissociation of isolated PVA CP using a number of complementary physicochemical methods. Analysis of the structure of PVA CP in solution was performed by limited proteolysis with MALDI-TOF mass spectrometry analysis, transmission electron microscopy, intrinsic fluorescence spectroscopy, and synchrotron small angle X-ray scattering (SAXS). Overall structural characteristics of PVA CP obtained by combination of these methods and ab initio shape reconstruction by SAXS show that PVA CP forms large multi-subunit particles. We demonstrate that a mixture of compact virus-like particles (VLP) longer than 30 nm is assembled on dialysis of isolated CP into neutral pH buffer (at low ionic strength). Under conditions of high ionic strength (0.5 M NaCl) and high pH (pH 10.5), PVA dissociates into low compactness oval-shaped particles of approximately 30 subunits (20-30 nm). The results of limited trypsinolysis of these particles (enzyme/substrate ratio 1:100, 30 min) showed the existence of non-cleavable core-fragment, consisting of 137 amino acid residues. Trypsin treatment removed only a short N-terminal fragment in the intact virions. These particles are readily reassembled into regular VLPs by changing pH back to neutral. It is possible that these particles may represent some kind of intermediate in PVA assembly in vitro and in vivo.

Production of the virus-like particles of nipah virus matrix protein in Pichia pastoris as diagnostic reagents.

PubMed

Joseph, Narcisse Ms; Ho, Kok Lian; Tey, Beng Ti; Tan, Chon Seng; Shafee, Norazizah; Tan, Wen Siang

2016-07-08

The matrix (M) protein of Nipah virus (NiV) is a peripheral protein that plays a vital role in the envelopment of nucleocapsid protein and acts as a bridge between the viral surface and the nucleocapsid proteins. The M protein is also proven to play an important role in production of virus-like particles (VLPs) and is essential for assembly and budding of NiV particles. The recombinant M protein produced in Escherichia coli assembled into VLPs in the absence of the viral surface proteins. However, the E. coli produced VLPs are smaller than the native virus particles. Therefore, the aims of this study were to produce NiV M protein in Pichia pastoris, to examine the structure of the VLPs formed, and to assess the potential of the VLPs as a diagnostic reagent. The M protein was successfully expressed in P. pastoris and was detected with anti-myc antibody using Western blotting. The VLPs formed by the recombinant M protein were purified with sucrose density gradient ultracentrifugation, high-performance liquid chromatography (HPLC), and Immobilized Metal Affinity Chromatography (IMAC). Immunogold staining and transmission electron microscopy confirmed that the M protein assembled into VLPs as large as 200 nm. ELISA revealed that the NiV M protein produced in P. pastoris reacted strongly with positive NiV sera demonstrating its potential as a diagnostic reagent. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1038-1045, 2016. © 2016 American Institute of Chemical Engineers.

Utility of Japanese encephalitis virus subgenomic replicon-based single-round infectious particles as antigens in neutralization tests for Zika virus and three other flaviviruses.

PubMed

Yamanaka, Atsushi; Moi, Meng Ling; Takasaki, Tomohiko; Kurane, Ichiro; Matsuda, Mami; Suzuki, Ryosuke; Konishi, Eiji

2017-05-01

The introduction of a foreign virus into an area may cause an outbreak, as with the Zika virus (ZIKV) outbreak in the Americas. Preparedness for handling a viral outbreak involves the development of tests for the serodiagnosis of foreign virus infections. We previously established a gene-based technology to generate some flaviviral antigens useful for functional antibody assays. The technology utilizes a Japanese encephalitis virus subgenomic replicon to generate single-round infectious particles (SRIPs) that possess designed surface antigens. In the present study, we successfully expanded the capacity of SRIPs to four human-pathogenic mosquito-borne flaviviruses that could potentially be introduced from endemic to non-endemic countries: ZIKV, Sepik virus, Wesselsbron virus, and Usutu virus. Flavivirus-crossreactive monoclonal antibodies dose-dependently neutralized these SRIPs. ZIKV-SRIPs also produced antibody-dose-dependent neutralization curves equivalent to those shown by authentic ZIKV particles using sera from a Zika fever patient. The faithful expression of designed surface antigens on SRIPs will allow their use in neutralization tests to diagnose foreign flaviviral infections. Copyright © 2017 Elsevier B.V. All rights reserved.

Recombinant Expression of Tandem-HBc Virus-Like Particles (VLPs).

PubMed

Stephen, Sam L; Beales, Lucy; Peyret, Hadrien; Roe, Amy; Stonehouse, Nicola J; Rowlands, David J

2018-01-01

The hepatitis B virus (HBV) core protein (HBc) has formed the building block for virus-like particle (VLP) production for more than 30 years. The ease of production of the protein, the robust ability of the core monomers to dimerize and assemble into intact core particles, and the strong immune responses they elicit when presenting antigenic epitopes all demonstrate its promise for vaccine development (reviewed in Pumpens and Grens (Intervirology 44: 98-114, 2001)). HBc has been modified in a number of ways in attempts to expand its potential as a novel vaccine platform. The HBc protein is predominantly α-helical in structure and folds to form an L-shaped molecule. The structural subunit of the HBc particle is a dimer of monomeric HBc proteins which together form an inverted T-shaped structure. In the assembled HBc particle the four-helix bundle formed at each dimer interface appears at the surface as a prominent "spike." The tips of the "spikes" are the preferred sites for the insertion of foreign sequences for vaccine purposes as they are the most highly exposed regions of the assembled particles. In the tandem-core modification two copies of the HBc protein are covalently linked by a flexible amino acid sequence which allows the fused dimer to fold correctly and assemble into HBc particles. The advantage of the modified structure is that the assembly of the dimeric subunits is defined and not formed by random association. This facilitates the introduction of single, larger sequences at the tip of each surface "spike," thus overcoming the conformational clashes contingent on insertion of large structures into monomeric HBc proteins.Differences in inserted sequences influence the assembly characteristics of the modified proteins, and it is important to optimize the design of each novel construct to maximize efficiency of assembly into regular VLPs. In addition to optimization of the construct, the expression system used can also influence the ability of

Structure of the immature HIV-1 capsid in intact virus particles at 8.8 Å resolution

NASA Astrophysics Data System (ADS)

Schur, Florian K. M.; Hagen, Wim J. H.; Rumlová, Michaela; Ruml, Tomáš; Müller, Barbara; Kräusslich, Hans-Georg; Briggs, John A. G.

2015-01-01

Human immunodeficiency virus type 1 (HIV-1) assembly proceeds in two stages. First, the 55 kilodalton viral Gag polyprotein assembles into a hexameric protein lattice at the plasma membrane of the infected cell, inducing budding and release of an immature particle. Second, Gag is cleaved by the viral protease, leading to internal rearrangement of the virus into the mature, infectious form. Immature and mature HIV-1 particles are heterogeneous in size and morphology, preventing high-resolution analysis of their protein arrangement in situ by conventional structural biology methods. Here we apply cryo-electron tomography and sub-tomogram averaging methods to resolve the structure of the capsid lattice within intact immature HIV-1 particles at subnanometre resolution, allowing unambiguous positioning of all α-helices. The resulting model reveals tertiary and quaternary structural interactions that mediate HIV-1 assembly. Strikingly, these interactions differ from those predicted by the current model based on in vitro-assembled arrays of Gag-derived proteins from Mason-Pfizer monkey virus. To validate this difference, we solve the structure of the capsid lattice within intact immature Mason-Pfizer monkey virus particles. Comparison with the immature HIV-1 structure reveals that retroviral capsid proteins, while having conserved tertiary structures, adopt different quaternary arrangements during virus assembly. The approach demonstrated here should be applicable to determine structures of other proteins at subnanometre resolution within heterogeneous environments.

Production of Japanese encephalitis virus-like particles in insect cells.

PubMed

Yamaji, Hideki; Konishi, Eiji

2013-01-01

Virus-like particles (VLPs) are composed of one or several recombinant viral surface proteins that spontaneously assemble into particulate structures without the incorporation of virus DNA or RNA. The baculovirus-insect cell system has been used extensively for the production of recombinant virus proteins including VLPs. While the baculovirus-insect cell system directs the transient expression of recombinant proteins in a batch culture, stably transformed insect cells allow constitutive production. In our recent study, a secretory form of Japanese encephalitis (JE) VLPs was successfully produced by Trichoplusia ni BTI-TN-5B1-4 (High Five) cells engineered to coexpress the JE virus (JEV) premembrane (prM) and envelope (E) proteins. A higher yield of E protein was attained with recombinant High Five cells than with the baculovirus-insect cell system. This study demonstrated that recombinant insect cells offer a promising approach to the high-level production of VLPs for use as vaccines and diagnostic antigens.

Defective-interfering particles of the human parvovirus adeno-associated virus. [uv radiation

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

Laughlin, C.A.; Myers, M.W.; Risin, D.L.

1979-04-15

We have previously shown that adeno-associated virus (AAV) grown in KB cells with a helper adenovirus, produced several classes of particles defined by their buoyant density in CsCl. The predominant density classes were referred to as AAV(1.45), AAV(1.41), AAV (1.35), and AAV(1.32), respectively, where the density of the particle was written in the parentheses. The AAV(1.45) and AAV(1.41) particles which contained standard genomes were the only infectious AAV these infectious AAV particles exhibited autointerference. The ligh-density AAV(1.35) and (1.32) particles contained aberrant (deleted and/or snap-back) genomes. We report here experiments which show that the light-density AAV particles were noninfectious butmore » interfered with the replication of AAV(1.41). The interference was intracellular and resulted in inhibition of synthesis of standard (14.5S) AAV genomes. In some cases there was also a concomitant increase in synthesis of aberrant, shorter AAV DNA. The inhibitory activity of the light-density particles was abolished by uv irradiation. These results show that the population of light AAV particles contained DI particles. The observed autointerference of AAV(1.45) or AAV(1.41) virus is postulated to be due to AAV DI particles. Replication of AAV DI genomes appeared to require the presence of replicating, standard AAV genomes. This is interpreted to mean that progeny strand replication of AAV requires an AAV-specified product, presumably the AAV capsid protein. In contrast to standard, infectious AAV, the AAV DI particles alone do not inhibit replication of the helper adenovirus.« less

Mutations in the Transmembrane Domain and Cytoplasmic Tail of Hendra Virus Fusion Protein Disrupt Virus-Like-Particle Assembly.

PubMed

Cifuentes-Muñoz, Nicolás; Sun, Weina; Ray, Greeshma; Schmitt, Phuong Tieu; Webb, Stacy; Gibson, Kathleen; Dutch, Rebecca Ellis; Schmitt, Anthony P

2017-07-15

Hendra virus (HeV) is a zoonotic paramyxovirus that causes deadly illness in horses and humans. An intriguing feature of HeV is the utilization of endosomal protease for activation of the viral fusion protein (F). Here we investigated how endosomal F trafficking affects HeV assembly. We found that the HeV matrix (M) and F proteins each induced particle release when they were expressed alone but that their coexpression led to coordinated assembly of virus-like particles (VLPs) that were morphologically and physically distinct from M-only or F-only VLPs. Mutations to the F protein transmembrane domain or cytoplasmic tail that disrupted endocytic trafficking led to failure of F to function with M for VLP assembly. Wild-type F functioned normally for VLP assembly even when its cleavage was prevented with a cathepsin inhibitor, indicating that it is endocytic F trafficking that is important for VLP assembly, not proteolytic F cleavage. Under specific conditions of reduced M expression, we found that M could no longer induce significant VLP release but retained the ability to be incorporated as a passenger into F-driven VLPs, provided that the F protein was competent for endocytic trafficking. The F and M proteins were both found to traffic through Rab11-positive recycling endosomes (REs), suggesting a model in which F and M trafficking pathways converge at REs, enabling these proteins to preassemble before arriving at plasma membrane budding sites. IMPORTANCE Hendra virus and Nipah virus are zoonotic paramyxoviruses that cause lethal infections in humans. Unlike that for most paramyxoviruses, activation of the henipavirus fusion protein occurs in recycling endosomal compartments. In this study, we demonstrate that the unique endocytic trafficking pathway of Hendra virus F protein is required for proper viral assembly and particle release. These results advance our basic understanding of the henipavirus assembly process and provide a novel model for the interplay between

Hantavirus Gn and Gc glycoproteins self-assemble into virus-like particles.

PubMed

Acuña, Rodrigo; Cifuentes-Muñoz, Nicolás; Márquez, Chantal L; Bulling, Manuela; Klingström, Jonas; Mancini, Roberta; Lozach, Pierre-Yves; Tischler, Nicole D

2014-02-01

How hantaviruses assemble and exit infected cells remains largely unknown. Here, we show that the expression of Andes (ANDV) and Puumala (PUUV) hantavirus Gn and Gc envelope glycoproteins lead to their self-assembly into virus-like particles (VLPs) which were released to cell supernatants. The viral nucleoprotein was not required for particle formation. Further, a Gc endodomain deletion mutant did not abrogate VLP formation. The VLPs were pleomorphic, exposed protrusions and reacted with patient sera.

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

PubMed

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

2008-03-28

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

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

PubMed Central

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

2009-01-01

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

The Measles Virus Receptor SLAMF1 Can Mediate Particle Endocytosis.

PubMed

Gonçalves-Carneiro, Daniel; McKeating, Jane A; Bailey, Dalan

2017-04-01

The signaling lymphocyte activation molecule F1 (SLAMF1) is both a microbial sensor and entry receptor for measles virus (MeV). Herein, we describe a new role for SLAMF1 to mediate MeV endocytosis that is in contrast with the alternative, and generally accepted, model that MeV genome enters cells only after fusion at the cell surface. We demonstrated that MeV engagement of SLAMF1 induces dramatic but transient morphological changes, most prominently in the formation of membrane blebs, which were shown to colocalize with incoming viral particles, and rearrangement of the actin cytoskeleton in infected cells. MeV infection was dependent on these dynamic cytoskeletal changes as well as fluid uptake through a macropinocytosis-like pathway as chemical inhibition of these processes inhibited entry. Moreover, we identified a role for the RhoA-ROCK-myosin II signaling axis in this MeV internalization process, highlighting a novel role for this recently characterized pathway in virus entry. Our study shows that MeV can hijack a microbial sensor normally involved in bacterial phagocytosis to drive endocytosis using a complex pathway that shares features with canonical viral macropinocytosis, phagocytosis, and mechanotransduction. This uptake pathway is specific to SLAMF1-positive cells and occurs within 60 min of viral attachment. Measles virus remains a significant cause of mortality in human populations, and this research sheds new light on the very first steps of infection of this important pathogen. IMPORTANCE Measles is a significant disease in humans and is estimated to have killed over 200 million people since records began. According to current World Health Organization statistics, it still kills over 100,000 people a year, mostly children in the developing world. The causative agent, measles virus, is a small enveloped RNA virus that infects a broad range of cells during infection. In particular, immune cells are infected via interactions between glycoproteins found

Different applications of virus-like particles in biology and medicine: Vaccination and delivery systems.

PubMed

Shirbaghaee, Zeinab; Bolhassani, Azam

2016-03-01

Virus-like particles (VLPs) mimic the whole construct of virus particles devoid of viral genome as used in subunit vaccine design. VLPs can elicit efficient protective immunity as direct immunogens compared to soluble antigens co-administered with adjuvants in several booster injections. Up to now, several prokaryotic and eukaryotic systems such as insect, yeast, plant, and E. coli were used to express recombinant proteins, especially for VLP production. Recent studies are also generating VLPs in plants using different transient expression vectors for edible vaccines. VLPs and viral particles have been applied for different functions such as gene therapy, vaccination, nanotechnology, and diagnostics. Herein, we describe VLP production in different systems as well as its applications in biology and medicine. © 2015 Wiley Periodicals, Inc.

Small-angle neutron scattering study of recombinant yeast-derived human hepatitis B virus surface antigen vaccine particle

NASA Astrophysics Data System (ADS)

Sato, M.; Ito, Y.; Kameyama, K.; Imai, M.; Ishikawa, N.; Takagi, T.

1995-02-01

The overall and internal structure of recombinant yeast-derived human hepatitis B virus surface antigen vaccine particles was investigated by small-angle neutron scattering using the contrast variation method. The vaccine is a nearly spherical particle, and its contrast-matching point was determined to be at about 24% D 2O content, indicating that a large part of the vaccine particle is occupied by lipids and carbohydrates from the yeast. The Stuhrmann plot suggests that the surface antigens exist predominantly in the peripheral region of the particle, which is favorable to the induction of anti-virus antibodies.

An enzymatic assay based on luciferase Ebola virus-like particles for evaluation of virolytic activity of antimicrobial peptides.

PubMed

Peskova, Marie; Heger, Zbynek; Janda, Petr; Adam, Vojtech; Pekarik, Vladimir

2017-02-01

Antimicrobial peptides are currently considered as promising antiviral compounds. Current assays to evaluate the effectivity of peptides against enveloped viruses based on liposomes or hemolysis are encumbered by the artificial nature of liposomes or distinctive membrane composition of used erythrocytes. We propose a novel assay system based on enzymatic Ebola virus-like particles containing sensitive luciferase reporter. The assay was validated with several cationic and anionic peptides and compared with lentivirus inactivation and hemolytic assays. The assay is sensitive and easy to perform in standard biosafety level laboratory with potential for high-throughput screens. The use of virus-like particles in the assay provides a system as closely related to the native viruses as possible eliminating some issues associated with other more artificial set ups. We have identified CAM-W (KWKLWKKIEKWGQGIGAVLKWLTTWL) as a peptide with the greatest antiviral activity against infectious lentiviral vectors and filoviral virus-like particles. Copyright © 2016 Elsevier Inc. All rights reserved.

The Respiratory Syncytial Virus Phosphoprotein, Matrix Protein, and Fusion Protein Carboxy-Terminal Domain Drive Efficient Filamentous Virus-Like Particle Formation

PubMed Central

Meshram, Chetan D.; Baviskar, Pradyumna S.; Ognibene, Cherie M.

2016-01-01

ABSTRACT Virus-like particles (VLPs) are attractive as a vaccine concept. For human respiratory syncytial virus (hRSV), VLP assembly is poorly understood and appears inefficient. Hence, hRSV antigens are often incorporated into foreign VLP systems to generate anti-RSV vaccine candidates. To better understand the assembly, and ultimately to enable efficient production, of authentic hRSV VLPs, we examined the associated requirements and mechanisms. In a previous analysis in HEp-2 cells, the nucleoprotein (N), phosphoprotein (P), matrix protein (M), and fusion protein (F) were required for formation of filamentous VLPs, which, similar to those of wild-type virus, were associated with the cell surface. Using fluorescence and electron microscopy combined with immunogold labeling, we examined the surfaces of transfected HEp-2 cells and further dissected the process of filamentous VLP formation. Our results show that N is not required. Coexpression of P plus M plus F, but not P plus M, M plus F, or P plus F, induced both viral protein coalescence and formation of filamentous VLPs that resembled wild-type virions. Despite suboptimal coalescence in the absence of P, the M and F proteins, when coexpressed, formed cell surface-associated filaments with abnormal morphology, appearing longer and thinner than wild-type virions. For F, only the carboxy terminus (Fstem) was required, and addition of foreign protein sequences to Fstem allowed incorporation into VLPs. Together, the data show that P, M, and the F carboxy terminus are sufficient for robust viral protein coalescence and filamentous VLP formation and suggest that M-F interaction drives viral filament formation, with P acting as a type of cofactor facilitating the process and exerting control over particle morphology. IMPORTANCE hRSV is responsible for >100,000 deaths in children worldwide, and a vaccine is not available. Among the potential anti-hRSV approaches are virus-like particle (VLP) vaccines, which, based on

Assembly and composition of intracellular particles formed by Moloney murine leukemia virus.

PubMed Central

Hansen, M; Jelinek, L; Jones, R S; Stegeman-Olsen, J; Barklis, E

1993-01-01

Assembly of type C retroviruses such as Moloney murine leukemia virus (M-MuLV) ordinarily occurs at the plasma membranes of infected cells and absolutely requires the particle core precursor protein, Pr65gag. Previously we have shown that Pr65gag is membrane associated and that at least a portion of intracellular Pr65gag protein appears to be routed to the plasma membrane by a vesicular transport pathway. Here we show that intracellular particle formation can occur in M-MuLV-infected cells. M-MuLV immature particles were observed by electron microscopy budding into and within rough endoplasmic reticulum, Golgi, and vacuolar compartments. Biochemical fractionation studies indicated that intracellular Pr65gag was present in nonionic detergent-resistant complexes of greater than 150S. Additionally, viral RNA and polymerase functions appeared to be associated with intracellular particles, as were Gag-beta-galactosidase fusion proteins which have the capacity to be incorporated into virions. Immature intracellular particles in postnuclear lysates could be proteolytically processed in vitro to mature forms, while extracellular immature M-MuLV particles remained immature as long as 10 h during incubations. The occurrence of M-MuLV-derived intracellular particles demonstrates that Pr65gag can associate with intracellular membranes and indicates that if a plasma membrane Pr65gag receptor exists, it also can be found in other membrane compartments. These results support the hypothesis that intracellular particles may serve as a virus reservoir during in vivo infections. Images PMID:8350394

Selective autophagy limits cauliflower mosaic virus infection by NBR1-mediated targeting of viral capsid protein and particles

PubMed Central

Hafrén, Anders; Macia, Jean-Luc; Love, Andrew J.; Milner, Joel J.; Drucker, Martin; Hofius, Daniel

2017-01-01

Autophagy plays a paramount role in mammalian antiviral immunity including direct targeting of viruses and their individual components, and many viruses have evolved measures to antagonize or even exploit autophagy mechanisms for the benefit of infection. In plants, however, the functions of autophagy in host immunity and viral pathogenesis are poorly understood. In this study, we have identified both anti- and proviral roles of autophagy in the compatible interaction of cauliflower mosaic virus (CaMV), a double-stranded DNA pararetrovirus, with the model plant Arabidopsis thaliana. We show that the autophagy cargo receptor NEIGHBOR OF BRCA1 (NBR1) targets nonassembled and virus particle-forming capsid proteins to mediate their autophagy-dependent degradation, thereby restricting the establishment of CaMV infection. Intriguingly, the CaMV-induced virus factory inclusions seem to protect against autophagic destruction by sequestering capsid proteins and coordinating particle assembly and storage. In addition, we found that virus-triggered autophagy prevents extensive senescence and tissue death of infected plants in a largely NBR1-independent manner. This survival function significantly extends the timespan of virus production, thereby increasing the chances for virus particle acquisition by aphid vectors and CaMV transmission. Together, our results provide evidence for the integration of selective autophagy into plant immunity against viruses and reveal potential viral strategies to evade and adapt autophagic processes for successful pathogenesis. PMID:28223514

Entrepreneurial Identity and Role Expectations in Nascent Entrepreneurship

ERIC Educational Resources Information Center

Lundqvist, Mats; Middleton, Karen Williams; Nowell, Pamela

2015-01-01

Entrepreneurship has been defined as an individual?new value creation dialogic. To study how entrepreneurial identity evolves, this article, drawing on entrepreneurial learning theory, adds an entrepreneurial role expectations dialogic. Longitudinal evidence from nascent entrepreneurs working in venture teams on invention disclosures offers an…

Evidences of Changes in Surface Electrostatic Charge Distribution during Stabilization of HPV16 Virus-Like Particles

PubMed Central

Vega, Juan F.; Vicente-Alique, Ernesto; Núñez-Ramírez, Rafael; Wang, Yang; Martínez-Salazar, Javier

2016-01-01

The stabilization of human papillomavirus type 16 virus-like particles has been examined by means of different techniques including dynamic and static light scattering, transmission electron microscopy and electrophoretic mobility. All these techniques provide different and often complementary perspectives about the aggregation process and generation of stabilized virus-like particles after a period of time of 48 hours at a temperature of 298 K. Interestingly, static light scattering results point towards a clear colloidal instability in the initial systems, as suggested by a negative value of the second virial coefficient. This is likely related to small repulsive electrostatic interactions among the particles, and in agreement with relatively small absolute values of the electrophoretic mobility and, hence, of the net surface charges. At this initial stage the small repulsive interactions are not able to compensate binding interactions, which tend to aggregate the particles. As time proceeds, an increase of the size of the particles is accompanied by strong increases, in absolute values, of the electrophoretic mobility and net surface charge, suggesting enhanced repulsive electrostatic interactions and, consequently, a stabilized colloidal system. These results show that electrophoretic mobility is a useful methodology that can be applied to screen the stabilization factors for virus-like particles during vaccine development. PMID:26885635

Hantavirus Gn and Gc Glycoproteins Self-Assemble into Virus-Like Particles

PubMed Central

Acuña, Rodrigo; Cifuentes-Muñoz, Nicolás; Márquez, Chantal L.; Bulling, Manuela; Klingström, Jonas; Mancini, Roberta; Lozach, Pierre-Yves

2014-01-01

How hantaviruses assemble and exit infected cells remains largely unknown. Here, we show that the expression of Andes (ANDV) and Puumala (PUUV) hantavirus Gn and Gc envelope glycoproteins lead to their self-assembly into virus-like particles (VLPs) which were released to cell supernatants. The viral nucleoprotein was not required for particle formation. Further, a Gc endodomain deletion mutant did not abrogate VLP formation. The VLPs were pleomorphic, exposed protrusions and reacted with patient sera. PMID:24335294

Accelerators as Authentic Training Experiences for Nascent Entrepreneurs

ERIC Educational Resources Information Center

Miles, Morgan P.; de Vries, Huibert; Harrison, Geoff; Bliemel, Martin; de Klerk, Saskia; Kasouf, Chick J.

2017-01-01

Purpose: The purpose of this paper is to address the role of accelerators as authentic learning-based entrepreneurial training programs. Accelerators facilitate the development and assessment of entrepreneurial competencies in nascent entrepreneurs through the process of creating a start-up venture. Design/methodology/approach: Survey data from…

RNA packaging of MRFV virus-like particles: The interplay between RNA pools and capsid coat protein

USDA-ARS?s Scientific Manuscript database

Virus-like particles (VLPs) can be produced through self-assembly of capsid protein (CP) into particles with discrete shapes and sizes and containing different types of RNA molecules. The general principle that governs particle assembly and RNA packaging is determined by unique interactions between ...

Nonenzymatic Role for WRN in Preserving Nascent DNA Strands after Replication Stress

DOE PAGES

Su, Fengtao; Mukherjee, Shibani; Yang, Yanyong; ...

2014-11-20

WRN, the protein defective in Werner syndrome (WS), is a multifunctional nuclease involved in DNA damage repair, replication, and genome stability maintenance. It was assumed that the nuclease activities of WRN were critical for these functions. Here, we report a nonenzymatic role for WRN in preserving nascent DNA strands following replication stress. We found that lack of WRN led to shortening of nascent DNA strands after replication stress. Furthermore, we discovered that the exonuclease activity of MRE11 was responsible for the shortening of newly replicated DNA in the absence of WRN. Mechanistically, the N-terminal FHA domain of NBS1 recruits WRNmore » to replication-associated DNA double-stranded breaks to stabilize Rad51 and to limit the nuclease activity of its C-terminal binding partner MRE11. Thus, this previously unrecognized nonenzymatic function of WRN in the stabilization of nascent DNA strands sheds light on the molecular reason for the origin of genome instability in WS individuals.« less

The 3.2 Angstrom Resolution Structure of the Polymorphic Cowpea Chlorotic Mottle Virus Ribonucleoprotein Particle

NASA Astrophysics Data System (ADS)

Speir, Jeffrey Alan

Structural studies of the polymorphic cowpea chlorotic mottle virus have resulted in high resolution structures for two distinct icosahedral ribonucleoprotein particle conformations dependent upon whether acidic or basic pH conditions prevail. CCMV is stable below pH 6.5, however metal-free particles maintain a 10% increase in hydrodynamic volume at pH >=q 7.5. Identification of this swollen' form of CCMV, which can easily be disrupted with 1M NaCl, led to the first reassembly of an icosahedral virus in vitro from purified viral protein and RNA to form infectious particles, and its assembly has been the subject of biochemical and biophysical investigations for over twenty-five years. Under well defined conditions of pH, ionic strength and divalent metal ion concentration, CCMV capsid protein or capsid protein and RNA will reassemble to form icosahedral particles of various sizes, sheets, tubes, rosettes, and a variety of laminar structures which resemble virion structures from non-related virus families. Analysis of native particles at 3.2A resolution and swollen particles at 28A resolution has suggested that the chemical basis for the formation of polymorphic icosahedral and anisometric structures is: (i) hexamers formed of beta-barrel subunits stabilized by an unusual hexameric parallel beta structure made up of their N-termini, (ii) the location of protein-RNA interactions, (iii) divalent metal cation binding sites that regulate quasi-symmetrical subunit associations, (iv) charge repulsion across the same interfaces when lacking divalent metal ions at basic pH, which induces the formation of sixty 20A diameter portals for RNA release, and (v) a novel, C-terminal-based, subunit dimer assembly unit. The use of C- and N-terminal arms in CCMV has not been observed in other icosahedral RNA virus structures determined at near atomic resolution, however, their detailed interactions and roles in stabilizing the quaternary organization of CCMV are related to that found

A Japanese Encephalitis Virus Peptide Present on Johnson Grass Mosaic Virus-Like Particles Induces Virus-Neutralizing Antibodies and Protects Mice against Lethal Challenge

PubMed Central

Saini, Manisha; Vrati, Sudhanshu

2003-01-01

Protection against Japanese encephalitis virus (JEV) is antibody dependent, and neutralizing antibodies alone are sufficient to impart protection. Thus, we are aiming to develop a peptide-based vaccine against JEV by identifying JEV peptide sequences that could induce virus-neutralizing antibodies. Previously, we have synthesized large amounts of Johnson grass mosaic virus (JGMV) coat protein (CP) in Escherichia coli and have shown that it autoassembled to form virus-like particles (VLPs). The envelope (E) protein of JEV contains the virus-neutralization epitopes. Four peptides from different locations within JEV E protein were chosen, and these were fused to JGMV CP by recombinant DNA methods. The fusion protein autoassembled to form VLPs that could be purified by sucrose gradient centrifugation. Immunization of mice with the recombinant VLPs containing JEV peptide sequences induced anti-peptide and anti-JEV antibodies. A 27-amino-acid peptide containing amino acids 373 to 399 from JEV E protein, present on JGMV VLPs, induced virus-neutralizing antibodies. Importantly, these antibodies were obtained without the use of an adjuvant. The immunized mice showed significant protection against a lethal JEV challenge. PMID:12610124

A Lagrangian particle model to predict the airborne spread of foot-and-mouth disease virus

NASA Astrophysics Data System (ADS)

Mayer, D.; Reiczigel, J.; Rubel, F.

Airborne spread of bioaerosols in the boundary layer over a complex terrain is simulated using a Lagrangian particle model, and applied to modelling the airborne spread of foot-and-mouth disease (FMD) virus. Two case studies are made with study domains located in a hilly region in the northwest of the Styrian capital Graz, the second largest town in Austria. Mountainous terrain as well as inhomogeneous and time varying meteorological conditions prevent from application of so far used Gaussian dispersion models, while the proposed model can handle these realistically. In the model, trajectories of several thousands of particles are computed and the distribution of virus concentration near the ground is calculated. This allows to assess risk of infection areas with respect to animal species of interest, such as cattle, swine or sheep. Meteorological input data like wind field and other variables necessary to compute turbulence were taken from the new pre-operational version of the non-hydrostatic numerical weather prediction model LMK ( Lokal-Modell-Kürzestfrist) running at the German weather service DWD ( Deutscher Wetterdienst). The LMK model provides meteorological parameters with a spatial resolution of about 2.8 km. To account for the spatial resolution of 400 m used by the Lagrangian particle model, the initial wind field is interpolated upon the finer grid by a mass consistent interpolation method. Case studies depict a significant influence of local wind systems on the spread of virus. Higher virus concentrations at the upwind side of the hills and marginal concentrations in the lee are well observable, as well as canalization effects by valleys. The study demonstrates that the Lagrangian particle model is an appropriate tool for risk assessment of airborne spread of virus by taking into account the realistic orographic and meteorological conditions.

Self-assembly of virus-like particles of canine parvovirus capsid protein expressed from Escherichia coli and application as virus-like particle vaccine.

PubMed

Xu, Jin; Guo, Hui-Chen; Wei, Yan-Quan; Dong, Hu; Han, Shi-Chong; Ao, Da; Sun, De-Hui; Wang, Hai-Ming; Cao, Sui-Zhong; Sun, Shi-Qi

2014-04-01

Canine parvovirus disease is an acute infectious disease caused by canine parvovirus (CPV). Current commercial vaccines are mainly attenuated and inactivated; as such, problems concerning safety may occur. To resolve this problem, researchers developed virus-like particles (VLPs) as biological nanoparticles resembling natural virions and showing high bio-safety. This property allows the use of VLPs for vaccine development and mechanism studies of viral infections. Tissue-specific drug delivery also employs VLPs as biological nanomaterials. Therefore, VLPs derived from CPV have a great potential in medicine and diagnostics. In this study, small ubiquitin-like modifier (SUMO) fusion motif was utilized to express a whole, naturalVP2 protein of CPV in Escherichia coli. After the cleavage of the fusion motif, the CPV VP2 protein has self-assembled into VLPs. The VLPs had a size and shape that resembled the authentic virus capsid. However, the self-assembly efficiency of VLPs can be affected by different pH levels and ionic strengths. The mice vaccinated subcutaneously with CPV VLPs and CPV-specific immune responses were compared with those immunized with the natural virus. This result showed that VLPs can effectively induce anti-CPV specific antibody and lymphocyte proliferation as a whole virus. This result further suggested that the antigen epitope of CPV was correctly present on VLPs, thereby showing the potential application of a VLP-based CPV vaccine.

Cancer Immunotherapy Using Virus-like Particles | NCI Technology Transfer Center | TTC

Cancer.gov

A considerable effort has been devoted to identifying and targeting specific extracellular cancer markers using antibody based therapies. However, diminished access to new cancer cell surface markers has limited the development of corresponding antibodies. NCI Technology Transfer Center is seeking to license cancer immunotherapy using virus-like particles.

Critical Role of the HTLV-1 Capsid N-Terminal Domain for Gag-Gag Interactions and Virus Particle Assembly.

PubMed

Martin, Jessica L; Mendonça, Luiza; Marusinec, Rachel; Zuczek, Jennifer; Angert, Isaac; Blower, Ruth J; Mueller, Joachim D; Perilla, Juan R; Zhang, Wei; Mansky, Louis M

2018-04-25

The retroviral Gag protein is the main structural protein responsible for virus particle assembly and release. Like human immunodeficiency virus type 1 (HIV-1) Gag, human T-cell leukemia virus type 1 (HTLV-1) has a structurally conserved capsid (CA) domain, including a β-hairpin turn and a centralized coiled-coil-like structure of six α helices in the CA amino-terminal domain (NTD) as well as four α-helices in the CA carboxy-terminal domain (CTD). CA drives Gag oligomerization, which is critical for both immature Gag lattice formation and particle production. The HIV-1 CA CTD has previously been shown to be a primary determinant for CA-CA interactions, and while both the HTLV-1 CA NTD and CTD have been implicated in Gag-Gag interactions, our recent observations have implicated the HTLV-1 CA NTD as encoding key determinants that dictate particle morphology. Here, we have conducted alanine-scanning mutagenesis in the HTLV-1 CA NTD nucleotide-encoding sequences spanning the loop regions and amino acids at the beginning and ends of α-helices due to their structural dissimilarity from the HIV-1 CA NTD structure. We analyzed both Gag subcellular distribution and efficiency of particle production for these mutants. We discovered several important residues (i.e., M17, Q47/F48, and Y61). Modeling implicated that these residues reside at the dimer interface (i.e., M17 and Y61) or at the trimer interface (i.e., Q47/F48). Taken together, these observations highlight the critical role of the HTLV-1 CA NTD in Gag-Gag interactions and particle assembly, which is, to the best of our knowledge, in contrast to HIV-1 and other retroviruses. Importance Retrovirus particle assembly and release from infected cells is driven by the Gag structural protein. Gag-Gag interactions, which form an oligomeric lattice structure at a particle budding site, are essential to the biogenesis of an infectious virus particle. The capsid (CA) domain of Gag is generally thought to possess the key

Cloning of nascent monkey DNA synthesized early in the cell cycle.

PubMed

Kaufmann, G; Zannis-Hadjopoulos, M; Martin, R G

1985-04-01

To study the structure and complexity of animal cell replication origins, we have isolated and cloned nascent DNA from the onset of S phase as follows: African green monkey kidney cells arrested in G1 phase were serum stimulated in the presence of the DNA replication inhibitor aphidicolin. After 18 h, the drug was removed, and DNA synthesis was allowed to proceed in vivo for 1 min. Nuclei were then prepared, and DNA synthesis was briefly continued in the presence of Hg-dCTP. The mercury-labeled nascent DNA was purified in double-stranded form by extrusion (M. Zannis-Hadjopoulos, M. Perisco, and R. G. Martin, Cell 27:155-163, 1981) followed by sulfhydryl-agarose affinity chromatography. Purified nascent DNA (ca. 500 to 2,000 base pairs) was treated with mung bean nuclease to remove single-stranded ends and inserted into the NruI site of plasmid pBR322. The cloned fragments were examined for their time of replication by hybridization to cellular DNA fractions synthesized at various intervals of the S phase. Among five clones examined, four hybridized preferentially with early replicating fractions.

Elastic Coupling of Nascent apCAM Adhesions to Flowing Actin Networks

PubMed Central

Mejean, Cecile O.; Schaefer, Andrew W.; Buck, Kenneth B.; Kress, Holger; Shundrovsky, Alla; Merrill, Jason W.; Dufresne, Eric R.; Forscher, Paul

2013-01-01

Adhesions are multi-molecular complexes that transmit forces generated by a cell’s acto-myosin networks to external substrates. While the physical properties of some of the individual components of adhesions have been carefully characterized, the mechanics of the coupling between the cytoskeleton and the adhesion site as a whole are just beginning to be revealed. We characterized the mechanics of nascent adhesions mediated by the immunoglobulin-family cell adhesion molecule apCAM, which is known to interact with actin filaments. Using simultaneous visualization of actin flow and quantification of forces transmitted to apCAM-coated beads restrained with an optical trap, we found that adhesions are dynamic structures capable of transmitting a wide range of forces. For forces in the picoNewton scale, the nascent adhesions’ mechanical properties are dominated by an elastic structure which can be reversibly deformed by up to 1 µm. Large reversible deformations rule out an interface between substrate and cytoskeleton that is dominated by a number of stiff molecular springs in parallel, and favor a compliant cross-linked network. Such a compliant structure may increase the lifetime of a nascent adhesion, facilitating signaling and reinforcement. PMID:24039928

A Proline-Rich N-Terminal Region of the Dengue Virus NS3 Is Crucial for Infectious Particle Production.

PubMed

Gebhard, Leopoldo G; Iglesias, Néstor G; Byk, Laura A; Filomatori, Claudia V; De Maio, Federico A; Gamarnik, Andrea V

2016-06-01

Dengue virus is currently the most important insect-borne viral human pathogen. Viral nonstructural protein 3 (NS3) is a key component of the viral replication machinery that performs multiple functions during viral replication and participates in antiviral evasion. Using dengue virus infectious clones and reporter systems to dissect each step of the viral life cycle, we examined the requirements of different domains of NS3 on viral particle assembly. A thorough site-directed mutagenesis study based on solvent-accessible surface areas of NS3 revealed that, in addition to being essential for RNA replication, different domains of dengue virus NS3 are critically required for production of infectious viral particles. Unexpectedly, point mutations in the protease, interdomain linker, or helicase domain were sufficient to abolish infectious particle formation without affecting translation, polyprotein processing, or RNA replication. In particular, we identified a novel proline-rich N-terminal unstructured region of NS3 that contains several amino acid residues involved in infectious particle formation. We also showed a new role for the interdomain linker of NS3 in virion assembly. In conclusion, we present a comprehensive genetic map of novel NS3 determinants for viral particle assembly. Importantly, our results provide evidence of a central role of NS3 in the coordination of both dengue virus RNA replication and particle formation. Dengue virus is an important human pathogen, and its prominence is expanding globally; however, basic aspects of its biology are still unclear, hindering the development of effective therapeutic and prophylactic treatments. Little is known about the initial steps of dengue and other flavivirus particle assembly. This process involves a complex interplay between viral and cellular components, making it an attractive antiviral target. Unpredictably, we identified spatially separated regions of the large NS3 viral protein as determinants for

Membrane Fluidity Sensing on the Single Virus Particle Level with Plasmonic Nanoparticle Transducers.

PubMed

Feizpour, Amin; Stelter, David; Wong, Crystal; Akiyama, Hisashi; Gummuluru, Suryaram; Keyes, Tom; Reinhard, Björn M

2017-10-27

Viral membranes are nanomaterials whose fluidity depends on their composition, in particular, the cholesterol (chol) content. As differences in the membrane composition of individual virus particles can lead to different intracellular fates, biophysical tools capable of sensing the membrane fluidity on the single-virus level are required. In this manuscript, we demonstrate that fluctuations in the polarization of light scattered off gold or silver nanoparticle (NP)-labeled virus-like-particles (VLPs) encode information about the membrane fluidity of individual VLPs. We developed plasmonic polarization fluctuation tracking microscopy (PFTM) which facilitated the investigation of the effect of chol content on the membrane fluidity and its dependence on temperature, for the first time on the single-VLP level. Chol extraction studies with different methyl-β-cyclodextrin (MβCD) concentrations yielded a gradual decrease in polarization fluctuations as a function of time. The rate of chol extraction for individual VLPs showed a broad spread, presumably due to differences in the membrane composition for the individual VLPs, and this heterogeneity increased with decreasing MβCD concentration.

Virus-like particle vaccine primes immune responses preventing inactivated-virus vaccine-enhanced disease against respiratory syncytial virus.

PubMed

Hwang, Hye Suk; Lee, Young-Tae; Kim, Ki-Hye; Ko, Eun-Ju; Lee, Youri; Kwon, Young-Man; Kang, Sang-Moo

2017-11-01

Formalin inactivated respiratory syncytial virus (FI-RSV) vaccination caused vaccine-enhanced respiratory disease (ERD) upon exposure to RSV in children. Virus-like particles presenting RSV F fusion protein (F VLP) are known to increase T helper type-1 (Th1) immune responses and avoid ERD in animal models. We hypothesized that F VLP would prime immune responses preventing ERD upon subsequent exposure to ERD-prone FI-RSV. Here, we demonstrated that heterologous F VLP priming and FI-RSV boosting of mice prevented FI-RSV vaccine-enhanced lung inflammation and eosinophilia upon RSV challenge. F VLP priming redirected pulmonary T cells toward effector CD8 T cells producing Th1 cytokines and significantly suppressed pulmonary Th2 cytokines. This study suggests that RSV F VLP priming would modulate and shift immune responses to subsequent exposure to ERD-prone FI-RSV vaccine and RSV infection, suppressing Th2 immune-mediated pulmonary histopathology and eosinophilia. Copyright © 2017. Published by Elsevier Inc.

Immunizations with chimeric hepatitis B virus-like particles to induce potential anti-hepatitis C virus neutralizing antibodies.

PubMed

Vietheer, Patricia T K; Boo, Irene; Drummer, Heidi E; Netter, Hans-Jürgen

2007-01-01

Virus-like particles (VLPs) are highly immunogenic and proven to induce protective immunity. The small surface antigen (HBsAg-S) of hepatitis B virus (HBV) self-assembles into VLPs and its use as a vaccine results in protective antiviral immunity against HBV infections. Chimeric HBsAg-S proteins carrying foreign epitopes allow particle formation and have the ability to induce anti-foreign humoral and cellular immune responses. The insertion of the hypervariable region 1 (HVR1) sequence derived from the envelope protein 2 (E2) of hepatitis C virus (HCV) into the major antigenic site of HBsAg-S ('a'-determinant) resulted in the formation of highly immunogenic VLPs that retained the antigenicity of the inserted HVR1 sequence. BALB/c mice were immunized with chimeric VLPs, which resulted in antisera with anti-HCV activity. The antisera were able to immunoprecipitate native HCV envelope complexes (E1E2) containing homologous or heterologous HVR1 sequences. HCV E1E2 pseudotyped HIV-1 particles (HCVpp) were used to measure entry into HuH-7 target cells in the presence or absence of antisera that were raised against chimeric VLPs. Anti-HVR1 VLP sera interfered with entry of entry-competent HCVpps containing either homologous or heterologous HVR1 sequences. Also, immunizations with chimeric VLPs induced antisurface antigen (HBsAg) antibodies, indicating that HBV-specific antigenicity and immunogenicity of the 'a'-determinant region is retained. A multivalent vaccine against different pathogens based on the HBsAg delivery platform should be possible. We hypothesize that custom design of VLPs with an appropriate set of HCV-neutralizing epitopes will induce antibodies that would serve to decrease the viral load at the initial infecting inoculum.

Expression and self-assembly of cowpea chlorotic mottle virus-like particles in Pseudomonas fluorescens.

PubMed

Phelps, Jamie P; Dao, Philip; Jin, Hongfan; Rasochova, Lada

2007-02-01

Coat protein of the cowpea chlorotic mottle virus (CCMV), a plant bromovirus, has been expressed in a soluble form in a prokaryote, Pseudomonas fluorescens, and assembled into virus-like particles (VLPs) in vivo that were structurally similar to the native CCMV particles derived from plants. The CCMV VLPs were purified by PEG precipitation followed by separation on a sucrose density gradient and analyzed by size exclusion chromatography, UV spectrometry, and transmission electron microscopy. DNA microarray experiments revealed that the VLPs encapsulated very large numbers of different host RNAs in a non-specific manner. The development of a P. fluorescens expression system now enables production of CCMV VLPs by bacterial fermentation for use in pharmaceutical or nanotechnology applications.

DNA Packaging Mutant: Repression of the Vaccinia Virus A32 Gene Results in Noninfectious, DNA-Deficient, Spherical, Enveloped Particles

PubMed Central

Cassetti, Maria Cristina; Merchlinsky, Michael; Wolffe, Elizabeth J.; Weisberg, Andrea S.; Moss, Bernard

1998-01-01

The vaccinia virus A32 open reading frame was predicted to encode a protein with a nucleoside triphosphate-binding motif and a mass of 34 kDa. To investigate the role of this protein, we constructed a mutant in which the original A32 gene was replaced by an inducible copy. The recombinant virus, vA32i, has a conditional lethal phenotype: infectious virus formation was dependent on isopropyl-β-d-thiogalactopyranoside (IPTG). Under nonpermissive conditions, the mutant synthesized early- and late-stage viral proteins, as well as viral DNA that was processed into unit-length genomes. Electron microscopy of cells infected in the absence of IPTG revealed normal-appearing crescents and immature virus particles but very few with nucleoids. Instead of brick-shaped mature particles with defined core structures, there were numerous electron-dense, spherical particles. Some of these spherical particles were wrapped with cisternal membranes, analogous to intracellular and extracellular enveloped virions. Mutant viral particles, purified by sucrose density gradient centrifugation, had low infectivity and transcriptional activity, and the majority were spherical and lacked DNA. Nevertheless, the particle preparation contained representative membrane proteins, cleaved and uncleaved core proteins, the viral RNA polymerase, the early transcription factor and several enzymes, suggesting that incorporation of these components is not strictly coupled to DNA packaging. PMID:9621036

Induction of neutralising antibodies by virus-like particles harbouring surface proteins from highly pathogenic H5N1 and H7N1 influenza viruses

PubMed Central

Szécsi, Judit; Boson, Bertrand; Johnsson, Per; Dupeyrot-Lacas, Pia; Matrosovich, Mikhail; Klenk, Hans-Dieter; Klatzmann, David; Volchkov, Viktor; Cosset, François-Loïc

2006-01-01

There is an urgent need to develop novel approaches to vaccination against the emerging, highly pathogenic avian influenza viruses. Here, we engineered influenza viral-like particles (Flu-VLPs) derived from retroviral core particles that mimic the properties of the viral surface of two highly pathogenic influenza viruses of either H7N1 or H5N1 antigenic subtype. We demonstrate that, upon recovery of viral RNAs from a field strain, one can easily generate expression vectors that encode the HA, NA and M2 surface proteins of either virus and prepare high-titre Flu-VLPs. We characterise these Flu-VLPs incorporating the HA, NA and M2 proteins and we show that they induce high-titre neutralising antibodies in mice. PMID:16948862

Enumerating virus-like particles in an optically concentrated suspension by fluorescence correlation spectroscopy.

PubMed

Hu, Yi; Cheng, Xuanhong; Daniel Ou-Yang, H

2013-01-01

Fluorescence correlation spectroscopy (FCS) is one of the most sensitive methods for enumerating low concentration nanoparticles in a suspension. However, biological nanoparticles such as viruses often exist at a concentration much lower than the FCS detection limit. While optically generated trapping potentials are shown to effectively enhance the concentration of nanoparticles, feasibility of FCS for enumerating field-enriched nanoparticles requires understanding of the nanoparticle behavior in the external field. This paper reports an experimental study that combines optical trapping and FCS to examine existing theoretical predictions of particle concentration. Colloidal suspensions of polystyrene (PS) nanospheres and HIV-1 virus-like particles are used as model systems. Optical trapping energies and statistical analysis are used to discuss the applicability of FCS for enumerating nanoparticles in a potential well produced by a force field.

Nascent body ego: metapsychological and neurophysiological aspects.

