Michele M. Fluck

Cellular transformation by Simian Virus 40 and Murine Polyoma Virus T antigens.

Simian Virus 40 (SV40) and Mouse Polyoma Virus (PY) are small DNA tumor viruses that have been used extensively to study cellular transformation. The SV40 early region encodes three tumor antigens, large T (LT), small T (ST) and 17KT that contribute to cellular transformation. While PY also encodes LT and ST, the unique middle T (MT) generates most of the transforming activity. SV40 LT mediated transformation requires binding to the tumor suppressor proteins Rb and p53 in the nucleus and ST binding to the protein phosphatase PP2A in the cytoplasm. SV40 LT also binds to several additional cellular proteins including p300, CBP, Cul7, IRS1, Bub1, Nbs1 and Fbxw7 that contribute to viral transformation. PY MT transformation is dependent on binding to PP2A and the Src family protein tyrosine kinases (PTK) and assembly of a signaling complex on cell membranes that leads to transformation in a manner similar to Her2/neu. Phosphorylation of MT tyrosine residues activates key signaling molecules including Shc/Grb2, PI3K and PLCgamma1. The unique contributions of SV40 LT and ST and PY MT to cellular transformation have provided significant insights into our understanding of tumor suppressors, oncogenes and the process of oncogenesis.

Lessons in Signaling and Tumorigenesis from Polyomavirus Middle T Antigen

SUMMARY The small DNA tumor viruses have provided a very long-lived source of insights into many aspects of the life cycle of eukaryotic cells. In recent years, the emphasis has been on cancer-related signaling. Here we review murine polyomavirus middle T antigen, its mechanisms, and its downstream pathways of transformation. We concentrate on the MMTV-PyMT transgenic mouse, one of the most studied models of breast cancer, which permits the examination of in situ tumor progression from hyperplasia to metastasis.

Introduction of oncogenes into mammary glands in vivo with an avian retroviral vector initiates and promotes carcinogenesis in mouse models

We have adapted the avian leukosis virus RCAS (replication-competent avian sarcoma-leukosis virus LTR splice acceptor)-mediated somatic gene transfer technique to introduce oncogenes into mammary cells in mice transgenic for the avian subgroup A receptor gene, tva, under control of the mouse mammary tumor virus (MMTV) promoter. Intraductal instillation of an RCAS vector carrying the polyoma middle T antigen (PyMT) gene (RCAS-PyMT) induced multiple, oligoclonal tumors within 3 weeks in infected mammary glands of MMTV-tva transgenic mice. The rapid appearance of these tumors from a relatively small pool of infected cells (estimated to be ≈2 × 103 cells per gland by infection with RCAS carrying a GFP gene; RCAS-GFP) was accompanied by a high fraction of cells positive for Ki67, Cyclin D1, and c-Myc, implying strong proliferation competence. Furthermore, the tumors displayed greater cellular heterogeneity than did tumors arising in MMTV-PyMT mice, suggesting that RCAS-PyMT transforms a relatively immature cell type. Infection of mice transgenic for both MMTV-Wnt-1 and MMTV-tva with RCAS virus carrying an activated Neu oncogene dramatically enhanced tumor formation over what is observed in uninfected bitransgenic animals. We conclude that infection of mammary glands with retrovirus vectors is an efficient means to screen candidate oncogenes for their capacity to initiate or promote mammary carcinogenesis in the mouse.

