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Zika Virus Non-Coding RNAs Antagonize Antiviral Responses by PKR-Mediated Translational Arrest (RNA-Seq)

ABSTRACT: Zika virus (ZIKV) is an emerging mosquito-borne flavivirus that causes severe outbreaks in human populations. ZIKV infection leads to the accumulation of small non-coding viral RNAs (known as sfRNAs) that are crucial for evasion of antiviral responses and for viral pathogenesis. However, the mechanistic understanding of how sfRNAs function remains incomplete. Here, we use recombinant ZIKVs and ribosome profiling of infected human cells to show that sfRNAs block translation of antiviral genes. Mechanistically, we demonstrate that specific RNA structures present in sfRNAs trigger PKR activation, which instead of limiting viral replication, enhances viral particle production. Although ZIKV infection induces mRNA expression of antiviral genes, translation efficiency of type I interferon and interferon stimulated genes were significantly downregulated by PKR activation. Our results reveal a novel viral adaptation mechanism mediated by sfRNAs, where ZIKV increases its fitness by repurposing the antiviral role of PKR into a proviral factor.

ORGANISM(S): Homo sapiens

PROVIDER: GSE254252 | GEO | 2024/04/28

REPOSITORIES: GEO

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Zika Virus Non-Coding RNAs Antagonize Antiviral Responses by PKR-Mediated Translational Arrest (Ribo-Seq)

Project description:Zika virus (ZIKV) is an emerging mosquito-borne flavivirus that causes severe outbreaks in human populations. ZIKV infection leads to the accumulation of small non-coding viral RNAs (known as sfRNAs) that are crucial for evasion of antiviral responses and for viral pathogenesis. However, the mechanistic understanding of how sfRNAs function remains incomplete. Here, we use recombinant ZIKVs and ribosome profiling of infected human cells to show that sfRNAs block translation of antiviral genes. Mechanistically, we demonstrate that specific RNA structures present in sfRNAs trigger PKR activation, which instead of limiting viral replication, enhances viral particle production. Although ZIKV infection induces mRNA expression of antiviral genes, translation efficiency of type I interferon and interferon stimulated genes were significantly downregulated by PKR activation. Our results reveal a novel viral adaptation mechanism mediated by sfRNAs, where ZIKV increases its fitness by repurposing the antiviral role of PKR into a proviral factor.

2024-04-28 | GSE254253 | GEO

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Project description:PKR is an interferon induced serine/threonine protein kinase, that is activated by double stranded RNA. PKR plays an important role in the antiviral defense by interferon. In addition to its role in translation, PKR participates in several signaling pathways to transcription. The goal of this experiment is to study the role of PKR in regulating gene expression in our NIH 3T3 inducible cell line, which could overexpress PKR wt protein after the removal of tetracycline (Donze O, Dostie J, Sonenberg N. (1999) Virology 256: 322-9).

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Expressed dsRNA provokes selective sequence-independent PKR response in transient co-transfection experiments

Project description:In mammals, double-stranded RNA (dsRNA) plays roles in sequence-specific RNA interference, sequence-independent interferon response, and RNA editing by adenosine deaminases. We have previously shown that long hairpin dsRNA expression in cultured cells does not activate the interferon response, it is poorly processed into siRNAs, and it is partially edited. Here, we demonstrate that dsRNA expressed from transiently transfected plasmids strongly inhibits expression of co-transfected reporter plasmids but not expression of endogenous genes or reporters stably integrated in the genome. The inhibition is concentration-dependent and independent of a cell type, transfection method, or dsRNA sequence. The inhibition occurs at the level of translation and is mediated by protein kinase R (PKR). PKR binds the expressed dsRNA, becomes phosphorylated and changes its distribution along polysome fractions. In conclusion, we demonstrate that expression from plasmids is selectively repressed if one of co transfected plasmids produces dsRNA. Our results highlight the importance of proper controls and careful interpretation of co-transfection experiments. HEK293 cells (human origin) were transiently transfected with hairpin dsRNA-expressing (MosIR) and control (pCag) plasmids.

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