PubMed

Lehtonen, Johannes; Partanen, Juhani; Purhonen, Maija; Valkonen-Korhonen, Minna; Kononen, Mervi; Saarikoski, Seppo; Launiala, Kari

2006-10-01

For Freud, body ego was the organizing basis of the structural theory. He defined it as a psychic projection of the body surface. Isakower's and Lewin's classical findings suggest that the body surface experiences of nursing provide the infant with sensory-affective stimulation that initiates a projection of sensory processes towards the psychic realm. During nursing, somato-sensory, gustatory and olfactory modalities merge with a primitive somatic affect of satiation, whereas auditory modality is involved more indirectly and visual contact more gradually. Repeated regularly, such nascent experiences are likely to play a part in the organization of the primitive protosymbolic mental experience. In support of this hypothesis, the authors review findings from a neurophysiological study of infants before, during and after nursing. Nursing is associated with a significant amplitude change in the newborn electroencephalogram (EEG), which wanes before the age of 3 months, and is transformed at the age of 6 months into rhythmic 3-5 Hz hedonic theta-activity. Sucking requires active physiological work, which is shown in a regular rise in heart rate. The hypothesis of a sensory-affective organization of the nascent body ego, enhanced by nursing and active sucking, seems concordant with neurophysiological phenomena related to nursing.

Perturbation of Human T-Cell Leukemia Virus Type 1 Particle Morphology by Differential Gag Co-Packaging

PubMed Central

Angert, Isaac; Cao, Sheng; Berk, Serkan; Zhang, Wei; Mueller, Joachim D.

2017-01-01

Human T-cell leukemia virus type 1 (HTLV-1) is an important cancer-causing human retrovirus that has infected approximately 15 million individuals worldwide. Many aspects of HTLV-1 replication, including virus particle structure and assembly, are poorly understood. Group-specific antigen (Gag) proteins labeled at the carboxy terminus with a fluorophore protein have been used extensively as a surrogate for fluorescence studies of retroviral assembly. How these tags affect Gag stoichiometry and particle morphology has not been reported in detail. In this study, we used an HTLV-1 Gag expression construct with the yellow fluorescence protein (YFP) fused to the carboxy-terminus as a surrogate for the HTLV-1 Gag-Pol to assess the effects of co-packaging of Gag and a Gag-YFP on virus-like particle (VLP) morphology and analyzed particles by cryogenic transmission electron microscopy (cryo-TEM). Scanning transmission electron microscopy (STEM) and fluorescence fluctuation spectroscopy (FFS) were also used to determine the Gag stoichiometry. We found that ratios of 3:1 (Gag:Gag-YFP) or greater resulted in a particle morphology indistinguishable from that of VLPs produced with the untagged HTLV-1 Gag, i.e., a mean diameter of ~113 nm and a mass of 220 MDa as determined by cryo-TEM and STEM, respectively. Furthermore, FFS analysis indicated that HTLV-1 Gag-YFP was incorporated into VLPs in a predictable manner at the 3:1 Gag:Gag-YFP ratio. Both STEM and FFS analyses found that the Gag copy number in VLPs produced with a 3:1 ratio of Gag:Gag-YFP was is in the range of 1500–2000 molecules per VLP. The observations made in this study indicate that biologically relevant Gag–Gag interactions occur between Gag and Gag-YFP at ratios of 3:1 or higher and create a Gag lattice structure in VLPs that is morphologically indistinguishable from that of VLPs produced with just untagged Gag. This information is useful for the quantitative analysis of Gag–Gag interactions that occur

Structure of nascent replicative form DNA of coliphage M13

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

Dasgupta, S.; Mitra, S.

Nascent replicative form type II (RFII) DNA of coliphage M13 synthesized in an Escherichia coli mutant deficient in the 5' ..-->.. 3' exonuclease associated with DNA polymerase I contains ribonucleotides that are retained in the covalently closed RFI DNA sealed in vitro by the joint action of T5 phage DNA polymerase and T4 phage DNA ligase. These RFI molecules are labile to alkali and RNase H, unlike the RFI produced either in vivo or from RFII with E. coli DNA polymerase I and E. coli DNA ligase. The ribonucleotides are located at one site and predominantly in one strand ofmore » the nascent RF DNA. Furthermore, these molecules contain multiple small gaps, randomly located, and one large gap in the intracistronic region.« less

Feline Tetherin Efficiently Restricts Release of Feline Immunodeficiency Virus but Not Spreading of Infection▿

PubMed Central

Dietrich, Isabelle; McMonagle, Elizabeth L.; Petit, Sarah J.; Vijayakrishnan, Swetha; Logan, Nicola; Chan, Chi N.; Towers, Greg J.; Hosie, Margaret J.; Willett, Brian J.

2011-01-01

Domestic cats endure infections by all three subfamilies of the retroviridae: lentiviruses (feline immunodeficiency virus [FIV]), gammaretroviruses (feline leukemia virus [FeLV]), and spumaretroviruses (feline foamy virus [FFV]). Thus, cats present an insight into the evolution of the host-retrovirus relationship and the development of intrinsic/innate immune mechanisms. Tetherin (BST-2) is an interferon-inducible transmembrane protein that inhibits the release of enveloped viruses from infected cells. Here, we characterize the feline homologue of tetherin and assess its effects on the replication of FIV. Tetherin was expressed in many feline cell lines, and expression was induced by interferons, including alpha interferon (IFN-α), IFN-ω, and IFN-γ. Like human tetherin, feline tetherin displayed potent inhibition of FIV and HIV-1 particle release; however, this activity resisted antagonism by either HIV-1 Vpu or the FIV Env and “OrfA” proteins. Further, as overexpression of complete FIV genomes in trans could not overcome feline tetherin, these data suggest that FIV lacks a functional tetherin antagonist. However, when expressed stably in feline cell lines, tetherin did not abrogate the replication of FIV; indeed, syncytium formation was significantly enhanced in tetherin-expressing cells infected with cell culture-adapted (CD134-independent) strains of FIV (FIV Fca-F14 and FIV Pco-CoLV). Thus, while tetherin may prevent the release of nascent viral particles, cell-to-cell spread remains efficient in the presence of abundant viral receptors and tetherin upregulation may enhance syncytium formation. Accordingly, tetherin expression in vivo may promote the selective expansion of viral variants capable of more efficient cell-to-cell spread. PMID:21490095

Feline tetherin efficiently restricts release of feline immunodeficiency virus but not spreading of infection.

PubMed

Dietrich, Isabelle; McMonagle, Elizabeth L; Petit, Sarah J; Vijayakrishnan, Swetha; Logan, Nicola; Chan, Chi N; Towers, Greg J; Hosie, Margaret J; Willett, Brian J

2011-06-01

Domestic cats endure infections by all three subfamilies of the retroviridae: lentiviruses (feline immunodeficiency virus [FIV]), gammaretroviruses (feline leukemia virus [FeLV]), and spumaretroviruses (feline foamy virus [FFV]). Thus, cats present an insight into the evolution of the host-retrovirus relationship and the development of intrinsic/innate immune mechanisms. Tetherin (BST-2) is an interferon-inducible transmembrane protein that inhibits the release of enveloped viruses from infected cells. Here, we characterize the feline homologue of tetherin and assess its effects on the replication of FIV. Tetherin was expressed in many feline cell lines, and expression was induced by interferons, including alpha interferon (IFN-α), IFN-ω, and IFN-γ. Like human tetherin, feline tetherin displayed potent inhibition of FIV and HIV-1 particle release; however, this activity resisted antagonism by either HIV-1 Vpu or the FIV Env and "OrfA" proteins. Further, as overexpression of complete FIV genomes in trans could not overcome feline tetherin, these data suggest that FIV lacks a functional tetherin antagonist. However, when expressed stably in feline cell lines, tetherin did not abrogate the replication of FIV; indeed, syncytium formation was significantly enhanced in tetherin-expressing cells infected with cell culture-adapted (CD134-independent) strains of FIV (FIV Fca-F14 and FIV Pco-CoLV). Thus, while tetherin may prevent the release of nascent viral particles, cell-to-cell spread remains efficient in the presence of abundant viral receptors and tetherin upregulation may enhance syncytium formation. Accordingly, tetherin expression in vivo may promote the selective expansion of viral variants capable of more efficient cell-to-cell spread.

Actin-myosin network is required for proper assembly of influenza virus particles

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

Kumakura, Michiko; Kawaguchi, Atsushi, E-mail: ats-kawaguchi@md.tsukuba.ac.jp; Nagata, Kyosuke, E-mail: knagata@md.tsukuba.ac.jp

Actin filaments are known to play a central role in cellular dynamics. After polymerization of actin, various actin-crosslinking proteins including non-muscle myosin II facilitate the formation of spatially organized actin filament networks. The actin-myosin network is highly expanded beneath plasma membrane. The genome of influenza virus (vRNA) replicates in the cell nucleus. Then, newly synthesized vRNAs are nuclear-exported to the cytoplasm as ribonucleoprotein complexes (vRNPs), followed by transport to the beneath plasma membrane where virus particles assemble. Here, we found that, by inhibiting actin-myosin network formation, the virus titer tends to be reduced and HA viral spike protein is aggregatedmore » on the plasma membrane. These results indicate that the actin-myosin network plays an important role in the virus formation. - Highlights: • Actin-myosin network is important for the influenza virus production. • HA forms aggregations at the plasma membrane in the presence of blebbistatin. • M1 is recruited to the budding site through the actin-myosin network.« less

[The true story and advantages of the famous Hepatitis B virus core particles: Outlook 2016].

PubMed

Pumpens, P; Grens, E

2016-01-01

This review article is a continuation of the paper "Hepatitis B core particles as a universal display model: a structure-function basis for development" written by Pumpens P. and Grens E., ordered by Professor Lev Kisselev and published in FEBS Letters, 1999, 442, 1-6. The past 17 years have strengthened the paper's finding that the human hepatitis B virus core protein, along with other Hepadnaviridae family member core proteins, is a mysterious, multifunctional protein. The core gene of the Hepadnaviridae genome encodes five partially collinear proteins. The most important of these is the HBV core protein p21, or HBc. It can self-assemble by forming viral HBc particles, but also plays a crucial role in the regulation of viral replication. Since 1986, the HBc protein has been one of the first and the most successful tools of the virus-like particle (VLP) technology. Later, the woodchuck hepatitis virus core protein (WHc) was also used as a VLP carrier. The Hepadnaviridae core proteins remain favourite VLP candidates for the knowledge-based design of future vaccines, gene therapy vectors, specifically targeted nanocontainers, and other modern nanotechnological tools for prospective medical use.

A role for the C terminus of Mopeia virus nucleoprotein in its incorporation into Z protein-induced virus-like particles.

PubMed

Shtanko, Olena; Imai, Masaki; Goto, Hideo; Lukashevich, Igor S; Neumann, Gabriele; Watanabe, Tokiko; Kawaoka, Yoshihiro

2010-05-01

Arenaviruses are enveloped, negative-strand RNA viruses. For several arenaviruses, virus-like particle (VLP) formation requires the viral matrix Z protein. However, the mechanism by which viral ribonucleoprotein complexes are incorporated into virions is poorly understood. Here, we show that the expression of the Z protein and nucleoprotein (NP) of Mopeia virus, a close relative of the pathogenic Lassa virus, resulted in the highly selective incorporation of the NP protein into Z protein-induced VLPs. Moreover, the Z protein promoted the association of NP with cellular membranes, suggesting that the association of NP, Z, and the cellular membranes may facilitate the efficient incorporation of NP into VLPs. By employing a series of NP deletion constructs and testing their VLP incorporation, we further demonstrated an important role for the C-terminal half of NP in its incorporation into VLPs.

Detection of virus-like particles in the liver of black and white ruffed lemurs with hepatitis.

PubMed

Worley, Michael B; Stalis, Ilse H

2002-04-01

Two young black and white ruffed lemurs (Varecia variegata variegata) died at the San Diego Zoo (San Diego, California, USA) with extensive liver lesions suggestive of acute viral infection. Immunoassays performed to detect hepatitis B virus (HBV) markers were negative. Polymerase chain reaction (PCR) primers overlapping the HBV core gene produced an amplicon of approximately 411 base pairs (bp) from serum DNA of a HBV-positive western lowland gorilla (Gorilla gorilla gorilla) but not from serum DNA of either lemur. Cesium chloride gradient fractions of liver homogenates from both lemurs contained a peak protein fraction with a density of 1.18 g/cm3. Electron microscopic analysis of fraction contents, concentrated by ultracentrifulgation, revealed numerous pleomorphic, spherical particles varying in diameter from 16-25 nm. In one of the lemurs, this peak fraction also contained a double-shelled virus-like particle 47-50 nm in diameter. The size, morphology, and density of these particles suggest they are members of the Hepadnaviridae, a group of hepatotropic DNA-genome viruses for which HBV is the prototype.

CAT-tailing as a fail-safe mechanism for efficient degradation of stalled nascent polypeptides

PubMed Central

Kostova, Kamena K.; Hickey, Kelsey L.; Osuna, Beatriz A.; Hussmann, Jeffrey A.; Frost, Adam; Weinberg, David E.; Weissman, Jonathan S.

2017-01-01

Ribosome stalling leads to recruitment of the Ribosome Quality control Complex (RQC), which targets the partially synthesized polypeptide for proteasomal degradation through the action of the ubiquitin ligase Ltn1p. A second core RQC component, Rqc2p, modifies the nascent polypeptide by adding a Carboxy-terminal Alanine and Threonine (CAT) tail through a non-canonical elongation reaction. Here we explore the role of CATtailing in nascent-chain degradation in budding yeast. We show that Ltn1p can efficiently access only nascent chain lysines immediately proximal to the ribosome exit tunnel. For substrates without Ltn1p-accessible lysines, CAT-tailing enables degradation by exposing lysines sequestered in the ribosome exit tunnel. Thus, CAT-tails do not serve as a degron, but rather provide a fail-safe mechanism that expands the range of RQC-degradable substrates. PMID:28751611

Replication-defective Friend murine leukemia virus particles containing uncleaved gag polyproteins and decreased levels of envelope glycoprotein.

PubMed Central

Collins, J K; Chesebro, B

1981-01-01

An erythroleukemia cell clone, 7C, which failed to produce reverse transcriptase-containing virions or infectious virus, was found to produce noninfectious virus particles by gradient banding of [3H]leucine- and [3H]uridine-labeled virions. The RNA from the 7C virus was shown to consist of the normal 70S size component, which converted to 35S upon heat denaturation. In contrast, the 7C virion proteins showed multiple defects. Analysis of the virion proteins by gel electrophoresis demonstrated that the pr65 gag precursor was incorporated into the 7C virus and that the processing of this precursor was severely diminished. Polymerase proteins pr180gag-pol and pr120pol were also detected in virions, and a third possible polymerase protein, p70, was reduced in size compared to its normal counterpart, p80. Incorporation of the viral gp70 glycoprotein into particles was also reduced 10-fold, despite synthesis and incorporation of gp70 into the 7C cell membrane in normal amounts. Pulse-chase analysis of the synthesis of the viral gag and env proteins in 7C cells showed greatly reduced amounts of pr180gag-pol, pr65gag, p80gag, and p42gag, whereas pr90env, gp70, and spleen focus-forming virus-specific gp55 were synthesized and processed normally. These results suggested that at least one defect in 7C virus was impaired cleavage of gag or pol proteins or both, most likely due to a lack of the appropriate viral protease, and that this lack of cleavage might affect incorporation of gp70 into virus particles. Images PMID:6163868

Exploiting virus-like particles as innovative vaccines against emerging viral infections.

PubMed

Jeong, Hotcherl; Seong, Baik Lin

2017-03-01

Emerging viruses pose a major threat to humans and livestock with global public health and economic burdens. Vaccination remains an effective tool to reduce this threat, and yet, the conventional cell culture often fails to produce sufficient vaccine dose. As an alternative to cell-culture based vaccine, virus-like particles (VLPs) are considered as a highpriority vaccine strategy against emerging viruses. VLPs represent highly ordered repetitive structures via macromolecular assemblies of viral proteins. The particulate nature allows efficient uptake into antigen presenting cells stimulating both innate and adaptive immune responses towards enhanced vaccine efficacy. Increasing research activity and translation opportunity necessitate the advances in the design of VLPs and new bioprocessing modalities for efficient and cost-effective production. Herein, we describe major achievements and challenges in this endeavor, with respect to designing strategies to harnessing the immunogenic potential, production platforms, downstream processes, and some exemplary cases in developing VLP-based vaccines.

Dynamic and Geometric Analyses of Nudaurelia capensis ωVirus Maturation Reveal the Energy Landscape of Particle Transitions

PubMed Central

Tang, Jinghua; Kearney, Bradley M.; Wang, Qiu; Doerschuk, Peter C.; Baker, Timothy S.; Johnson, John E.

2014-01-01

Quasi-equivalent viruses that infect animals and bacteria require a maturation process in which particles transition from initially assembled procapsids to infectious virions. Nudaurelia capensis ω virus (NωV) is a T=4, eukaryotic, ssRNA virus that has proved to be an excellent model system for studying the mechanisms of viral maturation. Structures of NωV procapsids (diam. = 480 Å), a maturation intermediate (410 Å), and the mature virion (410 Å) were determined by electron cryo-microscopy and three-dimensional image reconstruction (cryoEM). The cryoEM density for each particle type was analyzed with a recently developed Maximum Likelihood Variance (MLV) method for characterizing microstates occupied in the ensemble of particles used for the reconstructions. The procapsid and the mature capsid had overall low variance (i.e. uniform particle populations) while the maturation intermediate (that had not undergone post-assembly autocatalytic cleavage) had roughly 2-4 times the variance of the first two particles. Without maturation cleavage the particles assume a variety of microstates, as the frustrated subunits cannot reach a minimum energy configuration. Geometric analyses of subunit coordinates provided a quantitative description of the particle reorganization during maturation. Superposition of the four quasi-equivalent subunits in the procapsid had an average root mean square deviation (RMSD) of 3Å while the mature particle had an RMSD of 11Å, showing that the subunits differentiate from near equivalent environments in the procapsid to strikingly non-equivalent environments during maturation. Autocatalytic cleavage is clearly required for the reorganized mature particle to reach the minimum energy state required for stability and infectivity. PMID:24591180

Dynamic and geometric analyses of Nudaurelia capensis ω virus maturation reveal the energy landscape of particle transitions.

PubMed

Tang, Jinghua; Kearney, Bradley M; Wang, Qiu; Doerschuk, Peter C; Baker, Timothy S; Johnson, John E

2014-04-01

Quasi-equivalent viruses that infect animals and bacteria require a maturation process in which particles transition from initially assembled procapsids to infectious virions. Nudaurelia capensis ω virus (NωV) is a T = 4, eukaryotic, single-stranded ribonucleic acid virus that has proved to be an excellent model system for studying the mechanisms of viral maturation. Structures of NωV procapsids (diameter = 480 Å), a maturation intermediate (410 Å), and the mature virion (410 Å) were determined by electron cryo-microscopy and three-dimensional image reconstruction (cryoEM). The cryoEM density for each particle type was analyzed with a recently developed maximum likelihood variance (MLV) method for characterizing microstates occupied in the ensemble of particles used for the reconstructions. The procapsid and the mature capsid had overall low variance (i.e., uniform particle populations) while the maturation intermediate (that had not undergone post-assembly autocatalytic cleavage) had roughly two to four times the variance of the first two particles. Without maturation cleavage, the particles assume a variety of microstates, as the frustrated subunits cannot reach a minimum energy configuration. Geometric analyses of subunit coordinates provided a quantitative description of the particle reorganization during maturation. Superposition of the four quasi-equivalent subunits in the procapsid had an average root mean square deviation (RMSD) of 3 Å while the mature particle had an RMSD of 11 Å, showing that the subunits differentiate from near equivalent environments in the procapsid to strikingly non-equivalent environments during maturation. Autocatalytic cleavage is clearly required for the reorganized mature particle to reach the minimum energy state required for stability and infectivity. Copyright © 2014 John Wiley & Sons, Ltd.

Three-dimensional organization of nascent rod outer segment disk membranes.

PubMed

Volland, Stefanie; Hughes, Louise C; Kong, Christina; Burgess, Barry L; Linberg, Kenneth A; Luna, Gabriel; Zhou, Z Hong; Fisher, Steven K; Williams, David S

2015-12-01

The vertebrate photoreceptor cell contains an elaborate cilium that includes a stack of phototransductive membrane disks. The disk membranes are continually renewed, but how new disks are formed remains poorly understood. Here we used electron microscope tomography to obtain 3D visualization of the nascent disks of rod photoreceptors in three mammalian species, to gain insight into the process of disk morphogenesis. We observed that nascent disks are invariably continuous with the ciliary plasma membrane, although, owing to partial enclosure, they can appear to be internal in 2D profiles. Tomographic analyses of the basal-most region of the outer segment show changes in shape of the ciliary plasma membrane indicating an invagination, which is likely a first step in disk formation. The invagination flattens to create the proximal surface of an evaginating lamella, as well as membrane protrusions that extend between adjacent lamellae, thereby initiating a disk rim. Immediately distal to this initiation site, lamellae of increasing diameter are evident, indicating growth outward from the cilium. In agreement with a previous model, our data indicate that mature disks are formed once lamellae reach full diameter, and the growth of a rim encloses the space between adjacent surfaces of two lamellae. This study provides 3D data of nascent and mature rod photoreceptor disk membranes at unprecedented z-axis depth and resolution, and provides a basis for addressing fundamental questions, ranging from protein sorting in the photoreceptor cilium to photoreceptor electrophysiology.

Immunogenicity and safety of virus-like particle of the porcine encephalomyocarditis virus in pig

PubMed Central

2011-01-01

Background In this study, porcine encephalomyocarditis virus (EMCV) virus-like particles (VLPs) were generated using a baculovirus expression system and were tested for immunogenicity and protective efficacy in vivo. Results VLPs were successfully generated from Sf9 cells infected with recombinant baculovirus and were confirmed to be approximately 30-40 nm by transmission electron microscopy (TEM). Immunization of mice with 0.5 μg crude protein containing the VLPs resulted in significant protection from EMCV infection (90%). In swine, increased neutralizing antibody titers were observed following twice immunization with 2.0 μg crude protein containing VLPs. In addition, high levels of neutralizing antibodies (from 64 to 512 fold) were maintained during a test period following the second immunization. No severe injection site reactions were observed after immunization and all swine were healthy during the immunization period Conclusion Recombinant EMCV VLPs could represent a new vaccine candidate to protect against EMCV infection in pig farms. PMID:21492483

Prefoldin–Nascent Chain Complexes in the Folding of Cytoskeletal Proteins

PubMed Central

Hansen, William J.; Cowan, Nicholas J.; Welch, William J.

1999-01-01

In vitro transcription/translation of actin cDNA and analysis of the translation products by native-PAGE was used to study the maturation pathway of actin. During the course of actin synthesis, several distinct actin-containing species were observed and the composition of each determined by immunological procedures. After synthesis of the first ∼145 amino acids, the nascent ribosome-associated actin chain binds to the recently identified heteromeric chaperone protein, prefoldin (PFD). PFD remains bound to the relatively unfolded actin polypeptide until its posttranslational delivery to cytosolic chaperonin (CCT). We show that α- and β-tubulin follow a similar maturation pathway, but to date find no evidence for an interaction between PFD and several noncytoskeletal proteins. We conclude that PFD functions by selectively targeting nascent actin and tubulin chains pending their transfer to CCT for final folding and/or assembly. PMID:10209023

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

Long-read sequencing of nascent RNA reveals coupling among RNA processing events.

PubMed

Herzel, Lydia; Straube, Korinna; Neugebauer, Karla M

2018-06-14

Pre-mRNA splicing is accomplished by the spliceosome, a megadalton complex that assembles de novo on each intron. Because spliceosome assembly and catalysis occur cotranscriptionally, we hypothesized that introns are removed in the order of their transcription in genomes dominated by constitutive splicing. Remarkably little is known about splicing order and the regulatory potential of nascent transcript remodeling by splicing, due to the limitations of existing methods that focus on analysis of mature splicing products (mRNAs) rather than substrates and intermediates. Here, we overcome this obstacle through long-read RNA sequencing of nascent, multi-intron transcripts in the fission yeast Schizosaccharomyces pombe Most multi-intron transcripts were fully spliced, consistent with rapid cotranscriptional splicing. However, an unexpectedly high proportion of transcripts were either fully spliced or fully unspliced, suggesting that splicing of any given intron is dependent on the splicing status of other introns in the transcript. Supporting this, mild inhibition of splicing by a temperature-sensitive mutation in prp2 , the homolog of vertebrate U2AF65, increased the frequency of fully unspliced transcripts. Importantly, fully unspliced transcripts displayed transcriptional read-through at the polyA site and were degraded cotranscriptionally by the nuclear exosome. Finally, we show that cellular mRNA levels were reduced in genes with a high number of unspliced nascent transcripts during caffeine treatment, showing regulatory significance of cotranscriptional splicing. Therefore, overall splicing of individual nascent transcripts, 3' end formation, and mRNA half-life depend on the splicing status of neighboring introns, suggesting crosstalk among spliceosomes and the polyA cleavage machinery during transcription elongation. © 2018 Herzel et al.; Published by Cold Spring Harbor Laboratory Press.

Antigen vehiculization particles based on the Z protein of Junin virus.

PubMed

Borio, Cristina S; Bilen, Marcos F; Argüelles, Marcelo H; Goñi, Sandra E; Iserte, Javier A; Glikmann, Graciela; Lozano, Mario E

2012-11-02

Arenavirus matrix protein Z plays an important role in virus budding and is able to generate enveloped virus-like-particles (VLPs) in absence of any other viral proteins. In these VLPs, Z protein is associated to the plasma membrane inner surface by its myristoyl residue. Budding induction and vesicle formation properties can be exploited to generate enveloped VLPs platform. These structures can be designed to carry specific antigen in the inner side or on the surface of VLPs.Vaccines based on VLPs are a highly effective type of subunit vaccines that mimic the overall structure of virus particles in absence of viral nucleic acid, being noninfectious.In this work we assayed the capacity of Junin Z protein to produce VLPs carrying the green fluorescent protein (eGFP), as a model antigen. In this report the Junin Z protein ability to produce VLPs from 293T cells and its capacity to deliver a specific antigen (eGFP) fused to Z was evaluated. Confocal microscopy showed a particular membrane bending in cells expressing Z and a spot welded distribution in the cytoplasm. VLPs were detected by TEM (transmission electron microscopy) and were purified from cell supernatant. The proteinase protection assay demonstrated the VLPs integrity and the absence of degradation of the fused antigen, thus indicating its internal localization. Finally, immunization of mice with purified VLPs produced high titres of anti-eGFP antibodies compared to the controls. It was proved that VLPs can be generated from cells transfected with a fusion Junin virus Z-eGFP protein in absence of any other viral protein, and the capacity of Z protein to support fusions at the C-terminal, without impairing its budding activity, allowing vehiculization of specific antigens into VLPs.

The Use of NanoTrap Particles as a Sample Enrichment Method to Enhance the Detection of Rift Valley Fever Virus

PubMed Central

Shafagati, Nazly; Narayanan, Aarthi; Baer, Alan; Fite, Katherine; Pinkham, Chelsea; Bailey, Charles; Kashanchi, Fatah; Lepene, Benjamin; Kehn-Hall, Kylene

2013-01-01

Background Rift Valley Fever Virus (RVFV) is a zoonotic virus that is not only an emerging pathogen but is also considered a biodefense pathogen due to the threat it may cause to public health and national security. The current state of diagnosis has led to misdiagnosis early on in infection. Here we describe the use of a novel sample preparation technology, NanoTrap particles, to enhance the detection of RVFV. Previous studies demonstrated that NanoTrap particles lead to both 100 percent capture of protein analytes as well as an improvement of more than 100-fold in sensitivity compared to existing methods. Here we extend these findings by demonstrating the capture and enrichment of viruses. Results Screening of NanoTrap particles indicated that one particle, NT53, was the most efficient at RVFV capture as demonstrated by both qRT-PCR and plaque assays. Importantly, NT53 capture of RVFV resulted in greater than 100-fold enrichment from low viral titers when other diagnostics assays may produce false negatives. NT53 was also capable of capturing and enhancing RVFV detection from serum samples. RVFV that was inactivated through either detergent or heat treatment was still found bound to NT53, indicating the ability to use NanoTrap particles for viral capture prior to transport to a BSL-2 environment. Furthermore, both NP-40-lysed virus and purified RVFV RNA were bound by NT53. Importantly, NT53 protected viral RNA from RNase A degradation, which was not observed with other commercially available beads. Incubation of RVFV samples with NT53 also resulted in increased viral stability as demonstrated through preservation of infectivity at elevated temperatures. Finally, NanoTrap particles were capable of capturing VEEV and HIV, demonstrating the broad applicability of NanoTrap particles for viral diagnostics. Conclusion This study demonstrates NanoTrap particles are capable of capturing, enriching, and protecting RVFV virions. Furthermore, the use of NanoTrap particles

Affinity selection of Nipah and Hendra virus-related vaccine candidates from a complex random peptide library displayed on bacteriophage virus-like particles

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

Peabody, David S.; Chackerian, Bryce; Ashley, Carlee

The invention relates to virus-like particles of bacteriophage MS2 (MS2 VLPs) displaying peptide epitopes or peptide mimics of epitopes of Nipah Virus envelope glycoprotein that elicit an immune response against Nipah Virus upon vaccination of humans or animals. Affinity selection on Nipah Virus-neutralizing monoclonal antibodies using random sequence peptide libraries on MS2 VLPs selected peptides with sequence similarity to peptide sequences found within the envelope glycoprotein of Nipah itself, thus identifying the epitopes the antibodies recognize. The selected peptide sequences themselves are not necessarily identical in all respects to a sequence within Nipah Virus glycoprotein, and therefore may be referredmore » to as epitope mimics VLPs displaying these epitope mimics can serve as vaccine. On the other hand, display of the corresponding wild-type sequence derived from Nipah Virus and corresponding to the epitope mapped by affinity selection, may also be used as a vaccine.« less

Influence of solution chemistry on the inactivation of particle-associated viruses by UV irradiation.

PubMed

Feng, Zhe; Lu, Ruiqing; Yuan, Baoling; Zhou, Zhenming; Wu, Qingqing; Nguyen, Thanh H

2016-12-01

MS2 inactivation by UV irradiance was investigated with the focus on how the disinfection efficacy is influenced by bacteriophage MS2 aggregation and adsorption to particles in solutions with different compositions. Kaolinite and Microcystis aeruginosa were used as model inorganic and organic particles, respectively. In the absence of model particles, MS2 aggregates formed in either 1mM NaCl at pH=3 or 50-200mM ionic strength CaCl 2 solutions at pH=7 led to a decrease in the MS2 inactivation efficacy because the virions located inside the aggregate were protected from the UV irradiation. In the presence of kaolinite and Microcystis aeruginosa, MS2 adsorbed onto the particles in either 1mM NaCl at pH=3 or 50-200mM CaCl 2 solutions at pH=7. In contrast to MS2 aggregates formed without the presence of particles, more MS2 virions adsorbed on these particles were exposed to UV irradiation to allow an increase in MS2 inactivation. In either 1mM NaCl at pH from 4 to 8 or 2-200mM NaCl solutions at pH=7, the absence of MS2 aggregation and adsorption onto the model particles explained why MS2 inactivation was not influenced by pH, ionic strength, and the presence of model particles in these conditions. The influence of virus adsorption and aggregation on the UV disinfection efficiency found in this research suggests the necessity of accounting for particles and cation composition in virus inactivation for drinking water. Copyright © 2016 Elsevier B.V. All rights reserved.

Equine Rhinitis A Virus and Its Low pH Empty Particle: Clues Towards an Aphthovirus Entry Mechanism?

PubMed Central

Tuthill, Tobias J.; Harlos, Karl; Walter, Thomas S.; Knowles, Nick J.; Groppelli, Elisabetta; Rowlands, David J.; Stuart, David I.; Fry, Elizabeth E.

2009-01-01

Equine rhinitis A virus (ERAV) is closely related to foot-and-mouth disease virus (FMDV), belonging to the genus Aphthovirus of the Picornaviridae. How picornaviruses introduce their RNA genome into the cytoplasm of the host cell to initiate replication is unclear since they have no lipid envelope to facilitate fusion with cellular membranes. It has been thought that the dissociation of the FMDV particle into pentameric subunits at acidic pH is the mechanism for genome release during cell entry, but this raises the problem of how transfer across the endosome membrane of the genome might be facilitated. In contrast, most other picornaviruses form ‘altered’ particle intermediates (not reported for aphthoviruses) thought to induce membrane pores through which the genome can be transferred. Here we show that ERAV, like FMDV, dissociates into pentamers at mildly acidic pH but demonstrate that dissociation is preceded by the transient formation of empty 80S particles which have released their genome and may represent novel biologically relevant intermediates in the aphthovirus cell entry process. The crystal structures of the native ERAV virus and a low pH form have been determined via highly efficient crystallization and data collection strategies, required due to low virus yields. ERAV is closely similar to FMDV for VP2, VP3 and part of VP4 but VP1 diverges, to give a particle with a pitted surface, as seen in cardioviruses. The low pH particle has internal structure consistent with it representing a pre-dissociation cell entry intermediate. These results suggest a unified mechanism of picornavirus cell entry. PMID:19816570

Viruses, Artificial Viruses and Virus-Based Structures for Biomedical Applications.

PubMed

van Rijn, Patrick; Schirhagl, Romana

2016-06-01

Nanobiomaterials such as virus particles and artificial virus particles offer tremendous opportunities to develop new biomedical applications such as drug- or gene-delivery, imaging and sensing but also improve understanding of biological mechanisms. Recent advances within the field of virus-based systems give insights in how to mimic viral structures and virus assembly processes as well as understanding biodistribution, cell/tissue targeting, controlled and triggered disassembly or release and circulation times. All these factors are of high importance for virus-based functional systems. This review illustrates advances in mimicking and enhancing or controlling these aspects to a high degree toward delivery and imaging applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Virus-like Particles Containing Multiple M2 Extracellular Domains Confer Improved Cross-protection Against Various Subtypes of Influenza Virus

PubMed Central

Kim, Min-Chul; Song, Jae-Min; O, Eunju; Kwon, Young-Man; Lee, Youn-Jeong; Compans, Richard W; Kang, Sang-Moo

2013-01-01

The extracellular domain of M2 (M2e), a small ion channel membrane protein, is well conserved among different human influenza A virus strains. To improve the protective efficacy of M2e vaccines, we genetically engineered a tandem repeat of M2e epitope sequences (M2e5x) of human, swine, and avian origin influenza A viruses, which was expressed in a membrane-anchored form and incorporated in virus-like particles (VLPs). The M2e5x protein with the transmembrane domain of hemagglutinin (HA) was effectively incorporated into VLPs at a several 100-fold higher level than that on influenza virions. Intramuscular immunization with M2e5x VLP vaccines was highly effective in inducing M2e-specific antibodies reactive to different influenza viruses, mucosal and systemic immune responses, and cross-protection regardless of influenza virus subtypes in the absence of adjuvant. Importantly, immune sera were found to be sufficient for conferring protection in naive mice, which was long-lived and cross-protective. Thus, molecular designing and presenting M2e immunogens on VLPs provide a promising platform for developing universal influenza vaccines without using adjuvants. PMID:23247101

CAT-tailing as a fail-safe mechanism for efficient degradation of stalled nascent polypeptides.

PubMed

Kostova, Kamena K; Hickey, Kelsey L; Osuna, Beatriz A; Hussmann, Jeffrey A; Frost, Adam; Weinberg, David E; Weissman, Jonathan S

2017-07-28

Ribosome stalling leads to recruitment of the ribosome quality control complex (RQC), which targets the partially synthesized polypeptide for proteasomal degradation through the action of the ubiquitin ligase Ltn1p. A second core RQC component, Rqc2p, modifies the nascent polypeptide by adding a carboxyl-terminal alanine and threonine (CAT) tail through a noncanonical elongation reaction. Here we examined the role of CAT-tailing in nascent-chain degradation in budding yeast. We found that Ltn1p efficiently accessed only nascent-chain lysines immediately proximal to the ribosome exit tunnel. For substrates without Ltn1p-accessible lysines, CAT-tailing enabled degradation by exposing lysines sequestered in the ribosome exit tunnel. Thus, CAT-tails do not serve as a degron, but rather provide a fail-safe mechanism that expands the range of RQC-degradable substrates. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Biomedical and Catalytic Opportunities of Virus-Like Particles in Nanotechnology

PubMed Central

Schwarz, B.; Uchida, M.; Douglas, T.

2016-01-01

Within biology, molecules are arranged in hierarchical structures that coordinate and control the many processes that allow for complex organisms to exist. Proteins and other functional macromolecules are often studied outside their natural nanostructural context because it remains difficult to create controlled arrangements of proteins at this size scale. Viruses are elegantly simple nanosystems that exist at the interface of living organisms and nonliving biological machines. Studied and viewed primarily as pathogens to be combatted, viruses have emerged as models of structural efficiency at the nanoscale and have spurred the development of biomimetic nanoparticle systems. Virus-like particles (VLPs) are noninfectious protein cages derived from viruses or other cage-forming systems. VLPs provide incredibly regular scaffolds for building at the nanoscale. Composed of self-assembling protein subunits, VLPs provide both a model for studying materials’ assembly at the nanoscale and useful building blocks for materials design. The robustness and degree of understanding of many VLP structures allow for the ready use of these systems as versatile nanoparticle platforms for the conjugation of active molecules or as scaffolds for the structural organization of chemical processes. Lastly the prevalence of viruses in all domains of life has led to unique activities of VLPs in biological systems most notably the immune system. Here we discuss recent efforts to apply VLPs in a wide variety of applications with the aim of highlighting how the common structural elements of VLPs have led to their emergence as paradigms for the understanding and design of biological nanomaterials. PMID:28057256

Biomedical and Catalytic Opportunities of Virus-Like Particles in Nanotechnology.

PubMed

Schwarz, B; Uchida, M; Douglas, T

2017-01-01

Within biology, molecules are arranged in hierarchical structures that coordinate and control the many processes that allow for complex organisms to exist. Proteins and other functional macromolecules are often studied outside their natural nanostructural context because it remains difficult to create controlled arrangements of proteins at this size scale. Viruses are elegantly simple nanosystems that exist at the interface of living organisms and nonliving biological machines. Studied and viewed primarily as pathogens to be combatted, viruses have emerged as models of structural efficiency at the nanoscale and have spurred the development of biomimetic nanoparticle systems. Virus-like particles (VLPs) are noninfectious protein cages derived from viruses or other cage-forming systems. VLPs provide incredibly regular scaffolds for building at the nanoscale. Composed of self-assembling protein subunits, VLPs provide both a model for studying materials' assembly at the nanoscale and useful building blocks for materials design. The robustness and degree of understanding of many VLP structures allow for the ready use of these systems as versatile nanoparticle platforms for the conjugation of active molecules or as scaffolds for the structural organization of chemical processes. Lastly the prevalence of viruses in all domains of life has led to unique activities of VLPs in biological systems most notably the immune system. Here we discuss recent efforts to apply VLPs in a wide variety of applications with the aim of highlighting how the common structural elements of VLPs have led to their emergence as paradigms for the understanding and design of biological nanomaterials. © 2017 Elsevier Inc. All rights reserved.

Single-particle cryo-electron microscopy of Rift Valley fever virus

PubMed Central

Sherman, Michael B.; Freiberg, Alexander N.; Holbrook, Michael R.; Watowich, Stanley J.

2009-01-01

Rift Valley fever virus (RVFV; Bunyaviridae; Phlebovirus) is an emerging human veterinary pathogen causing acute hepatitis in ruminants and has the potential to Single-particle cryo-EM reconstruction of RVFV MP-12 hemorrhagic fever in humans. We report a three-dimensional reconstruction of RVFV vaccine strain MP-12 (RVFV MP-12) by cryo-electron microcopy using icosahedral symmetry of individual virions. Although the genomic core of RVFV MP-12 is apparently poorly ordered, the glycoproteins on the virus surface are highly symmetric and arranged on a T=12 icosahedral lattice. Our RVFV MP-12 structure allowed clear identification of inter-capsomer contacts and definition of possible glycoprotein arrangements within capsomers. This structure provides a detailed model for phleboviruses, opens new avenues for high-resolution structural studies of the bunyavirus family, and aids the design of antiviral diagnostics and effective subunit-vaccines. PMID:19304307

Human Norovirus Detection and Production, Quantification, and Storage of Virus-Like Particles

PubMed Central

Debbink, Kari; Costantini, Veronica; Swanstrom, Jesica; Agnihothram, Sudhakar; Vinjé, Jan; Baric, Ralph

2014-01-01

Human noroviruses constitute a significant worldwide disease burden. Each year noroviruses cause over 267 million infections, deaths in over 200,000 children under the age of five, and over 50% of U.S. food borne illness. Due to the absence of a tissue culture model or small animal model to study human norovirus, virus-like particles (VLPs) and ELISA-based biological assays have been used to answer questions about norovirus evolution and immunity as well provide a potential vaccine platform. This chapter outlines the protocols on norovirus detection in stool and norovirus VLP design, production, purification, and storage using a Venezuelan equine encephalitis virus (VEE)-based VRP expression system. PMID:24510290

Mutation of the TYTLE Motif in the Cytoplasmic Tail of the Sendai Virus Fusion Protein Deeply Affects Viral Assembly and Particle Production

PubMed Central

Essaidi-Laziosi, Manel; Shevtsova, Anastasia; Gerlier, Denis; Roux, Laurent

2013-01-01

Enveloped viruses contain glycoproteins protruding from the viral membrane. These proteins play a crucial role in the extra-cellular steps of the virus life cycle, namely attachment to and entry into cells. Their role during the intracellular late phase of virus multiplication has been less appreciated, overlooked by the documented central organizer role of the matrix M protein. Sendai virus, a member of the Paramyxoviridae family, expresses two trans-membrane proteins on its surface, HN and F. In previous work, we have shown that suppression of F in the context of an infection, results in about 70% reduction of virus particle production, a reduction similar to that observed upon suppression of the matrix M protein. Moreover, a TYTLE motif present in F cytoplasmic tail has been proposed essential for virus particle production. In the present work, using original alternate conditional siRNA suppression systems, we generated a double F gene recombinant Sendai virus expressing wt-F and a nonviable mutated TYTLE/5A F protein (F5A). Suppression of the wild type F gene expression in cells infected with this virus allowed the analysis of F5A properties in the context of the infection. Coupling confocal imaging analysis to biochemical characterization, we found that F5A i) was not expressed at the cell surface but restricted to the endoplasmic reticulum, ii) was still capable of interaction with M and iii) had profound effect on M and HN cellular distribution. On the basis of these data, we propose a model for SeV particle formation based on an M/F complex that would serve as nucleation site for virus particle assembly at the cell surface. PMID:24339863

Processing of the VP1/2A junction is not necessary for production of foot-and-mouth disease virus empty capsids and infectious viruses: characterization of "self-tagged" particles.

PubMed

Gullberg, Maria; Polacek, Charlotta; Bøtner, Anette; Belsham, Graham J

2013-11-01

The foot-and-mouth disease virus (FMDV) capsid protein precursor, P1-2A, is cleaved by 3C(pro) to generate VP0, VP3, VP1, and the peptide 2A. The capsid proteins self-assemble into empty capsid particles or viruses which do not contain 2A. In a cell culture-adapted strain of FMDV (O1 Manisa [Lindholm]), three different amino acid substitutions (E83K, S134C, and K210E) were identified within the VP1 region of the P1-2A precursor compared to the field strain (wild type [wt]). Expression of the O1 Manisa P1-2A (wt or with the S134C substitution in VP1) plus 3C(pro), using a transient expression system, resulted in efficient capsid protein production and self-assembly of empty capsid particles. Removal of the 2A peptide from the capsid protein precursor had no effect on capsid protein processing or particle assembly. However, modification of E83K alone abrogated particle assembly with no apparent effect on protein processing. Interestingly, the K210E substitution, close to the VP1/2A junction, completely blocked processing by 3C(pro) at this cleavage site, but efficient assembly of "self-tagged" empty capsid particles, containing the uncleaved VP1-2A, was observed. These self-tagged particles behaved like the unmodified empty capsids in antigen enzyme-linked immunosorbent assays and integrin receptor binding assays. Furthermore, mutant viruses with uncleaved VP1-2A could be rescued in cells from full-length FMDV RNA transcripts encoding the K210E substitution in VP1. Thus, cleavage of the VP1/2A junction is not essential for virus viability. The production of such engineered self-tagged empty capsid particles may facilitate their purification for use as diagnostic reagents and vaccines.

A heterologous prime-boosting strategy with replicating Vaccinia virus vectors and plant-produced HIV-1 Gag/dgp41 virus-like particles

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

Meador, Lydia R.

Showing modest efficacy, the RV144 HIV-1 vaccine clinical trial utilized a non-replicating canarypox viral vector and a soluble gp120 protein boost. Here we built upon the RV144 strategy by developing a novel combination of a replicating, but highly-attenuated Vaccinia virus vector, NYVAC-KC, and plant-produced HIV-1 virus-like particles (VLPs). Both components contained the full-length Gag and a membrane anchored truncated gp41 presenting the membrane proximal external region with its conserved broadly neutralizing epitopes in the pre-fusion conformation. We tested different prime/boost combinations of these components in mice and showed that the group primed with NYVAC-KC and boosted with both the viralmore » vectors and plant-produced VLPs have the most robust Gag-specific CD8 T cell responses, at 12.7% of CD8 T cells expressing IFN-γ in response to stimulation with five Gag epitopes. The same immunization group elicited the best systemic and mucosal antibody responses to Gag and dgp41 with a bias towards IgG1. - Highlights: • We devised a prime/boost anti HIV-1 vaccination strategy modeled after RV144. • We used plant-derived virus-like particles (VLPs) consisting of Gag and dgp41. • We used attenuated, replicating vaccinia virus vectors expressing the same antigens. • The immunogens elicited strong cellular and humoral immune responses.« less

Development of an Electrochemical Paper-Based Analytical Device for Trace Detection of Virus Particles.

PubMed

Channon, Robert B; Yang, Yuanyuan; Feibelman, Kristen M; Geiss, Brian J; Dandy, David S; Henry, Charles S

2018-06-19

Viral pathogens are a serious health threat around the world, particularly in resource limited settings, where current sensing approaches are often insufficient and slow, compounding the spread and burden of these pathogens. Here, we describe a label-free, point-of-care approach toward detection of virus particles, based on a microfluidic paper-based analytical device with integrated microwire Au electrodes. The device is initially characterized through capturing of streptavidin modified nanoparticles by biotin-modified microwires. An order of magnitude improvement in detection limits is achieved through use of a microfluidic device over a classical static paper-based device, due to enhanced mass transport and capturing of particles on the modified electrodes. Electrochemical impedance spectroscopy detection of West Nile virus particles was carried out using antibody functionalized Au microwires, achieving a detection limit of 10.2 particles in 50 μL of cell culture media. No increase in signal is found on addition of an excess of a nonspecific target (Sindbis). This detection motif is significantly cheaper (∼$1 per test) and faster (∼30 min) than current methods, while achieving the desired selectivity and sensitivity. This sensing motif represents a general platform for trace detection of a wide range of biological pathogens.

Completely assembled virus particles detected by transmission electron microscopy in proximal and mid-axons of neurons infected with herpes simplex virus type 1, herpes simplex virus type 2 and pseudorabies virus

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

Huang Jialing, E-mail: hjialing@mail.med.upenn.edu; Lazear, Helen M., E-mail: Hlazear@DOM.wustl.edu; Friedman, Harvey M., E-mail: hfriedma@mail.med.upenn.ed

2011-01-05

The morphology of alphaherpesviruses during anterograde axonal transport from the neuron cell body towards the axon terminus is controversial. Reports suggest that transport of herpes simplex virus type 1 (HSV-1) nucleocapsids and envelope proteins occurs in separate compartments and that complete virions form at varicosities or axon termini (subassembly transport model), while transport of a related alphaherpesvirus, pseudorabies virus (PRV) occurs as enveloped capsids in vesicles (assembled transport model). Transmission electron microscopy of proximal and mid-axons of primary superior cervical ganglion (SCG) neurons was used to compare anterograde axonal transport of HSV-1, HSV-2 and PRV. SCG cell bodies were infectedmore » with HSV-1 NS and 17, HSV-2 2.12 and PRV Becker. Fully assembled virus particles were detected intracellularly within vesicles in proximal and mid-axons adjacent to microtubules after infection with each virus, indicating that assembled virions are transported anterograde within axons for all three alphaherpesviruses.« less

Cryo-electron microscopy and three-dimensional reconstructions of hepatitis C virus particles

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

Yu Xuekui; Qiao Ming; Atanasov, Ivo

2007-10-10

The structural details of hepatitis C virus (HCV) have been elusive because of the lack of a robust tissue culture system for producing an adequate amount of virions from infectious sources for in-depth three-dimensional (3D) structural analysis. Using both negative-stain and cryo-electron microscopy (cryoEM), we show that HCV virions isolated from cell culture have a rather uniform size of 500 A in diameter and that recombinantly expressed HCV-like particles (HCV-LPs) have similar morphologic, biophysical and antigenic features in spite of the varying sizes of the particles. 3D reconstructions were obtained from HCV-LPs with the same size as the HCV virionsmore » in the presence and absence of monoclonal antibodies bound to the E1 glycoprotein. The 3D reconstruction of HCV-LP reveals a multilayered architecture, with smooth outer-layer densities arranged in a 'fishbone' configuration. Reconstruction of the particles in complex with anti-E1 antibodies shows that sites of the E1 epitope are exposed and surround the 5-, 3- and 2-fold axes. The binding pattern of the anti-E1 antibody and the fitting of the structure of the dengue virus E glycoprotein into our 3D reconstructions further suggest that the HCV-LP E1 and E2 proteins form a tetramer (or dimer of heterodimers) that corresponds morphologically and functionally to the flavivirus E homodimer. This first 3D structural analysis of HCV particles offers important insights into the elusive mechanisms of HCV assembly and maturation.« less

A strategy for co-translational folding studies of ribosome-bound nascent chain complexes using NMR spectroscopy.

