Pentapeptide-insertion scanning mutational analysis of turkey herpesvirus HVT063 reveals residues important for its RNA silencing suppression activity

HVT063, an RNA-binding protein encoded by turkey herpesvirus, has been shown previously to suppress RNA silencing. Here, a scanning library produced by pentapeptide-insertion scanning mutagenesis was used to identify key residues associated with its RNA silencing suppressor (RSS) activity. Forty-two in-frame insertion mutants of HVT063 protein were evaluated for their RSS activity using the dual-luciferase transient expressing assay system. Sixteen mutations resulted in a loss of RSS activity, 20 mutations resulted in decreased RSS activity, and six mutations exhibited high RSS activity similar to wild-type HVT063. Based on a three-dimensional structure prediction, most of the loss-of-function mutations were located around a predominantly α-helical region at the C-terminal end of HVT063. In particular, a conserved domain in this region, named herpes_UL69, showed low tolerance for five-amino-acid insertions. Combined with the results of our previous studies, basic amino acids could play a key role in RSS activity. These results also demonstrate that pentapeptide-insertion scanning mutagenesis combined with dual-luciferase assays is an effective method to functionally characterize RSSs.