The Experts below are selected from a list of 126 Experts worldwide ranked by ideXlab platform
Gregor Meyers - One of the best experts on this subject based on the ideXlab platform.
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The role of the Defective Interfering Particle DI9c in mucosal disease in cattle.
Archives of virology, 2003Co-Authors: Maria Stokstad, Gregor Meyers, M. E. Collins, R. Sorby, P. Barboni, T. Loken, Joe BrownlieAbstract:Mucosal disease occurs in cattle persistently infected with a noncytopathogenic strain of bovine viral diarrhoea virus (BVDVnc) following in utero infection. The disease can be initiated by superinfection with a cytopathogenic biotype (BVDVc) of the virus with antigenic “homology” to the persisting virus. A BVDVc isolated from a clinical case of mucosal disease has been discovered to consist of a Defective Interfering Particle, DI9, and an associated BVDVnc helper virus. A Defective virus corresponding to DI9 was recently recovered from an infectious cDNA clone and was named DI9c. To evaluate the role of DI9 in the pathogenesis of mucosal disease a two-part experimental study was carried out which included clinical, haematological, pathological and virological investigations. Eight of nine calves persistently infected with BVDVnc were experimentally inoculated with DI9c. The Defective virus was propagated in cells preinfected with the same strain of virus used to persistently infect the calves in utero. The calves were euthanased on days 4, 7, 14, 21, 28, 40, 40 or 87 post inoculation. None of the inoculated animals developed classical mucosal disease, neither clinically nor pathologically. DI9c was not found in serum, nasal swab or tissue samples from the calves by observing cytopathogenic effect and/or using a polymerase chain reaction after reverse transcription (RT-PCR) of viral RNA. DI9c did not replicate to a detectable extent in these assays, and its participation in the pathogenesis of mucosal disease could not be proven.
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characterization of an autonomous subgenomic pestivirus rna replicon
Journal of Virology, 1998Co-Authors: Svenerik Behrens, Gregor Meyers, H.j. Thiel, Claus W Grassmann, Norbert TautzAbstract:As an initial approach to define the requirements for the replication of bovine viral diarrhea virus (BVDV), a member of the Flaviviridae family with a positive-strand RNA genome, full-length genomic and subgenomic RNAs were originated by in vitro transcription of diverse BVDV cDNA constructs and transfected into eucaryotic host cells. RNA replication was measured either directly by an RNase protection method or by monitoring the synthesis of viral protein. When full-length BVDV cRNA was initially applied, the synthesis of negative-strand RNA intermediates as well as progeny positive-strand RNA was detected posttransfection in the cytoplasm of the host cells. Compared to the negative-strand RNA intermediate, an excess of positive-strand RNA was synthesized. Surprisingly, a subgenomic RNA molecule, DI9c, corresponding to a previously characterized Defective Interfering Particle, was found to support both steps of RNA replication in the absence of a helper virus as well, thus functioning as an autonomous replicon. DI9c comprises the 5' and 3' untranslated regions of the BVDV genome and the coding regions of the autoprotease Npro and the nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B. Most interestingly, the NS2 polypeptide was thus determined to be nonessential for RNA replication. As expected, deletion of the genomic 3' end as well as abolition of the catalytic function of the virus-encoded serine protease resulted in DI9c molecules that were unable to replicate. Deletion of the entire Npro gene also destroyed the ability of DI9c molecules to replicate. On the other hand, DI9c derivatives in which the 5' third of the Npro gene was fused to a ubiquitin gene, allowing the proteolytic release of NS3 in trans, turned out to be replication competent. These results suggest that the RNA sequence located at the 5' end of the open reading frame exerts an essential role during BVDV replication. Replication of DI9c and DI9c derivatives was found not to be limited to host cells of bovine origin, indicating that cellular factors functioning as potential parts of the viral replication machinery are well conserved between different mammalian cells. Our data provide an important step toward the ready identification and characterization of viral factors and genomic elements involved in the life cycle of pestiviruses. The implications for other Flaviviridae and, in particular, the BVDV-related human hepatitis C virus are discussed.
