Critical Role of the PA-X C-Terminal Domain of Influenza A Virus in Its Subcellular Localization and Shutoff Activity

Abstract

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...

Impacts of different expressions of PA-X protein on 2009 pandemic H1N1 virus replication, pathogenicity and host immune responses

“…4 and 8), which is consistent with previous findings (Gao et al, 2015a). However, two recent studies described that the first 15aa in the C-terminal region of PA-X are sufficient for maximum shutoff activity, while the addition of 20 aa of C-terminal end is not critical to shutoff activity of PA-X (Hayashi et al, 2016; Oishi, Yamayoshi, and Kawaoka, 2015). The reason for the different observations in shutoff activities of truncated PA-X between these studies could be due to differences of PA-X expression systems used, resulting in different protein expression levels, thereby showing a difference of the host shutoff activity.…”

“…The reason for the different observations in shutoff activities of truncated PA-X between these studies could be due to differences of PA-X expression systems used, resulting in different protein expression levels, thereby showing a difference of the host shutoff activity. Because the whole PA expression plasmid was used in our study and the former studies, which could generate full-length or truncated PA-X by ribosomal frameshifting (Gao et al , 2015a), while PA-X expression plasmids were used in latter studies (Gao et al, 2015a; Hayashi et al, 2016; Oishi, Yamayoshi, and Kawaoka, 2015). Comprehensive phylogenetic and evolutionary analysis revealed that truncated PA-X appears in particular hosts such as dogs and swine which indicates species specificity of PA-X protein (Shi et al, 2012).…”

“…Since PA-X shares the N-terminal endonuclease domain with PA, it provides the host shutoff activity for PA-X to destroy the host mRNA and suppress host protein synthesis (Desmet et al, 2013; Jagger et al, 2012). Although PA and PA-X have the same N-terminal domain, PA-X has been shown to have a stronger endonucleolytic activity than PA, indicating that the unique C-terminal part of PA-X is also responsible for shutoff activity (Bavagnoli et al, 2015; Desmet et al, 2013; Hayashi et al, 2016; Oishi, Yamayoshi, and Kawaoka, 2015). …”

PA-X: a key regulator of influenza A virus pathogenicity and host immune responses

“…It has been shown that multiple mechanisms are related to host shutoff in IAV-infected cells. However, recent studies have focused on the following three main mechanisms of global inhibition of host protein expression by IAV infection: (1) blockade of cellular mRNA processing and nuclear export by NS1 [16][17][18][19]; (2) degradation of host RNA Pol II by the viral RNA-dependent RNA polymerase RdRp complex (RdRP) [20][21][22]; and (3) wide-spread host mRNA degradation by the PA-X protein [11,[23][24][25][26][27][28][29][30][31]. In this review, we focus mainly on the shutoff activity of PA-X protein.…”

Cholesterol is required for stability and infectivity of influenza A and respiratory syncytial viruses

“…Recombinant A/California/04/2009 (H1N1) virus was rescued previously by a 12-plasmid rescue system (Hayashi et al, 2016) and propagated in eggs. A/Victoria/361/2011 (H3N2) virus was kindly provided by Dr. David Topham (University of Rochester) and propagated in MDCK cells in DMEM supplemented with 0.15% bovine serum albumin (BSA) and acetylated trypsin at 2 μg/ml.…”