Identification of FAM111A as an SV40 Host Range Restriction and Adenovirus Helper Factor

The small genome of polyomaviruses encodes a limited number of proteins that are highly dependent on interactions with host cell proteins for efficient viral replication. The SV40 large T antigen (LT) contains several discrete functional domains including the LXCXE or RB-binding motif, the DNA binding and helicase domains that contribute to the viral life cycle. In addition, the LT C-terminal region contains the host range and adenovirus helper functions required for lytic infection in certain restrictive cell types. To understand how LT affects the host cell to facilitate viral replication, we expressed full-length or functional domains of LT in cells, identified interacting host proteins and carried out expression profiling. LT perturbed the expression of p53 target genes and subsets of cell-cycle dependent genes regulated by the DREAM and the B-Myb-MuvB complexes. Affinity purification of LT followed by mass spectrometry revealed a specific interaction between the LT C-terminal region and FAM111A, a previously uncharacterized protein. Depletion of FAM111A recapitulated the effects of heterologous expression of the LT C-terminal region, including increased viral gene expression and lytic infection of SV40 host range mutants and adenovirus replication in restrictive cells. FAM111A functions as a host range restriction factor that is specifically targeted by SV40 LT.     

DJ-1, an oncogene and causative gene for familial Parkinson’s disease, is essential for SV40 transformation in mouse fibroblasts through up-regulation of c-Myc

Simian virus 40 (SV40) is a tumor virus and its early gene product large T-antigen (LT) is responsible for the transforming activity of SV40. Parkinson’s disease causative gene DJ-1 is also a ras-dependent oncogene, but the mechanism of its oncogene function is still not known. In this study, we found that there were no transformed foci when fibroblasts from DJ-1-knockout mice were transfected with LT. We also found that DJ-1 directly bound to LT and that the expression level of c-Myc in transformed cells was parallel to that of DJ-1. These findings indicate that DJ-1 is essential for SV40 transformation.

Structured summary

MINT-7988969: DJ-1 (uniprotkb:Q99497) binds (MI:0407) to LT SV40 (uniprotkb:P03070) by pull down (MI:0096) MINT-7988948: LT SV40 (uniprotkb:P03070) physically interacts (MI:0914) with DJ-1 (uniprotkb:Q99LX0) and p53 (uniprotkb:P02340) by anti bait coimmunoprecipitation (MI:0006)     

Vectors bicistronically linking a gene of interest to the SV40 large T antigen in combination with the SV40 origin of replication enhance transient protein expression and luciferase reporter activity

The simian virus 40 large T antigen (SVLT) induces replication of plasmids bearing the SV40 origin of replication (SV40 ori) within mammalian cells. The internal ribosomal entry site (IRES) is an element that allows for the cotranslation of proteins from one polycistronic mRNA. Through the combination of these elements, IRES-dependent coexpression of a protein of interest and the SVLT, either constitutive or regulated, on plasmids bearing the SV40 ori generates a positive feedback loop, resulting in enhanced expression. A vector linking red fluorescent protein (RFP) to the IRES-SVLT element enhances fluorescence ~10-fold over that demonstrated from a vector lacking this element. In transfection-resistant CV-1 cells, the RFP-IRES-SVLT vector substantially increases the number of cells expressing detectable levels of RFP. Furthermore, inclusion of the IRES-SVLT/SV40 ori elements in standard luciferase-based reporter gene constructs and associated effectors results in marked increases in luminescent output and sensitivity, using the β-catenin/TCF pathway and the mammalian two-hybrid assay as models. Ultimately, vector systems combining these well-established elements (IRES-SVLT/SV40 ori) will increase the utility of transient transfection for the production of recombinant proteins, the use of transfection-resistant cell lines, and the effectiveness of luciferase-based high-throughput screening assays.     

MDM2 is a target of simian virus 40 in cellular transformation and during lytic infection.

