Marburg virus VP35 can both fully coat the backbone and cap the ends of dsRNA for interferon antagonism

Abstract

Filoviruses, including Marburg virus (MARV) and Ebola virus (EBOV), cause fatal hemorrhagic fever in humans and non-human primates. All filoviruses encode a unique multi-functional protein termed VP35. The C-terminal double-stranded (ds)RNA-binding domain (RBD) of VP35 has been implicated in interferon antagonism and immune evasion. Crystal structures of the VP35 RBD from two ebolaviruses have previously demonstrated that the viral protein caps the ends of dsRNA. However, it is not yet understood how the expanses of dsRNA backbone, between the ends, are masked from immune surveillance during filovirus infection. Here, we report the crystal structure of MARV VP35 RBD bound to dsRNA. In the crystal structure, molecules of dsRNA stack end-to-end to form a pseudo-continuous oligonucleotide. This oligonucleotide is continuously and completely coated along its sugar-phosphate backbone by the MARV VP35 RBD. Analysis of dsRNA binding by dot-blot and isothermal titration calorimetry reveals that multiple copies of MARV VP35 RBD can indeed bind the dsRNA sugar-phosphate backbone in a cooperative manner in solution. Further, MARV VP35 RBD can also cap the ends of the dsRNA in solution, although this arrangement was not captured in crystals. Together, these studies suggest that MARV VP35 can both coat the backbone and cap the ends, and that for MARV, coating of the dsRNA backbone may be an essential mechanism by which dsRNA is masked from backbone-sensing immune surveillance molecules.

Figure 1. Structure of the MARV VP35 dsRNA binding domain (RBD).

(A) Cartoon representation of the MARV VP35 RBD showing the α-helical and β-sheet subdomains. Basic residues in the central basic patch are shown in ball-and-stick representation and colored orange. (B) Electrostatic surface representation of MARV VP35 RBD with limits of ±3 k_BT/e_c showing the highly conserved central basic patch. Key residues on the patch are labeled. (C) Superposition of VP35 RBD molecules from MARV (pale green), EBOV (yellow) and RESTV (dark blue) showing a conservation of the fold and secondary structure.

Figure 2. Structure of MARV VP35 in complex with 12-bp dsRNA.

(A) Top view of the contents of the asymmetric unit. (B) Side view of the long pseudo-helical arrangement of dsRNA coated by MARV VP35 RBD, as formed by crystal packing. The four monomers in the asymmetric unit are colored pale blue, pale green, pale yellow and ruby, respectively.

Figure 3. Key interactions of MARV VP35 RBD with dsRNA.

dsRNA is shown in ball-and-stick representation. Hydrogen bonds are shown as black dotted lines. A bridging water molecule is shown as a red sphere. A representative MARV VP35 RBD is drawn in pale green.

Figure 4. Binding of wild-type and mutant MARV VP35 RBD to dsRNA.

(A) MARV VP35 binds with similar affinity to 18-bp dsRNA with blunt ends or with a 3′ or 5′- overhang. (B) Binding of R301A, K311A, K298A, R271A, and F228 mutants of MARV VP35 RBD to 18-bp blunt ended dsRNA. Only the wild-type VP35 and the K311A mutant bind to dsRNA.

Figure 5. Isothermal calorimetry.

Shown are raw data and isotherms for binding of MARV VP35 RBD to each of 18-bp blunt-ended dsRNA, 18-bp dsRNA with 3′ overhang and 12-bp blunt-ended dsRNA.

Figure 6. IFN antagonism by EBOV, MARV and RESTV VP35.

HEK-293T cells were infected with Sendai virus and IFNβ promoter activity was determined by firefly luciferase expression driven by the human IFNβ promoter sequence in the presence of various versions of VP35. Uninfected cells and infected cells transfected with an empty vector were used as controls (blue bars). The inability of the RBD to reconstitute full inhibition of IFN signaling is the reason we call this region comprising residues 205–329 (and equivalent in EBOV) the RNA-binding domain (RBD) rather than the IFN inhibitory domain.

Figure 7. Coating and end-capping of filovirus VP35 RBDs.

The MARV-dsRNA crystal structure (green) is superimposed with that of EBOV (brick red). In the MARV crystals, all copies of VP35 in the asymmetric unit exhibit backbone binding, while in the EBOV crystals, at each end of the dsRNA, one VP35 binds the backbone while the other caps the end. Any of the four copies of MARV VP35 in the asymmetric unit superimpose with the backbone-binding VP35s of EBOV. Residues in MARV equivalent to those of the EBOV backbone-binding VP35 that form the dimer interface with the EBOV end-capping VP35, do not interact with another MARV VP35, but instead interact with the dsRNA backbone. Biophysical experiments indicate that in solution, MARV VP35 can both backbone-bind and end-cap, so the differences depicted here may be differences between complexes captured in different crystal packing arrangements, rather than inherent differences between MARV and EBOV.