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recovery of cytopathogenic and noncytopathogenic bovine viral diarrhea viruses from cdna constructs
Journal of Virology, 1996Co-Authors: Gregor Meyers, H.j. Thiel, Norbert Tautz, Paul Becher, B M KummererAbstract:After cDNA cloning of the genome of bovine viral diarrhea virus (BVDV) isolate CP7, a full-length cDNA clone was constructed. RNA transcribed in vitro from this construct was shown to direct the generation of infectious BVDV upon transfection into bovine cells. To confirm the de novo generation of infectious BVDV from cloned cDNA a genetically tagged virus was constructed. In comparison with parental BVDV, the recombinant virus was slightly retarded in growth. The NS2 coding region of the CP7 genome contains a duplication of 27 nucleotides which is not present in the genome of its noncytopathogenic counterpart, NCP7. Exchange of a small fragment harboring this insertion against the corresponding part of the NCP7 sequence led to recovery of noncytopathogenic BVDV. Alteration of the construct by introduction of a fragment derived from a cytopathogenic BVDV Defective Interfering Particle resulted in a chimeric Defective Interfering Particle which exhibits a cytopathogenic phenotype. These findings confirm the hypothesis that the recombination-induced alterations in the genomes of cytopathogenic BVDV are responsible for the induction of cell lysis.
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Bovine viral diarrhea virus: characterization of a cytopathogenic Defective Interfering Particle with two internal deletions.
Journal of virology, 1996Co-Authors: H. Kupfermann, H.j. Thiel, Edward J. Dubovi, Gregor MeyersAbstract:Molecular characterization of bovine viral diarrhea virus pair 13 revealed that isolate CP13 is composed of a cytopathogenic (cp) Defective Interfering Particle (DI13) and a noncytopathogenic (noncp) helper virus. The DI13 genome possesses two internal deletions of 1,611 and 3,102 nucleotides. Except for a small fragment of the gene coding for glycoprotein E1, all structural protein genes are deleted together with most of the Npro gene, the region coding for nonstructural proteins p7 and NS2. While the amino terminus of NS3 seems to be strictly conserved for all other cp bovine viral diarrhea viruses, NS3 of DI13 is amino-terminally truncated and fused to 23 amino acids derived from Npro and E1. Characterization of the DI-helper virus system revealed a striking discrepancy between RNA production and generation of infectious viruses.
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Bovine viral diarrhea virus: characterization of a cytopathogenic Defective Interfering Particle with two internal deletions
1996Co-Authors: Gregor Meyers, H. Kupfermann, H.j. Thiel, Edward J. Dubovi, J. VirolAbstract:Bovine viral diarrhea virus: characterization of a cytopathogenic Defective Interfering Particle with two internal deletions
Udo Reichl - One of the best experts on this subject based on the ideXlab platform.
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OP7, a novel influenza A virus Defective Interfering Particle: production, purification, and animal experiments demonstrating antiviral potential
Applied Microbiology and Biotechnology, 2021Co-Authors: Marc D Hein, Heike Kollmus, Sebastian Puttker, Dirk Benndorf, Yvonne Genzel, Klaus Schughart, Sascha Young Kupke, Pavel Marichal-gallardo, Udo ReichlAbstract:The novel influenza A virus (IAV) Defective Interfering Particle “OP7” inhibits IAV replication in a co-infection and was previously suggested as a promising antiviral agent. Here, we report a batch-mode cell culture-based production process for OP7. In the present study, a seed virus containing standard virus (STV) and OP7 was used. The yield of OP7 strongly depended on the production multiplicity of infection. To inactivate infectious STV in the OP7 material, which may cause harm in a potential application, UV irradiation was used. The efficacy of OP7 in this material was preserved, as shown by an in vitro interference assay. Next, steric