CHARACTERIZATION AND TEMPORAL REGULATION OF MESSENGER-RNAS ENCODED BYVACCINIA VIRUS INTERMEDIATE-STAGE GENES

The steady-state levels of mRNAs encoded by three intermediate-stage g enes of vaccinia virus, AIL, A2L, and G8R, were compared with those en coded by well-characterized early- and late-stage genes. After synchro nous infection of HeLa cells, the early mRNA was detected within 20 mi n and peaked at about 100 min; all three intermediate mRNAs were detec ted at 100 min and peaked at about 120 min; and the late mRNA was dete cted at 140 min and increased thereafter. Upon reaching maximum levels , the early and intermediate mRNAs declined at rates consistent with h alf-lives of about 30 min, providing the basis for rapid changes in ge ne expression. Intermediate mRNA was not detected when viral DNA synth esis was prevented, whereas its accumulation was enhanced by blocking translation after removal of the replication inhibitor. The 5' ends of the mRNAs initiated within a TAAAT or TAAAAT sequence in the coding D NA strand but contained a poly(A) leader of up to 30 additional bases. Diffuse bands of A1L and G8R RNA, equal to and longer than the coding region, were resolved by agarose gel electrophoresis, suggesting pref erred sites of 3'-end formation that did not correlate with early gene termination signals. The cis-regulatory sequences were investigated b y constructing recombinant viruses containing mutated intermediate pro moters preceding the beta-galactosidase reporter gene. The effects of mutations on expression were similar to those previously obtained by t ransfection studies (C. J. Baldick, Jr., J. G. Keck, and B. Moss, J. V irol. 66:4710-4719, 1992), providing further evidence for functional c ore, spacer, and initiator regions. In addition, an up-regulated bifun ctional early/intermediate promoter was created by making four single- base substitutions in the G8R promoter.