Sequences of the A-MuLV protein needed for fibroblast and lymphoid cell transformation

The role of various segments (gag or v-abl) of the Abelson murine leukemia virus (A-MuLV) genome in both lymphoid cell and fibroblast transformation was examined by deletion of areas from cloned, plasmid DNA representations of the genome. The deleted plasmids were tested by transfection into fibroblasts and by infection of bone marrow cells using virus stocks derived from the fibroblast transfectants. Deletion of gag coding sequence from the A-MuLV protein did not affect fibroblast transforming activity but abolished lymphoid transforming activity. The gag- A-MuLV genomes were very unstable in transformed fibroblasts leading to large secondary deletions in v-abl sequences. The gag- A-MuLV proteins also had lower autophosphorylation than their gag+ counterparts although cells transformed by gag- virus had a normal elevation of protein-linked phosphotyrosine. Systematic deletion of v-abl sequences showed that only 45,000 to the 130,000 molecular weight of v-abl sequence in the A-MuLV protein is needed for fibroblast transformation and, at most, slightly more is needed for lymphoid cell transformation.     

Biochemical activities of T-antigen proteins encoded by simian virus 40 A gene deletion mutants.

We have analyzed T antigens produced by a set of simian virus 40 (SV40) A gene deletion mutants for ATPase activity and for binding to the SV40 origin of DNA replication. Virus stocks of nonviable SV40 A gene deletion mutants were established in SV40-transformed monkey COS cells. Mutant T antigens were produced in mutant virus-infected CV1 cells. The structures of the mutant T antigens were characterized by immunoprecipitation with monoclonal antibodies directed against distinct regions of the T-antigen molecule. T antigens in crude extracts prepared from cells infected with 10 different mutants were immobilized on polyacrylamide beads with monoclonal antibodies, quantified by Coomassie blue staining, and then assayed directly for T antigen-specific ATPase activity and for binding to the SV40 origin of DNA replication. Our results indicate that the T antigen coding sequences required for origin binding map between 0.54 and 0.35 map units on the SV40 genome. In contrast, sequences closer to the C terminus of T antigen (between 0.24 and 0.20 map units) are required for ATPase activity. The presence of the ATPase activity correlated closely with the ability of the mutant viruses to replicate and to transform nonpermissive cells. The origin binding activity was retained, however, by three mutants that lacked these two functions, indicating that this activity is not sufficient to support either cellular transformation or viral replication. Neither the ATPase activity nor the origin binding activity correlated with the ability of the mutant DNA to activate silent rRNA genes or host cell DNA synthesis.     

Transformation by hamster polyomavirus: identification and functional analysis of the early genes.

A strategy involving polymerase chain reaction amplification of cDNAs was designed to study the expression of the hamster polyomavirus (HaPV) early region in HaPV-transformed rat fibroblasts, productively HaPV-infected cells, and HaPV-induced lymphoma. We identified three mRNAs resulting from alternative splicing of open reading frames leading to coding capacities for three polypeptides with molecular weights similar to those of the murine polyomavirus large T, middle T (MT), and small T (ST) antigens. The corresponding intronless cDNAs direct the in vitro synthesis of polypeptides with the expected electrophoretic mobilities. The biological activities carried by the HaPV early genes were assayed by transfection of appropriate cell systems. The fragment of genomic viral DNA that encodes the three early antigens contains all of the genetic information necessary for immortalization of primary rat embryo fibroblasts and transformation of F111 rat cells. The large T antigen is sufficient for immortalization, although the MT and ST antigens stimulate the growth and modify the phenotype of immortal cell lines. A stringent cooperative effect was observed in the transformation of F111 cells, which requires the simultaneous presence of the MT and ST antigens, as opposed to the transformation by murine polyomavirus, which can be carried out by the MT antigen alone.     

A plasmid vehicle suitable for the molecular cloning and characterization of mammalian promoters.

