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T L Benjamin

Publications and source records attributed to T L Benjamin.

At least 55 records · Page 3Linked to original sources
PubMed1988

The middle T proteins of high and low tumor strains of polyomavirus function equivalently in tumor induction.

The PTA strain of polyomavirus induces a variety of epithelial as well as mesenchymal tumors at high frequencies, while the RA strain induces only rare mesenchymal tumors following inoculation into newborn mice. DNA sequence analysis has revealed one amino acid difference between the middle T (mT) proteins encoded by these two virus strains. To test for possible biological differences between the mT proteins we constructed a recombinant virus carrying mT-coding sequences of RA on a PTA background. The tumor profile induced by this recombinant is like that of PTA, demonstrating that the transforming protein of the low tumor strain is competent to induce a high tumor profile. We conclude that a structural determinant(s) outside of mT in the PTA virus strain is important in induction of a broad spectrum of tumors and particularly those of epithelial origin.

Animals↗
PubMed1987

Expression of influenza hemagglutinin-polyoma T-antigen fusion proteins in a rat embryo fibroblast cell line.

Plasmids encoding the amino terminal portion of an influenza virus hemagglutinin (HA) fused to polyoma virus middle T (mT) or large T (lT) sequences have been constructed. Stable expression of the chimeric proteins was obtained in established rat embryo fibroblasts following plasmid co-transfection and selection for G418 resistance. The synthesis and localization of the proteins was followed by metabolic labeling with [35S]methionine and [3H]mannose, cell fractionation, and immunoprecipitation with anti-polyoma T antibody. The HA leader and amino terminal peptide direct the synthesis of the lT and mT proteins into the endoplasmic reticulum where they undergo glycosylation, but this occurs with a very low efficiency. Most of the HA-mT and HA-lT fusion protein molecules do not enter completely into the endoplasmic reticulum, but rather achieve their normal locations in the cell as slightly higher molecular weight proteins, presumably due to the extra sequences derived from HA at their amino termini. HA-mT fusion protein is found to have associated tyrosine-specific protein kinase activity precipitable with anti-src as well as anti-T antibody, and cells expressing this fusion protein have a transformed phenotype.

Animals↗
PubMed1987

Polyomavirus tumor induction in mice: influences of viral coding and noncoding sequences on tumor profiles.

We determined the DNA sequences of the noncoding regions of two polyomavirus strains that differ profoundly in their abilities to induce tumors in mice. Differences between strains were found, both on the late side of the replication origin in the region containing known enhancer elements and on the early side of the origin, affecting the number and location of large-T-antigen-binding sites. By constructing and analyzing recombinant viruses between these high- and low-tumor strains, we attempted to localize determinants which affect the frequency and histotype of tumors. Seven recombinants were constructed and propagated in vitro, and the tumor profile of each was established by inoculation into newborn C3H mice. Recombinants containing noncoding sequences from the high-tumor strain and coding sequences from the low-tumor strain behaved like the latter, inducing tumors at a low frequency and strictly of mesenchymal origin. Reciprocal recombinants with noncoding sequences of the low-tumor strain linked to structural determinants from the high-tumor strain induced several types of epithelial tumors typical of the high-tumor strain but at reduced frequency, in addition to mesenchymal tumors. A high frequency and full diversity of epithelial tumors required, in addition to structural regions from the high-tumor strain, noncoding sequences on the early side of the origin also present in this strain. A high-tumor profile thus resulted from the combined effects of structural and regulatory determinants in the high-tumor strain, with the former affecting primarily the tissue tropism and the latter affecting the frequency of tumors. No differential effects of the enhancer regions from the late side of the origin in the two virus strains were seen in this study.

Animals↗
PubMed1987

Variations in polyoma virus genotype in relation to tumor induction in mice. Characterization of wild type strains with widely differing tumor profiles.

