Sequence and transcriptional analysis of the guinea pig cytomegalovirus UL97 homolog

Guinea pig cytomegalovirus (GPCMV) displays a similar pathogenesis to human cytomegalovirus (HCMV), and the guinea pig has been used as a model system for testing anti-CMV therapies. However, not all agents active against HCMV share antiviral activity against GPCMV. For example, GPCMV appears resistant to the nucleoside analog, ganciclovir. The molecular basis for this discrepancy in antiviral susceptibility is unknown because to date there has been little analysis of the GPCMV genome. For HCMV, the antiviral effect of ganciclovir depends upon phosphorylation of the drug to its active form. This effect is mediated by the viral UL97 gene product. In order to begin to explore the molecular basis of the resistance of GPCMV to ganciclovir, experiments were undertaken to test whether the GPCMV genome encoded a homolog of the HCMV UL97 gene. Based on the prediction of co-linearity of UL97 homologs within the respective viral genomes, the EcoR I S and F fragments of the GPCMV genome were cloned and partially sequenced. A 1815 base pair open reading frame (ORF) capable of encoding a 604 amino acid (aa) protein was identified spanning portions of the EcoR I S and adjacent EcoR I F genome fragments. Computer-assisted matrix analyses revealed identity between this ORF and the HCMV UL97 gene. ORFs upstream of the GPCMV UL97 gene were identified which shared homology with the HCMV UL95 and 96 genes. Northern blot analyses identified a UL97-specific mRNA of 3.9 kb which was expressed at 'early' times post-infection. RNA transcripts of 6.0 and 4.6 kb were identified which corresponded to the UL95 and UL96 homolog coding sequences, respectively. Comparison of the GPCMV UL97 sequence to that of other herpesvirus homologs as well as that of ganciclovir-resistant clinical isolates of HCMV identified nonconservative aa substitutions in two domains involved in catalysis and substrate recognition.