RESUMO
Non-structural 5A protein (NS5A) has emerged as an important pharmacological target for hepatitis C virus (HCV). However, little is known about the conformation of NS5A intracellularly or how NS5A inhibitors achieve the picomolar (pM) inhibition of virus replication. Here, we have presented two structurally related small molecules, one that potently inhibits HCV replication and selects for resistance in NS5A, and another that is inactive. Resistance to this antiviral was greater in genotype 1a than in genotype 1b replicons and mapped to domain 1 of NS5A. Using a novel cell-based assay that measures the intracellular proximity of fluorescent tags covalently attached to NS5A, we showed that only the active antiviral specifically disrupted the close proximity of inter- and intramolecular positions of NS5A. The active antiviral, termed compound 1, caused a repositioning of both the N and C termini of NS5A, including disruption of the close approximation of the N termini of two different NS5A molecules in a multimolecular complex. These data provide the first study of how antivirals that select resistance in domain 1 of NS5A alter the cellular conformation of NS5A. This class of antiviral disrupts the close proximity of the N termini of domain 1 in a NS5A complex but also alters the conformation of domain 3, and leads to large aggregates of NS5A. Current models predict that a multicomponent cocktail of antivirals is needed to treat HCV infection, so a mechanistic understanding of what each component does to the viral machinery will be important.
Assuntos
Antivirais/metabolismo , Hepacivirus/efeitos dos fármacos , Proteínas não Estruturais Virais/metabolismo , Linhagem Celular , Farmacorresistência Viral , Hepatócitos/virologia , Humanos , Conformação Proteica/efeitos dos fármacos , Seleção Genética , Replicação Viral/efeitos dos fármacosRESUMO
Nonstructural protein 5A (NS5A) is essential for hepatitis C virus (HCV) replication and assembly and is a critical drug target. Biochemical data suggest large parts of NS5A are unfolded as an isolated protein, but little is known about its folded state in the cell. We used fluorescence resonance energy transfer (FRET) to probe whether or not different regions of NS5A are in close proximity within the cell. Twenty-three separate reporter constructs were created by inserting one or more fluorophores into different positions throughout the three domains of NS5A. FRET efficiency was maximal when donor and acceptor fluorophores were positioned next to each other but also could be observed when the two fluorophores flanked NS5A domain 1 or domain 3. Informatic and biochemical analysis suggests that large portions of the carboxy terminus of NS5A are in an unfolded and disordered state. Quercetin, a natural product known to disrupt NS5A function in cells, specifically disrupted a conformationally specific domain 3 FRET signal. Intermolecular FRET indicated that the NS5A amino termini, but not other regions, are in close proximity in multimeric complexes. Overall, this assay provides a new window on the intracellular conformation(s) of NS5A and how the conformation changes in response to cellular and viral components of the replication and assembly complex as well as antiviral drugs.
Assuntos
Antivirais/química , Sistemas de Liberação de Medicamentos , Transferência Ressonante de Energia de Fluorescência , Hepacivirus/química , Proteínas não Estruturais Virais/química , Antivirais/uso terapêutico , Linhagem Celular , Hepacivirus/genética , Hepacivirus/metabolismo , Hepatite C/tratamento farmacológico , Hepatite C/genética , Hepatite C/metabolismo , Humanos , Estrutura Terciária de Proteína , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismoRESUMO
Porcine reproductive and respiratory syndrome virus (PRRSV) contains the major glycoprotein, GP5, as well as three other minor glycoproteins, namely, GP2a, GP3, and GP4, on the virion envelope, all of which are required for generation of infectious virions. To study their interactions with each other and with the cellular receptor for PRRSV, we have cloned each of the viral glycoproteins and CD163 receptor in expression vectors and examined their expression and interaction with each other in transfected cells by coimmunoprecipitation (co-IP) assay using monospecific antibodies. Our results show that a strong interaction exists between the GP4 and GP5 proteins, although weak interactions among the other minor envelope glycoproteins and GP5 have been detected. Both GP2a and GP4 proteins were found to interact with all the other GPs, resulting in the formation of multiprotein complex. Our results further show that the GP2a and GP4 proteins also specifically interact with the CD163 molecule. The carboxy-terminal 223 residues of the CD163 molecule are not required for interactions with either the GP2a or the GP4 protein, although these residues are required for conferring susceptibility to PRRSV infection in BHK-21 cells. Overall, we conclude that the GP4 protein is critical for mediating interglycoprotein interactions and, along with GP2a, serves as the viral attachment protein that is responsible for mediating interactions with CD163 for virus entry into susceptible host cell.
Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/patogenicidade , Receptores de Superfície Celular/metabolismo , Proteínas do Envelope Viral/fisiologia , Sequência de Aminoácidos , Animais , Antígenos CD/química , Antígenos CD/genética , Antígenos de Diferenciação Mielomonocítica/química , Antígenos de Diferenciação Mielomonocítica/genética , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Cricetinae , Primers do DNA/genética , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multiproteicos , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Receptores Virais/química , Receptores Virais/genética , Receptores Virais/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sus scrofa , Suínos , Transfecção , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genéticaRESUMO
Serine/Threonine phosphorylation of the nonstructural protein 5 (NS5) is a conserved feature of flaviviruses, but the identity and function(s) of the responsible kinase(s) remain unknown. Serine 56 in the methyltransferase domain of NS5 can be phosphorylated intracellularly, is conserved in all flaviviruses, and is a critical residue in the catalytic mechanism. A negative charge at this residue inactivates the 2'-0 methyltransferase activity necessary to form a 5' cap structure of the viral RNA. Here we show pharmacologic inhibition of Casein Kinase 1 (CK1) suppresses yellow fever virus (YFV) production. We also demonstrate the alpha isoform of Casein Kinase 1 (CK1alpha), a kinase previously identified as phosphorylating Hepatitis C Virus NS5A protein, also phosphorylates serine 56 of YFV methyltransferase. Overall these results suggest CK1 activity can influence flaviviral replication.
Assuntos
Caseína Quinase I/metabolismo , Infecções por Flavivirus/enzimologia , Metiltransferases/metabolismo , Proteínas Virais/metabolismo , Vírus da Febre Amarela/enzimologia , Caseína Quinase I/química , Caseína Quinase I/genética , Linhagem Celular , Flavivirus/química , Flavivirus/enzimologia , Flavivirus/fisiologia , Humanos , Metiltransferases/química , Metiltransferases/genética , Fosforilação , Especificidade por Substrato , Proteínas Virais/química , Proteínas Virais/genética , Replicação Viral , Vírus da Febre Amarela/química , Vírus da Febre Amarela/fisiologiaRESUMO
A selective trans-packaging system was developed to produce and isolate bovine viral diarrhea virus (BVDV) pseudo-particles with complementing reporter replicons and their packaging proteins expressed in trans with recombinant vaccinia virus. The encapsidation of replicon rNS3-5B was dependent not only on the in trans expression of structural proteins C, E(rns), E1 and E2, but also the nonstructural proteins, p7 and contiguous precursor NS2-3-4A. Nonstructural p7, NS4B, NS5A or NS5B could be expressed in cis and in trans with precursor NS2-3-4A without significantly affecting virion assembly efficiency. NS2-3-4A was identified as an in trans functional precursor in virion assembly. BVDV genomes with mutant NS5B, which did not undergo active replication, were packaged 5-fold less efficiently than the intact genomes demonstrating the importance of replication in virion packaging. These results suggest that genome replication and assembly are closely associated, consistent with a model in which these two steps are coupled for maximum efficiency.
