Interaction of VP8 with mRNAs of bovine herpesvirus-1.

The UL47 gene product, VP8, is one of the most abundant tegument proteins of bovine herpesvirus-1 (BoHV-1). Deletion of VP8 leads to impaired growth in tissue culture, and VP8 is indispensable for BHV-1 replication in cattle. To elucidate the biological functions of VP8, we explored its interaction with mRNAs of immediate early (bICP0), early (gB, gD) and late (gC) genes of BoHV-1. FLAG-tagged VP8 was pulled down from COS-7 cells co-transfected with plasmids encoding VP8 and either gB, gC, gD or bICP0. This was followed by RNA extraction, cDNA synthesis and qPCR, which demonstrated binding of VP8 to bICP0, gB, gC and gD mRNAs in the cytoplasm and nucleus. These results were supported by co-localization of VP8 with bICP0, gB, gC and gD mRNAs in the nucleus as determined by confocal microscopy. Amino acids 259-342, located in the conserved portion of UL47 homologues, were found to contain the RNA binding region on VP8. To further characterize these interactions, Northwestern blotting was performed by immobilizing VP8 on a nitrocellulose membrane followed by hybridization with in vitro transcribed bICP0 mRNA. The results demonstrated binding of VP8 to intron-less mRNA but not intron-containing mRNA of bICP0. In addition, the interaction of VP8 with bICP0 mRNA was confirmed in vitro by RNA electrophoretic mobility shift assay, which also showed that the zinc finger and acidic domains both interact with VP8. Based on these results, we concluded that VP8 binds to intron-less mRNAs of bICP0, gB, gC and gD.