PubMed

Cassaignau, Anaïs M E; Launay, Hélène M M; Karyadi, Maria-Evangelia; Wang, Xiaolin; Waudby, Christopher A; Deckert, Annika; Robertson, Amy L; Christodoulou, John; Cabrita, Lisa D

2016-08-01

During biosynthesis on the ribosome, an elongating nascent polypeptide chain can begin to fold, in a process that is central to all living systems. Detailed structural studies of co-translational protein folding are now beginning to emerge; such studies were previously limited, at least in part, by the inherently dynamic nature of emerging nascent chains, which precluded most structural techniques. NMR spectroscopy is able to provide atomic-resolution information for ribosome-nascent chain complexes (RNCs), but it requires large quantities (≥10 mg) of homogeneous, isotopically labeled RNCs. Further challenges include limited sample working concentration and stability of the RNC sample (which contribute to weak NMR signals) and resonance broadening caused by attachment to the large (2.4-MDa) ribosomal complex. Here, we present a strategy to generate isotopically labeled RNCs in Escherichia coli that are suitable for NMR studies. Uniform translational arrest of the nascent chains is achieved using a stalling motif, and isotopically labeled RNCs are produced at high yield using high-cell-density E. coli growth conditions. Homogeneous RNCs are isolated by combining metal affinity chromatography (to isolate ribosome-bound species) with sucrose density centrifugation (to recover intact 70S monosomes). Sensitivity-optimized NMR spectroscopy is then applied to the RNCs, combined with a suite of parallel NMR and biochemical analyses to cross-validate their integrity, including RNC-optimized NMR diffusion measurements to report on ribosome attachment in situ. Comparative NMR studies of RNCs with the analogous isolated proteins permit a high-resolution description of the structure and dynamics of a nascent chain during its progressive biosynthesis on the ribosome.

Influenza Virus-Like Particles Containing M2 Induce Broadly Cross Protective Immunity

PubMed Central

Song, Jae-Min; Wang, Bao-Zhong; Park, Kyoung-Mi; Van Rooijen, Nico; Quan, Fu-Shi; Kim, Min-Chul; Jin, Hyun-Tak; Pekosz, Andrew; Compans, Richard W.; Kang, Sang-Moo

2011-01-01

Background Current influenza vaccines based on the hemagglutinin protein are strain specific and do not provide good protection against drifted viruses or emergence of new pandemic strains. An influenza vaccine that can confer cross-protection against antigenically different influenza A strains is highly desirable for improving public health. Methodology/Principal Findings To develop a cross protective vaccine, we generated influenza virus-like particles containing the highly conserved M2 protein in a membrane-anchored form (M2 VLPs), and investigated their immunogenicity and breadth of cross protection. Immunization of mice with M2 VLPs induced anti-M2 antibodies binding to virions of various strains, M2 specific T cell responses, and conferred long-lasting cross protection against heterologous and heterosubtypic influenza viruses. M2 immune sera were found to play an important role in providing cross protection against heterosubtypic virus and an antigenically distinct 2009 pandemic H1N1 virus, and depletion of dendritic and macrophage cells abolished this cross protection, providing new insight into cross-protective immune mechanisms. Conclusions/Significance These results suggest that presenting M2 on VLPs in a membrane-anchored form is a promising approach for developing broadly cross protective influenza vaccines. PMID:21267073

An optimized expression vector for improving the yield of dengue virus-like particles from transfected insect cells.

PubMed

Charoensri, Nicha; Suphatrakul, Amporn; Sriburi, Rungtawan; Yasanga, Thippawan; Junjhon, Jiraphan; Keelapang, Poonsook; Utaipat, Utaiwan; Puttikhunt, Chunya; Kasinrerk, Watchara; Malasit, Prida; Sittisombut, Nopporn

2014-09-01

Recombinant virus-like particles (rVLPs) of flaviviruses are non-infectious particles released from cells expressing the envelope glycoproteins prM and E. Dengue virus rVLPs are recognized as a potential vaccine candidate, but large scale production of these particles is hindered by low yields and the occurrence of cytopathic effects. In an approach to improve the yield of rVLPs from transfected insect cells, several components of a dengue serotype 2 virus prM+E expression cassette were modified and the effect of these modifications was assessed during transient expression. Enhancement of extracellular rVLP levels by simultaneous substitutions of the prM signal peptide and the stem-anchor region of E with homologous cellular and viral counterparts, respectively, was further augmented by codon optimization. Extensive formation of multinucleated cells following transfection with the codon-optimized expression cassette was abrogated by introducing an E fusion loop mutation. This mutation also helped restore the extracellular E levels affected negatively by alteration of a charged residue at the pr-M junction, which was intended to promote maturation of rVLPs during export. Optimized expression cassettes generated in this multiple add-on modification approach should be useful in the generation of stably expressing clones and production of dengue virus rVLPs for immunogenicity studies. Copyright © 2014 Elsevier B.V. All rights reserved.

Promising MS2 mediated virus-like particle vaccine against foot-and-mouth disease.

PubMed

Dong, Yan-mei; Zhang, Guo-guang; Huang, Xiao-jun; Chen, Liang; Chen, Hao-tai

2015-05-01

Foot-and-mouth disease (FMD) has caused severe economic losses to millions of farmers worldwide. In this work, the coding genes of 141-160 epitope peptide (EP141-160) of VP1 were inserted into the coat protein (CP) genes of MS2 in prokaryotic expression vector, and the recombinant protein self-assembled into virus-like particles (VLP). Results showed that the CP-EP141-160 VLP had a strong immunoreaction with the FMD virus (FMDV) antigen in vitro, and also had an effective immune response in mice. Further virus challenge tests were carried out on guinea pigs and swine, high-titer neutralizing antibodies were produced and the CP-EP141-160 VLP vaccine could protect most of the animals against FMDV. Copyright © 2015. Published by Elsevier B.V.

Virus-like particle expression and assembly in plants: hepatitis B and Norwalk viruses.

PubMed

Huang, Zhong; Elkin, Galina; Maloney, Bryan J; Beuhner, Norene; Arntzen, Charles J; Thanavala, Yasmin; Mason, Hugh S

2005-03-07

Expression of vaccine antigens in plants and delivery via ingestion of transgenic plant material has shown promise in numerous pre-clinical animal studies and in a few clinical trials. A number of different viral antigens have been tested, and among the most promising are those that can assemble virus-like particles (VLP), which mimic the form of authentic virions and display neutralizing antibody epitopes. We have extensively studied plant expression, VLP assembly, and immunogenicity of hepatitis B surface antigen (HBsAg) and Norwalk virus capsid protein (NVCP). The HBsAg small protein (S protein) was found by TEM to assemble tubular membrane complexes derived from endoplasmic reticulum in suspension cultured cells of tobacco and soybean, and in potato leaf and tuber tissues. The potato material was immunogenic in mice upon delivery by ingestion. Here we describe the plant expression and immunogenicity of HBsAg middle protein (M protein or pre-S2 + S) which contains additional 55 amino acid pre-S2 region at N-terminus of the S protein. Plant-derived recombinant M protein provoked stronger serum antibody responses against HBsAg than did S protein when injected systemically in mice. We discuss implications for use of fusion proteins for enhanced immunogenicity and mucosal targeting of HBsAg, as well as delivery of heterologous fused antigens. NVCP expressed in plants assembled 38 nm virion-size icosahedral (T = 3) VLP, similar to those produced in insect cells. The VLP stimulated serum IgG and IgA responses in mice and humans when they were delivered by ingestion of fresh potato tuber. Here we show that freeze-drying of transgenic NVCP tomato fruit yielded stable preparations that stimulated excellent IgG and IgA responses against NVCP when fed to mice. However, the predominant VLP form in tomato fruit was the small 23 nm particle also observed in insect cell-derived NVCP.

Maize rayado fino virus-like particles expressed in tobacco plants: a new platform for cysteine selective bioconjugation peptide display

USDA-ARS?s Scientific Manuscript database

The ability of plant virus coat proteins to self-assemble into virus-like particles (VLPs), coupled with unique properties including three-dimensional structures, orthogonal reactivities, suitability for genetic manipulation and chemical bio-conjugation, provide potential utility in nanotechnology a...

Chicken adenovirus (CELO virus) particles augment receptor-mediated DNA delivery to mammalian cells and yield exceptional levels of stable transformants.

PubMed Central

Cotten, M; Wagner, E; Zatloukal, K; Birnstiel, M L

1993-01-01

Delivery of genes via receptor-mediated endocytosis is severely limited by the poor exit of endocytosed DNA from the endosome. A large enhancement in delivery efficiency has been obtained by including human adenovirus particles in the delivery system. This enhancement is probably a function of the natural adenovirus entry mechanism, which must include passage through or disruption of the endosomal membrane. In an effort to identify safer virus particles useful in this application, we have tested the chicken adenovirus CELO virus for its ability to augment receptor-mediated gene delivery. We report here that CELO virus possesses pH-dependent, liposome disruption activity similar to that of human adenovirus type 5. Furthermore, the chicken adenovirus can be used to augment receptor-mediated gene delivery to levels comparable to those found for the human adenovirus when it is physically linked to polylysine ligand-condensed DNA particles. The chicken adenovirus has the advantage of being produced inexpensively in embryonated eggs, and the virus is naturally replication defective in mammalian cells, even in the presence of wild-type human adenovirus. Images PMID:8099627

RNase-Resistant Virus-Like Particles Containing Long Chimeric RNA Sequences Produced by Two-Plasmid Coexpression System▿

PubMed Central

Wei, Yuxiang; Yang, Changmei; Wei, Baojun; Huang, Jie; Wang, Lunan; Meng, Shuang; Zhang, Rui; Li, Jinming

2008-01-01

RNase-resistant, noninfectious virus-like particles containing exogenous RNA sequences (armored RNA) are good candidates as RNA controls and standards in RNA virus detection. However, the length of RNA packaged in the virus-like particles with high efficiency is usually less than 500 bases. In this study, we describe a method for producing armored L-RNA. Armored L-RNA is a complex of MS2 bacteriophage coat protein and RNA produced in Escherichia coli by the induction of a two-plasmid coexpression system in which the coat protein and maturase are expressed from one plasmid and the target RNA sequence with modified MS2 stem-loop (pac site) is transcribed from another plasmid. A 3V armored L-RNA of 2,248 bases containing six gene fragments—hepatitis C virus, severe acute respiratory syndrome coronavirus (SARS-CoV1, SARS-CoV2, and SARS-CoV3), avian influenza virus matrix gene (M300), and H5N1 avian influenza virus (HA300)—was successfully expressed by the two-plasmid coexpression system and was demonstrated to have all of the characteristics of armored RNA. We evaluated the 3V armored L-RNA as a calibrator for multiple virus assays. We used the WHO International Standard for HCV RNA (NIBSC 96/790) to calibrate the chimeric armored L-RNA, which was diluted by 10-fold serial dilutions to obtain samples containing 106 to 102 copies. In conclusion, the approach we used for armored L-RNA preparation is practical and could reduce the labor and cost of quality control in multiplex RNA virus assays. Furthermore, we can assign the chimeric armored RNA with an international unit for quantitative detection. PMID:18305135

Quantitation of influenza virus using field flow fractionation and multi-angle light scattering for quantifying influenza A particles

PubMed Central

Bousse, Tatiana; Shore, David A.; Goldsmith, Cynthia S.; Hossain, M. Jaber; Jang, Yunho; Davis, Charles T.; Donis, Ruben O.; Stevens, James

2017-01-01

Summary Recent advances in instrumentation and data analysis in field flow fractionation and multi-angle light scattering (FFF-MALS) have enabled greater use of this technique to characterize and quantitate viruses. In this study, the FFF-MALS technique was applied to the characterization and quantitation of type A influenza virus particles to assess its usefulness for vaccine preparation. The use of FFF-MALS for quantitation and measurement of control particles provided data accurate to within 5% of known values, reproducible with a coefficient of variation of 1.9 %. The methods, sensitivity and limit of detection were established by analyzing different volumes of purified virus, which produced a linear regression with fitting value R2 of 0.99. FFF-MALS was further applied to detect and quantitate influenza virus in the supernatant of infected MDCK cells and allantoic fluids of infected eggs. FFF fractograms of the virus present in these different fluids revealed similar distribution of monomeric and oligomeric virions. However, the monomer fraction of cell grown virus has greater size variety. Notably, β-propialactone (BPL) inactivation of influenza viruses did not influence any of the FFF-MALS measurements. Quantitation analysis by FFF-MALS was compared to infectivity assays and real-time RT-PCR (qRT-PCR) and the limitations of each assay were discussed. PMID:23916678

Mitigation strategies to reduce the generation and transmission of airborne highly pathogenic avian influenza virus particles during processing of infected poultry.

PubMed

Bertran, Kateri; Clark, Andrew; Swayne, David E

2018-06-08

Airborne transmission of H5N1 highly pathogenic avian influenza (HPAI) viruses has occurred among poultry and from poultry to humans during home or live-poultry market slaughter of infected poultry, and such transmission has been experimentally reproduced. In this study, we investigated simple, practical changes in the processing of H5N1 virus-infected chickens to reduce infectious airborne particles and their transmission. Our findings suggest that containing the birds during the killing and bleeding first step by using a disposable plastic bag, a commonly available cooking pot widely used in Egypt (halla), or a bucket significantly reduces generation of infectious airborne particles and transmission to ferrets. Similarly, lack of infectious airborne particles was observed when processing vaccinated chickens that had been challenged with HPAI virus. Moreover, the use of a mechanical defeatherer significantly increased total number of particles in the air compared to manual defeathering. This study confirms that simple changes in poultry processing can efficiently mitigate generation of infectious airborne particles and their transmission to humans. Published by Elsevier GmbH.

Creation of a bovine herpes virus 1 (BoHV-1) quantitative particle standard by transmission electron microscopy and comparison with established standards for use in real-time PCR.

PubMed

Hoferer, Marc; Braun, Anne; Sting, Reinhard

2017-07-01

Standards are pivotal for pathogen quantification by real-time PCR (qPCR); however, the creation of a complete and universally applicable virus particle standard is challenging. In the present study a procedure based on purification of bovine herpes virus type 1 (BoHV-1) and subsequent quantification by transmission electron microscopy (TEM) is described. Accompanying quantitative quality controls of the TEM preparation procedure using qPCR yielded recovery rates of more than 95% of the BoHV-1 virus particles on the grid used for virus counting, which was attributed to pre-treatment of the grid with 5% bovine albumin. To compare the value of the new virus particle standard for use in qPCR, virus counter based quantification and established pure DNA standards represented by a plasmid and an oligonucleotide were included. It could be shown that the numbers of virus particles, plasmid and oligonucleotide equivalents were within one log10 range determined on the basis of standard curves indicating that different approaches provide comparable quantitative values. However, only virus particles represent a complete, universally applicable quantitative virus standard that meets the high requirements of an RNA and DNA virus gold standard. In contrast, standards based on pure DNA have to be considered as sub-standard due to limited applications. Copyright © 2017 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

Characterization of Non-Infectious Virus-Like Particle Surrogates for Viral Clearance Applications.

PubMed

Johnson, Sarah; Brorson, Kurt A; Frey, Douglas D; Dhar, Arun K; Cetlin, David A

2017-09-01

Viral clearance is a critical aspect of biopharmaceutical manufacturing process validation. To determine the viral clearance efficacy of downstream chromatography and filtration steps, live viral "spiking" studies are conducted with model mammalian viruses such as minute virus of mice (MVM). However, due to biosafety considerations, spiking studies are costly and typically conducted in specialized facilities. In this work, we introduce the concept of utilizing a non-infectious MVM virus-like particle (MVM-VLP) as an economical surrogate for live MVM during process development and characterization. Through transmission electron microscopy, size exclusion chromatography with multi-angle light scattering, chromatofocusing, and a novel solute surface hydrophobicity assay, we examined and compared the size, surface charge, and hydrophobic properties of MVM and MVM-VLP. The results revealed that MVM and MVM-VLP exhibited nearly identical physicochemical properties, indicating the potential utility of MVM-VLP as an accurate and economical surrogate to live MVM during chromatography and filtration process development and characterization studies.

Bioorthogonal Metabolic Labeling of Nascent RNA in Neurons Improves the Sensitivity of Transcriptome-Wide Profiling.

PubMed

Zajaczkowski, Esmi L; Zhao, Qiong-Yi; Zhang, Zong Hong; Li, Xiang; Wei, Wei; Marshall, Paul R; Leighton, Laura J; Nainar, Sarah; Feng, Chao; Spitale, Robert C; Bredy, Timothy W

2018-06-15

Transcriptome-wide expression profiling of neurons has provided important insights into the underlying molecular mechanisms and gene expression patterns that transpire during learning and memory formation. However, there is a paucity of tools for profiling stimulus-induced RNA within specific neuronal cell populations. A bioorthogonal method to chemically label nascent (i.e., newly transcribed) RNA in a cell-type-specific and temporally controlled manner, which is also amenable to bioconjugation via click chemistry, was recently developed and optimized within conventional immortalized cell lines. However, its value within a more fragile and complicated cellular system such as neurons, as well as for transcriptome-wide expression profiling, has yet to be demonstrated. Here, we report the visualization and sequencing of activity-dependent nascent RNA derived from neurons using this labeling method. This work has important implications for improving transcriptome-wide expression profiling and visualization of nascent RNA in neurons, which has the potential to provide valuable insights into the mechanisms underlying neural plasticity, learning, and memory.

Prevalence of virus-like particles within a staghorn scleractinian coral ( Acropora muricata) from the Great Barrier Reef

NASA Astrophysics Data System (ADS)

Patten, N. L.; Harrison, P. L.; Mitchell, J. G.

2008-09-01

Transmission electron microscopy (TEM) was used to determine whether Acropora muricata coral colonies from the Great Barrier Reef (GBR), Australia, harboured virus-like particles (VLPs). VLPs were present in all coral colonies sampled at Heron Island (southern GBR) and in tagged coral colonies sampled in at least two of the three sampling periods at Lizard Island (northern GBR). VLPs were observed within gastrodermal and epidermal tissues, and on rarer occasions, within the mesoglea. These VLPs had similar morphologies to known prokaryotic and eukaryotic viruses in other systems. Icosahedral VLPs were observed most frequently, however, filamentous VLPs (FVLPs) and phage were also noted. There were no clear differences in VLP size, morphology or location within the tissues with respect to sample date, coral health status or site. The most common VLP morphotype exhibited icosahedral symmetry, 120-150 nm in diameter, with an electron-dense core and an electronlucent membrane. Larger VLPs of similar morphology were also common. VLPs occurred as single entities, in groups, or in dense clusters, either as free particles within coral tissues, or within membrane-bound vacuoles. VLPs were commonly observed within the perinuclear region, with mitochondria, golgi apparatus and crescent-shaped particles frequently observed within close proximity. The host(s) of these observed VLPs was not clear; however, the different sizes and morphologies of VLPs observed within A. muricata tissues suggest that viruses are infecting either the coral animal, zooxanthellae, intracellular bacteria and/or other coral-associated microbiota, or that the one host is susceptible to infection from more than one type of virus. These results add to the limited but emerging body of evidence that viruses represent another potentially important component of the coral holobiont.

IFIT1 Expression Patterns Induced by H9N2 Virus and Inactivated Viral Particle in Human Umbilical Vein Endothelial Cells and Bronchus Epithelial Cells.

PubMed

Feng, Bo; Zhang, Qian; Wang, Jianfang; Dong, Hong; Mu, Xiang; Hu, Ge; Zhang, Tao

2018-04-30

IFIT1 (also known as ISG56) is a member of the interferon-inducible protein with tetratricopeptide repeats (IFITs) family. IFITs are strongly induced by type I interferon (IFN), double-stranded RNA and virus infection. Here, we investigated IFIT1 expression in human umbilical vein endothelial cells (HUVECs) and in human bronchus epithelial cells (BEAS-2Bs) induced by the H9N2 virus and inactivated viral particle at different time points. We also investigated the effect of H9N2 virus and viral particle infection on IFN-α/β production, and assessed whether hemagglutinin or neuraminidase protein induced IFIT1 expression. Results showed that both H9N2 virus infection and viral particle inoculation induced the expression of IFIT1 at mRNA and protein levels in the two cell lines. Hemagglutinin or neuraminidase protein binding alone is not sufficient to induce IFIT1 expression. Surprisingly, the expression patterns of IFIT1 in response to H9N2 virus and viral particles in the two cell lines were opposite, and production kinetics of IFN-α/β also differed. An additional finding was that induction of IFIT1 in response to H9N2 virus infection or viral particle inoculation was more sensitive in HUVECs than in BEAS-2Bs. Our data offers new insight into the innate immune response of endothelial cells to H9N2 virus infection.

IFIT1 Expression Patterns Induced by H9N2 Virus and Inactivated Viral Particle in Human Umbilical Vein Endothelial Cells and Bronchus Epithelial Cells

PubMed Central

Feng, Bo; Zhang, Qian; Wang, Jianfang; Dong, Hong; Mu, Xiang; Hu, Ge; Zhang, Tao

2018-01-01

IFIT1 (also known as ISG56) is a member of the interferon-inducible protein with tetratricopeptide repeats (IFITs) family. IFITs are strongly induced by type I interferon (IFN), double-stranded RNA and virus infection. Here, we investigated IFIT1 expression in human umbilical vein endothelial cells (HUVECs) and in human bronchus epithelial cells (BEAS-2Bs) induced by the H9N2 virus and inactivated viral particle at different time points. We also investigated the effect of H9N2 virus and viral particle infection on IFN-α/β production, and assessed whether hemagglutinin or neuraminidase protein induced IFIT1 expression. Results showed that both H9N2 virus infection and viral particle inoculation induced the expression of IFIT1 at mRNA and protein levels in the two cell lines. Hemagglutinin or neuraminidase protein binding alone is not sufficient to induce IFIT1 expression. Surprisingly, the expression patterns of IFIT1 in response to H9N2 virus and viral particles in the two cell lines were opposite, and production kinetics of IFN-α/β also differed. An additional finding was that induction of IFIT1 in response to H9N2 virus infection or viral particle inoculation was more sensitive in HUVECs than in BEAS-2Bs. Our data offers new insight into the innate immune response of endothelial cells to H9N2 virus infection. PMID:29629559

Hepatitis C Virus Particle Assembly Involves Phosphorylation of NS5A by the c-Abl Tyrosine Kinase.

PubMed

Yamauchi, Shota; Takeuchi, Kenji; Chihara, Kazuyasu; Sun, Xuedong; Honjoh, Chisato; Yoshiki, Hatsumi; Hotta, Hak; Sada, Kiyonao

2015-09-04

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is thought to regulate the replication of viral RNA and the assembly of virus particles in a serine/threonine phosphorylation-dependent manner. However, the host kinases that phosphorylate NS5A have not been fully identified. Here, we show that HCV particle assembly involves the phosphorylation of NS5A by the c-Abl tyrosine kinase. Pharmacological inhibition or knockdown of c-Abl reduces the production of infectious HCV (J6/JFH1) particles in Huh-7.5 cells without markedly affecting viral RNA translation and replication. NS5A is tyrosine-phosphorylated in HCV-infected cells, and this phosphorylation is also reduced by the knockdown of c-Abl. Mutational analysis reveals that NS5A tyrosine phosphorylation is dependent, at least in part, on Tyr(330) (Tyr(2306) in polyprotein numbering). Mutation of this residue to phenylalanine reduces the production of infectious HCV particles but does not affect the replication of the JFH1 subgenomic replicon. These findings suggest that c-Abl promotes HCV particle assembly by phosphorylating NS5A at Tyr(330). © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Ferrets develop fatal influenza after inhaling small particle aerosols of highly pathogenic avian influenza virus A/Vietnam/1203/2004 (H5N1)

PubMed Central

2010-01-01

Background There is limited knowledge about the potential routes for H5N1 influenza virus transmission to and between humans, and it is not clear whether humans can be infected through inhalation of aerosolized H5N1 virus particles. Ferrets are often used as a animal model for humans in influenza pathogenicity and transmissibility studies. In this manuscript, a nose-only bioaerosol inhalation exposure system that was recently developed and validated was used in an inhalation exposure study of aerosolized A/Vietnam/1203/2004 (H5N1) virus in ferrets. The clinical spectrum of influenza resulting from exposure to A/Vietnam/1203/2004 (H5N1) through intranasal verses inhalation routes was analyzed. Results Ferrets were successfully infected through intranasal instillation or through inhalation of small particle aerosols with four different doses of Influenza virus A/Vietnam/1203/2004 (H5N1). The animals developed severe influenza encephalomyelitis following intranasal or inhalation exposure to 101, 102, 103, or 104 infectious virus particles per ferret. Conclusions Aerosolized Influenza virus A/Vietnam/1203/2004 (H5N1) is highly infectious and lethal in ferrets. Clinical signs appeared earlier in animals infected through inhalation of aerosolized virus compared to those infected through intranasal instillation. PMID:20843329

Vaccination with virus-like particles containing H5 antigens from three H5N1 clades protects chickens from H5N1 and H5N8 influenza viruses

USDA-ARS?s Scientific Manuscript database

Highly pathogenic avian influenza (HPAI) viruses, especially H5N1 strains, represent a public health threat and cause widespread morbidity and mortality in domestic poultry. Recombinant virus-like particles (VLPs) represent a promising novel vaccine approach to control avian influenza including HPAI...

The recruitment of the U5 snRNP to nascent transcripts requires internal loop 1 of U5 snRNA.

PubMed

Kim, Rebecca; Paschedag, Joshua; Novikova, Natalya; Bellini, Michel

2012-12-01

In this study, we take advantage of the high spatial resolution offered by the nucleus and lampbrush chromosomes of the amphibian oocyte to investigate the mechanisms that regulate the intranuclear trafficking of the U5 snRNP and its recruitment to nascent transcripts. We monitor the fate of newly assembled fluorescent U5 snRNP in Xenopus oocytes depleted of U4 and/or U6 snRNAs and demonstrate that the U4/U6.U5 tri-snRNP is not required for the association of U5 snRNP with Cajal bodies, splicing speckles, and nascent transcripts. In addition, using a mutational analysis, we show that a non-functional U5 snRNP can associate with nascent transcripts, and we further characterize internal loop structure 1 of U5 snRNA as a critical element for licensing U5 snRNP to target both nascent transcripts and splicing speckles. Collectively, our data support the model where the recruitment of snRNPs onto pre-mRNAs is independent of spliceosome assembly and suggest that U5 snRNP may promote the association of the U4/U6.U5 tri-snRNP with nascent transcripts.

Prevalence and stability of human serum antibodies to simian virus 40 VP1 virus-like particles.

PubMed

Lundstig, Annika; Eliasson, Linda; Lehtinen, Matti; Sasnauskas, Kestutis; Koskela, Pentti; Dillner, Joakim

2005-06-01

Possible human infection with simian virus 40 (SV40) has been of great concern ever since SV40 was discovered in polio vaccines. Human populations are SV40-seropositive, but because of serological cross-reactivity between SV40 and the human polyomaviruses BK virus (BKV) and JC virus (JCV), it is debatable whether these antibodies are specific. An SV40-specific serological assay was established, based on purified virus-like particles (VLPs), where the SV40 VLPs were blocked with hyperimmune sera to BKV and JCV. Competition with SV40 hyperimmune sera was used as a confirmatory test. Among 288 Swedish children of between 1 and 13 years of age, 7.6 % had SV40-specific antibodies. SV40 seroprevalence reached a peak of 14 % at 7-9 years of age. Among 100 control patients with benign tumours, 9 % were SV40-seropositive. However, SV40 DNA was not detectable in corresponding buffy-coat samples. In serial samples taken up to 5 years apart from 141 Finnish women participating in the population-based serological screening for congenital infections, only two of 141 women were SV40-seropositive in both samples. Six women seroconverted and eight women had a loss of antibodies over time. None of the SV40-seropositive samples contained detectable SV40 DNA. In conclusion, there is a low prevalence of SV40-specific antibodies in the Nordic population. The SV40 antibodies appear to have a low stability over time and their origin is not clear.

Ebola Virus VP35-VP40 Interaction Is Sufficient for Packaging 3E-5E Minigenome RNA into Virus-Like Particles

PubMed Central

Johnson, Reed F.; McCarthy, Sarah E.; Godlewski, Peter J.; Harty, Ronald N.

2006-01-01

The packaging of viral genomic RNA into nucleocapsids and subsequently into virions is not completely understood. Phosphoprotein (P) and nucleoprotein (NP) interactions link NP-RNA complexes with P-L (polymerase) complexes to form viral nucleocapsids. The nucleocapsid then interacts with the viral matrix protein, leading to specific packaging of the nucleocapsid into the virion. A mammalian two-hybrid assay and confocal microscopy were used to demonstrate that Ebola virus VP35 and VP40 interact and colocalize in transfected cells. VP35 was packaged into budding virus-like particles (VLPs) as observed by protease protection assays. Moreover, VP40 and VP35 were sufficient for packaging an Ebola virus minignome RNA into VLPs. Results from immunoprecipitation-reverse transcriptase PCR experiments suggest that VP35 confers specificity of the nucleocapsid for viral genomic RNA by direct VP35-RNA interactions. PMID:16698994

A Possible Role of the Full-Length Nascent Protein in Post-Translational Ribosome Recycling.

PubMed

Das, Debasis; Samanta, Dibyendu; Bhattacharya, Arpita; Basu, Arunima; Das, Anindita; Ghosh, Jaydip; Chakrabarti, Abhijit; Das Gupta, Chanchal

2017-01-01

Each cycle of translation initiation in bacterial cell requires free 50S and 30S ribosomal subunits originating from the post-translational dissociation of 70S ribosome from the previous cycle. Literature shows stable dissociation of 70S from model post-termination complexes by the concerted action of Ribosome Recycling Factor (RRF) and Elongation Factor G (EF-G) that interact with the rRNA bridge B2a/B2b joining 50S to 30S. In such experimental models, the role of full-length nascent protein was never considered seriously. We observed relatively slow release of full-length nascent protein from 50Sof post translation ribosome, and in that process, its toe prints on the rRNA in vivo and in in vitro translation with E.coli S30 extract. We reported earlier that a number of chemically unfolded proteins like bovine carbonic anhydrase (BCA), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), lysozyme, ovalbumin etc., when added to free 70Sin lieu of the full length nascent proteins, also interact with identical RNA regions of the 23S rRNA. Interestingly the rRNA nucleotides that slow down release of the C-terminus of full-length unfolded protein were found in close proximity to the B2a/B2b bridge. It indicated a potentially important chemical reaction conserved throughout the evolution. Here we set out to probe that conserved role of unfolded protein conformation in splitting the free or post-termination 70S. How both the RRF-EFG dependent and the plausible nascent protein-EFG dependent ribosome recycling pathways might be relevant in bacteria is discussed here.

A Possible Role of the Full-Length Nascent Protein in Post-Translational Ribosome Recycling

PubMed Central

Das, Debasis; Samanta, Dibyendu; Bhattacharya, Arpita; Basu, Arunima; Das, Anindita; Ghosh, Jaydip; Chakrabarti, Abhijit; Das Gupta, Chanchal

2017-01-01

Each cycle of translation initiation in bacterial cell requires free 50S and 30S ribosomal subunits originating from the post-translational dissociation of 70S ribosome from the previous cycle. Literature shows stable dissociation of 70S from model post-termination complexes by the concerted action of Ribosome Recycling Factor (RRF) and Elongation Factor G (EF-G) that interact with the rRNA bridge B2a/B2b joining 50S to 30S. In such experimental models, the role of full-length nascent protein was never considered seriously. We observed relatively slow release of full-length nascent protein from 50Sof post translation ribosome, and in that process, its toe prints on the rRNA in vivo and in in vitro translation with E.coli S30 extract. We reported earlier that a number of chemically unfolded proteins like bovine carbonic anhydrase (BCA), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), lysozyme, ovalbumin etc., when added to free 70Sin lieu of the full length nascent proteins, also interact with identical RNA regions of the 23S rRNA. Interestingly the rRNA nucleotides that slow down release of the C-terminus of full-length unfolded protein were found in close proximity to the B2a/B2b bridge. It indicated a potentially important chemical reaction conserved throughout the evolution. Here we set out to probe that conserved role of unfolded protein conformation in splitting the free or post-termination 70S. How both the RRF-EFG dependent and the plausible nascent protein–EFG dependent ribosome recycling pathways might be relevant in bacteria is discussed here. PMID:28099529

Role of the visual experience-dependent nascent proteome in neuronal plasticity

PubMed Central

Liu, Han-Hsuan; McClatchy, Daniel B; Schiapparelli, Lucio; Shen, Wanhua; Yates, John R

2018-01-01

Experience-dependent synaptic plasticity refines brain circuits during development. To identify novel protein synthesis-dependent mechanisms contributing to experience-dependent plasticity, we conducted a quantitative proteomic screen of the nascent proteome in response to visual experience in Xenopus optic tectum using bio-orthogonal metabolic labeling (BONCAT). We identified 83 differentially synthesized candidate plasticity proteins (CPPs). The CPPs form strongly interconnected networks and are annotated to a variety of biological functions, including RNA splicing, protein translation, and chromatin remodeling. Functional analysis of select CPPs revealed the requirement for eukaryotic initiation factor three subunit A (eIF3A), fused in sarcoma (FUS), and ribosomal protein s17 (RPS17) in experience-dependent structural plasticity in tectal neurons and behavioral plasticity in tadpoles. These results demonstrate that the nascent proteome is dynamic in response to visual experience and that de novo synthesis of machinery that regulates RNA splicing and protein translation is required for experience-dependent plasticity. PMID:29412139

Uncoupling cis-Acting RNA Elements from Coding Sequences Revealed a Requirement of the N-Terminal Region of Dengue Virus Capsid Protein in Virus Particle Formation

PubMed Central

Samsa, Marcelo M.; Mondotte, Juan A.; Caramelo, Julio J.

2012-01-01

Little is known about the mechanism of flavivirus genome encapsidation. Here, functional elements of the dengue virus (DENV) capsid (C) protein were investigated. Study of the N-terminal region of DENV C has been limited by the presence of overlapping cis-acting RNA elements within the protein-coding region. To dissociate these two functions, we used a recombinant DENV RNA with a duplication of essential RNA structures outside the C coding sequence. By the use of this system, the highly conserved amino acids FNML, which are encoded in the RNA cyclization sequence 5′CS, were found to be dispensable for C function. In contrast, deletion of the N-terminal 18 amino acids of C impaired DENV particle formation. Two clusters of basic residues (R5-K6-K7-R9 and K17-R18-R20-R22) were identified as important. A systematic mutational analysis indicated that a high density of positive charges, rather than particular residues at specific positions, was necessary. Furthermore, a differential requirement of N-terminal sequences of C for viral particle assembly was observed in mosquito and human cells. While no viral particles were observed in human cells with a virus lacking the first 18 residues of C, DENV propagation was detected in mosquito cells, although to a level about 50-fold less than that observed for a wild-type (WT) virus. We conclude that basic residues at the N terminus of C are necessary for efficient particle formation in mosquito cells but that they are crucial for propagation in human cells. This is the first report demonstrating that the N terminus of C plays a role in DENV particle formation. In addition, our results suggest that this function of C is differentially modulated in different host cells. PMID:22072762

Particle size effects on protein and virus-like particle adsorption on perfusion chromatography media.

PubMed

Wu, Yige; Abraham, Dicky; Carta, Giorgio

2015-01-02

The resin structure, chromatographic behavior, and adsorption kinetics of proteins and virus-like-particles (VLPs) are studied for POROS HS 20 and POROS HS 50 (23 and 52 μm mean diameter, respectively) to determine the effects of particle size on perfusion chromatography and to determine the predictive ability of available models. Transmission electron microscopy (TEM) and inverse size-exclusion chromatography (iSEC) show similar structures for the two resins, both containing 200-1000 nm pores that transect a network of much smaller pores. For non-binding conditions, trends of the height equivalent to a theoretical plate (HETP) as a function of reduced velocity are consistent with perfusion. The estimated intraparticle flow fractions for these conditions are 0.0018 and 0.00063 for POROS HS 20 and HS 50, respectively. For strong binding conditions, confocal laser scanning microscopy (CLSM) shows asymmetrical intraparticle concentrations profiles and enhanced rates of IgG adsorption on POROS HS 20 at 1000 cm/h. The corresponding effective diffusivity under flow is 2-3 times larger than for non-flow conditions and much larger than observed for POROS HS 50, consistent with available models. For VLPs, however, adsorption is confined to a thin layer near the particle surface for both resins, suggesting that the bound VLPs block the pores. Copyright © 2014 Elsevier B.V. All rights reserved.

Enhanced Influenza Virus-Like Particle Vaccination with a Structurally Optimized RIG-I Agonist as Adjuvant.

PubMed

Beljanski, Vladimir; Chiang, Cindy; Kirchenbaum, Greg A; Olagnier, David; Bloom, Chalise E; Wong, Terianne; Haddad, Elias K; Trautmann, Lydie; Ross, Ted M; Hiscott, John

2015-10-01

The molecular interaction between viral RNA and the cytosolic sensor RIG-I represents the initial trigger in the development of an effective immune response against infection with RNA viruses, resulting in innate immune activation and subsequent induction of adaptive responses. In the present study, the adjuvant properties of a sequence-optimized 5'-triphosphate-containing RNA (5'pppRNA) RIG-I agonist (termed M8) were examined in combination with influenza virus-like particles (VLP) (M8-VLP) expressing H5N1 influenza virus hemagglutinin (HA) and neuraminidase (NA) as immunogens. In combination with VLP, M8 increased the antibody response to VLP immunization, provided VLP antigen sparing, and protected mice from a lethal challenge with H5N1 influenza virus. M8-VLP immunization also led to long-term protective responses against influenza virus infection in mice. M8 adjuvantation of VLP increased endpoint and antibody titers and inhibited influenza virus replication in lungs compared with approved or experimental adjuvants alum, AddaVax, and poly(I·C). Uniquely, immunization with M8-VLP stimulated a TH1-biased CD4 T cell response, as determined by increased TH1 cytokine levels in CD4 T cells and increased IgG2 levels in sera. Collectively, these data demonstrate that a sequence-optimized, RIG-I-specific agonist is a potent adjuvant that can be utilized to increase the efficacy of influenza VLP vaccination and dramatically improve humoral and cellular mediated protective responses against influenza virus challenge. The development of novel adjuvants to increase vaccine immunogenicity is an important goal that seeks to improve vaccine efficacy and ultimately prevent infections that endanger human health. This proof-of-principle study investigated the adjuvant properties of a sequence-optimized 5'pppRNA agonist (M8) with enhanced capacity to stimulate antiviral and inflammatory gene networks using influenza virus-like particles (VLP) expressing HA and NA as immunogens

Characterization of self-assembled virus-like particles of dromedary camel hepatitis e virus generated by recombinant baculoviruses.

PubMed

Zhou, Xianfeng; Kataoka, Michiyo; Liu, Zheng; Takeda, Naokazu; Wakita, Takaji; Li, Tian-Cheng

2015-12-02

Dromedary camel hepatitis E virus (DcHEV), a novel hepatitis E virus, has been identified in dromedary camels in Dubai, United Arab Emirates. The antigenicity, pathogenicity and epidemiology of this virus have been unclear. Here we first used a recombinant baculovirus expression system to express the 13 and 111 N-terminus amino-acid-truncated DcHEV ORF2 protein in insect Tn5 cells, and we obtained two types of virus-like particles (VLPs) with densities of 1.300 g/cm(3) and 1.285 g/cm(3), respectively. The small VLPs (Dc4sVLPs) were estimated to be 24 nm in diameter, and were assembled by a protein with the molecular mass 53 kDa. The large VLPs (Dc3nVLPs and Dc4nVLPs) were 35 nm in diameter, and were assembled by a 64-kDa protein. An antigenic analysis demonstrated that DcHEV was cross-reactive with G1, G3-G6, ferret and rat HEVs, and DcHEV showed a stronger cross-reactivity to G1 G3-G6 HEV than it did to rat and ferret HEV. In addition, the antibody against DcHEV-LPs neutralized G1 and G3 HEV in a cell culture system, suggesting that the serotypes of these HEVs are identical. We also found that the amino acid residue Met-358 affects the small DcHEV-LPs assembly. Copyright © 2015 Elsevier B.V. All rights reserved.

Nipah virion entry kinetics, composition, and conformational changes determined by enzymatic virus-like particles and new flow virometry tools.

PubMed

Landowski, Matthew; Dabundo, Jeffrey; Liu, Qian; Nicola, Anthony V; Aguilar, Hector C

2014-12-01

Virus-cell membrane fusion is essential for enveloped virus infections. However, mechanistic viral membrane fusion studies have predominantly focused on cell-cell fusion models, largely due to the low availability of technologies capable of characterizing actual virus-cell membrane fusion. Although cell-cell fusion assays are valuable, they do not fully recapitulate all the variables of virus-cell membrane fusion. Drastic differences between viral and cellular membrane lipid and protein compositions and curvatures exist. For biosafety level 4 (BSL4) pathogens such as the deadly Nipah virus (NiV), virus-cell fusion mechanistic studies are notably cumbersome. To circumvent these limitations, we used enzymatic Nipah virus-like-particles (NiVLPs) and developed new flow virometric tools. NiV's attachment (G) and fusion (F) envelope glycoproteins mediate viral binding to the ephrinB2/ephrinB3 cell receptors and virus-cell membrane fusion, respectively. The NiV matrix protein (M) can autonomously induce NiV assembly and budding. Using a β-lactamase (βLa) reporter/NiV-M chimeric protein, we produced NiVLPs expressing NiV-G and wild-type or mutant NiV-F on their surfaces. By preloading target cells with the βLa fluorescent substrate CCF2-AM, we obtained viral entry kinetic curves that correlated with the NiV-F fusogenic phenotypes, validating NiVLPs as suitable viral entry kinetic tools and suggesting overall relatively slower viral entry than cell-cell fusion kinetics. Additionally, the proportions of F and G on individual NiVLPs and the extent of receptor-induced conformational changes in NiV-G were measured via flow virometry, allowing the proper interpretation of the viral entry kinetic phenotypes. The significance of these findings in the viral entry field extends beyond NiV to other paramyxoviruses and enveloped viruses. Virus-cell membrane fusion is essential for enveloped virus infections. However, mechanistic viral membrane fusion studies have predominantly

Theory of Force Regulation by Nascent Adhesion Sites

PubMed Central

Bruinsma, Robijn

2005-01-01

The mechanical coupling of a cell with the extracellular matrix relies on adhesion sites, clusters of membrane-associated proteins that communicate forces generated along the F-Actin filaments of the cytoskeleton to connecting tissue. Nascent adhesion sites have been shown to regulate these forces in response to tissue rigidity. Force-regulation by substrate rigidity of adhesion sites with fixed area is not possible for stationary adhesion sites, according to elasticity theory. A simple model is presented to describe force regulation by dynamical adhesion sites. PMID:15849245

Homologous and heterologous protection of nonhuman primates by Ebola and Sudan virus-like particles.

PubMed

Warfield, Kelly L; Dye, John M; Wells, Jay B; Unfer, Robert C; Holtsberg, Frederick W; Shulenin, Sergey; Vu, Hong; Swenson, Dana L; Bavari, Sina; Aman, M Javad

2015-01-01

Filoviruses cause hemorrhagic fever resulting in significant morbidity and mortality in humans. Several vaccine platforms that include multiple virus-vectored approaches and virus-like particles (VLPs) have shown efficacy in nonhuman primates. Previous studies have shown protection of cynomolgus macaques against homologous infection for Ebola virus (EBOV) and Marburg virus (MARV) following a three-dose vaccine regimen of EBOV or MARV VLPs, as well as heterologous protection against Ravn Virus (RAVV) following vaccination with MARV VLPs. The objectives of the current studies were to determine the minimum number of vaccine doses required for protection (using EBOV as the test system) and then demonstrate protection against Sudan virus (SUDV) and Taï Forest virus (TAFV). Using the EBOV nonhuman primate model, we show that one or two doses of VLP vaccine can confer protection from lethal infection. VLPs containing the SUDV glycoprotein, nucleoprotein and VP40 matrix protein provide complete protection against lethal SUDV infection in macaques. Finally, we demonstrate protective efficacy mediated by EBOV, but not SUDV, VLPs against TAFV; this is the first demonstration of complete cross-filovirus protection using a single component heterologous vaccine within the Ebolavirus genus. Along with our previous results, this observation provides strong evidence that it will be possible to develop and administer a broad-spectrum VLP-based vaccine that will protect against multiple filoviruses by combining only three EBOV, SUDV and MARV components.

Virus Assembly and Maturation

NASA Astrophysics Data System (ADS)

Johnson, John E.

2004-03-01

We use two techniques to look at three-dimensional virus structure: electron cryomicroscopy (cryoEM) and X-ray crystallography. Figure 1 is a gallery of virus particles whose structures Timothy Baker, one of my former colleagues at Purdue University, used cryoEM to determine. It illustrates the variety of sizes of icosahedral virus particles. The largest virus particle on this slide is the Herpes simplex virus, around 1200Å in diameter; the smallest we examined was around 250Å in diameter. Viruses bear their genomic information either as positive-sense DNA and RNA, double-strand DNA, double-strand RNA, or negative-strand RNA. Viruses utilize the various structure and function "tactics" seen throughout cell biology to replicate at high levels. Many of the biological principles that we consider general were in fact discovered in the context of viruses ...

Structures of Adenovirus Incomplete Particles Clarify Capsid Architecture and Show Maturation Changes of Packaging Protein L1 52/55k

PubMed Central

Condezo, Gabriela N.; Marabini, Roberto; Ayora, Silvia; Carazo, José M.; Alba, Raúl; Chillón, Miguel

2015-01-01

ABSTRACT Adenovirus is one of the most complex icosahedral, nonenveloped viruses. Even after its structure was solved at near-atomic resolution by both cryo-electron microscopy and X-ray crystallography, the location of minor coat proteins is still a subject of debate. The elaborated capsid architecture is the product of a correspondingly complex assembly process, about which many aspects remain unknown. Genome encapsidation involves the concerted action of five virus proteins, and proteolytic processing by the virus protease is needed to prime the virion for sequential uncoating. Protein L1 52/55k is required for packaging, and multiple cleavages by the maturation protease facilitate its release from the nascent virion. Light-density particles are routinely produced in adenovirus infections and are thought to represent assembly intermediates. Here, we present the molecular and structural characterization of two different types of human adenovirus light particles produced by a mutant with delayed packaging. We show that these particles lack core polypeptide V but do not lack the density corresponding to this protein in the X-ray structure, thereby adding support to the adenovirus cryo-electron microscopy model. The two types of light particles present different degrees of proteolytic processing. Their structures provide the first glimpse of the organization of L1 52/55k protein inside the capsid shell and of how this organization changes upon partial maturation. Immature, full-length L1 52/55k is poised beneath the vertices to engage the virus genome. Upon proteolytic processing, L1 52/55k disengages from the capsid shell, facilitating genome release during uncoating. IMPORTANCE Adenoviruses have been extensively characterized as experimental systems in molecular biology, as human pathogens, and as therapeutic vectors. However, a clear picture of many aspects of their basic biology is still lacking. Two of these aspects are the location of minor coat proteins in

Chimeric SV40 virus-like particles induce specific cytotoxicity and protective immunity against influenza A virus without the need of adjuvants.

PubMed

Kawano, Masaaki; Morikawa, Katsuma; Suda, Tatsuya; Ohno, Naohito; Matsushita, Sho; Akatsuka, Toshitaka; Handa, Hiroshi; Matsui, Masanori

2014-01-05

Virus-like particles (VLPs) are a promising vaccine platform due to the safety and efficiency. However, it is still unclear whether polyomavirus-based VLPs are useful for this purpose. Here, we attempted to evaluate the potential of polyomavirus VLPs for the antiviral vaccine using simian virus 40 (SV40). We constructed chimeric SV40-VLPs carrying an HLA-A*02:01-restricted, cytotoxic T lymphocyte (CTL) epitope derived from influenza A virus. HLA-A*02:01-transgenic mice were then immunized with the chimeric SV40-VLPs. The chimeric SV40-VLPs effectively induced influenza-specific CTLs and heterosubtypic protection against influenza A viruses without the need of adjuvants. Because DNase I treatment of the chimeric SV40-VLPs did not disrupt CTL induction, the intrinsic adjuvant property may not result from DNA contaminants in the VLP preparation. In addition, immunization with the chimeric SV40-VLPs generated long-lasting memory CTLs. We here propose that the chimeric SV40-VLPs harboring an epitope may be a promising CTL-based vaccine platform with self-adjuvant properties. © 2013 Elsevier Inc. All rights reserved.

Amphipathic α-helices in apolipoproteins are crucial to the formation of infectious hepatitis C virus particles.

PubMed

Fukuhara, Takasuke; Wada, Masami; Nakamura, Shota; Ono, Chikako; Shiokawa, Mai; Yamamoto, Satomi; Motomura, Takashi; Okamoto, Toru; Okuzaki, Daisuke; Yamamoto, Masahiro; Saito, Izumu; Wakita, Takaji; Koike, Kazuhiko; Matsuura, Yoshiharu

2014-12-01

Apolipoprotein B (ApoB) and ApoE have been shown to participate in the particle formation and the tissue tropism of hepatitis C virus (HCV), but their precise roles remain uncertain. Here we show that amphipathic α-helices in the apolipoproteins participate in the HCV particle formation by using zinc finger nucleases-mediated apolipoprotein B (ApoB) and/or ApoE gene knockout Huh7 cells. Although Huh7 cells deficient in either ApoB or ApoE gene exhibited slight reduction of particles formation, knockout of both ApoB and ApoE genes in Huh7 (DKO) cells severely impaired the formation of infectious HCV particles, suggesting that ApoB and ApoE have redundant roles in the formation of infectious HCV particles. cDNA microarray analyses revealed that ApoB and ApoE are dominantly expressed in Huh7 cells, in contrast to the high level expression of all of the exchangeable apolipoproteins, including ApoA1, ApoA2, ApoC1, ApoC2 and ApoC3 in human liver tissues. The exogenous expression of not only ApoE, but also other exchangeable apolipoproteins rescued the infectious particle formation of HCV in DKO cells. In addition, expression of these apolipoproteins facilitated the formation of infectious particles of genotype 1b and 3a chimeric viruses. Furthermore, expression of amphipathic α-helices in the exchangeable apolipoproteins facilitated the particle formation in DKO cells through an interaction with viral particles. These results suggest that amphipathic α-helices in the exchangeable apolipoproteins play crucial roles in the infectious particle formation of HCV and provide clues to the understanding of life cycle of HCV and the development of novel anti-HCV therapeutics targeting for viral assembly.

