Overproduction of the protein product of a nonselected foreign gene carried by an adenovirus vector.

We have constructed a recombinant adenovirus that carries the herpes simplex virus type I gene for thymidine kinase (EC 2.7.1.21) and expresses thymidine kinase under control of adenovirus major late promoter. A DNA fragment carrying thymidine kinase coding sequences but lacking the thymidine kinase promoter was sandwiched between a piece of adenoviral DNA and simian virus 40 early DNA on a plasmid. The aligned fragment was then inserted into the adenoviral genome, replacing internal adenoviral DNA. Hybrid viruses carrying the thymidine kinase gene were obtained by selecting for viruses that express simian virus 40 tumor antigen (T antigen) in monkey cells. The thymidine kinase gene was positioned in the third segment of the adenovirus tripartite leader downstream from the major late promoter by in vivo DNA recombination between the duplicated adenoviral sequences present in the plasmid insert and the viral vector. Levels of thymidine kinase activity in human or monkey cells infected with this hybrid virus were several times higher than in cells infected with herpes simplex virus. Infected cells produced thymidine kinase protein at very high levels, similar to those found for adenovirus late major capsid proteins. The thymidine kinase protein represented 10% of the newly synthesized protein in late infected cells and accumulated to represent 1% of total cell protein under optimal conditions. This vector system offers a procedure by which a variety of gene products that are biologically active and properly modified can be produced at high levels in mammalian cells.     

Interrupting the early region of polyoma virus DNA enhances tumorigenicity.

The tumorigenicity of DNA from polyoma virus after cleavage with a variety of restriction enzymes was evaluated in suckling hamsters. Cleavage with enzymes that interrupt the region of the genome coding for the large tumor (T) antigen of polyoma virus markedly enhanced the tumorigenicity above that observed with DNA I of the virus. Cell lines established in vitro from tumors induced by polyoma virions, polyoma virus DNA I, or polyoma virus DNA that had been cleaved with restriction endonucleases in the early region all contain the polyoma virus middle and small T antigens but not the large T antigen of polyoma virus is not required for maintenance of the transformed state and probably not for initiation of tumorigenesis by viral DNA.     

Expression of hepatitis B surface antigen with a recombinant adenovirus.

Early region 1 of the adenovirus type 5 genome was replaced with a DNA sequence containing the gene coding for the hepatitis B surface antigen (HBsAg) flanked by the major late promoter from adenovirus 2 and processing and polyadenylylation signals from simian virus 40. In one type of hybrid virus only the adenovirus 2 major late promoter, including just 33 base pairs of the adenovirus type 2 tripartite leader, preceded the coding region of the HBsAg gene. In another, this region was preceded by both the adenovirus major late promoter and almost the entire tripartite leader. The structure of the substituted sequence in each of the recombinant viral DNAs was identical to that in the plasmids used to construct the viruses. Approximately equivalent amounts of HBsAg-specific mRNA were produced late in infection with each recombinant virus. Although HBsAg production was detected late in infection of the hybrid virus not containing the full tripartite leader sequence, its level was 1/70th of that obtained with the hybrid virus containing this sequence. One likely interpretation is that the presence of the tripartite leader at the 5' end of this mRNA is critical for the synthesis of HBsAg polypeptide in the late stage of infection. HBsAg produced upon infection with the hybrid adenoviruses was glycosylated and secreted into the culture medium as particles that were essentially indistinguishable from the 22-nm particles found in human serum.     

DNA binding activity of polyoma virus large tumor antigen.

Polyoma virus large tumor antigen from productively infected mouse cells has been purified to greater than 50% homogeneity by a simple immunoaffinity procedure using monoclonal antibodies. A radioimmunoreaction was devised for assaying purity. The purified large tumor antigen retained its antigenicity and its ability to bind DNA specifically. The regions on the polyoma virus genome recognized by the protein were characterized. Three binding regions were localized within the portion of the genome between the viral origin of DNA replication and the protein coding sequence, overlapping the early promoter and the sites of initiation of mRNAs that specify the viral tumor antigens. The binding regions each contain direct repeats of the pentanucleotide sequence G-R-G-G-C.     

