SV40 Large T Antigen Is Not Responsible for the Loss of STING in 293T Cells but Can Inhibit cGAS-STING Interferon Induction

Several DNA viruses have evolved antagonists to inhibit the cyclic GMP–AMP synthase (cGAS)-stimulator of interferon genes (STING) DNA-sensing immune pathway. This includes DNA viral oncogenes that antagonize the cGAS-STING pathway by binding STING through the LxCxE motif. The 293T human cells are widely used in biology studies as they are highly transfectable. While parental 293 cells express high levels of STING, 293T cells lack STING and are unable to induce interferon antiviral responses to cytosolic DNA. Additionally, 293T cells express the SV40 polyomavirus large T antigen (LT) which enhances the replication of transfected DNA plasmids carrying the SV40 origin of replication. Since SV40 LT also encodes the LxCxE motif, the lack of STING expression in 293T cells is commonly assumed to be due to SV40 large T antigen. We find that SV40 LT does not alter exogenously expressed and endogenous levels of STING protein. We show that STING transcription is suppressed in 293T cells but is not driven by SV40. This study also revealed that SV40 LT does indeed inhibit cGAS-STING interferon induction, but through a mechanism distinct from other DNA virus oncogenes. Collectively, these results indicate that while SV40 LT can inhibit cGAS-STING interferon induction, it does so in an unanticipated manner.

Model for T-Antigen-Dependent Melting of the Simian Virus 40 Core Origin Based on Studies of the Interaction of the Beta-Hairpin with DNA

ABSTRACT The interaction of simian virus 40 (SV40) T antigen (T-ag) with the viral origin has served as a model for studies of site-specific recognition of a eukaryotic replication origin and the mechanism of DNA unwinding. These studies have revealed that a motif termed the “beta-hairpin” is necessary for assembly of T-ag on the SV40 origin. Herein it is demonstrated that residues at the tip of the “beta-hairpin” are needed to melt the origin-flanking regions and that the T-ag helicase domain selectively assembles around one of the newly generated single strands in a manner that accounts for its 3′-to-5′ helicase activity. Furthermore, T-ags mutated at the tip of the “beta-hairpin” are defective for oligomerization on duplex DNA; however, they can assemble on hybrid duplex DNA or single-stranded DNA (ssDNA) substrates provided the strand containing the 3′ extension is present. Collectively, these experiments indicate that residues at the tip of the beta-hairpin generate ssDNA in the core origin and that the ssDNA is essential for subsequent oligomerization events.

The Cap Region of Topoisomerase I Binds to Sites near Both Ends of Simian Virus 40 T Antigen

ABSTRACT Two independent binding sites on simian virus 40 (SV40) T antigen for topoisomerase I (topo I) were identified. One was mapped to the N-terminal domain (residues 83 to 160) by a combination of enzyme-linked immunosorbent assays (ELISAs) and glutathione S-transferase (GST) pull-down assays performed with various T antigen deletion mutants. The second was mapped to the C-terminal domain (residues 602 to 708). The region in human topo I that binds to both sites in T antigen was identified by ELISAs, GST pull-down assays, and double-hexamer binding assays with topo I deletion mutants. This region corresponds to a distinct domain on topo I known as the cap region that maps from residues 175 to 433. By combining these data with information about the structure of T-antigen double hexamers associated with origin DNA, we propose that the cap region of topo I associates specifically with both ends of the double hexamer bound to the SV40 origin to initiate DNA replication.

The Simian Virus 40 Core Origin Contains Two Separate Sequence Modules That Support T-Antigen Double-Hexamer Assembly

ABSTRACT Using subfragments of the simian virus 40 (SV40) core origin, we demonstrate that two alternative modules exist for the assembly of T-antigen (T-ag) double hexamers. Pentanucleotides 1 and 3 and the early palindrome (EP) constitute one assembly unit, while pentanucleotides 2 and 4 and the AT-rich region constitute a second, relatively weak, assembly unit. Related studies indicate that on the unit made up of pentanucleotide 1 and 3 and the EP assembly unit, the first hexamer forms on pentanucleotide 1 and that owing to additional protein-DNA and protein-protein interactions, the second hexamer is able to form on pentanucleotide 3. Oligomerization on the unit made up of pentanucleotide 2 and 4 and the AT-rich region is initiated by assembly of a hexamer on pentanucleotide 4; subsequent formation of the second hexamer takes place on pentanucleotide 2. Given that oligomerization on the SV40 origin is limited to double-hexamer formation, it is likely that only a single module is used for the initial assembly of T-ag double hexamers. Finally, we discuss the evidence that nucleotide hydrolysis is required for the remodeling events that result in the utilization of the second assembly unit.