The authors have explored the effects of variations in mouse polyoma virus genotype on patterns of tumor formation in the mouse. Four "wild type" virus strains were surveyed. Two were highly oncogenic, inducing multiple tumors of epithelial and mesenchymal origin, at high frequency and with short latency. The other two strains were weakly oncogenic, inducing fewer tumors, solely of mesenchymal origin, and after a long latency. These sharply contrasting tumor profiles were reproduced with virus stocks derived from molecularly cloned viral genomes. Though vastly different in their oncogenic properties, these cloned viruses proved equally effective in transforming established rat fibroblasts in culture and showed the same patterns of tumor antigen expression in cultured mouse cells. Complexes of polyoma middle T antigen and pp60c-src were demonstrated in extracts of epithelial tumors induced by a highly oncogenic virus strain. It is concluded that polyoma viral genetic determinants for tumor induction in the mouse are more complex than those previously defined by the use of cell transformation systems.

Animals↗
PubMed1987

Necrotizing arterial lesions in mice-bearing tumors induced by polyoma virus.

In the course of determining tumor profiles for wild-type and recombinant mouse polyoma viruses (MPyV's), we fortuitously discovered two types of necrotizing arterial disease in polyoma tumor-bearing C3H/BiDa mice. One type, designated BLAND, consisted of foci of necrosis unaccompanied by inflammatory reaction, in the muscular coat of aorta, pulmonary arterial trunk, and primary or occasionally secondary branches of these vessels. BLAND lesions contained MPyV capsid antigen VP1 as shown by immunocytochemistry, and appeared to be the result of viral cytolytic infection within artery walls. Lesions of the second type are designated PANoid in view of their resemblance to polyarteritis nodosa in humans. PANoid lesions had much the same distribution in the arterial tree as BLAND lesions and were also focal, but had the histologic properties of highly destructive acute inflammatory reactions. Specifically, there were dense infiltrations of polymorphonuclear leukocytes in any and often all coats of the arterial wall, acute fibrinoid necrosis, endothelial proliferations, intravascular thrombosis, and in one example, rupture of the intimal and medial coats with microaneurysm formation. In the acute phase, PANoid lesions exhibited attenuation, fragmentation, and loss of elastic laminae, and in the healing phase, intimal and medial fibrosis with varying degrees of lumenal occlusion. PANoid lesions gave negative immunocytochemical reactions for MPyV capsid antigen VP1, indicating either that the antigen was not present, or that it was masked in complexes with antibody and C3. BLAND lesions were found in 51% of 459 MPyV-infected mice, while PANoid lesions were found in 11%. There was no sex predilection for either type lesion, and practically all mice with lesions fell within ages 60-200 days. We suspect the PANoid lesions are examples of immune-complex arteritis related to persistent MPyV infection, but support for this hypothesis is presently tenuous, resting entirely on the coexistence of BLAND and PANoid lesions in MPyV-infected mice and the histological resemblance of PANoid lesions to naturally occurring and experimentally induced immune complex arteritis.

Animals↗
PubMed1986

12-O-tetradecanoylphorbol-13-acetate stimulates phosphorylation of the 58,000-Mr form of polyomavirus middle T antigen in vivo: implications for a possible role of protein kinase C in middle T function.

The 58,000-Mr form (58K form) of the polyomavirus middle T antigen (mT) is a minor species distinguished by its phosphorylation in vivo on serine and by its efficient phosphorylation on tyrosine in immune complexes (B.S. Schaffhausen and T.L. Benjamin, J. Virol. 40:184-196, 1981). Here we report that the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, rapidly stimulates phosphorylation of this mT species when added to cultures of wild-type polyomavirus-infected or polyomavirus-transformed 3T3 cells. Incubation with TPA leads to an accumulation of the 58K mT species to levels 1.5- to 5-fold higher than that in untreated cells within 15 min. TPA specifically stimulates phosphorylation of the 58K mT species without affecting that of the 56K species. Mapping by partial proteolysis shows that TPA-stimulated phosphorylation occurs at or near the site in 58K mT that is normally phosphorylated in the absence of TPA. A synthetic diacyl glycerol, 1-oleoyl-2-acetyl-glycerol, also specifically stimulates phosphorylation of 58K mT in vivo, while an inactive phorbol analog does not. TPA fails to induce phosphorylation of a 58K mT species encoded by certain nontransforming virus mutants with altered mT proteins that normally fail to undergo phosphorylation at the 58K site. These results indicate that the 58K form of mT is phosphorylated by or through the action of protein kinase C. TPA treatment of infected cells also leads to increased levels of 58K mT as measured in the immune complex kinase reaction, in which mT becomes phosphorylated on tyrosine by pp60c-src. These results are discussed in terms of a possible role for protein kinase C in activating mT function(s), including the formation of stable complexes with pp60c-src.