Infection of mouse preimplantation embryos with simian virus 40 and polyoma virus

Mouse two-cell embryos, morulae, and blastocysts were killed when infected in vitro with simian virus 40 (SV40) at high multiplicities of infection. Polyoma virus was not deleterious for preimplantation embryos, even at a very high multiplicity of infection; however, the outgrowths of polyoma-infected blastocysts disintegrated after several days of culture. Indirect immunofluorescence tests revealed the presence of SV40 T and V antigens and polyoma virus V antigen in the nuclei of trophoblastic cells. Virus-specific antigens were not found in the nuclei of cells forming inner cell masses of blastocysts or in inner cell mass-derived cells in blastocyst out-growths. The appearance of SV40 T and V antigens in the nuclei was inhibited by αamanitin, a RNA polymerase II inhibitor. The amount of infectious virus recovered from cultures of morulae or blastocysts on subsequent days after infection with SV40 initially declined but later increased. These points of evidence indicate that some cells of early mouse embryos are permissive for the expression of early and late functions of SV40 genome and that susceptibility to infection with polyoma virus and/or permissiveness for the expression of polyoma virus late functions develop gradually between the two-cell and blastocyst stages. Electron microscope observations showed the presence of specific complexes of membranes and virions in the cytoplasm of trophoblastic cells. Single viral particles could be found in the nuclei and also in mitochondria.     

Coding capacity of a 35 percent fragment of the polyoma virus genome is sufficient to initiate and maintain cellular transformation.

Rat-1 cells were transfected with the restriction enzyme fragment of polyoma virus DNA that extends clockwise from the Bcl I site ((65.4 map units) to the EcoRI site (0/100 map units). Six transformed cell lines were obtained and one of them (BE-1) has been investigated in detail. The viral DNA that is integrated into host DNA in this line appeared to consist of two fragments arranged in a "head-to-tail" tandem with no detectable intervening host sequences. BE-1 cells contained polyoma virus small and middle tumor antigens that were indistinguishable from the corresponding tumor antigens from lytically infected cells. No large tumor antigen was detected but a "new" Mr 34,000 protein, which proved to be a truncated version of large tumor antigen, was immunoprecipitated by anti-tumor-antigen antiserum. After injection of 10(6) BE-1 cells into young syngeneic Fischer rats, tumors appeared within 3--4 weeks. Thus, the coding capacity of the Bcl I/EcoRI fragment of polyoma virus DNA is sufficient to enable the cells to produce all of small and middle tumor antigens and about a third of large tumor antigen, to transform cells stably in culture, and to produce tumors in vivo.     

Antibodies specific for the polyoma virus middle-size tumor antigen.

We have obtained antibodies specific for the polyoma virus middle-size tumor antigen (middle T antigen) by immunizing rabbits with a synthetic peptide, Lys-Arg-Ser-Arg-His-Phe, corresponding to the six carboxy-terminal amino acids of the middle T antigen predicted from the nucleotide sequence of polyoma DNA. The antipeptide serum precipitates the polyoma middle T antigen but not the small or large tumor antigens, and precipitation is inhibited in the presence of the peptide. Two cellular proteins, 30,000 and 26,000 daltons, are also precipitated specifically by the antipeptide serum and may have amino acid sequences related to the peptide. Two other cellular proteins, 33,000 and 25,000 daltons, are precipitated only in the presence of the peptide and may associate with it in cell extracts. Antisera directed against synthetic peptides are likely to be important in various ways, including the production of antibodies directed against particular determinants and the recognition of unknown proteins whose genes have been analyzed.     

Differential subcellular localization of in vivo-phosphorylated and nonphosphorylated middle-sized tumor antigen of polyoma virus and its relationship to middle-sized tumor antigen phosphorylating activity in vitro.

A small fraction of polyoma virus middle-sized tumor (T) antigen is phosphorylated in vivo, resulting in a small amount of phosphotyrosine and phosphothreonine and significantly larger amounts of phosphoserine. When infected cells are separated into nuclear, plasma membrane, and low-speed supernatant fractions, 80-95% of in vivo-phosphorylated middle-sized T antigen is localized to the plasma membrane fraction, while 25-50% of [35S]methionine-labeled middle-sized T antigen is found in the nuclear fraction and the same amount is found in the plasma membrane fraction. Immunoprecipitated T antigens contain a protein kinase activity that phosphorylates middle-sized T antigen at tyrosine residues. Eighty to 90% of this activity is located in the plasma membrane fraction. When immunoprecipitated T antigens are treated with alkaline phosphatase, middle-sized T antigen-phosphorylating activity decreases as 32PO4 is lost from in vivo 32P-labeled middle-sized T antigen. The possibility that in vivo-phosphorylated middle-sized T antigen located in the plasma membrane is an active tyrosine-specific kinase is discussed.     