Assuntos
Vírus da Diarreia Viral Bovina/fisiologia , Precursores de Proteínas/fisiologia , Proteínas não Estruturais Virais/fisiologia , Vírion/fisiologia , Montagem de Vírus , Sequência de Aminoácidos , Animais , Bovinos , Linhagem Celular , Vírus da Diarreia Viral Bovina/isolamento & purificação , Teste de Complementação Genética/métodos , Humanos , Dados de Sequência Molecular , Precursores de Proteínas/genética , Replicon , Proteínas não Estruturais Virais/genética , Vírion/isolamento & purificaçãoRESUMO
In order to determine virulence associated genes in porcine reproductive and respiratory syndrome virus (PRRSV), a series of chimeric viruses were generated where specific genomic regions of a highly virulent PRRSV infectious clone (FL12) were replaced with their counterparts of an attenuated vaccine strain (Prime Pac). Initial genome-wide scanning using a sow reproductive failure model indicated that non-structural (ORF 1a and 1b) and structural (ORF2-7) genomic regions appear to be sites where virulence determinants of PRRSV may reside. These results thus confirm the multigenic character of PRRSV virulence. Additional chimeras containing each individual structural ORFs (2 through 7) of Prime Pac and ORF5 of Neb-1 (parental strain of Prime Pac) within the FL12 backbone were generated and tested individually for further mapping of virulence determinants. Our results allow to conclude that NSP3-8 and ORF5 are the location of major virulence determinants, while other virulence determinants may also be contained in NSP1-3, NSP10-12 and ORF2.
Assuntos
Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/patogenicidade , Proteínas Virais/fisiologia , Fatores de Virulência/fisiologia , Animais , Linhagem Celular , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/crescimento & desenvolvimento , Suínos , Proteínas Virais/genética , Fatores de Virulência/genéticaRESUMO
The availability of a DIVA (differentiating infected from vaccinated animals) vaccine is very important for the control and eradication of endemic infectious diseases such as porcine reproductive and respiratory syndrome (PRRS). Previous studies in our laboratory identified several B-cell linear epitopes consistently recognized by convalescent sera obtained from pigs infected with a North American porcine reproductive and respiratory syndrome virus (PRRSV) strain. To ascertain if one or more of these immunodominant epitopes can be used as the basis of DIVA differentiation, we selected two epitope markers previously identified on the non-structural protein 2 (PRRSV NSP2, predictably the viral protein most likely to tolerate large deletions). The choice of these epitopes was primarily based on their immunodominance and their deletion were performed along the backbone of the wild-type cDNA infectious clone (FL12). We were able to successfully rescue a mutant that fulfilled the requirements for a DIVA marker strain, such as: efficient growth of the deletion mutant in vitro and in vivo and induction of specific seroconversion as measured by a commercial ELISA kit, with absence of a marker-specific peptide-ELISA response in 100% (n=15) of the inoculated animals. In summary, our results provide proof of concept that DIVA PRRSV vaccines can potentially be developed by deletion of individual "marker" immunodominant epitopes.
Assuntos
Epitopos Imunodominantes/genética , Epitopos Imunodominantes/imunologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Animais , Anticorpos Antivirais/sangue , Biomarcadores , Síndrome Respiratória e Reprodutiva Suína/prevenção & controle , Vírus da Síndrome Respiratória e Reprodutiva Suína/crescimento & desenvolvimento , Deleção de Sequência , Suínos , Vacinas Virais/genética , Vacinas Virais/imunologia , ViremiaRESUMO
Porcine reproductive and respiratory syndrome virus (PRRSV) glycoprotein 5 (GP5) is the most abundant envelope glycoprotein and a major inducer of neutralizing antibodies in vivo. Three putative N-linked glycosylation sites (N34, N44, and N51) are located on the GP5 ectodomain, where a major neutralization epitope also exists. To determine which of these putative sites are used for glycosylation and the role of the glycan moieties in the neutralizing antibody response, we generated a panel of GP5 mutants containing amino acid substitutions at these sites. Biochemical studies with expressed wild-type (wt) and mutant proteins revealed that the mature GP5 contains high-mannose-type sugar moieties at all three sites. These mutations were subsequently incorporated into a full-length cDNA clone. Our data demonstrate that mutations involving residue N44 did not result in infectious progeny production, indicating that N44 is the most critical amino acid residue for infectivity. Viruses carrying mutations at N34, N51, and N34/51 grew to lower titers than the wt PRRSV. In serum neutralization assays, the mutant viruses exhibited enhanced sensitivity to neutralization by wt PRRSV-specific antibodies. Furthermore, inoculation of pigs with the mutant viruses induced significantly higher levels of neutralizing antibodies against the mutant as well as the wt PRRSV, suggesting that the loss of glycan residues in the ectodomain of GP5 enhances both the sensitivity of these viruses to in vitro neutralization and the immunogenicity of the nearby neutralization epitope. These results should have great significance for development of PRRSV vaccines of enhanced protective efficacy.