We have constructed a plasmid, pPSG4, that carries only the coding sequences for polyomavirus (Py) small, middle and truncated large T-antigens. A unique HindIII site allows the introduction of foreign promoters directly in front of viral coding sequences. The simian virus 40 (SV40) early and late, adenovirus-2 (Ad-2) major late and herpes simplex virus-1 (HSV-1) thymidine kinase (TK) promoters all confer on pPSG4 the ability to transform rat embryonic fibroblasts with high efficiency. Sequential deletion of the 72 bp repeats, the 21 bp repeats and the TATA box from the SV40 early region in pPSG4 produced a 50, then 30 and then a further 5 to 10-fold decrease in transformation efficiency, respectively. Thus pPSG4 is a convenient vector for the cloning and characterization of mammalian promoters.     

Rescue of silent integrated polyoma genomes suggests homologous recombination between resident and transfected DNA fragments

Two defective polyoma virus genomes, deleted in the nucleotide sequences coding the N-termini of the tumor antigens, were introduced into Fisher 3T3 rat cells by DNA-mediated gene transfer (transfection). The resulting integrated genomes were incapable of conferring a transformed phenotype to the cells. However, after transfection of these lines with small polyoma fragments overlapping the deleted sequences, transformed clones were isolated. These clones were analyzed by Southern genomic blot hybridization and by isolation in E. coli of plasmids containing viral sequences excised following fusion with mouse polyoma growth-permissive cells. In all cases at least one intact copy of the early region of the polyoma genome was found. Furthermore, restriction sites adjacent to the initial inactive insertion remained unchanged in many of the transformed lines. These results show that functional restoration of the defective polyoma early region involves homologous recombination between the deleted viral genomes integrated in the cellular DNA and the transfecting viral fragments.     

Polyoma viral middle T-antigen is required for transformation.

To determine whether small or middle T-antigen (or both) of polyoma virus is required for transformation, we constructed mutants of recombinant plasmids which bear the viral oncogene and measured the capacity of these mutants to transform rat cells in culture. Insertion and deletion mutations in sequences encoding small and middle T-antigens (79.7, 81.3, and 82.9 map units) rendered the DNA incapable of causing transformation by the focus assay. Similar mutations in sequences that encoded middle but not small T-antigen (89.7, 92.1, and 96.5 map units) generally abolished the transforming activity of the DNA. However, two mutants (pPdl1-4 and PPd12-7) that carried deletions at 92.1 map units retained the capacity to transform cells; pPdl1-4 did so at frequencies equal to those of the parental plasmid, whereas pPdl2-7 transformed at 10% the frequency of its antecedent. From these studies we conclude that small T-antigen alone is insufficient to cause transformation and that middle T-antigen is required for transformation, either in combination with small T-antigen or by itself.     

Replication-dependent transactivation of the polyomavirus late promoter.

When a plasmid containing the wild-type polyomavirus intergenic regulatory region fused to the bacterial cat gene was introduced into mouse NIH 3T3 cells along with a plasmid coding for the early viral proteins (T antigens), chloramphenicol transacetylase enzyme activity and mRNA levels were increased about 10-fold over levels observed in the absence of early proteins. To investigate this transactivation phenomenon further, 11 specific deletion mutant derivatives of the wild-type parent plasmid were constructed and studied. One mutant (NAL) with a minimal level of chloramphenicol transacetylase expression in the absence of T antigens was capable of being transactivated more than 40-fold. A number of other mutants, however, had little capacity for transactivation. Each of these mutants had in common a defect in large T-antigen-mediated DNA replication. Interestingly, one of the transactivation-defective mutants showed a basal late promoter activity fivefold higher than that of wild type and replicated in mouse cells in the absence of large T antigen. Subsequently, a small deletion abolishing viral DNA replication was introduced into those mutants capable of transactivation. The effect of the second deletion was to eliminate both replication and transactivation. Finally, wild-type and mutant constructs were transfected into Fisher rat F-111 cells in the presence or absence of early proteins. No transactivation or replication was ever observed in these cells. We concluded from these studies that the observed transactivation of the polyomavirus late promoter by one or more of the viral early proteins was due to either higher template concentration resulting from DNA replication or replication-associated changes in template conformation.     

Site-directed mutagenesis of the simian virus 40 large T-antigen gene: replication-defective amino acid substitution mutants that retain the ability to induce morphological transformation.

