Temperature-sensitive splicing defect of ts110 Moloney murine sarcoma virus is virus encoded.

ts110 Moloney murine sarcoma virus (Mo-MuSV)-nonproductively infected cells (6m2) have a transformed phenotype at 28 to 33 degrees C and a normal phenotype at 39 degrees C. At temperatures permissive for transformation, 6m2 cells contain P58gag produced from the 4.0-kilobase (kb) viral RNA genome and P85gag-mos translated from a 3.5-kb spliced mRNA. At 39 degrees C, only the 4.0-kb RNA and its product P58gag are detected. Two temperature-sensitive defects have been observed in ts110-infected 6m2 cells: (i) the splicing of the 4.0-kb RNA to the 3.5-kb RNA; and (ii) the thermolability of P85gag-mos and its kinase activity relative to the wild-type revertant protein, termed P100gag-mos (R.B. Arlinghaus, J. Gen. Virol. 66:1845-1853, 1985). In the present study, we examined the mos gene products of two cell lines (204-2F6 and 204-2F8) obtained by infection of normal rat kidney cells with ts110 Mo-MuSV as a simian sarcoma-associated virus pseudotype to see whether the temperature-sensitive splicing defect could be transferred by viral infection. Southern blot analysis of these two cell lines showed that viral DNAs containing restriction fragments from cellular DNA are different from those in 6m2 cells, indicating that 204-2F6 and 204-2F8 cells have different ts110 provirus integration sites from those of 6m2 cells. Northern blots, S1 mapping analyses, and immunoprecipitation experiments showed unequivocally that the splicing defect of ts110 Mo-MuSV is virus encoded and is independent of host cell factors.     

P85: a gag-mos polyprotein encoded by ts110 Moloney murine sarcoma virus

Antibody to a synthetic peptide (anti-C3 serum) with the predicted sequence of the C terminus of the Moloney murine sarcoma virus (strain 124) v-mos gene was used in immunoprecipitation experiments with cytoplasmic extracts of a clone of NRK cells infected with ts110 Moloney murine sarcoma virus, termed 6m2 cells. ts110 Moloney murine sarcoma virus codes for two viral proteins of 85,000 and 58,000 M_r, termed P85 and P58, respectively, in nonproducer 6m2 cells maintained at 33°C. Anti-C3 serum specifically recognized [³H]leucine-labeled P85, but not P58, from infected cells maintained at 33°C, whereas antiserum prepared against murine leukemia virus p12 recognized both proteins. Normal serum and anti-C3 serum pretreated with excess C3 peptide did not precipitate P85. Immunoprecipitation experiments after metabolic labeling of 6m2 cells with ³²P_i showed that P85 is phosphorylated. Both anti-C3 and anti-p12 sera specifically detected ³²P-labeled P85. Cell-free translation of ts110 murine sarcoma virus/murine lukemia virus RNA produces P85, P58, and helper virus protein Pr63^gag. Anti-C3 serum specifically precipitated P85 but neither P58 nor Pr63^gag. We conclude from these studies that P85 is a product of both the gag and mos genes of ts110 murine sarcoma virus, and, therefore, it is referred to as P85^gag-mos. We have not detected any other v-mos gene product in ts110-infected cells.     

Identification of proteins encoded by the Gazdar murine sarcoma virus genome by in vitro translation and comparison with Moloney murine sarcoma virus 124.

The gene products of Gazdar murine sarcoma virus (Gz-MuSV) were identified by in vitro translation of Gz-MuSV virion RNA. An overlapping set of proteins with approximate molecular weights of 37,000 (37K), 33K, 24K, and 18K were synthesized from the transforming gene of Gz-MuSV, v-mosGz. In addition, Gz-MuSV-specific RNA directed the in vitro synthesis of a 62K gag gene protein and a 37.5K env gene-related product. The Gz-MuSV-specific in vitro translation products were compared with the in vitro translation products of M-MuSV 124, an independent isolate with a similar v-mos gene. This analysis showed that the 62K Gz-MuSV gag gene protein and the 37K, 33K, 24K, and 18K v-mosGz proteins were almost identical to the M-MuSV 124 62K (gag) and 37K, 33K, 24K, and 18K (v-mosMo) proteins that we previously identified and characterized. The 37.5K env gene product from Gz-MuSV does not have a correlate in the M-MuSV 124 translation products. These results were analyzed in the context of expectations based on similarities and differences in genetic organization of these two viral genomes.     

