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    Essential Host Factors for Human Parvovirus B19 Replication

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    Issue Date
    2018-05-31
    Author
    Ganaie, Safder S.
    Publisher
    University of Kansas
    Format
    154 pages
    Type
    Dissertation
    Degree Level
    Ph.D.
    Discipline
    Microbiology, Molecular Genetics & Immunology
    Rights
    Copyright held by the author.
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    Abstract
    Parvovirus B19 (B19V) is a small, non-enveloped virus that contains a single-stranded DNA (ssDNA) genome of 5.6 kb in size. B19V is pathogenic to humans and causes bone marrow failure diseases and various other inflammatory disorders. B19V infection exhibits high tropism for human erythroid progenitor cells (EPCs) in the bone marrow and fetal liver. The exclusive restriction of B19V replication to erythroid lineage cells is partly due to the expression of receptor and co-receptor(s) on the cell surface of human EPCs and partly depends on the intracellular factors essential for virus replication. On this rationale, we tried to investigate the essential host factors for efficient virus replication. Our results demonstrated that signal transducer and activator of transcription 5 (STAT5) and RNA-binding motif protein-38 (RBM38) are two of these important host factors that ensure virus DNA replication and pre-mRNA processing, respectively, during B19V infection. The stages of erythropoiesis during which STAT5 is activated and RBM38 is expressed, are highly susceptible to B19V infection, thus suggesting that these two factors are among the key determinants of the B19V restriction to human EPCs. B19V requires erythropoietin (EPO) signaling and hypoxia for its efficient replication. EPO to EPO receptor signaling activates JAK2-STAT5 pathway that phosphorylates STAT5. In our first study, we show that phosphorylated STAT5 is critical for B19V replication. Upon in-silico analysis, we identified a consensus STAT5 binding element adjacent to the NS1-binding elements within the minimal origin of viral DNA replication (Ori) in the B19V genome. The phosphorylated STAT5 specifically interacts with viral Ori both in vivo and in vitro, and is actively recruited within the viral DNA replication centers. Furthermore, our study shows a novel interaction between STAT5 and the minichromosome maintenance (MCM) complex. Our proposed model suggests that STAT5 directly facilitates viral DNA replication by recruiting MCM complex into viral DNA replication centers. Interestingly, we found that pimozide, a STAT5 phosphorylation inhibitor and an FDA-approved drug, inhibits B19V replication in ex vivo expanded EPCs, suggesting that pimozide could be a promising antiviral drug for the treatment of B19V-related pathologies. B19V expresses a single precursor mRNA (pre-mRNA), which undergoes alternative splicing and alternate polyadenylation to generate 12 viral mRNA transcripts. Splicing at the second 5’ donor site (D2) of the B19V pre-mRNA is essential for the expression of VP1, VP2 and 11-kDa. We have previously identified that a cis-acting intronic splicing enhancer 2 (ISE2) that lies immediately after the D2 site facilitates recognition of the D2 donor for its efficient splicing. In our second study, we described that ISE2 harbors a consensus RBM38 binding sequence–5’-UGUGUG-3’. RBM38 is expressed during the middle stage of erythropoiesis. We first confirmed that the RBM38 binds specifically with the ISE2 element in vitro. Knockdown of RBM38 significantly decreases the level of the spliced mRNA at D2 that encodes 11-kDa protein and, thereafter, the expression of the 11-kDa protein. Importantly, we found that the 11-kDa protein enhances viral DNA replication and virion release. Accordingly, knockdown of RBM38 decreases virus replication via downregulating 11-kDa expression. Taken together, these results suggest that the 11-kDa protein facilitates B19V DNA replication, and that RBM38 is an essential host factor for the splicing of B19V pre-mRNA from D2 to A2-2 sites and for the expression of the 11-kDa protein. In conclusion, we identified two host factors, STAT5 and RBM38, which play important roles in B19V replication. We provide a mechanistic overview of how STAT5 facilitates virus DNA replication and RBM38 promotes the splicing of B19V pre-mRNA that ensures the expression of 11-kDa protein.
    URI
    http://hdl.handle.net/1808/27051
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    • KU Med Center Dissertations and Theses [464]
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    785-864-8983
    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    785-864-8983

    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
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    Contact KU ScholarWorks
    785-864-8983
    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    785-864-8983

    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    Image Credits
     

     

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