Simian Virus 40 Gene Expression Research Articles

Addition of m6A to SV40 late mRNAs enhances viral structural gene expression and replication.

Polyomaviruses are a family of small DNA tumor viruses that includes several pathogenic human members, including Merkel cell polyomavirus, BK virus and JC virus. As is characteristic of DNA tumor viruses, gene expression in polyomaviruses is temporally regulated into an early phase, consisting of the viral regulatory proteins, and a late phase, consisting of the viral structural proteins. Previously, the late transcripts expressed by the prototypic polyomavirus simian virus 40 (SV40) were reported to contain several adenosines bearing methyl groups at the N6 position (m6A), although the precise location of these m6A residues, and their phenotypic effects, have not been investigated. Here, we first demonstrate that overexpression of the key m6A reader protein YTHDF2 induces more rapid viral replication, and larger viral plaques, in SV40 infected BSC40 cells, while mutational inactivation of the endogenous YTHDF2 gene, or the m6A methyltransferase METTL3, has the opposite effect, thus suggesting a positive role for m6A in the regulation of SV40 gene expression. To directly test this hypothesis, we mapped sites of m6A addition on SV40 transcripts and identified two m6A sites on the viral early transcripts and eleven m6A sites on the late mRNAs. Using synonymous mutations, we inactivated the majority of the m6A sites on the SV40 late mRNAs and observed that the resultant viral mutant replicated more slowly than wild type SV40. Alternative splicing of SV40 late mRNAs was unaffected by the reduction in m6A residues and our data instead suggest that m6A enhances the translation of viral late transcripts. Together, these data argue that the addition of m6A residues to the late transcripts encoded by SV40 plays an important role in enhancing viral gene expression and, hence, replication.

Simian virus 40 tumor antigen expression and immunophenotypic profile of AIDS-related non-Hodgkin's lymphoma

Simian virus 40 (SV40) is associated with some systemic non-Hodgkin's lymphomas (NHL) among HIV-positive patients, based on assays for viral DNA sequences. To investigate the possible production of the viral transforming protein, we examined age-matched case–control specimens from patients with HIV/AIDS for the expression of SV40 large tumor antigen (T-ag). Masked specimens initially examined by polymerase chain reaction (PCR) for polyomavirus and herpesvirus DNA sequences were assessed for the expression of SV40 T-ag and phenotypic lymphocyte markers by immunohistochemistry (IHC). Fifty-five systemic NHL and 25 nonmalignant lymphoid and malignant nonlymphoid tissue control cases from two HIV community programs in Texas and New Jersey were scored for IHC positivity without knowledge of the PCR results. IHC showed expression of SV40 T-ag among B-cell lymphomas, whereas none of the control tissue samples were positive for T-ag (12/55, 22% vs. 0/25, 0%; P = 0.01). SV40 T-ag expression was detected only in B-cell lymphoma specimens that contained SV40 DNA sequences. Not all lymphoma cells in a positive specimen stained for T-ag, and the reaction was lower intensity than observed in SV40 hamster tumors. SV40 T-ag was demonstrated in both primary and recurrent tumors from one patient. A germinal center B-cell-like (GCB) profile was more frequently expressed by SV40-positive tumors than in Epstein–Barr virus (EBV)-related lymphomas (10/12, 83% vs. 6/13, 46%; P = 0.05), whereas a non-GCB phenotype was more frequent in EBV-positive than in SV40-positive lymphomas (7/13, 54% vs. 2/12, 17%; P = 0.05). This study shows that SV40 gene expression occurs in a fraction of cells in some B-cell lymphomas among patients with HIV/AIDS.

Suppression of Gene Expression on the Simian Virus 40 Major Late Promoter by Human TR4 Orphan Receptor: A MEMBER OF THE STEROID RECEPTOR SUPERFAMILY

The key expression of the simian virus 40 (SV40) major late promoter could be repressed by the human TR4 orphan receptor via the +55 region of the SV40 major late promoter (nucleotide numbers 368-389, 5' -GTTA-AGGTTCGTAGGTCATGGA-3'). Using the coupled in vitro transcribed and translated TR4 orphan receptor with a molecular mass of 67.3 kilodaltons, electrophoretic mobility shift assay showed specific binding with a dissociation constant of 1.09 nM between the TR4 orphan receptor and the SV40 +55 oligonucleotides. In addition, chloramphenicol acetyltransferase assay demonstrated that this SV40 +55 region can function as a repressor via the TR4 orphan receptor, suppressing the transcriptional activities of both SV40 early and late promoters. Together, our data suggest that the TR4 orphan receptor may play an important role for the suppression of the SV40 gene expression.

