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"Alphavirus-specific mechanisms of OAS/RNAse L pathway inhibition, study of non-structural proteins and search for inhibitors."

Final Report Summary - ALPHA (Alphavirus-specific mechanisms of OAS/RNAse L pathway inhibition, study of non-structural proteins and search for inhibitors.)

Emerging and re-emerging Infectious Diseases (EIDs) are defined as "infections that have newly appeared in a population or have existed previously but are rapidly increasing in incidence or geographic range". Over the last decades, a number of new emerging diseases, such as HIV/AIDS and SARS, as well as re-emerging diseases that increase in incidence or whose geographic range is changing, such as chikungunya or dengue fever, continue to pose a significant threat to populations in the world. Some of these EID are caused by the Alphavirus which are emergent arthropod borne viruses with a worldwide distribution.
At the level of the infected cell, several defense mechanisms exist against the virus invader. One of the most established antiviral pathways induced by viral dsRNAs is the Interferon inducible 2'5' oligoadenylate synthetase (OAS) / endoribonuclease L (RNAse L) system. OAS is activated upon binding to dsRNA and generates, from ATP, short 2'-5' linked oligoadenylates (25A) which activate a latent RNAse L. RNAse L cleaves ribosomal and viral RNA, inducing apoptosis and blocking viral infection. In turn, viruses developed means to escape the host antiviral response. Studies on Ebola, HCV, Influenza, Vaccinia and HIV showed that specific viral proteins (nsPs) can inhibit RNAse L/OAS system through direct binding to one of these enzymes or through the sequestration of dsRNA, 25A or other cellular mediators. However, mechanisms involved in the anti-apoptotic action of several RNA viruses including Alphaviruses are unknown.
I will unravel the molecular aspects of the viral evasion produced via the inhibition of OAS/ RNAse L pathway by non-structural proteins of Alphavirus. Subsequently, I will determine the structure and biophysical properties of these nsPs. The ultimate goal is to know which nsPs or subdomains of these proteins are important in the inhibition of OAS/ RNAse L pathway through molecular and cell biology experiments and relate to the structure obtained by crystallography making possibilities about the design of future inhibitors targeting these proteins or sub-domains.
The ALPHA project overall objective is to gain an insight into alphavirus mechanisms of OAS/RNAse L pathway inhibition and the role of non-structural proteins (nsPs). To reach this aim we will have to achieve the 2 following specific objectives:
a) Investigation of Alphavirus’s nsPs role in viral evasion.
b) Large scale production and functional, structural and biophysical characterization of nsPs.

To achieve its objectives ALPHA project has produced original research results of high quality and subsequently published them in top international conferences and several articles are being prepared for publications in high impact journals:
• We have achieved the first structure of a viral protein in complex with a 2-5A molecule. The crystal structure of a complex between ChikV macro domain and a molecule of 2-5A trimer at a resolution of 2.3 Å has been solved.
• Functional characterization of ChikV macro domain binding to 2-5A oligomer was measured by means of a novel biophysical technique; microscale thermophoresis (MST).
• We found that a mutant yielding CHIKV unable to grow in infectious CHIKV clones, doesn’t bind 2-5A, indicating that this residue might provide a critical contribution to 2-5A binding and subsequent viral escape.
• We have expressed and purified nsP1 of VEEV and it has been characterized enzymatically and biophysically.
• With the aim of search for new inhibitors, we have used the purified and enzymatically active VEEV nsP1 as a new antiviral target in alphavirus, validating known capping enzyme inhibitors for VEEV and finding a new scaffold of compounds for alphavirus inhibition.
• Interesting findings have been obtained about thermal sensitive mutants of VEEV macro domain important for viral replication.

To sum up, the results of research of ALPHA project has explored on a group of poorly characterized viruses like alphavirus. These results will integrate European scientific excellence and make Europe better prepared for emerging epidemics, developing better tools to search for possible inhibitors of this global threat.

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