FIG. 1.

pUL69 mediates the nuclear export of unspliced CAT mRNA. (A) Schematic view of the pDM128/CMV reporter plasmids showing locations of the splice donor (SD) and splice acceptor (SA) sites that flank the CAT reporter gene, the Rev (RRE) or Rex (RXRE) response element, and the SV40 late poly(A) signal. The localization of the antisense riboprobe used in the RNase protection assay is indicated. (B) HeLa cells were transfected with the respective pDM128/CMV reporter constructs and with plasmids expressing HIV Rev, human T-cell leukemia virus Rex, EBV EB2, HCMV UL69, and HCMV IE1 as indicated. CAT protein expression was measured in cell lysates 48 h after transfection using a CAT enzyme-linked immunosorbent assay. The relative amounts of CAT protein expressed were given as percentages of the amount obtained for the Rev positive control. One hundred percent represents an 8.2-fold increase of CAT protein in comparison to basal levels. (C) pUL69- and Rev-mediated cytoplasmic accumulation of CAT mRNA was monitored by RNase protection analysis of total (lanes 1 to 5) or cytoplasmic (lanes 6 to 10) RNA from 293T cells transfected with pDM128/CMV/RRE. In lanes 3, 4, 8, and 9, plasmids encoding pUL69 or Rev were cotransfected. As a control, the reporter plasmid was cotransfected either with empty vector (pCB6, lanes 2 and 7) or with a vector encoding Rex (lanes 5 and 10).