Structure of the transforming region of human cytomegalovirus AD169.

Abstract

The minimum size fragment of human cytomegalovirus AD169 required to initiate transformation was determined by transfection of primary rat embryo cells with deletion fragments constructed by digestion of a cloned fragment containing the transforming region (pCM4000) with exonuclease III and S1 nuclease. The results indicate that the left-hand boundary of the minimum size sequence for transformation must reside between 490 and 318 bases from the HindIII site of pCM4000. The right-hand boundary was defined by the EcoRI site which is 20 bases from the HindIII site. The nucleotide sequence of the transforming fragment of human cytomegalovirus was determined by the chemical degradation technique of Maxam and Gilbert and with the dideoxynucleoside triphosphate chain termination method. The sequence of pCM4000 comprises 2,848 base pairs and has an A X T composition of 59.5%. Reading frame analysis of the sequence indicated the longest open reading frame was 118 amino acids in length. Northern blot analysis of polyadenylated and non-polyadenylated RNA extracted from cells at immediate early and late times after infection with human cytomegalovirus indicate that one 5.0-kilobase RNA species hybridized to pCM4000 (Jahn et al., J. Virol, in press). The direction of transcription was determined by hybridization of this transcript with M13 single-stranded probes, and S1 analysis of this RNA did not detect introns.