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M J Tevethia

Publications and source records attributed to M J Tevethia.

At least 37 records · Page 2Linked to original sources
PubMed1993

Association of p53 binding and immortalization of primary C57BL/6 mouse embryo fibroblasts by using simian virus 40 T-antigen mutants bearing internal overlapping deletion mutations.

To more precisely map the immortalization and p53 binding domains of T antigen, a large series of overlapping deletion mutations were created between codons 251 to 651 by utilizing a combination of Bal 31 deletion and oligonucleotide-directed mutagenesis. Immortalization assay results indicated that amino acids (aa) 252 to 350, 400, and 451 to 532 could be removed without seriously compromising immortalization, although the appearance of immortal colonies was delayed in some cases. Western immunoblotting experiments indicated that the p53 binding capacities of T antigen produced by mutants missing aa 252 to 300, 301 to 350, 400, or 451 to 532 were only slightly reduced relative to that of wild-type T antigen. Within the limits of this deletion analysis, the immortalization and p53 binding domains appear to be colinear and, in fact, may represent two aspects of the same domain. This deletion analysis eliminates the entire zinc finger domain (aa 302 to 320), a small portion of the leucine-rich region (aa 345 to 350), and a large portion of the ATP binding domain (aa 451 to 528) as participants in p53 binding or in the immortalization process. The results also show that removal of T antigen amino acids within the region 451 to 532 appears to alter the capacity of newly synthesized but not older T antigen and p53 molecules to form complexes.

Animals↗
PubMed1992

Cytotoxic T lymphocytes (CTL) against a transforming gene product select for transformed cells with point mutations within sequences encoding CTL recognition epitopes.

The 94-kD large tumor (T) antigen specified by simian virus 40 (SV40) is sufficient to induce cell transformation. T antigen contains four H-2Db-restricted cytotoxic T lymphocyte (CTL) recognition epitopes that are targets for CTL clones Y-1, Y-2, Y-3, and Y-5. These epitopes have been mapped to T antigen amino acids 207-215 (site I), 223-231 (sites II and III), and 489-497 (site V), respectively. Antigenic site loss variant cells that had lost one or more CTL recognition epitopes were previously selected by coculturing SV40-transformed H-2Db cells with the site-specific Db-restricted CTL clones. The genetic bases for T antigen CTL recognition epitope loss from the variant cells were identified by DNA amplification and direct sequencing of epitope-coding regions from variant cell DNAs. Cells selected for resistance to CTL clone Y-1 (K-1; K-1,4,5; K-3,1) carry deleted SV40 genomes lacking site I, II, and III coding sequences. Point mutations present within the site II/III coding region of Y-2-/Y-3-resistant cell lines specify the substitution of asparagine for lysine as T antigen amino acid 228 (K-2) or phenylalanine for tyrosine at position 230 (K-3). Point mutations identified within independently selected Y-5 resistant populations (K-5 and K-1,4,5) direct the substitution of isoleucine for asparagine at position 496 (K-5) or the substitution of phenylalanine for isoleucine at position 491 (K-1,4,5) of T antigen. Each substitution causes loss of the relevant CTL recognition epitope, apparently by compromising CTL T cell receptor recognition. These experiments identify specific amino acid changes within a transforming protein that facilitate transformed cell escape from site-specific CTL clones while allowing maintenance of cellular transformation. This experimental model system provides unique opportunities for studying mechanisms of transformed cell escape from active immunosurveillance in vivo, and for analysis of differential host immune responses to wild-type and mutant cell-transforming proteins.

Amino Acid Sequence↗
PubMed1991

Localization of common cytotoxic T lymphocyte recognition epitopes on simian papovavirus SV40 and human papovavirus JC virus T antigens.

