DETECTION OF THE HUMAN CYTOMEGALOVIRUS 2.0-KB IMMEDIATE-EARLY GENE-1 TRANSCRIPTS IN PERMISSIVE AND NONPERMISSIVE INFECTIONS BY RMA IN-SITU HYBRIDIZATION

The immediate early gene 1 (IE1) is the first gene to be expressed fol lowing the entry of the human cytomegalovirus (HCMV) into the cell and it does not require prior protein synthesis for its expression. There fore, the IE1 gene is a potential candidate for the development of pro bes to detect HCMV in various states of infection. Using strand-specif ic P-32- Or digoxigenin-labeled riboprobes derived from an exon-specif ic subgenomic fragment of the HCMV Towne IE1 gene, we performed Northe rn blot analysis and RNA in situ hybridization on HCMV-infected human (permissive cells) and mouse (nonpermissive cells) fibroblasts and on 10 formalin-fixed paraffin-embedded sections of human tissue. By North ern blot analysis and by in situ hybridization, expression of the 2.0- kb IE1 gene was found in permissive as well as in nonpermissive infect ions. Specific nuclear and cytoplasmic hybridization was found at 5, 1 0, 24, and 72 h after infection in human fibroblasts. In comparison, h ybridization was first detected at 10 h after infection in mouse fibro blasts. Hybridization with the IE1 probe was detected in cells with an d without cytopathic changes in the formalin-fixed paraffin-embedded H CMV-infected human tissues. Hybridization patterns of the IE1 riboprob e were compared to those of the HCMV 2.7-kb major early P-riboprobe wh ich we have previously described [Am J Pathol 141: 1247-1254;1992]. Al though both riboprobes hybridize to their respective target sequences in the consecutive tissue sections, the patterns of hybridization are different. On occasion, sections of HCMV-infected human tissue showing no specific hybridization for the 2.7-kb riboprobe will show specific in situ hybridization when using the IE1 riboprobe. Our results sugge st that RNA in situ hybridization with a probe directed at the IE1 tra nscripts is an effective method of detecting early and late stages of both permissive and nonpermissive HCMV infections.