Ectopic expression and subcellular localization of the putative RNA-dependent RNA polymerase of Tulasnella ambivirus 4 in Saccharomyces cerevisiae

Ambiviruses are infectious agents possessing a circular single-stranded RNA genome folding in a compact secondary structure and with paired self-cleaving ribozymes, which replicates through a rolling circle mechanism. However, besides presenting typical features of viroid-like RNAs, ambiviruses share some characteristics of RNA viruses. They harbour two open reading frames (ORFs; ORFA and ORFB) which code for one protein in each polarity strand. ORFB encode a protein with unknown function. ORFA potentially code for an RNA-dependent RNA polymerase (RdRP), based on structural similarity to viral RdRPs, as shown by structural and phylogenetic analysis. Nevertheless, its role remains to be elucidated. Here to investigate the function of ORFA gene product, we used Tulasnella ambivirus 4 (TuAmV4) as a model, which infects Tulasnella sp., mycorrhizal fungi of orchid roots. The budding yeast Saccharomyces cerevisiae was selected as a surrogate model host, since it has been successfully used to express non-structural viral proteins to study their subcellular localization and the signals targeting to specific cell membranes. TuAmV4 ORFA fused to the Myc epitope was cloned under the control of a constitutive promoter in the yeast expression vector pA (pA-TuAmV4ORFAMyc) or of an inducible promoter in the yeast expression vector pYES2 (pYES2-TuAmV4ORFAMyc). Both constructs were used to transform S. cerevisiae YPH499 cells. The optimal culture conditions for the protein expression were determined. TuAmV4 ORFA was correctly expressed either constitutively or transiently, as shown by western blot analysis. The ectopic expression of ORFA did not affect yeast cell growth as a function of time. By differential centrifugation of protein extracts it was shown that ORFA expressed protein sedimented in a membrane-enriched fraction. Furthermore, TuAmV4 ORFA product resulted to be resistant to alkaline, urea or salt extraction, a property of integral membrane proteins. Finally, we observed the localization of the protein in yeast cells by immunofluorescence analysis. Results showed that the TuAmV4 ORFA was not dispersed in the cytosol, but it co-localized with a marker of the endoplasmic reticulum.