Amphipathic α-Helices in Apolipoproteins Are Crucial to the Formation of Infectious Hepatitis C Virus Particles

PubMed Central

Nakamura, Shota; Ono, Chikako; Shiokawa, Mai; Yamamoto, Satomi; Motomura, Takashi; Okamoto, Toru; Okuzaki, Daisuke; Yamamoto, Masahiro; Saito, Izumu; Wakita, Takaji; Koike, Kazuhiko; Matsuura, Yoshiharu

2014-01-01

Apolipoprotein B (ApoB) and ApoE have been shown to participate in the particle formation and the tissue tropism of hepatitis C virus (HCV), but their precise roles remain uncertain. Here we show that amphipathic α-helices in the apolipoproteins participate in the HCV particle formation by using zinc finger nucleases-mediated apolipoprotein B (ApoB) and/or ApoE gene knockout Huh7 cells. Although Huh7 cells deficient in either ApoB or ApoE gene exhibited slight reduction of particles formation, knockout of both ApoB and ApoE genes in Huh7 (DKO) cells severely impaired the formation of infectious HCV particles, suggesting that ApoB and ApoE have redundant roles in the formation of infectious HCV particles. cDNA microarray analyses revealed that ApoB and ApoE are dominantly expressed in Huh7 cells, in contrast to the high level expression of all of the exchangeable apolipoproteins, including ApoA1, ApoA2, ApoC1, ApoC2 and ApoC3 in human liver tissues. The exogenous expression of not only ApoE, but also other exchangeable apolipoproteins rescued the infectious particle formation of HCV in DKO cells. In addition, expression of these apolipoproteins facilitated the formation of infectious particles of genotype 1b and 3a chimeric viruses. Furthermore, expression of amphipathic α-helices in the exchangeable apolipoproteins facilitated the particle formation in DKO cells through an interaction with viral particles. These results suggest that amphipathic α-helices in the exchangeable apolipoproteins play crucial roles in the infectious particle formation of HCV and provide clues to the understanding of life cycle of HCV and the development of novel anti-HCV therapeutics targeting for viral assembly. PMID:25502789

The Impact of Aerosol Particle Mixing State on the Hygroscopicity of Sea Spray Aerosol.

PubMed

Schill, Steven R; Collins, Douglas B; Lee, Christopher; Morris, Holly S; Novak, Gordon A; Prather, Kimberly A; Quinn, Patricia K; Sultana, Camille M; Tivanski, Alexei V; Zimmermann, Kathryn; Cappa, Christopher D; Bertram, Timothy H

2015-06-24

Aerosol particles influence global climate by determining cloud droplet number concentrations, brightness, and lifetime. Primary aerosol particles, such as those produced from breaking waves in the ocean, display large particle-particle variability in chemical composition, morphology, and physical phase state, all of which affect the ability of individual particles to accommodate water and grow into cloud droplets. Despite such diversity in molecular composition, there is a paucity of methods available to assess how particle-particle variability in chemistry translates to corresponding differences in aerosol hygroscopicity. Here, an approach has been developed that allows for characterization of the distribution of aerosol hygroscopicity within a chemically complex population of atmospheric particles. This methodology, when applied to the interpretation of nascent sea spray aerosol, provides a quantitative framework for connecting results obtained using molecular mimics generated in the laboratory with chemically complex ambient aerosol. We show that nascent sea spray aerosol, generated in situ in the Atlantic Ocean, displays a broad distribution of particle hygroscopicities, indicative of a correspondingly broad distribution of particle chemical compositions. Molecular mimics of sea spray aerosol organic material were used in the laboratory to assess the volume fractions and molecular functionality required to suppress sea spray aerosol hygroscopicity to the extent indicated by field observations. We show that proper accounting for the distribution and diversity in particle hygroscopicity and composition are important to the assessment of particle impacts on clouds and global climate.

Effects of solution chemistry on the sunlight inactivation of particles-associated viruses MS2.

PubMed

Wu, Xueyin; Feng, Zhe; Yuan, Baoling; Zhou, Zhenming; Li, Fei; Sun, Wenjie

2018-02-01

The inactivation efficacy of bacteriophage MS2 by simulated sunlight irradiation was investigated to understand the effects of MS2 aggregation and adsorption to particles in solutions with different components. Kaolinite and Microcystis aeruginosa were used as model inorganic and organic particles, respectively. Lower pH and di-valent ions (Ca 2+ ) were main factors on the aggregation and inactivation of MS2. In the presence of both particles, there was no significant impact on the MS2 inactivation efficacy by kaolinite (10-200mM) or Microcystis aeruginosa (10 2 -10 5 Cells/mL) in 1mM NaCl at pH 7. However at lower pH 3, MS2 aggregates formed in the particle-free and kaolinite-containing solutions, caused lower inactivation since the outer viruses of aggregation protect the inner viruses. In addition, more MS2 adsorbed on Microcystis aeruginosa at lower pH (3 and 4). Microcystis aeruginosa would act as a potential photosensitizer for ROS production to inactivate the adsorbed MS2, since extracellular organic matter (EOM) of Microcystis aeruginosa was detected in this study, which has been reported to produce ROS under solar irradiation. At pH 7, Na + had no effect on the inactivation of MS2, because MS2 was stable and dispersed even at 200mM Na + . MS2 aggregated and adsorbed on particles even at 10mM Ca 2+ and led to lower inactivation. Kaolinite cannot offer enough protection to adsorbed MS2 as aggregation and Microcystis aeruginosa acts as potential photosensitizer to produce ROS and inactivate the adsorbed MS2 at high concentration of Ca 2+ . In particle-free solution, SRNOM inhibited MS2 inactivation by shielding the sunlight and coating MS2 to increase its survival. Copyright © 2017 Elsevier B.V. All rights reserved.

Investigation of antiviral state mediated by interferon-inducible transmembrane protein 1 induced by H9N2 virus and inactivated viral particle in human endothelial cells.

PubMed

Feng, Bo; Zhao, Lihong; Wang, Wei; Wang, Jianfang; Wang, Hongyan; Duan, Huiqin; Zhang, Jianjun; Qiao, Jian

2017-11-03

Endothelial cells are believed to play an important role in response to virus infection. Our previous microarray analysis showed that H9N2 virus infection and inactivated viral particle inoculation increased the expression of interferon-inducible transmembrane protein 1 (IFITM1) in human umbilical vein endothelial cells (HUVECs). In present study, we deeply investigated the expression patterns of IFITM1 and IFITM1-mediated antiviral response induced by H9N2 virus infection and inactivated viral particle inoculation in HUVECs. Epithelial cells that are considered target cells of the influenza virus were selected as a reference control. First, we quantified the expression levels of IFITM1 in HUVECs induced by H9N2 virus infection or viral particle inoculation using quantitative real-time PCR and western blot. Second, we observed whether hemagglutinin or neuraminidase affected IFITM1 expression in HUVECs. Finally, we investigated the effect of induced-IFITM1 on the antiviral state in HUVECs by siRNA and activation plasmid transfection. Both H9N2 virus infection and viral particle inoculation increased the expression of IFITM1 without elevating the levels of interferon-ɑ/β in HUVECs. HA or NA protein binding alone is not sufficient to increase the levels of IFITM1 and interferon-ɑ/β in HUVECs. IFITM1 induced by viral particle inoculation significantly decreased the virus titers in culture supernatants of HUVECs. Our results showed that inactivated viral particle inoculation increased the expression of IFITM1 at mRNA and protein levels. Moreover, the induction of IFITM1 expression mediated the antiviral state in HUVECs.

In Vitro Analysis of Virus Particle Subpopulations in Candidate Live-Attenuated Influenza Vaccines Distinguishes Effective from Ineffective Vaccines▿

PubMed Central

Marcus, Philip I.; Ngunjiri, John M.; Sekellick, Margaret J.; Wang, Leyi; Lee, Chang-Won

2010-01-01

Two effective (vac+) and two ineffective (vac−) candidate live-attenuated influenza vaccines (LAIVs) derived from naturally selected genetically stable variants of A/TK/OR/71-delNS1[1-124] (H7N3) that differed only in the length and kind of amino acid residues at the C terminus of the nonstructural NS1 protein were analyzed for their content of particle subpopulations. These subpopulations included total physical particles (measured as hemagglutinating particles [HAPs]) with their subsumed biologically active particles of infectious virus (plaque-forming particles [PFPs]) and different classes of noninfectious virus, namely, interferon-inducing particles (IFPs), noninfectious cell-killing particles (niCKPs), and defective interfering particles (DIPs). The vac+ variants were distinguished from the vac− variants on the basis of their content of viral subpopulations by (i) the capacity to induce higher quantum yields of interferon (IFN), (ii) the generation of an unusual type of IFN-induction dose-response curve, (iii) the presence of IFPs that induce IFN more efficiently, (iv) reduced sensitivity to IFN action, and (v) elevated rates of PFP replication that resulted in larger plaques and higher PFP and HAP titers. These in vitro analyses provide a benchmark for the screening of candidate LAIVs and their potential as effective vaccines. Vaccine design may be improved by enhancement of attributes that are dominant in the effective (vac+) vaccines. PMID:20739541

In vitro analysis of virus particle subpopulations in candidate live-attenuated influenza vaccines distinguishes effective from ineffective vaccines.

PubMed

Marcus, Philip I; Ngunjiri, John M; Sekellick, Margaret J; Wang, Leyi; Lee, Chang-Won

2010-11-01

Two effective (vac+) and two ineffective (vac-) candidate live-attenuated influenza vaccines (LAIVs) derived from naturally selected genetically stable variants of A/TK/OR/71-delNS1[1-124] (H7N3) that differed only in the length and kind of amino acid residues at the C terminus of the nonstructural NS1 protein were analyzed for their content of particle subpopulations. These subpopulations included total physical particles (measured as hemagglutinating particles [HAPs]) with their subsumed biologically active particles of infectious virus (plaque-forming particles [PFPs]) and different classes of noninfectious virus, namely, interferon-inducing particles (IFPs), noninfectious cell-killing particles (niCKPs), and defective interfering particles (DIPs). The vac+ variants were distinguished from the vac- variants on the basis of their content of viral subpopulations by (i) the capacity to induce higher quantum yields of interferon (IFN), (ii) the generation of an unusual type of IFN-induction dose-response curve, (iii) the presence of IFPs that induce IFN more efficiently, (iv) reduced sensitivity to IFN action, and (v) elevated rates of PFP replication that resulted in larger plaques and higher PFP and HAP titers. These in vitro analyses provide a benchmark for the screening of candidate LAIVs and their potential as effective vaccines. Vaccine design may be improved by enhancement of attributes that are dominant in the effective (vac+) vaccines.

Rqc2p and 60S ribosomal subunits mediate mRNA-independent elongation of nascent chains

PubMed Central

Shen, Peter S.; Park, Joseph; Qin, Yidan; Li, Xueming; Parsawar, Krishna; Larson, Matthew H.; Cox, James; Cheng, Yifan; Lambowitz, Alan M.; Weissman, Jonathan S.; Brandman, Onn; Frost, Adam

2015-01-01

In Eukarya, stalled translation induces 40S dissociation and recruitment of the Ribosome Quality control Complex (RQC) to the 60S subunit, which mediates nascent chain degradation. Here, we report cryoEM structures revealing that the RQC components Rqc2p (YPL009C/Tae2) and Ltn1p (YMR247C/Rkr1) bind to the 60S at sites exposed after 40S dissociation, placing the Ltn1p RING domain near the exit channel and Rqc2p over the P-site tRNA. We further demonstrate that Rqc2p recruits alanine and threonine charged tRNA to the A-site and directs elongation of nascent chains independently of mRNA or 40S subunits. Our work uncovers an unexpected mechanism of protein synthesis in which a protein—not an mRNA—determines tRNA recruitment and the tagging of nascent chains with Carboxy-terminal Ala and Thr extensions (“CAT tails”). PMID:25554787

Alphavirus vector-based replicon particles expressing multivalent cross-protective Lassa virus glycoproteins

PubMed Central

Wang, Min; Jokinen, Jenny; Tretyakova, Irina; Pushko, Peter; Lukashevich, Igor S.

2018-01-01

Lassa virus (LASV) is the most prevalent rodent-borne arenavirus circulated in West Africa. With population at risk from Senegal to Nigeria, LASV causes Lassa fever and is responsible for thousands of deaths annually. High genetic diversity of LASV is one of the challenges for vaccine R&D. We developed multivalent virus-like particle vectors (VLPVs) derived from the human Venezuelan equine encephalitis TC-83 IND vaccine (VEEV) as the next generation of alphavirus-based bicistronic RNA replicon particles. The genes encoding VEEV structural proteins were replaced with LASV glycoproteins (GPC) from distantly related clades I and IV with individual 26S promoters. Bicistronic RNA replicons encoding wild-type LASV GPC (GPCwt) and C-terminally deleted, non-cleavable modified glycoprotein (ΔGPfib), were encapsidated into VLPV particles using VEEV capsid and glycoproteins provided in trans. In transduced cells, VLPVs induced simultaneous expression of LASV GPCwt and ΔGPfib from 26S alphavirus promoters. LASV ΔGPfib was predominantly expressed as trimers, accumulated in the endoplasmic reticulum, induced ER stress and apoptosis promoting antigen cross-priming. VLPV vaccines were immunogenic and protective in mice and upregulated CD11c+/CD8+ dendritic cells playing the major role in cross-presentation. Notably, VLPV vaccination resulted in induction of cross-reactive multifunctional T cell responses after stimulation of immune splenocytes with peptide cocktails derived from LASV from clades I-IV. Multivalent RNA replicon-based LASV vaccines can be applicable for first responders, international travelers visiting endemic areas, military and lab personnel. PMID:29287681

Intramuscular and intranasal immunization with an H7N9 influenza virus-like particle vaccine protects mice against lethal influenza virus challenge.

PubMed

Ren, Zhiguang; Zhao, Yongkun; Liu, Jing; Ji, Xianliang; Meng, Lingnan; Wang, Tiecheng; Sun, Weiyang; Zhang, Kun; Sang, Xiaoyu; Yu, Zhijun; Li, Yuanguo; Feng, Na; Wang, Hualei; Yang, Songtao; Yang, Zhengyan; Ma, Yuanfang; Gao, Yuwei; Xia, Xianzhu

2018-05-01

The H7N9 influenza virus epidemic has been associated with a high mortality rate in China. Therefore, to prevent the H7N9 virus from causing further damage, developing a safe and effective vaccine is necessary. In this study, a vaccine candidate consisting of virus-like particles (VLPs) based on H7N9 A/Shanghai/2/2013 and containing hemagglutinin (HA), neuraminidase (NA), and matrix protein (M1) was successfully produced using a baculovirus (BV) expression system. Immunization experiments showed that strong humoral and cellular immune responses could be induced by the developed VLPs when administered via either the intramuscular (IM) or intranasal (IN) immunization routes. Notably, VLPs administered via both immunization routes provided 100% protection against lethal infection caused by the H7N9 virus. The IN immunization with 40μg of H7N9 VLPs induced strong lung IgA and lung tissue resident memory (TRM) cell-mediated local immune responses. These results provide evidence for the development of an effective preventive vaccine against the H7N9 virus based on VLPs administered through both the IM and IN immunization routes. Copyright © 2017. Published by Elsevier B.V.

Removal of virus to protozoan sized particles in point-of-use ceramic water filters.

PubMed

Bielefeldt, Angela R; Kowalski, Kate; Schilling, Cherylynn; Schreier, Simon; Kohler, Amanda; Scott Summers, R

2010-03-01

The particle removal performance of point-of-use ceramic water filters (CWFs) was characterized in the size range of 0.02-100 microm using carboxylate-coated polystyrene fluorescent microspheres, natural particles and clay. Particles were spiked into dechlorinated tap water, and three successive water batches treated in each of six different CWFs. Particle removal generally increased with increasing size. The removal of virus-sized 0.02 and 0.1 microm spheres were highly variable between the six filters, ranging from 63 to 99.6%. For the 0.5 microm spheres removal was less variable and in the range of 95.1-99.6%, while for the 1, 2, 4.5, and 10 microm spheres removal was >99.6%. Recoating four of the CWFs with colloidal silver solution improved removal of the 0.02 microm spheres, but had no significant effects on the other particle sizes. Log removals of 1.8-3.2 were found for natural turbidity and spiked kaolin clay particles; however, particles as large as 95 microm were detected in filtered water. Copyright 2009 Elsevier Ltd. All rights reserved.

Tests in mice of a dengue vaccine candidate made of chimeric Junin virus-like particles and conserved dengue virus envelope sequences.

PubMed

Mareze, Vania Aparecida; Borio, Cristina Silvia; Bilen, Marcos F; Fleith, Renata; Mirazo, Santiago; Mansur, Daniel Santos; Arbiza, Juan; Lozano, Mario Enrique; Bruña-Romero, Oscar

2016-01-01

Two new vaccine candidates against dengue virus (DENV) infection were generated by fusing the coding sequences of the self-budding Z protein from Junin virus (Z-JUNV) to those of two cryptic peptides (Z/DENV-P1 and Z/DENV-P2) conserved on the envelope protein of all serotypes of DENV. The capacity of these chimeras to generate virus-like particles (VLPs) and to induce virus-neutralizing antibodies in mice was determined. First, recombinant proteins that displayed reactivity with a Z-JUNV-specific serum by immunofluorescence were detected in HEK-293 cells transfected with each of the two plasmids and VLP formation was also observed by transmission electron microscopy. Next, we determined the presence of antibodies against the envelope peptides of DENV in the sera of immunized C57BL/6 mice. Results showed that those animals that received Z/DENV-P2 DNA coding sequences followed by a boost with DENV-P2 synthetic peptides elicited significant specific antibody titers (≥6.400). Finally, DENV plaque-reduction neutralization tests (PRNT) were performed. Although no significant protective effect was observed when using sera of Z/DENV-P1-immunized animals, antibodies raised against vaccine candidate Z/DENV-P2 (diluted 1:320) were able to reduce in over 50 % the number of viral plaques generated by infectious DENV particles. This reduction was comparable to that of the 4G2 DENV-specific monoclonal cross-reactive (all serotypes) neutralizing antibody. We conclude that Z-JUNV-VLP is a valid carrier to induce antibody-mediated immune responses in mice and that Z/DENV-P2 is not only immunogenic but also protective in vitro against infection of cells with DENV, deserving further studies. On the other side, DENV's fusion peptide-derived chimera Z/DENV-P1 did not display similar protective properties.

Homologous and Heterologous Protection of Nonhuman Primates by Ebola and Sudan Virus-Like Particles

PubMed Central

Warfield, Kelly L.; Dye, John M.; Wells, Jay B.; Unfer, Robert C.; Holtsberg, Frederick W.; Shulenin, Sergey; Vu, Hong; Swenson, Dana L.; Bavari, Sina; Aman, M. Javad

2015-01-01

Filoviruses cause hemorrhagic fever resulting in significant morbidity and mortality in humans. Several vaccine platforms that include multiple virus-vectored approaches and virus-like particles (VLPs) have shown efficacy in nonhuman primates. Previous studies have shown protection of cynomolgus macaques against homologous infection for Ebola virus (EBOV) and Marburg virus (MARV) following a three-dose vaccine regimen of EBOV or MARV VLPs, as well as heterologous protection against Ravn Virus (RAVV) following vaccination with MARV VLPs. The objectives of the current studies were to determine the minimum number of vaccine doses required for protection (using EBOV as the test system) and then demonstrate protection against Sudan virus (SUDV) and Taï Forest virus (TAFV). Using the EBOV nonhuman primate model, we show that one or two doses of VLP vaccine can confer protection from lethal infection. VLPs containing the SUDV glycoprotein, nucleoprotein and VP40 matrix protein provide complete protection against lethal SUDV infection in macaques. Finally, we demonstrate protective efficacy mediated by EBOV, but not SUDV, VLPs against TAFV; this is the first demonstration of complete cross-filovirus protection using a single component heterologous vaccine within the Ebolavirus genus. Along with our previous results, this observation provides strong evidence that it will be possible to develop and administer a broad-spectrum VLP-based vaccine that will protect against multiple filoviruses by combining only three EBOV, SUDV and MARV components. PMID:25793502

Conformational landscape of a virus by single-particle X-ray scattering

DOE PAGES

Hosseinizadeh, Ahmad; Mashayekhi, Ghoncheh; Copperman, Jeremy; ...

2017-08-14

Using a manifold-based analysis of experimental diffraction snapshots from an X-ray free electron laser, we determine the three-dimensional structure and conformational landscape of the PR772 virus to a detector-limited resolution of 9 nm. Our results indicate that a single conformational coordinate controls reorganization of the genome, growth of a tubular structure from a portal vertex and release of the genome. Furthermore, these results demonstrate that single-particle X-ray scattering has the potential to shed light on key biological processes.

Co-expression of HIV-1 virus-like particles and granulocyte-macrophage colony stimulating factor by GEO-D03 DNA vaccine.

PubMed

Hellerstein, Michael; Xu, Yongxian; Marino, Tracie; Lu, Shan; Yi, Hong; Wright, Elizabeth R; Robinson, Harriet L

2012-11-01

Here, we report on GEO-D03, a DNA vaccine that co-expresses non-infectious HIV-1 virus-like particles (VLPs) and the human cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF). The virus-like particles display the native gp160 form of the HIV-1 Envelope glycoprotein (Env) and are designed to elicit antibody against the natural form of Env on virus and virus-infected cells. The DNA-expressed HIV Gag, Pol and Env proteins also have the potential to elicit virus-specific CD4 and CD8 T cells. The purpose of the co-expressed GM-CSF is to target a cytokine that recruits, expands and differentiates macrophages and dendritic cells to the site of VLP expression. The GEO-D03 DNA vaccine is currently entered into human trials as a prime for a recombinant modified vaccinia Ankara (MVA) boost. In preclinical studies in macaques using an SIV prototype vaccine, this vaccination regimen elicited both anti-viral T cells and antibody, and provided 70% protection against acquisition during 12 weekly rectal exposures with a heterologous SIV. Higher avidity of the Env-specific Ab for the native form of the Env in the challenge virus correlated with lower likelihood of SIV infection.

Efficient Sensing of Infected Cells in Absence of Virus Particles by Blasmacytoid Dendritic Cells Is Blocked by the Viral Ribonuclease Erns

PubMed Central

Python, Sylvie; Gerber, Markus; Suter, Rolf; Ruggli, Nicolas; Summerfield, Artur

2013-01-01

Plasmacytoid dendritic cells (pDC) have been shown to efficiently sense HCV- or HIV-infected cells, using a virion-free pathway. Here, we demonstrate for classical swine fever virus, a member of the Flaviviridae, that this process is much more efficient in terms of interferon-alpha induction when compared to direct stimulation by virus particles. By employment of virus replicon particles or infectious RNA which can replicate but not form de novo virions, we exclude a transfer of virus from the donor cell to the pDC. pDC activation by infected cells was mediated by a contact-dependent RNA transfer to pDC, which was sensitive to a TLR7 inhibitor. This was inhibited by drugs affecting the cytoskeleton and membrane cholesterol. We further demonstrate that a unique viral protein with ribonuclease activity, the viral Erns protein of pestiviruses, efficiently prevented this process. This required intact ribonuclease function in intracellular compartments. We propose that this pathway of activation could be of particular importance for viruses which tend to be mostly cell-associated, cause persistent infection, and are non-cytopathogenic. PMID:23785283

Structure and organization of paramyxovirus particles.

PubMed

Cox, Robert M; Plemper, Richard K

2017-06-01

The paramyxovirus family comprises major human and animal pathogens such as measles virus (MeV), mumps virus (MuV), the parainfluenzaviruses, Newcastle disease virus (NDV), and the highly pathogenic zoonotic hendra (HeV) and nipah (NiV) viruses. Paramyxovirus particles are pleomorphic, with a lipid envelope, nonsegmented RNA genomes of negative polarity, and densely packed glycoproteins on the virion surface. A number of crystal structures of different paramyxovirus proteins and protein fragments were solved, but the available information concerning overall virion organization remains limited. However, recent studies have reported cryo-electron tomography-based reconstructions of Sendai virus (SeV), MeV, NDV, and human parainfluenza virus type 3 (HPIV3) particles and a surface assessment of NiV-derived virus-like particles (VLPs), which have yielded innovative hypotheses concerning paramyxovirus particle assembly, budding, and organization. Following a summary of the current insight into paramyxovirus virion morphology, this review will focus on discussing the implications of these particle reconstructions on the present models of paramyxovirus assembly and infection. Copyright © 2017 Elsevier B.V. All rights reserved.

Virus-Like Particle, Liposome, and Polymeric Particle-Based Vaccines against HIV-1

PubMed Central

Gao, Yong; Wijewardhana, Chanuka; Mann, Jamie F. S.

2018-01-01

It is acknowledged that vaccines remain the best hope for eliminating the HIV-1 epidemic. However, the failure to produce effective vaccine immunogens and the inability of conventional delivery strategies to elicit the desired immune responses remains a central theme and has ultimately led to a significant roadblock in HIV vaccine development. Consequently, significant efforts have been applied to generate novel vaccine antigens and delivery agents, which mimic viral structures for optimal immune induction. Here, we review the latest developments that have occurred in the nanoparticle vaccine field, with special emphasis on strategies that are being utilized to attain highly immunogenic, systemic, and mucosal anti-HIV humoral and cellular immune responses. This includes the design of novel immunogens, the central role of antigen-presenting cells, delivery routes, and biodistribution of nanoparticles to lymph nodes. In particular, we will focus on virus-like-particle formulations and their preclinical uses within the HIV prophylactic vaccine setting. PMID:29541072

An Annotation Agnostic Algorithm for Detecting Nascent RNA Transcripts in GRO-Seq.

PubMed

Azofeifa, Joseph G; Allen, Mary A; Lladser, Manuel E; Dowell, Robin D

2017-01-01

We present a fast and simple algorithm to detect nascent RNA transcription in global nuclear run-on sequencing (GRO-seq). GRO-seq is a relatively new protocol that captures nascent transcripts from actively engaged polymerase, providing a direct read-out on bona fide transcription. Most traditional assays, such as RNA-seq, measure steady state RNA levels which are affected by transcription, post-transcriptional processing, and RNA stability. GRO-seq data, however, presents unique analysis challenges that are only beginning to be addressed. Here, we describe a new algorithm, Fast Read Stitcher (FStitch), that takes advantage of two popular machine-learning techniques, hidden Markov models and logistic regression, to classify which regions of the genome are transcribed. Given a small user-defined training set, our algorithm is accurate, robust to varying read depth, annotation agnostic, and fast. Analysis of GRO-seq data without a priori need for annotation uncovers surprising new insights into several aspects of the transcription process.

Characterization of Protection Afforded by a Bivalent Virus-Like Particle Vaccine against Bluetongue Virus Serotypes 1 and 4 in Sheep

PubMed Central

Pérez de Diego, Ana Cristina; Athmaram, Thimmasandra N.; Stewart, Meredith; Rodríguez-Sánchez, Belén; Sánchez-Vizcaíno, José Manuel; Noad, Robert; Roy, Polly

2011-01-01

Background Bluetongue virus (BTV) is an economically important, arthropod borne, emerging pathogen in Europe, causing disease mainly in sheep and cattle. Routine vaccination for bluetongue would require the ability to distinguish between vaccinated and infected individuals (DIVA). Current vaccines are effective but are not DIVA. Virus-like particles (VLPs) are highly immunogenic structural mimics of virus particles, that only contain a subset of the proteins present in a natural infection. VLPs therefore offer the potential for the development of DIVA compatible bluetongue vaccines. Methodology/Principal Findings Merino sheep were vaccinated with either monovalent BTV-1 VLPs or a bivalent mixture of BTV-1 VLPs and BTV-4 VLPs, and challenged with virulent BTV-1 or BTV-4. Animals were monitored for clinical signs, antibody responses, and viral RNA. 19/20 animals vaccinated with BTV-1 VLPs either alone or in combination with BTV-4 VLPs developed neutralizing antibodies to BTV-1, and group specific antibodies to BTV VP7. The one animal that showed no detectable neutralizing antibodies, or group specific antibodies, had detectable viral RNA following challenge but did not display any clinical signs on challenge with virulent BTV-1. In contrast, all control animals' demonstrated classical clinical signs for bluetongue on challenge with the same virus. Six animals were vaccinated with bivalent vaccine and challenged with virulent BTV-4, two of these animals had detectable viral levels of viral RNA, and one of these showed clinical signs consistent with BTV infection and died. Conclusions There is good evidence that BTV-1 VLPs delivered as monovalent or bivalent immunogen protect from bluetongue disease on challenge with virulent BTV-1. However, it is possible that there is some interference in protective response for BTV-4 in the bivalent BTV-1 and BTV-4 VLP vaccine. This raises the question of whether all combinations of bivalent BTV vaccines are possible, or if

Protective immunity against nervous necrosis virus in convict grouper Epinephelus septemfasciatus following vaccination with virus-like particles produced in yeast Saccharomyces cerevisiae.

PubMed

Wi, Ga Ram; Hwang, Jee Youn; Kwon, Mun-Gyeong; Kim, Hyoung Jin; Kang, Hyun Ah; Kim, Hong-Jin

2015-05-15

Infection with nervous necrosis virus (NNV) causes viral nervous necrosis, which inflicts serious economic losses in marine fish cultivation. Virus-like particles (VLPs) are protein complexes consisting of recombinant virus capsid proteins, whose shapes are similar to native virions. VLPs are considered a novel vaccine platform because they are not infectious and have the ability to induce neutralizing antibodies efficiently. However, there have been few studies of protective immune responses employing virus challenge following immunization with NNV VLPs, and this is important for evaluating the utility of the vaccine. In the present study, we produced red-spotted grouper (Epinephelus akaara) NNV (RGNNV) VLPs in Saccharomyces cerevisiae and investigated protective immune responses in convict grouper (Epinephelus septemfasciatus) following intraperitoneal injection and oral immunization with the RGNNV VLPs. The parenterally administered VLPs elicited neutralizing antibody with high efficacy, and provided the fish with full protection against RGNNV challenge: 100% of the immunized fish survived compared with only 37% of the control fish receiving phosphate-buffered saline. RGNNV VLPs administered orally provoked neutralizing antibody systemically and conferred protective immunity against virus challenge: however only 57% of the fish survived. Our results demonstrate that RGNNV VLP produced in yeast has great potential as vaccine in fish. Copyright © 2015 Elsevier B.V. All rights reserved.

Visualizing Ebolavirus Particles Using Single-Particle Interferometric Reflectance Imaging Sensor (SP-IRIS).

PubMed

Carter, Erik P; Seymour, Elif Ç; Scherr, Steven M; Daaboul, George G; Freedman, David S; Selim Ünlü, M; Connor, John H

2017-01-01

This chapter describes an approach for the label-free imaging and quantification of intact Ebola virus (EBOV) and EBOV viruslike particles (VLPs) using a light microscopy technique. In this technique, individual virus particles are captured onto a silicon chip that has been printed with spots of virus-specific capture antibodies. These captured virions are then detected using an optical approach called interference reflectance imaging. This approach allows for the detection of each virus particle that is captured on an antibody spot and can resolve the filamentous structure of EBOV VLPs without the need for electron microscopy. Capture of VLPs and virions can be done from a variety of sample types ranging from tissue culture medium to blood. The technique also allows automated quantitative analysis of the number of virions captured. This can be used to identify the virus concentration in an unknown sample. In addition, this technique offers the opportunity to easily image virions captured from native solutions without the need for additional labeling approaches while offering a means of assessing the range of particle sizes and morphologies in a quantitative manner.

Atomic force microscopy investigation of Mason-Pfizer monkey virus and human immunodeficiency virus type 1 reassembled particles

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

Kuznetsov, Yu. G.; Ulbrich, P.; Institute of Molecular Genetics, Czech Academy of Sciences, 166 10 Prague

2007-04-10

Particles of {delta}ProCANC, a fusion of capsid (Canada) and nucleocapsid (NC) protein of Mason-Pfizer monkey virus (M-PMV), which lacks the amino terminal proline, were reassembled in vitro and visualized by atomic force microscopy (AFM). The particles, of 83-84 nm diameter, exhibited ordered domains based on trigonal arrays of prominent rings with center to center distances of 8.7 nm. Imperfect closure of the lattice on the spherical surface was affected by formation of discontinuities. The lattice is consistent only with plane group p3 where one molecule is shared between contiguous rings. There are no pentameric clusters nor evidence that the particlesmore » are icosahedral. Tubular structures were also reassembled, in vitro, from two HIV fusion proteins, {delta}ProCANC and CANC. The tubes were uniform in diameter, 40 nm, but varied in length to a maximum of 600 nm. They exhibited left handed helical symmetry based on a p6 hexagonal net. The organization of HIV fusion proteins in the tubes is significantly different than for the protein units in the particles of M-PMV {delta}ProCANC.« less

78 FR 18359 - Prospective Grant of Exclusive License: Papilloma Pseudovirus and Virus-Like Particles as a...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-03-26

... DEPARTMENT OF HEALTH AND HUMAN SERVICES National Institutes of Health Prospective Grant of Exclusive License: Papilloma Pseudovirus and Virus-Like Particles as a Delivery System for Human Cancer Therapeutics and Diagnostics AGENCY: National Institutes of Health, Public Health Service, HHS. ACTION: Notice...

Live attenuated measles vaccine expressing HIV-1 Gag virus like particles covered with gp160DELTAV1V2 is strongly immunogenic

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

Guerbois, Mathilde; Moris, Arnaud; Combredet, Chantal

Although a live attenuated HIV vaccine is not currently considered for safety reasons, a strategy inducing both T cells and neutralizing antibodies to native assembled HIV-1 particles expressed by a replicating virus might mimic the advantageous characteristics of live attenuated vaccine. To this aim, we generated a live attenuated recombinant measles vaccine expressing HIV-1 Gag virus-like particles (VLPs) covered with gp160DELTAV1V2 Env protein. The measles-HIV virus replicated efficiently in cell culture and induced the intense budding of HIV particles covered with Env. In mice sensitive to MV infection, this recombinant vaccine stimulated high levels of cellular and humoral immunity tomore » both MV and HIV with neutralizing activity. The measles-HIV virus infected human professional antigen-presenting cells, such as dendritic cells and B cells, and induced efficient presentation of HIV-1 epitopes and subsequent activation of human HIV-1 Gag-specific T cell clones. This candidate vaccine will be next tested in non-human primates. As a pediatric vaccine, it might protect children and adolescents simultaneously from measles and HIV.« less

Plant-derived virus-like particles as vaccines

PubMed Central

Chen, Qiang; Lai, Huafang

2013-01-01

Virus-like particles (VLPs) are self-assembled structures derived from viral antigens that mimic the native architecture of viruses but lack the viral genome. VLPs have emerged as a premier vaccine platform due to their advantages in safety, immunogenicity, and manufacturing. The particulate nature and high-density presentation of viral structure proteins on their surface also render VLPs as attractive carriers for displaying foreign epitopes. Consequently, several VLP-based vaccines have been licensed for human use and achieved significant clinical and economical success. The major challenge, however, is to develop novel production platforms that can deliver VLP-based vaccines while significantly reducing production times and costs. Therefore, this review focuses on the essential role of plants as a novel, speedy and economical production platform for VLP-based vaccines. The advantages of plant expression systems are discussed in light of their distinctive posttranslational modifications, cost-effectiveness, production speed, and scalability. Recent achievements in the expression and assembly of VLPs and their chimeric derivatives in plant systems as well as their immunogenicity in animal models are presented. Results of human clinical trials demonstrating the safety and efficacy of plant-derived VLPs are also detailed. Moreover, the promising implications of the recent creation of “humanized” glycosylation plant lines as well as the very recent approval of the first plant-made biologics by the U. S. Food and Drug Administration (FDA) for plant production and commercialization of VLP-based vaccines are discussed. It is speculated that the combined potential of plant expression systems and VLP technology will lead to the emergence of successful vaccines and novel applications of VLPs in the near future. PMID:22995837

Chimeric SV40 virus-like particles induce specific cytotoxicity and protective immunity against influenza A virus without the need of adjuvants

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

Kawano, Masaaki; Morikawa, Katsuma; Suda, Tatsuya

Virus-like particles (VLPs) are a promising vaccine platform due to the safety and efficiency. However, it is still unclear whether polyomavirus-based VLPs are useful for this purpose. Here, we attempted to evaluate the potential of polyomavirus VLPs for the antiviral vaccine using simian virus 40 (SV40). We constructed chimeric SV40-VLPs carrying an HLA-A{sup ⁎}02:01-restricted, cytotoxic T lymphocyte (CTL) epitope derived from influenza A virus. HLA-A{sup ⁎}02:01-transgenic mice were then immunized with the chimeric SV40-VLPs. The chimeric SV40-VLPs effectively induced influenza-specific CTLs and heterosubtypic protection against influenza A viruses without the need of adjuvants. Because DNase I treatment of the chimericmore » SV40-VLPs did not disrupt CTL induction, the intrinsic adjuvant property may not result from DNA contaminants in the VLP preparation. In addition, immunization with the chimeric SV40-VLPs generated long-lasting memory CTLs. We here propose that the chimeric SV40-VLPs harboring an epitope may be a promising CTL-based vaccine platform with self-adjuvant properties. - Highlights: • We constructed chimeric SV40-VLPs carrying an influenza virus-derived CTL epitope. • Chimeric SV40-VLPs induce influenza-specific CTLs in mice without adjuvants. • Chimeric SV40-VLPs induce heterosubtypic protection against influenza A viruses. • Chimeric SV40-VLPs induce long-lasting memory CTLs. • Chimeric SV40-VLPs is a promising vaccine platform with self-adjuvant properties.« less

Supplemented vaccination with tandem repeat M2e virus-like particles enhances protection against homologous and heterologous HPAI H5 viruses in chickens.

PubMed

Song, Byung-Min; Kang, Hyun-Mi; Lee, Eun-Kyoung; Jung, Suk Chan; Kim, Min-Chul; Lee, Yu-Na; Kang, Sang-Moo; Lee, Youn-Jeong

2016-01-27

Highly pathogenic avian influenza (HPAI) H5 viruses derived from A/Goose/Guangdong/1/96 have been continuously circulating globally, severely affecting the public health and poultry industries. The matrix 2 protein ectodomain (M2e) is considered a promising candidate for a universal cross-protective influenza vaccine that provides more effective control over HPAI H5 viruses harboring variant hemagglutinin (HA)-antigens. Here, we evaluated the protective efficacy of a tandem repeat construct of heterologous M2e presented on virus-like particles (M2e5x VLPs) either alone or as a supplement against HPAI H5 viruses in a chicken model. Chickens immunized with M2e5x VLPs alone induced M2e-specific antibodies but were not protected against HPAI H5. The homo- and cross-protective efficacy of M2e5x VLP-supplemented vaccination of chickens was also examined. Importantly, supplementation with M2e5x VLPs induced significantly higher levels of antibodies specific for M2e and different viruses as well as provided improved protection against homologous and heterologous HPAI H5 viruses. Considering the limited efficacy of inactivated vaccines, supplement vaccination with M2e5x VLPs may be an effective measure for preventing outbreaks of HPAI viruses that have the ability to constantly change their antigenic properties in poultry. Copyright © 2015 Elsevier Ltd. All rights reserved.

Proteins of Vasicular Stomatitis Virus

PubMed Central

Kang, C. Y.; Prevec, L.

1969-01-01

Infection of L cells with vesicular stomatitis virus results in the release, into the cell-free fluid, of four antigenic components separable by rate zonal centrifugation on sucrose gradients. The largest antigens are the infectious (B) particle and a shorter noninfectious, autointerfering (T) particle. The two small antigens are characterized by sedimentation coefficients of approximately 20S and 6S. Treatment of purified B or T particles with sodium deoxycholate results in the release from the particle of a nucleoprotein core which can be purified on sucrose gradient and which has a sedimentation coefficient characteristic of the virus from which it arose. Utilizing purified antigens labeled with 14C-amino acids during growth, we examined the protein constituents of each antigen by acrylamide-gel electrophoresis. The proteins of B and T particles are identical, each containing one minor (virus protein 1) and three major (virus proteins 2, 3, and 4) proteins, numbered in order of increasing mobility. Virus protein 3 originates from the nucleoprotein core, whereas proteins 2 and 4 come from the coat. The origin of virus protein 1 is not known. The 20S antigen contains a single protein equivalent to virus protein 3, whereas the 6S antigen shows a single protein which is similar to, but probably distinct from, virus protein 2. PMID:4306195

A host YB-1 ribonucleoprotein complex is hijacked by hepatitis C virus for the control of NS3-dependent particle production.

PubMed

Chatel-Chaix, Laurent; Germain, Marie-Anne; Motorina, Alena; Bonneil, Éric; Thibault, Pierre; Baril, Martin; Lamarre, Daniel

2013-11-01

Hepatitis C virus (HCV) orchestrates the different stages of its life cycle in time and space through the sequential participation of HCV proteins and cellular machineries; hence, these represent tractable molecular host targets for HCV elimination by combination therapies. We recently identified multifunctional Y-box-binding protein 1 (YB-1 or YBX1) as an interacting partner of NS3/4A protein and HCV genomic RNA that negatively regulates the equilibrium between viral translation/replication and particle production. To identify novel host factors that regulate the production of infectious particles, we elucidated the YB-1 interactome in human hepatoma cells by a quantitative mass spectrometry approach. We identified 71 YB-1-associated proteins that included previously reported HCV regulators DDX3, heterogeneous nuclear RNP A1, and ILF2. Of the potential YB-1 interactors, 26 proteins significantly modulated HCV replication in a gene-silencing screening. Following extensive interaction and functional validation, we identified three YB-1 partners, C1QBP, LARP-1, and IGF2BP2, that redistribute to the surface of core-containing lipid droplets in HCV JFH-1-expressing cells, similarly to YB-1 and DDX6. Importantly, knockdown of these proteins stimulated the release and/or egress of HCV particles without affecting virus assembly, suggesting a functional YB-1 protein complex that negatively regulates virus production. Furthermore, a JFH-1 strain with the NS3 Q221L mutation, which promotes virus production, was less sensitive to this negative regulation, suggesting that this HCV-specific YB-1 protein complex modulates an NS3-dependent step in virus production. Overall, our data support a model in which HCV hijacks host cell machinery containing numerous RNA-binding proteins to control the equilibrium between viral RNA replication and NS3-dependent late steps in particle production.

A Host YB-1 Ribonucleoprotein Complex Is Hijacked by Hepatitis C Virus for the Control of NS3-Dependent Particle Production

PubMed Central

Chatel-Chaix, Laurent; Germain, Marie-Anne; Motorina, Alena; Bonneil, Éric; Thibault, Pierre; Baril, Martin

2013-01-01

Hepatitis C virus (HCV) orchestrates the different stages of its life cycle in time and space through the sequential participation of HCV proteins and cellular machineries; hence, these represent tractable molecular host targets for HCV elimination by combination therapies. We recently identified multifunctional Y-box-binding protein 1 (YB-1 or YBX1) as an interacting partner of NS3/4A protein and HCV genomic RNA that negatively regulates the equilibrium between viral translation/replication and particle production. To identify novel host factors that regulate the production of infectious particles, we elucidated the YB-1 interactome in human hepatoma cells by a quantitative mass spectrometry approach. We identified 71 YB-1-associated proteins that included previously reported HCV regulators DDX3, heterogeneous nuclear RNP A1, and ILF2. Of the potential YB-1 interactors, 26 proteins significantly modulated HCV replication in a gene-silencing screening. Following extensive interaction and functional validation, we identified three YB-1 partners, C1QBP, LARP-1, and IGF2BP2, that redistribute to the surface of core-containing lipid droplets in HCV JFH-1-expressing cells, similarly to YB-1 and DDX6. Importantly, knockdown of these proteins stimulated the release and/or egress of HCV particles without affecting virus assembly, suggesting a functional YB-1 protein complex that negatively regulates virus production. Furthermore, a JFH-1 strain with the NS3 Q221L mutation, which promotes virus production, was less sensitive to this negative regulation, suggesting that this HCV-specific YB-1 protein complex modulates an NS3-dependent step in virus production. Overall, our data support a model in which HCV hijacks host cell machinery containing numerous RNA-binding proteins to control the equilibrium between viral RNA replication and NS3-dependent late steps in particle production. PMID:23986595

Development of recombinant Yarrowia lipolytica producing virus-like particles of a fish nervous necrosis virus.

PubMed

Luu, Van-Trinh; Moon, Hye Yun; Hwang, Jee Youn; Kang, Bo-Kyu; Kang, Hyun Ah

2017-08-01

Nervous necrosis virus (NNV) causes viral encephalopathy and retinopathy, a devastating disease of many species of cultured marine fish worldwide. In this study, we used the dimorphic non-pathogenic yeast Yarrowia lipolytica as a host to express the capsid protein of red-spotted grouper nervous necrosis virus (RGNNV-CP) and evaluated its potential as a platform for vaccine production. An initial attempt was made to express the codon-optimized synthetic genes encoding intact and N-terminal truncated forms of RGNNV-CP under the strong constitutive TEF1 promoter using autonomously replicating sequence (ARS)-based vectors. The full-length recombinant capsid proteins expressed in Y. lipolytica were detected not only as monomers and but also as trimers, which is a basic unit for formation of NNV virus-like particles (VLPs). Oral immunization of mice with whole recombinant Y. lipolytica harboring the ARS-based plasmids was shown to efficiently induce the formation of IgG against RGNNV-CP. To increase the number of integrated copies of the RGNNV-CP expression cassette, a set of 26S ribosomal DNA-based multiple integrative vectors was constructed in combination with a series of defective Ylura3 with truncated promoters as selection markers, resulting in integrants harboring up to eight copies of the RGNNV-CP cassette. Sucrose gradient centrifugation and transmission electron microscopy of this high-copy integrant were carried out to confirm the expression of RGNNV-CPs as VLPs. This is the first report on efficient expression of viral capsid proteins as VLPs in Y. lipolytica, demonstrating high potential for the Y. lipolytica expression system as a platform for recombinant vaccine production based on VLPs.

Guiding plant virus particles to integrin-displaying cells

NASA Astrophysics Data System (ADS)

Hovlid, Marisa L.; Steinmetz, Nicole F.; Laufer, Burkhardt; Lau, Jolene L.; Kuzelka, Jane; Wang, Qian; Hyypiä, Timo; Nemerow, Glen R.; Kessler, Horst; Manchester, Marianne; Finn, M. G.

2012-05-01

Viral nanoparticles (VNPs) are structurally regular, highly stable, tunable nanomaterials that can be conveniently produced in high yields. Unmodified VNPs from plants and bacteria generally do not show tissue specificity or high selectivity in binding to or entry into mammalian cells. They are, however, malleable by both genetic and chemical means, making them useful scaffolds for the display of large numbers of cell- and tissue-targeting ligands, imaging moieties, and/or therapeutic agents in a well-defined manner. Capitalizing on this attribute, we modified the genetic sequence of the Cowpea mosaic virus (CPMV) coat protein to display an RGD oligopeptide sequence derived from human adenovirus type 2 (HAdV-2). Concurrently, wild-type CPMV was modified via NHS acylation and Cu(i)-catalyzed azide-alkyne cycloaddition (CuAAC) chemistry to attach an integrin-binding cyclic RGD peptide. Both types of particles showed strong and selective affinity for several different cancer cell lines that express RGD-binding integrin receptors.Viral nanoparticles (VNPs) are structurally regular, highly stable, tunable nanomaterials that can be conveniently produced in high yields. Unmodified VNPs from plants and bacteria generally do not show tissue specificity or high selectivity in binding to or entry into mammalian cells. They are, however, malleable by both genetic and chemical means, making them useful scaffolds for the display of large numbers of cell- and tissue-targeting ligands, imaging moieties, and/or therapeutic agents in a well-defined manner. Capitalizing on this attribute, we modified the genetic sequence of the Cowpea mosaic virus (CPMV) coat protein to display an RGD oligopeptide sequence derived from human adenovirus type 2 (HAdV-2). Concurrently, wild-type CPMV was modified via NHS acylation and Cu(i)-catalyzed azide-alkyne cycloaddition (CuAAC) chemistry to attach an integrin-binding cyclic RGD peptide. Both types of particles showed strong and selective affinity

Viral fusion efficacy of specific H3N2 influenza virus reassortant combinations at single-particle level

PubMed Central

Hsu, Hung-Lun; Millet, Jean K.; Costello, Deirdre A.; Whittaker, Gary R.; Daniel, Susan

2016-01-01

Virus pseudotyping is a useful and safe technique for studying entry of emerging strains of influenza virus. However, few studies have compared different reassortant combinations in pseudoparticle systems, or compared entry kinetics of native viruses and their pseudotyped analogs. Here, vesicular stomatitis virus (VSV)-based pseudovirions displaying distinct influenza virus envelope proteins were tested for fusion activity. We produced VSV pseudotypes containing the prototypical X-31 (H3) HA, either alone or with strain-matched or mismatched N2 NAs. We performed single-particle fusion assays using total internal reflection fluorescence microscopy to compare hemifusion kinetics among these pairings. Results illustrate that matching pseudoparticles behaved very similarly to native virus. Pseudoparticles harboring mismatched HA-NA pairings fuse at significantly slower rates than native virus, and NA-lacking pseudoparticles exhibiting the slowest fusion rates. Relative viral membrane HA density of matching pseudoparticles was higher than in mismatching or NA-lacking pseudoparticles. An equivalent trend of HA expression level on cell membranes of HA/NA co-transfected cells was observed and intracellular trafficking of HA was affected by NA co-expression. Overall, we show that specific influenza HA-NA combinations can profoundly affect the critical role played by HA during entry, which may factor into viral fitness and the emergence of new pandemic influenza viruses. PMID:27752100

Viral fusion efficacy of specific H3N2 influenza virus reassortant combinations at single-particle level

NASA Astrophysics Data System (ADS)

Hsu, Hung-Lun; Millet, Jean K.; Costello, Deirdre A.; Whittaker, Gary R.; Daniel, Susan

2016-10-01

Virus pseudotyping is a useful and safe technique for studying entry of emerging strains of influenza virus. However, few studies have compared different reassortant combinations in pseudoparticle systems, or compared entry kinetics of native viruses and their pseudotyped analogs. Here, vesicular stomatitis virus (VSV)-based pseudovirions displaying distinct influenza virus envelope proteins were tested for fusion activity. We produced VSV pseudotypes containing the prototypical X-31 (H3) HA, either alone or with strain-matched or mismatched N2 NAs. We performed single-particle fusion assays using total internal reflection fluorescence microscopy to compare hemifusion kinetics among these pairings. Results illustrate that matching pseudoparticles behaved very similarly to native virus. Pseudoparticles harboring mismatched HA-NA pairings fuse at significantly slower rates than native virus, and NA-lacking pseudoparticles exhibiting the slowest fusion rates. Relative viral membrane HA density of matching pseudoparticles was higher than in mismatching or NA-lacking pseudoparticles. An equivalent trend of HA expression level on cell membranes of HA/NA co-transfected cells was observed and intracellular trafficking of HA was affected by NA co-expression. Overall, we show that specific influenza HA-NA combinations can profoundly affect the critical role played by HA during entry, which may factor into viral fitness and the emergence of new pandemic influenza viruses.

