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Theses and Dissertations

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Browsing Theses and Dissertations by Subject "alphavirus"

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    Commandeering of the cellular HuR protein by alphaviruses affects the regulation of host post-transcriptional gene expression
    (Colorado State University. Libraries, 2013) Barnhart, Michael D., author; Wilusz, Jeffrey, advisor; Wilusz, Carol J., committee member; Laybourn, Paul J., committee member
    It was previously shown that cellular HuR protein binds to a U-rich region in the 3'UTR of Sindbis virus RNA resulting in stabilization of viral transcripts and increased replication efficiency. While the presence of this U-rich region is generally conserved among alphaviruses, a subset lacks a typical U-rich region. The 3'UTR of two alphaviruses - Ross River virus and Chikungunya virus - that do not contain a typical U-rich region were tested for HuR interactions by Electrophoretic Mobility Shift Assay. HuR protein bound these 3'UTRs with nanomolar affinities, similar to what was observed for the U-rich region of Sindbis virus. These observations demonstrate that the critical role for HuR-mediated viral RNA stabilization is likely a conserved property of most, if not all, members of the virus family. By analyzing deletion derivatives, we mapped the novel HuR binding sites in these two viruses to specific regions in their 3'UTR. Next, we uncovered four novel aspects of virus-host interaction and pathogenesis related to the high affinity interaction between the 3'UTR of alphaviruses and the cellular HuR protein. First, HuR protein, which is usually localized predominantly to the nucleus, dramatically accumulates in the cytoplasm during Sindbis virus (SinV) infection. Studies involving the transfection of constructs that express viral 3'UTR RNA fragments indicated that the mechanism of induction of HuR accumulation to the cytoplasm in infected cells is due to the viral RNA acting as a sponge for the protein. Second, HuR interaction with numerous cellular mRNAs was found to be drastically decreased during a SinV infection and was associated with dramatic destabilization of the cellular transcripts as determined by mRNA half-life analysis. Third, we found that the reduced amounts of free HuR during a SinV infection results in the increased targeting of mRNAs by miRNAs. Together, these data indicate that in the process of commandeering the cellular HuR protein for its own use, alphaviruses are also effectively destabilizing numerous cellular mRNAs. Interestingly, many of the cellular mRNAs affected by alphaviruses play key roles in inflammation, innate immune responses and other fundamental cellular processes. Finally, we observed a novel effect of SinV infection on alternative polyadenylation of cellular transcripts. This is likely a direct result of sequestration of the HuR protein in the cytoplasm by the virus, preventing the protein from influencing nuclear polyadenylation site choice. Intriguingly, SinV infection influences the poly(A) site choice of the HuR pre-mRNA, favoring a more translatable isoform to promote the overexpression of this viral host factor. Therefore, the alphaviral-induced alterations in cellular mRNA stability and polyadenylation identified in this thesis may play a very important but underappreciated role in pathogenesis.
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    Development of novel alphavirus expression and detection systems and characterization of oxidation's effect on viral replication
    (Colorado State University. Libraries, 2014) Steel, J. Jordan, author; Geiss, Brian, advisor; Schenkel, Alan, committee member; Olson, Ken, committee member; Callan, Rob, committee member
    Infectious diseases cause significant global suffering and death each year. Specifically, arthropod-borne viruses are emerging and re-emerging around the world and infecting millions of people. Mosquitoes that transmit these viruses are spreading to new regions of the world with naïve populations to serve as viral hosts. Climate change and human encroachment of habitat has brought mosquitoes into close proximity with humans. The viruses are evolving and are expanding their vector compatibility to include more than one species of mosquitoes. The combination of these elements results in a serious global need to develop ways to control or prevent arthropod-borne viruses. In order to discover novel antivirals and ways to inhibit these arboviruses, a better understanding of viral infection and replication is needed. This dissertation will describe a combination of projects that all aim to provide enhanced knowledge or tools to prevent, control, or treat arbovirus infection. Specifically, we improved the ability to express recombinant infectious alphaviruses, developed a novel system to detect alphavirus infection in mosquito cell culture and transgenic mosquitoes, and discovered a new role for oxidation during flavivirus replication. First, we successfully developed and established a method for transcribing infectious alphavirus RNA from a plasmid DNA platform. This approach provides an efficient way for producing high titer infectious recombinant alphavirus in multiple cell types that robustly express foreign proteins. Secondly, we optimized a system for detecting alphavirus infection in mosquito cells using the virus dependent subgenomic promoter to transcribe a reporter gene only during active infection. We demonstrated that mosquito cells can be stably transformed to transcribe an engineered viral reporter RNA that expresses a fluorescent reporter protein (mCherry) only in the presence of wild-type virus infection. The reporter protein is not detected in uninfected controls, but significant expression is readily detected during infection. Transgenic mosquitoes were also developed to transcribe the reporter RNA, which amplifies and expresses the reporter protein during infection. The transgenic mosquitoes are able to express a fluorescent reporter protein only during Sindbis virus (Alphavirus) infection, providing a novel mechanism to detect infection of wild-type virus in living mosquitoes. This transgenic reporter system is the first of its kind and demonstrated that a system based on our reporter RNAs could be optimized and used to specifically detect infected mosquitoes. Finally, I was able to study and characterize several aspects of viral RNA replication within the cell. Specifically, we identified that viral RNA replication is dependent on oxidative conditions. We determined individual residues from the flavivirus NS5 capping protein that are specifically involved in the oxidative enhancement of viral replication. Our work provided significant advances to the arbovirus field. We now have a novel method for producing recombinant alphaviruses that is more time, cost, and resource effective. We understand the ability of the subgenomic promoter to act as a virus inducible promoter to express foreign proteins only during infection to help detect or manipulate infection in mosquitoes. Finally, we have made significant discoveries on how RNA replication works on a molecular level within the cell and better understand the important role of oxidation on virus infection. The work and discoveries described in this dissertation have enhanced multiple aspects of arbovirus research and will hopefully strengthen our ability to fight and control arbovirus infections around the world.
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    The DXO decapping exonuclease is a restriction factor for RNA viruses
    (Colorado State University. Libraries, 2019) Lynch, Erin R., author; Geiss, Brian, advisor; Wilusz, Jeffrey, committee member; Perera, Rushika, committee member; Stasevich, Tim, committee member
    Cellular RNA exonucleases, such as XRN1 and DXO, aid in the destruction of defective cellular mRNAs and help maintain overall cellular health. The RNA decay system, however, also serves another purpose – degrading viral RNAs. The XRN1 exonuclease is known to be a major antagonist of RNA virus genomes, but the role of other cellular RNA decay enzymes in controlling viral infection is less clear. The cellular 5' decapping exonuclease DXO is able to recognize, de-cap, and degrade RNAs lacking 2'-O-methylation on the first nucleotide after the 5' cap, helping the cell to discriminate self from non-self RNAs. Preliminary data we have developed indicate that flaviviruses and alphaviruses replicate to much higher levels in DXO deficient cells than in cells containing DXO, indicating that DXO may also act as a cellular viral restriction factor. Interestingly, flavivirus genomes contain a 5' cap that is generally 2'-O-methylated at the first base of the transcript, providing a potential mechanism to evade DXO degradation. Overall, our results indicate that the DXO decapping exonuclease helps control the replication of positive strand RNA viruses in cells and represents a new viral restriction factor.