The Ebola virus VP35 protein binds viral immunostimulatory and host RNAs identified through deep sequencing

<div><p>Ebola virus and Marburg virus are members of the <i>Filovirdae</i> family and causative agents of hemorrhagic fever with high fatality rates in humans. Filovirus virulence is partially attributed to the VP35 protein, a well-characterized inhibitor of the RIG-I-like receptor pathway that triggers the antiviral interferon (IFN) response. Prior work demonstrates the ability of VP35 to block potent RIG-I activators, such as Sendai virus (SeV), and this IFN-antagonist activity is directly correlated with its ability to bind RNA. Several structural studies demonstrate that VP35 binds short synthetic dsRNAs; yet, there are no data that identify viral immunostimulatory RNAs (isRNA) or host RNAs bound to VP35 in cells. Utilizing a SeV infection model, we demonstrate that both viral isRNA and host RNAs are bound to Ebola and Marburg VP35s in cells. By deep sequencing the purified VP35-bound RNA, we identified the SeV copy-back defective interfering (DI) RNA, previously identified as a robust RIG-I activator, as the isRNA bound by multiple filovirus VP35 proteins, including the VP35 protein from the West African outbreak strain (Makona EBOV). Moreover, RNAs isolated from a VP35 RNA-binding mutant were not immunostimulatory and did not include the SeV DI RNA. Strikingly, an analysis of host RNAs bound by wild-type, but not mutant, VP35 revealed that select host RNAs are preferentially bound by VP35 in cell culture. Taken together, these data support a model in which VP35 sequesters isRNA in virus-infected cells to avert RIG-I like receptor (RLR) activation.</p></div

VP35 proteins from Marburg virus and all five <i>Ebolavirus</i> species antagonize SeV induced promoter activity.

<p>The abilities of the VP35 proteins from Marburgvirus and the five species of Ebolavirus to antagonize the IFN response induced by virus infection were compared in the context of a luciferase reporter under the control of the IFN-β promoter. (A) 293T cells were transfected with increasing amounts (4 ng, 20 ng, or 100 ng) of pCAGGS-based plasmids expressing N-terminally FLAG-tagged filoviral proteins, an empty pCAGGS plasmid to transfect equal amounts between samples, and a plasmid expressing Renilla luciferase as a transfection control. The following day, cells were either mock-infected or infected with SeV to induce IFN-β promoter activity. The third day, cells were harvested and luciferase expression was measured. Error bars represent standard error of the mean of triplicates. MARV, Marburg Virus; EBOV, Ebola Virus (Mayinga); SUDV, Sudan Virus; BDBV, Bundibugyo Virus; RESTV, Reston Virus; TAFV, Taï Forest Virus. (B) Western blot analysis against the FLAG tag shows relative expression of filoviral proteins when 100 ng of each FLAG-tagged protein-expressing plasmid was transfected.</p

An analysis of host RNAs highlights transcripts bound by VP35.

<p>(A) Total number of sequencing reads from triplicate samples of wildtype and mutant VP35 proteins infected in the SeV infected groups. The pie charts depict the percentage of reads that map to the SeV DI genome and the percentage of reads that do not map to the SeV DI. (B) Multidimensional scaling (MDS) plot of triplicate samples from wild-type VP35 (black squares), wild-type VP35 infected with SeV (black circles), mutant VP35 (black triangle) and mutant VP35 infected with SeV (black diamond). Axes in the MDS plot (Leading logFC dim1 and Leading logFC dim2) are arbitrary, and the values on the axes are distance units. (C) Heat map showing the binding of the wild-type and mutant VP35 protein to human mRNA transcripts for all samples included in the analysis. Each row represents an experimental replicate, and each column represents a single transcript. Colors indicate relative abundance for each gene, where orange is low abundance and white is high abundance. (D) Sorting of the 62 most statistically significantly enriched mRNAs associated with VP35 (from the mock-infected wild-type VP35 samples). Y-axis denotes the p-value of each sample and X-axis denotes fold-change of transcript abundance between wild-type and mutant VP35.</p

Mutations in VP35 important for dsRNA binding abrogate its ability to bind the immunostimulatory SeV DI.

<p>(A) Schematic of the EBOV VP35 protein containing a coiled-coil domain important for oligomerization in the N-terminal half of the protein and dsRNA-binding domain in the C-terminal half. Residues K309 and R312 were mutated to alanine to generate the EBOV VP35 RNA-binding mutant. (B) Protein staining of immunoprecipitated FLAG-tagged wild-type and mutant VP35 from which the RNA transfected in (C) and sequenced in (D) were recovered. (C) Immunostimulatory activity of RNA following immunoprecipitation of the pCAGGS empty vector (EV), wild-type EBOV VP35, and mutant EBOV VP35 in cells infected with SeV or mock-infected. (D) Next-generation sequencing and read mapping to the SeV genome. RNA associated with the pCAGGS empty vector, wild-type EBOV VP35, and mutant EBOV VP35 was purified and subjected to Illumina sequencing and the resulting reads were mapped to the SeV genome. The graph depicts nucleotide coverage (Y-axis) at each position of the SeV genome (X-axis).</p