Browsing by Subject "Influenza A virus"
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Item Characterization of Host Factors Affecting Viral Entry(2019-04-10) Rinkenberger, Nicholas Ryan; Alto, Neal; Schoggins, John W.; Pfeiffer, Julie K.; Reese, Tiffany A.Viruses are obligate, intracellular parasites. For a virus to infect a host cell, it must gain access to the interior of the host cell by some means. In animals, this often involves the exploitation of host processes such as receptor-mediated endocytosis and vesicular trafficking. Zika virus is an emerging arbovirus with global health and economic impacts. Interestingly, while Asian lineage Zika virus causes human disease and has been associated with severe neurological complications, African lineage Zika virus has only rarely been reported to cause human disease. Large strides have been made in understanding Zika virus infection. However, the mechanism used by Zika virus to enter host cells remains somewhat obscure. In chapter 2, I delineate and compare the pathway utilized by both Asian and African lineage Zika virus to enter host cells. I find that these viruses require clathrin-mediated endocytosis and Rab5a function in a conserved manner. Additionally, all Zika virus strains tested were sensitive to pH in the range of 6.5-6.1 and were reliant on endosomal acidification for infection. I found that Zika virus preferentially fuses with late endosomes. Comparing lineages, Zika virus enters cells in a highly conserved manner. Just as viruses have evolved to exploit host factors to promote their entry and replication, hosts have developed mechanisms of defense against viral infection. Recognition of viral infection by vertebrate hosts results in the expression and secretion of interferon. Interferon signaling subsequently results in the induction of hundreds of interferon-stimulated genes (ISGs) which restrict pathogen infection. Some of these ISGs specifically block viral entry. Surprisingly, a small group of ISGs was previously identified which actually promote viral infection. In chapter 3, I characterize the mechanism of action of MCOLN2, one of the ISGs found to promote viral infection. I assign a role for MCOLN2 in modulating viral entry. I show that MCOLN2 specifically promotes viral vesicular trafficking and subsequent escape from endosomal compartments. This mechanism requires channel activity, occurs independently of antiviral signaling, and broadly applies to enveloped RNA viruses that require endosomal acidification for infection, including influenza A virus, yellow fever virus, and Zika virus.Item Chemical Intervention of Influenza Virus mRNA Nuclear Export(2020-07-15) Esparza, Matthew Aaron; Shay, Jerry W.; Fontoura, Beatriz; Grinnell, Frederick; Minna, John D.Influenza A viruses are human pathogens with limited therapeutic options, making it crucial to devise strategies for the identification of new classes of antiviral medications. The influenza A virus genome is constituted of 8 RNA segments. Two of these viral RNAs are transcribed into mRNAs that are alternatively spliced. The M1 mRNA encodes the M1 protein but is also alternatively spliced to yield the M2 mRNA during infection. M1 to M2 mRNA splicing occurs at nuclear speckles, and M1 and M2 mRNAs are exported to the cytoplasm for translation. M1 and M2 proteins are critical for viral trafficking, assembly, and budding. We show that influenza virus utilizes nuclear speckles to promote post-transcriptional splicing of its M1 mRNA. We assign previously unknown roles for the viral NS1 protein and cellular factors to an intranuclear trafficking pathway that targets the viral M1 mRNA to nuclear speckles, mediates splicing at these nuclear bodies, and exports the spliced M2 mRNA from the nucleus. In addition, gene knockout of the cellular protein NS1-BP, a constituent of the M mRNA speckle-export pathway, inhibits M mRNA nuclear export without significantly altering bulk cellular mRNA export, providing an avenue to preferentially target influenza virus. We performed a high-content, image-based chemical screen using single-molecule RNA-FISH to label viral M mRNAs followed by multistep quantitative approaches to assess cellular mRNA and cell toxicity. We identified inhibitors of viral mRNA biogenesis and nuclear export that exhibited no significant activity towards bulk cellular mRNA at non-cytotoxic concentrations. Among the hits is a small molecule that inhibits nuclear export of a subset of viral and cellular mRNAs via the mRNA export factor UAP56 without altering bulk cellular mRNA nuclear export. These findings underscore specific nuclear export requirements for viral mRNA nuclear export. This RNA export inhibitor also impaired replication of diverse influenza virus strains at non-toxic concentrations. Thus, this screening strategy yielded compounds that alone or in combination may serve as leads to new ways of treating influenza virus infection and are novel tools for studying viral RNA trafficking in the nucleus.