Mechanisms of nascent fiber formation during avian skeletal muscle hypertrophy

NASA Technical Reports Server (NTRS)

McCormick, K. M.; Schultz, E.

1992-01-01

This study examined two putative mechanisms of new fiber formation in postnatal skeletal muscle, namely longitudinal fragmentation of existing fibers and de novo formation. The relative contributions of these two mechanisms to fiber formation in hypertrophying anterior latissimus dorsi (ALD) muscle were assessed by quantitative analysis of their nuclear populations. Muscle hypertrophy was induced by wing-weighting for 1 week. All nuclei formed during the weighting period were labeled by continuous infusion of 5-bromo-2'-deoxyuridine (BrdU), a thymidine analog, and embryonic-like fibers were identified using an antibody to ventricular-like embryonic (V-EMB) myosin. The number of BrdU-labeled and unlabeled nuclei in V-EMB-positive fibers were counted. Wing-weighting resulted in significant muscle enlargement and the appearance of many V-EMB+ fibers. The majority of V-EMB+ fibers were completely independent of mature fibers and had a nuclear density characteristics of developing fibers. Furthermore, nearly 100% of the nuclei in independent V-EMB+ fibers were labeled. These findings strongly suggest that most V-EMB+ fibers were nascent fibers formed de novo during the weighting period by satellite cell activation and fusion. Nascent fibers were found primarily in the space between fascicles where they formed a complex anastomosing network of fibers running at angles to one another. Although wing-weighting induced an increase in the number of branched fibers, there was no evidence that V-EMB+ fibers were formed by longitudinal fragmentation. The location of newly formed fibers in wing-weighted and regenerating ALD muscle was compared to determine whether satellite cells in the ALD muscle were unusual in that, if stimulated to divide, they would form fibers in the inter- and intrafascicular space. In contrast to wing-weighted muscle, nascent fibers were always found closely associated with necrotic fibers. These results suggest that wing-weighting is not simply another

In vitro analysis of human immunodeficiency virus particle dissociation: gag proteolytic processing influences dissociation kinetics.

PubMed

Müller, Barbara; Anders, Maria; Reinstein, Jochen

2014-01-01

Human immunodeficiency virus particles undergo a step of proteolytic maturation, in which the main structural polyprotein Gag is cleaved into its mature subunits matrix (MA), capsid (CA), nucleocapsid (NC) and p6. Gag proteolytic processing is accompanied by a dramatic structural rearrangement within the virion, which is necessary for virus infectivity and has been proposed to proceed through a sequence of dissociation and reformation of the capsid lattice. Morphological maturation appears to be tightly regulated, with sequential cleavage events and two small spacer peptides within Gag playing important roles by regulating the disassembly of the immature capsid layer and formation of the mature capsid lattice. In order to measure the influence of individual Gag domains on lattice stability, we established Förster's resonance energy transfer (FRET) reporter virions and employed rapid kinetic FRET and light scatter measurements. This approach allowed us to measure dissociation properties of HIV-1 particles assembled in eukaryotic cells containing Gag proteins in different states of proteolytic processing. While the complex dissociation behavior of the particles prevented an assignment of kinetic rate constants to individual dissociation steps, our analyses revealed characteristic differences in the dissociation properties of the MA layer dependent on the presence of additional domains. The most striking effect observed here was a pronounced stabilization of the MA-CA layer mediated by the presence of the 14 amino acid long spacer peptide SP1 at the CA C-terminus, underlining the crucial role of this peptide for the resolution of the immature particle architecture.

Seroprevalence of sapovirus in dogs using baculovirus-expressed virus-like particles.

PubMed

Melegari, Irene; Marsilio, Fulvio; Di Profio, Federica; Sarchese, Vittorio; Massirio, Ivano; Palombieri, Andrea; D'Angelo, Anna Rita; Lanave, Gianvito; Diakoudi, Georgia; Cavalli, Alessandra; Martella, Vito; Di Martino, Barbara

2018-06-02

Caliciviruses of the Sapovirus genus have been recently detected in dogs. Canine sapoviruses (SaVs) have been identified in the stools of young or juvenile animals with gastro-enteric disease at low prevalence (2.0-2.2%), but whether they may have a role as enteric pathogens and to which extent dogs are exposed to SaVs remains unclear. Here, we report the expression in a baculovirus system of virus like-particles (VLPs) of a canine SaV strain, the prototype virus Bari/4076/2007/ITA. The recombinant antigen was used to develop an enzyme-linked immunosorbent assay (ELISA). By screening an age-stratified collection of serum samples from 516 dogs in Italy, IgG antibodies specific for the canine SaV VLPs were detected in 40.3% (208/516) of the sera. Also, as observed for SaV infection in humans, we observed a positive association between seropositivity and age, with the highest prevalence rates in dogs older than 4 years of age. Copyright © 2018 Elsevier B.V. All rights reserved.

Biophysical characterization and conformational stability of Ebola and Marburg virus-like particles.

PubMed

Hu, Lei; Trefethen, Jared M; Zeng, Yuhong; Yee, Luisa; Ohtake, Satoshi; Lechuga-Ballesteros, David; Warfield, Kelly L; Aman, M Javad; Shulenin, Sergey; Unfer, Robert; Enterlein, Sven G; Truong-Le, Vu; Volkin, David B; Joshi, Sangeeta B; Middaugh, C Russell

2011-12-01

The filoviruses, Ebola virus and Marburg virus, cause severe hemorrhagic fever with up to 90% human mortality. Virus-like particles of EBOV (eVLPs) and MARV (mVLPs) are attractive vaccine candidates. For the development of stable vaccines, the conformational stability of these two enveloped VLPs produced in insect cells was characterized by various spectroscopic techniques over a wide pH and temperature range. Temperature-induced aggregation of the VLPs at various pH values was monitored by light scattering. Temperature/pH empirical phase diagrams (EPDs) of the two VLPs were constructed to summarize the large volume of data generated. The EPDs show that both VLPs lose their conformational integrity above about 50°C-60°C, depending on solution pH. The VLPs were maximally thermal stable in solution at pH 7-8, with a significant reduction in stability at pH 5 and 6. They were much less stable in solution at pH 3-4 due to increased susceptibility of the VLPs to aggregation. The characterization data and conformational stability profiles from these studies provide a basis for selection of optimized solution conditions for further vaccine formulation and long-term stability studies of eVLPs and mVLPs. Copyright © 2011 Wiley-Liss, Inc.

Ebola virus-like particles produced in insect cells exhibit dendritic cell stimulating activity and induce neutralizing antibodies

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

Ye Ling; Lin Jianguo; Sun Yuliang

2006-08-01

Recombinant baculoviruses (rBV) expressing Ebola virus VP40 (rBV-VP40) or GP (rBV-GP) proteins were generated. Infection of Sf9 insect cells by rBV-VP40 led to assembly and budding of filamentous particles from the cell surface as shown by electron microscopy. Ebola virus-like particles (VLPs) were produced by coinfection of Sf9 cells with rBV-VP40 and rBV-GP, and incorporation of Ebola GP into VLPs was demonstrated by SDS-PAGE and Western blot analysis. Recombinant baculovirus infection of insect cells yielded high levels of VLPs, which were shown to stimulate cytokine secretion from human dendritic cells similar to VLPs produced in mammalian cells. The immunogenicity ofmore » Ebola VLPs produced in insect cells was evaluated by immunization of mice. Analysis of antibody responses showed that most of the GP-specific antibodies were of the IgG2a subtype, while no significant level of IgG1 subtype antibodies specific for GP was induced, indicating the induction of a Th1-biased immune response. Furthermore, sera from Ebola VLP immunized mice were able to block infection by Ebola GP pseudotyped HIV virus in a single round infection assay, indicating that a neutralizing antibody against the Ebola GP protein was induced. These results show that production of Ebola VLPs in insect cells using recombinant baculoviruses represents a promising approach for vaccine development against Ebola virus infection.« less

Reverse Transcriptase-Containing Particles Induced in Rous Sarcoma Virus-Transformed Rat Cells by Arginine Deprivation

PubMed Central

Kotler, Moshe; Weinberg, Eynat; Haspel, Osnat; Becker, Yechiel

1972-01-01

Incubation of rat cells transformed by Rous sarcoma virus (RSV) in an arginine-deficient medium resulted in accumulation of particles in the culture medium. Such particles did not appear when the transformed rat cells were incubated in a complete medium nor in the medium of primary rat cells which were incubated either in arginine-deficient or complete media. The particles which were released from the arginine-deprived transformed rat cells resemble C-type particles in their properties. These particles band in sucrose gradients at a density of 1.16 g/ml and contain 35S ribonucleic acid (RNA) molecules and a reverse transcriptase activity. Analysis of the cytoplasm of transformed and primary rat cells, deprived and undeprived of arginine, revealed the presence of reverse transcriptase-containing particles which banded in sucrose gradients at a density of 1.14 g/ml. These particles differed from the particles released into the medium by the arginine-deprived RSV-transformed rat cells. The deoxyribonucleic acid (DNA) molecules synthesized in vitro by the reverse transcriptase present in the particles isolated from the medium of arginine-deprived cells hybridized to RSV RNA, whereas the DNA synthesized by the cell-bound enzyme had no homology to RSV RNA. PMID:4116137

Mutations in the Basic Region of the Mason-Pfizer Monkey Virus Nucleocapsid Protein Affect Reverse Transcription, Genomic RNA Packaging, and the Virus Assembly Site.

PubMed

Dostálková, Alžběta; Kaufman, Filip; Křížová, Ivana; Kultová, Anna; Strohalmová, Karolína; Hadravová, Romana; Ruml, Tomáš; Rumlová, Michaela

2018-05-15

In addition to specific RNA-binding zinc finger domains, the retroviral Gag polyprotein contains clusters of basic amino acid residues that are thought to support Gag-viral genomic RNA (gRNA) interactions. One of these clusters is the basic K 16 NK 18 EK 20 region, located upstream of the first zinc finger of the Mason-Pfizer monkey virus (M-PMV) nucleocapsid (NC) protein. To investigate the role of this basic region in the M-PMV life cycle, we used a combination of in vivo and in vitro methods to study a series of mutants in which the overall charge of this region was more positive (RNRER), more negative (AEAEA), or neutral (AAAAA). The mutations markedly affected gRNA incorporation and the onset of reverse transcription. The introduction of a more negative charge (AEAEA) significantly reduced the incorporation of M-PMV gRNA into nascent particles. Moreover, the assembly of immature particles of the AEAEA Gag mutant was relocated from the perinuclear region to the plasma membrane. In contrast, an enhancement of the basicity of this region of M-PMV NC (RNRER) caused a substantially more efficient incorporation of gRNA, subsequently resulting in an increase in M-PMV RNRER infectivity. Nevertheless, despite the larger amount of gRNA packaged by the RNRER mutant, the onset of reverse transcription was delayed in comparison to that of the wild type. Our data clearly show the requirement for certain positively charged amino acid residues upstream of the first zinc finger for proper gRNA incorporation, assembly of immature particles, and proceeding of reverse transcription. IMPORTANCE We identified a short sequence within the Gag polyprotein that, together with the zinc finger domains and the previously identified RKK motif, contributes to the packaging of genomic RNA (gRNA) of Mason-Pfizer monkey virus (M-PMV). Importantly, in addition to gRNA incorporation, this basic region (KNKEK) at the N terminus of the nucleocapsid protein is crucial for the onset of reverse

Viruses of Entamoeba histolytica II. Morphogenesis of the Polyhedral Particle (ABRM2→HK-9)→HB-301 and the Filamentous Agent (ABRM)2→HK-9

PubMed Central

Mattern, Carl F. T.; Diamond, Louis S.; Daniel, Wendell A.

1972-01-01

The intracellular development of two morphologically different amoebal viruses has been studied by electron microscopy. One is a polyhedral agent which was observed as early as 24 hr after infection in the perinuclear cytoplasm. Subsequently, cell lysis occurred and particles were found in large number bound to membranes of disrupted amoebae. Other particles were found in phagocytic vacuoles suggesting a possible portal of entry into amoebae. The other virus is a filamentous particle which is first seen in small clusters in the nucleus after 24 hr of infection. The number of particles increases such that by 72 hr massive whorls of particles occupy a substantial part of the nucleus. After rupture of the nuclear membrane, clusters of filaments are widely dispersed throughout the cytoplasm. Still later, the cytoplasmic membrane disintegrates and clusters of filaments are found extracellularly, but free of cell membranes. The morphology of these agents is discussed in comparison with a variety of plant, animal, and bacterial viruses. Images PMID:4335523

Apolipoprotein E Likely Contributes to a Maturation Step of Infectious Hepatitis C Virus Particles and Interacts with Viral Envelope Glycoproteins

PubMed Central

Lee, Ji-Young; Acosta, Eliana G.; Stoeck, Ina Karen; Long, Gang; Hiet, Marie-Sophie; Mueller, Birthe; Fackler, Oliver T.; Kallis, Stephanie

2014-01-01

ABSTRACT The assembly of infectious hepatitis C virus (HCV) particles is tightly linked to components of the very-low-density lipoprotein (VLDL) pathway. We and others have shown that apolipoprotein E (ApoE) plays a major role in production of infectious HCV particles. However, the mechanism by which ApoE contributes to virion assembly/release and how it gets associated with the HCV particle is poorly understood. We found that knockdown of ApoE reduces titers of infectious intra- and extracellular HCV but not of the related dengue virus. ApoE depletion also reduced amounts of extracellular HCV core protein without affecting intracellular core amounts. Moreover, we found that ApoE depletion affected neither formation of nucleocapsids nor their envelopment, suggesting that ApoE acts at a late step of assembly, such as particle maturation and infectivity. Importantly, we demonstrate that ApoE interacts with the HCV envelope glycoproteins, most notably E2. This interaction did not require any other viral proteins and depended on the transmembrane domain of E2 that also was required for recruitment of HCV envelope glycoproteins to detergent-resistant membrane fractions. These results suggest that ApoE plays an important role in HCV particle maturation, presumably by direct interaction with viral envelope glycoproteins. IMPORTANCE The HCV replication cycle is tightly linked to host cell lipid pathways and components. This is best illustrated by the dependency of HCV assembly on lipid droplets and the VLDL component ApoE. Although the role of ApoE for production of infectious HCV particles is well established, it is still poorly understood how ApoE contributes to virion formation and how it gets associated with HCV particles. Here, we provide experimental evidence that ApoE likely is required for an intracellular maturation step of HCV particles. Moreover, we demonstrate that ApoE associates with the viral envelope glycoproteins. This interaction appears to be dispensable

The Fate of Nascent APP in Hippocampal Neurons: A Live Cell Imaging Study.

PubMed

DelBove, Claire E; Deng, Xian-Zhen; Zhang, Qi

2018-06-21

Amyloid precursor protein (APP) is closely associated with Alzheimer's disease (AD) because its proteolytic products form amyloid plaques and its mutations are linked to familial AD patients. As a membrane protein, APP is involved in neuronal development and plasticity. However, it remains unclear how nascent APP is distributed and transported to designated membrane compartments to execute its diverse functions. Here, we employed a dual-tagged APP fusion protein in combination with a synaptic vesicle marker to study the surface trafficking and cleavage of APP in hippocampal neurons immediately after its synthesis. Using long-term time-lapse imaging, we found that a considerable amount of nascent APP was directly transported to the somatodendritic surface, from which it propagates to distal neurites. Some APP in the plasma membrane was endocytosed and some was cleaved by α-secretase. Hence, we conclude that surface transportation of APP is a major step preceding its proteolytic processing and neuritic distribution.

RNA polymerase pausing and nascent RNA structure formation are linked through clamp domain movement

PubMed Central

Hein, Pyae P.; Kolb, Kellie E.; Windgassen, Tricia; Bellecourt, Michael J.; Darst, Seth A.; Mooney, Rachel A.; Landick, Robert

2014-01-01

The rates of RNA synthesis and nascent RNA folding into biologically active structures are linked via pausing by RNA polymerase (RNAP). Structures that form within the RNA exit channel can increase pausing by interacting with bacterial RNAP or decrease pausing by preventing backtracking. Conversely, pausing is required for proper folding of some RNAs. Opening of the RNAP clamp domain is proposed to mediate some effects of nascent RNA structures. However, the connections among RNA structure formation, clamp movement, and catalytic activity remain uncertain. We assayed exit-channel structure formation in Escherichia coli RNAP together with disulfide crosslinks that favor closed or open clamp conformations and found that clamp position directly influences RNA structure formation and catalytic activity. We report that exit-channel RNA structures slow pause escape by favoring clamp opening and through interactions with the flap that slow translocation. PMID:25108353

A method for rapid production of heteromultimeric protein complexes in plants: assembly of protective bluetongue virus-like particles.

PubMed

Thuenemann, Eva C; Meyers, Ann E; Verwey, Jeanette; Rybicki, Edward P; Lomonossoff, George P

2013-09-01

Plant expression systems based on nonreplicating virus-based vectors can be used for the simultaneous expression of multiple genes within the same cell. They therefore have great potential for the production of heteromultimeric protein complexes. This work describes the efficient plant-based production and assembly of Bluetongue virus-like particles (VLPs), requiring the simultaneous expression of four distinct proteins in varying amounts. Such particles have the potential to serve as a safe and effective vaccine against Bluetongue virus (BTV), which causes high mortality rates in ruminants and thus has a severe effect on the livestock trade. Here, VLPs produced and assembled in Nicotiana benthamiana using the cowpea mosaic virus-based HyperTrans (CPMV-HT) and associated pEAQ plant transient expression vector system were shown to elicit a strong antibody response in sheep. Furthermore, they provided protective immunity against a challenge with a South African BTV-8 field isolate. The results show that transient expression can be used to produce immunologically relevant complex heteromultimeric structures in plants in a matter of days. The results have implications beyond the realm of veterinary vaccines and could be applied to the production of VLPs for human use or the coexpression of multiple enzymes for the manipulation of metabolic pathways. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

The structure of Escherichia coli signal recognition particle revealed by scanning transmission electron microscopy.

PubMed

Mainprize, Iain L; Beniac, Daniel R; Falkovskaia, Elena; Cleverley, Robert M; Gierasch, Lila M; Ottensmeyer, F Peter; Andrews, David W

2006-12-01

Structural studies on various domains of the ribonucleoprotein signal recognition particle (SRP) have not converged on a single complete structure of bacterial SRP consistent with the biochemistry of the particle. We obtained a three-dimensional structure for Escherichia coli SRP by cryoscanning transmission electron microscopy and mapped the internal RNA by electron spectroscopic imaging. Crystallographic data were fit into the SRP reconstruction, and although the resulting model differed from previous models, they could be rationalized by movement through an interdomain linker of Ffh, the protein component of SRP. Fluorescence resonance energy transfer experiments determined interdomain distances that were consistent with our model of SRP. Docking our model onto the bacterial ribosome suggests a mechanism for signal recognition involving interdomain movement of Ffh into and out of the nascent chain exit site and suggests how SRP could interact and/or compete with the ribosome-bound chaperone, trigger factor, for a nascent chain during translation.

Recombinant H7 hemagglutinin forms subviral particles that protect mice and ferrets from challenge with H7N9 influenza virus

PubMed Central

Pushko, Peter; Pujanauski, Lindsey M.; Sun, Xiangjie; Pearce, Melissa; Hidajat, Rachmat; Kort, Thomas; Schwartzman, Louis M.; Tretyakova, Irina; Chunqing, Liu; Taubenberger, Jeffery K.; Tumpey, Terrence M.

2015-01-01

A novel avian-origin influenza A H7N9 virus emerged in China in 2013 and continues to cause sporadic human infections with mortality rates approaching 35%. Currently there are no approved human vaccines for H7N9 virus. Recombinant approaches including hemagglutinin (HA) and virus-like particles (VLPs) have resulted in experimental vaccines with advantageous safety and manufacturing characteristics. While high immunogenicity of VLP vaccines has been attributed to the native conformation of HA arranged in the regular repeated patterns within virus-like structures, there is limited data regarding molecular organization of HA within recombinant HA vaccine preparations. In this study, the full-length recombinant H7 protein (rH7) of A/Anhui/1/2013 (H7N9) virus was expressed in Sf9 cells. We showed that purified full-length rH7 retained functional ability to agglutinate red blood cells and formed oligomeric pleomorphic subviral particles (SVPs) of ~20 nm in diameter composed of approximately 10 HA0 molecules. No significant quantities of free monomeric HA0 were observed in rH7 preparation by size exclusion chromatography. Immunogenicity and protective efficacy of rH7 SVPs was confirmed in the mouse and ferret challenge models suggesting that SVPs can be used for vaccination against H7N9 virus. PMID:26207590

A novel phosphoserine motif in the LCMV matrix protein Z regulates the release of infectious virus and defective interfering particles.

PubMed

Ziegler, Christopher M; Eisenhauer, Philip; Bruce, Emily A; Beganovic, Vedran; King, Benjamin R; Weir, Marion E; Ballif, Bryan A; Botten, Jason

2016-09-01

We report that the lymphocytic choriomeningitis virus (LCMV) matrix protein, which drives viral budding, is phosphorylated at serine 41 (S41). A recombinant (r)LCMV bearing a phosphomimetic mutation (S41D) was impaired in infectious and defective interfering (DI) particle release, while a non-phosphorylatable mutant (S41A) was not. The S41D mutant was disproportionately impaired in its ability to release DI particles relative to infectious particles. Thus, DI particle production by LCMV may be dynamically regulated via phosphorylation of S41.

Substantial Goodness and Nascent Human Life.

PubMed

Floyd, Shawn

2015-09-01

Many believe that moral value is--at least to some extent--dependent on the developmental states necessary for supporting rational activity. My paper rejects this view, but does not aim simply to register objections to it. Rather, my essay aims to answer the following question: if a human being's developmental state and occurrent capacities do not bequeath moral standing, what does? The question is intended to prompt careful consideration of what makes human beings objects of moral value, dignity, or (to employ my preferred term) goodness. Not only do I think we can answer this question, I think we can show that nascent human life possesses goodness of precisely this sort. I appeal to Aquinas's metaethics to establish the conclusion that the goodness of a human being--even if that being is an embryo or fetus--resides at the substratum of her existence. If she possesses goodness, it is because human existence is good.

Real-time airborne particle analyzer

DOEpatents

Reilly, Peter T.A.

2012-10-16

An aerosol particle analyzer includes a laser ablation chamber, a gas-filled conduit, and a mass spectrometer. The laser ablation chamber can be operated at a low pressure, which can be from 0.1 mTorr to 30 mTorr. The ablated ions are transferred into a gas-filled conduit. The gas-filled conduit reduces the electrical charge and the speed of ablated ions as they collide and mix with buffer gases in the gas-filled conduit. Preferably, the gas filled-conduit includes an electromagnetic multipole structure that collimates the nascent ions into a beam, which is guided into the mass spectrometer. Because the gas-filled conduit allows storage of vast quantities of the ions from the ablated particles, the ions from a single ablated particle can be analyzed multiple times and by a variety of techniques to supply statistically meaningful analysis of composition and isotope ratios.

Sensing of Immature Particles Produced by Dengue Virus Infected Cells Induces an Antiviral Response by Plasmacytoid Dendritic Cells

PubMed Central

Hillaire, Marine L. B.; Dejnirattisai, Wanwisa; Mongkolsapaya, Juthathip; Screaton, Gavin R.; Davidson, Andrew D.; Dreux, Marlène

2014-01-01

Dengue virus (DENV) is the leading cause of mosquito-borne viral illness and death in humans. Like many viruses, DENV has evolved potent mechanisms that abolish the antiviral response within infected cells. Nevertheless, several in vivo studies have demonstrated a key role of the innate immune response in controlling DENV infection and disease progression. Here, we report that sensing of DENV infected cells by plasmacytoid dendritic cells (pDCs) triggers a robust TLR7-dependent production of IFNα, concomitant with additional antiviral responses, including inflammatory cytokine secretion and pDC maturation. We demonstrate that unlike the efficient cell-free transmission of viral infectivity, pDC activation depends on cell-to-cell contact, a feature observed for various cell types and primary cells infected by DENV, as well as West Nile virus, another member of the Flavivirus genus. We show that the sensing of DENV infected cells by pDCs requires viral envelope protein-dependent secretion and transmission of viral RNA. Consistently with the cell-to-cell sensing-dependent pDC activation, we found that DENV structural components are clustered at the interface between pDCs and infected cells. The actin cytoskeleton is pivotal for both this clustering at the contacts and pDC activation, suggesting that this structural network likely contributes to the transmission of viral components to the pDCs. Due to an evolutionarily conserved suboptimal cleavage of the precursor membrane protein (prM), DENV infected cells release uncleaved prM containing-immature particles, which are deficient for membrane fusion function. We demonstrate that cells releasing immature particles trigger pDC IFN response more potently than cells producing fusion-competent mature virus. Altogether, our results imply that immature particles, as a carrier to endolysosome-localized TLR7 sensor, may contribute to regulate the progression of dengue disease by eliciting a strong innate response. PMID

Quantitative nanoscale electrostatics of viruses

NASA Astrophysics Data System (ADS)

Hernando-Pérez, M.; Cartagena-Rivera, A. X.; Lošdorfer Božič, A.; Carrillo, P. J. P.; San Martín, C.; Mateu, M. G.; Raman, A.; Podgornik, R.; de Pablo, P. J.

2015-10-01

Electrostatics is one of the fundamental driving forces of the interaction between biomolecules in solution. In particular, the recognition events between viruses and host cells are dominated by both specific and non-specific interactions and the electric charge of viral particles determines the electrostatic force component of the latter. Here we probe the charge of individual viruses in liquid milieu by measuring the electrostatic force between a viral particle and the Atomic Force Microscope tip. The force spectroscopy data of co-adsorbed φ29 bacteriophage proheads and mature virions, adenovirus and minute virus of mice capsids is utilized for obtaining the corresponding density of charge for each virus. The systematic differences of the density of charge between the viral particles are consistent with the theoretical predictions obtained from X-ray structural data. Our results show that the density of charge is a distinguishing characteristic of each virus, depending crucially on the nature of the viral capsid and the presence/absence of the genetic material.Electrostatics is one of the fundamental driving forces of the interaction between biomolecules in solution. In particular, the recognition events between viruses and host cells are dominated by both specific and non-specific interactions and the electric charge of viral particles determines the electrostatic force component of the latter. Here we probe the charge of individual viruses in liquid milieu by measuring the electrostatic force between a viral particle and the Atomic Force Microscope tip. The force spectroscopy data of co-adsorbed φ29 bacteriophage proheads and mature virions, adenovirus and minute virus of mice capsids is utilized for obtaining the corresponding density of charge for each virus. The systematic differences of the density of charge between the viral particles are consistent with the theoretical predictions obtained from X-ray structural data. Our results show that the density of

Synthesis and assembly of Hepatitis B virus envelope protein-derived particles in Escherichia coli.

PubMed

Li, Hao; Onbe, Keisuke; Liu, Qiushi; Iijima, Masumi; Tatematsu, Kenji; Seno, Masaharu; Tada, Hiroko; Kuroda, Shun' Ichi

2017-08-19

Hepatitis B virus (HBV) envelope particles have been synthesized in eukaryotic cells (e.g., mammalian cells, insect cells, and yeast cells) as an HB vaccine immunogen and drug delivery system (DDS) nanocarrier. Many researchers had made attempts to synthesize the particles in Escherichia coli for minimize the cost and time for producing HBV envelope particles, but the protein was too deleterious to be synthesized in E. coli. In this study, we generated deletion mutants of HBV envelope L protein (389 amino acid residues (aa)) containing three transmembrane domains (TM1, TM2, TM3). The ΔNC mutant spanning from TM2 to N-terminal half of TM3 (from 237 aa to 335 aa) was found as a shortest form showing spontaneous particle formation. After the N-terminal end of ΔNC mutant was optimized by the N-end rule for E. coli expression, the modified ΔNC mutant (mΔNC) was efficiently expressed as particles in E. coli. The molecular mass of mΔNC particle was approx. 670 kDa, and the diameter was 28.5 ± 6.2 nm (mean ± SD, N = 61). The particle could react with anti-HBV envelope S protein antibody, indicating the particles exhibited S antigenic domain outside as well as HBV envelope particles. Taken together, the E. coli-derived mΔNC particles could be used as a substitute of eukaryotic cell-derived HBV envelope particles for versatile applications. Copyright © 2017 Elsevier Inc. All rights reserved.

Generation of Recombinant Porcine Parvovirus Virus-Like Particles in Saccharomyces cerevisiae and Development of Virus-Specific Monoclonal Antibodies

PubMed Central

Tamošiūnas, Paulius Lukas; Petraitytė-Burneikienė, Rasa; Lasickienė, Rita; Sereika, Vilimas; Lelešius, Raimundas; Žvirblienė, Aurelija; Sasnauskas, Kęstutis

2014-01-01

Porcine parvovirus (PPV) is a widespread infectious virus that causes serious reproductive diseases of swine and death of piglets. The gene coding for the major capsid protein VP2 of PPV was amplified using viral nucleic acid extract from swine serum and inserted into yeast Saccharomyces cerevisiae expression plasmid. Recombinant PPV VP2 protein was efficiently expressed in yeast and purified using density gradient centrifugation. Electron microscopy analysis of purified PPV VP2 protein revealed the self-assembly of virus-like particles (VLPs). Nine monoclonal antibodies (MAbs) against the recombinant PPV VP2 protein were generated. The specificity of the newly generated MAbs was proven by immunofluorescence analysis of PPV-infected cells. Indirect IgG ELISA based on the recombinant VLPs for detection of PPV-specific antibodies in swine sera was developed and evaluated. The sensitivity and specificity of the new assay were found to be 93.4% and 97.4%, respectively. In conclusion, yeast S. cerevisiae represents a promising expression system for generating recombinant PPV VP2 protein VLPs of diagnostic relevance. PMID:25045718

Tracking Virus Particles in Fluorescence Microscopy Images Using Multi-Scale Detection and Multi-Frame Association.

PubMed

Jaiswal, Astha; Godinez, William J; Eils, Roland; Lehmann, Maik Jorg; Rohr, Karl

2015-11-01

Automatic fluorescent particle tracking is an essential task to study the dynamics of a large number of biological structures at a sub-cellular level. We have developed a probabilistic particle tracking approach based on multi-scale detection and two-step multi-frame association. The multi-scale detection scheme allows coping with particles in close proximity. For finding associations, we have developed a two-step multi-frame algorithm, which is based on a temporally semiglobal formulation as well as spatially local and global optimization. In the first step, reliable associations are determined for each particle individually in local neighborhoods. In the second step, the global spatial information over multiple frames is exploited jointly to determine optimal associations. The multi-scale detection scheme and the multi-frame association finding algorithm have been combined with a probabilistic tracking approach based on the Kalman filter. We have successfully applied our probabilistic tracking approach to synthetic as well as real microscopy image sequences of virus particles and quantified the performance. We found that the proposed approach outperforms previous approaches.

Structural analyses of EBER1 and EBER2 ribonucleoprotein particles present in Epstein-Barr virus-infected cells.

PubMed Central

Glickman, J N; Howe, J G; Steitz, J A

1988-01-01

The ribonucleoprotein (RNP) particles containing the Epstein-Barr virus-associated small RNAs EBER1 and EBER2 were analyzed to determine their RNA secondary structures and sites of RNA-protein interaction. The secondary structures were probed with nucleases and by chemical modification with single-strand-specific reagents, and the sites of modification or cleavage were mapped by primer extension. These data were used to develop secondary structures for the two RNAs, and likely sites of close RNA-protein contact were identified by comparing modification patterns for naked RNA and RNA in RNP particles. In addition, sites of interaction between each Epstein-Barr virus-encoded RNA (EBER) and the La antigen were identified by analyzing RNA fragments resistant to digestion by RNase A or T1 after immunoprecipitation by an anti-La serum sample from a lupus patient. Our results confirm earlier findings that the La protein binds to the 3' terminus of each molecule. Possible functions for the EBER RNPs are discussed. Images PMID:2828685

β-structure of the coat protein subunits in spherical particles generated by tobacco mosaic virus thermal denaturation.

PubMed

Dobrov, Evgeny N; Nikitin, Nikolai A; Trifonova, Ekaterina A; Parshina, Evgenia Yu; Makarov, Valentin V; Maksimov, George V; Karpova, Olga V; Atabekov, Joseph G

2014-01-01

Conversion of the rod-like tobacco mosaic virus (TMV) virions into "ball-like particles" by thermal denaturation at 90-98 °C had been described by R.G. Hart in 1956. We have reported recently that spherical particles (SPs) generated by thermal denaturation of TMV at 94-98 °C were highly stable, RNA-free, and water-insoluble. The SPs were uniform in shape but varied widely in size (53-800 nm), which depended on the virus concentration. Here, we describe some structural characteristics of SPs using circular dichroism, fluorescence spectroscopy, and Raman spectroscopy. It was found that the structure of SPs protein differs strongly from that of the native TMV and is characterized by coat protein subunits transition from mainly (about 50%) α-helical structure to a structure with low content of α-helices and a significant fraction of β-sheets. The SPs demonstrate strong reaction with thioflavin T suggesting the formation of amyloid-like structures.

A Novel Self-Replicating Chimeric Lentivirus-Like Particle

PubMed Central

Young, Kelly R.; Madden, Victoria J.; Johnson, Philip R.; Johnston, Robert E.

2012-01-01

Successful live attenuated vaccines mimic natural exposure to pathogens without causing disease and have been successful against several viruses. However, safety concerns prevent the development of attenuated human immunodeficiency virus (HIV) as a vaccine candidate. If a safe, replicating virus vaccine could be developed, it might have the potential to offer significant protection against HIV infection and disease. Described here is the development of a novel self-replicating chimeric virus vaccine candidate that is designed to provide natural exposure to a lentivirus-like particle and to incorporate the properties of a live attenuated virus vaccine without the inherent safety issues associated with attenuated lentiviruses. The genome from the alphavirus Venezuelan equine encephalitis virus (VEE) was modified to express SHIV89.6P genes encoding the structural proteins Gag and Env. Expression of Gag and Env from VEE RNA in primate cells led to the assembly of particles that morphologically and functionally resembled lentivirus virions and that incorporated alphavirus RNA. Infection of CD4+ cells with chimeric lentivirus-like particles was specific and productive, resulting in RNA replication, expression of Gag and Env, and generation of progeny chimeric particles. Further genome modifications designed to enhance encapsidation of the chimeric virus genome and to express an attenuated simian immunodeficiency virus (SIV) protease for particle maturation improved the ability of chimeric lentivirus-like particles to propagate in cell culture. This study provides proof of concept for the feasibility of creating chimeric virus genomes that express lentivirus structural proteins and assemble into infectious particles for presentation of lentivirus immunogens in their native and functional conformation. PMID:22013035

A novel self-replicating chimeric lentivirus-like particle.

PubMed

Jurgens, Christy K; Young, Kelly R; Madden, Victoria J; Johnson, Philip R; Johnston, Robert E

2012-01-01

Successful live attenuated vaccines mimic natural exposure to pathogens without causing disease and have been successful against several viruses. However, safety concerns prevent the development of attenuated human immunodeficiency virus (HIV) as a vaccine candidate. If a safe, replicating virus vaccine could be developed, it might have the potential to offer significant protection against HIV infection and disease. Described here is the development of a novel self-replicating chimeric virus vaccine candidate that is designed to provide natural exposure to a lentivirus-like particle and to incorporate the properties of a live attenuated virus vaccine without the inherent safety issues associated with attenuated lentiviruses. The genome from the alphavirus Venezuelan equine encephalitis virus (VEE) was modified to express SHIV89.6P genes encoding the structural proteins Gag and Env. Expression of Gag and Env from VEE RNA in primate cells led to the assembly of particles that morphologically and functionally resembled lentivirus virions and that incorporated alphavirus RNA. Infection of CD4⁺ cells with chimeric lentivirus-like particles was specific and productive, resulting in RNA replication, expression of Gag and Env, and generation of progeny chimeric particles. Further genome modifications designed to enhance encapsidation of the chimeric virus genome and to express an attenuated simian immunodeficiency virus (SIV) protease for particle maturation improved the ability of chimeric lentivirus-like particles to propagate in cell culture. This study provides proof of concept for the feasibility of creating chimeric virus genomes that express lentivirus structural proteins and assemble into infectious particles for presentation of lentivirus immunogens in their native and functional conformation.

Defective RNA particles derived from Tomato black ring virus genome interfere with the replication of parental virus.

PubMed

Hasiów-Jaroszewska, Beata; Minicka, Julia; Zarzyńska-Nowak, Aleksandra; Budzyńska, Daria; Elena, Santiago F

2018-05-02

Tomato black ring virus (TBRV) is the only member of the Nepovirus genus that is known to form defective RNA particles (D RNAs) during replication. Here, de novo generation of D RNAs was observed during prolonged passages of TBRV isolates originated from Solanum lycopersicum and Lactuca sativa in Chenopodium quinoa plants. D RNAs of about 500 nt derived by a single deletion in the RNA1 molecule and contained a portion of the 5' untranslated region and viral replicase, and almost the entire 3' non-coding region. Short regions of sequence complementarity were found at the 5' and 3' junction borders, which can facilitate formation of the D RNAs. Moreover, in this study we analyzed the effects of D RNAs on TBRV replication and symptoms development of infected plants. C. quinoa, S. lycopersicum, Nicotiana tabacum, and L. sativa were infected with the original TBRV isolates (TBRV-D RNA) and those containing additional D RNA particles (TBRV + D RNA). The viral accumulation in particular hosts was measured up to 28 days post inoculation by RT-qPCR. Statistical analyses revealed that D RNAs interfere with TBRV replication and thus should be referred to as defective interfering particles. The magnitude of the interference effect depends on the interplay between TBRV isolate and host species. Copyright © 2018 Elsevier B.V. All rights reserved.

Virus templated metallic nanoparticles

NASA Astrophysics Data System (ADS)

Aljabali, Alaa A. A.; Barclay, J. Elaine; Lomonossoff, George P.; Evans, David J.

2010-12-01

Plant viruses are considered as nanobuilding blocks that can be used as synthons or templates for novel materials. Cowpea mosaic virus (CPMV) particles have been shown to template the fabrication of metallic nanoparticles by an electroless deposition metallization process. Palladium ions were electrostatically bound to the virus capsid and, when reduced, acted as nucleation sites for the subsequent metal deposition from solution. The method, although simple, produced highly monodisperse metallic nanoparticles with a diameter of ca. <=35 nm. CPMV-templated particles were prepared with cobalt, nickel, iron, platinum, cobalt-platinum and nickel-iron.Plant viruses are considered as nanobuilding blocks that can be used as synthons or templates for novel materials. Cowpea mosaic virus (CPMV) particles have been shown to template the fabrication of metallic nanoparticles by an electroless deposition metallization process. Palladium ions were electrostatically bound to the virus capsid and, when reduced, acted as nucleation sites for the subsequent metal deposition from solution. The method, although simple, produced highly monodisperse metallic nanoparticles with a diameter of ca. <=35 nm. CPMV-templated particles were prepared with cobalt, nickel, iron, platinum, cobalt-platinum and nickel-iron. Electronic supplementary information (ESI) available: Additional experimental detail, agarose gel electrophoresis results, energy dispersive X-ray spectra, ζ-potential measurements, dynamic light scattering data, nanoparticle tracking analysis and an atomic force microscopy image of Ni-CPMV. See DOI: 10.1039/c0nr00525h

Virus-like particles in venom of Meteorus pulchricornis induce host hemocyte apoptosis.

PubMed

Suzuki, M; Tanaka, T

2006-06-01

Ultrastructural studies on the reproductive tract and venom apparatus of a female braconid, Meteorus pulchricornis, revealed that the parasitoid lacks the calyx region in its oviduct, but possesses a venom gland with two venom gland filaments and a venom reservoir filled with white and cloudy fluid. Its venom gland cell is concaved and has a lumen filled with numerous granules. Transmisson electron microscopic (TEM) observation revealed that virus-like particles (VLPs) were produced in venom gland cells. The virus-like particle observed in M. pulchricornis (MpVLP) is composed of membranous envelopes with two different parts: a high-density core and a whitish low-density part. The VLPs of M. pulchricornis is also found assembling ultimately in the lumen of venom gland cell. Microvilli were found thrusting into the lumen of the venom gland cell and seem to aid in driving the matured MpVLPs to the common duct of the venom gland filament. Injection of MpVLPs into non-parasitized Pseudaletia separata hosts induced apoptosis in hemocytes, particularly granulocytes (GRs). Rate of apoptosis induced in GRs peaked 48h after VLP injection. While a large part of the GR population collapsed due to apoptosis caused by MpVLPs, the plasmatocyte population was minimally affected. The capacity of MpVLPs to cause apoptosis in host's hemocytes was further demonstrated by a decrease ( approximately 10-fold) in ability of host hemocytes to encapsulate fluorescent latex beads when MpVLPs were present. Apparently, the reduced encapsulation ability was due to a decrease in the GR population resulting from MpVLP-induced apoptosis.

Enhanced stability of a chimeric hepatitis B core antigen virus-like-particle (HBcAg-VLP) by a C-terminal linker-hexahistidine-peptide.

PubMed

Schumacher, Jens; Bacic, Tijana; Staritzbichler, René; Daneschdar, Matin; Klamp, Thorsten; Arnold, Philipp; Jägle, Sabrina; Türeci, Özlem; Markl, Jürgen; Sahin, Ugur

2018-04-13

Virus-like-particles (VLPs) are attractive nanoparticulate scaffolds for broad applications in material/biological sciences and medicine. Prior their functionalization, specific adaptations have to be carried out. These adjustments frequently lead to disordered particles, but the particle integrity is an essential factor for the VLP suitability. Therefore, major requirements for particle stabilization exist. The objective of this study was to evaluate novel stabilizing elements for functionalized chimeric hepatitis B virus core antigen virus-like particles (HBcAg-VLP), with beneficial characteristics for vaccine development, imaging or delivery. The effects of a carboxy-terminal polyhistidine-peptide and an intradimer disulfide-bridge on the stability of preclinically approved chimeric HBcAg-VLPs were assessed. We purified recombinant chimeric HBcAg-VLPs bearing different modified C-termini and compared their physical and chemical particle stability by quantitative protein-biochemical and biophysical techniques. We observed lower chemical resistance of T = 3- compared to T = 4-VLP (triangulation number) capsids and profound impairment of accessibility of hexahistidine-peptides in assembled VLPs. Histidines attached to the C-terminus were associated with superior mechanical and/or chemical particle stability depending on the number of histidine moieties. A molecular modeling approach based on cryo-electron microscopy and biolayer interferometry revealed the underlying structural mechanism for the strengthening of the integrity of VLPs. Interactions triggering capsid stabilization occur on a highly conserved residue on the basis of HBcAg-monomers as well as on hexahistidine-peptides of adjacent monomers. This new stabilization mechanism appears to mimic an evolutionary conserved stabilization concept for hepadnavirus core proteins. These findings establish the genetically simply transferable C-terminal polyhistidine-peptide as a general stabilizing element

A novel recombinant virus-like particle vaccine for prevention of porcine parvovirus-induced reproductive failure.

PubMed

Antonis, Adriaan F G; Bruschke, Christianne J M; Rueda, Paloma; Maranga, Luis; Casal, J Ignacio; Vela, Carmen; Hilgers, Luuk A Th; Belt, Peter B G M; Weerdmeester, Klaas; Carrondo, Manuel J T; Langeveld, Jan P M

2006-06-29

A novel vaccine against porcine parvovirus (PPV), composed of recombinant virus-like particles (PPV-VLPs) produced with the baculovirus expression vector system (BEVS) at industrial scale, was tested for its immunogenicity and protective potency. A formulation of submicrogram amounts of PPV-VLPs in a water-in-mineral oil adjuvant evoked high serum antibody titres in both guinea pigs, used as reference model, and target species, pigs. A single immunisation with 0.7microg of this antigen yielded complete foetal protection against PPV infection after challenge with a virulent strain of this virus. Furthermore, also in the presence of mild adjuvants the protective action of these PPV-VLPs is excellent. This recombinant subunit vaccine overcomes some of the drawbacks of classical PPV vaccines.

Silica Nanoparticles as the Adjuvant for the Immunisation of Mice Using Hepatitis B Core Virus-Like Particles

PubMed Central

Skrastina, Dace; Petrovskis, Ivars; Lieknina, Ilva; Bogans, Janis; Renhofa, Regina; Ose, Velta; Dishlers, Andris; Dekhtyar, Yuri; Pumpens, Paul

2014-01-01

Advances in nanotechnology and nanomaterials have facilitated the development of silicon dioxide, or Silica, particles as a promising immunological adjuvant for the generation of novel prophylactic and therapeutic vaccines. In the present study, we have compared the adjuvanting potential of commercially available Silica nanoparticles (initial particles size of 10–20 nm) with that of aluminium hydroxide, or Alum, as well as that of complete and incomplete Freund's adjuvants for the immunisation of BALB/c mice with virus-like particles (VLPs) formed by recombinant full-length Hepatitis B virus core (HBc) protein. The induction of B-cell and T-cell responses was studied after immunisation. Silica nanoparticles were able to adsorb maximally 40% of the added HBc, whereas the adsorption capacity of Alum exceeded 90% at the same VLPs/adjuvant ratio. Both Silica and Alum formed large complexes with HBc VLPs that sedimented rapidly after formulation, as detected by dynamic light scattering, spectrophotometry, and electron microscopy. Both Silica and Alum augmented the humoral response against HBc VLPs to the high anti-HBc level in the case of intraperitoneal immunisation, whereas in subcutaneous immunisation, the Silica-adjuvanted anti-HBc level even exceeded the level adjuvanted by Alum. The adjuvanting of HBc VLPs by Silica resulted in the same typical IgG2a/IgG1 ratios as in the case of the adjuvanting by Alum. The combination of Silica with monophosphoryl lipid A (MPL) led to the same enhancement of the HBc-specific T-cell induction as in the case of the Alum and MPL combination. These findings demonstrate that Silica is not a weaker putative adjuvant than Alum for induction of B-cell and T-cell responses against recombinant HBc VLPs. This finding may have an essential impact on the development of the set of Silica-adjuvanted vaccines based on a long list of HBc-derived virus-like particles as the biological component. PMID:25436773

Cryo-Electron Microscopy of Viruses Infecting Bacterium

NASA Astrophysics Data System (ADS)

Chiu, Wah

2010-03-01

Single particle cryo-EM can yield structures of infectious bacterial viruses with and without imposed icosahedral symmetry at subnanometer resolution. Reconstructions of infectious and empty phage particles show substantial differences in the portal vertex protein complex at one of the 12 pentameric vertices in the icosahedral virus particle through which the viral genomes are packaged or released. In addition, electron cryo-tomography of viruses during infecting its bacterial host cell displayed multiple conformations of the tail fiber of the virus. Our structural observations by single particle and tomographic reconstructions suggest a mechanism whereby the viral tail fibers, upon binding to the host cell, induce a cascade of structural alterations of the portal vertex protein complex that triggers DNA release.

West Nile virus infectious replicon particles generated using a packaging-restricted cell line is a safe reporter system.

PubMed

Li, Wei; Ma, Le; Guo, Li-Ping; Wang, Xiao-Lei; Zhang, Jing-Wei; Bu, Zhi-Gao; Hua, Rong-Hong

2017-06-12

West Nile virus (WNV) is a neurotropic pathogen which causes zoonotic disease in humans. Recently, there have been an increasing number of infected cases and there are no clinically approved vaccines or effective drugs to treat WNV infections in humans. The purpose of this study was to facilitate vaccine and antiviral drug discovery by developing a packaging cell line-restricted WNV infectious replicon particle system. We constructed a DNA-based WNV replicon lacking the C-prM-E coding region and replaced it with a GFP coding sequence. To produce WNV replicon particles, cell lines stably-expressing prM-E and C-prM-E were constructed. When the WNV replicon plasmid was co-transfected with a WNV C-expressing plasmid into the prM-E-expressing cell line or directly transfected the C-prM-E expressing cell line, the replicon particle was able to replicate, form green fluorescence foci, and exhibit cytopathic plaques similar to that induced by the wild type virus. The infectious capacity of the replicon particles was restricted to the packaging cell line as the replicons demonstrated only one round of infection in other permissive cells. Thus, this system provides a safe and convenient reporter WNV manipulating tool which can be used to study WNV viral invasion mechanisms, neutralizing antibodies and antiviral efficacy.

