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

Genetic stability of foot-and-mouth disease virus during long-term infections in natural hosts

<div><p>Foot-and-mouth disease (FMD) is a severe infection caused by a picornavirus that affects livestock and wildlife. Persistence in ruminants is a well-documented feature of Foot-and-mouth disease virus (FMDV) pathogenesis and a major concern for disease control. Persistently infected animals harbor virus for extended periods, providing a unique opportunity to study within-host virus evolution. This study investigated the genetic dynamics of FMDV during persistent infections of naturally infected Asian buffalo. Using next-generation sequencing (NGS) we obtained 21 near complete FMDV genome sequences from 12 sub-clinically infected buffalo over a period of one year. Four animals yielded only one virus isolate and one yielded two isolates of different serotype suggesting a serial infection. Seven persistently infected animals yielded more than one virus of the same serotype showing a long-term intra-host viral genetic divergence at the consensus level of less than 2.5%. Quasi-species analysis showed few nucleotide variants and non-synonymous substitutions of progeny virus despite intra-host persistence of up to 152 days. Phylogenetic analyses of serotype Asia-1 VP1 sequences clustered all viruses from persistent animals with Group VII viruses circulating in Pakistan in 2011, but distinct from those circulating on 2008–2009. Furthermore, signature amino acid (aa) substitutions were found in the antigenically relevant VP1 of persistent viruses compared with viruses from 2008–2009. Intra-host purifying selective pressure was observed, with few codons in structural proteins undergoing positive selection. However, FMD persistent viruses did not show a clear pattern of antigenic selection. Our findings provide insight into the evolutionary dynamics of FMDV populations within naturally occurring subclinical and persistent infections that may have implications to vaccination strategies in the region.</p></div

The 5' UTRs of EBOV do not function as internal ribosome entry sites.

<p><b>A</b>. Diagram of the bicistronic reporter constructs used. FF Luc, firefly luciferase; Ren Luc, <i>Renilla</i> luciferase; MCS, multiple cloning site; EMCV IRES, encephalomyocarditis virus internal ribosome entry site; EBOV UTR, EBOV-derived 5'-untranslated region <b>B</b>. Normalized data from transfected 293T cells indicating the ratio of <i>Renilla</i> luciferase to firefly luciferase for each bicistronic reporter construct. Each bar is the mean of three samples. Data is representative of three independent experiments.</p

Suppression of pORF translation by the uAUG in the L 5′-UTR is position dependent.

<p><b>A</b>. Diagram depicting wild-type and mutated L 5′-UTR-GFP reporter constructs in which the location of the uAUG was altered. For the L uORF, the black box represents the L uORF sequence, while the gray box depicts the remainder of the overlapping uORF that consists of GFP-derived sequences. Nucleotide changes that differ from the wildtype L 5′-UTR sequence are indicated in bold and the underlined sequences highlight the uORFs. Additional mutations were introduced to preserve the Kozak sequence at the −3 and +4 position such that they match that present in the wildtype L 5′-UTR. The reading frame of the uORF for each construct is indicated on the right. <b>B</b>. Equal amounts of in vitro transcribed mRNAs corresponding to the constructs depicted in panel A were transfected into 293T cells. At 2.5 hours post transfection, cells were harvested and analyzed by flow cytometry for GFP fluorescence. Each bar represents the mean of triplicate samples, and all values were calculated relative to the B-actin 5′-UTR GFP control mRNA which was set to 100%. <b>C</b>. A real time PCR measurement of GFP mRNA levels present in the transfected cells described in B was determined for each sample.</p

The L 5′-UTR uAUG suppresses the translation of the first 505 amino acids of L.

<p><b>A</b>. Schematic of the expression constructs used, where the L 5′-UTR is in its natural context, upstream of sequences encoding the first 505 amino acids of L and is FLAG-tagged at its C-terminus. The length of the 5′-UTR, the L uORF and the L pORF in nucleotides is indicated. <b>B</b>. Western blot analysis indicating that ablating the uAUG in the 5′-UTR of L enhances L (amino acids 1–505) protein expression. Left. Each L construct was coexpressed with GFP-FLAG. Right. Each L construct was coexpressed with VP35-FLAG, which enhances L expression.</p

pORF translation is suppressed by a strong uAUG Kozak sequence in the L 5′-UTR, but is not affected by a weak uAUG Kozak sequence.