Phosphopeptide analyses of the simian virus 40 (SV40) large tumor antigen (LT) in SV40-transformed rat cells, as well as in SV40 lytically infected monkey cells, showed that gel-purified LT that was not complexed to p53 (free LT) and p53-complexed LT differed substantially in their phosphorylation patterns. Most significantly, p53-complexed LT contained phosphopeptides not found in free LT. We show that these additional phosphopeptides were derived from MDM2, a cellular antagonist of p53, which coprecipitated with the p53-LT complexes, probably in a trimeric LT-p53-MDM2 complex. MDM2 also quantitatively bound the free p53 in SV40-transformed cells. Free LT, in contrast, was not found in complex with MDM2, indicating a specific targeting of the MDM2 protein by SV40. This specificity is underscored by significantly different phosphorylation patterns of the MDM2 proteins in normal and SV40-transformed cells. Furthermore, the MDM2 protein, like p53, becomes metabolically stabilized in SV40-transformed cells. This suggests the possibility that the specific targeting of MDM2 by SV40 is aimed at preventing MDM2-directed proteasomal degradation of p53 in SV40-infected and -transformed cells, thereby leading to metabolic stabilization of p53 in these cells.     

Plasmalemmal vesicle associated protein (PV1) modulates SV40 virus infectivity in CV-1 cells

Plasmalemmal vesicle associated protein (Plvap/PV1) is a structural protein required for the formation of the stomatal diaphragms of caveolae. Caveolae are plasma membrane invaginations that were implicated in SV40 virus entry in primate cells. Here we show that de novo Plvap/PV1 expression in CV-1 green monkey epithelial cells significantly reduces the ability of SV40 virus to establish productive infection, when cells are incubated with low concentrations of the virus. However, in presence of high viral titers PV1 has no effect on SV40 virus infectivity. Mechanistically, PV1 expression does not reduce the cell surface expression of known SV40 receptors such as GM1 ganglioside and MHC class I proteins. Furthermore, PV1 does not reduce the binding of virus-like particles made by SV40 VP1 protein to the CV-1 cell surface and does not impact their internalization when cells are incubated with either high or low VLP concentrations. These results suggest that PV1 protein is able to block SV40 infectivity at low but not at high viral concentration either by interfering with the infective internalization pathway at the cell surface or at a post internalization step.     

Simian virus 40 large T antigen disrupts genome integrity and activates a DNA damage response via Bub1 binding

Simian virus 40 (SV40) large T antigen (LT) is a multifunctional protein that is important for viral replication and oncogenic transformation. Previously, infection of monkey or human cells with SV40 was shown to lead to the induction of DNA damage response signaling, which is required for efficient viral replication. However, it was not clear if LT is sufficient to induce the damage response and, if so, what the genetic requirements and functional consequences might be. Here, we show that the expression of LT alone, without a replication origin, can induce key DNA damage response markers including the accumulation of γ-H2AX and 53BP1 in nuclear foci. Other DNA damage-signaling components downstream of ATM/ATR kinases were induced, including chk1 and chk2. LT also bound the Claspin mediator protein, which normally facilitates the ATR activation of chk1 and monitors cellular replication origins. Stimulation of the damage response by LT depends mainly on binding to Bub1 rather than to the retinoblastoma protein. LT has long been known to stabilize p53 despite functionally inactivating it. We show that the activation of a DNA damage response by LT via Bub1 appears to play a major role in p53 stabilization by promoting the phosphorylation of p53 at Ser15. Accompanying the DNA damage response, LT induces tetraploidy, which is also dependent on Bub1 binding. Taken together, our data suggest that LT, via Bub1 binding, breaches genome integrity mechanisms, leading to DNA damage responses, p53 stabilization, and tetraploidy.     

Targeting of p300/CREB binding protein coactivators by simian virus 40 is mediated through p53

The primary transforming functions of simian virus 40 large T antigen (SV40 LT) are conferred primarily through the binding and inactivation of p53 and the retinoblastoma family members. Normal p53 function requires an association with the CREB binding protein (CBP)/p300 coactivators, and a ternary complex containing SV40 LT, p53, and CBP/p300 has been identified previously. In this report, we have evaluated a secondary function of p53 bound to the SV40 LT complex in mediating the binding of human CBP/p300. We demonstrate that p53 associated with SV40 LT was posttranslationally modified in a manner consistent with the binding of CBP/p300. Furthermore, expression of SV40 LT induced the proportion of p53 phosphorylated on S15. An essential function for p53 in bridging the interaction between SV40 LT and CBP/p300 was identified through the reconstitution of the SV40 LT-CBP/p300 complex upon p53 reexpression in p53-null cells. In addition, the SV40 LT-CBP/p300 complex was disrupted through RNA interference-mediated depletion of endogenous p53. We also demonstrate that SV40 LT was acetylated in a p300- and p53-dependent manner, at least in part through the CH3 domain of p300. Therefore, the binding of p53 serves to modify SV40 LT by targeting CBP and p300 binding to direct the acetylation of SV40 LT.