exclusion chromatography was used to purify and to concentrate (~ 13-fold) the UV-treated material. Finally, administration of produced OP7 material in mice did not show any toxic effects. Furthermore, all mice infected with a lethal dose of IAV survived the infection upon OP7 co-treatment. Thus, the feasibility of a production workflow for OP7 and its potential for antiviral treatment was demonstrated. Key points • OP7 efficacy strongly depended on the multiplicity of infection used for production • Purification by steric exclusion chromatography increased OP7 efficacy • OP7-treated mice were protected against a lethal infection with IAV
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op7 a novel influenza a virus Defective Interfering Particle production purification and animal experiments demonstrating antiviral potential
Applied Microbiology and Biotechnology, 2020Co-Authors: Marc D Hein, Heike Kollmus, Pavel Marichalgallardo, Sebastian Puttker, Dirk Benndorf, Yvonne Genzel, Klaus Schughart, Sascha Young Kupke, Udo ReichlAbstract:The novel influenza A virus (IAV) Defective Interfering Particle “OP7” inhibits IAV replication in a co-infection and was previously suggested as a promising antiviral agent. Here, we report a batch-mode cell culture-based production process for OP7. In the present study, a seed virus containing standard virus (STV) and OP7 was used. The yield of OP7 strongly depended on the production multiplicity of infection. To inactivate infectious STV in the OP7 material, which may cause harm in a potential application, UV irradiation was used. The efficacy of OP7 in this material was preserved, as shown by an in vitro interference assay. Next, steric exclusion chromatography was used to purify and to concentrate (~ 13-fold) the UV-treated material. Finally, administration of produced OP7 material in mice did not show any toxic effects. Furthermore, all mice infected with a lethal dose of IAV survived the infection upon OP7 co-treatment. Thus, the feasibility of a production workflow for OP7 and its potential for antiviral treatment was demonstrated. • OP7 efficacy strongly depended on the multiplicity of infection used for production • Purification by steric exclusion chromatography increased OP7 efficacy • OP7-treated mice were protected against a lethal infection with IAV
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Multiscale modeling of influenza A virus replication in cell cultures predicts infection dynamics for highly different infection conditions
2019Co-Authors: Daniel Rüdiger, Sascha Young Kupke, Pawel Zmora, Tanja Laske, Udo ReichlAbstract:Influenza A viruses (IAV) are commonly used to infect animal cell cultures for research purposes and vaccine production. Their replication is influenced strongly by the multiplicity of infection (MOI), which ranges over several orders of magnitude depending on the respective application. So far, mathematical models of IAV replication have paid little attention to the impact of the MOI on infection dynamics and virus yields. To address this issue, we extended an existing model of IAV replication in adherent MDCK cells with kinetics that explicitly consider the time point of cell infection. This modification does not only enable the fitting of high MOI measurements, but also the successful prediction of viral release dynamics of low MOI experiments using the same set of parameters. Furthermore, this model allows the investigation of Defective Interfering Particle (DIP) propagation in different MOI regimes. The key difference between high and low MOI conditions is the percentage of infectious virions among the total virus Particle release. Simulation studies show that DIP interference at a high MOI is determined exclusively by the DIP content of the seed virus while, in low MOI conditions, it is predominantly controlled by the de novo generation of DIPs. Overall, the extended model provides an ideal framework for the prediction and optimization of cell culture-derived IAV manufacturing and the production of DIPs for therapeutic use.
