Molecular characterization of the guinea pig cytomegalovirus UL83 (pp65) protein homolog.
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.
Schleiss
M R
MR
Division of Infectious Diseases, Children's Hospital Research Foundation, Cincinnati, Ohio 45229, USA.
McGregor
A
A
Jensen
N J
NJ
Erdem
G
G
Aktan
L
L
D000595:Amino Acid Sequence; D000818:Animals; D000956:Antigens, Viral / Q000032:analysis / Q000235:genetics*; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003587:Cytomegalovirus / Q000235:genetics*; D005455:Fluorescent Antibody Technique; D006168:Guinea Pigs; D006801:Humans; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000032:analysis / Q000235:genetics*; D016415:Sequence Alignment; D014158:Transcription, Genetic; D014763:Viral Matrix Proteins / Q000032:analysis / Q000235:genetics*
D000956:Antigens, Viral; D010750:Phosphoproteins; D014763:Viral Matrix Proteins; C065418:cytomegalovirus matrix protein 65kDa