Peroral infection of nuclear polyhedrosis virus budded particles in the host, Bombyx mori l., enabled by an optical brightener, Tinopal UNPA-GX.

PubMed

Arakawa, T; Kamimura, M; Furuta, Y; Miyazawa, M; Kato, M

2000-08-01

Perorally inoculated budded particles of a nuclear polyhedrosis virus was used to infect Bombyx mori (BmNPV) (Lepidoptera; Bombycidae), aided by an optical brightener, Tinopal UNPA-GX (Tinopal). BmNPV budded particles not occluded in the occlusion body do not infect successfully the host, B. mori, when administered perorally. It was found that feeding the host Tinopal enabled perorally delivered BmNPV budded particles to infect the host. B. mori larvae ingesting BmNPV budded particles (1.3 x 10(6) TCID(50) units per larva) after they consumed an artificial diet containing 0. 3% Tinopal died of the viral infection. Peroral administration of these particles to host larvae with 1% Tinopal also resulted in virus infection. Tinopal is a candidate for viral activity enhancing agent promoting viral insecticide infection in hosts. The results suggest that B. mori-BmNPV budded particles are convenient for detecting viral infection enhancement activity of a chemical of interest. Since recombinant baculovirus vectors are constructed by replacing the polyhedrin gene with the foreign gene of interest, they do not produce occlusion bodies, i.e. polyhedra. Budded particles of a baculovirus vector not occluded in polyhedra cannot infect their hosts when administered perorally. The peroral inoculation of BmNPV budded particles by Tinopal leads to industrial pharmaceutics production using a baculovirus vector for a huge number of insect hosts, i.e. an 'insect factory'.

Ferrets develop fatal influenza after inhaling small particle aerosols of highly pathogenic avian influenza virus A/Vietnam/1203/2004 (H5N1)

USDA-ARS?s Scientific Manuscript database

There is limited knowledge about the potential routes for H5N1 influenza virus transmission to and between humans, and it is not clear whether humans can be infected through inhalation of aerosolized H5N1 virus particles. Ferrets are often used as a surrogate for humans in influenza pathogenicity a...

Rad51 recombinase prevents Mre11 nuclease-dependent degradation and excessive PrimPol-mediated elongation of nascent DNA after UV irradiation

PubMed Central

Vallerga, María Belén; Mansilla, Sabrina F.; Federico, María Belén; Bertolin, Agustina P.; Gottifredi, Vanesa

2015-01-01

After UV irradiation, DNA polymerases specialized in translesion DNA synthesis (TLS) aid DNA replication. However, it is unclear whether other mechanisms also facilitate the elongation of UV-damaged DNA. We wondered if Rad51 recombinase (Rad51), a factor that escorts replication forks, aids replication across UV lesions. We found that depletion of Rad51 impairs S-phase progression and increases cell death after UV irradiation. Interestingly, Rad51 and the TLS polymerase polη modulate the elongation of nascent DNA in different ways, suggesting that DNA elongation after UV irradiation does not exclusively rely on TLS events. In particular, Rad51 protects the DNA synthesized immediately before UV irradiation from degradation and avoids excessive elongation of nascent DNA after UV irradiation. In Rad51-depleted samples, the degradation of DNA was limited to the first minutes after UV irradiation and required the exonuclease activity of the double strand break repair nuclease (Mre11). The persistent dysregulation of nascent DNA elongation after Rad51 knockdown required Mre11, but not its exonuclease activity, and PrimPol, a DNA polymerase with primase activity. By showing a crucial contribution of Rad51 to the synthesis of nascent DNA, our results reveal an unanticipated complexity in the regulation of DNA elongation across UV-damaged templates. PMID:26627254

Vaccination with Combination DNA and Virus-Like Particles Enhances Humoral and Cellular Immune Responses upon Boost with Recombinant Modified Vaccinia Virus Ankara Expressing Human Immunodeficiency Virus Envelope Proteins.

PubMed

Gangadhara, Sailaja; Kwon, Young-Man; Jeeva, Subbiah; Quan, Fu-Shi; Wang, Baozhong; Moss, Bernard; Compans, Richard W; Amara, Rama Rao; Jabbar, M Abdul; Kang, Sang-Moo

2017-12-19

Heterologous prime boost with DNA and recombinant modified vaccinia virus Ankara (rMVA) vaccines is considered as a promising vaccination approach against human immunodeficiency virus (HIV-1). To further enhance the efficacy of DNA-rMVA vaccination, we investigated humoral and cellular immune responses in mice after three sequential immunizations with DNA, a combination of DNA and virus-like particles (VLP), and rMVA expressing HIV-1 89.6 gp120 envelope proteins (Env). DNA prime and boost with a combination of VLP and DNA vaccines followed by an rMVA boost induced over a 100-fold increase in Env-specific IgG antibody titers compared to three sequential immunizations with DNA and rMVA. Cellular immune responses were induced by VLP-DNA and rMVA vaccinations at high levels in CD8 T cells, CD4 T cells, and peripheral blood mononuclear cells secreting interferon (IFN)-γ, and spleen cells producing interleukin (IL)-2, 4, 5 cytokines. This study suggests that a DNA and VLP combination vaccine with MVA is a promising strategy in enhancing the efficacy of DNA-rMVA vaccination against HIV-1.

Intranasal Immunization with Influenza Virus-Like Particles Containing Membrane-Anchored Cholera Toxin B or Ricin Toxin B Enhances Adaptive Immune Responses and Protection against an Antigenically Distinct Virus.

PubMed

Ji, Xianliang; Ren, Zhiguang; Xu, Na; Meng, Lingnan; Yu, Zhijun; Feng, Na; Sang, Xiaoyu; Li, Shengnan; Li, Yuanguo; Wang, Tiecheng; Zhao, Yongkun; Wang, Hualei; Zheng, Xuexing; Jin, Hongli; Li, Nan; Yang, Songtao; Cao, Jinshan; Liu, Wensen; Gao, Yuwei; Xia, Xianzhu

2016-04-21

Vaccination is the most effective means to prevent influenza virus infection, although current approaches are associated with suboptimal efficacy. Here, we generated virus-like particles (VLPs) composed of the hemagglutinin (HA), neuraminidase (NA) and matrix protein (M1) of A/Changchun/01/2009 (H1N1) with or without either membrane-anchored cholera toxin B (CTB) or ricin toxin B (RTB) as molecular adjuvants. The intranasal immunization of mice with VLPs containing membrane-anchored CTB or RTB elicited stronger humoral and cellular immune responses when compared to mice immunized with VLPs alone. Administration of VLPs containing CTB or RTB significantly enhanced virus-specific systemic and mucosal antibody responses, hemagglutination inhibiting antibody titers, virus neutralizing antibody titers, and the frequency of virus-specific IFN-γ and IL-4 secreting splenocytes. VLPs with and without CTB or RTB conferred complete protection against lethal challenge with a mouse-adapted homologous virus. When challenged with an antigenically distinct H1N1 virus, all mice immunized with VLPs containing CTB or RTB survived whereas mice immunized with VLPs alone showed only partial protection (80% survival). Our results suggest that membrane-anchored CTB and RTB possess strong adjuvant properties when incorporated into an intranasally-delivered influenza VLP vaccine. Chimeric influenza VLPs containing CTB or RTB may represent promising vaccine candidates for improved immunological protection against homologous and antigenically distinct influenza viruses.

Intranasal Immunization with Influenza Virus-Like Particles Containing Membrane-Anchored Cholera Toxin B or Ricin Toxin B Enhances Adaptive Immune Responses and Protection against an Antigenically Distinct Virus

PubMed Central

Ji, Xianliang; Ren, Zhiguang; Xu, Na; Meng, Lingnan; Yu, Zhijun; Feng, Na; Sang, Xiaoyu; Li, Shengnan; Li, Yuanguo; Wang, Tiecheng; Zhao, Yongkun; Wang, Hualei; Zheng, Xuexing; Jin, Hongli; Li, Nan; Yang, Songtao; Cao, Jinshan; Liu, Wensen; Gao, Yuwei; Xia, Xianzhu

2016-01-01

Vaccination is the most effective means to prevent influenza virus infection, although current approaches are associated with suboptimal efficacy. Here, we generated virus-like particles (VLPs) composed of the hemagglutinin (HA), neuraminidase (NA) and matrix protein (M1) of A/Changchun/01/2009 (H1N1) with or without either membrane-anchored cholera toxin B (CTB) or ricin toxin B (RTB) as molecular adjuvants. The intranasal immunization of mice with VLPs containing membrane-anchored CTB or RTB elicited stronger humoral and cellular immune responses when compared to mice immunized with VLPs alone. Administration of VLPs containing CTB or RTB significantly enhanced virus-specific systemic and mucosal antibody responses, hemagglutination inhibiting antibody titers, virus neutralizing antibody titers, and the frequency of virus-specific IFN-γ and IL-4 secreting splenocytes. VLPs with and without CTB or RTB conferred complete protection against lethal challenge with a mouse-adapted homologous virus. When challenged with an antigenically distinct H1N1 virus, all mice immunized with VLPs containing CTB or RTB survived whereas mice immunized with VLPs alone showed only partial protection (80% survival). Our results suggest that membrane-anchored CTB and RTB possess strong adjuvant properties when incorporated into an intranasally-delivered influenza VLP vaccine. Chimeric influenza VLPs containing CTB or RTB may represent promising vaccine candidates for improved immunological protection against homologous and antigenically distinct influenza viruses. PMID:27110810

Murine Sarcoma Virus Gene Expression: Transformants Which Express Viral Envelope Glycoprotein In The Absence Of The Major Internal Protein And Infectious Particles

PubMed Central

Bilello, John A.; Strand, Mette; August, J. T.

1974-01-01

Expression of the major internal protein and the envelope glycoprotein of murine C-type viruses in focus-derived lines of normal rat kidney cells infected with Kirsten murine sarcoma virus was measured by radioimmunoassay. Of the clones selected, which do not produce virus particles or the major viral structural protein, approximately half express the viral envelope glycoprotein at concentrations found in productively infected cells. Expression of the envelope glycoprotein did not appear to alter significantly the properties of the transformed cells in culture. PMID:4370209

GB Virus Type C Envelope Protein E2 Elicits Antibodies That React with a Cellular Antigen on HIV-1 Particles and Neutralize Diverse HIV-1 Isolates

PubMed Central

Mohr, Emma L.; Xiang, Jinhua; McLinden, James H.; Kaufman, Thomas M.; Chang, Qing; Montefiori, David C.; Klinzman, Donna; Stapleton, Jack T.

2012-01-01

Broadly neutralizing Abs to HIV-1 are well described; however, identification of Ags that elicit these Abs has proven difficult. Persistent infection with GB virus type C (GBV-C) is associated with prolonged survival in HIV-1–infected individuals, and among those without HIV-1 viremia, the presence of Ab to GBV-C glycoprotein E2 is also associated with survival. GBV-C E2 protein inhibits HIV-1 entry, and an antigenic peptide within E2 interferes with gp41-induced membrane perturbations in vitro, suggesting the possibility of structural mimicry between GBV-C E2 protein and HIV-1 particles. Naturally occurring human and experimentally induced GBV-C E2 Abs were examined for their ability to neutralize infectious HIV-1 particles and HIV-1–enveloped pseudovirus particles. All GBV-C E2 Abs neutralized diverse isolates of HIV-1 with the exception of rabbit anti-peptide Abs raised against a synthetic GBV-C E2 peptide. Rabbit anti–GBV-C E2 Abs neutralized HIV-1–pseudotyped retrovirus particles but not HIV-1–pseudotyped vesicular stomatitis virus particles, and E2 Abs immune-precipitated HIV-1 gag particles containing the vesicular stomatitis virus type G envelope, HIV-1 envelope, GBV-C envelope, or no viral envelope. The Abs did not neutralize or immune-precipitate mumps or yellow fever viruses. Rabbit GBV-C E2 Abs inhibited HIV attachment to cells but did not inhibit entry following attachment. Taken together, these data indicate that the GBV-C E2 protein has a structural motif that elicits Abs that cross-react with a cellular Ag present on retrovirus particles, independent of HIV-1 envelope glycoproteins. The data provide evidence that a heterologous viral protein can induce HIV-1–neutralizing Abs. PMID:20826757

Monovalent virus-like particle vaccine protects guinea pigs and nonhuman primates against infection with multiple Marburg viruses.

PubMed

Swenson, Dana L; Warfield, Kelly L; Larsen, Tom; Alves, D Anthony; Coberley, Sadie S; Bavari, Sina

2008-05-01

Virus-like particle (VLP)-based vaccines have the advantage of being morphologically and antigenically similar to the live virus from which they are derived. Expression of the glycoprotein and VP40 matrix protein from Lake Victoria marburgvirus (MARV) results in spontaneous production of VLPs in mammalian cells. Guinea pigs vaccinated with Marburg virus VLPs (mVLPs) or inactivated MARV (iMARV) develop homologous humoral and T-cell responses and are completely protected from a lethal homologous MARV challenge. To determine whether mVLPs based on the Musoke (aka Lake Victoria) isolate of MARV could broadly protect against diverse isolates of MARV, guinea pigs were vaccinated with mVLPs or iMARV-Musoke and challenged with MARV-Musoke, -Ravn or -Ci67. Prior to challenge, the mVLP- and iMARV-vaccinated guinea pigs had high levels of homologous MARV-Musoke and heterologous MARV-Ravn and -Ci67 antibodies. The Musoke-based mVLPs and iMARV vaccines provided complete protection in guinea pigs against viremia, viral replication and pathological changes in tissues, and lethal disease following challenge with MARV-Musoke, -Ravn or -Ci67. Guinea pigs vaccinated with RIBI adjuvant alone and infected with guinea pig-adapted MARV-Musoke, -Ravn or -Ci67 had histopathologic findings similar to those seen in the nonhuman primate model for MARV infection. Based on the strong protection observed in guinea pigs, we next vaccinated cynomolgus macaques with Musoke-based mVLPs and showed the VLP-vaccinated monkeys were broadly protected against three isolates of MARV (Musoke, Ravn and Ci67). Musoke mVLPs are effective at inducing broad heterologous immunity and protection against multiple MARV isolates.

Immunogenicity of plant-produced African horse sickness virus-like particles: implications for a novel vaccine.

PubMed

Dennis, Susan J; Meyers, Ann E; Guthrie, Alan J; Hitzeroth, Inga I; Rybicki, Edward P

2018-02-01

African horse sickness (AHS) is a debilitating and often fatal viral disease affecting horses in much of Africa, caused by the dsRNA orbivirus African horse sickness virus (AHSV). Vaccination remains the single most effective weapon in combatting AHS, as there is no treatment for the disease apart from good animal husbandry. However, the only commercially available vaccine is a live-attenuated version of the virus (LAV). The threat of outbreaks of the disease outside its endemic region and the fact that the LAV is not licensed for use elsewhere in the world, have spurred attempts to develop an alternative safer, yet cost-effective recombinant vaccine. Here, we report the plant-based production of a virus-like particle (VLP) AHSV serotype five candidate vaccine by Agrobacterium tumefaciens-mediated transient expression of all four capsid proteins in Nicotiana benthamiana using the cowpea mosaic virus-based HyperTrans (CPMV-HT) and associated pEAQ plant expression vector system. The production process is fast and simple, scalable, economically viable, and most importantly, guinea pig antiserum raised against the vaccine was shown to neutralize live virus in cell-based assays. To our knowledge, this is the first report of AHSV VLPs produced in plants, which has important implications for the containment of, and fight against the spread of, this deadly disease. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Interferon-induced Sus scrofa Mx1 blocks endocytic traffic of incoming influenza A virus particles.

PubMed

Palm, Mélanie; Garigliany, Mutien-Marie; Cornet, François; Desmecht, Daniel

2010-01-01

The interferon-induced Mx proteins of vertebrates are dynamin-like GTPases, some isoforms of which can additionally inhibit the life cycle of certain RNA viruses. Here we show that the porcine Mx1 protein (poMx1) inhibits replication of influenza A virus and we attempt to identify the step at which the viral life cycle is blocked. In infected cells expressing poMx1, the level of transcripts encoding the viral nucleoprotein is significantly lower than normal, even when secondary transcription is prevented by exposure to cycloheximide. This reveals that a pretranscriptional block participates to the anti-influenza activity. Binding and internalization of incoming virus particles are normal in the presence of poMx1 but centripetal traffic to the late endosomes is interrupted. Surprisingly but decisively, poMx1 significantly alters binding of early endosome autoantigen 1 to early endosomes and/or early endosome size and spatial distribution. This is compatible with impairment of traffic of the endocytic vesicles to the late endosomes. INRA, EDP Sciences, 2010.

Norovirus Narita 104 Virus-Like Particles Expressed in Nicotiana benthamiana Induce Serum and Mucosal Immune Responses

PubMed Central

Mathew, Lolita George; Herbst-Kralovetz, Melissa M.; Mason, Hugh S.

2014-01-01

Narita 104 virus is a human pathogen belonging to the norovirus (family Caliciviridae) genogroup II. Noroviruses cause epidemic gastroenteritis worldwide. To explore the potential of developing a plant-based vaccine, a plant optimized gene encoding Narita 104 virus capsid protein (NaVCP) was expressed transiently in Nicotiana benthamiana using a tobacco mosaic virus expression system. NaVCP accumulated up to approximately 0.3 mg/g fresh weight of leaf at 4 days postinfection. Initiation of hypersensitive response-like symptoms followed by tissue necrosis necessitated a brief infection time and was a significant factor limiting expression. Transmission electron microscopy of plant-derived NaVCP confirmed the presence of fully assembled virus-like particles (VLPs). In this study, an optimized method to express and partially purify NaVCP is described. Further, partially purified NaVCP was used to immunize mice by intranasal delivery and generated significant mucosal and serum antibody responses. Thus, plant-derived Narita 104 VLPs have potential for use as a candidate subunit vaccine or as a component of a multivalent subunit vaccine, along with other genotype-specific plant-derived VLPs. PMID:24949472

Venezuelan Equine Encephalitis Virus Replicon Particle Vaccine Protects Nonhuman Primates from Intramuscular and Aerosol Challenge with Ebolavirus

PubMed Central

Herbert, Andrew S.; Kuehne, Ana I.; Barth, James F.; Ortiz, Ramon A.; Nichols, Donald K.; Zak, Samantha E.; Stonier, Spencer W.; Muhammad, Majidat A.; Bakken, Russell R.; Prugar, Laura I.; Olinger, Gene G.; Groebner, Jennifer L.; Lee, John S.; Pratt, William D.; Custer, Max; Kamrud, Kurt I.; Smith, Jonathan F.; Hart, Mary Kate

2013-01-01

There are no vaccines or therapeutics currently approved for the prevention or treatment of ebolavirus infection. Previously, a replicon vaccine based on Venezuelan equine encephalitis virus (VEEV) demonstrated protective efficacy against Marburg virus in nonhuman primates. Here, we report the protective efficacy of Sudan virus (SUDV)- and Ebola virus (EBOV)-specific VEEV replicon particle (VRP) vaccines in nonhuman primates. VRP vaccines were developed to express the glycoprotein (GP) of either SUDV or EBOV. A single intramuscular vaccination of cynomolgus macaques with VRP expressing SUDV GP provided complete protection against intramuscular challenge with SUDV. Vaccination against SUDV and subsequent survival of SUDV challenge did not fully protect cynomolgus macaques against intramuscular EBOV back-challenge. However, a single simultaneous intramuscular vaccination with VRP expressing SUDV GP combined with VRP expressing EBOV GP did provide complete protection against intramuscular challenge with either SUDV or EBOV in cynomolgus macaques. Finally, intramuscular vaccination with VRP expressing SUDV GP completely protected cynomolgus macaques when challenged with aerosolized SUDV, although complete protection against aerosol challenge required two vaccinations with this vaccine. PMID:23408633

Venezuelan equine encephalitis virus replicon particle vaccine protects nonhuman primates from intramuscular and aerosol challenge with ebolavirus.

PubMed

Herbert, Andrew S; Kuehne, Ana I; Barth, James F; Ortiz, Ramon A; Nichols, Donald K; Zak, Samantha E; Stonier, Spencer W; Muhammad, Majidat A; Bakken, Russell R; Prugar, Laura I; Olinger, Gene G; Groebner, Jennifer L; Lee, John S; Pratt, William D; Custer, Max; Kamrud, Kurt I; Smith, Jonathan F; Hart, Mary Kate; Dye, John M

2013-05-01

There are no vaccines or therapeutics currently approved for the prevention or treatment of ebolavirus infection. Previously, a replicon vaccine based on Venezuelan equine encephalitis virus (VEEV) demonstrated protective efficacy against Marburg virus in nonhuman primates. Here, we report the protective efficacy of Sudan virus (SUDV)- and Ebola virus (EBOV)-specific VEEV replicon particle (VRP) vaccines in nonhuman primates. VRP vaccines were developed to express the glycoprotein (GP) of either SUDV or EBOV. A single intramuscular vaccination of cynomolgus macaques with VRP expressing SUDV GP provided complete protection against intramuscular challenge with SUDV. Vaccination against SUDV and subsequent survival of SUDV challenge did not fully protect cynomolgus macaques against intramuscular EBOV back-challenge. However, a single simultaneous intramuscular vaccination with VRP expressing SUDV GP combined with VRP expressing EBOV GP did provide complete protection against intramuscular challenge with either SUDV or EBOV in cynomolgus macaques. Finally, intramuscular vaccination with VRP expressing SUDV GP completely protected cynomolgus macaques when challenged with aerosolized SUDV, although complete protection against aerosol challenge required two vaccinations with this vaccine.

Quantitative nanoscale electrostatics of viruses.

PubMed

Hernando-Pérez, M; Cartagena-Rivera, A X; Lošdorfer Božič, A; Carrillo, P J P; San Martín, C; Mateu, M G; Raman, A; Podgornik, R; de Pablo, P J

2015-11-07

Electrostatics is one of the fundamental driving forces of the interaction between biomolecules in solution. In particular, the recognition events between viruses and host cells are dominated by both specific and non-specific interactions and the electric charge of viral particles determines the electrostatic force component of the latter. Here we probe the charge of individual viruses in liquid milieu by measuring the electrostatic force between a viral particle and the Atomic Force Microscope tip. The force spectroscopy data of co-adsorbed ϕ29 bacteriophage proheads and mature virions, adenovirus and minute virus of mice capsids is utilized for obtaining the corresponding density of charge for each virus. The systematic differences of the density of charge between the viral particles are consistent with the theoretical predictions obtained from X-ray structural data. Our results show that the density of charge is a distinguishing characteristic of each virus, depending crucially on the nature of the viral capsid and the presence/absence of the genetic material.

POWASSAN VIRUS: MORPHOLOGY AND CYTOPATHOLOGY.

PubMed

ABDELWAHAB, K S; ALMEIDA, J D; DOANE, F W; MCLEAN, D M

1964-05-02

Powassan virus, a North American tickborne group B arbovirus, multiplied after simultaneous inoculation into bottles or tubes of virus and trypsinized suspension of continuous-line cultures of rhesus monkey kidney cells, strain LLC-MK2. Cytopathic effects comprising cell rounding and cytoplasmic vacuolation were first observed five days after inoculation. Mixture of Powassan antiserum with virus before inoculation into tissue cultures inhibited the appearance of cytopathic effects. Hemagglutinins for rooster erythrocytes, optimally at pH 6.4 and 22 degrees C., first appeared in tissue culture supernatant fluids four days after inoculation.Electron microscopic observation of thin sections of infected tissue culture cells showed virus particles 360-380 A.U. along outer cell membranes and edges of cytoplasmic vacuoles. In phosphotungstic acid negatively stained preparations, intact virus particles, 400-450 A.U. total diameter, were observed inside infected cells. In particles in which the peripheral layer became discontinuous, geometrically arranged subunits compatible with cubic symmetry were observed.

A novel H6N1 virus-like particle vaccine induces long-lasting cross-clade antibody immunity against human and avian H6N1 viruses.

PubMed

Yang, Ji-Rong; Chen, Chih-Yuan; Kuo, Chuan-Yi; Cheng, Chieh-Yu; Lee, Min-Shiuh; Cheng, Ming-Chu; Yang, Yu-Chih; Wu, Chia-Ying; Wu, Ho-Sheng; Liu, Ming-Tsan; Hsiao, Pei-Wen

2016-02-01

Avian influenza A(H6N1) virus is one of the most common viruses isolated from migrating birds and domestic poultry in many countries. The first and only known case of human infection by H6N1 virus in the world was reported in Taiwan in 2013. This led to concern that H6N1 virus may cause a threat to public health. In this study, we engineered a recombinant H6N1 virus-like particle (VLP) and investigated its vaccine effectiveness compared to the traditional egg-based whole inactivated virus (WIV) vaccine. The H6N1-VLPs exhibited similar morphology and functional characteristics to influenza viruses. Prime-boost intramuscular immunization in mice with unadjuvanted H6N1-VLPs were highly immunogenic and induced long-lasting antibody immunity. The functional activity of the VLP-elicited IgG antibodies was proved by in vitro seroprotective hemagglutination inhibition and microneutralization titers against the homologous human H6N1 virus, as well as in vivo viral challenge analyses which showed H6N1-VLP immunization significantly reduced viral load in the lung, and protected against human H6N1 virus infection. Of particular note, the H6N1-VLPs but not the H6N1-WIVs were able to confer cross-reactive humoral immunity; antibodies induced by H6N1-VLP vaccine robustly inhibited the hemagglutination activities and in vitro replication of distantly-related heterologous avian H6N1 viruses. Furthermore, the H6N1-VLPs were found to elicit significantly greater anti-HA2 antibody responses in immunized mice than H6N1-WIVs. Collectively, we demonstrated for the first time a novel H6N1-VLP vaccine that effectively provides broadly protective immunity against both human and avian H6N1 viruses. These results, which uncover the underlying mechanisms for induction of wide-range immunity against influenza viruses, may be useful for future influenza vaccine development. Copyright © 2015 Elsevier B.V. All rights reserved.

Nascent starbursts: a missing link in galaxy evolution

NASA Astrophysics Data System (ADS)

Roussel, Helene; Beck, Rainer; Condon, Jim; Helou, George; Smith, John-David

2005-06-01

We have identified a rare category of galaxies characterized by an extreme deficiency in synchro- tron radiation, relative to dust emission, and very high dust temperatures. We studied in detail the most extreme such object, and concluded in favor of a starburst just breaking out, less than one megayear old, in a galaxy having undergone no major star formation episode in the last 100 Myr. Such systems offer a perfect setting to study the initial conditions and early dynamics of starbursts and understand better the regulation of the infrared-radio continuum correlation in galaxies. For the prototypical nascent starburst, the mid-infrared spectrum is quite peculiar, suggesting tran- sient dust species and high optical depth; tracers of dust and molecular gas are the only indicators of unusual activity, and the active regions are likely very compact and dust-bounded, suppressing ionization. Only Spitzer data can provide the needed physical diagnostics for such regions. A sample of 25 nascent starbursts was drawn from the cross-correlation of the IRAS Faint Source Catalog and the NVSS VLA radio survey, and carefully selected based on our multi-wavelength VLA maps to span a range of infrared to radio ratios and luminosities. This sample allows a first step beyond studying prototypes toward a statistical analysis addressing systematic physical pro- perties, classification and search for starburst development sequences. We propose imaging and spectroscopic observations from 3 to 160 microns to characterize the state of the interstellar medium and the gas and dust excitation origin. Our aim is to learn from these unique systems how a star formation burst may develop in its very earliest phases, how it affects the fueling material and the host galaxy. Acquired observations of the radio continuum, cold molecular gas and tracers of shocks and HII regions will help us interpret the rich Spitzer data set and extract a coherent picture of the interstellar medium in our targets.

Fullerene-like organization of HIV gag-protein shell in virus-like particles produced by recombinant baculovirus.

PubMed

Nermut, M V; Hockley, D J; Jowett, J B; Jones, I M; Garreau, M; Thomas, D

1994-01-01

Virus-like particles produced by a recombinant baculovirus containing the HIV gag gene were examined by negative staining after delipidization. This technique demonstrated that the gag-protein shell consisted of radially arranged short rods which formed a network of ring-like structures. Similar structures were observed at the plasma membrane of infected cells which had been opened by wet-cleaving. Occasionally five or six subunits were observed forming a ring. These findings suggest that the gag-encoded precursor (pr55) is a rod-like molecule about 34 A in diameter and 85 A in length. A protein cylinder of such dimensions would have a molecular weight of 56K. The center-to-center distance of two neighboring rings formed by the rods was 66 +/- 8 A (N = 200) by direct measurements and 65 A as obtained from averaged images. This morphology and these dimensions indicate that the virus-like particles contain the gag precursor in the form of a near-spherical "fullerene-like" icosahedral shell. Our data indicate that the triangulation number of the rings equals 63. However, since one rod of pr55 is shared by two rings, the number of copies of the precursor will be 1890 as opposed to 2522 if the molecules were closely packed. The particle diameter of 102 nm deduced from the proposed model was close to the diameter obtained from thin sections of low-temperature-embedded specimens (103-108 nm).

Engineering RNA phage MS2 virus-like particles for peptide display

NASA Astrophysics Data System (ADS)

Jordan, Sheldon Keith

Phage display is a powerful and versatile technology that enables the selection of novel binding functions from large populations of randomly generated peptide sequences. Random sequences are genetically fused to a viral structural protein to produce complex peptide libraries. From a sufficiently complex library, phage bearing peptides with practically any desired binding activity can be physically isolated by affinity selection, and, since each particle carries in its genome the genetic information for its own replication, the selectants can be amplified by infection of bacteria. For certain applications however, existing phage display platforms have limitations. One such area is in the field of vaccine development, where the goal is to identify relevant epitopes by affinity-selection against an antibody target, and then to utilize them as immunogens to elicit a desired antibody response. Today, affinity selection is usually conducted using display on filamentous phages like M13. This technology provides an efficient means for epitope identification, but, because filamentous phages do not display peptides in the high-density, multivalent arrays the immune system prefers to recognize, they generally make poor immunogens and are typically useless as vaccines. This makes it necessary to confer immunogenicity by conjugating synthetic versions of the peptides to more immunogenic carriers. Unfortunately, when introduced into these new structural environments, the epitopes often fail to elicit relevant antibody responses. Thus, it would be advantageous to combine the epitope selection and immunogen functions into a single platform where the structural constraints present during affinity selection can be preserved during immunization. This dissertation describes efforts to develop a peptide display system based on the virus-like particles (VLPs) of bacteriophage MS2. Phage display technologies rely on (1) the identification of a site in a viral structural protein that is

Molecular packing in virus crystals: geometry, chemistry, and biology.

PubMed

Natarajan, P; Johnson, J E

1998-01-01

An automated procedure was developed to determine the geometrical and chemical interactions of crystalline virus particles using the crystal parameters, particle position, orientation, and atomic coordinates for an icosahedral asymmetric unit. Two applications of the program are reported: (1) An analysis of a novel pseudo-rhombohedral (R32) symmetry present in the monoclinic crystal lattices of both Nodamura Virus (NOV) and Coxsackie virus B3 (CVB3). The study shows that in both cases the interactions between particles is substantially increased by minor deviations from exact R32 symmetry and that only particles with the proper ratio of dimensions along twofold and fivefold symmetry axes (such as southern bean mosaic virus) can achieve comparable buried surface area in the true R32 space group. (2) An attempt was made to correlate biological function with remarkably conserved interparticle contact regions found in different crystal forms of three members of the nodavirus family, NOV, Flock House Virus (FHV), and Black Beetle Virus (BBV). Mutational evidence implicates the quasi-threefold region on the viral surface in receptor binding in nodaviruses and this region is dominant in particle contacts in all three virus crystals. Examination of particle contacts in numerous crystal structures of viruses in the picornavirus super-family showed that portions of the capsid surface known to interact with a receptor or serve as an epitope for monoclonal antibodies frequently stabilize crystal contacts.

Giant Syncytia and Virus-Like Particles in Ovarian Carcinoma Cells Isolated from Ascites Fluid

PubMed Central

Rakowicz-Szulczynska, Eva M.; McIntosh, David G.; Smith, McClure L.

1999-01-01

Ovarian cancer cells were isolated from ascites fluid of 30 different patients diagnosed with cystadenocarcinoma of ovaries. Large colonies of malignant ASC cells were observed during the first week of cell growth in vitro. Colony formation was followed by fusion of cells and formation of large multinucleated and highly vacuolated syncytia. In contrast, cells isolated from the ascites fluid produced by patients with benign mucinous cystadenoma of ovaries did not form syncytia. Nonmalignant Brenner tumor cells, isolated from the ascites fluid, also did not form syncytia. Syncytia, but not the nonmalignant tumor cells, were immunofluorescence stained with an anti-human immunodeficiency virus type 1 (HIV-1) gp120 monoclonal antibody (MAb) and MAb RAK-BrI. Both MAbs recognized cancer-associated antigens RAK (for Rakowicz markers) p120, p42, and p25. Exposure of ASC cells to either the anti-HIV-1 gp120 MAb or MAb RAK-BrI inhibited syncytium formation. PCR with HIV-1 Env-derived primers revealed DNA sequences with over 90% homology to HIV-1 gp41 in syncytia and in ovarian cancer cells but not in normal ovary cells. Electron microscopic analysis revealed viral particles, hexagonal in shape (90 nm in diameter), with a dense central core surrounded by an inner translucent capsid and dense outer shell with projections. Negative staining detected membrane-covered particles (100 to 110 nm in diameter) in the cell culture medium. Incubation of normal breast cells with viral particles resulted in drastic morphological changes and syncytium formation by the transformed breast cells. The cytopathic effects of the identified virus resembled those of spumaviruses, which, in addition to their epitopic and genetic homology to HIV-1, might suggest a common phylogeny. PMID:9874674

Giant syncytia and virus-like particles in ovarian carcinoma cells isolated from ascites fluid.

PubMed

Rakowicz-Szulczynska, E M; McIntosh, D G; Smith, M L

1999-01-01

Ovarian cancer cells were isolated from ascites fluid of 30 different patients diagnosed with cystadenocarcinoma of ovaries. Large colonies of malignant ASC cells were observed during the first week of cell growth in vitro. Colony formation was followed by fusion of cells and formation of large multinucleated and highly vacuolated syncytia. In contrast, cells isolated from the ascites fluid produced by patients with benign mucinous cystadenoma of ovaries did not form syncytia. Nonmalignant Brenner tumor cells, isolated from the ascites fluid, also did not form syncytia. Syncytia, but not the nonmalignant tumor cells, were immunofluorescence stained with an anti-human immunodeficiency virus type 1 (HIV-1) gp120 monoclonal antibody (MAb) and MAb RAK-BrI. Both MAbs recognized cancer-associated antigens RAK (for Rakowicz markers) p120, p42, and p25. Exposure of ASC cells to either the anti-HIV-1 gp120 MAb or MAb RAK-BrI inhibited syncytium formation. PCR with HIV-1 Env-derived primers revealed DNA sequences with over 90% homology to HIV-1 gp41 in syncytia and in ovarian cancer cells but not in normal ovary cells. Electron microscopic analysis revealed viral particles, hexagonal in shape (90 nm in diameter), with a dense central core surrounded by an inner translucent capsid and dense outer shell with projections. Negative staining detected membrane-covered particles (100 to 110 nm in diameter) in the cell culture medium. Incubation of normal breast cells with viral particles resulted in drastic morphological changes and syncytium formation by the transformed breast cells. The cytopathic effects of the identified virus resembled those of spumaviruses, which, in addition to their epitopic and genetic homology to HIV-1, might suggest a common phylogeny.

Immunogenic Subviral Particles Displaying Domain III of Dengue 2 Envelope Protein Vectored by Measles Virus

PubMed Central

Harahap-Carrillo, Indira S.; Ceballos-Olvera, Ivonne; Reyes-del Valle, Jorge

2015-01-01

Vaccines against dengue virus (DV) are commercially nonexistent. A subunit vaccination strategy may be of value, especially if a safe viral vector acts as biologically active adjuvant. In this paper, we focus on an immunoglobulin-like, independently folded domain III (DIII) from DV 2 envelope protein (E), which contains epitopes that elicits highly specific neutralizing antibodies. We modified the hepatitis B small surface antigen (HBsAg, S) in order to display DV 2 DIII on a virus-like particle (VLP), thus generating the hybrid antigen DIII-S. Two varieties of measles virus (MV) vectors were developed to express DIII-S. The first expresses the hybrid antigen from an additional transcription unit (ATU) and the second additionally expresses HBsAg from a separate ATU. We found that this second MV vectoring the hybrid VLPs displaying DIII-S on an unmodified HBsAg scaffold were immunogenic in MV-susceptible mice (HuCD46Ge-IFNarko), eliciting robust neutralizing responses (averages) against MV (1:1280 NT90), hepatitis B virus (787 mIU/mL), and DV2 (1:160 NT50) in all of the tested animals. Conversely, the MV vector expressing only DIII-S induced immunity against MV alone. In summary, DV2 neutralizing responses can be generated by displaying E DIII on a scaffold of HBsAg-based VLPs, vectored by MV. PMID:26350592

Robust production of virus-like particles and monoclonal antibodies with geminiviral replicon vectors in lettuce

PubMed Central

Lai, Huafang; He, Junyun; Engle, Michael; Diamond, Michael S.; Chen, Qiang

2011-01-01

Summary Pharmaceutical protein production in plants has been greatly promoted by the development of viral-based vectors and transient expression systems. Tobacco and related Nicotiana species are currently the most common host plants for generation of plant-made pharmaceutical proteins (PMPs). Downstream processing of target PMPs from these plants, however, is hindered by potential technical and regulatory difficulties due to the presence of high levels of phenolics and toxic alkaloids. Here, we explored the use of lettuce, which grows quickly yet produces low levels of secondary metabolites, and viral vector-based transient expression systems to develop a robust PMP production platform. Our results showed that a geminiviral replicon system based on the bean yellow dwarf virus permits high-level expression in lettuce of virus-like particles (VLP) derived from the Norwalk virus capsid protein and therapeutic monoclonal antibodies (mAbs) against Ebola and West Nile viruses. These vaccine and therapeutic candidates can be readily purified from lettuce leaves with scalable processing methods while fully retaining functional activity. Furthermore, this study also demonstrated the feasibility of using commercially produced lettuce for high-level PMP production. This allows our production system to have access to unlimited quantities of inexpensive plant material for large-scale production. These results establish a new production platform for biological pharmaceutical agents that is effective, safe, low-cost, and amenable to large-scale manufacturing. PMID:21883868

A Replication-incompetent Rift Valley Fever Vaccine: Chimeric Virus-like Particles Protect Mice and Rats Against Lethal Challenge

PubMed Central

Mandell, Robert B.; Koukuntla, Ramesh; Mogler, Laura J. K.; Carzoli, Andrea K.; Freiberg, Alexander N.; Holbrook, Michael R.; Martin, Brian K.; Staplin, William R.; Vahanian, Nicholas N.; Link, Charles J.; Flick, Ramon

2009-01-01

Virus-like particles (VLPs) present viral antigens in a native conformation and are effectively recognized by the immune system and therefore are considered as suitable and safe vaccine candidates against many viral diseases. Here we demonstrate that chimeric VLPs containing Rift Valley fever virus (RVFV) glycoproteins GN and GC, nucleoprotein N and the gag protein of Moloney murine leukemia virus represent an effective vaccine candidate against Rift Valley fever, a deadly disease in humans and livestock. Long-lasting humoral and cellular immune responses are demonstrated in a mouse model by the analysis of neutralizing antibody titers and cytokine secretion profiles. Vaccine efficacy studies were performed in mouse and rat lethal challenge models resulting in high protection rates. Taken together, these results demonstrate that replication-incompetent chimeric RVF VLPs are an efficient RVFV vaccine candidate. PMID:19932911

Effect of Shock Waves Generated by Pulsed Electric Discharges in Water on Yeast Cells and Virus Particles

NASA Astrophysics Data System (ADS)

Girdyuk, A. E.; Gorshkov, A. N.; Egorov, V. V.; Kolikov, V. A.; Snetov, V. N.; Shneerson, G. A.

2018-02-01

The aim of this study is to determine the optimal parameters of the electric pulses and shock waves generated by them for the soft destruction of the virus and yeast envelopes with no changes in the structure of antigenic surface albumin and in the cell morphology in order to use them to produce antivirus vaccines and in biotechnology. The pulse electric discharges in water have been studied for different values of amplitude, pulse duration and the rate of the rise in the current. A mathematical model has been developed to estimate the optimal parameters of pulsed electric charges and shock waves for the complete destruction of the yeast cell envelopes and virus particles at a minimum of pulses.

Electron microscopy of two viruses of deadly nightshade (Atropa belladonna L.).

PubMed

Fránová, J

2000-02-01

Deadly nightshade plants showing severe necrotic lesions on leaves were observed in southern Bohemia. In negatively stained preparations of spontaneously infected deadly nightshade, artificially inoculated host plants and purified preparations two types of virus particles, isometric ones of about 26 nm in diameter and flexuous ones with length of 765 nm were seen by electron microscopy. The virus with isometric particles was identified as belladonna mottle virus (BMV), indistinguishable serologically from the Hungarian isolate of this virus. Identification of the virus with flexuous particles is discussed. Observations of the ultrastructure revealed the presence of filamentous virus particle aggregates and chloroplasts with peripheral vesicles bounded by double membranes, a feature typical for tymoviruses.

Templated Formation of Luminescent Virus-like Particles by Tailor-Made Pt(II) Amphiphiles

PubMed Central

2018-01-01

Virus-like particles (VLPs) have been created from luminescent Pt(II) complex amphiphiles, able to form supramolecular structures in water solutions, that can be encapsulated or act as templates of cowpea chlorotic mottle virus capsid proteins. By virtue of a bottom-up molecular design, icosahedral and nonicosahedral (rod-like) VLPs have been constructed through diverse pathways, and a relationship between the molecular structure of the complexes and the shape and size of the VLPs has been observed. A deep insight into the mechanism for the templated formation of the differently shaped VLPs was achieved, by electron microscopy measurements (TEM and STEM) and bulk analysis (FPLC, DLS, photophysical investigations). Interestingly, the obtained VLPs can be visualized by their intense emission at room temperature, generated by the self-assembly of the Pt(II) complexes. The encapsulation of the luminescent species is further verified by their higher emission quantum yields inside the VLPs, which is due to the confinement effect of the protein cage. These hybrid materials demonstrate the potential of tailor-made supramolecular systems able to control the assembly of biological building blocks. PMID:29357236

Templated Formation of Luminescent Virus-like Particles by Tailor-Made Pt(II) Amphiphiles.

PubMed

Sinn, Stephan; Yang, Liulin; Biedermann, Frank; Wang, Di; Kübel, Christian; Cornelissen, Jeroen J L M; De Cola, Luisa

2018-02-14

Virus-like particles (VLPs) have been created from luminescent Pt(II) complex amphiphiles, able to form supramolecular structures in water solutions, that can be encapsulated or act as templates of cowpea chlorotic mottle virus capsid proteins. By virtue of a bottom-up molecular design, icosahedral and nonicosahedral (rod-like) VLPs have been constructed through diverse pathways, and a relationship between the molecular structure of the complexes and the shape and size of the VLPs has been observed. A deep insight into the mechanism for the templated formation of the differently shaped VLPs was achieved, by electron microscopy measurements (TEM and STEM) and bulk analysis (FPLC, DLS, photophysical investigations). Interestingly, the obtained VLPs can be visualized by their intense emission at room temperature, generated by the self-assembly of the Pt(II) complexes. The encapsulation of the luminescent species is further verified by their higher emission quantum yields inside the VLPs, which is due to the confinement effect of the protein cage. These hybrid materials demonstrate the potential of tailor-made supramolecular systems able to control the assembly of biological building blocks.

New perspectives on virus detection in shellfish: hemocytes as a source of concentrated virus

USDA-ARS?s Scientific Manuscript database

USDA ARS research indicates that circulating phagocytic cells (hemocytes) within oysters retain virus particles. We find that persistence of hepatitis A virus (HAV) within oyster hemocytes correlates with the presence of virus within whole oysters. Since bivalve shellfish have no self-nonself immun...

Crystal structures of the reverse transcriptase-associated ribonuclease H domain of xenotropic murine leukemia-virus related virus

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

Zhou, Dongwen; Chung, Suhman; Miller, Maria

2012-06-19

The ribonuclease H (RNase H) domain of retroviral reverse transcriptase (RT) plays a critical role in the life cycle by degrading the RNA strands of DNA/RNA hybrids. In addition, RNase H activity is required to precisely remove the RNA primers from nascent (-) and (+) strand DNA. We report here three crystal structures of the RNase H domain of xenotropic murine leukemia virus-related virus (XMRV) RT, namely (i) the previously identified construct from which helix C was deleted, (ii) the intact domain, and (iii) the intact domain complexed with an active site {alpha}-hydroxytropolone inhibitor. Enzymatic assays showed that the intactmore » RNase H domain retained catalytic activity, whereas the variant lacking helix C was only marginally active, corroborating the importance of this helix for enzymatic activity. Modeling of the enzyme-substrate complex elucidated the essential role of helix C in binding a DNA/RNA hybrid and its likely mode of recognition. The crystal structure of the RNase H domain complexed with {beta}-thujaplicinol clearly showed that coordination by two divalent cations mediates recognition of the inhibitor.« less

TTSV1, a new virus-like particle isolated from the hyperthermophilic crenarchaeote Thermoproteus tenax.

PubMed

Ahn, Dae-Gyun; Kim, Se-Il; Rhee, Jin-Kyu; Kim, Kwang Pyo; Pan, Jae-Gu; Oh, Jong-Won

2006-08-01

A new virus-like particle TTSV1 was isolated from the hyperthermophilic crenarchaeote Thermoproteus tenax sampled at a hot spring region in Indonesia. TTSV1 had a spherical shape with a diameter of approximately 70 nm and was morphologically similar to the PSV isolated from a strain of Pyrobaculum. The 21.6 kb linear double-stranded DNA genome of TTSV1 had 38 open reading frames (ORFs), of which 15 ORFs were most similar to those of PSV. The remaining 23 ORFs showed little similarity to proteins in the public databases. Southern blot analysis demonstrated that the viral genome is not integrated into the host chromosome. TTSV1 consisted of three putative structural proteins of 10, 20, and 35 kDa in size, and the 10-kDa major protein was identified by mass spectrometry as a TTSV1 gene product. TTSV1 could be assigned as a new member of the newly emerged Globuloviridae family that includes so far only one recently characterized virus PSV.

The Fluid Dynamics of Nascent Biofilms

NASA Astrophysics Data System (ADS)

Farthing, Nicola; Snow, Ben; Wilson, Laurence; Bees, Martin

2017-11-01

Many anti-biofilm approaches target mature biofilms with biochemical or physio-chemical interventions. We investigate the mechanics of interventions at an early stage that aim to inhibit biofilm maturation, focusing on hydrodynamics as cells transition from planktonic to surface-attached. Surface-attached cells generate flow fields that are relatively long-range compared with cells that are freely-swimming. We look at the effect of these flows on the biofilm formation. In particular, we use digital inline holographic microscopy to determine the three-dimensional flow due to a surface-attached cell and the effect this flow has on both tracers and other cells in the fluid. We compare experimental data with two models of cells on boundaries. The first approach utilizes slender body theory and captures many of the features of the experimental field. The second model develops a simple description in terms of singularity solutions of Stokes' flow, which produces qualitatively similar dynamics to both the experiments and more complex model but with significant computational savings. The range of validity of multiple cell arrangements is investigated. These two descriptions can be used to investigate the efficacy of actives developed by Unilever on nascent biofilms.

Characterization of a small (25-kilodalton) derivative of the Rous sarcoma virus Gag protein competent for particle release.

PubMed Central

Weldon, R A; Wills, J W

1993-01-01

Retroviral Gag proteins have the ability to induce budding and particle release from the plasma membrane when expressed in the absence of all of the other virus-encoded components; however, the locations of the functional domains within the Gag protein that are important for this process are poorly understood. It was shown previously that the protease sequence of the Rous sarcoma virus (RSV) Gag protein can be replaced with a foreign polypeptide, iso-1-cytochrome c from a yeast, without disrupting particle assembly (R. A. Weldon, Jr., C. R. Erdie, M. G. Oliver, and J. W. Wills, J. Virol. 64:4169-4179, 1990). An unexpected product of the chimeric gag gene is a small, Gag-related protein named p25C. This product was of interest because of its high efficiency of packaging into particles. The goal of the experiments described here was to determine the mechanism by which p25C is synthesized and packaged into particles. The results demonstrate that it is not the product of proteolytic processing of the Gag-cytochrome precursor but is derived from an unusual spliced mRNA. cDNA clones of the spliced mRNA were obtained, and each expressed a product of approximately 25 kDa, designated p25M1, which was released into the growth medium in membrane-enclosed particles that were much lighter than authentic retrovirions as measured in sucrose density gradients. DNA sequencing revealed that the clones encode the first 180 of the 701 amino acids of the RSV Gag protein and no residues from iso-1-cytochrome c. This suggested that a domain in the carboxy-terminal half of Gag is important for the packaging of Gag proteins into dense arrays within the particles. In support of this hypothesis, particles of the correct density were obtained when a small segment from the carboxy terminus of the RSV Gag protein (residues 417 to 584) was included on the end of p25. Images PMID:8394460

Powassan Virus: Morphology and Cytopathology

PubMed Central

Abdelwahab, K. S. E.; Almeida, J. D.; Doane, F. W.; McLean, D. M.

