Abstract
The UL47 gene product, VP8, is a major tegument protein of BoHV-1. While VP8 is not essential for virus replication in cell culture, a UL47-deleted virus exhibits a smaller tegument structure and is avirulent in cattle. To obtain pure VP8 protein for structural analysis, we expressed a N-terminally truncated version of VP8 in Eschericia coli. However, the recombinant VP8 was consistently co-purified with a tightly associated bacterial protein; this protein was identified by mass spectrometry as GroEL, which has considerable homology with mammalian heat shock protein-60 (HSP60), thus suggesting a new role for VP8 in virus-host interaction. A physical interaction of HSP60 and VP8 in both VP8-transfected and BoHV-1-infected cells was demonstrated by immunoprecipitation. Analysis of different truncated VP8 constructs revealed that amino acids 259-482 and 632-741 are involved in binding to HSP60. Full-length VP8 and VP8 219-741 (containing both interacting domains, 259-482 and 632-741) co-localized with HSP60 and mitochondria. VP8 was localized in the mitochondria from 2 to 14 h post infection in BoHV-1-infected cells. The mitochondrial membrane potential was reduced in both VP8-transfected and BoHV-1-infected cells and was further diminished by overexpression of HSP60 in the presence of VP8. In addition, VP8 expression decreased the ATP concentration during transfection, as well as BoHV-1 infection. Thus, VP8 may play a role in the deregulation of mitochondrial function through interaction with HSP60. This is consistent with the fact that BoHV-1 infection is known to promote mitochondrial dysfunction.