Download or read book Mechanistic Analysis of Influenza A Virus Assembly and Host Gene Regulation written by Chutikarn Chaimayo and published by . This book was released on 2018 with total page 201 pages. Available in PDF, EPUB and Kindle. Book excerpt: Influenza A virus contains eight single-stranded, negative-sense RNA segmented genomes, which allow genetic reassortment between human and animal strains, leading to many virus pandemics. In addition to genetic reassortment, efficient regulation of host response is required for virus adaptation and transmission to different hosts. In this study, we investigated the processes of influenza genome packaging and assembly, as well as how the virus modulates host antiviral gene expression to unveil the mechanism of host adaptation. Influenza viruses replicate in the nucleus, where both genomic nucleocapsids (vRNPs) and the positive-sense complementary RNPs (cRNPs) are produced. Despite the structural similarity to vRNPs, cRNPs are not incorporated into progeny virions. To determine the mechanism by which cRNP packaging is restricted, we analyzed cRNP intracellular localization using strand-specific qRT-PCR. Our findings demonstrated that cRNPs were exported from the nucleus in a chromosome region maintenance 1 (CRM1) -independent manner. However, viral matrix (M1) protein explicitly interacted with cytosolic vRNPs, but not cRNPs, at the plasma membrane. Hence, our data indicate that a specific interaction with M1 during viral assembly determines selective incorporation of vRNPs into progeny virions. Influenza A viruses regulate host gene expression through two accessory proteins, NS1 and a novel PA-X protein expressed from ribosomal frameshifting of PA mRNA. Their shutoff activities vary between viruses from different host origins, suggesting a functional interplay between these two proteins. To analyze the effect of PA-X and NS1 on host gene expression and identify their target mRNAs, we generated four recombinant A/California/04/2009 viruses with various PA-X and NS1 shutoff activities. Our transcriptome analysis of infected human lung epithelial cells indicates that PA-X induced general gene shutoff, while NS1 specifically inhibited genes involved in interferon and cytokine signaling pathways. The virus expressing active NS1 with reduced PA-X activity most efficiently suppressed antiviral and innate immune responses in human cells, suggesting that NS1 and PA-X shutoff activities are precisely adjusted to achieve productive viral replication in human hosts. The mechanism of action and functional domains of PA-X have not been fully elucidated. Our data showed that PA-X was localized equally in the nucleus and cytoplasm, and degraded host mRNAs at both sites. By characterizing PA-X Cterminal deletion mutants, we found that the first 15 residues in the unique C-terminal domain were sufficient for host mRNA degradation and required for nuclear localization of PA-X. Our co-immunoprecipitation result showed that PA-X interacted with host factors that are either involved in pre-mRNA processing or associated with mature mRNAs. Collectively, this study sheds light on virus-host interaction and mechanisms by which influenza A viruses regulate viral assembly and host gene expression.