Assuntos
Anticorpos Antivirais/sangue , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Proteínas do Envelope Viral/metabolismo , Animais , Linhagem Celular , Cricetinae , Epitopos , Glicosilação , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/farmacologia , Mutação , Suínos , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologiaRESUMO
Understanding of the molecular basis of virulence and attenuation of porcine reproductive and respiratory syndrome virus (PRRSV) is important for the development of a safe and efficacious vaccine. Prime Pac (PP) is an attenuated vaccine strain of PRRSV which is being used in our laboratories as a source of gene(s) for the generation of chimeric constructs in the background of a highly virulent PRRSV derived from an infectious clone (FL12) to examine the molecular determinants of virulence and attenuation. To facilitate these studies, we generated a full-length cDNA clone of the PP vaccine strain by serially replacing the genomic fragments of the FL12 with the corresponding regions from the PP strain. The virus rescued from this newly assembled cDNA clone (PP18) exhibited in vitro growth properties and in vivo apathogenic characteristics of the parental PP virus. Using pregnant sows as the experimental model of reproductive pathogenesis, we have been able to unequivocally demonstrate the clearly contrasting phenotypes of the virulent and the attenuated viruses derived from the infectious clones (FL12 and PP18). The development of an infectious clone derived from a bona fide attenuated PRRSV vaccine strain should significantly facilitate ongoing studies to determine the molecular basis of virulence and attenuation.
Assuntos
Síndrome Respiratória e Reprodutiva Suína/imunologia , Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/patogenicidade , Vacinas Virais/imunologia , Animais , Células Cultivadas , Clonagem de Organismos , Primers do DNA , DNA Complementar/genética , DNA Complementar/imunologia , DNA Viral/genética , DNA Viral/imunologia , Eletroporação , Feminino , Fenótipo , Vírus da Síndrome Respiratória e Reprodutiva Suína/isolamento & purificação , Gravidez , RNA Viral/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sobrevida , Suínos , Transcrição Gênica , Vacinas Atenuadas/imunologiaRESUMO
Hepatitis C virus (HCV) non-structural protein 5A (NS5A) plays an essential role in viral genome replication. A series of transposon-mediated insertion mutants and deletion mutants of NS5A was used to examine the colony-forming ability of HCV subgenomic replicons encoding the mutant proteins. The results reveal that two regions of NS5A can tolerate insertions: one spanning residues 240-314, which contain the interferon sensitivity-determining region (ISDR), and the other spanning residues 349-417 at the carboxy terminus. The majority of these sites also tolerated insertion of enhanced green fluorescent protein. Furthermore, replicons encoding NS5A with deletions in ISDR or in the carboxy-terminal regions were replication-competent, indicating that these regions of NS5A are not necessary for replication. Taken together, the results suggest that the central region spanning the ISDR and the carboxy-terminal region of the molecule are dispensable for the functions of NS5A in viral genome replication.
