Recombination resulting in unusual features in the polyomavirus genome isolated from a murine tumor cell line.

Polyomavirus-induced tumor formation in the adult natural mouse host has been investigated. Tumors were produced in nude mice with the transformation-defective mutant strain NG18 after a long latency period by apparent activation of a cryptic endogenous transforming viral function. A tumor cell line, designated ScB, was established and characterized. Cells from this morphologically distinct line were unusual in that they grew in soft agar but did not form foci. They were highly tumorigenic. They had a 3.1-kilobase major viral transcript that hybridized to probes derived from regions encoding both the T antigens and the structural proteins. ScB cells expressed polyomavirus small T antigen, a slightly altered middle T antigen, and a truncated large T antigen but no capsid proteins. Middle T antigen preserved its interactions with host proteins of 60 and 37 kilodaltons and with c-src. Analysis of cDNA and genomic clones indicated that the stable viral insert in the ScB genome contained multiple copies of the viral B-enhancer. The genome contained two intragenic inversions which created novel early- to late-strand switches. A simple model for the generation of one inversion is proposed that involves the juxtaposition of two stem-loop structures at an illegitimate recombination site; the location of the inverted segment within the integrated sequence permits use of the viral late polyadenylation signal in early-region transcripts, as confirmed by DNA sequence. A repetitive sequence may facilitate recombination at the other inversion site. Both the biological consequences of the observed rearrangements and the structure of the integrated viral DNA suggest that the recombination events are nonrandom

Myristylated polyomavirus VP2: role in the life cycle of the virus.

The double-stranded genome of the small DNA tumor virus, polyomavirus, is enclosed in a capsid composed of a major protein, VP1, which associates as pentameric capsomeres into an icosahedral structure, and two minor proteins, VP2 and VP3, whose functions and positions within the structure are unknown. The N-terminal glycine of the VP2 coat protein has been shown to be cotranslationally acylated with myristic acid. To study the function of this modification and the role of VP2 in the life cycle of polyomavirus, the N-terminal glycine, critical to the myristylation consensus sequence, has been altered to a glutamic acid or a valine residue by site-directed oligonucleotide mutagenesis. The glycine----glutamic acid mutant DNA has been further studied. When transfected into cells permissive for the polyomavirus full lytic life cycle, this mutant DNA replicated at levels comparable to those of wild-type viral DNA, and small amounts of nonrevertant (mutant) virus could be harvested from the cultures. The virus particles viewed by electron microscopy appeared slightly distorted, but the ratio of full to empty particles was similar to that produced in a wild-type viral infection. Mutant virus was capable of reinfecting permissive cells but with a considerably reduced efficiency

Medullary thyroid carcinomas in transgenic mice expressing a polyoma carboxyl-terminal truncated middle-T and wild type small-T antigens

Medullary thyroid carcinoma (MTC) is a rare human tumor affecting the calcitonin-secreting c-cells of the thyroid. Here me report that two independent strains of transgenic mice expressing a Polyomavirus (Py) truncated middle-T antigen (Delta MT), consisting of the aminoterminal 304 amino acids, and the full length Py small-T antigen, developed multifocal bilateral MTCs with 100% penetrance, Occasionally one strain also developed mammary and bone tumors. Furthermore, offspring from both transgenic lines displayed pronounced waviness of the whiskers and fur, previously associated,vith defective epidermal growth factor receptor signaling, Transgene transcription, driven by the homologous early promoter/enhancer, and the corresponding translation products mere detected in tumors and in many other organs which did not develop pathologies. The subcellular distribution of Delta MT and its interactions with the adapter proteins of the SHC family have also been analysed. Our study describes a novel murine model of MTC and provides evidence that the N-terminal 304 amino acid fragment of Py middle-T antigen, possibly in co-operation with small-T antigen, acts as a potent oncogene in c-cells of the thyroid

Scanning surface confocal microscopy for simultaneous topographical and fluorescence imaging: Application to single virus-like particle entry into a cell

We have developed a method for simultaneous recording of high-resolution topography and cell surface fluorescence in a single scan which we call scanning surface confocal microscopy. The resolution of the system allows imaging of individual fluorescent particles in the nanometer range on fixed or live cells. We used this technique to record the interaction of single virus-like particles with the cell surface and demonstrated that single particles sink into the membrane in invaginations reminiscent of caveolae or pinocytic vesicles. This method provides a technique for elucidating the interaction of individual viruses and other nanoparticles, such as gene therapy vectors, with target cells. Furthermore, this technique should find widespread application for studying the relationship of fluorescently tagged molecules with components of the cell plasma membrane