Human papovavirus JC virus (JCV) and Simian virus 40 (SV40) tumor or T antigens demonstrate considerable sequence homology which is reflected by antibody cross-reactivity. This similarity raised the possibility that JCV and SV40 T antigen also might contain common cytotoxic T lymphocyte (CTL) recognition epitopes. In this study we identified and mapped such sites on the JCV T antigen. C57Bl/6 cell lines transformed by JCV/SV40 T antigen chimeras were generated and tested for susceptibility to lysis by five H-2b restricted SV40-specific CTL clones: Y-1, Y-2, Y-3, Y-4, and Y-5. These CTL clones recognize specific epitopes within amino acids 205-219 (site I), 220-233 (sites II and III), 369-511 (site IV), and 489-503 (site V) on SV40 T Ag, respectively. The results show that sites I, II, III, and IV (recognized by CTL clones Y-1, Y-2, Y-3, and Y-4, respectively) represent common epitopes on SV40 and JCV T antigens. CTL clone Y-5 failed to recognize JCV T antigen indicating that CTL can discriminate between the two antigenically related T antigens.

Amino Acid Sequence↗
PubMed1990

Dissociation of Rb-binding and anchorage-independent growth from immortalization and tumorigenicity using SV40 mutants producing N-terminally truncated large T antigens.

The large T antigen of SV40 is both necessary and sufficient for conversion of primary mouse cells to cells with fully transformed phenotype. In this investigation, the influence of the N-terminal portion of T antigen on individual transformed cell characteristics was probed by using mutants bearing deletions in the 5'T antigen coding sequence. Specifically, DNA constructs expected to produce T antigens missing the first 109, 127, 150, or 176 amino acids or internal amino acid segments between 117 and 250 were tested for the ability to immortalize C57Bl/6 mouse embryo fibroblasts. The transformed cell properties displayed by clonally derived cell lines were then examined. The results indicated that neither the first 127 amino acids nor amino acids 127-250 of T antigen were necessary for efficient immortalization of primary cells or for their tumorigenicity. Functions mapped within these regions, including binding of the retinoblastoma susceptibility gene product (Rb) and transactivation of heterologous promoters, therefore, were not required to confer either of these growth properties. In addition the results showed that anchorage-independent growth was separable genetically from tumorigenicity and that removal of amino acids within the first 250 residues of T antigen compromised other transformed cell growth properties.

Animals↗
PubMed1989

Temperature-sensitive mutants identify crucial structural regions of simian virus 40 large T antigen.

We have completed the cloning and sequencing of all known temperature-sensitive, amino acid substitution mutants of simian virus 40 large T antigen (tsA mutants). Surprisingly, many of the mutants isolated from distinct viral strains by different laboratories are identical. Thus, 17 independently isolated mutants represent only eight distinct genotypes. This remarkable clustering of tsA mutations in a few "hot spots" in the amino acid sequence of T antigen and the temperature-sensitive phenotypes of the mutations strongly suggest that these amino acids play crucial roles in organizing the structure of one or more functional domains. Most of the mutations are located in highly conserved regions of T antigen that correlate with DNA binding, protein-protein interactions, or ATP binding. With the exception of one mutant with a lesion in the putative ATP-binding region, all the mutants are temperature sensitive for DNA replication.

Adenosine Triphosphate↗
PubMed1988

Requirements for immortalization of primary mouse embryo fibroblasts probed with mutants bearing deletions in the 3' end of SV40 gene A.

The influence of specific contiguous stretches of amino acids predominantly in the carboxy terminal third of the SV40 large T antigen on the immortalization of cells in culture was investigated. Mutants that bear either small in-phase or frameshift deletions in the large T antigen coding sequence were transfected into primary mouse embryo fibroblasts of C57Bl/6 origin (B6/MEF). The frequency of immortalization was determined as the number of colonies that developed from cells escaping senescence. The results indicated that the terminal 81 amino acids of large T antigen are not needed for efficient immortalization or tumorigenicity. In contrast removal of as few as three amino acids encoded in the vicinity of the Dde-1 site at 0.234 map units (m.u.) severely restricted immortalization, suggesting that this region of the coding sequence either structurally or functionally is essential to at least one parameter of the transformed cell phenotype. The T antigen produced by dlA2433 which bears a deletion of nine nucleotides at 0.234 m.u. fails to associate stably with the cellular protein p53. The results showed that the addition of long stretches of amino acids (96 or 97 residues) from the open reading frame at the 3' end of the early region inactivated immortalizing functions, although the addition of as many as 18 amino acids from other reading frames was not detrimental. The evidence presented also confirmed that wild-type levels of ATPase activity are not necessary for immortalization or tumorigenicity of B6/MEF. Finally, we show that one of the mutants that immortalized primary cells did not produce dense foci on a cell monolayer. This last result indicated that independent functions are required for these two parameters of the transformed cell phenotype.

