Degree Type

Dissertation

Date of Award

2012

Degree Name

Doctor of Philosophy

Department

Biochemistry, Biophysics and Molecular Biology

First Advisor

Gaya K. Amarasinghe

Abstract

Filoviruses are among the most deadly pathogens that cause acute disease in humans. Ebolavirus (EBOV) and marburgvirus (MARV) are the two members of this family, which have been documented to cause infrequent but severe outbreaks of hemorrhagic fever in humans. The severe pathogenesis and high lethality associated with filoviral infections, is in part, due to the suppression of host innate immune responses by virus-encoded proteins. Hence, structural and biochemical studies of filoviral proteins, to uncover the immune evasion mechanisms employed by filoviral proteins are an intense area of investigation. Previous studies on EBOV, have shown that one of the viral proteins called VP35 plays a key role in virus replication by functioning as a cofactor in the viral replication complex, and immune suppression by antagonizing the type I interferon (IFN) pathway. The C-terminal region of EBOV VP35 was implicated in dsRNA binding and IFN antagonism, although the mechanisms for immune evasion remained poorly defined. Recent work from our lab has resulted in crystal structures of Zaire ebolavirus (ZEBOV) and Reston ebolavirus (REBOV) VP35 C-terminal domain, and ZEBOV VP35 C-terminal domain bound to dsRNA. These studies gave new insights into the role of conserved basic residues in the C-terminal domain in both viral replication and immune evasion functions of VP35. These studies also established that mutation of residues mediating dsRNA binding also resulted in diminished IFN-inhibition using in vivo assays. In addition, the dsRNA bound structure suggested a potential mechanism by which EBOV VP35 hides viral dsRNA from detection by host RIG-I like receptors (RLRs). Studies addressing immune evasion mechanisms by filoviruses have predominantly been done on ZEBOV, and functions of MARV proteins are largely uncharacterized and are inferred through homology to EBOV. Moreover recent reports on MARV proteins have shown that there are important differences in cell entry, host tropism, replication complex formation, and immune evasion mechanisms between the two viruses. The goal of my thesis work was to develop a comparative understanding of EBOV and MARV VP35, by characterizing MARV VP35 mediated immune evasion mechanisms using structural, biochemical, and cell biological studies. During the course of this study, we solved the crystal structure of MARV VP35 interferon inhibitory domain (IID) bound to dsRNA. This structure revealed several similarities with ZEBOV VP35 IID, but importantly there are several striking differences. Similar to ZEBOV, mutation of residues involved in dsRNA contacts in the MARV VP35 IID-dsRNA structure results in diminished dsRNA binding and IFN inhibition in vivo. While both MARV and ZEBOV VP35 IID bind to dsRNA in a sequence independent manner, MARV VP35 IID binds long(er) dsRNA compared to ZEBOV. We did not observe any interactions of MARV VP35 IID with the dsRNA blunt-ends, as in the case ZEBOV VP35. We biochemically validated these structural differences by in vitro dsRNA binding assays and show that MARV VP35 IID binds preferentially to longer dsRNA. Moreover MARV VP35 IID is insensitive to the presence of 5' or 3' overhangs in dsRNA, whereas ZEBOV VP35 IID binds preferentially to blunt-end dsRNA compared to overhang containing dsRNA. In this study, for the first time, using in vitro ATPase assays, we show that while both MARV and ZEBOV VP35 IID can inhibit RIG-I activation by overhang containing dsRNA, only ZEBOV VP35 IID can inhibit RIG-I activation by short blunt-end dsRNA. In addition we show that both MARV and ZEBOV VP35 IID can inhibit MDA5 activation by poly I:C, a long dsRNA mimic, mediated ATPase activation. The results from this study supports a model based on both structural and biochemical data, in which MARV and ZEBOV VP35 IID inhibit host immune responses by sequestration of overlapping (double-strandedness) and distinct (blunt-ends) RNA PAMPs from being detected by host RIG-I like receptors. This work provides new insight into the structure and function of MARV VP35 IID, and advances our understanding of the structural basis for dsRNA binding by MARV VP35 IID and its role in IFN antagonism and immune evasion.

Copyright Owner

Parameshwaran Ramanan

Language

en

File Format

application/pdf

File Size

154 pages

Included in

Biochemistry Commons

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