Molecular characterization of the guinea pig cytomegalovirus UL83 (pp65) protein homolog

The tegument phosphoproteins of human cytomegalovirus (HCMV) elicit cytotox ic T-lymphocyte (CTL) responses and are hence candidates for subunit vaccin e development. Little is known, however, about the tegument proteins of non human cytomegaloviruses, such as guinea pig CMV (GPCMV). DNA sequence analy sis of the Eco R I "C" fragment of the GPCMV genome identified an open read ing frame (ORF) which is colinear with that of the HCMV tegument phosphopro tein, UL83 (pp65). This ORF was found to have identity to HCMV UL83 and was predicted to encode a 565-amino-acid (aa) protein with a molecular mass of 62.3 kDa. Transcriptional analyses revealed that a GPCMV UL83 probe hybrid ized with both 2.2 kb and 4.2 kb mRNA species at 48 h post-infection (p.i.) ; synthesis of these messages was blocked by phosphonoacetic acid (PAA), de fining these as "late" gene transcripts. In vitro translation of the UL83 O RF in reticulocyte lysate resulted in synthesis of a 65 kDa protein. Immuno fluorescence experiments revealed that the putative GPCMV UL83 homolog exhi bited a predominantly nuclear localization pattern. Polyclonal antisera wer e raised against a UL83/glutathione-S-transferase (GST) fusion protein. Thi s antibody identified a 70-kDa virion-associated protein, the putative GPCM V UL83 homolog, in immunoblot and radioimmunoprecipitation experiments. Lab eling experiments with P-32-orthophosphate indicated that the GPCMV UL83 pr otein is phosphorylated. Western blot analysis of glycerol tartrate gradien t-purified virions and dense bodies confirmed that the putative GPCMV UL83 homolog was a constituent of both fractions.