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CHARACTERIZING THE IMPACT OF VIRAL PROTEIN BINDING ON THE FUNCTION OF THE DEAD-BOX RNA HELICASE DDX3X

Venus, Sarah L
http://orcid.org/0000-0002-3450-3816
http://rave.ohiolink.edu/etdc/view?acc_num=case1662842513440799

Abstract Details

2022, Doctor of Philosophy, Case Western Reserve University, Biochemistry.
The DEAD-box RNA helicase DDX3X is targeted by proteins from diverse viruses, including K7 from vaccinia virus, core protein from Hepatitis C virus, NS1 and NP proteins from influenza A virus, and capsid protein from dengue virus. These interactions have been shown to disrupt DDX3X-mediated promotion of innate immune signaling; however, it is unknown whether and how these viral proteins impact the biochemical and cellular functions of DDX3X. Biochemically, DDX3X hydrolyzes ATP and unwinds RNA duplexes. In the cell, this biochemical activity enables DDX3X to promote translation initiation by remodeling structured mRNAs. Vaccinia virus K7 protein binds to the N-terminus of DDX3X that contains large intrinsically disordered regions. These intrinsically disordered regions mediate DDX3X’s oligomerization required for optimal helicase activity in vitro and DDX3X’s association with stress granules in the cell. Here I show that K7 binding to DDX3X’s N-terminus inhibits the ATP-dependent RNA unwinding and RNA-dependent ATP hydrolysis activities of DDX3X by reducing the functional RNA affinity of DDX3X. K7 also reduces the formation of recombinant DDX3X phase-separated droplets in vitro. In cells, K7 inhibits formation of DDX3X-containing stress granules. In addition, K7 reduces the formation of aberrant DDX3X foci induced by mutations in DDX3X that are associated with medulloblastoma and intellectual disability. Finally, K7 reduces global translation in HCT 116 cells by approximately 14%, suggesting that K7- mediated inhibition of DDX3X helicase activity may reduce translation of structured DDX3X-dependent transcripts. Our data reveal that K7 binding to the intrinsically disordered N-terminus of DDX3X is an effective way to interfere with the ability of DDX3X to resolve RNA structure and to undergo liquid-liquid phase separation. Dual inhibition of DDX3X enzymatic activity and condensate formation by K7 binding suggests that the N-terminal extension of DDX3X participates in inter-DDX3X interactions important for both DDX3X oligomerization and DDX3X liquid-liquid phase separation. Our findings also provide mechanistic insight into viral strategies that target DDX3X to alter host RNA metabolism, as well as highlight potential strategies to therapeutically target aberrant liquid-liquid phase separation linked to DDX3X mutations in medulloblastoma and intellectual disability.
Eckhard Jankowsky (Advisor)
Donny Licatalosi (Committee Member)
Derek Abbott (Committee Member)
William Merrick (Committee Chair)
200 p.
Biochemistry
DDX3X; vaccinia; RNA helicase

Recommended Citations

Citations

  • Venus, S. L. (2022). CHARACTERIZING THE IMPACT OF VIRAL PROTEIN BINDING ON THE FUNCTION OF THE DEAD-BOX RNA HELICASE DDX3X [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1662842513440799

    APA Style (7th edition)

  • Venus, Sarah. CHARACTERIZING THE IMPACT OF VIRAL PROTEIN BINDING ON THE FUNCTION OF THE DEAD-BOX RNA HELICASE DDX3X. 2022. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1662842513440799.

    MLA Style (8th edition)

  • Venus, Sarah. "CHARACTERIZING THE IMPACT OF VIRAL PROTEIN BINDING ON THE FUNCTION OF THE DEAD-BOX RNA HELICASE DDX3X." Doctoral dissertation, Case Western Reserve University, 2022. http://rave.ohiolink.edu/etdc/view?acc_num=case1662842513440799

    Chicago Manual of Style (17th edition)

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This open access ETD is published by Case Western Reserve University School of Graduate Studies and OhioLINK.
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