Transformation-defective mutants of Snyder-Theilen feline sarcoma virus lack tyrosine-specific protein kinase activity.

Four phenotypically normal mink cell clones, each containing a transformation-defective provirus of the Snyder-Theilen strain of feline sarcoma virus (ST-FeSV), synthesized an 85,000-dalton viral polyprotein (P85) indistinguishable in size and antigenic complexity from that encoded by wild-type transforming ST-FeSV. An additional transformation-defective, ST-FeSV-containing flat cell clone produced a polyprotein of 88,000 daltons (P88). The viral polyproteins immunoprecipitated from cytoplasmic extracts of these cells lacked the tyrosine-specific protein kinase activity associated with the wild-type ST-FeSV gene product. In addition, the products encoded by representative transformation-defective ST-FeSV genomes were poorly phosphorylated in vivo and lacked detectable phosphotyrosine residues. Whereas proteins of ST-FeSV transformants contained elevated levels of phosphotyrosine, those of mink cells containing transformation-defective ST-FeSV exhibited phosphotyrosine levels no higher than those found in uninfected cells. These findings provide genetic evidence that the tyrosine-specific protein kinase activity associated with ST-FeSV P85 is required for virus-induced transformation.     

Identification of simian virus 40 protein A.

A large simian virus 40 (SV40)-specific protein can be efficiently immunoprecipitated from infected cell extracts with antisera obtained from hamsters bearing SV40-induced tumors. The protein has an apparent molecular weight of 88,000 to 100,000 with respect to markers with known molecular weights, but behaves anomalously on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Cell lines infected by two different strains of SV40 synthesize immunoreactive proteins that differ slightly in mobility during SDS-polyacrylamide gel electrophoresis, evidence that the protein is coded for by the virus. These differences in protein size correlate with differences in the electrophoretic mobility of viral DNA fragments obtained by digestion with HindII and III restriction enzymes. The size of the viral capsid proteins VP2 and VP3 also varies with the strain of virus. dl-1001, a constructed deletion mutant that lacks part of the SV40A gene, directs the synthesis of a 33,000-dalton polypeptide that is not detected in cells infected with wild-type virus. The deletion fragment, like the larger protein, is phosphorylated. Maps of tryptic peptides from the 88,000- to 100,000-dalton protein and the 33,000-dalton fragment show common peptides and provide strong direct evidence that the proteins are products of the SV40 A gene. The deletion fragment reacts with antitumor sera and binds to double-stranded DNA in the presence of the complete A protein.     

Immunological characterization of proteins detected by phosphotyrosine antibodies in cells transformed by Rous sarcoma virus.

Phosphotyrosine antibodies were used to identify tyrosine-phosphorylated proteins in Rous sarcoma virus (RSV)-transformed chicken embryo fibroblasts. A large number of tyrosine phosphoproteins were detected. A similar set of proteins was observed in RSV-transformed murine cells. An 85,000-dalton protein, however, was present in transformed avian cells but missing in transformed murine cells. Neither the 85,000-dalton protein nor any of the other tyrosine phosphoproteins appeared to be viral structural proteins. Use of RSV mutants encoding partially deleted src gene products enabled us to identify a 60,000-dalton cellular tyrosine phosphoprotein that comigrated with wild-type pp60v-src. With the exception of calpactin I, the major tyrosine phosphoproteins detected in immunoblots appeared to be different from several previously characterized substrates of pp60v-src with similar molecular masses (ezrin, vinculin, and the fibronectin receptor).     

Snyder-Theilen feline sarcoma virus P85 contains a single phosphotyrosine acceptor site recognized by its associated protein kinase.

Cells nonproductively transformed by a variant of the Snyder-Theilen strain of feline sarcoma virus (FeSV) expressed an 85,000-dalton polyprotein (P85) with associated tyrosine-specific protein kinase activity. We identified within this polyprotein a single tyrosine acceptor site for its enzyme activity. This acceptor site, as well as two serine phosphorylation sites localized with the p12 structural component of Snyder-Theilen FeSv P85, was phosphorylated in cells nonproductively transformed by Snyder-Theilen FeSv. In contrast, infection by Snyder-Theilen FeSV transformation-defective mutants resulted in phosphorylation only of the two serine acceptor sites, indicating phosphorylation of the tyrosine acceptor site to be transformation specific. In addition, we describe in vitro labeling conditions, using unfractionated cell extracts, which resulted in preferential phosphorylation of the single Snyder-Theilen FeSV tyrosine-specific acceptor site.     