Comparison of myeloproliferative sarcoma virus with Moloney murine sarcoma virus variants by nucleotide sequencing and heteroduplex analysis.

The myeloproliferative sarcoma virus (MPSV) was derived by passage of Moloney sarcoma virus (Mo-MuSV) in adult mice. Mo-MuSV variants transform fibroblasts. However, MPSV also affects erythroid, myeloid, and hematopoietic stem cells. The MPSV proviral genome, two temperature-sensitive mutants derived from it, Mo-MuSV variant M1, and Moloney murine leukemia virus (Mo-MuLV) were compared by heteroduplex mapping. MPSV wild type was found to have 1 kilobase pair deleted from the pol gene and to contain v-mos-related sequences. The 3' end of MPSV, including the oncogene-helper junctions, the v-mos gene, and the 3' long terminal repeat, was sequenced and compared with sequences of Mo-MuLV, MSV-124, and the mouse oncogene c-mos. From these data, MPSV appears to be either closely related to the original Mo-MuSV or an independent recombinant of Mo-MuLV and c-mos. Five possible explanations of the altered specificity of MPSV are considered. (i) The MPSV mos protein has properties inherent in c-mos but lost by other Mo-MuSV mos proteins. (ii) The MPSV mos protein has altered characteristics due to amino acid changes. (iii) Due to a frameshift, MPSV codes for a mos protein truncated at the amino terminal and also a novel peptide. (iv) A second novel peptide may be encoded from the 3' env region. (v) MPSV has long terminal repeats and an enhancer sequence more like Mo-MuLV than Mo-MuSV, with a consequently altered target cell specificity.     

Phosphorylation of the nonstructural proteins encoded by three avian acute leukemia viruses and by avian fujinami sarcoma virus.

The gag gene-related, nonstructural proteins of three avian acute leukemia viruses (namely, myelocytomatosis viruses MC29 and CMII and avian erythroblastosis virus) and of avian Fujinami sarcoma virus (FSV) isolated by immunoprecipitation from cellular lysates with anti-gag serum were shown to be phosphoproteins in vivo. The specific 32P radioactivity of the nonstructural proteins of MC29, CMII, and FSV was significantly higher than that of helper viral, intracellular gag proteins. Two of these proteins, i.e., the 140,000-dalton FSV and the 110,000-dalton MC29 proteins, were also phosphorylated in vitro by a kinase activity associated with immunocomplexes. This kinase activity is either separated from these proteins or inactivated by incubation of cellular lysates with normal serum followed by adsorption to staphylococcal protein A or sedimentation at 100,000 x g or both. It remains to be resolved whether the 110,000-dalton MC29 and 140,000-dalton FV proteins, in addition to being substrates for phosphorylation, also have intrinsic kinase activity.     

Identification of proteins encoded by Epstein-Barr virus trans-activator genes.

Specific antisera were generated to characterize Epstein-Barr virus proteins reported to have trans-activating properties. Open reading frame BRLF1 was found to be expressed in two modifications in vivo, with molecular sizes ranging from 94 to 98 kilodaltons (kDa) depending on the cell line, whereas only one protein (Raji cells, 96 kDa) was detected by in vitro translation. Open reading frame BZLF1 encoded polypeptides of 38 and 35 kDa and additional smaller forms. A BZLF1-encoded 30-kDa protein could be detected under conditions in which expression was restricted to immediate early genes. Nuclear localization could be detected under conditions in which expression was restricted to immediate early genes. Nuclear localization could be shown for the proteins derived from reading frames BZLF1 and BMLF1. BMLF1 expression gave a heterogeneous protein pattern, with molecular sizes between 45 and 70 kDa, including a predominant 60-kDa protein detected in different B-cell lines.     