We used a heteroduplex deletion loop mutagenesis procedure for directing sodium bisulfite-induced mutations to specific sites on viral or plasmid DNA to generate a series of SV40 large T-antigen point mutants. The mutations were directed to a region of the T-antigen gene, 0.5 map units, that is thought to be important for interaction of the protein with the viral origin of DNA replication. Of the 16 mutants reported here, 10 had lost the ability to replicate their DNA, and 3 others showed a reduced level of replication compared to wild type. All of the mutants tested were capable of transforming rat cells in culture by the dense focus assay. We conclude that the sequences of the early region around 0.5 map units are critical for the replication of viral DNA but not for the transformation function of T antigen.     

N-terminal amino acid sequences of the polyoma middle-size T antigen are important for protein kinase activity and cell transformation.

We constructed deletion mutations which removed N-terminal coding sequences of various lengths from a cloned polyoma middle-size T antigen (MT antigen) gene. We introduced the MT antigen genes into a simian virus 40 expression vector so that they were expressed at high levels under the control of the simian virus 40 late promoter in COS-1 cells. The deletion mutant genes synthesized truncated MT antigens whose size was consistent with the deletion of either 70 or 106 amino acids from N termini, owing to initiation of translation at internal methionine codons in the MT antigen-coding region. The truncated MT antigens were found in cell membrane fractions but failed to show MT antigen-associated protein kinase activity. The cloned deletion mutant DNAs failed to transform rat F2408 or mouse NIH 3T3 cells. Therefore, N-terminal amino acid sequences of the polyoma MT antigen, as well as C-terminal sequences, are important for protein kinase activity and cell transformation.     

Definition of the human raf amino-terminal regulatory region by deletion mutagenesis.

Activation of transforming potential of the cellular raf gene has uniformly been associated with the deletion of amino-terminal coding sequences. In order to determine whether 5' truncation alone could activate cellular raf, we constructed 21 human c-raf-1 cDNAs with variable BAL 31-generated deletions distal to a Moloney murine sarcoma virus long terminal repeat and a consensus translation initiation sequence. The deletions ranged from 136 to 1,399 nucleotides of coding sequence and shortened the 648-amino-acid raf protein by 44 to 465 amino acids. The full-length c-raf-1 cDNA was nontransforming upon transfection of NIH 3T3 cells, as were four mutants with deletions of 142 or fewer amino acids. Seven of nine mutants with deletions of 154 to 273 amino acids induced transformation with efficiencies ranging from 0.25 to 70 foci per micrograms of DNA. Mutants with deletions of 303 to 324 amino acids displayed high transforming activities (comparable with that of v-raf), with a peak activity of 2,400 foci per microgram of DNA when 305 amino acids were deleted. Deletions of greater than 383 amino acids, extending into the raf kinase domain, lacked transforming activity. Northern (RNA) blotting and immunoprecipitation assays indicated that transfected NIH cells expressed raf RNAs and proteins of the expected sizes. Thus, 5' truncation alone can activate raf transforming potential, with a sharp peak of activation around amino acid 300. Analysis of three raf genes previously detected by transfection of tumor DNAs indicated that these genes were activated by recombination in raf intron 7 and encoded fusion proteins containing amino-terminal non-raf sequences. The extend of deletion of raf sequences in these recombinant genes corresponded to BAL 31 mutants which did not display high transforming activity, suggesting that the fused non-raf coding sequences may also contribute to biological activity.     

Molecular basis of retrovirus adaptation: nucleotide sequence of Rous sarcoma virus adapted to duck cells

The host range of retroviruses is rather complex and specific. It is controlled by the products of viral structural genes that interact with the determinants both on the surface and within the cell. The possibility to infect and transform duck embryo fibroblasts is shown for the Prague strain of chicken Rous sarcoma virus (subgroup C), though virus production in these cells is restricted. However, after the 6th passage the "adapted" virus gave the titre practically the same as it was for chicken embryo fibroblasts. Provirus of RSV adapted to the duck embryo fibroblasts and integrated into host DNA was isolated from the library of nucleotide sequences of duck embryo fibroblasts transformed by this virus. The nucleotide sequence of such provirus was determined. The alterations in gp85 coding region of the env gene which proved to be the result of recombination with endogeneous RAV-0 sequences were shown. The formation of viral particles with rather high titre was induced by the proviral transfection on both chicken and duck embryo fibroblasts. The contribution of the revealed alterations in the genome of transformation active virus and possible participation of its td mutant in the adaptation to the new host are discussed.     