Animals↗
PubMed1985

Expression of polyoma early gene products in E. coli.

The three products of the early region of polyoma virus have been cloned for expression in E. coli using the Tac promoter. Although the identical promoter and ribosome binding site are used in each final construction, the observed level of protein expression is different for each protein. While plasmids expressing wild type T antigens as well as a plasmid expressing the truncated Py-1387T middle T antigen lacking the membrane-anchoring sequence give rise to synthesis of proteins readily detectible by 35S-methionine labeling and immunoprecipitation, only small T and the middle T of Py-1387T are made in amounts sufficient for ready detection in total cell protein. Unlike middle T expressed in animal cells, middle T produced in E. coli is not detectibly phosphorylated. Further, the E. coli protein lacks tyrosine kinase activity.

Antigens, Viral, Tumor↗
PubMed1985

Phosphorylation of polyoma middle T antigen and cellular proteins in purified plasma membranes of polyoma virus-infected cells.

We have studied phosphorylation carried out by purified plasma membranes from polyoma virus-infected cells. When isolated membranes are incubated with [gamma-32P]ATP, polyoma virus middle T antigen (mT) becomes phosphorylated on tyrosine. Partial proteolysis mapping shows the same pattern as previously noted for mT labeled in immune complexes. Membranes labeled in vitro were also extracted and immunoprecipitated with anti-T or anti-src antibody. With either antibody, both mT and pp60c-src were brought down and shown to be labeled on tyrosine. The mT of an hr-t mutant (NG59) showed only a trace amount of labeling in membranes under the same conditions. Proteins from infected and uninfected cell membranes labeled in vitro were separated on two-dimensional gels. An acidic 40-kd phosphoprotein was labeled in uninfected cell membranes, but was not seen using membranes from wild-type virus-infected cells. Neither NG59, which encodes a defective but membrane-associated mT, nor a mutant encoding a truncated mT that fails to associate with membranes, alters the level of the 40-kd phosphoprotein in membranes labeled in vitro. These results suggest that mT, acting through pp60c-src and possibly other cellular kinases and phosphatases, can affect cell protein phosphorylation as part of the transformation process.

Adenosine Triphosphate↗
PubMed1985

Residual transforming activity of PY1178T, a mutant lacking the principal in vitro tyrosine phosphorylation site, is not affected by removal of the secondary tyrosine phosphorylation site at residue 322.

Polyoma virus mutants lacking one or both tyrosines at position 315 and 322 of wild-type middle T antigen have been constructed. The effects of the removal of these tyrosines are additive for middle T phosphorylation in immune complexes, with tyrosine 315 being the major acceptor site and 322 a secondary site. Previous studies have shown little or no effect of deletion of tyrosine 322 on transforming ability, whereas a strong effect has been seen by substitution of phenylalanine for tyrosine 315. In contrast to the phosphokinase results, there is no additive effect of combining these mutations on the viruses' transforming ability. Thus the double mutant lacking both tyrosines has the same weak transforming activity as the single mutant containing tyrosine 322 and phenylalanine 315. Phosphorylation of middle T antigen at tyrosine 322 by pp60c-src or other tyrosine-specific cellular protein kinase is therefore unimportant for transformation.

Antigens, Polyomavirus Transforming↗
PubMed1985

Virion assembly defect of polyomavirus hr-t mutants: underphosphorylation of major capsid protein VP1 before viral DNA encapsidation.

