Analysis of an activatable promoter: sequences in the simian virus 40 late promoter required for T-antigen-mediated trans activation.

The late promoter of simian virus 40 (SV40) is activated in trans by the viral early gene product, T antigen. We inserted the wild-type late-promoter region, and deletion mutants of it, into chloramphenicol acetyltransferase transient expression vectors to identify promoter sequences which are active in the presence of T antigen. We defined two promoter activities. One activity was mediated by a promoter element within simian virus 40 nucleotides 200 to 270. The activity of this element was detectable only in the presence of an intact, functioning origin of replication and accounted for 25 to 35% of the wild-type late-promoter activity in the presence of T antigen. The other activity was mediated by an element located within a 33-base-pair sequence (simian virus nucleotides 168 to 200) which spans the junction of the 72-base-pair repeats. This element functioned in the absence of both the origin of replication and the T-antigen-binding sites and appeared to be responsible for trans-activated gene expression. When inserted into an essentially promoterless plasmid, the 33-base-pair element functioned in an orientation-dependent manner. Under wild-type conditions in the presence of T antigen, the activity of this element accounted for 65 to 75% of the late-promoter activity. The roles of the 33-base-pair element and T antigen in trans-activation are discussed.     

Minimal transcriptional enhancer of simian virus 40 is a 74-base-pair sequence that has interacting domains.

We have assayed the ability of segments of the simian virus 40 (SV40) 72-base-pair (bp) repeat enhancer region to activate gene expression under the control of the SV40 early promoter and to compete for trans-acting enhancer-binding factors of limited availability in vivo in monkey CV-1 or human HeLa cells. The bacterial chloramphenicol acetyltransferase and the herpes simplex virus type 1 thymidine kinase genes were used as reporters in these assays. A 94-bp sequence located between SV40 nucleotides 179 and 272, including one copy of the 72-bp repeat, has been termed the minimal enhancer in previous studies. In the present study, we found that the 20-bp origin-proximal region located between nucleotides 179 and 198 was dispensable, since its removal caused only a slight reduction in enhancer activity. However, the deletion of another 4 bp up to nucleotide 202 abolished the enhancer activity. We propose that the minimal enhancer is a 74-bp sequence located between nucleotides 199 and 272, including 52 bp of one copy of the 72-bp repeat and a 22-bp adjacent sequence up to the PvuII site at 272. The nonamer 5'-AAGT/CATGCA-3', which we term the K core, occurred as a tandem duplication around the SphI site at nucleotide 200, and we found that this duplication was essential for enhancement and factor-binding activities. A heterologous core element (which we term the C core), 5'-GTGGA/TA/TA/TG-3', identified earlier (G. Khoury and P. Gruss, Cell 33:313-314, 1983; Weiher et al., Science 219:626-631, 1983) also occurred in duplicate, with one of the copies located within the 22-bp sequence near nucleotide 272 present outside the 72-bp repeat. We provide direct evidence that this 22-bp sequence augments enhancer activity considerably. We also found that in addition to the heterologous interaction occurring normally between the K and C cores within the minimal enhancer, certain homologous interactions were also permitted provided there was proper spacing between the elements.     

Cell type-specific replication of simian virus 40 conferred by hormone response elements in the late promoter

The late genes of SV40 are not expressed at significant levels until after the onset of viral DNA replication. We previously identified two hormone response elements (HREs) in the late promoter that contribute to this delay. Mutants defective in these HREs overexpress late RNA at early, but not late, times after transfection of CV-1PD cells. Overexpression of nuclear receptors (NRs) that recognize these HREs leads to repression of the late promoter in a sequence-specific and titratable manner, resulting in a delay in late gene expression. These observations led to a model in which the late promoter is repressed at early times after infection by NRs, with this repression being relieved by titration of these repressors through simian virus 40 (SV40) genome replication to high copy number. Here, we tested this model in the context of the viral life cycle. SV40 genomes containing mutations in either or both HREs that significantly reduce NR binding without altering the coding of any proteins were constructed. Competition for replication between mutant and wild-type viruses in low-multiplicity coinfections indicated that the +1 HRE offered a significant selective advantage to the virus within a few cycles of infection in African green monkey kidney cell lines CV-1, CV-1P, TC-7, MA-134, and Vero but not in CV-1PD' cells. Interestingly, the +55 HRE offered a selective disadvantage in MA-134 cells but had no effect in CV-1, CV-1P, TC-7, Vero, and CV-1PD' cells. Thus, we conclude that these HREs are biologically important to the virus, but in a cell type-specific manner.     