Construction of replication-competent Herpesvirus saimiri deletion mutants

DNA fragments derived from the left end of Herpesvirus saimiri 11 L-DNA were cloned in Escherichia coli by using vector pBR322. Deletions were introduced within a cloned 7.4-kilobase-pair sequence by using restriction endonucleases that cut once or twice within this sequence. Permissive owl monkey kidney-cultured cells were transfected with parental strain 11 viral DNA plus cloned DNA with specific sequences deleted. By screening the progeny of these transfections with a limiting-dilution spot hybridization assay, we isolated recombinant viruses containing deletions in this region. A contiguous 4.5-kilobase-pair sequence representing 4.1% of the coding capacity of the virus was found to be unnecessary for virus replication in cultured cells. These deletion mutants will allow us to test whether sequences in this region are required for the lymphoma-inducing capacity of H. saimiri. These same procedures should also allow us to introduce foreign DNA sequences into this region for studying their expression.     

A fps gene without gag gene sequences transforms cells in culture and induces tumors in chickens.

From molecularly cloned DNAs of Fujinami sarcoma virus (FSV) and the Schmidt-Ruppin-A strain of Rous sarcoma virus (SRA), viral DNA was constructed in which fps-specific sequences encoded in FSV replaced the src gene of SRA. A 3' fragment of FSV DNA, from an ATG methionine coding sequence 148 base pairs downstream from the gag-fps junction through the long terminal repeat, was joined to cloned SRA DNA at the translation start site for the src gene. The resultant DNA clone contained the splice acceptor site for src mRNA processing in SRA, but contained no src coding sequences from SRA nor any gag sequences from FSV. All genes for the replication of SRA were retained. Transfection of this cloned viral DNA genome into chicken embryo fibroblasts induced morphological transformation of the cells in culture. However, the morphology of the transformed cells was distinct from that observed in cells infected with wild-type FSV. The transformed cells produced a nondefective transforming virus called F36 which contained a hybrid FSV-SRA long terminal repeat. F36-infected cells produced a protein with the expected molecular weight of 91,000, which had an associated protein kinase activity and was immunoprecipitated by antibodies raised against fps gene determinants but not by antibodies raised against gag or src proteins. Injection of F36 virus into 8-day-old chicks produced tumors at the site of inoculation, detectable within 7 days. These results demonstrated that the gag portion of the gag-fps fusion protein of FSV is not required for transformation or tumorigenesis.     

Structural and biological analysis of integrated polyoma virus DNA and its adjacent host sequences cloned from transformed rat cells.

EcoRI fragments containing integrated viral and adjacent host sequences were cloned from two polyoma virus-transformed cell lines (7axT and 7axB) which each contain a single insert of polyoma virus DNA. Cloned DNA fragments which contained a complete coding capacity for the polyoma virus middle and small T-antigens were capable of transforming rat cells in vitro. Analysis of the flanking sequences indicated that rat DNA had been reorganized or deleted at the sites of polyoma virus integration, but none of the hallmarks of retroviral integration, such as the duplication of host DNA, were apparent. There was no obvious similarity of DNA sequences in the four virus-host joins. In one case the virus-host junction sequence predicted the virus-host fusion protein which was detected in the transformed cell line. DNA homologous to the flanking sequences of three out of four of the joins was present in single copy in untransformed cells. One copy of the flanking host sequences existed in an unaltered form in the two transformed cell lines, indicating that a haploid copy of the viral transforming sequences is sufficient to maintain transformation. The flanking sequences from one cell line were further used as a probe to isolate a target site (unoccupied site) for polyoma virus integration from uninfected cellular DNA. The restriction map of this DNA was in agreement with that of the flanking sequences, but the sequence of the unoccupied site indicated that viral integration did not involve a simple recombination event between viral and cellular sequences. Instead, sequence rearrangements or alterations occurred immediately adjacent to the viral insert, possibly as a consequence of the integration of viral DNA.