1964-01-01

Powassan virus, a North American tickborne group B arbovirus, multiplied after simultaneous inoculation into bottles or tubes of virus and trypsinized suspension of continuous-line cultures of rhesus monkey kidney cells, strain LLC-MK2. Cytopathic effects comprising cell rounding and cytoplasmic vacuolation were first observed five days after inoculation. Mixture of Powassan antiserum with virus before inoculation into tissue cultures inhibited the appearance of cytopathic effects. Hemagglutinins for rooster erythrocytes, optimally at pH 6.4 and 22° C., first appeared in tissue culture supernatant fluids four days after inoculation. Electron microscopic observation of thin sections of infected tissue culture cells showed virus particles 360-380 A.U. along outer cell membranes and edges of cytoplasmic vacuoles. In phosphotungstic acid negatively stained preparations, intact virus particles, 400-450 A.U. total diameter, were observed inside infected cells. In particles in which the peripheral layer became discontinuous, geometrically arranged subunits compatible with cubic symmetry were observed. ImagesFig. 1Fig. 2Fig. 3Fig. 4Fig. 5 PMID:14146854

Impact of Ebola mucin-like domain on antiglycoprotein antibody responses induced by Ebola virus-like particles.

PubMed

Martinez, Osvaldo; Tantral, Lee; Mulherkar, Nirupama; Chandran, Kartik; Basler, Christopher F

2011-11-01

Ebola virus (EBOV) glycoprotein (GP), responsible for mediating host-cell attachment and membrane fusion, contains a heavily glycosylated mucin-like domain hypothesized to shield GP from neutralizing antibodies. To test whether the mucin-like domain inhibits the production and function of anti-GP antibodies, we vaccinated mice with Ebola virus-like particles (VLPs) that express vesicular stomatitis virus G, wild-type EBOV GP (EBGP), EBOV GP without its mucin-like domain (ΔMucGP), or EBOV GP with a Crimean-Congo hemorrhagic fever virus mucin-like domain substituted for the EBOV mucin-like domain (CMsubGP). EBGP-VLP immunized mice elicited significantly higher serum antibody titers toward EBGP or its mutants, as detected by western blot analysis, than did VLP-ΔMucGP. However, EBGP-, ΔMucGP- and CMsubGP-VLP immunized mouse sera contained antibodies that bound to cell surface-expressed GP at similar levels. Furthermore, low but similar neutralizing antibody titers, measured against a vesicular stomatitis virus (VSV) expressing EBGP or ΔMucGP, were present in EBGP, ΔMucGP, and CMsubGP sera, although a slightly higher neutralizing titer (2- to 2.5-fold) was detected in ΔMucGP sera. We conclude that the EBOV GP mucin-like domain can increase relative anti-GP titers, however these titers appear to be directed, at least partly, to denatured GP. Furthermore, removing the mucin-like domain from immunizing VLPs has modest impact on neutralizing antibody titers in serum.

A transgenic plant cell-suspension system for expression of epitopes on chimeric Bamboo mosaic virus particles.

PubMed

Muthamilselvan, Thangarasu; Lee, Chin-Wei; Cho, Yu-Hsin; Wu, Feng-Chao; Hu, Chung-Chi; Liang, Yu-Chuan; Lin, Na-Sheng; Hsu, Yau-Heiu

2016-01-01

We describe a novel strategy to produce vaccine antigens using a plant cell-suspension culture system in lieu of the conventional bacterial or animal cell-culture systems. We generated transgenic cell-suspension cultures from Nicotiana benthamiana leaves carrying wild-type or chimeric Bamboo mosaic virus (BaMV) expression constructs encoding the viral protein 1 (VP1) epitope of foot-and-mouth disease virus (FMDV). Antigens accumulated to high levels in BdT38 and BdT19 transgenic cell lines co-expressing silencing suppressor protein P38 or P19. BaMV chimeric virus particles (CVPs) were subsequently purified from the respective cell lines (1.5 and 2.1 mg CVPs/20 g fresh weight of suspended biomass, respectively), and the resulting CVPs displayed VP1 epitope on the surfaces. Guinea pigs vaccinated with purified CVPs produced humoral antibodies. This study represents an important advance in the large-scale production of immunopeptide vaccines in a cost-effective manner using a plant cell-suspension culture system. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Duck hepatitis A virus structural proteins expressed in insect cells self-assemble into virus-like particles with strong immunogenicity in ducklings.

PubMed

Wang, Anping; Gu, Lingling; Wu, Shuang; Zhu, Shanyuan

2018-02-01

Duck hepatitis A virus (DHAV), a non-enveloped ssRNA virus, can cause a highly contagious disease in young ducklings. The three capsid proteins of VP0, VP1 and VP3 are translated within a single large open reading frame (ORF) and hydrolyzed by protease 3CD. However, little is known on whether the recombinant viral structural proteins (VPs) expressed in insect cells could spontaneously assemble into virus-like particles (VLPs) and whether these VLPs could induce protective immunity in young ducklings. To address these issues, the structural polyprotein precursor gene P1 and the protease gene 3CD were amplified by PCR, and the recombinant proteins were expressed in insect cells using a baculovirus expression system for the characterization of their structures and immunogenicity. The recombinant proteins expressed in Sf9 cells were detected by indirect immunofluorescence assay and Western blot analysis. Electron microscopy showed that the recombinant proteins spontaneously assembled into VLPs in insect cells. Western blot analysis of the purified VLPs revealed that the VLPs were composed with the three structural proteins. In addition, vaccination with the VLPs induced high humoral immune response and provided strong protection. Therefore, our findings may provide a framework for development of new vaccines for the prevention of duck viral hepatitis. Copyright © 2018 Elsevier B.V. All rights reserved.

SWI/SNF Associates with Nascent Pre-mRNPs and Regulates Alternative Pre-mRNA Processing

PubMed Central

Tyagi, Anu; Ryme, Jessica; Brodin, David; Östlund Farrants, Ann Kristin; Visa, Neus

2009-01-01

The SWI/SNF chromatin remodeling complexes regulate the transcription of many genes by remodeling nucleosomes at promoter regions. In Drosophila, SWI/SNF plays an important role in ecdysone-dependent transcription regulation. Studies in human cells suggest that Brahma (Brm), the ATPase subunit of SWI/SNF, regulates alternative pre-mRNA splicing by modulating transcription elongation rates. We describe, here, experiments that study the association of Brm with transcribed genes in Chironomus tentans and Drosophila melanogaster, the purpose of which was to further elucidate the mechanisms by which Brm regulates pre-mRNA processing. We show that Brm becomes incorporated into nascent Balbiani ring pre-mRNPs co-transcriptionally and that the human Brm and Brg1 proteins are associated with RNPs. We have analyzed the expression profiles of D. melanogaster S2 cells in which the levels of individual SWI/SNF subunits have been reduced by RNA interference, and we show that depletion of SWI/SNF core subunits changes the relative abundance of alternative transcripts from a subset of genes. This observation, and the fact that a fraction of Brm is not associated with chromatin but with nascent pre-mRNPs, suggest that SWI/SNF affects pre-mRNA processing by acting at the RNA level. Ontology enrichment tests indicate that the genes that are regulated post-transcriptionally by SWI/SNF are mostly enzymes and transcription factors that regulate postembryonic developmental processes. In summary, the data suggest that SWI/SNF becomes incorporated into nascent pre-mRNPs and acts post-transcriptionally to regulate not only the amount of mRNA synthesized from a given promoter but also the type of alternative transcript produced. PMID:19424417

Probing the Role of Nascent Helicity in p27 Function as a Cell Cycle Regulator

PubMed Central

Otieno, Steve; Kriwacki, Richard

2012-01-01

p27 regulates the activity of Cdk complexes which are the principal governors of phase transitions during cell division. Members of the p27 family of proteins, which also includes p21 and p57, are called the Cip/Kip cyclin-dependent kinase regulators (CKRs). Interestingly, the Cip/Kip CKRs play critical roles in cell cycle regulation by being intrinsically unstructured, a characteristic contrary to the classical structure-function paradigm. They exhibit nascent helicity which has been localized to a segment referred to as sub-domain LH. The nascent helicity of this sub-domain is conserved and we hypothesize that it is an important determinant of their functional properties. To test this hypothesis, we successfully designed and prepared p27 variants in which domain LH was either more or less helical with respect to the wild-type protein. Thermal denaturation experiments showed that the ternary complexes of the p27 variants bound to Cdk2/Cyclin A were less stable compared to the wild-type complex. Isothermal titration calorimetry experiments showed a decrease in the enthalpy of binding for all the mutants with respect to p27. The free energies of binding varied within a much narrower range. In vitro Cdk2 inhibition assays showed that the p27 variants exhibited disparate inhibitory potencies. Furthermore, when over-expressed in NIH 3T3 mouse fibroblast cells, the less helical p27 variants were less effective in causing cell cycle arrest relative to the wild-type p27. Our results indicate that the nascent helicity of sub-domain LH plays a key role mediating the biological function of p27. PMID:23071750

Live Cell Imaging of the Nascent Inactive X Chromosome during the Early Differentiation Process of Naive ES Cells towards Epiblast Stem Cells

PubMed Central

Guyochin, Aurélia; Maenner, Sylvain; Chu, Erin Tsi-Jia; Hentati, Asma; Attia, Mikael; Avner, Philip; Clerc, Philippe

2014-01-01

Random X-chromosome inactivation ensures dosage compensation in mammals through the transcriptional silencing of one of the two X chromosomes present in each female cell. Silencing is initiated in the differentiating epiblast of the mouse female embryos through coating of the nascent inactive X chromosome by the non-coding RNA Xist, which subsequently recruits the Polycomb Complex PRC2 leading to histone H3-K27 methylation. Here we examined in mouse ES cells the early steps of the transition from naive ES cells towards epiblast stem cells as a model for inducing X chromosome inactivation in vitro. We show that these conditions efficiently induce random XCI. Importantly, in a transient phase of this differentiation pathway, both X chromosomes are coated with Xist RNA in up to 15% of the XX cells. In an attempt to determine the dynamics of this process, we designed a strategy aimed at visualizing the nascent inactive X-chromosome in live cells. We generated transgenic female XX ES cells expressing the PRC2 component Ezh2 fused to the fluorescent protein Venus. The fluorescent fusion protein was expressed at sub-physiological levels and located in nuclei of ES cells. Upon differentiation of ES cell towards epiblast stem cell fate, Venus-fluorescent territories appearing in interphase nuclei were identified as nascent inactive X chromosomes by their association with Xist RNA. Imaging of Ezh2-Venus for up to 24 hours during the differentiation process showed survival of some cells with two fluorescent domains and a surprising dynamics of the fluorescent territories across cell division and in the course of the differentiation process. Our data reveal a strategy for visualizing the nascent inactive X chromosome and suggests the possibility for a large plasticity of the nascent inactive X chromosome. PMID:25546018

Infectious Hepatitis C Virus Pseudo-particles Containing Functional E1–E2 Envelope Protein Complexes

PubMed Central

Bartosch, Birke; Dubuisson, Jean; Cosset, François-Loïc

2003-01-01

The study of hepatitis C virus (HCV), a major cause of chronic liver disease, has been hampered by the lack of a cell culture system supporting its replication. Here, we have successfully generated infectious pseudo-particles that were assembled by displaying unmodified and functional HCV glycoproteins onto retroviral and lentiviral core particles. The presence of a green fluorescent protein marker gene packaged within these HCV pseudo-particles allowed reliable and fast determination of infectivity mediated by the HCV glycoproteins. Primary hepatocytes as well as hepato-carcinoma cells were found to be the major targets of infection in vitro. High infectivity of the pseudo-particles required both E1 and E2 HCV glycoproteins, and was neutralized by sera from HCV-infected patients and by some anti-E2 monoclonal antibodies. In addition, these pseudo-particles allowed investigation of the role of putative HCV receptors. Although our results tend to confirm their involvement, they provide evidence that neither LDLr nor CD81 is sufficient to mediate HCV cell entry. Altogether, these studies indicate that these pseudo-particles may mimic the early infection steps of parental HCV and will be suitable for the development of much needed new antiviral therapies. PMID:12615904

Suitability and perspectives on using recombinant insect cells for the production of virus-like particles.

PubMed

Yamaji, Hideki

2014-03-01

Virus-like particles (VLPs) can be produced in recombinant protein production systems by expressing viral surface proteins that spontaneously assemble into particulate structures similar to authentic viral or subviral particles. VLPs serve as excellent platforms for the development of safe and effective vaccines and diagnostic antigens. Among various recombinant protein production systems, the baculovirus-insect cell system has been used extensively for the production of a wide variety of VLPs. This system is already employed for the manufacture of a licensed human papillomavirus-like particle vaccine. However, the baculovirus-insect cell system has several inherent limitations including contamination of VLPs with progeny baculovirus particles. Stably transformed insect cells have emerged as attractive alternatives to the baculovirus-insect cell system. Different types of VLPs, with or without an envelope and composed of either single or multiple structural proteins, have been produced in stably transformed insect cells. VLPs produced by stably transformed insect cells have successfully elicited immune responses in vivo. In some cases, the yield of VLPs attained with recombinant insect cells was comparable to, or higher than, that obtained by baculovirus-infected insect cells. Recombinant insect cells offer a promising approach to the development and production of VLPs.

Recombinant Hepatitis E virus like particles can function as RNA nanocarriers.

PubMed

Panda, Subrat Kumar; Kapur, Neeraj; Paliwal, Daizy; Durgapal, Hemlata

2015-06-24

Assembled virus-like particles (VLPs) without genetic material, with structure similar to infectious virions, have been successfully used as vaccines. We earlier described in vitro assembly, characterisation and tissue specific receptor dependent Clathrin mediated entry of empty HEV VLPs, produced from Escherichia coli expressed HEV capsid protein (pORF2). Similar VLP's have been described as a potential candidate vaccine (Hecolin) against HEV. We have attempted to use such recombinant assembled Hepatitis E virus (HEV) VLPs as a carrier for heterologous RNA with protein coding sequence fused in-frame with HEV 5' region (containing cap and encapsidation signal) and investigated, if the relevant protein could be expressed and elicit an immune response in vivo. In vitro transcribed red fluorescent protein (RFP)/Hepatitis B virus surface antigen (HBsAg) RNA, fused to 5'-HEV sequence with cap and encapsidation signal (1-249 nt), was packaged into the recombinant HEV-VLPs and incubated with five different cell lines (Huh7, A549, Vero, HeLa and SiHa). The pORF2-VLPs could specifically transfer exogenous coding RNA into Huh7 and A549 cells. In vivo, Balb/c mice were immunized (intramuscular injections) with 100 µg pORF2-VLP encapsidated with 5'-methyl-G-HEV (1-249 nt)-HBsAg RNA, blood samples were collected and screened by ELISA for anti-pORF2 and anti-HBsAg antibodies. Humoral immune response could be elicited in Balb/c mice against both HEV capsid protein and cargo RNA encoded HBsAg protein. These findings suggest that other than being a possible vaccine, HEV pORF2-VLPs can be used as a promising non-replicative tissue specific gene delivery system.

Studies on Human Hepatitis A Virus in Chimpanzees

PubMed Central

Thornton, A.; Tsiquaye, K. N.; Zuckerman, A. J.

1977-01-01

Several chimpanzees found to be seronegative for hepatitis A by immune electron microscopy and by radioimmunoassay were inoculated with known infective faecal extracts from several sources, including the MS-1 strain of hepatitis A virus, first passage material from chimpanzee George and a strain of hepatitis A virus obtained during a naturally occurring outbreak of infection in Germany. Elevated serum transaminase levels were found in the chimpanzees about 19 days after inoculation. Virus particles were found in faeces as early as 9 days after infection in one chimpanzee and by days 12 and 14 in the others. Excretion of virus continued for 9 to 19 days, and the maximum numbers of virus particles were found on the 17th to the 19th day after inoculation. The buoyant density in caesium chloride of virus particles separated from faeces was 1.31 to 1.43 g/ml. Most of the particles were found in the fraction with a density of 1.34 g/ml. Hepatitis A antibody was detected in the serum by immune electron microscopy and by radioimmunoassay during the period of incubation while virus particles were still being excreted in the faeces and coinciding approximately with the onset of biochemical evidence of liver damage. A very close or identical morphological and serological relationship was demonstrated between hepatitis A virus isolated from man and from the experimentally infected chimpanzees. ImagesFigs. 1-3 PMID:199227

L Particles Transmit Viral Proteins from Herpes Simplex Virus 1-Infected Mature Dendritic Cells to Uninfected Bystander Cells, Inducing CD83 Downmodulation.

PubMed

Heilingloh, Christiane S; Kummer, Mirko; Mühl-Zürbes, Petra; Drassner, Christina; Daniel, Christoph; Klewer, Monika; Steinkasserer, Alexander

2015-11-01

Mature dendritic cells (mDCs) are known as the most potent antigen-presenting cells (APCs) since they are also able to prime/induce naive T cells. Thus, mDCs play a pivotal role during the induction of antiviral immune responses. Remarkably, the cell surface molecule CD83, which was shown to have costimulatory properties, is targeted by herpes simplex virus 1 (HSV-1) for viral immune escape. Infection of mDCs with HSV-1 results in downmodulation of CD83, resulting in reduced T cell stimulation. In this study, we report that not only infected mDCs but also uninfected bystander cells in an infected culture show a significant CD83 reduction. We demonstrate that this effect is independent of phagocytosis and transmissible from infected to uninfected mDCs. The presence of specific viral proteins found in these uninfected bystander cells led to the hypothesis that viral proteins are transferred from infected to uninfected cells via L particles. These L particles are generated during lytic replication in parallel with full virions, called H particles. L particles contain viral proteins but lack the viral capsid and DNA. Therefore, these particles are not infectious but are able to transfer several viral proteins. Incubation of mDCs with L particles indeed reduced CD83 expression on uninfected bystander DCs, providing for the first time evidence that functional viral proteins are transmitted via L particles from infected mDCs to uninfected bystander cells, thereby inducing CD83 downmodulation. HSV-1 has evolved a number of strategies to evade the host's immune system. Among others, HSV-1 infection of mDCs results in an inhibited T cell activation caused by degradation of CD83. Interestingly, CD83 is lost not only from HSV-1-infected mDCs but also from uninfected bystander cells. The release of so-called L particles, which contain several viral proteins but lack capsid and DNA, during infection is a common phenomenon observed among several viruses, such as human

Long lasting immunity in chickens induced by a single shot of influenza vaccine prepared from inactivated non-pathogenic H5N1 virus particles against challenge with a highly pathogenic avian influenza virus.

PubMed

Sasaki, Takashi; Kokumai, Norihide; Ohgitani, Toshiaki; Sakamoto, Ryuichi; Takikawa, Noriyasu; Lin, Zhifeng; Okamatsu, Masatoshi; Sakoda, Yoshihiro; Kida, Hiroshi

2009-08-20

An influenza vaccine was prepared from inactivated whole particles of the non-pathogenic strain A/duck/Hokkaido/Vac-1/04 (H5N1) virus using an oil adjuvant containing anhydromannitol-octadecenoate-ether (AMOE). The vaccine was injected intramuscularly into five 4-week-old chickens, and 138 weeks after vaccination, they were challenged intranasally with 100 times 50% chicken lethal dose of the highly pathogenic avian influenza (HPAI) virus A/chicken/Yamaguchi/7/04 (H5N1). All 5 chickens survived without exhibiting clinical signs of influenza, although 2 days post-challenge, 3 vaccinated chickens shed limited titres of viruses in laryngopharyngeal swabs.

Morphology of certain viruses of Salmonid fishes. II. In vivo studies of infectious Hematopoietic Necrosis Virus

USGS Publications Warehouse

Amend, Donald F.; Chambers, Velma C.

1970-01-01

Juvenile sockeye salmon (Oncorhynchus nerka) were injected with the infectious hematopoietic necrosis (IHN) virus, and tissue samples from the anterior kidney, spleen, liver, intestine, and pyloric caeca of moribund fish were prepared for electron microscopy. Bullet-shaped virus particles measuring 158 × 90 mμ were observed in the hematopoietic tissues of the anterior kidney and spleen. Virus particles were also observed in the outer connective tissues of the pancreas or pyloric caeca, or both. No virus was found in the intestine or liver. The healthy appearance of erythrocytes, reticular cells, and endothelial cells in necrotic areas of the spleen and anterior kidney, and the absence of lymphocytes in these areas, suggested that lymphocytes might be one source of the virus.

Induction of ebolavirus cross-species immunity using retrovirus-like particles bearing the Ebola virus glycoprotein lacking the mucin-like domain.

PubMed

Ou, Wu; Delisle, Josie; Jacques, Jerome; Shih, Joanna; Price, Graeme; Kuhn, Jens H; Wang, Vivian; Verthelyi, Daniela; Kaplan, Gerardo; Wilson, Carolyn A

2012-01-25

The genus Ebolavirus includes five distinct viruses. Four of these viruses cause hemorrhagic fever in humans. Currently there are no licensed vaccines for any of them; however, several vaccines are under development. Ebola virus envelope glycoprotein (GP1,2) is highly immunogenic, but antibodies frequently arise against its least conserved mucin-like domain (MLD). We hypothesized that immunization with MLD-deleted GP1,2 (GPΔMLD) would induce cross-species immunity by making more conserved regions accessible to the immune system. To test this hypothesis, mice were immunized with retrovirus-like particles (retroVLPs) bearing Ebola virus GPΔMLD, DNA plasmids (plasmo-retroVLP) that can produce such retroVLPs in vivo, or plasmo-retroVLP followed by retroVLPs. Cross-species neutralizing antibody and GP1,2-specific cellular immune responses were successfully induced. Our findings suggest that GPΔMLD presented through retroVLPs may provide a strategy for development of a vaccine against multiple ebolaviruses. Similar vaccination strategies may be adopted for other viruses whose envelope proteins contain highly variable regions that may mask more conserved domains from the immune system.

Particle-based vaccines for HIV-1 infection.

PubMed

Young, Kelly R; Ross, Ted M

2003-06-01

The use of live-attenuated viruses as vaccines has been successful for the control of viral infections. However, the development of an effective vaccine against the human immunodeficiency virus (HIV) has proven to be a challenge. HIV infects cells of the immune system and results in a severe immunodeficiency. In addition, the ability of the virus to adapt to immune pressure and the ability to reside in an integrated form in host cells present hurdles for vaccinologists to overcome. A particle-based vaccine strategy has promise for eliciting high titer, long-lived, immune responses to a diverse number of viral epitopes from different HIV antigens. Live-attenuated viruses are effective at generating both cellular and humoral immunity, however, a live-attenuated vaccine for HIV is problematic. The possibility of a live-attenuated vaccine to revert to a pathogenic form or recombine with a wild-type or defective virus in an infected individual is a drawback to this approach. Therefore, these vaccines are currently only being tested in non-human primate models. Live-attenuated vaccines are effective in stimulating immunity, however challenged animals rarely clear viral infection and the degree of attenuation directly correlates with the protection of animals from disease. Another particle-based vaccine approach for HIV involves the use of virus-like particles (VLPs). VLPs mimic the viral particle without causing an immunodeficiency disease. HIV-like particles (HIV-LP) are defined as self-assembling, non-replicating, nonpathogenic, genomeless particles that are similar in size and conformation to intact virions. A variety of VLPs for both HIV and SIV are currently in pre-clinical and clinical trials. This review focuses on the current knowledge regarding the immunogenicity and safety of particle-based vaccine strategies for HIV-1.

A DNA replicon system for rapid high-level production of virus-like particles in plants.

PubMed

Huang, Zhong; Chen, Qiang; Hjelm, Brooke; Arntzen, Charles; Mason, Hugh

2009-07-01

Recombinant virus-like particles (VLPs) represent a safe and effective vaccine strategy. We previously described a stable transgenic plant system for inexpensive production and oral delivery of VLP vaccines. However, the relatively low-level antigen accumulation and long-time frame to produce transgenic plants are the two major roadblocks in the practical development of plant-based VLP production. In this article, we describe the optimization of geminivirus-derived DNA replicon vectors for rapid, high-yield plant-based production of VLPs. Co-delivery of bean yellow dwarf virus (BeYDV)-derived vector and Rep/RepA-supplying vector by agroinfiltration of Nicotiana benthamiana leaves resulted in efficient replicon amplification and robust protein production within 5 days. Co-expression of the P19 protein of tomato bush stunt virus, a gene silencing inhibitor, further enhanced VLP accumulation by stabilizing the mRNA. With this system, hepatitis B core antigen (HBc) and Norwalk virus capsid protein (NVCP) were produced at 0.80 and 0.34 mg/g leaf fresh weight, respectively. Sedimentation analysis and electron microscopy of transiently expressed antigens verified the efficient assembly of VLPs. Furthermore, a single replicon vector containing a built-in Rep/RepA cassette without P19 drove protein expression at similar levels as the three-component system. These results demonstrate the advantages of fast and high-level production of VLP-based vaccines using the BeYDV-derived DNA replicon system for transient expression in plants. (c) 2009 Wiley Periodicals, Inc.

A DNA replicon system for rapid high-level production of virus-like particles in plants

PubMed Central

Huang, Zhong; Chen, Qiang; Hjelm, Brooke; Arntzen, Charles

2009-01-01

Recombinant virus-like particles (VLPs) represent a safe and effective vaccine strategy. We previously described a stable transgenic plant system for inexpensive production and oral delivery of VLP vaccines. However, the relatively low level antigen accumulation and long time frame to produce transgenic plants are the two major roadblocks in the practical development of plant-based VLP production. In this paper, we describe the optimization of geminivirus-derived DNA replicon vectors for rapid, high-yield plant-based production of VLPs. Co-delivery of bean yellow dwarf virus (BeYDV)-derived vector and Rep/RepA-supplying vector by agroinfiltration of Nicotiana benthamiana leaves resulted in efficient replicon amplification and robust protein production within five days. Co-expression of the P19 protein of tomato bush stunt virus, a gene silencing inhibitor, further enhanced VLP accumulation by stabilizing the mRNA. With this system, hepatitis B core antigen (HBc) and Norwalk virus capsid protein (NVCP) were produced at 0.80 and 0.34 mg/g leaf fresh weight, respectively. Sedimentation analysis and electron microscopy of transiently expressed antigens verified the efficient assembly of VLPs. Furthermore, a single replicon vector containing a built-in Rep/RepA cassette without p19 drove protein expression at similar levels as the three-component system. These results demonstrate the advantages of fast and high-level production of VLP-based vaccines using the BeYDV-derived DNA replicon system for transient expression in plants. PMID:19309755

A Primer on the Pathway to Scholarly Writing: Helping Nascent Writers to Unlearn Conditioned Habits

ERIC Educational Resources Information Center

McDougall, Dennis; Ornelles, Cecily; Rao, Kavita

2015-01-01

In this article, we identify eight common error patterns of nascent writers when they attempt to navigate the pathway to scholarly writing. We illustrate each error pattern via examples and counter-examples (corrections). We also describe how to identify such patterns, why those patterns might occur and persist, and why each pattern is…

Encapsidated Host Factors in Alphavirus Particles Influence Midgut Infection of Aedes aegypti.

PubMed

Mackenzie-Liu, David; Sokoloski, Kevin J; Purdy, Sarah; Hardy, Richard W

2018-05-16

Transmission of mosquito-borne viruses requires the efficient infection of both a permissive vertebrate host and a competent mosquito vector. The infectivity of Sindbis virus (SINV), the type species of the Alphavirus genus, is influenced by both the original and new host cell. We have shown that infection of vertebrate cells by SINV, chikungunya virus (CHIKV), and Ross River virus (RRV) produces two subpopulations of virus particles separable based on density. In contrast, a single population of viral particles is produced by mosquito cells. Previous studies demonstrated that the denser vertebrate-derived particles and the mosquito-derived particles contain components of the small subunit of the host cell ribosome, whereas the less dense vertebrate-derived particles do not. Infection of mice with RRV showed that both particle subpopulations are produced in an infected vertebrate, but in a tissue specific manner with serum containing only the less dense version of the virus particles. Previous infectivity studies using SINV particles have shown that the denser particles (SINV Heavy ) and mosquito derived particles SINV C6/36 are significantly more infectious in vertebrate cells than the less dense vertebrate derived particles (SINV Light ). The current study shows that SINV Light particles, initiate the infection of the mosquito midgut more efficiently than SINV Heavy particles and that this enhanced infectivity is associated with an exacerbated immune response to SINV Light infection in midgut tissues. The enhanced infection of SINV Light is specific to the midgut as intrathoracically injected virus do not exhibit the same fitness advantage. Together, our data indicate a biologically significant role for the SINV Light subpopulation in the efficient transmission from infected vertebrates to the mosquito vector.

Enhanced assembly and colloidal stabilization of primate erythroparvovirus 1 virus-like particles for improved surface engineering.

PubMed

Sánchez-Rodríguez, Sandra Paola; Morán-García, Areli del Carmen; Bolonduro, Olurotimi; Dordick, Jonathan S; Bustos-Jaimes, Ismael

2016-04-15

Virus-like particles (VLPs) are the product of the self-assembly, either in vivo or in vitro, of structural components of viral capsids. These particles are excellent scaffolds for surface display of biomolecules that can be used in vaccine development and tissue-specific drug delivery. Surface engineering of VLPs requires structural stability and chemical reactivity. Herein, we report the enhanced assembly, colloidal stabilization and fluorescent labeling of primate erythroparvovirus 1 (PE1V), generally referred to as parvovirus B19. In vitro assembly of the VP2 protein of PE1V produces VLPs, which are prone to flocculate and hence undergo limited chemical modification by thiol-specific reagents like the fluorogenic monobromobimane (mBBr). We determined that the addition of 0.2M l-arginine during the assembly process produced an increased yield of soluble VLPs with good dispersion stability. Fluorescent labeling of VLPs suspended in phosphate buffered saline (PBS) added with 0.2M l-Arg was achieved in significantly shorter times than the flocculated VLPs assembled in only PBS buffer. Finally, to demonstrate the potential application of this approach, mBBr-labeled VLPs were successfully used to tag human hepatoma HepG2 cells. This new method for assembly and labeling PE1V VLPs eases its applications and provides insights on the manipulation of this biomaterial for further developments. Application of virus-derived biomaterials sometimes requires surface modification for diverse purposes, including enhanced cell-specific interaction, the inclusion of luminescent probes for bioimaging, or the incorporation of catalytic properties for the production of enzyme nanocarriers. In this research, we reported for the first time the colloidal stabilization of the primate erythroparvovirus 1 (PE1V) virus-like particles (VLPs). Also, we report the chemical modification of the natural Cys residues located on the surface of these VLPs with a fluorescent probe, as well as its

Sharp Transition from Nonmetallic Au246 to Metallic Au279 with Nascent Surface Plasmon Resonance.

PubMed

Higaki, Tatsuya; Zhou, Meng; Lambright, Kelly J; Kirschbaum, Kristin; Sfeir, Matthew Y; Jin, Rongchao

2018-05-02

The optical properties of metal nanoparticles have attracted wide interest. Recent progress in controlling nanoparticles with atomic precision (often called nanoclusters) provide new opportunities for investigating many fundamental questions, such as the transition from excitonic to plasmonic state, which is a central question in metal nanoparticle research because it provides insights into the origin of surface plasmon resonance (SPR) as well as the formation of metallic bond. However, this question still remains elusive because of the extreme difficulty in preparing atomically precise nanoparticles larger than 2 nm. Here we report the synthesis and optical properties of an atomically precise Au 279 (SR) 84 nanocluster. Femtosecond transient absorption spectroscopic analysis reveals that the Au 279 nanocluster shows a laser power dependence in its excited state lifetime, indicating metallic state of the particle, in contrast with the nonmetallic electronic structure of the Au 246 (SR) 80 nanocluster. Steady-state absorption spectra reveal that the nascent plasmon band of Au 279 at 506 nm shows no peak shift even down to 60 K, consistent with plasmon behavior. The sharp transition from nonmetallic Au 246 to metallic Au 279 is surprising and will stimulate future theoretical work on the transition and many other relevant issues.

A manual and an automatic TERS based virus discrimination

NASA Astrophysics Data System (ADS)

Olschewski, Konstanze; Kämmer, Evelyn; Stöckel, Stephan; Bocklitz, Thomas; Deckert-Gaudig, Tanja; Zell, Roland; Cialla-May, Dana; Weber, Karina; Deckert, Volker; Popp, Jürgen

2015-02-01

Rapid techniques for virus identification are more relevant today than ever. Conventional virus detection and identification strategies generally rest upon various microbiological methods and genomic approaches, which are not suited for the analysis of single virus particles. In contrast, the highly sensitive spectroscopic technique tip-enhanced Raman spectroscopy (TERS) allows the characterisation of biological nano-structures like virions on a single-particle level. In this study, the feasibility of TERS in combination with chemometrics to discriminate two pathogenic viruses, Varicella-zoster virus (VZV) and Porcine teschovirus (PTV), was investigated. In a first step, chemometric methods transformed the spectral data in such a way that a rapid visual discrimination of the two examined viruses was enabled. In a further step, these methods were utilised to perform an automatic quality rating of the measured spectra. Spectra that passed this test were eventually used to calculate a classification model, through which a successful discrimination of the two viral species based on TERS spectra of single virus particles was also realised with a classification accuracy of 91%.Rapid techniques for virus identification are more relevant today than ever. Conventional virus detection and identification strategies generally rest upon various microbiological methods and genomic approaches, which are not suited for the analysis of single virus particles. In contrast, the highly sensitive spectroscopic technique tip-enhanced Raman spectroscopy (TERS) allows the characterisation of biological nano-structures like virions on a single-particle level. In this study, the feasibility of TERS in combination with chemometrics to discriminate two pathogenic viruses, Varicella-zoster virus (VZV) and Porcine teschovirus (PTV), was investigated. In a first step, chemometric methods transformed the spectral data in such a way that a rapid visual discrimination of the two examined viruses

Analysis of Particle Content of Recombinant Adeno-Associated Virus Serotype 8 Vectors by Ion-Exchange Chromatography

PubMed Central

Lock, Martin; Alvira, Mauricio R.

2012-01-01

Abstract Advances in adeno-associated virus (AAV)-mediated gene therapy have brought the possibility of commercial manufacturing of AAV vectors one step closer. To realize this prospect, a parallel effort with the goal of ever-increasing sophistication for AAV vector production technology and supporting assays will be required. Among the important release assays for a clinical gene therapy product, those monitoring potentially hazardous contaminants are most critical for patient safety. A prominent contaminant in many AAV vector preparations is vector particles lacking a genome, which can substantially increase the dose of AAV capsid proteins and lead to possible unwanted immunological consequences. Current methods to determine empty particle content suffer from inconsistency, are adversely affected by contaminants, or are not applicable to all serotypes. Here we describe the development of an ion-exchange chromatography-based assay that permits the rapid separation and relative quantification of AAV8 empty and full vector particles through the application of shallow gradients and a strong anion-exchange monolith chromatography medium. PMID:22428980

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

Core protein cleavage by signal peptide peptidase is required for hepatitis C virus-like particle assembly

PubMed Central

Ait-Goughoulte, Malika; Hourioux, Christophe; Patient, Romuald; Trassard, Sylvie; Brand, Denys; Roingeard, Philippe

2006-01-01

SUMMARY Hepatitis C virus (HCV) core protein, expressed with a Semliki forest virus (SFV) replicon, self-assembles into HCV-like particles (HCV-LP) at the endoplasmic reticulum (ER) membrane, providing an opportunity to study HCV assembly and morphogenesis by electron microscopy. We used this model to investigate whether the processing of the HCV core protein by the signal peptide peptidase (SPP) is required for the HCV-LP assembly. We designed several mutants as there are conflicting reports concerning the cleavage of mutant proteins by SPP. Production of the only core mutant protein that escaped SPP processing led to the formation of multiple layers of electron-dense ER membrane, with no evidence of HCV-LP assembly. Our data shed light on the HCV core residues involved in SPP cleavage and suggest that this cleavage is essential for HCV assembly. PMID:16528035

Biophysical characterization of influenza virus subpopulations using field flow fractionation and multiangle light scattering: correlation of particle counts, size distribution and infectivity.

PubMed

Wei, Ziping; McEvoy, Matt; Razinkov, Vladimir; Polozova, Alla; Li, Elizabeth; Casas-Finet, Jose; Tous, Guillermo I; Balu, Palani; Pan, Alfred A; Mehta, Harshvardhan; Schenerman, Mark A

2007-09-01

Adequate biophysical characterization of influenza virions is important for vaccine development. The influenza virus vaccines are produced from the allantoic fluid of developing chicken embryos. The process of viral replication produces a heterogeneous mixture of infectious and non-infectious viral particles with varying states of aggregation. The study of the relative distribution and behavior of different subpopulations and their inter-correlation can assist in the development of a robust process for a live virus vaccine. This report describes a field flow fractionation and multiangle light scattering (FFF-MALS) method optimized for the analysis of size distribution and total particle counts. The FFF-MALS method was compared with several other methods such as transmission electron microscopy (TEM), atomic force microscopy (AFM), size exclusion chromatography followed by MALS (SEC-MALS), quantitative reverse transcription polymerase chain reaction (RT Q-PCR), median tissue culture dose (TCID(50)), and the fluorescent focus assay (FFA). The correlation between the various methods for determining total particle counts, infectivity and size distribution is reported. The pros and cons of each of the analytical methods are discussed.

Characterization of virus-like particles by atomic force microscopy in ambient conditions

NASA Astrophysics Data System (ADS)

Oropesa, Reinier; Ramos, Jorge R.; Falcón, Viviana; Felipe, Ariel

2013-06-01

Recombinant virus-like particles (VLPs) are attractive candidates for vaccine design since they resemble native viroids in size and morphology, but they are non-infectious due to the absence of a viral genome. The visualization of surface morphologies and structures can be used to deepen the understanding of physical, chemical, and biological phenomena. Atomic force microscopy (AFM) is a useful tool for the visualization of soft biological samples in a nanoscale resolution. In this work we have investigated the morphology of recombinant surface antigens of hepatitis B (rHBsAg) VLPs from Cuban vaccine against hepatitis B. The rHBsAg VLPs sizes estimated by AFM between 15 and 30 nm are similar to those reported on previous transmission electron microscopy (TEM) studies.

Hepatitis C Virus Lipoviroparticles Assemble in the Endoplasmic Reticulum (ER) and Bud off from the ER to the Golgi Compartment in COPII Vesicles.

PubMed

Syed, Gulam H; Khan, Mohsin; Yang, Song; Siddiqui, Aleem

2017-08-01

Hepatitis C virus (HCV) exists as a lipoprotein-virus hybrid lipoviroparticle (LVP). In vitro studies have demonstrated the importance of apolipoproteins in HCV secretion and infectivity, leading to the notion that HCV coopts the secretion of very-low-density lipoprotein (VLDL) for its egress. However, the mechanisms involved in virus particle assembly and egress are still elusive. The biogenesis of VLDL particles occurs in the endoplasmic reticulum (ER), followed by subsequent lipidation in the ER and Golgi compartment. The secretion of mature VLDL particles occurs through the Golgi secretory pathway. HCV virions are believed to latch onto or fuse with the nascent VLDL particle in either the ER or the Golgi compartment, resulting in the generation of LVPs. In our attempt to unravel the collaboration between HCV and VLDL secretion, we studied HCV particles budding from the ER en route to the Golgi compartment in COPII vesicles. Biophysical characterization of COPII vesicles fractionated on an iodixanol gradient revealed that HCV RNA is enriched in the highly buoyant COPII vesicle fractions and cofractionates with apolipoprotein B (ApoB), ApoE, and the HCV core and envelope proteins. Electron microscopy of immunogold-labeled microsections revealed that the HCV envelope and core proteins colocalize with apolipoproteins and HCV RNA in Sec31-coated COPII vesicles. Ultrastructural analysis also revealed the presence of HCV structural proteins, RNA, and apolipoproteins in the Golgi stacks. These findings support the hypothesis that HCV LVPs assemble in the ER and are transported to the Golgi compartment in COPII vesicles to embark on the Golgi secretory route. IMPORTANCE HCV assembly and release accompany the formation of LVPs that circulate in the sera of HCV patients and are also produced in an in vitro culture system. The pathway of HCV morphogenesis and secretion has not been fully understood. This study investigates the exact site where the association of HCV

Hepatitis C Virus Lipoviroparticles Assemble in the Endoplasmic Reticulum (ER) and Bud off from the ER to the Golgi Compartment in COPII Vesicles

PubMed Central

Khan, Mohsin; Yang, Song

2017-01-01

ABSTRACT Hepatitis C virus (HCV) exists as a lipoprotein-virus hybrid lipoviroparticle (LVP). In vitro studies have demonstrated the importance of apolipoproteins in HCV secretion and infectivity, leading to the notion that HCV coopts the secretion of very-low-density lipoprotein (VLDL) for its egress. However, the mechanisms involved in virus particle assembly and egress are still elusive. The biogenesis of VLDL particles occurs in the endoplasmic reticulum (ER), followed by subsequent lipidation in the ER and Golgi compartment. The secretion of mature VLDL particles occurs through the Golgi secretory pathway. HCV virions are believed to latch onto or fuse with the nascent VLDL particle in either the ER or the Golgi compartment, resulting in the generation of LVPs. In our attempt to unravel the collaboration between HCV and VLDL secretion, we studied HCV particles budding from the ER en route to the Golgi compartment in COPII vesicles. Biophysical characterization of COPII vesicles fractionated on an iodixanol gradient revealed that HCV RNA is enriched in the highly buoyant COPII vesicle fractions and cofractionates with apolipoprotein B (ApoB), ApoE, and the HCV core and envelope proteins. Electron microscopy of immunogold-labeled microsections revealed that the HCV envelope and core proteins colocalize with apolipoproteins and HCV RNA in Sec31-coated COPII vesicles. Ultrastructural analysis also revealed the presence of HCV structural proteins, RNA, and apolipoproteins in the Golgi stacks. These findings support the hypothesis that HCV LVPs assemble in the ER and are transported to the Golgi compartment in COPII vesicles to embark on the Golgi secretory route. IMPORTANCE HCV assembly and release accompany the formation of LVPs that circulate in the sera of HCV patients and are also produced in an in vitro culture system. The pathway of HCV morphogenesis and secretion has not been fully understood. This study investigates the exact site where the association of

Virus-mimetic polyplex particles for systemic and inflammation-specific targeted delivery of large genetic contents.

PubMed

Kang, S; Lu, K; Leelawattanachai, J; Hu, X; Park, S; Park, T; Min, I M; Jin, M M

2013-11-01

Systemic and target-specific delivery of large genetic contents has been difficult to achieve. Although viruses effortlessly deliver kilobase-long genome into cells, its clinical use has been hindered by serious safety concerns and the mismatch between native tropisms and desired targets. Nonviral vectors, in contrast, are limited by low gene transfer efficiency and inherent cytotoxicity. Here we devised virus-mimetic polyplex particles (VMPs) based on electrostatic self-assembly among polyanionic peptide (PAP), cationic polymer polyethyleneimine (PEI) and nucleic acids. We fused PAP to the engineered ligand-binding domain of integrin αLβ2 to target intercellular adhesion molecule-1 (ICAM-1), an inducible marker of inflammation. Fully assembled VMPs packaged large genetic contents, bound specifically to target molecules, elicited receptor-mediated endocytosis and escaped endosomal pathway, resembling intracellular delivery processes of viruses. Unlike conventional PEI-mediated transfection, molecular interaction-dependent gene delivery of VMPs was unaffected by the presence of serum and achieved higher efficiency without toxicity. By targeting overexpressed ICAM-1, VMPs delivered genes specifically to inflamed endothelial cells and macrophages both in vitro and in vivo. Simplicity and versatility of the platform and inflammation-specific delivery may open up opportunities for multifaceted gene therapy that can be translated into the clinic and treat a broad range of debilitating immune and inflammatory diseases.

Nucleic Acid Binding by Mason-Pfizer Monkey Virus CA Promotes Virus Assembly and Genome Packaging

PubMed Central

Füzik, Tibor; Píchalová, Růžena; Schur, Florian K. M.; Strohalmová, Karolína; Křížová, Ivana; Hadravová, Romana; Rumlová, Michaela; Briggs, John A. G.

2016-01-01

ABSTRACT The Gag polyprotein of retroviruses drives immature virus assembly by forming hexameric protein lattices. The assembly is primarily mediated by protein-protein interactions between capsid (CA) domains and by interactions between nucleocapsid (NC) domains and RNA. Specific interactions between NC and the viral RNA are required for genome packaging. Previously reported cryoelectron microscopy analysis of immature Mason-Pfizer monkey virus (M-PMV) particles suggested that a basic region (residues RKK) in CA may serve as an additional binding site for nucleic acids. Here, we have introduced mutations into the RKK region in both bacterial and proviral M-PMV vectors and have assessed their impact on M-PMV assembly, structure, RNA binding, budding/release, nuclear trafficking, and infectivity using in vitro and in vivo systems. Our data indicate that the RKK region binds and structures nucleic acid that serves to promote virus particle assembly in the cytoplasm. Moreover, the RKK region appears to be important for recruitment of viral genomic RNA into Gag particles, and this function could be linked to changes in nuclear trafficking. Together these observations suggest that in M-PMV, direct interactions between CA and nucleic acid play important functions in the late stages of the viral life cycle. IMPORTANCE Assembly of retrovirus particles is driven by the Gag polyprotein, which can self-assemble to form virus particles and interact with RNA to recruit the viral genome into the particles. Generally, the capsid domains of Gag contribute to essential protein-protein interactions during assembly, while the nucleocapsid domain interacts with RNA. The interactions between the nucleocapsid domain and RNA are important both for identifying the genome and for self-assembly of Gag molecules. Here, we show that a region of basic residues in the capsid protein of the betaretrovirus Mason-Pfizer monkey virus (M-PMV) contributes to interaction of Gag with nucleic acid. This

Infection and Proliferation of Giant Viruses in Amoeba Cells.

PubMed

Takemura, Masaharu

2016-01-01

Acanthamoeba polyphaga mimivirus, the first discovered giant virus with genome size and particle size much larger than previously discovered viruses, possesses several genes for translation and CRISPER Cas system-like defense mechanism against virophages, which co-infect amoeba cells with the giant virus and which inhibit giant virus proliferation. Mimiviruses infect amoeba cells by phagocytosis and release their DNA into amoeba cytoplasm through their stargate structure. After infection, giant virion factories (VFs) form in amoeba cytoplasm, followed by DNA replication and particle formation at peripheral regions of VF. Marseilleviruses, the smallest giant viruses, infect amoeba cells by phagocytosis or endocytosis, form larger VF than Mimivirus's VF in amoeba cytoplasm, and replicate their particles. Pandoraviruses found in 2013 have the largest genome size and particle size among all viruses ever found. Pandoraviruses infect amoeba cells by phagocytosis and release their DNA into amoeba cytoplasm through their mouth-like apical pores. The proliferation of Pandoraviruses occurs along with nucleus disruption. New virions form at the periphery of the region formerly occupied by the amoeba cell nucleus.

Yeast-produced recombinant virus-like particles of coxsackievirus A6 elicited protective antibodies in mice.

PubMed

Zhou, Yu; Shen, Chaoyun; Zhang, Chao; Zhang, Wei; Wang, Lili; Lan, Ke; Liu, Qingwei; Huang, Zhong

2016-08-01

Coxsackievirus A6 (CA6) has recently emerged as the predominant pathogen of hand, foot and mouth disease (HFMD), causing significant morbidity in children and adults. The increasing prevalence of CA6 infection and its associated disease burden underscore the need for effective CA6 vaccines. However, CA6 grows poorly in cultured cells, making it difficult to develop inactivated whole-virus or live attenuated vaccines. Here we report the development of a recombinant virus-like particle (VLP) based CA6 vaccine. CA6 VLPs were produced in Pichia pastoris yeast transformed with a vector encoding both P1 and 3CD proteins of CA6. Immunization with CA6 VLPs elicited CA6-specific serum antibodies in mice. Passive transfer of anti-VLP antisera protected recipient mice against lethal CA6 challenge. Collectively, these results demonstrate that CA6 VLPs represent a viable CA6 vaccine candidate which warrants further preclinical and clinical development. Copyright © 2016 Elsevier B.V. All rights reserved.

Virus replicon particles expressing porcine reproductive and respiratory syndrome virus proteins elicit immune priming but do not confer protection from viremia in pigs.

PubMed

Eck, Melanie; Durán, Margarita García; Ricklin, Meret E; Locher, Samira; Sarraseca, Javier; Rodríguez, María José; McCullough, Kenneth C; Summerfield, Artur; Zimmer, Gert; Ruggli, Nicolas

2016-02-19

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of one of the most devastating and economically significant viral disease of pigs worldwide. The vaccines currently available on the market elicit only limited protection. Recombinant vesicular stomatitis virus (VSV) replicon particles (VRP) have been used successfully to induce protection against influenza A virus (IAV) in chickens and bluetongue virus in sheep. In this study, VSV VRP expressing the PRRSV envelope proteins GP5, M, GP4, GP3, GP2 and the nucleocapsid protein N, individually or in combination, were generated and evaluated as a potential vector vaccine against PRRSV infection. High level expression of the recombinant PRRSV proteins was demonstrated in cell culture. However, none of the PRRSV antigens expressed from VRP, with the exception of the N protein, did induce any detectable antibody response in pigs before challenge infection with PRRSV. After challenge however, the antibody responses against GP5, GP4 and GP3 appeared in average 2 weeks earlier than in pigs vaccinated with the empty control VRP. No reduction of viremia was observed in the vaccinated group compared with the control group. When pigs were co-vaccinated with VRP expressing IAV antigens and VRP expressing PRRSV glycoproteins, only antibody responses to the IAV antigens were detectable. These data show that the VSV replicon vector can induce immune responses to heterologous proteins in pigs, but that the PRRSV envelope proteins expressed from VSV VRP are poorly immunogenic. Nevertheless, they prime the immune system for significantly earlier B-cell responses following PRRSV challenge infection.