Search for viruses in rheumatoid macrophage-rich synovial cell populations.

The adherent cells remaining after short-term culture of synovial fluid and synovial membrane cells from rheumatoid and non-rheumatoid patients were examined for the presence of a productive virus infection and for various viral antigens. Labelling was carried out with 3H-thymidine and 3H-uridine followed by sucrose density gradient centrifugation of the culture supernatant. Only in 1 case was there incorporation of 3H-uridine into material of density 1 . 21 g/cm3. Viral antigens were tested for by indirect immunofluorescence with antisera to rubella virus, the retroviruses RD-114 and simian sarcoma associated virus, early adenovirus type 2 antigens, late adenovirus type 2 antigens, SV-40 T antigen, and in 1 case measles virus. No cell showed immunofluorescence with any antiserum except the early adenovirus type 2 antiserum, which stained the cytoplasm of about half the synovial cell cultures, some from rheumatoid and some from non-rheumatoid patients.     

Polyoma large tumor antigen is not required for tumorigenesis mediated by viral DNA.

The arrangement of viral DNA sequences in a hamster cell line derived from a tumor induced by a recombinant plasmid DNA preparation containing the entire polyoma virus genome was examined. In the recombinant plasmid employed, viral DNA sequences specifying the large species of polyoma tumor antigen but not the small and middle tumor antigens were interrupted by the insertion of plasmid DNA at the EcoRI restriction endonuclease site. Blot-hybridization analyses of tumor cell DNA indicated that the "joints" linking viral and plasmid DNAs in the original recombinant plasmid used in animal inoculation had been preserved. Integration into the hamster cell genome had apparently occurred within plasmid DNA sequences. These results indicate that polyoma large tumor antigen is not required for tumorigenesis mediated by viral DNA.     

DNAs of simian virus 40 and polyoma direct the synthesis of viral tumor antigens and capsid proteins in Xenopus oocytes.

Purified simian virus 40 and polyoma DNAs injected into nuclei of Xenopus oocytes were transcribed and subsequently translated into virus-specific tumor antigens and capsid proteins. Simian virus 40 large and small tumor antigens synthesized in the oocytes were indistinguishable, by gel electrophoresis and [35S]methionine-labeled tryptic peptide mapping, from the corresponding polypeptides synthesized in CV-1 African green monkey cells. The synthesis of large simian virus 40 tumor antigen implies the correct splicing of its mRNA, which is complementary to nonadjacent nucleotide sequences in the early region of the viral genome. Polyoma DNA directed synthesis of two polyoma tumor antigen polypeptides, 57,000 Mr and small tumor antigen, and of the main capsid protein.     

Recombinant retroviruses that transduce individual polyoma tumor antigens: effects on growth and differentiation.

We have constructed infectious retroviral vectors, derived from Moloney murine leukemia virus, that efficiently transduce the polyoma virus tumor (T) antigens individually. The parental vector we have chosen [pZIP-NeoSV(X)1] expresses a dominant selectable marker for neomycin resistance and is a shuttle vector capable of propagation in both eukaryotic and prokaryotic cells, thus facilitating its use in structure-function studies. To address the relationship between polyoma T-antigen tumorigenesis and the effects of individual T antigens on growth control and differentiation, we used these vectors to introduce and stably express large, middle-sized, or small T antigens into mouse fibroblasts and preadipocytes. All cDNAs introduced into the vector are expressed stably even in the absence of selective pressure. The stable expression of small T antigen is noted particularly because cell lines expressing small T antigen have not been readily available prior to the use of retroviral vectors. Small T antigen-induced increase in saturation density of NIH 3T3 cells can be scored on the basis of the morphology of drug-resistant colonies. Middle-sized T antigen eliminates the growth requirement of NIH 3T3 cells for epidermal growth factor in a defined medium and permits growth in platelet-poor plasma, indicating elimination of the platelet-derived growth factor requirement as well. Large T antigen suppresses mouse preadipocyte (3T3-F442A) differentiation. These vectors and these functional assays of T-antigen activity permit genetic analysis of the relationship between tumorigenesis by T antigens and the alteration of cellular growth and differentiation.     