Phosphorylation of Simian Virus 40 T Antigen on Thr 124 Selectively Promotes Double-Hexamer Formation on Subfragments of the Viral Core Origin

ABSTRACT Cell cycle-dependent phosphorylation of simian virus 40 (SV40) large tumor antigen (T-ag) on threonine 124 is essential for the initiation of viral DNA replication. A T-ag molecule containing a Thr→Ala substitution at this position (T124A) was previously shown to bind to the SV40 core origin but to be defective in DNA unwinding and initiation of DNA replication. However, exactly what step in the initiation process is defective as a result of the T124A mutation has not been established. Therefore, to better understand the control of SV40 replication, we have reinvestigated the assembly of T124A molecules on the SV40 origin. Herein it is demonstrated that hexamer formation is unaffected by the phosphorylation state of Thr 124. In contrast, T124A molecules are defective in double-hexamer assembly on subfragments of the core origin containing single assembly units. We also report that T124A molecules are inhibitors of T-ag double hexamer formation. These and related studies indicate that phosphorylation of T-ag on Thr 124 is a necessary step for completing the assembly of functional double hexamers on the SV40 origin. The implications of these studies for the cell cycle control of SV40 DNA replication are discussed.

Interference of the Simian Virus 40 Origin of Replication by the Cytomegalovirus Immediate Early Gene Enhancer: Evidence for Competition of Active Regulatory Chromatin Conformation in a Single Domain

ABSTRACT Replication origins are often found closely associated with transcription regulatory elements in both prokaryotic and eukaryotic cells. To examine the relationship between these two elements, we studied the effect of a strong promoter-enhancer on simian virus 40 (SV40) DNA replication. The human cytomegalovirus (CMV) immediate early gene enhancer-promoter was found to exert a strong inhibitory effect on SV40 origin-based plasmid replication in Cos-1 cells in a position- and dose-dependent manner. Deletion analysis indicated that the effect was exerted by sequences located in the enhancer portion of the CMV sequence, thus excluding the mechanism of origin occlusion by transcription. Insertion of extra copies of the SV40 origin only partially alleviated the inhibition. Analysis of nuclease-sensitive cleavage sites of chromatin containing the transfected plasmids indicate that the chromatin was cleaved at one of the regulatory sites in the plasmids containing more than one regulatory site, suggesting that only one nuclease-hypersensitive site existed per chromatin. A positive correlation was found between the degree of inhibition of DNA replication and the decrease of P1 cleavage frequency at the SV40 origin. The CMV enhancer was also found to exhibit an inhibitory effect on the CMV enhancer-promoter driving chloramphenicol acetyltransferase expression in a dose-dependent manner. Together these results suggest that inhibition of SV40 origin-based DNA replication by the CMV enhancer is due to intramolecular competition for the formation of active chromatin structure.

Topoisomerase I Associates Specifically with Simian Virus 40 Large-T-Antigen Double Hexamer–Origin Complexes

ABSTRACT Topoisomerase I (topo I) is required for releasing torsional stress during simian virus 40 (SV40) DNA replication. Recently, it has been demonstrated that topo I participates in initiation of replication as well as in elongation. Although T antigen and topo I can bind to one another in vitro, there is no direct evidence that topo I is a component of the replication initiation complex. We demonstrate in this report that topo I associates with T-antigen double hexamers bound to SV40 origin DNA (TDH) but not to single hexamers. This association has the same nucleotide and DNA requirements as those for the formation of double hexamers on DNA. Interestingly, topo I prefers to bind to fully formed TDH complexes over other oligomerized forms of T antigen associated with the origin. High ratios of topo I to origin DNA destabilize TDH. The partial unwinding of a small-circular-DNA substrate is dependent on the presence of both T antigen and topo I but is inhibited at high topo I concentrations. Competition experiments with a topo I-binding fragment of T antigen indicate that an interaction between T antigen and topo I occurs during the unwinding reaction. We propose that topo I is recruited to the initiation complex after the assembly of TDH and before unwinding to facilitate DNA replication.

Large T-Antigen Double Hexamers Imaged at the Simian Virus 40 Origin of Replication

ABSTRACTThe initial step of simian virus 40 (SV40) DNA replication is the binding of the large tumor antigen (T-Ag) to the SV40 core origin. In the presence of Mg2+and ATP, T-Ag forms a double-hexamer complex covering the complete core origin. By using electron microscopy and negative staining, we visualized for the first time T-Ag double hexamers bound to the SV40 origin. Image processing of side views of these nucleoprotein complexes revealed bilobed particles 24 nm long and 8 to 12 nm wide, which indicates that the two T-Ag hexamers are oriented head to head. Taking into account all of the biochemical data known on the T-Ag–DNA interactions at the replication origin, we present a model in which the DNA passes through the inner channel of both hexamers. In addition, we describe a previously undetected structural domain of the T-Ag hexamer and thereby amend the previously published dimensions of the T-Ag hexamer. This domain we have determined to be the DNA-binding domain of T-Ag.