Transcription analysis and sequence of the putative murine cytomegalovirus DNA polymerase gene

Abstract

The conservation of the herpesvirus DNA polymerases has allowed cross-hybridization studies to be used for their identification and mapping on the viral genome. With the use of a DNA fragment containing the DNA polymerase gene of human cytomegalovirus (HCMV) as a hybridization probe, we were able to localize the DNA polymerase gene of murine cytomegalovirus (MCMV) to a region within MCMV EcoRI fragment B which spans the HindIII site separating HindIII fragments D and H. This site is colinear with the HCMV strain AD169 DNA polymerase gene. To confirm that this region encoded the MCMV DNA polymerase gene, we sequenced a 5131 nucleotide fragment from the Pstl site in HindIII fragment D to a Bg/II site in HindIII fragment H. Initiating in HindIII fragment D and extending into HindIII fragment H was a long open reading frame (ORF) 1097 amino acids in length with extensive homology to the DNA polymerases of HCMV, herpes simplex virus, and Epstein-Barr virus. Upstream of the polymerase ORF was a reading frame with considerable homology to the carboxy terminal half of the glycoprotein B gene of human herpesviruses. At early times in the infection, we could detect with a probe representing part of the polymerase ORF two 3′ coterminal transcripts, 3.9 kb and 1.7 kb in length. S1 nuclease and exonuclease VII analyses indicated that both transcripts were unspliced and initiated at independent sites in HindIII fragment D. By primer extension, we were able to map precisely the 5′ end of the 3.9-kb RNA to a site 186 nucleotides upstream of the beginning of the DNA polymerase ORF.