Abstract
Influenza A virus (IAV) polymerase complexes function in the nucleus of infected cells, generating mRNAs that bear 5′ caps and poly(A) tails, and which are exported to the cytoplasm and translated by host machinery. Host antiviral defences include mechanisms that detect the stress of virus infection and arrest cap-dependent mRNA translation, which normally results in the formation of cytoplasmic aggregates of translationally stalled mRNA-protein complexes known as stress granules (SGs). It remains unclear how IAV ensures preferential translation of viral gene products while evading stress-induced translation arrest. Here, we demonstrate that at early stages of infection both viral and host mRNAs are sensitive to drug-induced translation arrest and SG formation. By contrast, at later stages of infection, IAV becomes partially resistant to stress-induced translation arrest, thereby maintaining ongoing translation of viral gene products. To this end, the virus deploys multiple proteins that block stress-induced SG formation: 1) non-structural protein 1 (NS1) inactivates the antiviral double-stranded RNA (dsRNA)-activated kinase PKR, thereby preventing eIF2α phosphorylation and SG formation; 2) nucleoprotein (NP) inhibits SG formation without affecting eIF2α phosphorylation; 3) host-shutoff protein polymerase-acidic protein-X (PA-X) strongly inhibits SG formation concomitant with dramatic depletion of cytoplasmic poly(A) RNA and nuclear accumulation of poly(A)-binding protein. Recombinant viruses with disrupted PA-X host shutoff function fail to effectively inhibit stress-induced SG formation. The existence of three distinct mechanisms of IAV-mediated SG blockade reveals the magnitude of the threat of stress-induced translation arrest during viral replication.
Highlights
Transcription of Influenza A virus (IAV) genes is performed by a viral polymerase that generates 59-capped and polyadenylated messenger RNAs structurally similar to host mRNAs [1]
We observed that stress granules (SGs) do not form at any point during the IAV replication cycle and efficient translation of viral gene products is maintained in the later stages of infection, when a variety of eIF2akinase activating signals might be anticipated
Our finding that PA-X inhibits SG formation led us to explore PA-X host shutoff function, and our findings support the recently proposed model; we find that PA-X causes striking depletion of poly(A) RNA from the cytoplasm of cells transfected with a PA-X expression vector, or in IAV-infected cells late in infection, but not in cells infected with a mutant virus with defects in PA-X protein production
Summary
Transcription of Influenza A virus (IAV) genes is performed by a viral polymerase that generates 59-capped and polyadenylated (poly[A]) messenger RNAs (mRNAs) structurally similar to host mRNAs [1]. Despite this similarity, IAV transcripts gain preferential access to cellular translation machinery through a host shutoff mechanism executed by the viral non-structural protein 1 (NS1) [2,3] and the recently discovered viral PA-X protein [4]. The reliance on cap-dependent translation initiation makes viral mRNAs susceptible to host-cell stress-induced translation inhibition mechanisms. This inhibition results from phosphorylation of eukaryotic translation initiation factor-2a (eIF2a) by any of four kinases activated by distinct types of stress [5]. Many viruses have evolved specific mechanisms that modulate SG responses (reviewed in [7])
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