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

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 construc...

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Main Authors: Huining Pang, Ling Li, Hongru Liu, Zishu Pan
Format: Article
Language:English
Published: MDPI AG 2021-08-01
Series:Viruses
Subjects:
Online Access:https://www.mdpi.com/1999-4915/13/8/1523
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author Huining Pang
Ling Li
Hongru Liu
Zishu Pan
author_facet Huining Pang
Ling Li
Hongru Liu
Zishu Pan
author_sort Huining Pang
collection DOAJ
description 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′UTR<sub>P162T</sub> replication in rabbits. From RNA-dependent RNA polymerase (RdRp) assays in vitro, the NS5B containing P162T mutation (NS5B<sub>P162T</sub>) 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 <i>pestivirus</i> replication.
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spelling doaj.art-ee8666ae95234c9ca19e28386f7647282023-11-22T10:10:41ZengMDPI AGViruses1999-49152021-08-01138152310.3390/v13081523Proline 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 SynthesisHuining Pang0Ling Li1Hongru Liu2Zishu Pan3State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, ChinaState Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, ChinaState Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, ChinaState Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, ChinaThe 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′UTR<sub>P162T</sub> replication in rabbits. From RNA-dependent RNA polymerase (RdRp) assays in vitro, the NS5B containing P162T mutation (NS5B<sub>P162T</sub>) 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 <i>pestivirus</i> replication.https://www.mdpi.com/1999-4915/13/8/1523classical swine fever virus3′untranslated regionNS5BRNA-dependent RNA polymerasereplicationinitiation
spellingShingle Huining Pang
Ling Li
Hongru Liu
Zishu Pan
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
classical swine fever virus
3′untranslated region
NS5B
RNA-dependent RNA polymerase
replication
initiation
title 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
title_full 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
title_fullStr 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
title_full_unstemmed 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
title_short 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
title_sort 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
topic classical swine fever virus
3′untranslated region
NS5B
RNA-dependent RNA polymerase
replication
initiation
url https://www.mdpi.com/1999-4915/13/8/1523
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