Assuntos
Genoma Viral , Hepacivirus/fisiologia , Proteínas não Estruturais Virais/fisiologia , Replicação Viral/genética , Deleção de Genes , Proteínas de Fluorescência Verde , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , Interferons/farmacologia , Mutagênese Insercional , Proteínas não Estruturais Virais/genéticaRESUMO
The alpha/beta interferon (IFN-alpha/beta) system is the first line of defense against viral infection and a critical link between the innate and adaptive immune responses. IFN-alpha/beta secretion is the hallmark of cellular responses to acute RNA virus infections. As part of their survival strategy, many viruses have evolved mechanisms to counteract the host IFN-alpha/beta response. Bovine viral diarrhea virus (BVDV) (genus Pestivirus) was reported to trigger interferon production in infected cultured cells under certain circumstances or to suppress it under others. Our studies with various cultured fibroblasts and epithelial bovine cells indicated that cytopathic (cp) BVDV induces IFN-alpha/beta very inefficiently. Using a set of engineered cp BVDVs expressing mutant Npro and appropriate controls, we found that the IFN-alpha/beta response to infection was dependent on Npro expression and independent of viral replication efficiency. In order to investigate whether the protease activity of Npro is required for IFN-alpha/beta antagonism, we engineered Npro mutants lacking protease activity by replacement of amino acid E22, H49, or C69. We found that E22 and H49 substitutions abolished the ability of Npro to suppress IFN, whereas C69 had no effect, suggesting that the structural integrity of the N terminus of Npro was more important than its catalytic activity for IFN-alpha/beta suppression. A catalytically active mutant with a change at a conserved Npro region near the N terminus (L8P) in both BVDV biotypes did not antagonize IFN-alpha/beta production, confirming its involvement in this process. Taken together, these results not only provide direct evidence for the role of Npro in blocking IFN-alpha/beta induction, but also implicate the amino-terminal domain of the protein in this function.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina/metabolismo , Vírus da Diarreia Viral Bovina/fisiologia , Interferon-alfa/metabolismo , Interferon beta/metabolismo , Proteínas Virais/fisiologia , Aminoácidos/genética , Animais , Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Bovinos , Células Cultivadas , Vírus da Diarreia Viral Bovina/genética , Regulação para Baixo , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Interferon-alfa/antagonistas & inibidores , Interferon beta/antagonistas & inibidores , Mutação , Proteínas Virais/genéticaRESUMO
We report on the highly potent and selective antipestivirus activity of 5-[(4-bromophenyl)methyl]-2-phenyl-5H-imidazo[4,5-c]pyridine (BPIP). The 50% effective concentration (EC50) for inhibition of bovine viral diarrhea virus (BVDV)-induced cytopathic effect formation was 0.04 +/- 0.01 microM. Comparable reduction of viral RNA synthesis (EC50 = 0.12 +/- 0.02 microM) and production of infectious virus (EC50= 0.074 +/- 0.003 microM) were observed. The selectivity index (ratio of 50% cytostatic concentration/EC50) of BPIP was approximately 2,000. BPIP was inactive against the hepatitis C virus subgenomic replicon and yellow fever virus but demonstrated weak activity against GB virus. Drug-resistant mutants were at least 300-fold less susceptible to BPIP than wild-type virus; showed cross-resistance to N-propyl-N-[2-(2H-1,2,4-triazino[5,6-b]indol-3-ylthio)ethyl]-1-propanamine (VP32947), and carried the F224S mutation in the viral RNA-dependent RNA polymerase (RdRp). When the F224S mutation was introduced into an infectious clone, the drug-resistant phenotype was obtained. BPIP did not inhibit the in vitro activity of recombinant BVDV RdRp, but did inhibit the activity of replication complexes (RCs). Computational docking revealed that F224 is located at the top of the finger domain of the polymerase. Docking of BPIP in the crystal structure of the BVDV RdRp revealed aromatic ring stacking, some hydrophobic contacts, and a hydrogen bond. Since two structurally unrelated compounds, i.e., BPIP and VP32947, target the same region of the BVDV RdRp, this position may be expected to be critical in the functioning of the polymerase or assembly of the RC. The potential of BPIP for the treatment of pestivirus and hepacivirus infections is discussed.
