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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - StressEBOV (Ebola virus manipulation of the cellular stress responses)

Teaser

Summary

Ebola virus (EBOV) is a highly pathogenic filovirus causing severe haemorrhagic fever. It causes recurrent epidemics, like in 2014 in Western Africa where about 28,000 people were infected of which over 11,000 died or the current ongoing epidemic in Democratic Republic of Congo where so far 1926 people were infected of which 1287 died. Although potential vaccines and drugs are being used, no treatment has been approved. Therefore, understanding the molecular and cellular regulation of EBOV replication is fundamental to the development of novel treatments.
EBOV is a nonsegmented negative-strand (NNS) RNA virus encoding nine proteins. After entry, EBOV replicates in the cytoplasm of infected cells in inclusion bodies, non-membranous organelles that separate by density from the rest of the cytoplasm. However, the exact mechanism of EBOV transcription and replication is mostly assumed based on other NNS viruses.
Viruses replicate in host cells, hijacking the cell machinery as well as evading the antiviral response. Likewise, host cells have evolved mechanisms to sense the presence of foreign nucleic acids (e.g. vRNA). The synthesis of vRNAs triggers a cellular antiviral response via three distinct but linked mechanisms: (1) RNA-sensing by RIG-I and MDA5 leading to interferon (IFN) production, the host antiviral protein to activate innate cells, (2) RNA-sensing by the RNA-dependent protein kinase (PKR), inhibiting translation, leading to a host cell shut-off, and (3) formation of stress granules (SGs) to inhibit viral replication and store cellular mRNAs during stress to prevent them from degradation. However, the impact of SGs on EBOV still remains poorly understood.
SGs are non-membrane-bound cytosolic mRNA-protein granules that form in response to stress, such as viral infection. Stress can lead to the activation of PKR which phosphorylates eIF2, inhibiting translation and leading to sequestration of mRNAs. The assembly and disassembly of SGs is dynamic and modulates the cellular stress response as well as viral replication. Moreover, antiviral SGs have been reported to form in response to viral infection serving as amplification sites for antiviral IFN induction. Therefore, RNA viruses have evolved mechanisms to dysregulate SGs in three ways: (1) inhibition of SG formation by PKR, (2) cleavage of SG proteins or (3) sequestration of SG components. Recently, EBOV has been reported to partially inhibit the formation of SGs by expression of the viral VP35. In addition, EBOV recruits some SG-inducing proteins to its inclusion bodies, whereas redistributing others. However, the impact of the cellular stress response on EBOV replication and how EBOV and VP35 regulate this response is poorly defined.
Therefore, this proposal aims to investigate the anti-immunity and pro-replication functions of VP35 in the context of the polymerase complex and viral replication using a transcription- and replication-competent virus-like particle system (EBOV trVLP) by addressing the following aim:
To investigate the impact of SG proteins on EBOV transcription/replication and manipulation by VP35

Work performed

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.

Final results

The project involved the establishment of several protocols for analysis of EBOV replication and transcription in vitro. Moreover, the results of this project will inform future studies on EBOV by clarifying cellular processes during infection. In addition, the established protocols will be made available for a wider audience by publication in open access journals. Therefore, these protocols can be further employed to analyse the impact of EBOV on host cells and to further develop treatments for EBOV infection.