Detection of an 85,000-dalton phosphoprotein in ts110 murine sarcoma virus-infected cells with antiserum against a v-mos peptide.

We have used an antiserum directed against a synthetic v-mos peptide (anti-C3 serum) to screen ts110 murine sarcoma virus (MuSV)-infected cells for the presence of v-mos-encoded proteins. Anti-C3 serum specifically recognized an 85,000-dalton protein doublet (P85) from [35S]methionine-labeled ts110 MuSV-infected producer cells grown at 32 degrees C, the permissive temperature for transformation. The P85 doublet was also recognized by an antiserum directed against the viral gag protein p15. P85 was present but at 2- to 10-fold-lower levels in ts110 MuSV-infected producer cells grown at 39 degrees C, the restrictive temperature for transformation. The P85gag-mos fusion product was the only v-mos protein reproducibly detected in this ts110 MuSV-transformed cell line. Immunoprecipitation of 32P-labeled cells with anti-C3 serum revealed that the upper band of the P85 doublet is phosphorylated, containing mostly phosphoserine and some phosphothreonine. Cells acutely infected with ts110 MuSV contained slightly higher levels of P85 than did the ts110 MuSV-infected producer cell line. Anti-C3 serum specifically recognized a 33,000-dalton protein (p33) in the acutely infected cells labeled with [35S]methionine. p33 was present in trace amounts and may represent a previously unidentified ts110 MuSV-encoded v-mos protein.     

Biochemical characterization of cells transformed via transfection by feline sarcoma virus proviral DNA.

Murine fibroblasts transformed by transfection with DNA from mink cells infected with the Snyder-Theilen strain of feline sarcoma virus and subgroup B feline leukemia virus were analyzed for the presence of integrated proviral DNA and the expression of feline leukemia virus- and feline sarcoma virus-specific proteins. The transformed murine cells harbored at least one intact feline sarcoma virus provirus, but did not contain feline leukemia virus provirus. The transformed murine cells expressed an 85,000-dalton protein that was precipitated by antisera directed against feline leukemia virus p12, p15, and p30 proteins. No feline oncornavirus-associated cell membrane antigen reactivity was detected on the surfaces of the transformed murine cells by indirect membrane immunofluorescence techniques. The 85,000-dalton feline sarcoma virus-specific protein was also found in feline cells transformed by transfection. However, these cells also contained env gene products. The results of this study demonstrate that the feline sarcoma virus genome is sufficient to transform murine cells and that expression of the 85,000-dalton gag-x protein is associated with transformation of both murine and feline cells transformed by transfection.     

Identification of multiple forms of the noncapsid parvovirus protein NCVP1 in H-1 parvovirus-infected cells.

Analysis of extracts of H-1 parvovirus-infected cells with virus-specific antiserum led to the identification of two forms of the noncapsid virus protein NCVP1. These two proteins had apparent molecular weights of 84,000 (NCVP1) and 92,000 (NCVP1') and were structurally related, based on their immunological reactivity and on peptide map analysis. Both of these proteins appeared early in the virus infection, about the same time that capsid proteins appeared. NCVP1' was a highly phosphorylated protein which was apparently derived from NCVP1 via a post-translational event. Phosphoserine was the predominant phosphorylated amino acid in NCVP1' and appeared to be localized in one site or a few sites on the protein. The possible involvement of these noncapsid proteins in parvovirus DNA replication is discussed.     

