Aim 1: Identification of SG proteins that affect EBOV transcription and replication
To identify SG proteins that affect EBOV transcription and replication, I performed a targeted overexpression screen in an EBOV replication system. Herein, I could identify five candidate proteins, that inhibited EBOV propagation by at least 50%, without affecting the controls.
Aim 2: Validation and functional analysis of SG proteins during EBOV life cycle
To validate the results from the screen, the five chosen proteins plus their known orthologs, proteins that have similar sequences and functions in the cell, were further analysed by virus titration as well as plasmid titration. Herein, I could show that the inhibitory effect of the SG proteins could not be overcome by saturation of virus infection by increased viral input. The plasmid titrations showed that the antiviral effect of the candidate proteins was dose-dependent. Moreover, the experiments showed that the orthologs presented similar antiviral functions, suggesting similar modes of action.
Next, I performed quantitative PCR experiments to further map the inhibition by the identified candidates to the viral life cycle. All tested candidates presented similar inhibitory effects on transcription (mRNA) and replication (vRNA and cRNA) in transfected and infected cells, suggesting an early block in the viral life cycle. To further specify the analysis, I established protocols to specifically identify the affected viral RNA species. Interestingly, one candidate affected mRNA levels to about 50-fold compared to vRNA and cRNA levels being reduced by 10-fold.
To further characterize the identified candidates, I generated cell lines in which I depleted the candidate proteins using genome editing with CRISPR/Cas9. Herein, I could show that the knockout of two candidate proteins resulted in increased viral propagation compared to wildtype cells.
To further analyse the impact of the viral protein VP35 on EBOV replication and transcription, I generated a set of VP35 single amino acid mutants. These mutations have been chosen based on their described loss of function in replication and dsRNA binding. We tested these mutants and showed that all mutants are expressed to a similar level. Tested in the EBOV replication system, we could not detect any impairment of replication or transcription.
Aim 3: Analysis of SG formation and manipulation by VP35
To analyse the impact of the candidate proteins, I overexpressed the proteins and performed immunofluorescence analysis using microscopy in which I visualised SGs by using specific markers. Herein, I could show that overexpression of three of the candidates leads to formation of SGs. To further analyse the formation of SGs, I established a protocol for SG induction by transfecting polyIC, an artificial RNA species mimicking viral infection, to analyse the physiological pathway of SG induction in virus infection, namely through the activation of PKR. Herein, I could show that PKR knockout cells do not form SGs in response to polyIC, but are still able to form SGs in response to Sodium-arsenite, a stressor activating a different pathway for SG formation. I further analysed the impact of VP35 to inhibit SG formation in response to polyIC and could show that VP35, but not a mutant VP35 protein, counteracts polyIC-induced PKR activation and subsequent SG formation. Notably, this effect is dose-dependent on polyIC as well as VP35, suggesting that the incoming VP35 is necessary to counteract the early cellular antiviral response through PKR.
Therefore, we conclude that VP35 counteracts PKR early in infection to prevent formation of SGs and antiviral effects of SG proteins on viral replication and transcription.
