Structural basis for Marburg virus VP35–mediated immune evasion mechanisms

Abstract

Filoviruses, marburgvirus (MARV) and ebolavirus (EBOV), are causative agents of highly lethal hemorrhagic fever in humans. MARV and EBOV share a common genome organization but show important differences in replication complex formation, cell entry, host tropism, transcriptional regulation, and immune evasion. Multifunctional filoviral viral protein (VP) 35 proteins inhibit innate immune responses. Recent studies suggest double-stranded (ds)RNA sequestration is a potential mechanism that allows EBOV VP35 to antagonize retinoic-acid inducible gene-I (RIG-I) like receptors (RLRs) that are activated by viral pathogen-associated molecular patterns (PAMPs), such as double-strandedness and dsRNA blunt ends. Here, we show that MARV VP35 can inhibit IFN production at multiple steps in the signaling pathways downstream of RLRs. The crystal structure of MARV VP35 IID in complex with 18-bp dsRNA reveals that despite the similar protein fold as EBOV VP35 IID, MARV VP35 IID interacts with the dsRNA backbone and not with blunt ends. Functional studies show that MARV VP35 can inhibit dsRNA-dependent RLR activation and interferon (IFN) regulatory factor 3 (IRF3) phosphorylation by IFN kinases TRAF family member-associated NFkb activator (TANK) binding kinase-1 (TBK-1) and IFN kB kinase e (IKKe) in cell-based studies. We also show that MARV VP35 can only inhibit RIG-I and melanoma differentiation associated gene 5 (MDA5) activation by double strandedness of RNA PAMPs (coating backbone) but is unable to inhibit activation of RLRs by dsRNA blunt ends (end capping). In contrast, EBOV VP35 can inhibit activation by both PAMPs. Insights on differential PAMP recognition and inhibition of IFN induction by a similar filoviral VP35 fold, as shown here, reveal the structural and functional plasticity of a highly conserved virulence factor.T he Filoviridae family of viruses, which includes marburgvirus (MARV) and ebolavirus (EBOV), can cause intermittent outbreaks that often result in high fatality rates (1). The family consists of five species of EBOV, Zaire, Reston, Sudan, Taï Forest, and Bundibugyo; one species of MARV; and a proposed genus Cuevavirus possessing a single species Lloviu cuevavirus (2). Despite overall similarities in genome size and organization, virion structure, and disease characteristics (3), EBOV and MARV exhibit important differences, including their strategies for immune evasion (4). For example, although EBOV viral protein (VP) 24 and MARV VP40 counter IFN signaling, neither MARV VP24 nor EBOV VP40 appears to function similarly to its corresponding counterparts with regard to immune evasion (5-10).Filoviruses also counteract innate immunity through the multifunctional VP35 proteins, which perform critical roles in viral RNA synthesis, virus assembly, and virus structure (reviewed in refs. 11 and 12). EBOV VP35 interacts with several components of innate antiviral defenses, including retinoic-acid inducible gene-I (RIG-I)-like receptor (RLR) pathways that lead to IFN production (13-24). These include inhibition of ...

The Lack of Maturation of Ebola Virus-Infected Dendritic Cells Results from the Cooperative Effect of at Least Two Viral Domains

“…Third, Marburg virus, which is another virus of the family Filoviridae, is also known to infect DC without inducing their maturation (9). The mechanism by which Marburg virus evades innate immunity is different from that of EBOV and is associated with its VP40 protein (79), in addition to the VP35 protein (80). It will be important to know whether the virus suppresses DC maturation in a manner similar to that described here for EBOV.…”

Crystal Structure of the Marburg Virus VP35 Oligomerization Domain

“…Unexpectedly, the DXMS data indicate rapid exchange in the ultimate C terminus (residues 299 to 329) of MARV VP35 but not that of EBOV VP35. This is surprising, given that ordered density is observed in this region of all of the MARV CTD crystal structures solved to date, including RNA-bound and unbound structures (15,16).…”

“…Further, differences in the oligomeric states of the MARV and EBOV VP35 proteins may contribute to the observed variations in RNA binding preferences. EBOV VP35 binds RNA in a cooperative manner, utilizing an asymmetric dimer of CTDs to cap blunt-ended RNA termini (20,21), but MARV VP35 has not been shown to form these dimers in crystal structures (15,16). It is possible that a tetrameric VP35 is better adapted to form these CTD dimers because of the slightly increased avidity that would be facilitated by the higher oligomeric state of the overall protein.…”

“…By binding dsRNA, VP35 blocks viral detection by cellular immune sensors such as RIG-I. Several crystal structures of the CTD, alone and in complex with dsRNA, have been solved (14)(15)(16)(17)(18)(19)(20)(21). Though the folds of the CTDs of the Marburgvirus and Ebolavirus VP35 proteins are nearly identical, there are clear differences in the RNA-binding preferences of the two VP35 proteins.…”

VP24-Karyopherin Alpha Binding Affinities Differ between Ebolavirus Species, Influencing Interferon Inhibition and VP24 Stability

“…While the determinants of filovirus pathogenesis are incompletely defined, robust viral replication and modulation of the innate immune responses are important (14,28,29). For example, VP35 mutations that abrogate its inhibition of IFN production result in attenuation of the virus in vivo and in vitro (28,(30)(31)(32). In addition, adaptation of EBOV and Marburg virus (MARV) to animal models involves mutations within their respective IFN signaling antagonists, VP24 and VP40, to enhance virulence (15, 16, 19, 29, 33).…”