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.     

cis-active elements from mouse chromosomal DNA suppress simian virus 40 DNA replication.

Simian virus 40 (SV40)-containing DNA was rescued after the fusion of SV40-transformed VLM cells with permissive COS1 monkey cells and cloned, and prototype plasmid clones were characterized. A 2-kilobase mouse DNA fragment fused with the rescued SV40 DNA, and derived from mouse DNA flanking the single insert of SV40 DNA in VLM cells, was sequenced. Insertion of the intact rescued mouse sequence, or two nonoverlapping fragments of it, into wild-type SV40 plasmid DNA suppressed replication of the plasmid in TC7 monkey cells, although the plasmids expressed replication-competent T antigen. Rat cells were transformed with linearized wild-type SV40 plasmid DNA with or without fragments of the mouse DNA in cis. Although all of the rat cell lines expressed approximately equal amounts of T antigen and p53, transformants carrying SV40 DNA linked to either of the same two replication suppressor fragments produced significantly less free SV40 DNA after fusion with permissive cells than those transformed by SV40 DNA without a cellular insert or with a cellular insert lacking suppressor activity. The results suggest that two independent segments of cellular DNA act in cis to suppress SV40 replication in vivo, either as a plasmid or integrated in chromosomal DNA.     

RNA replication from the simian virus 5 antigenomic promoter requires three sequence-dependent elements separated by sequence-independent spacer regions

We have previously shown for the paramyxovirus simian virus 5 (SV5) that a functional promoter for RNA replication requires proper spacing between two discontinuous elements: a 19-base segment at the 3' terminus (conserved region I [CRI]) and an 18-base internal region (CRII) that is contained within the coding region of the L protein gene. In the work described here, we have used a reverse-genetics system to determine if the 53-base segment between CRI and CRII contains additional sequence-specific signals required for optimal replication or if this segment functions solely as a sequence-independent spacer region. A series of copyback defective interfering minigenome analogs were constructed to contain substitutions of nonviral sequences in place of bases 21 to 72 of the antigenomic promoter, and the relative level of RNA replication was measured by Northern blot analysis. The results from our mutational analysis indicate that in addition to CRI and CRII, optimal replication from the SV5 antigenomic promoter requires a third sequence-dependent element located 51 to 66 bases from the 3' end of the RNA. Minigenome RNA replication was not affected by changes in the either the position of this element in relation to CRI and CRII or the predicted hexamer phase of NP encapsidation. Thus, optimal RNA replication from the SV5 antigenomic promoter requires three sequence-dependent elements, CRI, CRII and bases 51 to 66.     

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.     

Protein-DNA interactions at the simian virus 40 origin of replication

Simian Virus 40 (SV40)-encoded large T antigen has an intrinsic ATP-dependent DNA-unwinding activity which is necessary for an early step in the activation of the viral origin of replication. Isolated T antigen unwinds any double-stranded DNA, regardless of whether it is linear or circularly closed. However, initiation of DNA replication depends on an intact origin of replication, and even minor deviations from the wild-type origin sequence abolish the template activity of an origin-bearing plasmid. This discrepancy suggests that T antigen may not be sufficient for origin activation and that other, probably cellular, functions are involved. We have isolated a cellular protein, the LOB protein, which specifically interacts with the AT-rich region of the SV40 origin and which induces a pronounced bending of the bound DNA.     

Initiation of simian virus 40 DNA replication in vitro.

Exogenously added simian virus 40 (SV40) DNA can be replicated semiconservatively in vitro by a mixture of a soluble extract of HeLa cell nuclei and the cytoplasm from SV40-infected CosI cells. When cloned DNA was used as a template, the clone containing the SV40 origin of DNA replication was active, but a clone lacking the SV40 origin was inactive. The major products of the in vitro reaction were form I and form II SV40 DNAs and a small amount of form III. DNA synthesis in extracts began at or near the in vivo origin of SV40 DNA synthesis and proceeded bidirectionally. The reaction was inhibited by the addition of anti-large T hamster serum, aphidicolin, or RNase but not by ddNTP. Furthermore, this system was partially reconstituted between HeLa nuclear extract and the semipurified SV40 T antigen instead of the CosI cytoplasm. It is clear from these two systems that the proteins containing SV40 T antigen change the nonspecific repair reaction performed by HeLa nuclear extract alone to the specific semiconservative DNA replication reaction. These results show that these in vitro systems closely resemble SV40 DNA replication in vivo and provide an assay that should be useful for the purification and subsequent characterization of viral and cellular proteins involved in DNA replication.     

Denaturation of the simian virus 40 origin of replication mediated by human replication protein A.

The initiation of simian virus 40 (SV40) replication requires recognition of the viral origin of replication (ori) by SV40 T antigen, followed by denaturation of ori in a reaction dependent upon human replication protein A (hRPA). To understand how origin denaturation is achieved, we constructed a 48-bp SV40 "pseudo-origin" with a central 8-nucleotide (nt) bubble flanked by viral sequences, mimicking a DNA structure found within the SV40 T antigen-ori complex. hRPA bound the pseudo-origin with similar stoichiometry and an approximately fivefold reduced affinity compared to the binding of a 48-nt single-stranded DNA molecule. The presence of hRPA not only distorted the duplex DNA flanking the bubble but also resulted in denaturation of the pseudo-origin substrate in an ATP-independent reaction. Pseudo-origin denaturation occurred in 7 mM MgCl2, distinguishing this reaction from Mg2+-independent DNA-unwinding activities previously reported for hRPA. Tests of other single-stranded DNA-binding proteins (SSBs) revealed that pseudo-origin binding correlates with the known ability of these SSBs to support the T-antigen-dependent origin unwinding activity. Our results suggest that hRPA binding to the T antigen-ori complex induces the denaturation of ori including T-antigen recognition sequences, thus releasing T antigen from ori to unwind the viral DNA. The denaturation activity of hRPA has the potential to play a significant role in other aspects of DNA metabolism, including DNA repair.