Activity of simian virus 40 late promoter elements in the absence of large T antigen: evidence for repression of late gene expression.

We used chloramphenicol acetyltransferase transient expression to examine the activity of the promoter elements of the simian virus 40 late promoter in the absence of large T antigen. Since the experiments were done in permissive CV-1 cells, these conditions mimic the state which exists early in the viral lytic cycle before the onset of replication and T-antigen-mediated trans activation. Our data, using deletion analysis, indicate that removal of the 21-base-pair (bp) repeat region causes as much as a 10-fold increase in activity of the late promoter elements. This result suggests that the 21-bp repeat sequences may be involved in repression of the late promoter elements during the early phase of the lytic infection. This is supported by competition analysis which indicates that increasing amounts of competitor containing only the 21-bp repeat region results in increased activity of the intact promoter. A model for the activity of the late promoter through the course of lytic infection is presented.     

Activation of mutated simian virus 40 enhancers by amplification of wild-type enhancer elements.

We show that duplication of any one of three separate simian virus 40 enhancer elements, A, B, or C, can compensate for loss of function in the remaining two. Simian virus 40 revertants containing point mutations within the A and C (dpm16) or B and C (dpm26) enhancer elements contain tandem duplications that include the remaining wild-type element. These simple tandem duplications can create enhancers 25-fold more active than that of the parental mutant. These revertants can arise by illegitimate recombination between heterologous viral genomes. This was demonstrated by the recombinants resulting from a mixed infection with the viruses dpm16 and dpm2, which contain mutations in the A and C elements and the B element, respectively.     

Characterization of enhancer elements in the long terminal repeat of Moloney murine sarcoma virus

A series of recombinant molecules were constructed which direct the expression of the easily assayed gene chloramphenicol acetyltransferase. We have used these recombinants to show that the 73/72-base-pair tandem repeat unit from the Moloney murine sarcoma virus long terminal repeat shares a number of properties with the prototypic enhancer element, the simian virus 40 72-base-pair repeat. Specifically, the Moloney murine sarcoma virus sequence significantly enhances the level of gene expression at both 5' and 3' locations and in either orientation relative to the test gene. It is able to enhance gene activity both from its own promoter and from a heterologous (simian virus 40) promoter. The 73/72-base-pair subunits of the Moloney murine sarcoma virus enhancer differ in sequence by four nucleotides and also in the strength of their enhancer function. The promoter distal A repeat is at least three times as active as the promoter proximal B repeat in enhancing chloramphenicol acetyltransferase expression. Results of these studies also show that the enhancer sequence alone is unable to induce gene activity but requires other promoter elements, including a proximal GC-rich sequence and the Goldberg-Hogness box.     

Characterization of the 5''-terminal capped structures of late simian virus 40-specific mRNA.

32P-labeled, late simian virus 40-specific RNA was isoalted from infected CV1 cells and completely degraded with RNase T2 and bacterial alkaline phosphatase. The RNase-resistant material was fractionated two dimensionally and further characterized with Penicillium nuclease and nucleotide pyrophosphatase. Two major 5' termini were identified in late simian virus 40 RNA, namely, 7-methyl Gppp 2',6-dimethyl ApUp and 7-methyl Gppp 2',6-dimethyl Ap 2'-methyl, UpUp. Both 5' termini are present in unfractionated viral RNA as well as in the separated 16S and 19S species. As both caps differ only in secondary modification, it is possible that they are derived from the same site on the DNA. The relatively higher cap II content of the 16S mRNA may be related to its slower rate of turnover.