The tegument phosphoproteins of human cytomegalovirus (HCMV) elicit cytotoxic T-lymphocyte (CTL) responses and are hence candidates for subunit vaccine development. Little is known, however, about the tegument proteins of nonhuman cytomegaloviruses, such as guinea pig CMV (GPCMV). DNA sequence analysis of the Eco R I 'C' fragment of the GPCMV genome identified an open reading frame (ORF) which is colinear with that of the HCMV tegument phosphoprotein, 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 hybridized 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), defining these as 'late' gene transcripts. In vitro translation of the UL83 ORF in reticulocyte lysate resulted in synthesis of a 65 kDa protein. Immunofluorescence experiments revealed that the putative GPCMV UL83 homolog exhibited a predominantly nuclear localization pattern. Polyclonal antisera were raised against a UL83/glutathione-S-transferase (GST) fusion protein. This antibody identified a 70-kDa virion-associated protein, the putative GPCMV UL83 homolog, in immunoblot and radioimmunoprecipitation experiments. Labeling experiments with 32P-orthophosphate indicated that the GPCMV UL83 protein is phosphorylated. Western blot analysis of glycerol tartrate gradient-purified virions and dense bodies confirmed that the putative GPCMV UL83 homolog was a constituent of both fractions.
Bibliographical noteFunding Information:
We thank Dr. Nigel Bourne for assistance with animal studies and for helpful discussions. We thank Fernando Bravo for technical assistance. This research was supported by NIH grants AI 01276-01 and HD 28827-01, March of Dimes Basic Research Grant 6-FY98-0416, and by a grant from the Pediatric Infectious Diseases Society supporting G.E. (sponsored by Merck Pharmaceuticals, Inc.).
- CMV vaccines
- Guinea pig cytomegalovirus
- UL83 gene