Abstract
Eukaryotic translation initiation factor 4A (eIF4A) is a helicase that facilitates assembly of the translation preinitiation complex by unwinding structured mRNA 5′ untranslated regions. Pateamine A (PatA) and silvestrol are natural products that disrupt eIF4A function and arrest translation, thereby triggering the formation of cytoplasmic aggregates of stalled preinitiation complexes known as stress granules (SGs). Here we examined the effects of eIF4A inhibition by PatA and silvestrol on influenza A virus (IAV) protein synthesis and replication in cell culture. Treatment of infected cells with either PatA or silvestrol at early times post-infection resulted in SG formation, arrest of viral protein synthesis and failure to replicate the viral genome. PatA, which irreversibly binds to eIF4A, sustained long-term blockade of IAV replication following drug withdrawal, and inhibited IAV replication at concentrations that had minimal cytotoxicity. By contrast, the antiviral effects of silvestrol were fully reversible; drug withdrawal caused rapid SG dissolution and resumption of viral protein synthesis. IAV inhibition by silvestrol was invariably associated with cytotoxicity. PatA blocked replication of genetically divergent IAV strains, suggesting common dependence on host eIF4A activity. This study demonstrates that the core host protein synthesis machinery can be targeted to block viral replication.
Highlights
Influenza A viruses (IAV) are enveloped viruses with segmented, negative-sense, single-strandedRNA genomes that primarily infect epithelial cells in a diverse range of hosts
We have shown that IAV mRNAs are sensitive to pateamine A (PatA) [11], a natural product that selectively inhibits the Asp-Glu-Ala-Asp (DEAD)-box RNA helicase Eukaryotic translation initiation factor 4A (eIF4A) which assembles with eIF4E and eIF4G, to form the eIF4F complex [13,14]
PatA was a kind gift from Dr Jerry Pelletier (McGill University, Montreal, QC, Canada), silvestrol was obtained from MedChem Express (Princeton, NJ, USA); 1 mM stocks of silvestrol and pateamine A were prepared in dimethyl sulfoxide (DMSO; MilliporeSigma, Etobicoke, ON, Canada) and stored at −80 ◦ C
Summary
RNA genomes (vRNAs) that primarily infect epithelial cells in a diverse range of hosts. Endosomal acidification aids pH-dependent fusion of viral and endosome membranes and acidification of the virion core, thereby allowing uncoating of viral genome segments These genome segments are imported into the cell nucleus, where viral mRNA synthesis is initiated by the viral RNA-dependent RNA polymerase (RdRp) complex, which cleaves nascent host RNA polymerase II (pol II)-transcribed RNAs at sites 10–14 nucleotides downstream of 50 7-methyl-guanosine (m7G) caps [1]. Template-directed viral mRNA synthesis is terminated by reiterative decoding of short uridine-rich sequences, which generates 30 -poly-adenylate (polyA)
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