Search

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.

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.

The sophisticated anti-viral functions of nuclear domain 10 (ND10) are revealed by identifying the role of each component and the countermeasures applied by viruses. Several ND10 proteins suppress herpesviruses at initial and early phases of infection. Herpesviruses need to antagonize these anti-viral proteins to start a productive infection. In this review the recently identified similarities and differences among the strategies adopted by the three subfamilies of herpesviruses are discussed, highlighting that one of the significant purposes of incorporating tegument proteins into the viral particles might be to counteract ND10 proteins immediately after the viral genome enters the host nucleus. Once the infection progresses, a sufficient amount of immediate early proteins is expressed to disperse and hydrolyze ND10 proteins, accelerating the development of infection.

VP8 is the most abundant tegument protein of bovine herpesvirus-1 (BHV-1). In the present study DNA damage binding protein 1 (DDB1) was identified as interacting partner of VP8. MALDI-TOF mass spectroscopy analysis of proteins co-immunoprecipitated with VP8 identified DDB1 as a protein interacting with VP8. The interaction between VP8 and DDB1 was confirmed based on co-immunoprecipitation and co-localization in both VP8-transfected and BHV-1 infected cells. DDB1 was distributed both in the nucleus and the cytoplasm with some nuclear speckles prior to BHV-1 infection, became perinuclear by 4h and was predominantly nuclear at 5h post infection, where it co-localized with VP8. In contrast, in cells infected with a U(L)47 deletion mutant DDB1 remained cytoplasmic throughout the course of infection. This suggests that VP8 mediates nuclear re-localization of DDB1. Finally, VP8 was shown to be monoubiquitinated both in VP8-transfected and BHV-1-infected cells. These data suggest that BHV-1 VP8 interacts with DDB1-CUL4 E3 ubiquitin ligase, which correlates to monoubiquitination of this viral protein.

Elk herpesvirus (ElkHV) from North American elk (wapiti, Cervus elaphus nelsoni) is a recently identified alphaherpesvirus related to bovine herpesvirus-1 (BHV-1). In this study, we determined its relationship with European cervid herpesviruses: cervid herpesvirus-1 (CerHV-1) from red deer and rangiferine herpesvirus (RanHV) from reindeer. For phylogenetic analysis, genes for the gC and gD proteins of these viruses were sequenced. These genes demonstrated an extremely high GC content (76-79%). Genetically, ElkHV was found to be closely related to CerHV-1 and both viruses are more closely related to BHV-1 than to RanHV. Antigenically, the same relationships were found. ElkHV shares common neutralizing epitopes with both CerHV-1 and RanHV. A total of 10 epitopes were defined on the gB, gC and gD proteins of these viruses, including a shared neutralizing epitope on gD. The results indicate that ElkHV and CerHV-1 have diverged from a common ancestor virus. Cervid herpesviruses may be useful in determination of evolutionary rates of change for alphaherpesvirus genes.