<p><b>A</b>. Diagram depicting the in vitro generated L 5′-UTR GFP mRNA reporter constructs with permutations surrounding the L uAUG. For the L uORF, the black box represents the L uORF sequence, while the gray box depicts the remainder of the overlapping uORF that consists of GFP-derived sequences. Each sequence surrounding the uAUG above the diagram represents a reporter construct. The top is the wildtype nucleotide sequence surrounding the L uAUG (WT L 5′-UTR). The next construct has a strong Kozak sequence (A at the −3 position and a G at the +4 position, where the A of the AUG is designated as +1). Two constructs predicted to have a weak Kozak sequence (uAUG WK1 and WK2) have mutations at the +4 and +4/+5 positions, respectively. The construct labeled “uAUG STOP” has a point mutation that incorporates a stop codon directly after the uAUG start codon. Finally, uUUG and uUCG both ablate the uAUG start codon. Each construct is analyzed in panel B. <b>B</b>. Equal amounts of each in vitro transcribed mRNA were transfected into 293T cells. At 2.5 hours post transfection, cells were harvested and analyzed by flow cytometry. Each bar represents the mean and standard deviation of triplicate samples, and all values were calculated relative to the B-actin 5′-UTR GFP control mRNA, which was set to 100%. <b>C</b>. A real time PCR measurement of GFP mRNA levels present in the transfected cells described in B was determined for each sample.</p

Efficiency of translation initiation at L uAUG is determined by its sequence context.

<p><b>A</b>. L 5′-UTR GFP mRNA reporter constructs were generated such that the uORF is fused in frame to GFP. The nucleotide sequence immediately surrounding the uAUG of each construct is displayed. The first construct (uORF-GFP) includes the first 46 nucleotides of the L 5′-UTR up to the uAUG followed by the entire L uORF sequence placed in frame with the GFP ORF (labeled uORF-GFP). The middle construct (uORF-GFP SK) is identical to the first, but the uAUG is surrounded by a strong Kozak sequence (A at the −3 position and a G at the +4 position, where the A of the AUG is designated as +1). The bottom construct includes the L 5′-UTR, through the uAUG and the second codon of the uORF which was placed in frame with the GFP ORF, but lacks the rest of the uORF. In each case, the start codon for GFP was removed. The number of nucleotides in each construct is indicated and the features of each construct are summarized in the box above the diagram. <b>B</b>. Equal amounts of each in vitro transcribed mRNA were transfected into 293T cells. At 2.5 hours post transfection, cells were harvested and analyzed by flow cytometry. The experiment was performed in triplicate, and a representative sample is displayed for each group. <b>C</b>. GFP mRNA levels, as determined by real time PCR, present in the transfected cells described in B were determined for each sample.</p

The L uAUG modulates L translation and maintains EBOV replication in response to eIF2α phosphorylation in A549 cells.

<p><b>A</b>. Diagram of the EBOV L 5′-UTR firefly luciferase fusion reporter construct with the EBOV L 5′-UTR upstream of L (amino acids 1–13) in frame with firefly luciferase (L-FF), a construct with a strong Kozak sequence (A at the −3 position and G at the +4) surrounding the uAUG (Lsk-FF) and a construct lacking the uAUG codon (Lns-FF). <b>B</b>. A549 cells were transfected with pRLTK and either L-FF, Lns-FF or Lsk-FF. An established stress reporter, the ATF4 5′-UTR upstream of firefly luciferase, was separately transfected and serves as a control to monitor activation of a stress response. At 24 hpt, cells were untreated (U), treated with DMSO (D) or with three doses of Thapsigargin (TG) and harvested at 6.5 hours post treatment. The luciferase ratio of untreated cells transfected with L-FF was normalized to 1, and this value was also used to normalize the values in the Lsk-FF and Lns-FF transfected cells. The untreated ATF4 transfected sample was also set to 1. <b>C</b>. Effect of TG on wt and 5′-UTR mutant virus replication. A549 cells were infected with either WT EBOV or the L 5′-UTR mutant virus an MOI of 0.1, followed by TG treatment 4 hours post infection. Twenty four hours post-infection, infectious virus present in the cell supernatants was quantified by TCID50 assay. <b>D</b>. TCID50 values of both viruses treated with either DMSO or with TG at 24 hours post-infection. <b>E</b>. Model proposing how eIF2α phosphorylation modulates translation initiation at the L uAUG versus the primary AUG in the L mRNA. During low stress conditions, translation initiation is efficient, resulting in more ribosomes initiating at the L uORF. During times of high stress, translation initiation is inefficient, resulting in a ribosome scanning past the uAUG and initiating at the pAUG to maintain L translation.</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

Bayesian phylogenetic analysis of the VP1 nucleotide sequence of Asia-1 isolates from Pakistan and related published regional sequences.

<p>Posterior probability values are color coded.</p