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a novel type of influenza a virus derived Defective Interfering Particle with nucleotide substitutions in its genome
Journal of Virology, 2018Co-Authors: Sascha Young Kupke, Udo Reichl, Dietmar Riedel, Timo Frensing, Pawel ZmoraAbstract:Defective Interfering Particles (DIPs) replicate at the expense of coinfecting, fully infectious homologous virus. Typically, they contain a highly deleted form of the viral genome. Utilizing single-cell analysis, here we report the discovery of a yet-unknown DIP type, derived from influenza A viruses (IAVs), termed OP7 virus. Instead of deletions, the genomic viral RNA (vRNA) of segment 7 (S7) carried 37 point mutations compared to the reference sequence, affecting promoter regions, encoded proteins, and genome packaging signals. Coinfection experiments demonstrated strong interference of OP7 virus with IAV replication, manifested by a dramatic decrease in the infectivity of released virions. Moreover, an overproportional quantity of S7 in relation to other genome segments was observed, both intracellularly and in the released virus population. Concurrently, OP7 virions lacked a large fraction of other vRNA segments, which appears to constitute its defect in virus replication. OP7 virus might serve as a promising candidate for antiviral therapy. Furthermore, this novel form of DIP may also be present in other IAV preparations.IMPORTANCE Defective Interfering Particles (DIPs) typically contain a highly deleted form of the viral genome, rendering them Defective in virus replication. Yet upon complementation through coinfection with fully infectious standard virus (STV), interference with the viral life cycle can be observed, leading to suppressed STV replication and the release of mainly noninfectious DIPs. Interestingly, recent research indicates that DIPs may serve as an antiviral agent. Here we report the discovery of a yet-unknown type of influenza A virus-derived DIP (termed "OP7" virus) that contains numerous point mutations instead of large deletions in its genome. Furthermore, the underlying principles that render OP7 virions Interfering and apparently Defective seem to differ from those of conventional DIPs. In conclusion, we believe that OP7 virus might be a promising candidate for antiviral therapy. Moreover, it exerts strong effects, both on virus replication and on the host cell response, and may have been overlooked in other IAV preparations.
H.j. Thiel - One of the best experts on this subject based on the ideXlab platform.
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characterization of an autonomous subgenomic pestivirus rna replicon
Journal of Virology, 1998Co-Authors: Svenerik Behrens, Gregor Meyers, H.j. Thiel, Claus W Grassmann, Norbert TautzAbstract:As an initial approach to define the requirements for the replication of bovine viral diarrhea virus (BVDV), a member of the Flaviviridae family with a positive-strand RNA genome, full-length genomic and subgenomic RNAs were originated by in vitro transcription of diverse BVDV cDNA constructs and transfected into eucaryotic host cells. RNA replication was measured either directly by an RNase protection method or by monitoring the synthesis of viral protein. When full-length BVDV cRNA was initially applied, the synthesis of negative-strand RNA intermediates as well as progeny positive-strand RNA was detected posttransfection in the cytoplasm of the host cells. Compared to the negative-strand RNA intermediate, an excess of positive-strand RNA was synthesized. Surprisingly, a subgenomic RNA molecule, DI9c, corresponding to a previously characterized Defective Interfering Particle, was found to support both steps of RNA replication in the absence of a helper virus as well, thus functioning as an autonomous replicon. DI9c comprises the 5' and 3' untranslated regions of the BVDV genome and the coding regions of the autoprotease Npro and the nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B. Most interestingly, the NS2 polypeptide was thus determined to be nonessential for RNA replication. As expected, deletion of the genomic 3' end as well as abolition of the catalytic function of the virus-encoded serine protease resulted in DI9c molecules that were unable to replicate. Deletion of the entire Npro gene also destroyed the ability of DI9c molecules to replicate. On the other hand, DI9c derivatives in which the 5' third of the Npro gene was fused to a ubiquitin gene, allowing the proteolytic release of NS3 in trans, turned out to be replication competent. These results suggest that the RNA sequence located at the 5' end of the open reading frame exerts an essential role during BVDV replication. Replication of DI9c and DI9c derivatives was found not to be limited to host cells of bovine origin, indicating that cellular factors functioning as potential parts of the viral replication machinery are well conserved between different mammalian cells. Our data provide an important step toward the ready identification and characterization of viral factors and genomic elements involved in the life cycle of pestiviruses. The implications for other Flaviviridae and, in particular, the BVDV-related human hepatitis C virus are discussed.
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recovery of cytopathogenic and noncytopathogenic bovine viral diarrhea viruses from cdna constructs
Journal of Virology, 1996Co-Authors: Gregor Meyers, H.j. Thiel, Norbert Tautz, Paul Becher, B M KummererAbstract:After cDNA cloning of the genome of bovine viral diarrhea virus (BVDV) isolate CP7, a full-length cDNA clone was constructed. RNA transcribed in vitro from this construct was shown to direct the generation of infectious BVDV upon transfection into bovine cells. To confirm the de novo generation of infectious BVDV from cloned cDNA a genetically tagged virus was constructed. In comparison with parental BVDV, the recombinant virus was slightly retarded in growth. The NS2 coding region of the CP7 genome contains a duplication of 27 nucleotides which is not present in the genome of its noncytopathogenic counterpart, NCP7. Exchange of a small fragment harboring this insertion against the corresponding part of the NCP7 sequence led to recovery of noncytopathogenic BVDV. Alteration of the construct by introduction of a fragment derived from a cytopathogenic BVDV Defective Interfering Particle resulted in a chimeric Defective Interfering Particle which exhibits a cytopathogenic phenotype. These findings confirm the hypothesis that the recombination-induced alterations in the genomes of cytopathogenic BVDV are responsible for the induction of cell lysis.
