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, 27, 2040206619889220

Relevance of Ebola Virus VP35 Homo-Dimerization on the Type I Interferon Cascade Inhibition

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Relevance of Ebola Virus VP35 Homo-Dimerization on the Type I Interferon Cascade Inhibition

Francesco Di Palma et al. Antivir Chem Chemother.

Abstract

Ebola virus high lethality relies on its ability to efficiently bypass the host innate antiviral response, which senses the viral dsRNA through the RIG-I receptor and induces type I interferon α/β production. In the bypassing action, the Ebola virus protein VP35 plays a pivotal role at multiple levels of the RIG-I cascade, masking the viral 5′-triphosphorylated dsRNA from RIG-I, and interacting with other cascade components. The VP35 type I interferon inhibition is exerted by the C-terminal domain, while the N-terminal domain, containing a coiled-coil region, is primarily required for oligomerization. However, mutations at key VP35 residues L90/93/107A (VP35-3m) in the coiled-coil region were reported to affect oligomerization and reduce type I interferon antagonism, indicating a possible but unclear role of homo-oligomerization on VP35 interaction with the RIG-I pathway components. In this work, we investigated the VP35 dimerization thermodynamics and its contribution to type I interferon antagonism by computational and biological methods. Focusing on the coiled-coil region, we combined coarse-grained and all-atom simulations on wild type VP35 and VP35-3m homo-dimerization. According to our results, wild type VP35 coiled-coil is able to self-assemble into dimers, while VP35-3m coiled-coil shows poor propensity to even dimerize. Free-energy calculations confirmed the key role of L90, L93 and L107 in stabilizing the coiled-coil homo-dimeric structure. In vitro type I interferon antagonism studies, using full-length wild type VP35 and VP35-3m, revealed that VP35 homo-dimerization is an essential preliminary step for dsRNA binding, which appears to be the main factor of the VP35 RIG-I cascade inhibition, while it is not essential to block the other steps.

Keywords: Filoviridae; Models/projections; mutations.

Figures

Figure 1.
Figure 1.
EBOV VP35 overview. (a) Sequence position and corresponding known function(s) of the protein domains: the NP-chaperoning domain (navy), the homo-olimerization domain (pale red) and the IID (green); the amino acid numbering, the N- and C-terminals are noted. (b) CC region model super-imposition of Zaire EBOV VP35-wt and VP35-3m. Structures (image pair with a stereo angle of 90°) are shown as cartoon colored by residue type (non-polar, polar, basic and acidic side chains, respectively, in white, green, blue and red) and as surface representation. The mutated amino acids (90, 93 and 107) are highlighted in cyan (VP35-wt) and magenta (VP35-3m) licorice. The two sequences are aligned along the structure with the associated CC heptad-register.
Figure 2.
Figure 2.
Final conformations for VP35-wt (top) and VP35-3m (bottom) variants from 12 independent replicas of CG simulations. Dimers resulting in a proper CC configuration are shown in green boxes, improper CC and uncoiled dimers are in yellow boxes, disordered/anti-parallel dimers are in red boxes. The two monomers are shown in red and blue beads with the mutated residues in cyan and pink, respectively; yellow terminal prolines give the relative monomer orientation (parallel/anti-parallel).
Figure 3.
Figure 3.
Coiled-coil timeline for the CG (a) and AA (b) simulations. (a) Twelve replicas for VP35-wt (top) and VP35-3m (bottom) variants (numbered on the left side of the box) of CG simulations. (b) Seven AA replicas for both variants (VP35-wt top, VP35-3m bottom). The occurrence of the CC conformation over time is evaluated by means of SOCKET. The time-percentage is indicated on the right border of the box. In the presence of CC fraying of the terminal three residues at the 5′- and/or 3′-end, the bars are pale-colored.
Figure 4.
Figure 4.
Representative final structures from the AA MD simulations. The systems, VP35-wt (left) in perfect CC conformation and VP35-3m (right) as disordered oligomer, are shown in cartoon representation with the three mutated residues highlighted as licorice.
Figure 5.
Figure 5.
Per-residue-based free-energies for VP35-wt complexes. The residues contributing to the total free-energy with at least 3.0 kcal/mol in at least one replica are included. The value resulting from the weighted mean and the associated standard deviation of the five replicas are reported emphasizing in red the contributions from leucine 90, 93 and 107.
Figure 6.
Figure 6.
Biochemical assays. (a) Comparison between rVP35-wt (red column) and rVP35-3m (gray column) ability to bind to dsRNA, performed with a nickel-coated plate assay; 700 ng/well of rVP35 or vVP35 3 m were incubated with 7.5 nM of 30 bp 5′-fluorescein-dsRNA and incubate for 60′. Unbound dsRNA was removed and fluorescence signals of samples were read. All experiments were repeated three times in duplicate. The bound dsRNA has been quantified (in femtomoles) interpolating the obtained values in calibration curve. (b) Comparison of the inhibitory effect of EBOV VP35-wt and VP35-3m in the luciferase reporter gene assay. A549 cells were co-transfected with pGL interferon β (IFN-β) luc plus different amounts of the pcDNA3-ZEBOV-VP35wt (red column) or pcDNA3-ZEBOV-VP353m (gray column), using pcDNA3 as empty vector control (EV) (black column). Twenty-four hours after transfection, cells were additionally transfected with influenza A virus (IAV) RNA. Six hours after transfection, cells were lysed and luciferase activity was measured. Results show the percentage of luciferase expression over the unstimulated control. Significant at: p-value < 0.05 (*); p-value <0.01 (**).

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