Alternative replication sites in tombusvirus infections

Positive-strand RNA viruses replicate in association with intracellular membranes of host cells, rearranged to form vesicles. The membranes can derive from diverse organelles, i.e. endoplasmic reticulum (ER), tonoplast, mitochondria, chloroplasts or peroxisomes. The confined environment protects the viral replication machinery from host cell defence. Targeting of the viral proteins required for replication to a specific membrane is genetically determined by the viral genome. For instance, tombusviruses evolved to direct their replication machinery either to mitochondria or peroxisome membrane. However, it is matter of debate whether the requirement for a specific membrane is an absolute requirement for a given virus or alternate sites of replication are possible under particular conditions of the host cells. The issue is relevant not only for comprehension of the basic mechanism of plus-strand virus replication but also to validate antiviral strategies based on altering the composition of a particular host membrane. The tombusvirus Cymbidium ringspot virus (CymRSV) replicase proteins normally associate to the peroxisomal membrane both in plant and yeast (Saccharomyces cerevisiae) cells where genomic or DI RNA takes place. Taking advantage of the availability of yeast strains defective in peroxisome biogenesis, CymRSV replicase proteins were expressed along with the replication template DI RNA in one of these (YPH499) which contains only primordial forms of peroxisomes unable to import any of peroxisomal or viral proteins Biochemical, immunofluorescence and immunoelectron microscopy demonstrated that replication took place on ER-derived membrane, thus confirming the adaptability of plus-strand viruses to changes of the membranous composition of host cells.