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.     

Molecular cloning and characterization of a leukemia-inducing myeloproliferative sarcoma virus and two of its temperature-sensitive mutants.

The myeloproliferative sarcoma virus (MPSV) induces extensive hematopoietic changes, including spleen foci in adult mice, and transforms fibroblasts in vitro. NRK nonproducer cell lines of MPSV and ts temperature-sensitive mutants were analyzed by restriction enzyme digestion and Southern blotting. EcoRI fragments containing the proviral DNAs of MPSV and two temperature-sensitive mutants and rat cellular sequences homologous to c-mos were molecularly cloned. By comparing restriction enzyme cleavage sites, it was shown that the MPSV genome consists only of sequences related either to Moloney murine leukemia virus or to the c-mos mouse oncogenic sequences. Two regions of fragment heterogeneity were observed: (i) in the defective pol gene, where MPSV and the two cloned temperature-sensitive mutants were different from Moloney murine sarcoma virus and from each other, although MPSV wild-type retained more of the pol gene than any of the Moloney murine sarcoma virus isolates; (ii) in the area 3' to the mos gene, which was identical in MPSV and its temperature-sensitive mutants but different from other Moloney murine sarcoma virus variants. Transfection of cloned MPSV DNA in RAT4 cells and virus rescue on infection with Friend murine leukemia virus yielded MPSV which transformed fibroblasts in vitro and also induced spleen foci in adult mice, thus proving that both properties are coded by the same viral genome.     

Inheritance of susceptibility to the myeloproliferative sarcoma virus: effect of the Fv-2 locus and evidence for a myeloproliferative sarcoma virus resistance locus.

Myeloproliferative sarcoma virus (MPSV) causes a generalized stem cell leukemia with erythroid and myeloid hyperplasia in adult mice. MPSV also transforms fibroblasts. Mice congenic for the Fv-2 locus showed marked differences in susceptibility to MPSV according to the Fv-2 genotype. MPSV was injected into C57BL/6 Fvs and C57BL/6 Fv-2r mice congenic except for the Fv-2 locus. C57BL/6 mice with the Fvs genotype were much more susceptible to MPSV than were those with the Fvr genotype. Both DDD Fv-2r mice congenic with DDD Fv-2s mice except for the Fv-2 locus and DDD Fv-2s mice, however, were sensitive to spleen focus formation by MPSV. These data indicate that at least one additional resistance locus to MPSV is present in C57BL/6 mice but not in DDD mice. Both the Fv-2 locus and the putative MSPV resistance locus (loci) Mpsvr appear to be epistatic to either of the sensitivity loci. Fibroblast focus formation by MPSV was obtained well in C57BL/6 Fv-2r and C57BL/6 Fvs fibroblasts, indicating that the genes for MPSV resistance (Fv-2r and Mpsvr) were not operating in fibroblast cells. A model is proposed which may account for the differences in response of genetically different mice to MPSV and Friend spleen focus-forming virus.     

Envelope assembly mutant of rous sarcoma virus.

The properties of a novel nonconditional envelope mutant of Rous sarcoma virus, rdPN3/2-SR-D, defective in the assembly of viral glycoproteins into mature virions, are described.     

Characterization of ts 16, a temperature-sensitive mutant of vaccinia virus.

