CORDIS - Forschungsergebnisse der EU

Elucidating how XRN1-mediated RNA degradation controls virus infection

Periodic Reporting for period 1 - RNAdeg-Virus (Elucidating how XRN1-mediated RNA degradation controls virus infection)

Berichtszeitraum: 2020-05-01 bis 2022-04-30

Every living organism is in a constant and fierce battle with viruses, which are obligate intracellular pathogens that require the cellular resources of the host to replicate and generate new viral progeny. These sub-living entities constitute a major threat for health and a burden for countries’ economies.
Arboviruses are transmitted to human by arthropods and represent one of the most prominent biomedically relevant group of viruses. Due to global warming and increased urbanization, arthropods habitats are expanding and arboviruses are reaching non-endemic areas causing new epidemies.

Viruses have been intensively studied since many years to understand the origin of their existence, their biological cycles, and the consequences of their infection. Due to the potential threat caused by these entities it is critical to understand the replication steps of these viruses in order to develop more effective therapies, since relatively few therapeutic options are available and only for a limited scope of viruses.

Since viruses only possess very limited genetic instructions on their own, they strongly rely on cellular resources and tools, thus it bears extreme importance to study the virus-host interaction. RNA binding proteins (RBPs) mediate numerous critical steps for the codification of the genetic information. In the past few years RBPs emerged as central regulators of infection, controlling virtually every step of the viral cycle, and it is essential to deeply understand their interconnection with viruses.

To investigate the virus-host interaction and gain a better understanding of the viral replication, I employed Sindbis virus (SINV), an arthropod-borne virus transmitted to human through the bite of mosquitoes, as a discovery model.

In a recent scientific breakthrough, it has been highlighted that SINV requires the presence of a host RBP called 5’-3’ exoribonuclease 1 (XRN1), a master regulator of gene expression, in order to infect cells. Also, SINV infection causes a global downregulation of gene expression concomitant with a pervasive degradation of cellular RNA.

With RNAdeg-Virus project, I aim to understand the molecular mechanism of SINV infection regulated by XRN1.
(i) I set to understand if XRN1 is the driving force rewiring the transcriptome of a cell, (ii) what is the trigger for XRN1 activity, and (iii) the biological significance of its role during infection.

By the use of a multidisciplinary approach I am clarifying the molecular mechanism of XRN1 regulation of SINV infection, and also generating numerous datasets that will spark the creativity of several future scientific projects. The knowledge generated by this work will be the stepping stone for future discoveries which will improve human health and have a strong impact on society. In addition, this project brought us a step closer to the development of specific antiviral drugs potentially acting on a broad range of viruses.
To assess the role of XRN1 in SINV infection I have depleted this enzyme from cells, via CRISPR/Cas9 system, generating XRN1-KO cell lines, and confirmed the requirement of XRN1 for SINV infection via near real-time measurements of viral gene expression with a fluorescent-based plate reader method. Since XRN1 is an exoribonuclease enzyme which degrades cellular RNA, and SINV infection causes a pervasive downregulation of the majority of cellular RNA, one of the main objective aimed to understand whether the catalytic exonuclease activity of XRN1 is important for viral infection.
To answer this question I have generated rescue cell lines, where I complemented the XRN1-KO cell line previously generated with the addition of inducible expressed XRN1-WT, XRN1 catalytic mutant. With these experiments I could show not only that XRN1-WT restores SINV infection, confirming a direct role of this enzyme, but also that the catalytic activity is important for sustaining infection. Microscopy experiments revealed that XRN1 colocalises with SINV non-structural protein 3 and non-structural protein 1, which are part of the viral replication complex, and with viral RNA, suggesting a direct recruitment of XRN1 to viral factories, areas of the cells where the virus replicates.
Interestingly, XRN1 does not regulate infection only of SINV but is also required for the replication of Ross river virus and Semliki forest virus, suggesting a conserved mechanism of action of this enzyme in the viral cycles of alphaviruses.
RNA sequencing experiments further recapitulate the importance of XRN1 in SINV infection, as in the absence of this enzyme the characteristic degradation of cellular transcripts does not occur, supporting the hypothesis that XRN1 is important to eliminate competitor transcripts and provide free nucleotides and ribosomes to support the robust viral replication.

This project bring us a step closer to the understanding of the complete SINV replication cycle, however further experiments are required to pinpoint the details of XRN1-mediated regulation of alphaviruses infection.
These results will set the basis for new scientific projects aiming to understand the role of XRN1 and the RNA degradation machinery in viral infection, with the long term goal to test new antiviral drugs which target this important cellular pathway. This project has been selected for a talk at the RNA UK 2022 conference. In addition, this project has been presented in different events such as the interdepartmental postdoc poster session at the University of Oxford. The resulting manuscripts will be published in peer-reviewed open access journals and the datasets generated by this project will be uploaded to public repository to allow the re-analysis by the whole scientific community.
RNAdeg-Virus employs next generation sequencing and data analysis combined with virology and RNA and protein biochemistry to study the role of a cellular enzyme in the regulation of SINV infection. The datasets and the molecular details generated by this study will be crucial for the design of follow up projects, because bring in the scientific field unprecedently reached viral infection details. Such in-deep knowledge are pre-requisites for the design of new antiviral drugs, since it has the potential to amplify the pool of the so far known players involved in infection. Moreover, since this process seems to be conserved among alphaviruses, the new drugs might have multi-viral targets acting on a large spectrum of infectious agents.
The work obtained here on model viruses will be expanded to other more pathological viruses, potentially greatly impacting our society and improving human life. This work pioneer the understanding of the steps required for viral replication. In details, this project provides (i) in-depth knowledge of SINV infection mechanism, (ii) offering new insights into the multiple players which sustain viral replication, (iii) it fuels creativity toward the design of follow-up projects and (iv) presents a list of candidates for antiviral targets. Moreover, this project triggered various international scientific collaborations between outstanding scientific groups, ensuring the success of this work and enhancing European excellence and world-class research.
Schematic representation of Sindbis replication cycle in human cell lines