The major capsid protein of polyomavirus, VP1, was separated into at least four subspecies by isoelectric focusing. One of these subspecies was selectively extracted from purified virions by mild treatment with sodium dodecyl sulfate, leaving a 140S particle enriched in the other three forms. The two most acidic subspecies were labeled in vivo with [32P]phosphate, and these subspecies are among those identified as being deficient in nontransforming host range (hr-t) mutant virus nonpermissive infection of NIH3T3 cells. Quantitation of VP1 phosphorylation revealed that hr-t mutant virus VP1 is phosphorylated to about 40 to 50% the level of the wild type in NIH3T3 cells, and two-dimensional phosphoamino acid analysis suggested that threonine phosphorylation was affected more than serine phosphorylation. Two results indicate that the VP1 modifications occur before and independent of virus assembly: modified subspecies were detected during wild-type infection within a 2-min pulse-label with [32S]methionine, and VP1 modifications of temperature-sensitive VP1 mutants were the same at both restrictive and permissive temperatures for virus assembly. We conclude that most VP1 modification occurs before viral DNA encapsidation, and that one defect in hr-t mutant virus assembly is in VP1 phosphorylation, primarily affecting threonine.

DNA, Viral↗
PubMed1985

Regulation of cellular phenotype and expression of polyomavirus middle T antigen in rat fibroblasts.

Polyoma middle T antigen (mT) was expressed in rat F-111 cells under control of the dexamethasone-regulatable mouse mammary tumor virus promoter. Graded phenotypic responses to levels of mT induction by the hormone were seen, with morphological transformation, focus formation, and anchorage-independent growth requiring increasing levels of mT expression. The ability of different clones to form tumors reflected their maximum level of induction of mT-associated kinase and their ability to grow in soft agar. Expression of transformation parameters and tumorigenicity correlates with the level of mT phosphorylated by pp60c-src in immune complexes and not with the total amount of mT determined by metabolic labeling. We suggest that cellular factors regulate mT activity by forming a kinase-active fraction of mT molecules that controls the transformed state.

Animals↗
PubMed1984

Transformation by polyoma virus is drastically reduced by substitution of phenylalanine for tyrosine at residue 315 of middle-sized tumor antigen.

We used an oligonucleotide to introduce an A----T transversion at nucleotide position 1178 in polyoma virus DNA. The single effect of this mutation is to substitute phenylalanine for tyrosine at residue 315 of the middle-sized tumor (mT) protein (antigen). This site was previously identified as a major phosphate acceptor in the protein kinase reaction of immunocomplexes containing mT antigen. Reconstituted polyoma virus with the transversion, Py-1178-T, produces an altered mT protein that shows about 20% of the activity of wild-type mT antigen in the immunocomplex kinase assay. This residual activity appears to be directed primarily at another tyrosine at position 322 in the mT protein. The transforming ability of Py-1178-T is drastically reduced compared to wild-type virus. The efficiency of transformation by the mutant is less than 1% of that of wild type in focus assays and less than 0.1% in soft-agar growth assays. Cells identified in focus assays with Py-1178-T are generally less transformed in their phenotype than wild-type transformed cells.

Amino Acid Sequence↗
PubMed1984

A polyoma mutant that encodes small T antigen but not middle T antigen demonstrates uncoupling of cell surface and cytoskeletal changes associated with cell transformation.

The hr-t gene of polyoma virus encodes both the small and middle T (tumor) antigens and exerts pleiotropic effects on cells. By mutating the 3' splice site for middle T mRNA, we have constructed a virus mutant, Py808A, which fails to express middle T but encodes normal small and large T proteins. The mutant failed to induce morphological transformation or growth in soft agar, but did stimulate postconfluent growth of normal cells. Cells infected by Py808A became fully agglutinable by lectins while retaining normal actin cable architecture and normal levels of extracellular fibronectin. These properties of Py808A demonstrated the separability of structural changes at the cell surface from those in the cytoskeleton and extracellular matrix, parameters which have heretofore been linked in the action of the hr-t and other viral oncogenes.

Actins↗
PubMed1983

An analysis of transformed clones obtained by coinfections with hr-t and ts-a mutants of polyoma virus.

