Influenza virus is a major human and animal pathogen causing seasonal epidemics and occasional pandemics in the human population that are associated with significant morbidity and mortality. ...Influenza A virus, a member of the orthomyxovirus family, contains an RNA genome with a coding capacity for a limited number of proteins. In addition to ensuring the structural integrity of virions, these viral proteins facilitate the replication of virus in the host cell. Consequently, viral proteins often evolve to perform multiple functions, the influenza A virus nuclear export protein (NEP) (also referred to as non-structural protein 2, or NS2) being an emerging example. NEP was originally implicated in mediating the nuclear export of viral ribonucleoprotein (RNP) complexes, which are synthesized in the infected cell nucleus and are assembled into progeny virions at the cell membrane. However, since then, new and unexpected roles for NEP during the influenza virus life cycle have started to emerge. These recent studies have shown NEP to be involved in regulating the accumulation of viral genomic vRNA and antigenomic cRNA as well as viral mRNA synthesized by the viral RNA-dependent RNA polymerase. Subsequently, this regulation of viral RNA transcription and replication by NEP was shown to be an important factor in the adaptation of highly pathogenic avian H5N1 influenza viruses to the mammalian host. Unexpectedly, NEP has also been implicated in recruiting a cellular ATPase to the cell membrane to aid the efficient release of budding virions. Accordingly, NEP is proposed to play multiple biologically important roles during the influenza virus life cycle.
The influenza virus RNA-dependent RNA polymerase (RdRP) cleaves the 5′ end of nascent capped host RNAs and uses the capped RNA fragment to prime viral transcription in a mechanism called ‘cap ...snatching’. Cap snatching requires an intimate association between influenza RdRP and cellular RNA polymerase II (Pol II), which is the source of nascent capped host RNAs targeted by influenza virus. Recent structural studies have revealed how influenza RdRP binds to Pol II and how this binding promotes the initiation of viral transcription by influenza RdRP. In this review we focus on these recent insights into the mechanism of cap snatching by influenza virus and the impact of cap snatching on host gene expression during influenza virus infection.
Influenza virus RdRP associates with cellular Pol II. This provides influenza RdRP with a source of nascent capped host RNAs which it targets in a process called ‘cap snatching’ to generate primers for viral transcription.
Influenza RdRP makes direct interactions with the Pol II C terminal domain (CTD). Molecular details of these interactions have recently been determined, and there is evidence that Pol II CTD binding promotes viral transcription by modulating the conformation of influenza RdRP.
Influenza RdRP cap snatches small nuclear/nucleolar RNAs most frequently. When cap snatching generates suboptimal capped RNA fragments, influenza RdRP can use a prime-and-realign mechanism to promote transcription initiation.
Influenza virus infection reduces Pol II abundance in gene bodies, suggesting that cap snatching contributes to host shut-off.
Transcription and replication of the influenza virus RNA genome is catalyzed by the viral heterotrimeric RNA-dependent RNA polymerase in the context of viral ribonucleoprotein (vRNP) complexes. ...Atomic resolution structures of the viral RNA synthesis machinery have offered insights into the initiation mechanisms of viral transcription and genome replication, and the interaction of the viral RNA polymerase with host RNA polymerase II, which is required for the initiation of viral transcription. Replication of the viral RNA genome by the viral RNA polymerase depends on host ANP32A, and host-specific sequence differences in ANP32A underlie the poor activity of avian influenza virus polymerases in mammalian cells. A failure to faithfully copy the viral genome segments can lead to the production of aberrant viral RNA products, such as defective interfering (DI) RNAs and mini viral RNAs (mvRNAs). Both aberrant RNA types have been implicated in innate immune responses against influenza virus infection. This review discusses recent insights into the structure-function relationship of the viral RNA polymerase and its role in determining host range and virulence.
RNA viruses include many important human and animal pathogens, such as the influenza viruses, respiratory syncytial virus, Ebola virus, measles virus and rabies virus. The genomes of these viruses ...consist of single or multiple RNA segments that assemble with oligomeric viral nucleoprotein into ribonucleoprotein complexes. Replication and transcription of the viral genome is performed by ~250-450 kDa viral RNA-dependent RNA polymerases that also contain capping or cap-snatching activity. In this Review, we compare recent high-resolution X-ray and cryoelectron microscopy structures of RNA polymerases of negative-sense RNA viruses with segmented and non-segmented genomes, including orthomyxoviruses, peribunyaviruses, phenuiviruses, arenaviruses, rhabdoviruses, pneumoviruses and paramyxoviruses. In addition, we discuss how structural insights into these enzymes contribute to our understanding of the molecular mechanisms of viral transcription and replication, and how we can use these insights to identify targets for antiviral drug design.
