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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 ( IF 5.818 ) Pub Date : 2021-08-02 , DOI: 10.3390/v13081523
Huining Pang 1 , Ling Li 1 , Hongru Liu 1 , Zishu Pan 1
Affiliation  

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.

中文翻译:

经典猪瘟病毒疫苗 C 株 NS5B 162 位脯氨酸到苏氨酸突变通过促进 RNA 合成的启动促进基因组复制和感染性病毒生产

经典猪瘟病毒 (CSFV) 的 3'非翻译区 (3'UTR) 和 NS5B 在病毒基因组复制中起重要作用。本研究以猪瘟病毒感染性cDNA克隆为基础,构建了猪瘟病毒石门毒株或牛病毒性腹泻病毒(BVDV)NADL毒株3'UTR替代的vC/SM3'UTR和vC/b3'UTR两种嵌合病毒。疫苗C株,分别。病毒拯救后,每个重组嵌合病毒在PK-15细胞中连续传代。对具有代表性的传代病毒进行表征和测序。连续传代导致突变的产生,与亲本病毒相比,传代的病毒表现出显着提高的基因组复制效率和感染性病毒产量。在传代的 vC/SM3'UTR 和 vC/b3'UTR 中都鉴定到 NS5B 的第 162 位的脯氨酸到苏氨酸突变。我们分别使用反向遗传学系统生成了两个嵌合体的 P162T 突变体。vC/SM3'UTR 或 vC/b3'UTR 的 NS5B 中的单个 P162T 突变在增加病毒基因组复制和感染性病毒产生中起关键作用。P162T 突变增加了 vC/SM3'UTR兔中的P162T复制。从体外 RNA 依赖性 RNA 聚合酶 (RdRp) 测定中,含有 P162T 突变的 NS5B (NS5B P162T ) 对不同的 RNA 模板表现出增强的 RdRp 活性。我们进一步确定增强的 RdRp 活性源于 RNA 合成起始效率的提高。这些发现揭示了 NS5B 残基 162 在调节瘟病毒复制中的新功能。
更新日期:2021-08-03
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