Hayashi Tsuyoshi, MacDonald Leslie A, Takimoto Toru
Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York, USA.
Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York, USA
J Virol. 2015 Jun;89(12):6442-52. doi: 10.1128/JVI.00319-15. Epub 2015 Apr 8.
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 β-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-β) 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-β mRNA in their lungs than mice infected with Cal WT. Importantly, more antihemagglutinin and neutralizing antibodies were produced in Cal PA-XFS-infected 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.
Virus-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.
流感病毒感染会导致受感染细胞中宿主蛋白质合成的全面抑制。这种宿主关闭现象被认为能使病毒逃避宿主抗病毒反应，而宿主抗病毒反应会限制病毒的复制和传播。尽管宿主关闭的机制尚不清楚，但发现一种由核糖体移码表达的新型病毒蛋白PA-X在流感病毒诱导的宿主关闭中起主要作用。然而，关于PA-X表达对当前流行的甲型流感病毒致病性和宿主抗病毒反应的影响知之甚少。在本研究中，我们拯救了一种重组甲型流感病毒A/California/04/09（H1N1，Cal），其聚合酶PA基因的移码基序存在突变（Cal PA-XFS）。Cal PA-XFS表达的PA-X明显少于Cal野生型（WT）。Cal WT能诱导宿主β-肌动蛋白mRNA降解并抑制宿主蛋白质合成，但Cal PA-XFS则不能，这支持了PA-X通过mRNA降解诱导宿主关闭的观点。此外，在人呼吸道细胞中，Cal WT抑制β干扰素（IFN-β）表达，且比Cal PA-XFS复制得更快。感染Cal PA-XFS的小鼠肺部病毒生长水平显著低于感染Cal WT的小鼠，且其IFN-β mRNA表达更高。重要的是，尽管Cal PA-XFS感染小鼠肺部的病毒复制水平较低，但与Cal WT感染小鼠相比，前者产生了更多的抗血凝素和中和抗体。我们的数据表明，大流行H1N1病毒的PA-X对病毒生长以及宿主对流感病毒的先天性和获得性免疫反应有强烈影响。
病毒诱导的宿主蛋白质关闭被认为是病毒逃避先天性和获得性免疫识别的主要因素。我们提供的证据表明，2009年甲型H1N1流感病毒蛋白PA-X通过其在体外和体内的宿主蛋白质合成关闭活性，在病毒复制和抑制宿主抗病毒反应中发挥作用。我们还证明，虽然Cal PA-XFS在受感染动物肺部的生长减弱，但该突变体诱导的体液反应比Cal WT更强。我们的研究结果清楚地突出了PA-X在对抗宿主对重要全球病原体流感病毒的先天性和获得性免疫反应中的重要性。这项工作表明，抑制流感病毒疫苗株中PA-X的表达可能提供一种新方法，既能安全地减弱病毒生长，又能诱导更强有力的免疫反应。