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Bovine viral diarrhea virus: characterization of a cytopathogenic Defective Interfering Particle with two internal deletions.
Journal of virology, 1996Co-Authors: H. Kupfermann, H.j. Thiel, Edward J. Dubovi, Gregor MeyersAbstract:Molecular characterization of bovine viral diarrhea virus pair 13 revealed that isolate CP13 is composed of a cytopathogenic (cp) Defective Interfering Particle (DI13) and a noncytopathogenic (noncp) helper virus. The DI13 genome possesses two internal deletions of 1,611 and 3,102 nucleotides. Except for a small fragment of the gene coding for glycoprotein E1, all structural protein genes are deleted together with most of the Npro gene, the region coding for nonstructural proteins p7 and NS2. While the amino terminus of NS3 seems to be strictly conserved for all other cp bovine viral diarrhea viruses, NS3 of DI13 is amino-terminally truncated and fused to 23 amino acids derived from Npro and E1. Characterization of the DI-helper virus system revealed a striking discrepancy between RNA production and generation of infectious viruses.
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Bovine viral diarrhea virus: characterization of a cytopathogenic Defective Interfering Particle with two internal deletions
1996Co-Authors: Gregor Meyers, H. Kupfermann, H.j. Thiel, Edward J. Dubovi, J. VirolAbstract:Bovine viral diarrhea virus: characterization of a cytopathogenic Defective Interfering Particle with two internal deletions
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An L (polymerise)-deficient rabies virus Defective Interfering Particle RNA is replicated and transcribed by heterologous helper virus L proteins
Virology, 1991Co-Authors: Karl-klaus Conzelmann, James H. Cox, H.j. ThielAbstract:Abstract A rabies virus-derived Defective Interfering Particle (DI) was isolated and characterized. The DI genome contained an internal deletion of 6.4 kb spanning the 3′ moiety of the pseudogene region (Ψ) and most of the L gene. DI-specific monocistronic N, NS, and M mRNAs as well as a G/L fusion mRNA were transcribed in cells coinfected with DI and helper virus. In addition, polycistronic DI RNAs and standard virus RNAs with internal A stretches and intergenic regions were found. Superinfection experiments showed that heterologous rabies-related viruses (Lyssavirus serotypes 2, 3, and 4) can complement the L deficiency of the DI genome. The heterologous polymerase proteins recognize correctly the replicational and transcriptional signal sequences of the Lyssavirus serotype 1-derived DI.
Sue A. Moyer - One of the best experts on this subject based on the ideXlab platform.
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Measles virus nucleocapsid protein can function in Sendai virus Defective InterferingParticle genome synthesis in vitro
Virology, 1995Co-Authors: R. Chandrika, Tina M. Myers, Sue A. MoyerAbstract:The Sendai virus P and L proteins, the viral RNA polymerase, and the nucleocapsid protein, NP, synthesized in a transient mammalian expression system support the replication of Sendai virus Defective Interfering Particle (DI) genome RNA in vitro. We have shown that the measles virus nucleocapsid protein, N, can substitute for the Sendai NP protein in genome synthesis. The chimeric product nucleocapsids, which contained Sendai RNA encapsidated with measles N protein, were atypical since they were sensitive to micrococcal nuclease digestion, unlike wild-type Sendai or measles nucleocapsids. The utilization of measles N protein required the endogenous Sendai virus RNA polymerase, since DI nucleocapsids free of polymerase were not replicated. Although both Sendai virus NP and P proteins and measles N and P proteins formed complexes when they were coexpressed, sedimentation analysis showed that measles N protein self-assembled and did not form a complex when expressed with the Sendai P protein. Furthermore, when the Sendai P-L polymerase complex was provided separately, measles N protein alone synthesized DI genome RNA in the absence of Sendai P protein. These data suggest that the self-assembled form of measles N protein functions in Sendai DI genome synthesis.
