The Plum Pox Virus 6K1 Protein Is Required for Viral Replication and Targets the Viral Replication Complex at the Early Infection Stage.
Cui, H. and Wang, A.M. (2016). "The Plum Pox Virus 6K1 Protein Is Required for Viral Replication and Targets the Viral Replication Complex at the Early Infection Stage.", Journal of Virology, 90(10), pp. 5119-5131. doi : 10.1128/JVI.00024-16 Access to full text
The potyviral RNA genome encodes two polyproteins that are proteolytically processed by three viral protease domains into 11 mature proteins. Extensive molecular studies have identified functions for the majority of the viral proteins. For example, 6K2, one of the two smallest potyviral proteins, is an integral membrane protein, and induces the endoplasmic reticulum (ER)-originated replication vesicles that target chloroplast for robust viral replication. However, the functional role of 6K1, the other smallest protein, remains uncharacterized. In this study, we developed a series of recombinant full-length viral cDNA clones derived from a Canadian Plum pox virus (PPV) isolate. We found that deletion of any of short motifs of 6K1 (ranging from 5 to 13 amino acids) or most of 6K1 (keeping the conserved sequence of cleavage sites) or entire 6K1 in the PPV infectious clone abolished viral replication. The trans expression of 6K1 or cis expression of a dislocated 6K1 failed to rescue the loss-of-replication phenotype, suggesting the temporal and spatial requirement of 6K1 in viral replication. Disruption of the N- or C-terminal cleavage site of 6K1 preventing the release of 6K1 from the polyprotein either partially or completely inhibited viral replication, suggesting the functional importance of the mature 6K1. We further found that 6K1-GFP formed punctate inclusions at the viral early infection stage, and co-localized with chloroplast-bound viral replicase elements 6K2 and NIb. Taken together, our results suggest that 6K1 is required for viral replication as an important viral element of the viral replication complex at the early infection stage. IMPORTANCE Potyviruses account for more than 30% known plant viruses and consist of many agriculturally important viruses. Potyviruses encode two polyproteins that are proteolytically processed into 11 mature proteins, with the majority of them having been functionally characterized at least partially. However, the functional role of a small protein named 6K1 remains obscure. In this study, we show that deletion of 6K1 or a short motif/region of 6K1 in the full-length cDNA clones of plum pox virus abolished viral replication, and mutation of the N- or C-terminal cleavage sites of 6K1 to prevent its release from the polyprotein greatly attenuated or completely inhibited viral replication, suggesting its important role in potyviral infection. We report that 6K1 formed punctate structures and targeted the replication vesicles in PPV-infected plant leaf cells at the early infection stage. Our data reveals that 6K1 is an important viral protein of the potyviral replication complex.
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