Aquatic birds represent a vast reservoir from which new pandemic influenza A viruses can emerge
. Influenza viruses contain a negative-sense segmented RNA genome that is transcribed and replicated by ...the viral heterotrimeric RNA polymerase (FluPol) in the context of viral ribonucleoprotein complexes
. RNA polymerases of avian influenza A viruses (FluPolA) replicate viral RNA inefficiently in human cells because of species-specific differences in acidic nuclear phosphoprotein 32 (ANP32), a family of essential host proteins for FluPol activity
. Host-adaptive mutations, particularly a glutamic-acid-to-lysine mutation at amino acid residue 627 (E627K) in the 627 domain of the PB2 subunit, enable avian FluPolA to overcome this restriction and efficiently replicate viral RNA in the presence of human ANP32 proteins. However, the molecular mechanisms of genome replication and the interplay with ANP32 proteins remain largely unknown. Here we report cryo-electron microscopy structures of influenza C virus polymerase (FluPolC) in complex with human and chicken ANP32A. In both structures, two FluPolC molecules form an asymmetric dimer bridged by the N-terminal leucine-rich repeat domain of ANP32A. The C-terminal low-complexity acidic region of ANP32A inserts between the two juxtaposed PB2 627 domains of the asymmetric FluPolA dimer, suggesting a mechanism for how the adaptive PB2(E627K) mutation enables the replication of viral RNA in mammalian hosts. We propose that this complex represents a replication platform for the viral RNA genome, in which one of the FluPol molecules acts as a replicase while the other initiates the assembly of the nascent replication product into a viral ribonucleoprotein complex.
Influenza A viruses cause seasonal epidemics and global pandemics, representing a considerable burden to healthcare systems. Central to the replication cycle of influenza viruses is the viral ...RNA-dependent RNA polymerase which transcribes and replicates the viral RNA genome. The polymerase undergoes conformational rearrangements and interacts with viral and host proteins to perform these functions. Here we determine the structure of the 1918 influenza virus polymerase in transcriptase and replicase conformations using cryo-electron microscopy (cryo-EM). We then structurally and functionally characterise the binding of single-domain nanobodies to the polymerase of the 1918 pandemic influenza virus. Combining these functional and structural data we identify five sites on the polymerase which are sensitive to inhibition by nanobodies. We propose that the binding of nanobodies at these sites either prevents the polymerase from assuming particular functional conformations or interactions with viral or host factors. The polymerase is highly conserved across the influenza A subtypes, suggesting these sites as effective targets for potential influenza antiviral development.
Viruses use virions to spread between hosts, and virion composition is therefore the primary determinant of viral transmissibility and immunogenicity. However, the virions of many viruses are complex ...and pleomorphic, making them difficult to analyse in detail. Here we address this by identifying and quantifying virion proteins with mass spectrometry, producing a complete and quantified model of the hundreds of host-encoded and viral proteins that make up the pleomorphic virions of influenza viruses. We show that a conserved influenza virion architecture is maintained across diverse combinations of virus and host. This 'core' architecture, which includes substantial quantities of host proteins as well as the viral protein NS1, is elaborated with abundant host-dependent features. As a result, influenza virions produced by mammalian and avian hosts have distinct protein compositions. Finally, we note that influenza virions share an underlying protein composition with exosomes, suggesting that influenza virions form by subverting microvesicle production.
