Crystal Structure of the Marburg Virus VP35 Oligomerization Domain

Abstract

Marburg virus (MARV) is a highly pathogenic filovirus that is classified in a genus distinct from that of Ebola virus (EBOV) (genera Marburgvirus and Ebolavirus, respectively). Both viruses produce a multifunctional protein termed VP35, which acts as a polymerase cofactor, a viral protein chaperone, and an antagonist of the innate immune response. VP35 contains a central oligomerization domain with a predicted coiled-coil motif. This domain has been shown to be essential for RNA polymerase function. Here we present crystal structures of the MARV VP35 oligomerization domain. These structures and accompanying biophysical characterization suggest that MARV VP35 is a trimer. In contrast, EBOV VP35 is likely a tetramer in solution. Differences in the oligomeric state of this protein may explain mechanistic differences in replication and immune evasion observed for MARV and EBOV.IMPORTANCE Marburg virus can cause severe disease, with up to 90% human lethality. Its genome is concise, only producing seven proteins. One of the proteins, VP35, is essential for replication of the viral genome and for evasion of host immune responses. VP35 oligomerizes (self-assembles) in order to function, yet the structure by which it assembles has not been visualized. Here we present two crystal structures of this oligomerization domain. In both structures, three copies of VP35 twist about each other to form a coiled coil. This trimeric assembly is in contrast to tetrameric predictions for VP35 of Ebola virus and to known structures of homologous proteins in the measles, mumps, and Nipah viruses. Distinct oligomeric states of the Marburg and Ebola virus VP35 proteins may explain differences between them in polymerase function and immune evasion. These findings may provide a more accurate understanding of the mechanisms governing VP35's functions and inform the design of therapeutics. KEYWORDS VP35, filovirus, RNA-dependent RNA polymerase, phosphoprotein, coiled coil, oligomerization, Marburg virus, Ebola virus, X-ray crystallography M arburg virus (MARV) can cause severe hemorrhagic fever in humans with high case fatality rates. Though less well known than its relative Ebola virus (EBOV), MARV was the first filovirus identified. MARV has caused sporadic outbreaks since its identification in 1968, including the 1998 to 2000 outbreak in the Democratic Republic of the Congo, which occurred with 83% lethality, and the 2004 to 2005 outbreak in Angola, which occurred with 90% lethality (1). Currently, there are no approved vaccines or therapeutics available to treat individuals infected with MARV. Despite sharing ϳ50% nucleotide sequence identity, MARV and EBOV have several striking functional differences. MARV does not produce sGP or ssGP protein and does not require VP30 for transcription. MARV and EBOV also exhibit differences in their immune evasion strategies (2).

Cryo-EM structure of the respiratory syncytial virus RNA polymerase

“…P is the most variable protein and shares the lowest sequence identities among NNS RNA viruses. Strikingly, despite the low sequence conservation, all P (or VP35 in Filoviridae) share a common feature and exist as an oligomer (dimer, trimer, or tetramer) in solution [53][54][55][56][57][58][59][60] ( Fig. 3g and Supplementary Fig.…”

“…Further investigations are required to discover whether the RSV L alone exhibits the same way as VSV L. Interestingly, all RNA polymerases of NNS RNA viruses require P (or VP35 in Filoviridae), but P exists as diverse oligomeric protein ( Supplementary Fig. 9) and shares low sequence identity [53][54][55][56][57][58][59][60] . In most cases, the protomers, the basic building blocks of oligomeric proteins, assemble into a higher-order oligomer following a defined repeating or symmetry rule.…”

Conservation of Structure and Immune Antagonist Functions of Filoviral VP35 Homologs Present in Microbat Genomes

“…A co-immunoprecipitation assay demonstrated that FLAG-tagged batVP35 can co-precipitate with hemagglutinin (HA)-tagged batVP35, consistent with oligomerization (Figure 6A). Both eVP35 and mVP35 form tetramers, as demonstrated by light scattering analyses, although work has described the crystal structure of the N terminus of mVP35 forming a trimer instead (Bruhn et al, 2017; Edwards et al, 2016). To determine the oligomeric state of batVP35, we used multi-angle light scattering coupled to size exclusion chromatography and found that it also forms a tetramer (Figure 6B).…”

Multi-pass transmission electron microscopy

“…Multi-pass TEM simulations. In the following we show multi-pass TEM simulations of three model systems of known structure: graphene 33,34 , the hexameric unit of the immature HIV-1 Gag CTD-SP1 lattice (HIV-1 Gag, PDB ID: 5I4T) 35 , and the Marburg Virus 7 VP35 Oligomerization Domain P4222 (MARV VP35, PDB ID: 5TOI) 36 . In the simulations (see methods for details) an electron wave passes through the sample of an aberration-free multi-pass TEM multiple times.…”