Animals↗
PubMed1988

Recognition of simian virus 40 T antigen synthesized during viral lytic cycle in monkey kidney cells expressing mouse H-2Kb- and H-2Db-transfected genes by SV40-specific cytotoxic T lymphocytes leads to the abrogation of virus lytic cycle.

Simian virus 40 (SV40)-encoded tumor or T antigen localizes in the membranes in addition to the nucleus of SV40-infected permissive monkey cells and SV40-transformed nonpermissive cells. The surface T antigen in SV40-transformed mouse cells provides a target for the cytotoxic T lymphocytes (CTL) which recognize SV40 T antigen in association with murine K/D, class I H-2 antigens. In order to demonstrate that SV40 T antigen synthesized in SV40-infected permissive monkey kidney cells (TC-7) may also function as a target for CTL, cloned murine H-2Db and H-2Kb genes were expressed in TC-7 cells by DNA transfection and TC-7 cell lines expressing high levels of either H-2Kb or H-Db antigens were established after cell sorting. SV40-infected TC-7/H-2Kb and TC-7/H-2Db cells became susceptible to lysis by SV40-specific H-2b restricted CTL. The susceptibility of these transfected SV40-infected monkey cells to anti-SV40 bulk culture CTL and SV40-specific H-2Db- and H-2Db-restricted CTL clones depended upon the synthesis of SV40 T antigen and the expression of the appropriate H-2Kb or H-2Db restriction elements. Treatment of SV40-infected TC-7/H-2Db and TC-7/H-2Kb with CTL clones abrogated the virus lytic cycle indicating that CTL may play an important role in limiting papovavirus infection in the natural host.

Animals↗
PubMed1988

Clustering of antigenic sites recognized by cytotoxic T lymphocyte clones in the amino terminal half of SV40 T antigen.

The distribution of antigenic sites recognized by cytotoxic T lymphocytes (CTL) in the amino terminal half of SV40 T antigen was studied using SV40-specific CTL clones. Spleen cells of C57BL/6 (B6) mice immunized with B6/pSV3T3-20GV cells, which synthesize a truncated SV40 T antigen of amino acids 1-368, were restimulated in vitro with B6/pPVU-5-70K cells expressing SV40 T antigen of amino acids 109-708 and then cloned. The recognition sequence for all 10 CTL clones established mapped in the amino terminal half of SV40 T antigen between amino acids 109 and 271. Fine mapping of these 10 CTL clones defined three distinct antigenic sites. These three sites were abolished by the deletion of SV40 T antigen amino acids 193-211, 220-223, and 220-228, respectively. Additional CTL clones were established from spleen cells of B6 mice immunized with B6-K/S11-S24 cells, which synthesize a SV40 T antigen missing amino acids 127-250. None of these CTL clones reacted with B6/pSV3T3-20GV cells. These CTL clones recognized an antigenic site(s) which mapped in the carboxy terminal half of SV40 T antigen. Our results indicate that the antigenic sites in the amino terminal half of SV40 T antigen are tightly clustered between amino acids 193 and 271 and most probably between 193 and 228.

Animals↗
PubMed1988

Fine mapping two distinct antigenic sites on simian virus 40 (SV40) T antigen reactive with SV40-specific cytotoxic T-cell clones by using SV40 deletion mutants.

The existence of two distinct antigenic sites at the surface of simian virus 40 (SV40)-transformed H-2b cells has been previously demonstrated (A. E. Campbell, L. F. Foley, and S. S. Tevethia, J. Immunol. 130:490-492, 1983) by using two independently isolated SV40-specific cytotoxic T-lymphocyte (CTL) clones, K11 and K19. We identified amino acids in the amino-terminal half of SV40 T antigen that are essential for the recognition of antigenic sites by these CTL clones by using H-2b cells transformed by mutants that produce T antigen truncated from the amino-terminal or carboxy-terminal end or carrying overlapping internal deletions in the amino-terminal regions of SV40 T antigen. The results show that CTL clone K11 failed to recognize and lyse target cells missing SV40 T-antigen amino acids 189 to 211, whereas CTL clone K19 lysed these cells. The cell lines missing SV40 T-antigen amino acids 220 to 223 and 220 to 228 were not lysed by CTL clone K19 but were susceptible to lysis by CTL clone K11. Two other cell lines missing amino acids 189 to 223 and 189 to 228 of SV40 T antigen were not lysed by either of the CTL clones but were lysed by SV40-specific bulk-culture CTL if sufficient amounts of relevant restriction elements were expressed at the cell surface. The SV40 T-antigen amino acids critical for the recognition of an antigenic site by CTL clone K11 were identified to be 193 to 211; 220 to 223 were identified as critical for recognition by CTL clone K19. The deletion of these amino acids from the T antigen resulted in the loss of antigenic sites specific for CTL clones K11 and K19.

