MOLECULAR CHARACTERIZATION OF GUINEA PIG CYTOMEGALOVIRUS

The genome of GPCMV has been characterized and compared with that of HCMV. Purified GPCMV DNA was infectious when transfected into guinea pig embryo fibroblast cells. The number, size, and molarity of GPCMV DNA fragments generated by restriction endonucleases were determined. Fragments of GPCMV DNA produced by HindIII or EcoRI digestions were cloned, and recombinants representing approximately 97% of the genome were constructed. Hybridization of ('32)P-labeled fragments to Southern blots of GPCMV DNA verified the virus origin of cloned fragments and allowed construction of HindIII, EcoRi, and XbaI restriction maps. The size of the GPCMV genome was 239 kilobase pairs (Kb), corresponding to a molecular weight (MW) of 158 x 106. GPCMV genome consists of a long unique sequence with a terminal repeat sequence but without internal repeat regions. In addition, GPCMV DNA molecules exist in two forms. In the predominant form, the molecules demonstrate sequence homology between the terminal fragments; in the minor population, one terminal fragment is smaller and is not homologous with the fragment at the other end of the physical map.;DNA sequence homology was localized on both GPCMV genome and HCMV AD169 genomes. DNA sequence homology between the cloned GPCMV HindIII-D and HCMV HindIII-E fragments was not simply due to the high guanine plus cytosine (G + C)-containing DNA regions binding to each other. Hybridization experiments in which conditions were varied to alter the stringency further indicated that sequence homology does exist between the GPCMV HinIII-D and HCMV Hin-III-E fragments.;The regions of the virus genome that encode the immediate early (IE), early, and late virus RNAs were mapped. The IE RNAs were transcribed predominantly from HindIII-D, -G, and -B; recombinant DNAs representing approximately 55% of the virus genome detected IE transcripts. Before the onset of virus DNA replication, recombinant DNAs representing approximately 93% of the genome detected early transcripts. GPCMV DNA replication was initiated at 16 hr postinfection (p.i.) and reached its maximal level at 28-32 hr. At 72 hr p.i. recombinant DNA representing approximately 99% of the genome detected late transcripts. Different patterns of transcription at early and late times were identified, indicating that expression of GPCMV genome is temporally regulated.