scholarly journals Proline to Threonine Mutation at Position 162 of NS5B of Classical Swine Fever Virus Vaccine C Strain Promoted Genome Replication and Infectious Virus Production by Facilitating Initiation of RNA Synthesis

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1523
Author(s):  
Huining Pang ◽  
Ling Li ◽  
Hongru Liu ◽  
Zishu Pan
Keyword(s):  
Classical Swine Fever Virus ◽  
Viral Genome ◽  
Rna Synthesis ◽  
Infectious Virus ◽  
Virus Production ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Genome Replication ◽  
Rdrp Activity ◽  
Viral Genome Replication

The 3′untranslated region (3′UTR) and NS5B of classical swine fever virus (CSFV) play vital roles in viral genome replication. In this study, two chimeric viruses, vC/SM3′UTR and vC/b3′UTR, with 3′UTR substitution of CSFV Shimen strain or bovine viral diarrhea virus (BVDV) NADL strain, were constructed based on the infectious cDNA clone of CSFV vaccine C strain, respectively. After virus rescue, each recombinant chimeric virus was subjected to continuous passages in PK-15 cells. The representative passaged viruses were characterized and sequenced. Serial passages resulted in generation of mutations and the passaged viruses exhibited significantly increased genomic replication efficiency and infectious virus production compared to parent viruses. A proline to threonine mutation at position 162 of NS5B was identified in both passaged vC/SM3′UTR and vC/b3′UTR. We generated P162T mutants of two chimeras using the reverse genetics system, separately. The single P162T mutation in NS5B of vC/SM3′UTR or vC/b3′UTR played a key role in increased viral genome replication and infectious virus production. The P162T mutation increased vC/SM3′UTRP162T replication in rabbits. From RNA-dependent RNA polymerase (RdRp) assays in vitro, the NS5B containing P162T mutation (NS5BP162T) exhibited enhanced RdRp activity for different RNA templates. We further identified that the enhanced RdRp activity originated from increased initiation efficiency of RNA synthesis. These findings revealed a novel function for the NS5B residue 162 in modulating pestivirus replication.

scholarly journals Characterization of the N-terminal domain of classical swine fever virus RNA-dependent RNA polymerase

2006 ◽  
Vol 87 (2) ◽  
pp. 347-356 ◽  
Author(s):  
Ming Xiao ◽  
Huaibo Li ◽  
Yujing Wang ◽  
Xiaohui Wang ◽  
Wei Wang ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Classical Swine Fever Virus ◽  
Rna Synthesis ◽  
De Novo ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Rdrp Activity ◽  
Rna Dependent Rna Polymerase ◽  
Terminal Domain ◽  
Ns5b Protein

To investigate RNA-dependent RNA polymerase (RdRp) further, mutational analysis of the N-terminal domain of the NS5B protein of Classical swine fever virus was performed. Results show that the N-terminal domain (positions 1–300) of the protein might be divided artificially into four different regions, N1–N4. The N1 region (positions 1–61) contained neither conserved lysine nor conserved arginine residues. NS5B protein with deletion of the N1 region has the capacity for elongative RNA synthesis, but not for de novo RNA synthesis on natural templates. All substitutions of the conserved lysines and arginines in the N2 region (positions 63–216) destroyed RdRp activity completely. Substitutions of the conserved arginines in the N3 region (positions 217–280) seriously reduced RdRp activity. However, all substitutions of the conserved lysines in this region enhanced RNA synthesis and made the mutants synthesize RNA on any template. Substitutions of the conserved arginines in the N4 region (positions 281–300) reduced elongative synthesis and destroyed de novo RNA synthesis. In contrast, substitutions of lysines in this region did not affect RdRp activity significantly. These data indicate that the N3 region might be related to the enzymic specificity for templates, and the conserved lysines and arginines in different regions have different effects on RdRp activity. In combination with the published crystal structure of bovine viral diarrhea virus NS5B, these results define the important role of the N-terminal domain of NS5B for template recognition and de novo RNA synthesis.

scholarly journals Specific interaction between the classical swine fever virus NS5B protein and the viral genome

2004 ◽  
Vol 271 (19) ◽  
pp. 3888-3896 ◽  
Author(s):  
Ming Xiao ◽  
Jufang Gao ◽  
Wei Wang ◽  
Yujing Wang ◽  
Jun Chen ◽  
...  
Keyword(s):  
Classical Swine Fever Virus ◽  
Viral Genome ◽  
Specific Interaction ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Ns5b Protein

The Necessary Site for Initiation of RNA Synthesis in the 3 -Noncoding Region of Classical Swine Fever Virus Genome

2004 ◽  
Vol 38 (2) ◽  
pp. 289-297 ◽  
Author(s):  
Ming Xiao ◽  
Wenwei Lu ◽  
Jun Chen ◽  
Yujing Wang ◽  
Yamei Zhen ◽  
...  
Keyword(s):  
Classical Swine Fever Virus ◽  
Rna Synthesis ◽  
Classical Swine Fever ◽  
Virus Genome ◽  
Fever Virus ◽  
Noncoding Region

scholarly journals Analysis of classical swine fever virus RNA replication determinants using replicons

2013 ◽  
Vol 94 (8) ◽  
pp. 1739-1748 ◽  
Author(s):  
Peter Christian Risager ◽  
Ulrik Fahnøe ◽  
Maria Gullberg ◽  
Thomas Bruun Rasmussen ◽  
Graham J. Belsham
Keyword(s):  
Classical Swine Fever Virus ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Genome Replication ◽  
Coding Region ◽  
Reporter Protein ◽  
Coding Sequence ◽  
Replication Efficiency ◽  
Chimeric Rnas

Self-replicating RNAs (replicons), with or without reporter gene sequences, derived from the genome of the Paderborn strain of classical swine fever virus (CSFV) have been produced. The full-length viral cDNA, propagated within a bacterial artificial chromosome, was modified by targeted recombination within Escherichia coli. RNA transcripts were produced in vitro and introduced into cells by electroporation. The translation and replication of the replicon RNAs could be followed by the accumulation of luciferase (from Renilla reniformis or Gaussia princeps) protein expression (where appropriate), as well as by detection of CSFV NS3 protein production within the cells. Inclusion of the viral E2 coding region within the replicon was advantageous for replication efficiency. Production of chimeric RNAs, substituting the NS2 and NS3 coding regions (as a unit) from the Paderborn strain with the equivalent sequences from the highly virulent Koslov strain or the vaccine strain Riems, blocked replication. However, replacing the Paderborn NS5B coding sequence with the RNA polymerase coding sequence from the Koslov strain greatly enhanced expression of the reporter protein from the replicon. In contrast, replacement with the Riems NS5B sequence significantly impaired replication efficiency. Thus, these replicons provide a system for determining specific regions of the CSFV genome required for genome replication without the constraints of maintaining infectivity.

Classical swine fever virus NS5A protein interacts with 3′-untranslated region and regulates viral RNA synthesis

2012 ◽  
Vol 163 (2) ◽  
pp. 636-643 ◽  
Author(s):  
Chun Sheng ◽  
Yan Chen ◽  
Jun Xiao ◽  
Jing Xiao ◽  
Jing Wang ◽  
...  
Keyword(s):  
Classical Swine Fever Virus ◽  
Untranslated Region ◽  
Rna Synthesis ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Viral Rna ◽  
Ns5a Protein
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