Amino Acid Sequence↗
PubMed1987

Participation of two human cytomegalovirus immediate early gene regions in transcriptional activation of adenovirus promoters.

The participation of human cytomegalovirus (HCMV) immediate early genes in the activation of the expression of adenovirus genes in trans (trans-activation) was examined. The initial strategy used was to determine the ability of HCMV genes to complement mutants of adenovirus E1a, an immediate early gene which encodes a trans-activator. The HCMV immediate early gene regions IE1 and IE2 complemented E1a-deficient mutants in three separate assays. IE1 and IE2 substituted for E1a in the synthesis of infectious adenovirus, late adenovirus RNA, and adenovirus DNA. Complementation by the IE2 gene region alone, but not by IE1 alone, was observed using the most discriminating assay, that for late adenovirus RNA synthesis. A role for both HCMV gene regions in positive transcriptional control was indicated by their ability to increase expression of chloramphenicol acetyltransferase (CAT) mediated by the adenovirus E2a promoter. The IE2 region alone activated CAT synthesis but IE1 alone had no detectable activity. Moreover, the activity of both gene regions was about 10-fold higher than that of IE2 alone. These data indicate that efficient complementation of E1a-deficient mutants and trans-activation of adenovirus early promoters involved the participation of both HCMV immediate early gene regions.

Adenoviruses, Human↗
PubMed1986

Influence of amino acids encoded in the 3' open reading frame of the SV40 early region on transformation and antigenicity of large T antigen.

The mutant dlA2414 bears a frame-shift deletion of nucleotides 2936-2927 in the coding sequence for the simian virus 40 (SV40) large T antigen. Based on its nucleotide sequence, this mutant should produce a T antigen containing the first 627 authentic large T antigen amino acids followed by 97 amino acids encoded in the alternate open reading frame at the 3' end of the early region. This protein resembles the hypothetical T* protein that would be translated from an early SV40 mRNA if it were spliced to permit utilization of the open reading frame. We show that stable mouse cell lines can be generated that express the T antigen produced by dlA2414 and that this T antigen has an altered carboxy terminus. In addition, the expected tryptic peptides were missing from the large T antigen and replaced by more hydrophobic peptides. The T*-like protein produced by dlA2414 was apparently less stable than wild-type T antigen and did not stably complex with the cellular phosphoprotein p53. This protein retained the ability to immunize mice against a challenge of syngeneic SV40-tumor cells. The dlA2414 T antigen was expressed at the surface of cells as shown by in vitro lymphocyte-mediated cytotoxicity assay. The results presented here also showed that the expression of a T*-like protein at the cell surface is not likely to be essential for tumorigenesis of cells transformed by SV40.

Amino Acids↗
PubMed1986

Identification of a human cytomegalovirus virus DNA segment that complements an adenovirus 5 immediate early mutant.

Human cytomegalovirus (HCMV) complements adenovirus mutant dl312, which is completely defective for expression of the adenovirus immediate early E1a gene region, for lytic growth (Tevethia and Spector, 1984). This assay defines at least one HCMV function, activation of the transcription of adenovirus early genes in trans, that must be provided for dl312 replication in coinfected cells. We show here that trans-activation depended on the expression of one or more HCMV gene. Human embryonic lung cells were transfected with dl312 DNA-protein complex and either HCMV DNA or recombinant plasmids containing the adenovirus E1a gene region or HCMV (Towne strain) DNA fragments. Replication of dl312 occurred only in cells that received both DPC and either the E1a gene, HCMV DNA, or the XbaI-E HCMV DNA fragment (0.68 to 0.77 map units). In addition, we show that HCMV also complemented adenovirus mutant pm975 for growth in 0.2% serum. Since pm975 grows poorly in low concentrations of serum due to a defect in an E1a gene product, this assay identified a second HCMV E1a-like function.