Clones of stably transformed rat cells from the F-111 (Fisher rat embryo) and NRK (normal rat kidney) cell lines have been obtained by complementation using pairs of nontransforming polyoma mutants of the hr-t and ts-a classes. A total of 78 clones were isolated and studied, 21 from the NRK line, and 57 from the F-111 line. These "complementation transformed clones" were then examined for the presence and expression of each of the parental mutant viruses. The expression of viral T (tumor) antigens was analyzed by immunoprecipitation. Every one of 54 clones examined express the 22K (small) and 56K (middle) T antigens which are the products of the hr-t viral gene. This indicates a requirement for the presence of a wild-type hr-t allele contributed by the ts-a mutant parent. Ten clones lack detectable large T antigen, while four show thermolabile large T antigen. These results support the conclusion that middle and small but not large T antigen(s) are essential for the maintenance of the transformed phenotype. Most of the 57 F-111 clones are virogenic when fused to mouse cells under permissive conditions and yield both mutant types. Surprisingly, wild-type recombinants have been recovered from 40 of the F-111 clones. Three clones show evidence of retention of only the ts-a mutant genome. Virus cannot be rescued from the majority of the 21 NRK clones. The few clones which are virogenic, however, yield both hr-t and ts-a mutants. Integration patterns of several clones analyzed by Southern blotting confirm the expectations based on viral T antigen(s) and virus rescue analyses. Hr-t mutant genomes, though not required for the maintenance of the transformed state, are frequently retained in complementation transformed clones. Tandem integration of the two parental mutants is clearly demonstrated in one clone and implicated in the other eight out of nine virogenic F-111 complementation transformed clones examined. This observation provides the basis of a model for the generation of wild-type recombinants following fusion of F-111 clones.

Animals↗
PubMed1983

Isolation and characterization of polyoma nucleoprotein complexes.

A method for the isolation of polyoma nucleoprotein complexes has been developed using neuraminidase treatment of infected cell lysates. At least three distinct forms of polyoma virion intermediates were identified by their [3H]thymidine labeling kinetics and sedimentation coefficients: a rapidly labeled 95 S "replicating complex" which chases to a 75 S minichromosome and then to a 240 S virion structure. The general properties of these distinct intermediates were similar to those found for SV40. In contrast to SV40, however, a continuum of labeled polyoma viral DNA sedimented between 240 S and 95 S. These complexes were characterized by their release from cell debris with neuraminidase, precipitation with antivirion antibody, complete disruption in 1 M NaCl, and association with hemagglutinating (HA) activity. These intermediates may represent incremental capsid protein additions to the 75 S minichromosome, hypothesized in the current models for SV40 assembly. The ability to isolate a complete complement of polyoma subviral complexes provides a basis for studying the growth defect of polyoma host-range mutants, and the properties of neuraminidase release, hemagglutination, and specific immunoprecipitation suggest purification steps for further characterization of these virion assembly intermediates.

Animals↗
PubMed1983

Host range transforming gene of polyoma virus plays a role in virus assembly.

Polyoma virus host range transforming (hr-t) mutants are blocked in virion assembly. In normal 3T3 cells, a nonpermissive host, these mutants synthesize 30-40% as much viral DNA and 80-100% as much capsid proteins as does wild-type virus and yet produce only 1-2% as much infectious virus. Intermediates in virion assembly have been followed by [3H]thymidine incorporation. hr-t mutants synthesize 95S replicating minichromosomes, which accumulate as 75S forms. However, the latter fail to undergo efficient transition to 240S virion structures. This block in encapsidation is overcome in permissive hosts such as primary baby mouse kidney (BMK) epithelial cells. The block in assembly of 240S particles is accompanied by a failure to induce a series of acidic isoelectric forms of the major capsid protein, VP1. Multiple species of post-translationally modified VP1 are seen by two-dimensional gel electrophoresis in wild-type virus-infected cells. These acidic VP1 subspecies are decreased 6- to 10-fold in hr-t mutant-infected 3T3 cells but are produced in normal amounts when the same mutants infect BMK cells. When 3T3 cells are coinfected with hr-t mutant and wild-type viruses, normal amounts of the VP1 subspecies are present, and hr-t mutant viral DNA is efficiently packaged into virions. These studies demonstrate an important role of the hr-t gene of polyoma virus in virus assembly. Specifically, we propose that VP1 is a target for hr-t gene-controlled modification and that modified forms of VP1 are essential for encapsidation of viral minichromosomes.

Animals↗