A Unique Role for the Host ESCRT Proteins in Replication of Tomato bushy stunt virus

Abstract

Plus-stranded RNA viruses replicate in infected cells by assembling viral replicase complexes consisting of viral- and host-coded proteins. Previous genome-wide screens with Tomato bushy stunt tombusvirus (TBSV) in a yeast model host revealed the involvement of seven ESCRT ( endosomal sorting complexes required for transport) proteins in viral replication. In this paper, we show that the expression of dominant negative Vps23p, Vps24p, Snf7p, and Vps4p ESCRT factors inhibited virus replication in the plant host, suggesting that tombusviruses co-opt selected ESCRT proteins for the assembly of the viral replicase complex. We also show that TBSV p33 replication protein interacts with Vps23p ESCRT-I and Bro1p accessory ESCRT factors. The interaction with p33 leads to the recruitment of Vps23p to the peroxisomes, the sites of TBSV replication. The viral replicase showed reduced activity and the minus-stranded viral RNA in the replicase became more accessible to ribonuclease when derived from vps23Δ or vps24Δ yeast, suggesting that the protection of the viral RNA is compromised within the replicase complex assembled in the absence of ESCRT proteins. The recruitment of ESCRT proteins is needed for the precise assembly of the replicase complex, which might help the virus evade recognition by the host defense surveillance system and/or prevent viral RNA destruction by the gene silencing machinery.

Author Summary

Plus-stranded RNA viruses, which are important pathogens of humans, animals and plants, replicate in infected cells by assembling viral replicase complexes consisting of viral- and host-coded proteins. In this paper, we show that a group of host factors called ESCRT proteins ( endosomal sorting complexes required for transport) play important roles in tombusvirus replication. The expression of dominant negative mutants of ESCRT factors inhibited virus replication in the plant host, suggesting that tombusviruses co-opt selected ESCRT proteins for the assembly of the viral replicase complex. In addition, we show direct interaction between the viral p33 replication protein and Vps23p ESCRT-I and Bro1p accessory ESCRT factors. The interaction with p33 leads to the recruitment of Vps23p to the peroxisomes, the sites of tombusvirus replication. We also showed that the viral RNA within the viral replicase complex became more sensitive to ribonuclease in the absence of ESCRT factors, suggesting that the protection of the viral RNA is compromised within the replicase complex assembled in the absence of ESCRT proteins. Intriguingly, the host ESCRT factors also affect the budding of several enveloped viruses, intracellular transport of proteins and cytokinesis. Overall, this work demonstrates that a plus-stranded RNA virus uses the endosomal sorting pathway in a unique way.