We have characterized a temperature-sensitive mutant of vaccinia virus, ts16, originally isolated by Condit et al. (Virology 128:429-443, 1983), at the permissive and nonpermissive temperatures. In a previous study by Kane and Shuman (J. Virol 67:2689-2698, 1993), the mutation of ts16 was mapped to the I7 gene, encoding a 47-kDa protein that shows partial homology to the type II topoisomerase of Saccharomyces cerevisiae. The present study extends previous electron microscopy analysis, showing that in BSC40 cells infected with ts16 at the restrictive temperature (40 degrees C), the assembly was arrested at a stage between the spherical immature virus and the intracellular mature virus (IMV). In thawed cryosections, a number of the major proteins normally found in the IMV were subsequently localized to these mutant particles. By using sucrose density gradients, the ts16 particles were purified from cells infected at the permissive and nonpermissive temperatures. These were analyzed by immunogold labelling and negative-staining electron microscopy, and their protein composition was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. While the ts16 virus particles made at the permissive temperature appeared to have a protein pattern identical to that of wild-type IMV, in the mutant particles the three core proteins, p4a, p4b, and 28K, were not proteolytically processed. Consistent with previous data the sucrose-purified particles could be labelled with [3H]thymidine. In addition, anti-DNA labelling on thawed cryosections suggested that most of the mutant particles had taken up DNA. On thawed cryosections of cells infected at the permissive temperature, antibodies to I7 labelled the virus factories, the immature viruses, and the IMVs, while under restrictive conditions these structures were labelled much less, if at all. Surprisingly, however, by Western blotting (immunoblotting) the I7 protein was present in similar amounts in the defective particles and in the IMVs isolated at the permissive temperature. Finally, our data suggest that at the nonpermissive temperature the assembly of ts16 is irreversibly arrested in a stage at which the DNA is in the process of entering but before the particle has completely sealed, as monitored by protease experiments.     

Two related Epstein-Barr virus membrane proteins are encoded by separate genes.

The structures of the 2.3- and 2.0-kilobase Epstein-Barr virus (EBV) mRNAs, partially encoded within the EcoRI J fragment DNA of the viral genome, were determined by analysis of their cDNAs. Both mRNAs are transcribed across the fused terminal repeats of the EBV episome and consist of nine exons. The mRNAs are transcribed from different promoters and have a unique 5' exon from the U5 region of the genome but eight common exons from the U1 region. One principal open reading frame is present in each mRNA and is predicted to encode 54,000- and 40,000-dalton integral membrane proteins. This result was confirmed by in vitro translation of RNAs in the presence of canine pancreatic microsomes. The 2.3-kilobase mRNA is not expressed in Raji cells, owing to the deletion of the 5' regulatory and coding region of this gene, whereas neither mRNA is expressed in Namalwa cells, owing to inactivation as a result of integration of the EBV genome via the terminal repeats. Since these mRNAs are readily detected in largely latently infected cells and do not increase in abundance with EBV replication, these putative latent-infection membrane proteins are tentatively designated LMP-2A and LMP-2B, respectively.     

Recombination between a temperature-sensitive mutant and a deletion mutant of Rous sarcoma virus.

Cells doubly infected with two mutants of the Schmidt-Ruppin strain of Rous sarcoma virus (RSV), ts68, which is temperature sensitive for cell transformation (srcts), and a deletion mutant, N8, which is deficient in the envelope glycoprotein (env-), produced a recombinant which carried the defects of both parents. The frequency of formation of such a recombinant was exceptionally high and made up 45 to 55% of the progeny carrying the srcts marker. By contrast, the reciprocal recombinant, which is wild type in transformation (srcts) and contains the subgroup A envelope glycoprotein (envA), was almost undetectable. This remarkable difference in the frequency of the formation of the two possible recombinants suggests that a unique mechanism may be involved in the genetic interaction of the two virus genomes, one of which has a large deletion. When an RNA-dependent DNA polymerase-negative variant of the N8 (N8alpha) was crinants also became deficient in the polymerase. Cells infected by the srctsenv- recombinant were morphologically normal at the nonpermissive temperature (41 degrees C) and susceptible to all subgroups of RSV. The rate by which the wild-type RSV transformed the recombinant-preinfected cells was indistinguishable from that of transformation of uninfected chicken cells by the same wild-type virus. This indicates that no detectable interference exists at postpenetration stages between the preinfected and superinfecting virus genomes and confirms that the expression of the transformed state is dominant over the suppressed state.     