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complexes of sendai virus np p and p l proteins are required for Defective Interfering Particle genome replication in vitro
Journal of Virology, 1992Co-Authors: S M Horikami, J Curran, D Kolakofsky, Sue A. MoyerAbstract:We present evidence that the formation of NP-P and P-L protein complexes is essential for replication of the genome of Sendai Defective Interfering (DI-H) virus in vitro, using extracts of cells expressing these viral proteins from plasmids. Optimal replication of DI-H nucleocapsid RNA required extracts of cells transfected with critical amounts and ratios of each of the plasmids and was three- to fivefold better than replication with a control extract prepared from a natural virus infection. Extracts in which NP and P proteins were coexpressed supported replication of the genome of purified DI-H virus which contained endogenous polymerase proteins, but extracts in which NP and P were expressed separately and then mixed were inactive. Similarly, the P and L proteins must be coexpressed for biological activity. The replication data thus suggest that two protein complexes, NP-P and P-L, are required for nucleocapsid RNA replication and that these complexes must form during or soon after synthesis of the proteins. Biochemical evidence in support of the formation of each complex includes coimmunoprecipitation of both proteins of each complex with an antibody specific for one component and cosedimentation of the subunits of each complex. We propose that the P-L complex serves as the RNA polymerase and NP-P is required for encapsidation of newly synthesized RNA.
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Required for Defective Interfering Particle Genome Replication In Vitro
1992Co-Authors: Sue A. Moyer, S M Horikami, J Curran, D Kolakofsky, J. VirolAbstract:Complexes of Sendai virus NP-P and P-L proteins are required for Defective Interfering Particle genome replication in vitro
Tomas Kirchhausen - One of the best experts on this subject based on the ideXlab platform.
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oligomerization of the vesicular stomatitis virus phosphoprotein is dispensable for mrna synthesis but facilitates rna replication
Journal of Virology, 2020Co-Authors: Louis Marie Bloyet, Benjamin Morin, Vesna Brusic, Erica Gardner, Robin A Ross, Tegy Vadakkan, Tomas Kirchhausen, Sean P J WhelanAbstract:Nonsegmented negative-strand (NNS) RNA viruses possess a ribonucleoprotein template in which the genomic RNA is sequestered within a homopolymer of nucleocapsid protein (N). The viral RNA-dependent RNA polymerase (RdRP) resides within an approximately 250-kDa large protein (L), along with unconventional mRNA capping enzymes: a GDP:polyribonucleotidyltransferase (PRNT) and a dual-specificity mRNA cap methylase (MT). To gain access to the N-RNA template and orchestrate the LRdRP, LPRNT, and LMT, an oligomeric phosphoprotein (P) is required. Vesicular stomatitis virus (VSV) P is dimeric with an oligomerization domain (OD) separating two largely disordered regions followed by a globular C-terminal domain that binds the template. P is also responsible for bringing new N protomers onto the nascent RNA during genome replication. We show VSV P lacking the OD (PΔOD) is monomeric but is indistinguishable from wild-type P in supporting mRNA transcription in vitro Recombinant virus VSV-PΔOD exhibits a pronounced kinetic delay in progeny virus production. Fluorescence recovery after photobleaching demonstrates that PΔOD diffuses 6-fold more rapidly than the wild type within viral replication compartments. A well-characterized Defective Interfering Particle of VSV (DI-T) that is only competent for RNA replication requires significantly higher levels of N to drive RNA replication in the presence of PΔOD We conclude P oligomerization is not required for mRNA synthesis but enhances genome replication by facilitating RNA encapsidation.IMPORTANCE All NNS RNA viruses, including the human pathogens rabies, measles, respiratory syncytial virus, Nipah, and Ebola, possess an essential L-protein cofactor, required to access the N-RNA template and coordinate the various enzymatic activities of L. The polymerase cofactors share a similar modular organization of a soluble N-binding domain and a template-binding domain separated by a central oligomerization domain. Using a prototype of NNS RNA virus gene expression, vesicular stomatitis virus (VSV), we determined the importance of P oligomerization. We find that oligomerization of VSV P is not required for any step of viral mRNA synthesis but is required for efficient RNA replication. We present evidence that this likely occurs through the stage of loading soluble N onto the nascent RNA strand as it exits the polymerase during RNA replication. Interfering with the oligomerization of P may represent a general strategy to interfere with NNS RNA virus replication.
