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Non-replicating Vaccinia Virus TianTan Strain (NTV) Translation Arrest of Viral Late Protein Synthesis Associated With Anti-viral Host Factor SAMD9.

ABSTRACT: NTV is a highly attenuated virus that was created by genetically deleting 26 genes related to host range and virulence from TianTan strain. Since NTV is highly attenuated, it has been used widely as an optimizing viral vector. In this study, we explored the biological characteristics in vitro and the host restriction mechanism of NTV. Most cell lines do not support sufficient dissemination and replication of NTV, and in non-permissive cell line HeLa, the replication block of NTV occurred at the translation stage of viral late protein expression. Lack of PKR activity was not sufficient to rescue expression of viral late proteins and replication, even though the phosphorylation level of eIF2? increased in NTV-infected HeLa cells. Moreover, the translation inhibition of NTV in HeLa cells was dependent upon a SAMD9 signaling pathway, as demonstrated by silencing SAMD9 expression with siRNA and observing the colocalization of SAMD9 and AVGs. Reinserting C7L or K1L into NTV rescued the late viral protein expression and replication of NTV in HeLa cells. Among the genes deleted in NTV, C7L or/and K1L gene was mainly responsible for its replication defect. Protein C7 interacted with SAMD9, which antagonized the antiviral response of SAMD9 to ensure viral protein translation and replication of NTV in non-permissive cell lines. Our finding will serve as a baseline for modification of NTV in future application.

SUBMITTER: Zhao Y

PROVIDER: S-EPMC7098914 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Non-replicating Vaccinia Virus TianTan Strain (NTV) Translation Arrest of Viral Late Protein Synthesis Associated With Anti-viral Host Factor SAMD9.

Zhao Ying Y Zhao Li L Huang Panpan P Ren Jiao J Zhang Peng P Tian Houwen H Tan Wenjie W

Frontiers in cellular and infection microbiology 20200320

NTV is a highly attenuated virus that was created by genetically deleting 26 genes related to host range and virulence from TianTan strain. Since NTV is highly attenuated, it has been used widely as an optimizing viral vector. In this study, we explored the biological characteristics <i>in vitro</i> and the host restriction mechanism of NTV. Most cell lines do not support sufficient dissemination and replication of NTV, and in non-permissive cell line HeLa, the replication block of NTV occurred ...[more]

PMID: 32266167

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Project description:UnlabelledRNA interference (RNAi) screens intended to identify host factors that restrict virus replication may fail if the virus already counteracts host defense mechanisms. To overcome this limitation, we are investigating the use of viral host range mutants that exhibit impaired replication in nonpermissive cells. A vaccinia virus (VACV) mutant with a deletion of both the C7L and K1L genes, K1L(-)C7L(-), which abrogates replication in human cells at a step prior to late gene expression, was chosen for this strategy. We carried out a human genome-wide small interfering RNA (siRNA) screen in HeLa cells infected with a VACV K1L(-)C7L(-) mutant that expresses the green fluorescent protein regulated by a late promoter. This positive-selection screen had remarkably low background levels and resulted in the identification of a few cellular genes, notably SAMD9 and WDR6, from approximately 20,000 tested that dramatically enhanced green fluorescent protein expression. Replication of the mutant virus was enabled by multiple siRNAs to SAMD9 or WDR6. Moreover, SAMD9 and WDR6 clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 knockout HeLa cell lines were permissive for replication of the K1L(-)C7L(-) mutant, in agreement with the siRNA data. Expression of exogenous SAMD9 or interferon regulatory factor 1 restricted replication of the K1L(-)C7L(-) mutant in the SAMD9(-/-) cells. Independent interactions of SAMD9 with the K1 and C7 proteins were suggested by immunoprecipitation. Knockout of WDR6 did not reduce the levels of SAMD9 and interactions of WDR6 with SAMD9, C7, and K1 proteins were not detected, suggesting that these restriction factors act independently but possibly in the same innate defense pathway.ImportanceThe coevolution of microbial pathogens with cells has led to an arms race in which the invader and host continuously struggle to gain the advantage. For this reason, traditional siRNA screens may fail to uncover important immune mechanisms if the pathogens have already developed effective responses. However, host-restricted viral mutants have lost one or more defense genes needed for their replication in nonpermissive cells. By screening human genome libraries of short RNAs that inhibit the expression of individual host genes in nonpermissive cells, we identified SAMD9 and WDR6 as major restriction factors that prevented replication of a vaccinia virus mutant and suggest that host range screening can be generally useful for the investigation of host-pathogen interactions.

Dataset Information

Non-replicating Vaccinia Virus TianTan Strain (NTV) Translation Arrest of Viral Late Protein Synthesis Associated With Anti-viral Host Factor SAMD9.

Publications

Non-replicating Vaccinia Virus TianTan Strain (NTV) Translation Arrest of Viral Late Protein Synthesis Associated With Anti-viral Host Factor SAMD9.

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