Complex regulation of simian virus 40 early-region transcription from different overlapping promoters.

Abstract

During simian virus 40 lytic infection there is a shift in initiation sites used to transcribe the early region, which encodes large T and small t antigens. Early in infection, transcription is initiated almost exclusively from sites that are downstream of the origin of DNA replication, whereas transcripts produced later are initiated mainly from sites on the upstream side. We have used mutant virus and specially constructed plasmid DNAs to investigate the factors regulating this transcriptional shift. In our studies simian virus 40 large T antigen appears to mediate the shift in transcription in two ways: first, T antigen represses transcription at the downstream sites late in infection by binding to the region where these RNAs are initiated; second, T antigen promotes transcription from sites on the upstream side by its ability to initiate replication or amplification, or both, of the template DNA. In addition, transcription from the downstream sites is heavily dependent on enhancer sequences located in the 72-base-pair repeat region, whereas transcription from the upstream sites late in infection does not require enhancer sequences. Thus, different overlapping promoters regulate simian virus 40 early-region expression in a manner that apparently coordinates the production of large T antigen with the increase in viral DNA.At present, the molecular mechanisms controlling gene expression in higher eucaryotes are only poorly understood. One of the best characterized systems which has been used to approach this problem is the early region of simian virus 40 (SV40) (1, 24, 56). The early region codes for two known proteins, large T antigen and small t antigen. Although the function of small t antigen remains obscure, large T antigen is known to be required for the initiation of viral DNA synthesis. These proteins are translated from differentially spliced RNAs which are transcribed, using the same promoter and polyadenylation signals. The SV40 early-region promoter is contained within a set of repeated segments adjacent to the origin of viral DNA replication (ori) (Fig. 1). Extensive analysis of this region has demonstrated the presence of three separate elements involved in SV40 early transcription (5, 16). At least one copy of a set of six short guanine-cytosine-rich repeats is necessary for early transcription. In addition, a sequence within the 72-base-pair (bp) repeat has been shown to be an essential element. Finally, a Goldberg-Hogness or TATA box sequence (19; M. Goldberg, Ph.D. thesis, Stanford University, Stanford, Calif., 1978), imbedded within a larger 17-bp adenine-thymine (AT)-rich block, is responsible for positioning the 5' ends of early RNA.The SV40 early region is also one of the best characterized examples of regulation mediated by a defined protein. Large T antigen regulates its own synthesis by controlling the level of early-region transcription (33, 53, 57). Furthermore, T antigen binds to SV40 DNA at three adjacent sites overlapping ori and the early promoter (Fig. 1) that T-antigen binding a...

Unusual regulation of simian virus 40 early-region transcription in genomes containing two origins of DNA replication.

“…These findings with double-ori recombinants are consistent with our ideas regarding the regulation of SV40 early-region transcription (6,12). In addition, the peculiar behavior of these recombinants provides some revelations regarding normal early-region expression.…”

“…The fact that the 299-bp deletion in IVS2-6 and SVIV-6 inactivates both the downstream and the upstream promoters suggests that sequences within the 72-bp and GC-rich repeat regions are important for upRNA information. Since our earlier study (6) showed that the enhancer sequences are not required for upRNA synthesis, it seems likely that transcription from the upstream sites requires the GC-rich repeats. The recent findings of Baty et al (2) also indicate that the GC-rich repeat region is involved in upRNA formation.…”

“…One consequence of the shift in the site of early-region transcription is that the upRNAs contain a small open reading frame 5' to the coding region of the T antigens (6). Quite possibly, the upstream coding region modulates the translation of the T-antigen coding sequences (22); specifically, upRNA might be a less efficient template for T-antigen synthesis.…”

“…With both IVS2-6 and IVS2-9, substantial quantities of 3-globin were produced 48 h after infection. Moreover, just as for small t-antigen production form SVIV-9, P-globin formation with IVS2-9 was higher at 48 h than at 24 h after infection, (6). Because genomes with two ori's -globin by IVS2-9 shows the replicate more rapidly than ones with a single ori, it seemed Iy-region expression as does possible that the unusual transcription from the early-region promoter was caused by the altered rate of DNA replication.…”

Sequences involved in initiation of simian virus 40 late transcription in the absence of T antigen.

“…The early promoter is composed of at least three spatially distinct elements: (i) the TATA box; (ii) a G-C-rich region; and (iii) the enhancer element. The TATA box, which is contained within a 17-bp A-T-rich sequence, dictates the position of the major early initiation sites (at nucleotides [nt] 5230 to 5237, 21 to 28 bp downstream from the TATA box) used preferentially before viral DNA replication (3,8,17). The G-C-rich region contains six copies of the sequence 5'-PyPyCCGCCC-3' in two direct repeat sequences of 21 bp and a third, almost perfect repeat of 22 bp.…”