Simian virus 40 late gene expression is regulated by members of the steroid/thyroid hormone receptor superfamily.

Transcription of the late genes of simian virus 40 (SV40) is repressed during the early phase of the lytic cycle of infection of binding of cellular factors, called IBP-s, to the SV40 late promoter; repression is relieved after the onset of viral DNA replication by titration of these repressors. Preliminary data indicated that one of the major components of IBP-s was human estrogen-related receptor 1 (hERR1). We show here that several members of the steroid/thyroid hormone receptor superfamily, including testis receptor 2, thyroid receptor alpha 1 in combination with retinoid X receptor alpha, chicken ovalbumin upstream promoter transcription factors 1 and 2 (COUP-TF1 and COUP-TF2), as well as hERR1, possess the properties of IBP-s. These receptors bind specifically to hormone receptor binding sites present in the SV40 major late promoter. Recombinant COUP-TF1 specifically represses transcription from the SV40 major late promoter in a cell-free transcription system. Expression of COUP-TF1, COUP-TF2, or hERR1 in monkey cells results in repression of the SV40 late promoter, but not the early promoter, in the absence of the virally encoded large tumor antigen. Overexpression of COUP-TF1 leads to a delay in the early-to-late switch in SV40 gene expression during the lytic cycle of infection. Thus, members of this superfamily can play major direct roles in regulating expression of SV40. Possibly, natural or synthetic ligands to these receptors can serve as antiviral drugs. Our findings also provide the basis for the development of assays to screen for the ligands to testis receptor 2 and hERR1.

Identification of Human TR2 Orphan Receptor Response Element in the Transcriptional Initiation Site of the Simian Virus 40 Major Late Promoter

A DNA response element (TR2RE-SV40) for the TR2 orphan receptor, a member of the steroid-thyroid hormone receptor superfamily, has been identified in the simian virus 40 (SV40) +55 region (nucleotide numbers 368-389, 5'-GTTAAGGTTCGTAGGTCATGGA-3'). Electrophoretic mobility shift assay, using in vitro translated TR2 orphan receptor with a molecular mass of 67 kilodaltons, showed a specific binding with high affinity (dissociation constant = 9 nM) for this DNA sequence. DNA-swap experiments using chloramphenicol acetyl-transferase assay demonstrated that androgen can suppress the transcriptional activities of SV40 early promoter via the interaction between this TR2RE-SV40 and the chimeric receptor AR/TR2/AR with the DNA-binding domain of the TR2 orphan receptor flanked by the N-terminal and androgen-binding domains of the androgen receptor. In addition, this TR2RE-SV40 can function as a repressor to suppress the transcriptional activities of both SV40 early and late promoters. Together, these data suggest the TR2RE-SV40 may represent the first identified natural DNA response element for the TR2 orphan receptor that may function as a repressor for the SV40 gene expression.

Losses of 3p, 11p, and 13q in EJ/ras-transformable simian virus 40-immortalized human uroepithelial cells.

Five independent clones of Simian virus 40 (SV40)-immortalized human uroepithelial cells (CK/SV-HUC) were established after transfection of HUC cultures from the same tissue donor with plasmids encoding SV40 large T and small t antigen genes. Each CK/SV-HUC clone contained a unique SV40 integration site, and all expressed similar levels of SV40 mRNA. All five clones were nontumorigenic, but clones 2, 4, and 5 tumorigenically transformed after transfection at P19 with mutant EJ/ras and also spontaneously after 40 serial passages in vitro. In contrast, CK/SV-HUC clones 1 and 3 did not transform when either approach was used. These differences in transformability among CK/SV-HUC clones could not be predicted based on differences in SV40 gene expression nor on any in vitro growth property tested. In cytogenetic analyses, a transformable clone showed losses of three chromosome arms containing putative cancer suppressor gene regions, including 3p14----pter, 13q, and 11p, whereas the nontransformable clones showed none of these losses. Thus these data indicate that genetic losses on 3p, 11p, and 13q may contribute to tumorigenic transformation of SV40-immortalized human uroepithelial cells.

