Mycovirus Cryphonectria Hypovirus 1 Elements Cofractionate with trans-Golgi Network Membranes of the Fungal Host Cryphonectria parasitica

The mycovirus cryphonectria hypovirus 1 (CHV1) causes proliferation of vesicles in its host, Cryphonectria parasitica, the causal agent of chestnut blight. These vesicles have previously been shown to contain both CHV1 genomic double-stranded RNA (dsRNA) and RNA polymerase activity. To determine the cellular origins of these virus-induced membrane structures, we compared the fractionation of several cellular and viral markers. Results showed that viral dsRNA, helicase, polymerase, and protease p29 copurify with C. parasitica trans-Golgi network (TGN) markers, suggesting that the virus utilizes the fungal TGN for replication. We also show that the CHV1 protease p29 associates with vesicle membranes and is resistant to treatments that would release peripheral membrane proteins. Thus, p29 behaves as an integral membrane protein of the vesicular fraction derived from the fungal TGN. Protease p29 was also found to be fully susceptible to proteolytic digestion in the absence of detergent and, thus, is wholly or predominantly on the cytoplasmic face of the vesicles. Fractionation analysis of p29 deletion variants showed that sequences in the C terminal of p29 mediate membrane association. In particular, the C-terminal portion of the protein (Met-135-Gly-248) is sufficient for membrane association and is enough to direct p29 to the TGN vesicles in the absence of other viral elements.