Characterization Of Influenza Virus Messenger Rna

Author : Mitchell Ledwith
Publisher :
ISBN 13 :
Total Pages : 0 pages
Book Rating : 4.:/5 (134 download)

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Download or read book RNA-binding Proteins Regulate Influenza Virus Infection written by Mitchell Ledwith and published by . This book was released on 2022 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Viruses are obligate intracellular parasites that depend on host resources andmacromolecular machinery to selfishly replicate and spread genetic material. This genetic material can exist in multiple forms, with viruses utilizing RNA, DNA, and diverse combinations thereof to stably transmit between hosts. These diverse strategies require diverse replication schemes, e.g., DNA viruses must either encode or co-opt host DNA-dependent DNA polymerases to replicate a genome, and negative sense RNA viruses must encode and package RNA-dependent RNA polymerases in virons in order to replicate an RNA genome in the next host. Regardless of scheme, all viruses converge and are absolutely reliant on RNA, and more specifically the host ability to translate RNA molecules into proteins. Thus, both viruses and hosts have devoted significant coding capital in regulating RNA, with emphasis on the evolutionary struggle that exists at this nexus. The over-arching theme of this thesis is to illuminate how and why influenza viruses co- opt, manipulate, and embrace RNA regulation strategies of human hosts. In the first chapter of this thesis, we profiled the RNA-binding characteristics of IFIT2, a canonically anti-viral and interferon-induced host protein, which influenza virus utilizes in a pro- viral manner. Through genome-wide cross-linking immunopreciptation sequencing (CLIP-seq) experiments, we found that IFIT2 binds mRNAs including those of influenza virus. We validated IFIT2 as an mRNA-binding protein, and found that IFIT2 specifically binds AU-rich regions in human RNAs with a preference for binding the mRNAs of other interferon-induced proteins. To accomplish a broadly anti-viral and selectively pro-viral function, we found that IFIT2 binds mRNAs to enhance their translation. Polysome and ribosome-profiling revealed that IFIT2-bound RNAs are poorly translated in the absence of IFIT2, including influenza virus mRNAs. This links the RNA-binding ability of IFIT2 to a functional role in translation, potentially explaining thexi contradiction between the previously observed anti-viral phenotypic role for IFIT2 and the mechanistically observed pro-viral role for IFIT2 in the context of influenza virus. Altogether, these data describe a new node for the regulation of translation during interferon responses and highlight the regulatory volatility that exists at the mRNA interface during infection. In the second chapter of this thesis, we sought to understand the non-specific RNA-binding capability of the influenza virus nucleoprotein, which binds and protects the negative-sense RNA genome to facilitate gene expression and genome replication. Despite this well-established role, the incongruity of the specificity of NP for the viral genome and known biochemical promiscuity of NP is not reconciled. To address this, we perfomed cross-linking immunoprecipitation sequencing to unbiasedly determine the identity of NP-bound RNAs during infection. NP binds the viral genome and anti-genome, but also engages with a number of discrete classes of host small non-coding RNAs. Many of these RNAs have been shown to be host-derived RIG-I agonists during DNA virus infections, or structurally resemble validated RIG-I agonists. We profiled the RNAs bound by RIG-I during infection with quantitative CLIP-seq and found that host RNAs are bound and sampled by RIG-I, and NP likely interferes with the ability of RIG-I to sample endogenous and exogenous agonists. These data show that NP not only acts as a structural component of the influenza gene expression and replication machinery, but also acts as a viral countermeasure for innate sensing of immunogenic RNAs. As a whole, this thesis describes two dissimilar instances by which influenza manipulates host RNA regulation. While the context of these host protein:viral mRNA and viral protein:host non-coding RNA interactions are different, they collectively suggest that RNA regulation during the host innate response to viral infection is an essential and commonplace strategy, but often leaves the host exposed to quickly adapting viral pathogens.