Expression of simian virus 40 early and late genes in mouse oocytes and embryos

Simian virus 40 (SV40) large- and small-tumor antigens (T-Ag, t-Ag) are normally synthesized early after infection of either permissive (monkey) or nonpermissive (mouse) fibroblasts, whereas an equivalent amount of viral coat protein (V-Ag) is observed late after infection of permissive cells and only after viral DNA replication has occurred. To determine whether or not expression of these genes is regulated in the same manner during early mammalian development, SV40 DNA was injected into the nuclei of mouse oocytes and one- and two-cell embryos. In oocytes, about three times more V-Ag was produced than T-Ag, and both were synthesized concomitantly in the same cells. Viral mRNA and proteins synthesized in oocytes comigrated during gel electrophoresis with the same products synthesized in SV40-infected monkey cells. Viral gene expression required circular DNA molecules injected into the nuclei of transcriptionally and translationally active cells. Injected DNA was stable and underwent conformational changes consistent with chromatin assembly. Oocytes did not replicate either polyomavirus or SV40 DNA. Thus, the temporal order of viral gene expression is circumvented in mouse germ cells, allowing these proteins to be expressed concurrently and in equivalent amounts with no requirement for DNA replication. However, in preimplantation embryos, neither T-Ag nor V-Ag was detected by immunoprecipitation although T-Ag synthesis was demonstrated as a specific requirement for SV40 DNA replication. Thus, viral gene expression in mouse embryos as early as the one-cell stage was reduced at least 500-fold relative to that in oocytes. Similarities between SV40 gene expression in mouse oocytes and that in Xenopus oocytes suggest that germ cells in higher animals share common regulatory mechanisms.

Trans Activation of the simian virus 40 late transcription unit by T-antigen.

We have investigated the role of simian virus 40 (SV40) T-antigen in the induction of late gene expression independent of its function in amplifying templates through DNA replication. Northern blot and S1 nuclease analyses showed that stimulation occurred at the transcriptional level. At least two template elements, the T-antigen-binding sites and the 72-base-pair repeats, appeared to be important for this induction. Using template mutants, we demonstrated that deletions within T-antigen-binding site II decreased T-antigen-mediated late gene expression approximately 10- to 20-fold. In addition, multiple point mutations within a single retained copy of the SV40 72-base-pair repeat decreased T-antigen-mediated late gene expression. Using in vivo competition studies, we demonstrated that competitor DNA fragments containing the SV40 control region (nucleotides 5171 through 272) quantitatively decreased SV40 late gene expression in COS-1 cells. In contrast, competition with a plasmid containing SV40 nucleotides 1 through 294 (which removes all of T-antigen-binding site I and half of site II) was much less efficient. Finally, we demonstrated that in vivo competition experiments employing competitor fragments distal to the T-antigen-binding sites within the late template region (SV40 nucleotides 180 through 2533) resulted in superinduction of late gene expression in COS-1 cells. This finding suggests that negative factors such as repressors or attenuators may modulate late SV40 gene expression before induction. Our results are consistent with a model in which induction of late gene expression involves an interaction of the SV40 origin region with DNA-binding proteins, one of which may be T-antigen. Activation of the SV40 late transcription unit may involve induction of the SV40 enhancer or removal of a repressor-like protein or both.

Trans Activation of the simian virus 40 late transcription unit by T-antigen.