A NONDEFECTIVE (COMPETENT) ADENOVIRUS-SV40 HYBRID ISOLATED FROM THE AD.2-SV40 HYBRID POPULATION

A new nondefective hybrid virus has been plaque-isolated from the Ad.2-SV40 hybrid population. This virus replicates efficiently with one-hit kinetics in both human embryonic kidney and African green monkey kidney cells, induces an SV40 specific antigen which is detectable by immunofluorescence and complement-fixation using sera from SV40 tumor-bearing hamsters, and produces SV40-specific RNA detectable by DNA-RNA hybridization. The SV40-specific antigen induced by this virus is heat-stable, sensitive to inhibitors of DNA synthesis, serologically different from SV40 T and viral antigens, and is an unrecognized SV40 antigen.     

Isolation and characterization of polyoma host range mutants that replicate in nullipotential embryonal carcinoma cells.

Polyoma wild-type virus replicates in most murine differentiated cells but fails to produce virus in murine embryonal carcinoma cells. Polyoma host range mutants have been isolated that replicate in several nullipotential embryonal carcinoma cell lines but fail to replicate in a pluripotential cell line. The final virus yield of these host range mutants is dependent upon the multiplicity of infection in both differentiated cells and nullipotent embryonal carcinoma cells. Two independently derived host range mutants contain the same single base pair change (A.T to G.C) and a 33- and 67-base pair duplication of those viral DNA sequences containing this point mutation. This duplication of viral DNA is located at 69 map units on the polyoma genome on the late gene side of the origin of viral DNA replication (70.5 map units). This type of mutation suggests several models to explain the polyoma host range restriction in embryonal carcinoma cells.     

Species-specific in vitro synthesis of DNA containing the polyoma virus origin of replication.

In vitro replication of DNA containing the polyoma (Py) virus origin of replication has been carried out with cell-free extracts prepared from mouse FM3A cells. The in vitro system required the Py virus-encoded large tumor (T) antigen, DNA containing the Py virus origin of replication, ATP, and an ATP-regenerating system. The replication reaction was inhibited by aphidicolin, suggesting the involvement of DNA polymerase alpha in this system. Simian virus 40 (SV40) T antigen could not substitute for the Py T antigen. Cell extracts prepared from HeLa cells, a source that replicates SV40 DNA in the presence of SV40 T antigen, replicated Py DNA poorly. The addition of purified DNA polymerase alpha-primase complex isolated from FM3A cells enabled HeLa cell extracts to replicate Py DNA with the same efficiency as FM3A cell extracts. Complementary experiments have shown that FM3A cell extracts do not support SV40 DNA replication unless supplemented with DNA polymerase alpha-primase complex from HeLa cells [Murakami, Y., Wobbe, C.R., Weissbach, L., Dean, F.B. & Hurwitz, J. (1986) Proc. Natl. Acad. Sci. USA 83, 2869-2873]. These results indicate that the host-cell source of the DNA polymerase alpha-primase complex plays an important role in discriminating between SV40 T antigen- and Py T antigen-dependent replication of their homologous DNA in vitro. This may explain the host-range specificity of these viruses in vivo.     

Polyoma virus early-late switch: regulation of late RNA accumulation by DNA replication.

Early in infection of permissive mouse cells, messages from the early region of the polyoma virus genome accumulate preferentially over those from the late region. After initiation of DNA replication, the balance between early and late gene expression is reversed in favor of the late products. In previous work from our laboratory, we showed that viral early proteins do not activate the polyoma late promoter in the absence of DNA replication. Here we show that activation of the late genes in replication-incompetent viral genomes can occur if actively replicating genomes are present in the same cell. A low level of DNA replication, however, is insufficient to induce the early-late switch. Furthermore, replication-competent genomes that fail to accumulate late RNA molecules are defective in the transactivation of replication-incompetent genomes. We suggest that titration of an unknown diffusible factor(s) after DNA replication relieves the block to late RNA accumulation seen in the early phase, with most of this titration being attributable to late-strand RNA molecules themselves.