Structure-based identification of functional residues in the nucleoside-2'-O-methylase domain of Bluetongue virus VP4 capping enzyme

Stewart, Meredith E.; Roy, Polly

In order to determine if these two mutant viruses could be rescued when a native VP4 was provided in trans, a stable cell line (BSR4) constitutively expressing WT VP4 was generated. The BSR4 cells were transfected with each mutant S4 transcripts together with 9 BTV-1 transcripts and monitored for CPE and plaque formation. Both mutant viruses were successfully rescued in the complementary cell line with plaque morphology similar to that of the WT BTV virus (Fig. 6A) . The disruption of the catalytic site could be compensated by the normal VP4 supplied in trans. Further, viruses grown in the complementary BSR4 cells were used to infect both normal BSR and BSR4 in parallel, at an MOI $ 1 and viral structural and non-structural proteins (VP3 and NS1) synthesis were examined. The expression of BTV proteins was detected in both cell lines at 24 h post infection indicating that the mutant viruses were able to infect and express the viral proteins in both cell lines (Fig. 6B) . Progeny mutant viruses grown in normal BSR cells failed to replicate further (data not shown). The dsRNA genome of the mutant viruses were extracted from BSR4, infected cells, but not from normal cells, were purified and had a typical BTV genome profile indicating that there were no abnormal segments and genome replication was occurring (Fig. 6C) .