Structural and biochemical characterization of marburgvirus VP35 and its role in immune evasion

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\u27 or 3\u27 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

Structural mechanisms of DREAM complex assembly and regulation

The DREAM complex represses cell cycle genes during quiescence through scaffolding MuvB proteins with E2F4/5 and the Rb tumor suppressor paralog p107 or p130. Upon cell cycle entry, MuvB dissociates from p107/p130 and recruits B-Myb and FoxM1 for up-regulating mitotic gene expression. To understand the biochemical mechanisms underpinning DREAM function and regulation, we investigated the structural basis for DREAM assembly. We identified a sequence in the MuvB component LIN52 that binds directly to the pocket domains of p107 and p130 when phosphorylated on the DYRK1A kinase site S28. A crystal structure of the LIN52–p107 complex reveals that LIN52 uses a suboptimal LxSxExL sequence together with the phosphate at nearby S28 to bind the LxCxE cleft of the pocket domain with high affinity. The structure explains the specificity for p107/p130 over Rb in the DREAM complex and how the complex is disrupted by viral oncoproteins. Based on insights from the structure, we addressed how DREAM is disassembled upon cell cycle entry. We found that p130 and B-Myb can both bind the core MuvB complex simultaneously but that cyclin-dependent kinase phosphorylation of p130 weakens its association. Together, our data inform a novel target interface for studying MuvB and p130 function and the design of inhibitors that prevent tumor escape in quiescence

Mutual Antagonism between the Ebola Virus VP35 Protein and the RIG-I Activator PACT Determines Infection Outcome

SummaryThe cytoplasmic pattern recognition receptor RIG-I is activated by viral RNA and induces type I IFN responses to control viral replication. The cellular dsRNA binding protein PACT can also activate RIG-I. To counteract innate antiviral responses, some viruses, including Ebola virus (EBOV), encode proteins that antagonize RIG-I signaling. Here, we show that EBOV VP35 inhibits PACT-induced RIG-I ATPase activity in a dose-dependent manner. The interaction of PACT with RIG-I is disrupted by wild-type VP35, but not by VP35 mutants that are unable to bind PACT. In addition, PACT-VP35 interaction impairs the association between VP35 and the viral polymerase, thereby diminishing viral RNA synthesis and modulating EBOV replication. PACT-deficient cells are defective in IFN induction and are insensitive to VP35 function. These data support a model in which the VP35-PACT interaction is mutually antagonistic and plays a fundamental role in determining the outcome of EBOV infection

Structural and biochemical characterization of marburgvirus VP35 and its role in immune evasion

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.</p

Structural mechanisms of DREAM complex assembly and regulation.

The DREAM complex represses cell cycle genes during quiescence through scaffolding MuvB proteins with E2F4/5 and the Rb tumor suppressor paralog p107 or p130. Upon cell cycle entry, MuvB dissociates from p107/p130 and recruits B-Myb and FoxM1 for up-regulating mitotic gene expression. To understand the biochemical mechanisms underpinning DREAM function and regulation, we investigated the structural basis for DREAM assembly. We identified a sequence in the MuvB component LIN52 that binds directly to the pocket domains of p107 and p130 when phosphorylated on the DYRK1A kinase site S28. A crystal structure of the LIN52-p107 complex reveals that LIN52 uses a suboptimal LxSxExL sequence together with the phosphate at nearby S28 to bind the LxCxE cleft of the pocket domain with high affinity. The structure explains the specificity for p107/p130 over Rb in the DREAM complex and how the complex is disrupted by viral oncoproteins. Based on insights from the structure, we addressed how DREAM is disassembled upon cell cycle entry. We found that p130 and B-Myb can both bind the core MuvB complex simultaneously but that cyclin-dependent kinase phosphorylation of p130 weakens its association. Together, our data inform a novel target interface for studying MuvB and p130 function and the design of inhibitors that prevent tumor escape in quiescence

Filoviral Immune Evasion Mechanisms

The Filoviridae family of viruses, which includes the genera Ebolavirus (EBOV) and Marburgvirus (MARV), causes severe and often times lethal hemorrhagic fever in humans. Filoviral infections are associated with ineffective innate antiviral responses as a result of virally encoded immune antagonists, which render the host incapable of mounting effective innate or adaptive immune responses. The Type I interferon (IFN) response is critical for establishing an antiviral state in the host cell and subsequent activation of the adaptive immune responses. Several filoviral encoded components target Type I IFN responses, and this innate immune suppression is important for viral replication and pathogenesis. For example, EBOV VP35 inhibits the phosphorylation of IRF-3/7 by the TBK-1/IKKε kinases in addition to sequestering viral RNA from detection by RIG-I like receptors. MARV VP40 inhibits STAT1/2 phosphorylation by inhibiting the JAK family kinases. EBOV VP24 inhibits nuclear translocation of activated STAT1 by karyopherin-α. The examples also represent distinct mechanisms utilized by filoviral proteins in order to counter immune responses, which results in limited IFN-α/β production and downstream signaling

The MuvB complex binds and stabilizes nucleosomes downstream of the transcription start site of cell-cycle dependent genes.

The chromatin architecture in promoters is thought to regulate gene expression, but it remains uncertain how most transcription factors (TFs) impact nucleosome position. The MuvB TF complex regulates cell-cycle dependent gene-expression and is critical for differentiation and proliferation during development and cancer. MuvB can both positively and negatively regulate expression, but the structure of MuvB and its biochemical function are poorly understood. Here we determine the overall architecture of MuvB assembly and the crystal structure of a subcomplex critical for MuvB function in gene repression. We find that the MuvB subunits LIN9 and LIN37 function as scaffolding proteins that arrange the other subunits LIN52, LIN54 and RBAP48 for TF, DNA, and histone binding, respectively. Biochemical and structural data demonstrate that MuvB binds nucleosomes through an interface that is distinct from LIN54-DNA consensus site recognition and that MuvB increases nucleosome occupancy in a reconstituted promoter. We find in arrested cells that MuvB primarily associates with a tightly positioned +1 nucleosome near the transcription start site (TSS) of MuvB-regulated genes. These results support a model that MuvB binds and stabilizes nucleosomes just downstream of the TSS on its target promoters to repress gene expression

Mutations Abrogating VP35 Interaction with Double-Stranded RNA Render Ebola Virus Avirulent in Guinea Pigs ▿

Ebola virus (EBOV) protein VP35 is a double-stranded RNA (dsRNA) binding inhibitor of host interferon (IFN)-α/β responses that also functions as a viral polymerase cofactor. Recent structural studies identified key features, including a central basic patch, required for VP35 dsRNA binding activity. To address the functional significance of these VP35 structural features for EBOV replication and pathogenesis, two point mutations, K319A/R322A, that abrogate VP35 dsRNA binding activity and severely impair its suppression of IFN-α/β production were identified. Solution nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography reveal minimal structural perturbations in the K319A/R322A VP35 double mutant and suggest that loss of basic charge leads to altered function. Recombinant EBOVs encoding the mutant VP35 exhibit, relative to wild-type VP35 viruses, minimal growth attenuation in IFN-defective Vero cells but severe impairment in IFN-competent cells. In guinea pigs, the VP35 mutant virus revealed a complete loss of virulence. Strikingly, the VP35 mutant virus effectively immunized animals against subsequent wild-type EBOV challenge. These in vivo studies, using recombinant EBOV viruses, combined with the accompanying biochemical and structural analyses directly correlate VP35 dsRNA binding and IFN inhibition functions with viral pathogenesis. Moreover, these studies provide a framework for the development of antivirals targeting this critical EBOV virulence factor