SUMMARY Adaptation of viruses to their hosts can result in specialization and a restricted host range. Species specific polymorphisms in the influenza virus polymerase restrict its host range during transmission from birds to mammals. ANP32A was recently identified as a cellular co-factor affecting polymerase adaption and activity. Avian influenza polymerases require ANP32A containing an insertion resulting from an exon duplication uniquely encoded in birds. Here we find that natural splice variants surrounding this exon create avian ANP32A proteins with distinct effects on polymerase activity. We demonstrate species-independent direct interactions between all ANP32A variants and the PB2 polymerase subunit. This interaction is enhanced in the presence of viral genomic RNA. In contrast, only avian ANP32A restored ribonucleoprotein complex assembly for a restricted polymerase by enhancing RNA synthesis. Our data suggest that ANP32A splicing variation among birds differentially affects viral replication, polymerase adaption, and the potential of avian hosts to be reservoirs.

Cells infected by influenza virus mount a large-scale antiviral response and most cells ultimately initiate cell-death pathways in an attempt to suppress viral replication. We performed a CRISPR/Cas9-knockout selection designed to identify host factors required for replication following viral entry. We identified a large class of presumptive antiviral factors that unexpectedly act as important pro-viral enhancers during influenza virus infection. One of these, IFIT2, is an interferon-stimulated gene with well-established antiviral activity but limited mechanistic understanding. As opposed to suppressing infection, we show here that IFIT2 is instead repurposed by influenza virus to promote viral gene expression. CLIP‐seq demonstrated that IFIT2 binds directly to viral and cellular mRNAs in AU‐rich regions, with bound cellular transcripts enriched in interferon‐stimulated mRNAs. Polysome and ribosome profiling revealed that IFIT2 prevents ribosome pausing on bound mRNAs. Together, the data link IFIT2 binding to enhanced translational efficiency for viral and cellular mRNAs and ultimately viral replication. Our findings establish a model for the normal function of IFIT2 as a protein that increases translation of cellular mRNAs to support antiviral responses and explain how influenza virus uses this same activity to redirect a classically antiviral protein into a pro-viral effector.

Gammaherpesviruses are ubiquitous pathogens that establish lifelong infection in Ͼ95% of adults worldwide and are associated with a variety of malignancies. Coevolution of gammaherpesviruses with their hosts has resulted in an intricate relationship between the virus and the host immune system, and perturbation of the virus-host balance results in pathology. Interferon regulatory factor 1 (IRF-1) is a tumor suppressor that is also involved in the regulation of innate and adaptive immune responses. Here, we show that type I interferon (IFN) and IRF-1 cooperate to control acute gammaherpesvirus infection. Specifically, we demonstrate that a combination of IRF-1 and type I IFN signaling ensures host survival during acute gammaherpesvirus infection and supports IFN gamma-mediated suppression of viral replication. Thus, our studies reveal an intriguing cross talk between IRF-1 and type I and II IFNs in the induction of the antiviral state during acute gammaherpesvirus infection.IMPORTANCE Gammaherpesviruses establish chronic infection in a majority of adults, and this long-term infection is associated with virus-driven development of a range of malignancies. In contrast, a brief period of active gammaherpesvirus replication during acute infection of a naive host is subclinical in most individuals. Here, we discovered that a combination of type I interferon (IFN) signaling and interferon regulatory factor 1 (IRF-1) expression is required to ensure survival of a gammaherpesvirus-infected host past the first 8 days of infection. Specifically, both type I IFN receptor and IRF-1 expression potentiated antiviral effects of type II IFN to restrict gammaherpesvirus replication in vivo, in the lungs, and in vitro, in primary macrophage cultures.KEYWORDS IRF-1, acute infection, gammaherpesvirus, interferon, viral replication G ammaherpesviruses are ubiquitous pathogens that establish lifelong infection in a majority of the adult population and are associated with cancer (1-3). Similar to replication of other viruses, replication of both human (Epstein-Barr virus [EBV] and Kaposi's sarcoma-associated herpesvirus [KSHV]) and murine (mouse gammaherpesvirus 68[MHV68]) gammaherpesviruses is suppressed by type I and type II interferons (IFNs), two partially overlapping yet distinct host networks that are critical for the control of gammaherpesvirus infection (4-10). In the case of MHV68, both acute and chronic MHV68 infection is attenuated by type I and type II IFNs (4,6,11,12). While the antiviral role of IFNs in the context of gammaherpesvirus infection, including in vivo, is firmly established, the mechanism by which this restriction is imposed and the molecular players involved in this response are still being defined.Interferon regulatory factor 1 (IRF-1) is a broadly antiviral transcription factor that restricts replication of diverse RNA and DNA viruses in cell culture via a poorly understood mechanism (13, 14). While initially IRF-1 was thought to induce type I interferon (IFN) expression (15)

Adaptation of viruses to their host can result in specialization and a restricted host range. Species-specific polymorphisms in the influenza virus polymerase restrict its host range during transmission from birds to mammals. ANP32A was recently been identified as a cellular co-factor impacting polymerase adaption and activity. Avian influenza polymerases require ANP32A containing an insertion resulting from an exon duplication uniquely encoded in birds. Here we find that natural splice variants surrounding this exon create avian ANP32A proteins with distinct effects on polymerase activity. We demonstrate species-independent direct interactions between all ANP32A variants and the PB2 polymerase subunit. This interaction is enhanced in the presence of viral genomic RNA. In contrast, only avian ANP32A restored ribonucleoprotein complex assembly for a restricted polymerase by enhancing RNA synthesis. Our data suggest that ANP32A splicing variation amongst birds differentially impacts viral replication, polymerase adaption, and the potential of avian hosts to be reservoirs.

Viruses must balance their reliance on host cell machinery for replication while avoiding host defense. Influenza A viruses are zoonotic agents that frequently switch hosts, causing localized outbreaks with the potential for larger pandemics. The host range of influenza virus is limited by the need for successful interactions between the virus and cellular partners. Here we used immunocompetitive capture-mass spectrometry to identify cellular proteins that interact with human- and avian-style viral polymerases. We focused on the proviral activity of heterogenous nuclear ribonuclear protein U-like 1 (hnRNP UL1) and the antiviral activity of mitochondrial enoyl CoA-reductase (MECR). MECR is localized to mitochondria where it functions in mitochondrial fatty acid synthesis (mtFAS). While a small fraction of the polymerase subunit PB2 localizes to the mitochondria, PB2 did not interact with full-length MECR. By contrast, a minor splice variant produces cytoplasmic MECR (cMECR). Ectopic expression of cMECR shows that it binds the viral polymerase and suppresses viral replication by blocking assembly of viral ribonucleoprotein complexes (RNPs). MECR ablation through genome editing or drug treatment is detrimental for cell health, creating a generic block to virus replication. Using the yeast homolog Etr1 to supply the metabolic functions of MECR in MECR-null cells, we showed that specific antiviral activity is independent of mtFAS and is reconstituted by expressing cMECR. Thus, we propose a strategy where alternative splicing produces a cryptic antiviral protein that is embedded within a key metabolic enzyme.