Inactivated Recombinant Rabies Viruses Displaying Canine Distemper Virus Glycoproteins Induce Protective Immunity against Both Pathogens.

PubMed

da Fontoura Budaszewski, Renata; Hudacek, Andrew; Sawatsky, Bevan; Krämer, Beate; Yin, Xiangping; Schnell, Matthias J; von Messling, Veronika

2017-04-15

The development of multivalent vaccines is an attractive methodology for the simultaneous prevention of several infectious diseases in vulnerable populations. Both canine distemper virus (CDV) and rabies virus (RABV) cause lethal disease in wild and domestic carnivores. While RABV vaccines are inactivated, the live-attenuated CDV vaccines retain residual virulence for highly susceptible wildlife species. In this study, we developed recombinant bivalent vaccine candidates based on recombinant vaccine strain rabies virus particles, which concurrently display the protective CDV and RABV glycoprotein antigens. The recombinant viruses replicated to near-wild-type titers, and the heterologous glycoproteins were efficiently expressed and incorporated in the viral particles. Immunization of ferrets with beta-propiolactone-inactivated recombinant virus particles elicited protective RABV antibody titers, and animals immunized with a combination of CDV attachment protein- and fusion protein-expressing recombinant viruses were protected from lethal CDV challenge. However, animals that were immunized with only a RABV expressing the attachment protein of CDV vaccine strain Onderstepoort succumbed to infection with a more recent wild-type strain, indicating that immune responses to the more conserved fusion protein contribute to protection against heterologous CDV strains. IMPORTANCE Rabies virus and canine distemper virus (CDV) cause high mortality rates and death in many carnivores. While rabies vaccines are inactivated and thus have an excellent safety profile and high stability, live-attenuated CDV vaccines can retain residual virulence in highly susceptible species. Here we generated recombinant inactivated rabies viruses that carry one of the CDV glycoproteins on their surface. Ferrets immunized twice with a mix of recombinant rabies viruses carrying the CDV fusion and attachment glycoproteins were protected from lethal CDV challenge, whereas all animals that received

Inactivated Recombinant Rabies Viruses Displaying Canine Distemper Virus Glycoproteins Induce Protective Immunity against Both Pathogens

PubMed Central

da Fontoura Budaszewski, Renata; Hudacek, Andrew; Sawatsky, Bevan; Krämer, Beate; Yin, Xiangping

2017-01-01

ABSTRACT The development of multivalent vaccines is an attractive methodology for the simultaneous prevention of several infectious diseases in vulnerable populations. Both canine distemper virus (CDV) and rabies virus (RABV) cause lethal disease in wild and domestic carnivores. While RABV vaccines are inactivated, the live-attenuated CDV vaccines retain residual virulence for highly susceptible wildlife species. In this study, we developed recombinant bivalent vaccine candidates based on recombinant vaccine strain rabies virus particles, which concurrently display the protective CDV and RABV glycoprotein antigens. The recombinant viruses replicated to near-wild-type titers, and the heterologous glycoproteins were efficiently expressed and incorporated in the viral particles. Immunization of ferrets with beta-propiolactone-inactivated recombinant virus particles elicited protective RABV antibody titers, and animals immunized with a combination of CDV attachment protein- and fusion protein-expressing recombinant viruses were protected from lethal CDV challenge. However, animals that were immunized with only a RABV expressing the attachment protein of CDV vaccine strain Onderstepoort succumbed to infection with a more recent wild-type strain, indicating that immune responses to the more conserved fusion protein contribute to protection against heterologous CDV strains. IMPORTANCE Rabies virus and canine distemper virus (CDV) cause high mortality rates and death in many carnivores. While rabies vaccines are inactivated and thus have an excellent safety profile and high stability, live-attenuated CDV vaccines can retain residual virulence in highly susceptible species. Here we generated recombinant inactivated rabies viruses that carry one of the CDV glycoproteins on their surface. Ferrets immunized twice with a mix of recombinant rabies viruses carrying the CDV fusion and attachment glycoproteins were protected from lethal CDV challenge, whereas all animals that

Chimeric human parainfluenza virus bearing the Ebola virus glycoprotein as the sole surface protein is immunogenic and highly protective against Ebola virus challenge

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

Bukreyev, Alexander; Marzi, Andrea; Feldmann, Friederike

2009-01-20

We generated a new live-attenuated vaccine against Ebola virus (EBOV) based on a chimeric virus HPIV3/{delta}F-HN/EboGP that contains the EBOV glycoprotein (GP) as the sole transmembrane envelope protein combined with the internal proteins of human parainfluenza virus type 3 (HPIV3). Electron microscopy analysis of the virus particles showed that they have an envelope and surface spikes resembling those of EBOV and a particle size and shape resembling those of HPIV3. When HPIV3/{delta}F-HN/EboGP was inoculated via apical surface of an in vitro model of human ciliated airway epithelium, the virus was released from the apical surface; when applied to basolateral surface,more » the virus infected basolateral cells but did not spread through the tissue. Following intranasal (IN) inoculation of guinea pigs, scattered infected cells were detected in the lungs by immunohistochemistry, but infectious HPIV3/{delta}F-HN/EboGP could not be recovered from the lungs, blood, or other tissues. Despite the attenuation, the virus was highly immunogenic, and a single IN dose completely protected the animals against a highly lethal intraperitoneal challenge of guinea pig-adapted EBOV.« less

Morphology of certain viruses of Salmonid Fishes. I. in vitro studies of some viruses causing Hematopoietic Necrosis

USGS Publications Warehouse

Amend, Donald F.; Chambers, Velma C.

1970-01-01

An electron microscope study was performed on three virus isolates that caused hematopoietic necrosis in salmonid fishes: infectious hematopoietic necrosis (IHN), Oregon Sockeye Disease (OSD), and Sacramento River Chinook Salmon Disease (SRCD). All three isolates were examined by negative staining of fathead minnow (FHM) monolayer tissue culture concentrates and IHN virus was also examined in thin sections of FHM cells. Viruslike particles were observed in infected tissues, but similar structures were not found in uninfected cultures. All three isolates were bullet-shaped, but oval and truncated forms were also observed. Mean measurements of particles from IHN-virus-infected tissue were 158 × 90 mμ. They consisted of an outer coat 15 mμ thick, a core 60 mμ in diameter, subunits about 5 mμ, and an axial pore about 20 mμ in diameter. These particles also were seen budding from the cytoplasmic membrane. Similar particles from SRCD were 159 × 90 mμ and isolates from OSD were 181 × 91 mμ. The three isolates were morphologically indistinguishable from one another and the greater length of OSD was considered insignificant. IHN, SRCD, and OSD viruses were tentatively placed in the rhabdovirus group, but serological studies are needed to determine if they are antigenically identical or should be included as separate members. Biochemical and physical characteristics of these viruses and a comparison with other salmonid viruses is also discussed.

Induction of ebolavirus cross-species immunity using retrovirus-like particles bearing the Ebola virus glycoprotein lacking the mucin-like domain

PubMed Central

2012-01-01

Background The genus Ebolavirus includes five distinct viruses. Four of these viruses cause hemorrhagic fever in humans. Currently there are no licensed vaccines for any of them; however, several vaccines are under development. Ebola virus envelope glycoprotein (GP1,2) is highly immunogenic, but antibodies frequently arise against its least conserved mucin-like domain (MLD). We hypothesized that immunization with MLD-deleted GP1,2 (GPΔMLD) would induce cross-species immunity by making more conserved regions accessible to the immune system. Methods To test this hypothesis, mice were immunized with retrovirus-like particles (retroVLPs) bearing Ebola virus GPΔMLD, DNA plasmids (plasmo-retroVLP) that can produce such retroVLPs in vivo, or plasmo-retroVLP followed by retroVLPs. Results Cross-species neutralizing antibody and GP1,2-specific cellular immune responses were successfully induced. Conclusion Our findings suggest that GPΔMLD presented through retroVLPs may provide a strategy for development of a vaccine against multiple ebolaviruses. Similar vaccination strategies may be adopted for other viruses whose envelope proteins contain highly variable regions that may mask more conserved domains from the immune system. PMID:22273269

Single-virus tracking approach to reveal the interaction of Dengue virus with autophagy during the early stage of infection

NASA Astrophysics Data System (ADS)

Chu, Li-Wei; Huang, Yi-Lung; Lee, Jin-Hui; Huang, Long-Ying; Chen, Wei-Jun; Lin, Ya-Hsuan; Chen, Jyun-Yu; Xiang, Rui; Lee, Chau-Hwang; Ping, Yueh-Hsin

2014-01-01

Dengue virus (DENV) is one of the major infectious pathogens worldwide. DENV infection is a highly dynamic process. Currently, no antiviral drug is available for treating DENV-induced diseases since little is known regarding how the virus interacts with host cells during infection. Advanced molecular imaging technologies are powerful tools to understand the dynamics of intracellular interactions and molecular trafficking. This study exploited a single-virus particle tracking technology to address whether DENV interacts with autophagy machinery during the early stage of infection. Using confocal microscopy and three-dimensional image analysis, we showed that DENV triggered the formation of green fluorescence protein-fused microtubule-associated protein 1A/1B-light chain 3 (GFP-LC3) puncta, and DENV-induced autophagosomes engulfed DENV particles within 15-min postinfection. Moreover, single-virus particle tracking revealed that both DENV particles and autophagosomes traveled together during the viral infection. Finally, in the presence of autophagy suppressor 3-methyladenine, the replication of DENV was inhibited and the location of DENV particles spread in cytoplasma. In contrast, the numbers of newly synthesized DENV were elevated and the co-localization of DENV particles and autophagosomes was detected while the cells were treated with autophagy inducer rapamycin. Taken together, we propose that DENV particles interact with autophagosomes at the early stage of viral infection, which promotes the replication of DENV.

Self-assembly and release of peste des petits ruminants virus-like particles in an insect cell-baculovirus system and their immunogenicity in mice and goats.

PubMed

Li, Wenchao; Jin, Hongyan; Sui, Xiukun; Zhao, Zhanzhong; Yang, Chenghuai; Wang, Wenquan; Li, Junping; Li, Gang

2014-01-01

Peste des petits ruminants (PPR) is an acute, febrile, viral disease of small ruminants that has a significant economic impact. For many viral diseases, vaccination with virus-like particles (VLPs) has shown considerable promise as a prophylactic approach; however, the processes of assembly and release of peste des petits ruminants virus (PPRV) VLPs are not well characterized, and their immunogenicity in the host is unknown. In this study, VLPs of PPRV were generated in a baculovirus system through simultaneous expression of PPRV matrix (M) protein and hemaglutin in (H) or fusion (F) protein. The released VLPs showed morphology similar to that of the native virus particles. Subcutaneous injection of these VLPs (PPRV-H, PPRV-F) into mice and goats elicited PPRV-specific IgG production, increased the levels of virus neutralizing antibodies, and promoted lymphocyte proliferation. Without adjuvants, the immune response induced by the PPRV-H VLPs was comparable to that obtained using equivalent amounts of PPRV vaccine. Thus, our results demonstrated that VLPs containing PPRV M protein and H or F protein are potential "differentiating infected from vaccinated animals" (DIVA) vaccine candidates for the surveillance and eradication of PPR.

Purification of lymphocystis disease virus (LDV) grown in tissue culture. Evidences for the presence of two types of viral particles.

PubMed

Robin, J; Berthiaume, L

1981-12-01

Lymphocystis disease virus was highly purified from host cells by precipitation with PEG-6000 and isopycnic centrifugation in a metrizamide gradient. Metrizamide gradient centrifugation produce two distinct bands at equilibrium. As calculated from reconstruction experiments, only 4 and 0.3% respectively of the host DNA and the host proteins were recovered at the position of the bands. The final recovery of infectivity was about 41%. Electron microscopy of the bands showed two types of particles: small and dense particles measuring 100-150 nm and lymphocystis virions that measured about 300-350 nm in diameter.

Quantitative real-time single particle analysis of virions.

PubMed

Heider, Susanne; Metzner, Christoph

2014-08-01

Providing information about single virus particles has for a long time been mainly the domain of electron microscopy. More recently, technologies have been developed-or adapted from other fields, such as nanotechnology-to allow for the real-time quantification of physical virion particles, while supplying additional information such as particle diameter concomitantly. These technologies have progressed to the stage of commercialization increasing the speed of viral titer measurements from hours to minutes, thus providing a significant advantage for many aspects of virology research and biotechnology applications. Additional advantages lie in the broad spectrum of virus species that may be measured and the possibility to determine the ratio of infectious to total particles. A series of disadvantages remain associated with these technologies, such as a low specificity for viral particles. In this review we will discuss these technologies by comparing four systems for real-time single virus particle analysis and quantification. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

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.

Splicing-independent loading of TREX on nascent RNA is required for efficient expression of dual-strand piRNA clusters in Drosophila

PubMed Central

Hur, Junho K.; Luo, Yicheng; Moon, Sungjin; Ninova, Maria; Marinov, Georgi K.; Chung, Yun D.; Aravin, Alexei A.

2016-01-01

The conserved THO/TREX (transcription/export) complex is critical for pre-mRNA processing and mRNA nuclear export. In metazoa, TREX is loaded on nascent RNA transcribed by RNA polymerase II in a splicing-dependent fashion; however, how TREX functions is poorly understood. Here we show that Thoc5 and other TREX components are essential for the biogenesis of piRNA, a distinct class of small noncoding RNAs that control expression of transposable elements (TEs) in the Drosophila germline. Mutations in TREX lead to defects in piRNA biogenesis, resulting in derepression of multiple TE families, gametogenesis defects, and sterility. TREX components are enriched on piRNA precursors transcribed from dual-strand piRNA clusters and colocalize in distinct nuclear foci that overlap with sites of piRNA transcription. The localization of TREX in nuclear foci and its loading on piRNA precursor transcripts depend on Cutoff, a protein associated with chromatin of piRNA clusters. Finally, we show that TREX is required for accumulation of nascent piRNA precursors. Our study reveals a novel splicing-independent mechanism for TREX loading on nascent RNA and its importance in piRNA biogenesis. PMID:27036967

Directional Spread of Surface-Associated Retroviruses Regulated by Differential Virus-Cell Interactions▿ †

PubMed Central

Sherer, Nathan M.; Jin, Jing; Mothes, Walther

2010-01-01

The spread of viral infections involves the directional progression of virus particles from infected cells to uninfected target cells. Prior to entry, the binding of virus particles to specific cell surface receptors can trigger virus surfing, an actin-dependent lateral transport of viruses toward the cell body (M. J. Lehmann et al., J. Cell Biol. 170:317-325, 2005; M. Schelhaas, et al., PLoS Pathog. 4:e1000148, 2008; J. L. Smith, D. S. Lidke, and M. A. Ozbun, Virology 381:16-21, 2008). Here, we have used live-cell imaging to demonstrate that for cells chronically infected with the gammaretrovirus murine leukemia virus in which receptor has been downregulated, a significant portion of completely assembled virus particles are not immediately released into the supernatant but retain long-term association with the cell surface. Retention can be attributed, at least in part, to nonspecific particle attachment to cell surface glycosylaminoglycans. In contrast to virus surfing, viruses retained at the surface of infected cells undergo a lateral motility that is random and actin independent. This diffusive motility can be abruptly halted and converted into inward surfing after treatment with Polybrene, a soluble cation that increases virus-cell adsorption. In the absence of Polybrene, particle diffusion allows for an outward flow of viruses to the infected cell periphery. Peripheral particles are readily captured by and transmitted to neighboring uninfected target cells in a directional fashion. These data demonstrate a surface-based mechanism for the directional spread of viruses regulated by differential virus-cell interactions. PMID:20089647

Biomimetic structural engineering of P22 virus-like particles for catalysis and immune modulation

NASA Astrophysics Data System (ADS)

Schwarz, Benjamin

Within biology molecules are arranged in hierarchical structures that coordinate and control the many processes that allow for complex organisms to exist. Proteins and other functional macromolecules are often studied outside their natural nanostructural context because it remains difficult to create controlled arrangements of proteins at this size scale. Viruses are elegantly simple nano-systems that exist at the interface of living organisms and non-living biological machines. Studied and viewed primarily as pathogens to be combatted, viruses have emerged as models of structural efficiency at the nanoscale and have spurred the development of biomimetic nanoparticle systems. Virus-like particles (VLPs) are noninfectious protein cages derived from viruses or other cage-forming systems. VLPs provide incredibly regular scaffolds for building at the nanoscale. In this work I have utilized the VLP derived from the bacteriophage P22 as a platform for the organization of enzymes, antigens, and immune-stimulating proteins inside and outside the capsid through purely genetic means. In the case of enzymes, encapsulation of a two-enzyme pathway has led to the development of metabolic nanoparticle catalysts and an expanded understanding of the control that structure exerts on metabolic flux. These same structural elements applied to the delivery of protein subunit antigens directed at cytotoxic T cell immunity result in drastically enhanced antigen processing and lasting immunological memory. Lastly, presentation of immune-stimulating proteins from the Tumor Necrosis Factor Super Family on the surface of the P22 VLP enhances the cell signaling efficiency of these compounds 50-fold and provides strategies for the application of these proteins as immune modulatory oncology therapeutics. In all of these cases, the reintroduction of nanostructure to these protein systems, reminiscent of their natural environment, has led to both new technologies and a better understanding of the

Lipids and RNA virus replication.

PubMed

Konan, Kouacou V; Sanchez-Felipe, Lorena

2014-12-01

Most viruses rely heavily on their host machinery to successfully replicate their genome and produce new virus particles. Recently, the interaction of positive-strand RNA viruses with the lipid biosynthetic and transport machinery has been the subject of intense investigation. In this review, we will discuss the contribution of various host lipids and related proteins in RNA virus replication and maturation. Copyright © 2014 Elsevier B.V. All rights reserved.

High serum levels of pregenomic RNA reflect frequently failing reverse transcription in hepatitis B virus particles.

PubMed

Prakash, Kasthuri; Rydell, Gustaf E; Larsson, Simon B; Andersson, Maria; Norkrans, Gunnar; Norder, Heléne; Lindh, Magnus

2018-05-15

Hepatocytes infected by hepatitis B virus (HBV) produce different HBV RNA species, including pregenomic RNA (pgRNA), which is reverse transcribed during replication. Particles containing HBV RNA are present in serum of infected individuals, and quantification of this HBV RNA could be clinically useful. In a retrospective study of 95 patients with chronic HBV infection, we characterised HBV RNA in serum in terms of concentration, particle association and sequence. HBV RNA was detected by real-time PCR at levels almost as high as HBV DNA. The HBV RNA was protected from RNase and it was found in particles of similar density as particles containing HBV DNA after fractionation on a Nycodenz gradient. Sequencing the epsilon region of the RNA did not reveal mutations that would preclude its binding to the viral polymerase before encapsidation. Specific quantification of precore RNA and pgRNA by digital PCR showed almost seven times lower ratio of precore RNA/pgRNA in serum than in liver tissue, which corresponds to poorer encapsidation of this RNA as compared with pgRNA. The serum ratio between HBV DNA and HBV RNA was higher in genotype D as compared with other genotypes. The results suggest that HBV RNA in serum is present in viral particles with failing reverse transcription activity, which are produced at almost as high rates as viral particles containing DNA. The results encourage further studies of the mechanisms by which these particles are produced, the impact of genotype, and the potential clinical utility of quantifying HBV RNA in serum.

Paramyxovirus Assembly and Budding: Building Particles that Transmit Infections

PubMed Central

Harrison, Megan S.; Sakaguchi, Takemasa; Schmitt, Anthony P.

2010-01-01

The paramyxoviruses define a diverse group of enveloped RNA viruses that includes a number of important human and animal pathogens. Examples include human respiratory syncytial virus and the human parainfluenza viruses, which cause respiratory illnesses in young children and the elderly; measles and mumps viruses, which have caused recent resurgences of disease in developed countries; the zoonotic Hendra and Nipah viruses, which have caused several outbreaks of fatal disease in Australia and Asia; and Newcastle disease virus, which infects chickens and other avian species. Like other enveloped viruses, paramyxoviruses form particles that assemble and bud from cellular membranes, allowing the transmission of infections to new cells and hosts. Here, we review recent advances that have improved our understanding of events involved in paramyxovirus particle formation. Contributions of viral matrix proteins, glycoproteins, nucleocapsid proteins, and accessory proteins to particle formation are discussed, as well as the importance of host factor recruitment for efficient virus budding. Trafficking of viral structural components within infected cells is described, together with mechanisms that allow for the selection of specific sites on cellular membranes for the coalescence of viral proteins in preparation of bud formation and virion release. PMID:20398786

Differentiation of West Nile and St. Louis Encephalitis Virus Infections by Use of Noninfectious Virus-Like Particles with Reduced Cross-Reactivity▿ †

PubMed Central

Roberson, Jill A.; Crill, Wayne D.; Chang, Gwong-Jen J.

2007-01-01

Differential diagnosis of St. Louis encephalitis virus (SLEV) and West Nile virus (WNV) infections can be complicated due to the high degree of cross-reactivity observed in most serodiagnostic assays. In an effort to provide a more specific diagnostic test, we developed virus-like particle (VLP) antigens with reduced cross-reactivity for both SLEV and WNV by identifying and mutating envelope protein amino acids within the cross-reactive epitopes of VLP expression plasmids. To determine the serodiagnostic discriminatory ability of the antigens with reduced cross-reactivity, a panel of 134 human serum samples collected predominately from North American patients with SLEV or WNV infections was used to evaluate the performance of these novel antigens in imunoglobulin M antibody-capture enzyme-linked immunosorbent assays. Positive/negative ratios and the resulting diagnostic classifications were compared between the mutant and the wild-type (WT) VLPs. The mutant VLP antigens were more specific, with higher positive predictive values and higher likelihood ratios than the WT VLP antigens. Both the SLEV and WNV mutant VLPs greatly reduced the observed cross-reactivity, significantly increasing the specificity and sensitivity of the assay. The use of these novel VLP antigens with reduced cross-reactivity in these serodiagnostic assays and others should lead to more accurate diagnoses of current infections, thereby reducing the need for time-consuming and cumbersome confirmatory plaque-reduction neutralization tests to differentiate between SLEV and WNV infections in North America. PMID:17715375

Natural protection from zoonosis by alpha-gal epitopes on virus particles in xenotransmission.

PubMed

Kim, Na Young; Jung, Woon-Won; Oh, Yu-Kyung; Chun, Taehoon; Park, Hong-Yang; Lee, Hoon-Taek; Han, In-Kwon; Yang, Jai Myung; Kim, Young Bong

2007-03-01

Clinical transplantation has become one of the preferred treatments for end-stage organ failure, and one of the novel approaches being pursued to overcome the limited supply of human organs involves the use of organs from other species. The pig appears to be a near ideal animal due to proximity to humans, domestication, and ability to procreate. The presence of Gal-alpha1,3-Gal residues on the surfaces of pig cells is a major immunological obstacle to xenotransplantation. Alpha1,3galactosyltransferase (alpha1,3GT) catalyzes the synthesis of Gal alpha 1-3Gal beta 1-4GlcNAc-R (alpha-gal epitope) on the glycoproteins and glycolipids of non-primate mammals, but this does not occur in humans. Moreover, the alpha-gal epitope causes hyperacute rejection of pig organs in humans, and thus, the elimination of this antigen from pig tissues is highly desirable. Recently, concerns have been raised that the risk of virus transmission from such pigs may be increased due to the absence of alpha-gal on their viral particles. In this study, transgenic cells expressing alpha1,3GT were selected using 1.25 mg/ml neomycin. The development of HeLa cells expressing alpha1,3GT now allows accurate studies to be conducted on the function of the alpha-gal epitope in xenotransmission. The expressions of alpha-gal epitopes on HeLa/alpha-gal cells were demonstrated by flow cytometry and confocal microscopy using cells stained with IB4-fluorescein isothiocyanate lectin. Vaccinia viruses propagated in HeLa/alpha-gal cells also expressed alpha-gal on their viral envelopes and were more sensitive to inactivation by human sera than vaccinia virus propagated in HeLa cells. Moreover, neutralization of vaccinia virus was inhibited in human serum by 10 mm ethylene glycol bis(beta-aminoethylether)tetraacetic acid (EDTA) treatment. Our data indicated that alpha-gal epitopes are one of the major barriers to zoonosis via xenotransmission.

The influence of ligand charge and length on the assembly of Brome mosaic virus derived virus-like particles with magnetic core

NASA Astrophysics Data System (ADS)

Mieloch, Adam A.; Krecisz, Monika; Rybka, Jakub D.; Strugała, Aleksander; Krupiński, Michał; Urbanowicz, Anna; Kozak, Maciej; Skalski, Bohdan; Figlerowicz, Marek; Giersig, Michael

2018-03-01

Virus-like particles (VLPs) have sparked a great interest in the field of nanobiotechnology and nanomedicine. The introduction of superparamagnetic nanoparticles (SPIONs) as a core, provides potential use of VLPs in the hyperthermia therapy, MRI contrast agents and magnetically-powered delivery agents. Magnetite NPs also provide a significant improvement in terms of VLPs stability. Moreover employing viral structural proteins as self-assembling units has opened a new paths for targeted therapy, drug delivery systems, vaccines design, and many more. In many cases, the self-assembly of a virus strongly depends on electrostatic interactions between positively charged groups of the capsid proteins and negatively charged nucleic acid. This phenomenon imposes the negative net charge as a key requirement for the core nanoparticle. In our experiments, Brome mosaic virus (BMV) capsid proteins isolated from infected plants Hordeum vulgare were used. Superparamagnetic iron oxide nanoparticles (Fe3O4) with 15 nm in diameter were synthesized by thermal decomposition and functionalized with COOH-PEG-PL polymer or dihexadecylphosphate (DHP) in order to provide water solubility and negative charge required for the assembly. Nanoparticles were characterized by Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS), Zeta Potential, Fourier Transformed Infrared Spectroscopy (FTIR) and Superconducting Quantum Interference Device (SQUID) magnetometry. TEM and DLS study were conducted to verify VLPs creation. This study demonstrates that the increase of negative surface charge is not a sufficient factor determining successful assembly. Additional steric interactions provided by longer ligands are crucial for the assembly of BMV SPION VLPs and may enhance the colloidal stability.

Bioprocessing of plant-derived virus-like particles of Norwalk virus capsid protein under current Good Manufacture Practice regulations

PubMed Central

Lai, Huafang; Chen, Qiang

2012-01-01

Despite the success in expressing a variety of subunit vaccine proteins in plants and the recent stride in improving vaccine accumulation levels by transient expression systems, there is still no plant-derived vaccine that has been licensed for human use. The lack of commercial success of plant-made vaccines lies in several technical and regulatory barriers that remain to be overcome. These challenges include the lack of scalable downstream processing procedures, the uncertainty of regulatory compliance of production processes, and the lack of demonstration of plant-derived products that meet the required standards of regulatory agencies in identity, purity, potency and safety. In this study, we addressed these remaining challenges and successfully demonstrate the ability of using plants to produce a pharmaceutical grade Norwalk virus (NV) vaccine under current Good Manufacture Practice (cGMP) guidelines at multiple gram scales. Our results demonstrate that an efficient and scalable extraction and purification scheme can established for processing virus-like particles (VLP) of NV capsid protein (NVCP). We successfully operated the upstream and downstream NVCP production processes under cGMP regulations. Furthermore, plant-derived NVCP VLP demonstrates the identity, purity, potency and safety that meet the preset release specifications. This material is being tested in a Phase I human clinical trial. This research provides the first report of producing a plant-derived vaccine at scale under cGMP regulations in an academic setting and an important step for plant-produced vaccines to become a commercial reality. PMID:22134876

Synthesis of human parainfluenza virus 4 nucleocapsid-like particles in yeast and their use for detection of virus-specific antibodies in human serum.

PubMed

Bulavaitė, Aistė; Lasickienė, Rita; Tamošiūnas, Paulius Lukas; Simanavičius, Martynas; Sasnauskas, Kęstutis; Žvirblienė, Aurelija

2017-04-01

The aim of this study was to produce human parainfluenza virus type 4 (HPIV4) nucleocapsid (N) protein in yeast Saccharomyces cerevisiae expression system, to explore its structural and antigenic properties and to evaluate its applicability in serology. The use of an optimized gene encoding HPIV4 N protein amino acid (aa) sequence GenBank AGU90031.1 allowed high yield of recombinant N protein forming nucleocapsid-like particles (NLPs) in yeast. A substitution L332D disrupted self-assembly of NLPs, confirming the role of this position in the N proteins of Paramyxovirinae. Three monoclonal antibodies (MAbs) were generated against the NLP-forming HPIV4 N protein. They recognised HPIV4-infected cells, demonstrating the antigenic similarity between the recombinant and virus-derived N proteins. HPIV4 N protein was used as a coating antigen in an indirect IgG ELISA with serum specimens of 154 patients with respiratory tract infection. The same serum specimens were tested with previously generated N protein of a closely related HPIV2, another representative of genus Rubulavirus. Competitive ELISA was developed using related yeast-produced viral antigens to deplete the cross-reactive serum antibodies. In the ELISA either without or with competition using heterologous HPIV (2 or 4) N or mumps virus N proteins, the seroprevalence of HPIV4 N-specific IgG was, respectively, 46.8, 39.6 and 40.3% and the seroprevalence of HPIV2 N-specific IgG-47.4, 39.0 and 37.7%. In conclusion, yeast-produced HPIV4 N protein shares structural and antigenic properties of the native virus nucleocapsids. Yeast-produced HPIV4 and HPIV2 NLPs are prospective tools in serology.

High-yield production of canine parvovirus virus-like particles in a baculovirus expression system.

PubMed

Jin, Hongli; Xia, Xiaohong; Liu, Bing; Fu, Yu; Chen, Xianping; Wang, Huihui; Xia, Zhenqiang

2016-03-01

An optimized VP2 gene from the current prevalent CPV strain (new CPV-2a) in China was expressed in a baculovirus expression system. It was found that the VP2 proteins assembled into virus-like particles (VLPs) with antigenic properties similar to those of natural CPV and with an especially high hemagglutination (HA) titer (1:2(20)). Dogs intramuscularly or orally immunized with VLPs produced antibodies against CPV with >1:80 hemagglutination inhibition (HI) units for at least 3 months. The CPV VLPs could be considered for use as a vaccine against CPV or as a platform for research on chimeric VLP vaccines against other diseases.

The African swine fever virus virion membrane protein pE248R is required for virus infectivity and an early postentry event.

PubMed

Rodríguez, Irene; Nogal, María L; Redrejo-Rodríguez, Modesto; Bustos, María J; Salas, María L

2009-12-01

The African swine fever virus (ASFV) protein pE248R, encoded by the gene E248R, is a late structural component of the virus particle. The protein contains intramolecular disulfide bonds and has been previously identified as a substrate of the ASFV-encoded redox system. Its amino acid sequence contains a putative myristoylation site and a hydrophobic transmembrane region near its carboxy terminus. We show here that the protein pE248R is myristoylated during infection and associates with the membrane fraction in infected cells, behaving as an integral membrane protein. Furthermore, the protein localizes at the inner envelope of the virus particles in the cytoplasmic factories. The function of the protein pE248R in ASFV replication was investigated by using a recombinant virus that inducibly expresses the gene E248R. Under repressive conditions, the ASFV polyproteins pp220 and pp62 are normally processed and virus particles with morphology indistinguishable from that of those produced in a wild-type infection or under permissive conditions are generated. Moreover, the mutant virus particles can exit the cell as does the parental virus. However, the infectivity of the pE248R-deficient virions was reduced at least 100-fold. An investigation of the defect of the mutant virus indicated that neither virus binding nor internalization was affected by the absence of the protein pE248R, but a cytopathic effect was not induced and early and late gene expression was impaired, indicating that the protein is required for some early postentry event.

Novel mechanism of antibodies to hepatitis B virus in blocking viral particle release from cells.

PubMed

Neumann, Avidan U; Phillips, Sandra; Levine, Idit; Ijaz, Samreen; Dahari, Harel; Eren, Rachel; Dagan, Shlomo; Naoumov, Nikolai V

2010-09-01

Antibodies are thought to exert antiviral activities by blocking viral entry into cells and/or accelerating viral clearance from circulation. In particular, antibodies to hepatitis B virus (HBV) surface antigen (HBsAg) confer protection, by binding circulating virus. Here, we used mathematical modeling to gain information about viral dynamics during and after single or multiple infusions of a combination of two human monoclonal anti-HBs (HepeX-B) antibodies in patients with chronic hepatitis B. The antibody HBV-17 recognizes a conformational epitope, whereas antibody HBV-19 recognizes a linear epitope on the HBsAg. The kinetic profiles of the decline of serum HBV DNA and HBsAg revealed partial blocking of virion release from infected cells as a new antiviral mechanism, in addition to acceleration of HBV clearance from the circulation. We then replicated this approach in vitro, using cells secreting HBsAg, and compared the prediction of the mathematical modeling obtained from the in vivo kinetics. In vitro, HepeX-B treatment of HBsAg-producing cells showed cellular uptake of antibodies, resulting in intracellular accumulation of viral particles. Blocking of HBsAg secretion also continued after HepeX-B was removed from the cell culture supernatants. These results identify a novel antiviral mechanism of antibodies to HBsAg (anti-HBs) involving prolonged blocking of the HBV and HBsAg subviral particles release from infected cells. This may have implications in designing new therapies for patients with chronic HBV infection and may also be relevant in other viral infections.

Novel functions of prototype foamy virus Gag glycine- arginine-rich boxes in reverse transcription and particle morphogenesis.

PubMed

Müllers, Erik; Uhlig, Tobias; Stirnnagel, Kristin; Fiebig, Uwe; Zentgraf, Hanswalter; Lindemann, Dirk

2011-02-01

Prototype foamy virus (PFV) Gag lacks the characteristic orthoretroviral Cys-His motifs that are essential for various steps of the orthoretroviral replication cycle, such as RNA packaging, reverse transcription, infectivity, integration, and viral assembly. Instead, it contains three glycine-arginine-rich boxes (GR boxes) in its C terminus that putatively represent a functional equivalent. We used a four-plasmid replication-deficient PFV vector system, with uncoupled RNA genome packaging and structural protein translation, to analyze the effects of deletion and various substitution mutations within each GR box on particle release, particle-associated protein composition, RNA packaging, DNA content, infectivity, particle morphology, and intracellular localization. The degree of viral particle release by all mutants was similar to that of the wild type. Only minimal effects on Pol encapsidation, exogenous reverse transcriptase (RT) activity, and genomic viral RNA packaging were observed. In contrast, particle-associated DNA content and infectivity were drastically reduced for all deletion mutants and were undetectable for all alanine substitution mutants. Furthermore, GR box I mutants had significant changes in particle morphology, and GR box II mutants lacked the typical nuclear localization pattern of PFV Gag. Finally, it could be shown that GR boxes I and III, but not GR box II, can functionally complement each other. It therefore appears that, similar to the orthoretroviral Cys-His motifs, the PFV Gag GR boxes are important for RNA encapsidation, genome reverse transcription, and virion infectivity as well as for particle morphogenesis.

Construction and immunological characterization of CD40L or GM-CSF incorporated Hantaan virus like particle

PubMed Central

Zhang, Xiaoxiao; Truax, Agnieszka D.; Ma, Ruixue; Liu, Ziyu; Lei, Yingfeng; Zhang, Liang; Ye, Wei; Zhang, Fanglin; Xu, Zhikai; Shang, Lei; Liu, Rongrong; Wang, Fang; Wu, Xingan

2016-01-01

Infection of Hantaan virus (HTNV) usually causes hemorrhagic fever with renal syndrome (HFRS). China has the worst epidemic incidence of HFRS as well as high fatality. Inactivated whole virus has been used for HFRS vaccination, however there are still problems such as safety concerns. CD40 ligand (CD40L) and granulocyte macrophage colony-stimulating factor (GM-CSF) are well-known immune stimulating molecules that can enhance antigen presenting, lymphocytes activation and maturation, incorporation of CD40L and GM-CSF to the surface of virus like particles (VLPs) can greatly improve the vaccination effect. We constructed eukaryotic vectors expressing HTNV M segment and S segment, as well as vectors expressing HTNV M segment with CD40L or GM-CSF, our results showed successful production of CD40L or GM-CSF incorporated HTNV VLPs. In vitro stimulation with CD40L or GM-CSF anchored HTNV VLP showed enhanced activation of macrophages and DCs. CD40L/GM-CSF incorporated VLP can induce higher level of HTNV specific antibody and neutralizing antibody in mice. Immunized mice splenocytes showed higher ability of secreting IFN-γ and IL-2, as well as enhancing CTL activity. These results suggest CD40L/GM-CSF incorporated VLP can serve as prospective vaccine candidate. PMID:27542281

Defining the sizes of airborne particles that mediate influenza transmission in ferrets.

PubMed

Zhou, Jie; Wei, Jianjian; Choy, Ka-Tim; Sia, Sin Fun; Rowlands, Dewi K; Yu, Dan; Wu, Chung-Yi; Lindsley, William G; Cowling, Benjamin J; McDevitt, James; Peiris, Malik; Li, Yuguo; Yen, Hui-Ling

2018-03-06

Epidemics and pandemics of influenza are characterized by rapid global spread mediated by non-mutually exclusive transmission modes. The relative significance between contact, droplet, and airborne transmission is yet to be defined, a knowledge gap for implementing evidence-based infection control measures. We devised a transmission chamber that separates virus-laden particles by size and determined the particle sizes mediating transmission of influenza among ferrets through the air. Ferret-to-ferret transmission was mediated by airborne particles larger than 1.5 µm, consistent with the quantity and size of virus-laden particles released by the donors. Onward transmission by donors was most efficient before fever onset and may continue for 5 days after inoculation. Multiple virus gene segments enhanced the transmissibility of a swine influenza virus among ferrets by increasing the release of virus-laden particles into the air. We provide direct experimental evidence of influenza transmission via droplets and fine droplet nuclei, albeit at different efficiency. Copyright © 2018 the Author(s). Published by PNAS.

Immunogenicity of Newcastle disease virus vectors expressing Norwalk virus capsid protein in the presence or absence of VP2 protein.

PubMed

Kim, Shin-Hee; Chen, Shun; Jiang, Xi; Green, Kim Y; Samal, Siba K

2015-10-01

Noroviruses are the most common cause of acute gastroenteritis in humans. Development of an effective vaccine is required for reducing their outbreaks. In order to develop a GI norovirus vaccine, Newcastle disease virus vectors, rLaSota and modified rBC, were used to express VP1 protein of Norwalk virus. Co-expression of VP1 and VP2 proteins by Newcastle disease virus vectors resulted in enhanced expression of Norwalk virus VP1 protein and self-assembly of VP1 protein into virus-like particles. Furthermore, the Norwalk virus-specific IgG response induced in mice by Newcastle disease virus vectors was similar to that induced by baculovirus-expressed virus-like particles in mice. However, the modified rBC vector in the presence of VP2 protein induced significantly higher levels of cellular and mucosal immune responses than those induced by baculovirus-expressed VLPs. These results indicate that Newcastle disease virus has great potential for developing a live Norwalk virus vaccine by inducing humoral, cellular and mucosal immune responses in humans. Copyright © 2015 Elsevier Inc. All rights reserved.

Immunogenicity of Newcastle Disease Virus Vectors Expressing Norwalk Virus Capsid Protein in the Presence or Absence of VP2 Protein

PubMed Central

Kim, Shin-Hee; Chen, Shun; Jiang, Xi; Green, Kim Y.; Samal, Siba K.

2015-01-01

Noroviruses are the most common cause of acute gastroenteritis in humans. Development of an effective vaccine is required for reducing their outbreaks. In order to develop a GI norovirus vaccine, Newcastle disease virus vectors, rLaSota and modified rBC, were used to express VP1 protein of Norwalk virus. Co-expression of VP1 and VP2 proteins by Newcastle disease virus vectors resulted in enhanced expression of Norwalk virus VP1 protein and self-assembly of VP1 protein into virus-like particles. Furthermore, the Norwalk virus-specific IgG response induced in mice by Newcastle disease virus vectors was similar to that induced by baculovirs-expressed virus-like particles in mice. However, the modified rBC vector in the presence of VP2 protein induced significantly higher levels of cellular and mucosal immune responses than those induced by baculovirus-expressed VLPs. These results indicate that Newcastle disease virus has great potential for developing a live Norwalk virus vaccine by inducing humoral, cellular and mucosal immune responses in humans. PMID:26099695

The preparation of nanosized polyethylene particles via novel heterogeneous non-metallocene catalyst (m-CH3PhO)TiCl3/CNTs/AlEt3

NASA Astrophysics Data System (ADS)

Wang, J.; Guo, J. P.; Yi, J. J.; Huang, Q. G.; Li, H. M.; Li, Y. F.; Gao, K. J.; Yang, W. T.

2014-08-01

This paper reports the preparation of coral-shaped topological morphology nascent polyethylene (PE) particles promoted by the novel heterogeneous non-metallocene catalyst (m-CH3PhO)TiCl3/carbon nanotubes (CNTs), with AlEt3 used as a cocatalyst. Scanning electron microscope (SEM), high resolution transmission electron microscope (HR-TEM) and inductively coupled plasma (ICP) emission spectroscopy were used to determine the morphology of the catalyst particles and the content of (m-CH3PhO)TiCl3. The carbon nanotube surface was treated with Grignard Reagent prior to reacting with (m-CH3PhO)TiCl3. The catalyst system could effectively catalyze ethylene polymerization and ethylene with 1- hexene copolymerization, the catalytic activity could reach up to 5.8 kg/((gTi)h). Morphology of the obtained polymer particles by SEM and HR-TEM technique revealed that the nascent polyethylene particles looked like coral shape in micro-size. The multiwalled carbon nanotubes (MWCNTs) supported catalysts polymerized ethylene to form polymer nanocomposite in situ. The microscopic examination of this nanocomposite revealed that carbon nanoparticles in PE matrix had a good distribution and the cryogenically fractured surface was ductile-like when polymerization time was 2 min.

Hepatitis E virus ORF3 is a functional ion channel required for release of infectious particles.

PubMed

Ding, Qiang; Heller, Brigitte; Capuccino, Juan M V; Song, Bokai; Nimgaonkar, Ila; Hrebikova, Gabriela; Contreras, Jorge E; Ploss, Alexander

2017-01-31

Hepatitis E virus (HEV) is the leading cause of enterically transmitted viral hepatitis globally. Of HEV's three ORFs, the function of ORF3 has remained elusive. Here, we demonstrate that via homophilic interactions ORF3 forms multimeric complexes associated with intracellular endoplasmic reticulum (ER)-derived membranes. HEV ORF3 shares several structural features with class I viroporins, and the function of HEV ORF3 can be maintained by replacing it with the well-characterized viroporin influenza A virus (IAV) matrix-2 protein. ORF3's ion channel function is further evidenced by its ability to mediate ionic currents when expressed in Xenopus laevis oocytes. Furthermore, we identified several positions in ORF3 critical for its formation of multimeric complexes, ion channel activity, and, ultimately, release of infectious particles. Collectively, our data demonstrate a previously undescribed function of HEV ORF3 as a viroporin, which may serve as an attractive target in developing direct-acting antivirals.

Quantitative live-cell imaging of human immunodeficiency virus (HIV-1) assembly.

PubMed

Baumgärtel, Viola; Müller, Barbara; Lamb, Don C

2012-05-01

Advances in fluorescence methodologies make it possible to investigate biological systems in unprecedented detail. Over the last few years, quantitative live-cell imaging has increasingly been used to study the dynamic interactions of viruses with cells and is expected to become even more indispensable in the future. Here, we describe different fluorescence labeling strategies that have been used to label HIV-1 for live cell imaging and the fluorescence based methods used to visualize individual aspects of virus-cell interactions. This review presents an overview of experimental methods and recent experiments that have employed quantitative microscopy in order to elucidate the dynamics of late stages in the HIV-1 replication cycle. This includes cytosolic interactions of the main structural protein, Gag, with itself and the viral RNA genome, the recruitment of Gag and RNA to the plasma membrane, virion assembly at the membrane and the recruitment of cellular proteins involved in HIV-1 release to the nascent budding site.

Quantitative Live-Cell Imaging of Human Immunodeficiency Virus (HIV-1) Assembly

PubMed Central

Baumgärtel, Viola; Müller, Barbara; Lamb, Don C.

2012-01-01

Advances in fluorescence methodologies make it possible to investigate biological systems in unprecedented detail. Over the last few years, quantitative live-cell imaging has increasingly been used to study the dynamic interactions of viruses with cells and is expected to become even more indispensable in the future. Here, we describe different fluorescence labeling strategies that have been used to label HIV-1 for live cell imaging and the fluorescence based methods used to visualize individual aspects of virus-cell interactions. This review presents an overview of experimental methods and recent experiments that have employed quantitative microscopy in order to elucidate the dynamics of late stages in the HIV-1 replication cycle. This includes cytosolic interactions of the main structural protein, Gag, with itself and the viral RNA genome, the recruitment of Gag and RNA to the plasma membrane, virion assembly at the membrane and the recruitment of cellular proteins involved in HIV-1 release to the nascent budding site. PMID:22754649

Herpes Simplex Virus Dances with Amyloid Precursor Protein while Exiting the Cell

PubMed Central

Cheng, Shi-Bin; Ferland, Paulette; Webster, Paul; Bearer, Elaine L.

2011-01-01

Herpes simplex type 1 (HSV1) replicates in epithelial cells and secondarily enters local sensory neuronal processes, traveling retrograde to the neuronal nucleus to enter latency. Upon reawakening newly synthesized viral particles travel anterograde back to the epithelial cells of the lip, causing the recurrent cold sore. HSV1 co-purifies with amyloid precursor protein (APP), a cellular transmembrane glycoprotein and receptor for anterograde transport machinery that when proteolyzed produces A-beta, the major component of senile plaques. Here we focus on transport inside epithelial cells of newly synthesized virus during its transit to the cell surface. We hypothesize that HSV1 recruits cellular APP during transport. We explore this with quantitative immuno-fluorescence, immuno-gold electron-microscopy and live cell confocal imaging. After synchronous infection most nascent VP26-GFP-labeled viral particles in the cytoplasm co-localize with APP (72.8+/−6.7%) and travel together with APP inside living cells (81.1+/−28.9%). This interaction has functional consequences: HSV1 infection decreases the average velocity of APP particles (from 1.1+/−0.2 to 0.3+/−0.1 µm/s) and results in APP mal-distribution in infected cells, while interplay with APP-particles increases the frequency (from 10% to 81% motile) and velocity (from 0.3+/−0.1 to 0.4+/−0.1 µm/s) of VP26-GFP transport. In cells infected with HSV1 lacking the viral Fc receptor, gE, an envelope glycoprotein also involved in viral axonal transport, APP-capsid interactions are preserved while the distribution and dynamics of dual-label particles differ from wild-type by both immuno-fluorescence and live imaging. Knock-down of APP with siRNA eliminates APP staining, confirming specificity. Our results indicate that most intracellular HSV1 particles undergo frequent dynamic interplay with APP in a manner that facilitates viral transport and interferes with normal APP transport and distribution. Such dynamic

Tandem Fusion of Hepatitis B Core Antigen Allows Assembly of Virus-Like Particles in Bacteria and Plants with Enhanced Capacity to Accommodate Foreign Proteins

PubMed Central

Peyret, Hadrien; Gehin, Annick; Thuenemann, Eva C.; Blond, Donatienne; El Turabi, Aadil; Beales, Lucy; Clarke, Dean; Gilbert, Robert J. C.; Fry, Elizabeth E.; Stuart, David I.; Holmes, Kris; Stonehouse, Nicola J.; Whelan, Mike; Rosenberg, William; Lomonossoff, George P.; Rowlands, David J.

2015-01-01

The core protein of the hepatitis B virus, HBcAg, assembles into highly immunogenic virus-like particles (HBc VLPs) when expressed in a variety of heterologous systems. Specifically, the major insertion region (MIR) on the HBcAg protein allows the insertion of foreign sequences, which are then exposed on the tips of surface spike structures on the outside of the assembled particle. Here, we present a novel strategy which aids the display of whole proteins on the surface of HBc particles. This strategy, named tandem core, is based on the production of the HBcAg dimer as a single polypeptide chain by tandem fusion of two HBcAg open reading frames. This allows the insertion of large heterologous sequences in only one of the two MIRs in each spike, without compromising VLP formation. We present the use of tandem core technology in both plant and bacterial expression systems. The results show that tandem core particles can be produced with unmodified MIRs, or with one MIR in each tandem dimer modified to contain the entire sequence of GFP or of a camelid nanobody. Both inserted proteins are correctly folded and the nanobody fused to the surface of the tandem core particle (which we name tandibody) retains the ability to bind to its cognate antigen. This technology paves the way for the display of natively folded proteins on the surface of HBc particles either through direct fusion or through non-covalent attachment via a nanobody. PMID:25830365

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

In Vitro Morphology and Maturation of Lymphocystis Virus 1

PubMed Central

Midlige, F. H.; Malsberger, R. G.

1968-01-01

The temporal sequence of development of lymphocystis disease virus (LDV) was studied by electron microscopy of thin sections of infected tissue-culture monolayers. Neither the typical cytoplasmic inclusion nor virus was detected at 4 days postinfection (PI). Inclusions, but no viruses, were detected at 8 days PI. Inclusions and associated virions were detected at 15 days PI, and by 28 days PI the undisrupted cells were filled with the typical virions. No release mechanism was detected, and severe clumping of particles was noted. Negatively stained preparations revealed particles 200 nm in diameter with no capsomere structure and apparent spikes associated with the particle. The relationship of LDV to the well-defined deoxyribonucleic acid virus groups is discussed. Images PMID:5701821