Identification and characterization of cellular targets for tyrosine protein kinases

Protein kinases associated with the transforming proteins of a number of retroviruses are specific for tyrosine. Several proteins in cells transformed by these viruses are phosphorylated at tyrosine. We have now identified three unrelated abundant nonphosphorylated cellular proteins of 46,000, 39,000 and 28,000 daltons in chick embryo cells, which are the unphosphorylated forms of phosphotyrosine-containing proteins and thus are substrates for tyrosine protein kinases. By two-dimensional gel analysis, we have found that the 46,000-dalton protein exists in two unphosphorylated forms of which the more acidic is a minor species. This latter form is phosphorylated, chiefly at serine, in both normal and transformed cells, generating a yet more acidic species. In transformed but not normal cells, the major form is phosphorylated at tyrosine and serine, yielding a fourth isoelectric variant. The 46,000-dalton unphosphorylated protein has been partially purified, and antiserum to it recognizes all four isoelectric variants of the protein. The 39,000-dalton protein has two unphosphorylated forms of which the more acidic is a minor species. The major form is phosphorylated at tyrosine and serine in transformed cells only. The 39,000-dalton unphosphorylated protein has been partially purified, and antiserum raised to it recognizes all three isoelectric variants. The 28,000-dalton protein has a single phosphorylated form which contains serine in normal cells, but both serine and tyrosine in transformed cells.     

Isolation and characterization of a heat-inducible simian virus 40 mutant.

We have isolated a new type of temperature-sensitive mutant of simian virus 40 (SV40) that is capable of productive infection in permissive cells but not of maintenance of viral DNA integration in transformed cells at the conditional temperature. Virus development is induced when cells transformed by this mutant are shifted to temperatures above 39 degrees C, but is not induced below this temperature. The plaque-purified, temperature-sensitive mutant virus confers heat inducibility to new host cells, indicating that the conditional function is a property of the viral genome. Unlike previously described temperature-sensitive SV40 mutants, in (ts)-1501 is capable of productive infection in permissive cells at the conditional temperature. The morphology, growth, and oncogenicity of in (ts)-1501-transformed cells at 37 degrees C are similar to those of cell lines transformed by wild-type SV40. HK10-c2(in(ts)-1501), a cloned cell line, transformed at 37 degrees C by the mutant virus, exhibits a transient increase in DNA synthesis before cell death at the conditional temperature. Many properties of in(ts)-1501 are analogous to those of the heat-inducible mutants of bacteriophages in which a heat-inactivated protein is responsible for the stable integration of the prophage in the bacterial chromosome.     

Phosphorylation and transformation sensitivity of a major collagen-binding protein of fibroblasts

Using affinity chromatography with immobilized gelatin and native type I collagen, we have identified the major collagen-binding proteins in Nonidet P-40 extracts of chick embryo fibroblasts labeled with [35S] methionine. After washing the gelatin- or collagen-Sepharose beads with high ionic strength buffer, a 47,000-dalton protein was the only major protein besides fibronectin found to bind to these affinity beads. The isoelectric point of this protein was approximately 9.0, with a closely spaced minor spot. The total amount and the synthesis of this collagen-binding protein were both decreased in Rous sarcoma virus-transformed cells. This collagen-binding protein was found to be phosphorylated by incubating intact cells with [32P]orthophosphate. Phosphoamino acid analysis revealed that serine and threonine residues were phosphorylated, but tyrosine was not. Although quantities of the 47,000-dalton protein labeled with [35S]methionine were decreased by a factor 2.5 after transformation, the incorporation of [32P]orthophosphate/unit of protein was 5-7-fold higher in transformed cells. In temperature-sensitive mutant virus-infected cells, the amount of the 47,000-dalton protein was also decreased at the temperature permissive for transformation, and the incorporation of [32P]orthophosphate/protein was also increased. These studies establish that a major membrane-associated collagen-binding protein of fibroblasts is phosphorylated and that it is altered in both total quantity and degree of phosphorylation after malignant transformation.     

Identification of the Gross cell surface antigen associated with murine leukemia virus-infected cells.

The Gross cell surface antigen (GCSA) is produced by cells that are either exogenously infected with murine leukemia virus (MuLV) or are expressing endogenous MuLV genomes. In immune precipitation assays, GCSA was resolved into two serologically distinct 85,000- and 95,000-dalton viral proteins. These antigenic components are glycosylated forms of the polyprotein precursors of the MuLV internal structural proteins.     

The growth of simian virus 40 (SV40) host range/adenovirus helper function mutants in an African green monkey cell line that constitutively expresses the SV40 agnoprotein.

