DNA-SEQUENCES OUTSIDE THE SIMIAN-VIRUS-40 EARLY REGION CAUSE DOWN-REGULATION OF T-ANTIGEN PRODUCTION IN PERMISSIVE SIMIAN CELLS

Using a series of modified wtSV40 and early region SV40 DNAs, we assay ed the effect of viral late region sequences on T-antigen production b y the SV40 early region. We found that SV40 late region (L-SV40) DNA s equences reduced T-antigen (T-Ag) production by the SV40 early region (E-SV40) when both viral regions were linked as they are in wtSV40 DNA . This was demonstrated by Western analysis which showed that E-SV40 D NA produced 10 times more T-Ag than wtSV40 DNA L-SV40, with its own pr omoter but unlinked to E-SV40 DNA, also greatly inhibited T-Ag product ion when it was cotransfected with E-SV40. Therefore, L-SV40 DNA inhib ited T-Ag production by E-SV40 DNA when present in cis or in trans. We have shown that expression of the SV40 late transcription unit domina ted that of the early (T-Ag gene) transcription unit because late regi on RNA accumulated to much higher levels than early viral RNA. However . in cotransfected cells L-SV40 DNA did not replicate to higher levels than E-SV40 DNA. We offer a model for control of T-Ag expression in w hich a relatively small amount of T-Ag activates tate transcription at the expense of T-Ag gene transcription and that this represents a swi tch from early to late viral gene expression. We suggest that when act ivation of the late transcription unit occurs at the late promoter, ex pression of the T-Ag gene is greatly reduced. The L-SV40 promoter may inhibit T-Ag gene transcription by sequestering cellular factors requi red for early transcription, factors which may be present in limited a mounts. We suggest further that activation of late transcription allow s for the necessary production of large amounts of capsomeres and viri ons and downregulation of early transcription prevents the early regio n from interfering with capsid synthesis. We tested the model using a construct with a wild-type T-Ag gene but with mutations in the SV40 ma jor late promoter which prevent the promoter from being bound by cellu lar repressors of late transcription. We found that this construct, wh ich overproduces late SV40 RNA, was defective for T-Ag production. Thi s indicates that activation of the late promoter results in repression of T-Ag gene expression. (C) 1998 Academic Press.