The influence of the reading context upon the suppression of nonsense codons

SummaryWe have examined the response of phage T4 nonsense mutations located at various sites within the same cistron to different suppression agents. A wide range of suppression efficiency is found for both ochre (UAA) and amber (UAG) mutations under conditions where suppression provides a measurement of the amount of chain propagation past the mutated site. We have established a relationship between our measurement-the size of the phage yield-and the amount of rIIB product present in the infection. Our data suggest that the 1000-fold range of variations in yields observed in the rIIB cistron corresponds to a 30-fold range of variation in the level of rIIB product, i.e. in the relative frequency of chain propagation past the various nonsense codons included in our test.From the parallelism of response of any particular mutant to very different suppression mechanisms we conclude that the efficiency of suppression is site specific, that is to say, that the main factor determining the frequency of chain propagation at a nonsense codon by any type of suppression mechanism is the nucleotide sequence adjacent to the nonsense codon (reading context).We propose that the recognition of a natural termination signal involves a sequence longer than a nonsense codon and that nonsense codons outside of their natural environment induce variable termination rates which are reflected in the suppression potential.

Comparisons of two early gene functions essential for transformation in polyoma virus and SV-40

Abstract We have compared ts-a mutants of polyoma virus with ts-A mutants of SV-40, and hr-t mutants of polyoma virus with the viable deletion mutants of SV-40 mapping between 0.54 and 0.59 map unit (referred to as dl). All four groups of mutants are either totally or partially defective in inducing stable transformation as assayed by anchorage-independent growth. In each virus system, two classes of mutants—hr-t and ts-a of polyoma virus and dl and tsA of SV-40—complement to induce stable transformation. Two distinct functions essential for transformation are therefore encoded within the early regions of these papova viruses. Two approaches have been taken in attempts to define the roles of these early viral genes in cell transformation. In the first approach, a clonal analysis was made of cells transformed at the permissive temperature by ts-a/A mutants. Selections were carried out either for anchorage-independent growth or for focus formation. Although the variation in expression of the selected parameter of transformation among multiple clones derived with the same virus and cell line is often high, the majority of clones show no temperature dependence of either selected or unselected properties when compared to wild-type virus-transformed clones. In some instances, temperature-sensitive clones are observed. No correlation is seen between the appearance of a temperature-sensitive phenotype in individual clones and the expression of T-antigen species at permissive and nonpermissive temperatures determined by immunofluorescence or immunoprecipitation of [35Slmethio-nine-labeled proteins. In the second approach, mutants of all four groups were tested for their ability to induce abortive transformation measured as the transient loss of anchorage-dependent growth. This assay circumvents the problem of clonal variation and gives a clearcut result. ts-a/A mutants retain the ability to induce abortive transformation, behaving like wild-type virus at the nonpermissive temperature. hr-t mutants are virtually negative, while the dl mutants show a reduced ability to induce abortive transformation. The simplest explanation which is adequate for the majority of the results is that the ts-a/A function is required only transiently to carry out an initiation event which stabilizes transformation, while the hr-t/dl function acts to induce parameters of the transformed phenotype in the manner of a maintenance function. Additional interpretations are put forward to explain the results with temperature-sensitive clones transformed by ts-a/A mutants.

Host range selection of transformation-defective hr-t mutants of polyoma virus

Abstract Nineteen independent mutants have been isolated by host range selection using polyoma transformed 3T3 cells as a permissive host and normal 3T3 cells as a nonpermissive host. All nineteen mutants fail to transform rat and hamster fibroblasts. Complementation experiments indicate that these mutants belong to a single group. This group is designated “hr-t,” indicating defects in host range and transformation. In both productive and nonproductive infections, hr-t mutants induce the synthesis of polyoma-specific T antigen(s), and are therefore not blocked prior to uncoating. Some of the mutants bear small deletions in their DNAs, notably in the proximal (5′) part of the early region. The ensemble of results to date shows a uniform biological behavior for all mutants isolated by this procedure, and suggests that single mutations are responsible for the reduced host range and the inability to induce cell transformation. These and earlier results are discussed in terms of a model in which the hr-t viral function acts to alter the expression of cellular genes.