The simian virus 40 T-antigen carboxy-terminal mutants, dlA2459 and dlA2475, are cell line and temperature dependent for growth and plaque formation in monkey kidney cells. Although these mutants did form plaques on BSC-1 cells at 37 degrees C, they were about fivefold less efficient for plaque formation than wild-type simian virus 40. These mutants did not grow in CV-1 cells and did not synthesize agnoprotein in those cells. CV-1 cells which constitutively express the agnoprotein were permissive for mutant plaque formation. However, late mRNAs, virion proteins, and progeny virion yields did not accumulate to wild-type levels during mutant infection of the agnoprotein-producing cells.     

Properties of T antigens induced by wild-type SV40 and tsA mutants in lytic infection

T antigen in extracts of cells infected with tsA mutants is 2 to 6 times more labile at 32°C or 41°C than the antigen in extracts of cells infected with wild-type SV40, as assayed by complement fixation. The stabilities of wild-type and mutant antigens are not altered by mixing the extracts, and thus the stability is an intrinsic property of each antigen and is not determined by another component of the extract. This observation indicates that T antigen is probably the virus-coded product of the A gene. In cells infected at the permissive temperature of 32°C with a high multiplicity of either wild-type or tsA mutant virus, the amounts of T antigen are approximately equivalent and increase logarithmically during the entire period of infection, up to 96 hr. Cells infected at 32°C for 96 hr with mixtures of wild-type and tsA virus produce T antigen with the stability of wild-type, even when the infection is carried out with up to a 5 fold excess of the mutant. The more stable wild-type antigen may repress, directly or indirectly, the synthesis of the more labile mutant antigen.     

Host range and cell cycle activation properties of polyomavirus large T-antigen mutants defective in pRB binding.

We have examined the growth properties of polyomavirus large T-antigen mutants that are unable to bind pRB, the product of the retinoblastoma tumor suppressor gene. These mutants grow poorly on primary mouse cells yet grow well on NIH 3T3 and other established mouse cell lines. Preinfection of primary baby mouse kidney (BMK) epithelial cells with wild-type simian virus 40 renders these cells permissive to growth of pRB-binding polyomavirus mutants. Conversely, NIH 3T3 cells transfected by and expressing wild-type human pRB become nonpermissive. Primary fibroblasts from mouse embryos that carry a homozygous knockout of the RB gene are permissive, while those from normal littermates are nonpermissive. The host range of polyomavirus pRB-binding mutants is thus determined by expression or lack of expression of functional pRB by the host. These results demonstrate the importance of pRB binding by large T antigen for productive viral infection in primary cells. Failure of pRB-binding mutants to grow well in BMK cells correlates with their failure to induce progression from G0 or G1 through the S phase of the cell cycle. Time course studies show delayed synthesis and lower levels of accumulation of large T antigen, viral DNA, and VP1 in mutant compared with wild-type virus-infected BMK cells. These results support a model in which productive infection by polyomavirus in normal mouse cells is tightly coupled to the induction and progression of the cell cycle.     

Small and middle T antigens contribute to lytic and abortive polyomavirus infection.

Using three different polyomavirus hr-t mutants and two polyomavirus mlT mutants, we studied induction of S-phase by mutants and wild-type virus in quiescent mouse kidney cells, mouse 3T6 cells, and FR 3T3 cells. At different times after infection, we measured the proportion of T-antigen-positive cells, the incorporation of [3H]thymidine, the proportion of DNA-synthesizing cells, and the increase in total DNA, RNA, and protein content of the cultures. In permissive mouse cells, we also determined the amount of viral DNA and the proportion of viral capsid-producing cells. In polyomavirus hr-t mutant-infected cultures, onset of host DNA replication was delayed by several hours, and a smaller proportion of T-antigen-positive cells entered S-phase than in wild-type-infected cultures. Of the two polyomavirus mlT mutants studied, dl-23 behaved similarly to wild-type virus in many, but not all, parameters tested. The poorly replicating but well-transforming mutant dl-8 was able to induce S-phase, and (in permissive cells) progeny virus production, in only about one-third of the T-antigen-positive cells. From our experiments, we conclude that mutations affecting small and middle T-antigen cause a reduction in the proportion of cells responding to virus infection and a prolongation of the early phase, i.e., the period before cells enter S-phase. In hr-t mutant-infected mouse 3T6 cells, production of viral DNA was less than 10% of that in wild-type-infected cultures; low hr-t progeny production in 3T6 cells was therefore largely due to poor viral DNA replication.