THE HUMAN CYTOMEGALOVIRUS US3 IMMEDIATE-EARLY PROTEIN LACKING THE PUTATIVE TRANSMEMBRANE DOMAIN REGULATES GENE-EXPRESSION

Through alternative transcript splicing, the human cytomegalovirus (HC MV) US3 immediate-early (IE) locus encodes multiple products including potential membrane-bound glycoproteins. To characterize the US3 produ cts and determine which encode regulatory activity, individual cDNAs w ere cloned and expressed. Three transcript species were confirmed thro ugh the isolation of cDNAs; an unspliced transcript, a transcript spli ced once from exon 3 to exon 5 and a transcript spliced both at exon 1 to exon 3 and at exon 3 to exon 5. The predicted signal sequences and N-linked glycosylation sites in the US3 products were confirmed using expression in reticulocyte lysates containing microsomal membranes. R egulatory activity of the individual US3 products was demonstrated usi ng transient transfection assays. The unspliced cDNA and the cDNA cont aining the exon 3 to exon 5 splice, encoded products which increased e xpression of the human heat shock protein 70 (hsp70) promoter, while t he product of the doubly-spliced US3 cDNA did not. Transactivation was synergistically increased by coexpression with the HCMV UL37 protein. We conclude that the first 132 amino acids common to the unspliced an d the singly-spliced US3 gene products are sufficient for hsp70 transa ctivation; while the amino-terminal 28 amino acids, encoded by the dou bly-spliced US3 cDNA, are not. These results demonstrate that a US3 IE protein lacking the putative transmembrane domain has regulatory acti vity.