Polymorphism of avian sarcoma virus src proteins.

The src gene products of seven different avian sarcoma viruses were compared. In vitro translation of virion RNA yielded products identified unambiguously as p60src in the case of two stocks of the Schmidt-Ruppin strain, three stocks of the Prague strain, the Bryan strain, and the Bratislava 77 strain of avian sarcoma virus. Differences in the electrophoretic mobility of these seven p60src proteins in sodium dodecyl sulfate-polyacrylamide gels, corresponding to variation in the apparent molecular weights ranging from 56,000 to 60,500, were observed. Antigenic variability was also found; only three of the seven viruses tested encoded a p60src, which was precipitated by antisera derived from rabbits bearing tumors induced by the Schmidt-Ruppin strain of Rous sarcoma virus. Examination of the methionine-containing tryptic peptides of the seven ;60src proteins by two-dimensional mapping revealed four common peptides but marked variability in the five to eight other peptides in each protein. Clear differences in the peptide maps of p60src were observed, both between different strains of virus and within strains. In the three cases examined, p60src synthesized in transformed cells was found to be essentially identical to that synthesized in vitro. We conclude that there is significant polymorphism in the p60src proteins of the avian sarcoma viruses.     

Monoclonal antibodies specific to transforming polyproteins encoded by independent isolates of feline sarcoma virus.

Hybridomas secreting monoclonal antibodies directed against polyprotein gene products of the Gardner, Snyder-Theilen, and McDonough strain of feline sarcoma virus have been isolated. Antibody produced by one hybridoma recognizes immunological determinants localized within a feline leukemia virus gag gene structural component (p15) common to polyproteins encoded by each feline sarcoma virus isolate while antibody produced by a second is specific for p30 determinants unique to P170gag-fms. Additional hybridomas secrete antibody directed against v-fes specific determinants common to the Gardner and Snyder-Theilen feline sarcoma virus-encoded polyproteins and to v-fms determinants unique to P170gas-fms polyprotein. GA P110gas-fes and ST P85gas-fes immunoprecipitated by antibody directed against p15 exhibit readily detectable levels of protein kinase activity but lack such activity when precipitated by antibody specific for their acquired sequence (v-fes) components. P170gas-fms immunoprecipitated by monoclonal antibody to either p15 or p30 lacks detectable levels of autophosphorylation but represents a substrate for the GA P110gag-fes and ST P85gag-fes enzymatic activities. These findings argue that the v-fes-associated protein kinase represents an intrinsic property of the v-fes gene product and recognizes tyrosine acceptor sites within polyprotein gene products of all three strains of feline sarcoma virus.     

Transfer of defective avian tumor virus genomes by a Rous sarcoma virus RNA packaging mutant.

SE21Q1b, a Rous sarcoma virus mutant which packages cellular rather than viral RNA, is competent for infection of quail cells and can transmit defective transforming retrovirus genes. Stably transformed recipient clones have been obtained by using this mutant.     

Temperature-sensitive viral RNA expression in Moloney murine sarcoma virus ts110-infected cells.