We have investigated the role of simian virus 40 (SV40) T-antigen in the induction of late gene expression independent of its function in amplifying templates through DNA replication. Northern blot and S1 nuclease analyses showed that stimulation occurred at the transcriptional level. At least two template elements, the T-antigen-binding sites and the 72-base-pair repeats, appeared to be important for this induction. Using template mutants, we demonstrated that deletions within T-antigen-binding site II decreased T-antigen-mediated late gene expression approximately 10- to 20-fold. In addition, multiple point mutations within a single retained copy of the SV40 72-base-pair repeat decreased T-antigen-mediated late gene expression. Using in vivo competition studies, we demonstrated that competitor DNA fragments containing the SV40 control region (nucleotides 5171 through 272) quantitatively decreased SV40 late gene expression in COS-1 cells. In contrast, competition with a plasmid containing SV40 nucleotides 1 through 294 (which removes all of T-antigen-binding site I and half of site II) was much less efficient. Finally, we demonstrated that in vivo competition experiments employing competitor fragments distal to the T-antigen-binding sites within the late template region (SV40 nucleotides 180 through 2533) resulted in superinduction of late gene expression in COS-1 cells. This finding suggests that negative factors such as repressors or attenuators may modulate late SV40 gene expression before induction. Our results are consistent with a model in which induction of late gene expression involves an interaction of the SV40 origin region with DNA-binding proteins, one of which may be T-antigen. Activation of the SV40 late transcription unit may involve induction of the SV40 enhancer or removal of a repressor-like protein or both.

Control of late Simian virus 40 transcription by the attenuation mechanism and transcriptionally active ternary complexes are associated with the nuclear matrix

Isolated nuclei derived from simian virus 40 (SV40)-infected cells and incubated with [alpha-32P]UTP can elongate the in vivo preinitiated SV40 late RNA, synthesizing a viral RNA species 94 nucleotides long (attenuator RNA) as well as longer RNA molecules. In contrast to newly synthesized SV40 RNA, the attenuator RNA is not associated with the nuclear matrix. Pretreating the cells with 5,6-dichloro-1-beta-ribofuranosylbenzimidazole before the incubation of isolated nuclei in vitro, enhances the accumulation of the attenuator RNA, but again it is removed from nuclei by DNase and high salt. In contrast, pretreating the cells with proflavine, an intercalating drug that interferes with RNA secondary structure, prevents the accumulation of the attenuator RNA and increases the amount of the long RNA molecules. These RNA molecules become associated with the nuclear matrix. Isolated nuclear matrices from SV40-infected cells are highly enriched in transcriptionally active ternary complexes. Thus, isolated nuclear matrices that contain from 2 to 6% of SV40 DNA are capable of synthesizing at least 35% of the viral RNA synthesized in isolated nuclei after 2 to 15 minutes incubation with [alpha-32P]UTP. The RNA synthesized in vitro on purified nuclear matrices and isolated nuclei is derived from the same regions of the viral genome, suggesting that there is an association between transcribed DNA sequences and the nuclear matrix. The results suggest a major role for the nuclear matrix in controlling SV40 gene expression.

Regulation of SV40 Gene Expression

Publisher Summary Papovaviruses simian virus 40 (SV40) and polyoma virus (PV) are the smallest DNA tumor viruses known. The constituents of the viruses are proteins, cell and virus coded, and one double-stranded DNA molecule. Mouse cells infected by the conventional virus adsorption method or by DNA transfection are nonpermissive for SV40. Semipermissive cells support SV40 or PV gene expression with a low efficiency exhibiting mostly an abortive type of response. Resistance to virus infection can be of a species-specific quality acquired during the process of cell differentiation, or a feature gained upon cell transformation. SV40 stimulates not only cell DNA but also rRNA synthesis. The purpose of this chapter is to emphasize the applicability of the microinjection technique as a tool to study the relationship between eukaryotic genes and their functions, to unravel phenotypic changes exerted by purified gene products, to investigate the regulation of DNA replication and transcription, and to get some deeper understanding of the process of DNA integration and malignant cell transformation.

DNA-transformed murine teratocarcinoma cells: regulation of expression of simian virus 40 tumor antigen in stem versus differentiated cells.

Thymidine kinase-deficient (TK-; ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21)F9 teratocarcinoma stem cells have been transformed with a recombinant plasmid genome consisting of the pBR322 genome linked to a herpes simplex virus type 1 thymidine kinase gene (HSV-1 tk) and a simian virus 40 (SV40) genome. A clonal line of stem cells was obtained that contains only one copy of plasmid DNA, which is integrated into murine chromosomal DNA through a site on the pBRR322 genome. The HSV-1 tk gene, which is adjacent to the SV40 genome, is expressed in stem cells, whereas SV40 gene expression is not detectable. If differentiation of these stem cells is induced, the differentiated cells express SV40 early gene products. Thus, we have constructed a stem cell which contains a set of genes (SV40), the expression of which is regulated differently in stem and differentiated cells. This cell line could be used to determine the mechanism of suppression of expression of these genes in stem cells.

