Influenza virus infection causes global inhibition of host protein synthesis in infected cells. This host shutoff is thought to allow viruses to escape from the host antiviral response, which restricts virus replication and spread. Although the mechanism of host shutoff is unclear, a novel viral protein expressed by ribosomal frameshifting, PA-X, was found to play a major role in influenza virus-induced host shutoff. However, little is known about the impact of PA-X expression on currently circulating influenza A virus pathogenicity and the host antiviral response. In this study, we rescued a recombinant influenza A virus, A/California/ 04/09 (H1N1, Cal), containing mutations at the frameshift motif in the polymerase PA gene (Cal PA-XFS). Cal PA-XFS expressed significantly less PA-X than Cal wild type (WT). Cal WT, but not Cal PA-XFS, induced degradation of host ␤-actin mRNA and suppressed host protein synthesis, supporting the idea that PA-X induces host shutoff via mRNA decay. Moreover, Cal WT inhibited beta interferon (IFN-␤) expression and replicated more rapidly than Cal PA-XFS in human respiratory cells. Mice infected with Cal PA-XFS had significantly lower levels of viral growth and greater expression of IFN-␤ mRNA in their lungs than mice infected with Cal WT. Importantly, more antihemagglutinin and neutralizing antibodies were produced in Cal PA-XFSinfected mice than in Cal WT-infected mice, despite the lower level of virus replication in the lungs. Our data indicate that PA-X of the pandemic H1N1 virus has a strong impact on viral growth and the host innate and acquired immune responses to influenza virus. IMPORTANCEVirus-induced host protein shutoff is considered to be a major factor allowing viruses to evade innate and acquired immune recognition. We provide evidence that the 2009 H1N1 influenza A virus protein PA-X plays a role in virus replication and inhibition of host antiviral response by means of its host protein synthesis shutoff activity both in vitro and in vivo. We also demonstrated that, while the growth of Cal PA-XFS was attenuated in the lungs of infected animals, this mutant induced a stronger humoral response than Cal WT. Our findings clearly highlight the importance of PA-X in counteracting the host innate and acquired immune responses to influenza virus, an important global pathogen. This work demonstrates that inhibition of PA-X expression in influenza virus vaccine strains may provide a novel way of safely attenuating viral growth while inducing a more robust immune response.

PA-X is a recently identified influenza virus protein that is composed of the PA N-terminal 191 amino acids and unique C-terminal 41 or 61 residues. We and others showed that PA-X has a strong ability to suppress host protein synthesis via host mRNA decay, which is mediated by endonuclease activity in its N-terminal domain (B. -12). However, the mechanism of host mRNA degradation, especially where and how PA-X targets mRNAs, has not been analyzed. In this study, we determined the localization of PA-X and the role of the C-terminal unique region in shutoff activity. Quantitative subcellular localization analysis revealed that PA-X was located equally in both cytoplasm and nucleus. By characterizing a series of PA-X C-terminal deletion mutants, we found that the first 9 amino acids were sufficient for nuclear localization, but an additional 6 residues were required to induce the maximum shutoff activity observed with intact PA-X. Importantly, forced nuclear localization of the PA-X C-terminal deletion mutant enhanced shutoff activity, highlighting the ability of nuclear PA-X to degrade host mRNAs more efficiently. However, PA-X also inhibited luciferase expression from transfected mRNAs synthesized in vitro, suggesting that PA-X also degrades mRNAs in the cytoplasm. Among the basic amino acids in the PA-X C-terminal region, 3 residues, 195K, 198K, and 199R, were identified as key residues for inducing host shutoff and nuclear localization. Overall, our data indicate a critical role for the 15 residues in the PA-X C-terminal domain in degrading mRNAs in both the cytoplasm and nucleus.W IMPORTANCEInfluenza A viruses express PA-X proteins to suppress global host gene expression, including host antiviral genes, to allow efficient viral replication in infected cells. However, little is known about how PA-X induces host shutoff. In this study, we showed that PA-X localized equally in both the cytoplasm and nucleus of the cells, but the nuclear localization of PA-X mediated by its C-terminal region has a significant impact on shutoff activity. Three basic residues at the C-terminal region play a critical role in nuclear localization, but additional basic residues were required for maximum shutoff activity. Our findings indicate that PA-X targets and degrades mRNAs in both the nucleus and cytoplasm, and that the first 15 residues of the PA-X unique C-terminal region play a critical role in shutoff activity. Influenza virus infection induces suppression of host protein synthesis, called "host shutoff," during viral replication in infected cells. This host shutoff activity has been thought to contribute to efficient virus replication in the cells by dampening host antiviral response. Earlier studies have suggested that the capsnatching activity and degradation of RNA polymerase II mediated by viral RNA polymerase (PA, PB1, and PB2) play a role in host shutoff (1, 2). In addition, the NS1 protein, a well-known antagonist against host antiviral interferon responses, has been shown to induce host shutoff in infected c...

Cholesterol-rich lipid raft microdomains in the plasma membrane are considered to play a major role in the enveloped virus lifecycle. However, the functional role of cholesterol in assembly, infectivity and stability of respiratory RNA viruses is not fully understood. We previously reported that depletion of cellular cholesterol by cholesterol-reducing agents decreased production of human parainfluenza virus type 1 (hPIV1) particles by inhibiting virus assembly. In this study, we analyzed the role of cholesterol on influenza A virus (IAV) and respiratory syncytial virus (RSV) production. Unlike hPIV1, treatment of human airway cells with the agents did not decrease virus particle production. However, the released virions were less homogeneous in density and unstable. Addition of exogenous cholesterol to the released virions restored virus stability and infectivity. Collectively, these data indicate a critical role of cholesterol in maintaining IAV and RSV membrane structure that is essential for sustaining viral stability and infectivity.

Mammalian melanoma differentiation-associated gene-5 (MDA5) and retinoic acid-inducible gene-I (RIG-I) selectively sense double-stranded RNA (dsRNA) according to length, as well as various RNA viruses to induce an antiviral response. RIG-I, which plays a predominant role in the induction of antiviral responses against influenza virus infection, has been considered to be lacking in chicken, putting the function of chicken MDA5 (chMDA5) under the spotlight. Here, we show that chMDA5, unlike mammalian MDA5, preferentially senses shorter dsRNA synthetic analogues, poly(I:C), in chicken DF-1 fibroblasts. A requirement for caspase activation and recruitment domains for chMDA5-mediated chicken interferon beta (chIFNβ) induction and its interaction with mitochondrial antiviral signaling proteins were demonstrated. We also found that chMDA5 is involved in chIFNβ induction against avian influenza virus infection. Our findings imply that chMDA5 compensates in part the function of RIG-I in chicken, and highlights the importance of chMDA5 in the innate immune response in chicken.