Involvement of a Bovine Viral Diarrhea Virus NS5B Locus in Virion Assembly

Abstract

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-toproline 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.Virion assembly and release from the host cell constitute the final stages of the viral life cycle. These events are as important for viral pathogenesis in the host as the widely studied mechanisms of virus entry or genome replication. Enveloped virion assembly represents the convergence of viral proteins, nucleic acid, and host lipids to build an infectious particle. The process of virus budding, which characterizes enveloped viruses, is difficult to study because it involves a living cell. No cell-free virus budding systems have been developed to dissect this process.Hepatitis C virus (HCV) is an important human pathogen that causes persistent infections leading to chronic hepatitis, which can result in hepatocellular carcinoma (1, 2). To avoid the late sequelae of chronic HCV infections, millions of infected individuals await the development of effective antivirals (29). Efforts to explore all the possible drug targets for HCV, including virion assembly, have been hampered by the lack of robust in vitro cell culture systems that yield infectious virions (60). Much of our current knowledge on HCV virion assembly is derived from the study of virus-like particles (VLPs). Numerous reports support the concept that viral structural proteins carry enough structural information to direct the as...

Characterization of the Determinants of NS2-3-Independent Virion Morphogenesis of Pestiviruses

“…The findings in this study are the first indication for an involvement of the N-terminal NS5B domain in virion morphogenesis. So far, only the C terminus of NS5B could be identified experimentally as being critical for infectious particle formation (16). An effect of the mutation 5B/EG on RNA replication efficiency has been excluded (Fig.…”

“…It is known for the members of the Flaviviridae that besides the structural proteins, the nonstructural proteins also are critical for virion morphogenesis (14). For pestiviruses, almost all nonstructural proteins have been shown to be essential for the production of infectious viral particles (15)(16)(17)(18)(19). Pestiviral NS4B has not yet been characterized with regard to its role in virion morphogenesis but has been shown to be critical in HCV virion morphogenesis, suggesting a similar function in pestiviruses (20).…”

The Amino-Terminal Domain of Bovine Viral Diarrhea Virus NproProtein Is Necessary for Alpha/Beta Interferon Antagonism

“…The NCL1-ISRE-Luc-Hygro cell line (L. Gil and R. Donis, unpublished data) is a stable bovine cell line selected for integration of the luciferase reporter under transcriptional control of an IFN-stimulated response element (ISRE); it was cultured in the same medium supplemented with 300 g/ml hygromycin (A. G. Scientific, Inc.). pNADLp15 (cp NADL), N-dINS (ncp NADL), N-H/B, and 5B-1HR are modified viruses derived from a plasmid containing the full-length cDNA of the BVDV strain NADL genome, as described elsewhere (3,40,76). The mutant 5B-1HR was derived by reverse genetics from the NADL strain of BVDV by insertion of a 19-amino-acid epitope tag at position 704 of NS5B (insertion of 57 nucleotides at position 12303) (3).…”

“…The in vitro-transcribed RNAs were transfected into bovine uterine cells by electroporation as described previously (3). Briefly, bovine uterine cells were trypsinized, washed two times with MEM, and resuspended at 2 ϫ 10 7 cells/ml in cytomix (120 mM KCl, 0.15 mM CaCl 2 , 10 mM K 2 HPO 4 /KH 2 PO 4 , pH 7.6, 25 mM HEPES, pH 7.6, 2.0 mM EGTA, 5.0 mM MgCl 2 ) plus 2.0 mM of ATP and 5.0 mM of glutathione (72).…”

“…Briefly, bovine uterine cells (90% confluent) were trypsinized, washed twice with MEM, and resuspended at 2 ϫ 10 7 cells/ml in cytomix (72). Unless otherwise indicated, 3 g of reporter DNA plasmid was mixed with 0.1 ml of cells in suspension and immediately pulsed with a BTX-600 ElectroSquarePorator (320 V; 100 F; R3) as previously described (3). The electroporated cells were diluted in 12 ml of MEM-ES and seeded (1 ml per well) into 12-well plates.…”