(+)RNA VIRUS REPLICATION-ASSOCIATED PROTEIN EXPRESSION IN SACCHAROMYCES CEREVISIAE

Background and aims: Positive-strand RNA [(+)RNA] viruses are agents of important diseases in humans, animals and plants, including COVID-19. Regardless of the host, the replication of all (+) RNA viruses occurs in association with the host endomembrane system. Based on this common replication mechanism, we have used the yeast model to express the replication-associated proteins of one plant and one human virus to decipher virusmembrane interactions, with the final aim of identifying host factors co-opted for viral replication, to develope new antiviral strategies. Methods: We expressed the (+)RNA carnation Italian ringspot virus (CIRV) replicationassociated protein p36 under the control of the inducible GAL1 promoter in Saccharomyces cerevisiae. Similarly, we cloned and expressed in yeast the SARS-CoV-2 non-structural proteins nsp3, nsp4 and nsp6, having a role in the formation of the double-membrane vesicles, which likely represent the virus replication site. Protein expression was analyzed by Western blot and immunofluorescence analyses. Results and conclusions: It was shown that p36 expression in S. cerevisiae strain YPH499 (i) increased necrotic cell death and concomitantly decreased regulated cell death in response to acetic acid; (ii) decreased respiratory yeast cell growth; (iii) altered the mitochondrial network; (iv) decreased oxygen consumption due to respiratory chain complex impairment. SARS-CoV-2 non-structural proteins nsp3, nsp4 and nsp6 expression was obtained in S. cerevisiae strain W303-1B and viability was measured as a function of time. Cells expressing nsp3 and nsp4 showed significantly reduced viability compared to control cells at all time points, whereas the viability of yeast cells expressing nsp6 was reduced only up to 24 h. Cell growth rate was determined and shown to be reduced. The expression of SARSCoV- 2 nsp3, nsp4 and nsp6 was verified at all time points by Western blotting and the intracellular localization of the expressed proteins was determined by immunofluorescence analysis. We show that the yeast model system can be successfully employed to study both plant and human virus replication-associated proteins. S. cerevisiae was confirmed as an invaluable model host to study the molecular pathogenesis of (+)RNA viruses and the evasion of the host immune system. Elucidating virus-host cell interaction complexity is crucial to the identification of novel druggable targets for the development of broad-spectrum antivirals.