Hr-t and ts-a: Two early gene functions of polyoma virus

Abstract Complementation in productive infection occurs between hr-t mutants of polyoma virus and all previously defined classes of temperature-sensitive mutants. Hr-t and ts-a mutants, both of which map in the early region of the viral DNA and are defective in cell transformation, have been compared in detail. The growth of ts-a mutants fails to respond to host factors or murine leukemia virus infection in the manner of hr-t mutants. Optimal complementation for virus growth occurs at high ts-a:hr-t input ratios, and under these conditions the complementation is symmetric and efficient. Results of gene dosage experiments suggest a catalytic role for the ts-a (wild type) product, and reveal a partial dominant lethal effect of hr-t mutant products. Hr-t and ts-a mutants also complement one another for cell transformation. These results indicate that the hr-t and ts-a viral genes govern different steps in the processes of virus growth and cell transformation.

Kinetic Analysis of the Steps of the Polyomavirus Lytic Cycle

ABSTRACT Kinetic studies of the accumulation of early and late transcripts, early and late proteins, genomes, and live virus, during the lytic cycle of murine polyomavirus wild-type A2, were carried out in synchronized NIH 3T3 cells released from G0 by the addition of serum after infection. This first-time simultaneous analysis of all parameters of the virus life cycle led to new insights concerning the transcriptional control at the early-to-late transition. During the early phase, early transcripts were synthesized at very low levels, detectable only by reverse transcription-PCR, from 6 h postinfection (hpi). Large T protein could be detected by 8 hpi (while infected cells were in the G1 phase). The level of expression of the middle T and small T proteins was lower than that of large T at all times, due, at least in part, to a splicing preference for the large-T 5′ splice site at nucleotide 411. A large increase in the level of both early and late transcripts coincided closely with the detection in mid-S phase of viral genome amplification. Thereafter, both classes of transcripts continued to further accumulate up to the end of the experiments (48 hpi). In addition, during the late phase, “giant” multigenomic transcripts were synthesized from the early as well as the late promoter. Thus, a major type of transcriptional control appears to be applied similarly to the transcription of both early and late genes. This view differs from that in the literature, which highlights the enhancement of late transcription and the repression of early transcription. However, despite this parallel transcriptional control, additional regulations are applied which result in higher levels of late compared to early transcripts, as previously described. In the accompanying article, a key role for middle T and/or small T in this late-phase enhancement of early and late transcription is demonstrated (16). Other novel findings, e.g., the synthesis of a very abundant short early promoter proximal RNA, are also described.

Mammary tumors induced by polyomavirus

SummaryThe first known member of the Polyomavirus family, murine Polyomavirus (MPyV), was discovered because of its oncogenic properties. The genetic simplicity of MPyV (shared with all members of the Py family), the wide spectrum of tumors induced by MPyV, and the convenient properties of its natural host, the mouse, make it a particularly interesting model system to study oncogenesis. This paper briefly reviews the virus infectious cycle and our current understanding of the viral proteins that are involved in oncogenesis, and focuses on recent studies on oncogenesis of the mammary gland. Mammary gland ductal adenocarcinomas develop at high frequency and with short latency in infected immunoincompetent adult female or normal neonatal mice or in transgenic mice expressing the viral oncogene, middle T. These tumors provide excellent model systems for the study of human breast cancer.

Isolation and characterization of context mutations affecting the suppressibility of nonsense mutations

SummarySecondary mutations which increase the efficiency of suppression of nonsense mutations in the rHB cistron of bacteriophage T4 have been isolated. These secondary mutations, called context mutations, map at sites very close to the nonsense codon, possibly on the promotor distal side. In context-nonsense double mutants, the amount of suppressed gene product is increased approximately 10-fold. The context mutations examined can act on the UAA (ochre) nonsense allele as well as on the UAG (amber) nonsense allele at a given site. These context mutations affect all suppression mechanisms analyzed (genetic suppressors. 5-fluorouracil suppression and spontaneous suppression).We suggest that context mutations affect information which is significant to the termination of polypeptide chains. According to our view, context mutations change the immediate neighborhood of nonsense mutations and so reduce the degree of resemblance to the sequences normally used for the termination of translation.