Adenoviruses, Human↗
PubMed1985

Abrogation of simian virus 40 DNA-mediated transformation of primary C57BL/6 mouse embryo fibroblasts by exposure to a simian virus 40-specific cytotoxic T-lymphocyte clone.

Primary mouse embryo fibroblasts of C57BL/6 origin (B6/MEF) were transformed in vitro by transfection with simian virus 40 (SV40) DNA. The transformation frequency was markedly reduced if the SV40 DNA-transfected cultures were briefly exposed to K11 cells, an SV40-specific clone of cytotoxic T lymphocytes. This abrogation of SV40 transformation in vitro by cytotoxic T-lymphocyte clone K11 was specific, since transformation of B6/MEF cells by adenovirus type 5 DNA was not affected. The approach described here should serve as an ideal model of dissecting immunological events during in vivo tumorigenesis.

Adenoviruses, Human↗
PubMed1985

Cytotoxic T-lymphocyte recognition of simian virus 40 antigens.

During the transformation of mammalian cells by SV40, the cells acquire new antigenicity at the cell surface that acts as a target for cytotoxic T lymphocytes. Evidence is discussed which leads to the conclusion that the product of the SV40 tsA gene, the T antigen, is directly involved in converting a cell to malignant phenotype, is largely localized in the nucleus of transformed cells and is also present at the cell surface. Therefore, it appears that the T antigen serves a dual function, i.e. it is involved in the initiation and maintenance of transformation and provides a target for the immune effector mechanisms in the host undergoing viral carcinogenesis.

Animals↗
PubMed1984

Immortalization of primary mouse embryo fibroblasts with SV40 virions, viral DNA, and a subgenomic DNA fragment in a quantitative assay.

In order to investigate the capacity of the N-terminal half of large T antigen to convert primary cells to continuous cell lines conditions were standardized for transforming primary mouse embryo fibroblasts of C57Bl/6 origin (B6/MEF) with DNA. Using these conditions the transforming capability of SV40 virions, viral DNA, and a plasmid, pSV3T3-20-GV (C.E. Clayton, D. Murphey, M. Lovett, and P.W.J. Rigby, Nature (London), 299, 59-61, 1982) containing the 5' half of the SV40 early region, was determined in a quantitative immortalization assay. The plasmid pSV3T3-20-GV transformed B6/MEF at only 1/16 the frequency of a plasmid containing the entire early region. These results suggest that the 3' half of large T antigen which cannot be produced by this plasmid strongly influences the frequency of immortalization. It is not known whether this influence reflects the presence of a transformation domain in the carboxy terminus of large T antigen or, alternatively, results from an altered conformation or stability of the truncated polypeptide.

Animals↗
PubMed1984

Complementation of an adenovirus 5 immediate early mutant by human cytomegalovirus.

The initiation of the lytic cycle of adenovirus 5 requires synthesis of a transcriptional activator encoded by the viral early genetic unit, E1a. Mutant viruses lacking E1a are defective. Human cytomegalovirus activated transcription of early adenovirus genes and complemented an E1a- adenovirus mutant in cells permissive for both viruses.

Adenoviruses, Human↗
PubMed1984

Simian virus 40 specific cytotoxic lymphocyte clones localize two distinct TSTA sites on cells synthesizing a 48 kD SV40 T antigen.

Simian virus 40 specific antigenic sites (TSTA) which react with SV40 specific cytotoxic lymphocytes (CTL) were localized on the surface of mouse embryo fibroblasts (H-2b) transformed with a recombinant plasmid which contain SV40 large T antigen coding DNA sequences (0.65-0.37 map units). These cells (B6/pSV-20-GV) synthesize a large T antigen polypeptide of 48 kD and could be lysed with two independently isolated CTL clones which recognize distinct antigenic sites on SV40 transformed cell surface. These results suggest that at least two distinct TSTA sites are present in cells synthesizing only the amino terminal half of SV40 T antigen.

Animals↗