Assuntos
Antivirais/farmacologia , Pestivirus/efeitos dos fármacos , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Triazinas/farmacologia , Replicação Viral/efeitos dos fármacos , Vírus da Diarreia Viral Bovina Tipo 1/efeitos dos fármacos , Farmacorresistência Viral , Imidazóis/farmacologia , Dose Letal Mediana , Mutação , Pestivirus/fisiologia , Piridinas/farmacologia , RNA Viral/biossíntese , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , Triazinas/química , Células Tumorais CultivadasRESUMO
Bovine viral diarrhoea virus (BVDV) isolates infect cultured Madin-Darby bovine kidney (MDBK) cells as efficiently as sheep kidney cells. In contrast, border disease virus (BDV) propagates poorly in MDBK cells but infects sheep cells very efficiently. The envelope glycoprotein E2 has been shown to be essential for virus infectivity. To explore the potential role of E2 in pestivirus host range in cell cultures, we engineered a chimeric BVDV with the E2 coding region from BDV. As expected, the BVDV-E2(bdv) chimera retained the ability of BDV to multiply in sheep cells but experienced a remarkable reduction in its ability to propagate and form plaques in MDBK, a phenotype that is characteristic of the E2 donor, BDV31 virus. Control chimeric BVDV bearing a type II E2 demonstrated that the heterologous E2 does not impair replication in MDBK or lamb cells. These results establish a role for E2 in determining the tropism of a pestivirus in cell culture.
Assuntos
Vírus da Doença da Fronteira/patogenicidade , Vírus da Diarreia Viral Bovina/patogenicidade , Glicoproteínas/metabolismo , Ruminantes/virologia , Proteínas do Envelope Viral/metabolismo , Proteínas Estruturais Virais/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Linhagem Celular , Glicoproteínas/química , Glicoproteínas/genética , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ovinos , Especificidade da Espécie , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Ensaio de Placa Viral , Proteínas Estruturais Virais/química , Proteínas Estruturais Virais/genética , VirulênciaRESUMO
A novel mutant of bovine viral diarrhea virus (BVDV) was found with a virion assembly phenotype attributable to an insertion into the NS5B polymerase locus. This mutant, termed 5B-741, was engineered by reverse genetics to express NS5B with a C-terminal peptide tag of 22 amino acids. Electroporation of bovine cells with genomic RNA from this mutant showed levels RNA synthesis which were regarded as sufficient for infectivity, yet infectious virions were not produced. Pseudorevertants of mutant 5B-741 that released infectious virions and formed plaques revealed a single nucleotide change (T12369C). This change resulted in a leucine-to-proline substitution within the NS5B tag (L726P). Genetic analysis revealed that indeed a single nucleotide change encoding proline at NS5B position 726 in the pseudorevertant polyprotein mediated recovery of virion assembly function without improving genomic RNA accumulation levels. A subgenomic BVDV reporter replicon (rNS3-5B) was used to analyze the consequences of alterations of the genomic region encoding the NS5B C terminus on replication and assembly. Interestingly, rNS3-5B-L726P (revertant) replicated with the same efficiency as the rNS3-5B-741 mutant but produced 10 times more virions in a trans-packaging assay. These results indicated that impairment of assembly function in 5B-741 was independent of RNA accumulation levels and agreed with the observations from the full-length mutant and revertant genomes. Finally, we recapitulated the packaging defect of 5B-741 with a vaccinia virus expression system to eliminate possible unwanted interactions between the helper virus and the packaged replicon. Taken together, these studies revealed an unexpected role of NS5B in infectious virion assembly.