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The length of vesicular stomatitis virus Particles dictates a need for actin assembly during clathrin-dependent endocytosis. PLoS Pathog
2013Co-Authors: David K Cureton, Sean P J Whelan, Ramiro Massol, Tomas KirchhausenAbstract:Microbial pathogens exploit the clathrin endocytic machinery to enter host cells. Vesicular stomatitis virus (VSV), an enveloped virus with bullet-shaped virions that measure 706200 nm, enters cells by clathrin-dependent endocytosis. We showed previously that VSV Particles exceed the capacity of typical clathrin-coated vesicles and instead enter through endocytic carriers that acquire a partial clathrin coat and require local actin filament assembly to complete vesicle budding and internalization. To understand why the actin system is required for VSV uptake, we compared the internalization mechanisms of VSV and its shorter (75 nm long) Defective Interfering Particle, DI-T. By imaging the uptake of individual Particles into live cells, we found that, as with parental virions, DI-T enters via the clathrin endocytic pathway. Unlike VSV, DI-T internalization occurs through complete clathrin-coated vesicles and does not require actin polymerization. Since VSV and DI-T Particles display similar surface densities of the same attachment glycoprotein, we conclude that the physical properties of the Particle dictate whether a virus-containing clathrin pit engages the actin system. We suggest that the elongated shape of a VSV Particle prevents full enclosure by the clathrin coat and that stalling of coat assembly triggers recruitment of the actin machinery to finish the internalization process. Since some enveloped viruses have pleomorphic Particle shapes and sizes, our work suggests that they may use altered modes of endocytic uptake. More generally, our findings show the importance of cargo geometry for specifying cellular entry modes, even when the receptor recognitio
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the length of vesicular stomatitis virus Particles dictates a need for actin assembly during clathrin dependent endocytosis
PLOS Pathogens, 2010Co-Authors: David K Cureton, Sean P J Whelan, Ramiro Massol, Tomas KirchhausenAbstract:Microbial pathogens exploit the clathrin endocytic machinery to enter host cells. Vesicular stomatitis virus (VSV), an enveloped virus with bullet-shaped virions that measure 70×200 nm, enters cells by clathrin-dependent endocytosis. We showed previously that VSV Particles exceed the capacity of typical clathrin-coated vesicles and instead enter through endocytic carriers that acquire a partial clathrin coat and require local actin filament assembly to complete vesicle budding and internalization. To understand why the actin system is required for VSV uptake, we compared the internalization mechanisms of VSV and its shorter (75 nm long) Defective Interfering Particle, DI-T. By imaging the uptake of individual Particles into live cells, we found that, as with parental virions, DI-T enters via the clathrin endocytic pathway. Unlike VSV, DI-T internalization occurs through complete clathrin-coated vesicles and does not require actin polymerization. Since VSV and DI-T Particles display similar surface densities of the same attachment glycoprotein, we conclude that the physical properties of the Particle dictate whether a virus-containing clathrin pit engages the actin system. We suggest that the elongated shape of a VSV Particle prevents full enclosure by the clathrin coat and that stalling of coat assembly triggers recruitment of the actin machinery to finish the internalization process. Since some enveloped viruses have pleomorphic Particle shapes and sizes, our work suggests that they may use altered modes of endocytic uptake. More generally, our findings show the importance of cargo geometry for specifying cellular entry modes, even when the receptor recognition properties of a ligand are maintained.