The negative-sense RNA genome of influenza A virus is transcribed and replicated by the viral RNA-dependent RNA polymerase (RdRP). The viral RdRP is an important host range determinant, indicating ...that its function is affected by interactions with cellular factors. However, the identities and the roles of most of these factors remain unknown. Here, we employed affinity purification followed by mass spectrometry to identify cellular proteins that interact with the influenza A virus RdRP in infected human cells. We purified RdRPs using a recombinant influenza virus in which the PB2 subunit of the RdRP is fused to a Strep-tag. When this tagged subunit was purified from infected cells, copurifying proteins included the other RdRP subunits (PB1 and PA) and the viral nucleoprotein and neuraminidase, as well as 171 cellular proteins. Label-free quantitative mass spectrometry revealed that the most abundant of these host proteins were chaperones, cytoskeletal proteins, importins, proteins involved in ubiquitination, kinases and phosphatases, and mitochondrial and ribosomal proteins. Among the phosphatases, we identified three subunits of the cellular serine/threonine protein phosphatase 6 (PP6), including the catalytic subunit PPP6C and regulatory subunits PPP6R1 and PPP6R3. PP6 was found to interact directly with the PB1 and PB2 subunits of the viral RdRP, and small interfering RNA (siRNA)-mediated knockdown of the catalytic subunit of PP6 in infected cells resulted in the reduction of viral RNA accumulation and the attenuation of virus growth. These results suggest that PP6 interacts with and positively regulates the activity of the influenza virus RdRP.
Influenza A viruses are serious clinical and veterinary pathogens, causing substantial health and economic impacts. In addition to annual seasonal epidemics, occasional global pandemics occur when viral strains adapt to humans from other species. To replicate efficiently and cause disease, influenza viruses must interact with a large number of host factors. The reliance of the viral RNA-dependent RNA polymerase (RdRP) on host factors makes it a major host range determinant. This study describes and quantifies host proteins that interact, directly or indirectly, with a subunit of the RdRP. It increases our understanding of the role of host proteins in viral replication and identifies a large number of potential barriers to pandemic emergence. Identifying host factors allows their importance for viral replication to be tested. Here, we demonstrate a role for the cellular phosphatase PP6 in promoting viral replication, contributing to our emerging knowledge of regulatory phosphorylation in influenza virus biology.
RIG-I is a key mediator of antiviral immunity, able to couple detection of infection by RNA viruses to the induction of interferons. Natural RIG-I stimulatory RNAs have variously been proposed to ...correspond to virus genomes, virus replication intermediates, viral transcripts, or self-RNA cleaved by RNase L. However, the relative contribution of each of these RNA species to RIG-I activation and interferon induction in virus-infected cells is not known. Here, we use three approaches to identify physiological RIG-I agonists in cells infected with influenza A virus or Sendai virus. We show that RIG-I agonists are exclusively generated by the process of virus replication and correspond to full-length virus genomes. Therefore, nongenomic viral transcripts, short replication intermediates, and cleaved self-RNA do not contribute substantially to interferon induction in cells infected with these negative strand RNA viruses. Rather, single-stranded RNA viral genomes bearing 5′-triphosphates constitute the natural RIG-I agonists that trigger cell-intrinsic innate immune responses during infection.
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► RIG-I is a sensor for virus infection that triggers antiviral responses ► RIG-I binds replicated viral genomes in influenza- and Sendai virus-infected cells ► These single-stranded viral genomes are the natural RIG-I agonists ► 5′-triphosphate groups on viral genomes are critical for RIG-I activation
The flow of genetic information from sites of transcription within the nucleus to the cytoplasmic translational machinery of eukaryotic cells is obstructed by a physical blockade, the nuclear double ...membrane, which must be overcome in order to adhere to the central dogma of molecular biology, DNA makes RNA makes protein. Advancement in the field of cellular and molecular biology has painted a detailed picture of the molecular mechanisms from transcription of genes to mRNAs and their processing that is closely coupled to export from the nucleus. The rules that govern delivering messenger transcripts from the nucleus must be obeyed by influenza A virus, a member of the Orthomyxoviridae that has adopted a nuclear replication cycle. The negative-sense genome of influenza A virus is segmented into eight individual viral ribonucleoprotein (vRNP) complexes containing the viral RNA-dependent RNA polymerase and single-stranded RNA encapsidated in viral nucleoprotein. Influenza A virus mRNAs fall into three major categories, intronless, intron-containing unspliced and spliced. During evolutionary history, influenza A virus has conceived a way of negotiating the passage of viral transcripts from the nucleus to cytoplasmic sites of protein synthesis. The major mRNA nuclear export NXF1 pathway is increasingly implicated in viral mRNA export and this review considers and discusses the current understanding of how influenza A virus exploits the host mRNA export pathway for replication.
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