A small RNA induced late in simian virus 40 infection can associate with early viral mRNAs.

Analysis of total cytoplasmic and polyadenylylated cytoplasmic RNA from cells lytically infected with simian virus 40 (SV40) has demonstrated the presence of a small RNA, approximately 65 nucleotides long, that is induced late in lytic infection. This small RNA is apparently specific in size and sequence and is not selected on columns of oligo(dT)-cellulose. It is homologous to a region of the early SV40 mRNAs (and to the late DNA strand), starting approximately 250 nucleotides from the 3' end of the early mRNAs (SV40 map position 0.21). The function of this RNA in the viral cycle and its source are unknown at this time; however, its temporal expression, unique sequence, and interesting region of homology within the SV40 genome suggest a possible role in the control of SV40 gene expression.

Mapping of early SV40-specific functions by microinjection of different early viral DNA fragments

DNA sequences within the early simian virus 40 (SV40) genome region responsible for virus-specific tumor (T) and U antigenicity, stimulation of cell DNA synthesis, initiation of late SV40 gene expression and helper function for human adenovirus 2 were confined by experiments using microinjection of different early SV40 DNA fragments. All these early functions were detected in cells supplied with complete early viral information. Cells microinjected with DNA fragments containing about 80, 70 or 60% of the early SV40 genome were still stained positively when challenged with antibody against T antigen, and the same cells were stimulated for cellular DNA synthesis when about 70% or more of the early viral information was available. DNA sequences at map position 0.67 are required for intracellular early viral transcription.

Regulatory function of simian virus 40 DNA replication for late viral gene expression.

Inhibition of simian virus 40 (SV40) DNA synthesis prevents late but not early viral gene expression in infected cells. To test whether the late SV40 template specificity is elicited by replicative intermediate DNA molecules (RI-DNA), we isolated subcellular fractions containing RI-DNA from SV-40-infected monkey cells and microinjected these preparations into various cell lines under conditions in which viral DNA synthesis was blocked. Late SV40 gene expression (V-antigen synthesis) was obtained by microinjection of wild-type RI-DNA or temperature-sensitive mutant A7 RI-DNA preparations at 37 degrees or 41.5 degrees, respectively, while SV40 native superhelical DNA (DNA I) at a 10-fold higher concentration failed to induce V-antigen synthesis under the restrictive conditions. V-antigen synthesis was also obtained by microinjection of partially denatured SV40 DNA or a high number of randomly nicked DNA molecules (DNA II) in the absence of DNA synthesis; both single-stranded regions and nicks are specific features of RI-DNA molecules.

Regulatory mechanism of simian virus 40 gene expression in permissive and in nonpermissive cells

Primary or continuous lines of mouse cells (3T3) are nonpermissive for simian virus 40 (SV40). Abortively infected cells synthesize tumor antigen (T antigen but not viral DNA and virus capsid protein (V antigen). V antigen, however, was obtained when SV40 DNA was injected into 3T3 cells. This late gene expression also appears to be correlated with the quantity of injected DNA molecules per 3T3 cell. T antigen formation can be detected after microinjection of only 1 to 2 DNA molecules, but the intensity of intranuclear T antigen fluorescence is significantly brighter with injection of higher concentrations of viral DNA. In permissive cells (TC7), early and late SV40 gene expression is directly related to the number of injected molecules. Microinjection of 1DNA molecule induced T and V antigen formation with the same efficiency as microinjection of 2,000 to 4,000 molecules. The question of weather late SV40 gene expression is directly related to the quantity of an early virus-specific product was approached by microinjection of early SV40 complementary RNA together with small amounts of viral DNA. V antigen was obtained in a high proportion of recipient 3T3 cells at conditions where microinjection of viral DNA alone induced T but not V antigen synthesis.