We examined the mos-specific intracellular RNA species in 6m2 cells, an NRK cell line nonproductively infected with the ts110 mutant of Moloney murine sarcoma virus. These cells present a normal phenotype at 39 degrees C and a transformed phenotype at 28 or 33 degrees C, expressing two viral proteins, termed P85gag-mos and P58gag, at 28 to 33 degrees C, whereas only P58gag is expressed at 39 degrees C. It has been previously shown that 6m2 cells contain two virus-specific RNA species, a 4.0-kilobase (kb) RNA coding for P58gag and a 3.5-kb RNA coding for P85gag-mos. Using both Northern blot and S1 nuclease analyses, we show here that the 3.5-kb RNA is the predominant viral RNA species in 6m2 cells grown at 28 degrees C, whereas only the 4.0-kb RNA is detected at 39 degrees C. During temperature shift experiments, the 3.5-kb RNA species disappears after a shift from 28 to 39 degrees C and is detected again after a shift back from 39 to 28 degrees C. By Southern blot analysis, we have detected only one ts110 proviral DNA in the 6m2 genome. This observation, as well as previously published heteroduplex and S1 nuclease analyses which showed that the 3.5-kb RNA species lacks about 430 bases found at the gag gene-mos gene junction in the 4.0-kb RNA, suggests that the 3.5-kb RNA is a splicing product of the 4.0-kb RNA. The absence of the 3.5-kb RNA when 6m2 cells are grown at 39 degrees C indicates that the splicing reaction is thermosensitive. The splicing defect of the ts110 Moloney murine sarcoma virus viral RNA in 6m2 cells cannot be complemented by acute Moloney murine leukemia virus superinfection, since no 3.5-kb ts110 RNA was detected in acutely superinfected 6m2 cells maintained at 39 degrees C. The spliced Moloney murine leukemia virus env mRNA, however, is found in acutely infected cells maintained at 39 degrees C, suggesting that the lack of ts110 viral RNA splicing at 39 degrees C is not due to an obvious host defect. In sharp contrast, however, 6m2 cells chronically superinfected with Moloney murine leukemia virus produce a 3.5-kb RNA species at 39 degrees C as well as at 28 degrees C and contain proviral DNAs corresponding to the two viral RNA species.(ABSTRACT TRUNCATED AT 400 WORDS)     

A selective temperature-sensitive defect in viral RNA expression in cells infected with a ts transformation mutant of murine sarcoma virus

We investigated the nature of the defect in the temperature-sensitive mutant of Moloney murine sarcoma virus (Mo-MuSV), termed ts110. This mutant has a temperature-sensitive defect in a function required for maintenance of the transformed state. A nonproducer cell clone, 6m2, infected with ts110 expresses P85 and P58 at 33°C, the transformed temperature, but only P58 is detected at the restrictive temperature of 39°C. Shift-up (33°C → 39°C) and in vitro experiments have established that P85 is not thermolabile for immunoprecipitation. Previous temperature-shift experiments (39°C → 33°C) have shown that P85 synthesis resumes after a 2–3 hr lag period. Temperature shifts (39°C → 33°C) performed in the presence of actinomycin D prevented the synthesis of P85, whereas P58 synthesis did not decline for 5 hr, suggesting that P58 and P85 are translated from different mRNAs. The shift-up experiments also indicated that, once made, the RNA coding for P85 can function at the restrictive temperature for several hours. MuSV-ts110-infected cells superinfected with Mo-MuLV produced a ts110 MuSV-MuLV mixture. Sucrose gradient analysis of virus subunit RNAs revealed a ～28S and a ～35S peak. Electrophoresis of the ～28S poly(A)-containing RNA from ts110 virus in methyl mercuric hydroxide gels resolved two RNAs with estimated sizes of 1.9 × 10⁶ and 1.6 × 10⁶ daltons, both smaller than the wild type MuSV-349 genomic RNA (2.2 × 10⁶ daltons). RNA in the ～28S size class from virus preparations harvested at 33°C was found to translate from P85 and P58, whereas, the ～35S RNA yielded helper virus Pr63^gag. In contrast, virus harvested at 39°C was deficient in P85 coding RNA only. Peptide mapping experiments indicate that P85 contains P23 sequences, a candidate Moloney mouse sarcoma virus src gene product. Taken together, these results suggest that two virus-specific RNAs are present in ts 110-infected 6m2 cells and rescued ts110 pseudotype virions at 33°C, one coding for P85, whose expression can be interfered with by shifting the culture to 39°C; the other coding for P58, whose expression is unaffected by temperature shifts. P85 is a candidate gag-src fusion protein, while P58 contains gag sequences only.