Novel nonconditional mutants in the src gene of Rous sarcoma virus: isolation and preliminary characterization

We have isolated a number of nonconditional transformation-defective (td) mutants of Prague strain Rous sarcoma virus, subgroup A (PR-RSV-A). Many of these resembled td mutants reported previously, but 11 isolates from low-passage stocks of PR--A showed unusual properties and were designated partially td (ptd) mutants. In mixed infections with temperature-sensitive (ts) transformation-defective RSV mutants the ptd viruses produced cell transformation at restrictive temperature (41°), probably by genetic recombination to yield wild-type virus. In tests with a panel of 4 ts mutants, we found that different ptd isolates varied in the number and pattern of ts mutants with which they showed this effect. In mixed infections with one another the ptd viruses yielded transforming virus. Again, the pattern shown by different ptd viruses varied, and on the basis of this variation the 11 ptd isolates appear to comprise at least 10 distinct mutants. The possibility of genome deletions in some of the viruses was examined in Southern blots of EcoRI digests of proviral DNA. Two ptd viruses, which recombined with all 4 ts mutants tested, had EcoRI restriction fragments identical to those of wild-type PR-A. Three isolates which recombined with either 3, 2, or none of the ts mutants, showed deletions in the EcoRI fragment containing the src gene. These deletions corresponded to losses of 1.0, 1.5, and 1.6 kilobases, respectively, from the RNA genome. We conclude that these ptd viruses bear either point mutations or deletions of varying size but all retain part of the src gene. These mutants are stable and should be useful for further genetic and physiological studies on the src gene and its product.     

Continuous production of Rous sarcoma virus free of transformation defective virus in clones of established RSV-transformed quail cells

Quail embryo fibroblasts were infected with a Schmidt-Ruppin strain RSV × chf recombinant virus. Virus-transformed cells were established as a permanent line and then cloned in methyl cellulose. Out of 140 clones isolated four clones were capable of indefinite growth. These clones were examined for (i) production of sarcoma and td virus particles, (ii) number of integrated virus genome equivalents, and (iii) deletions of the src gene in the provirus. We found that the clones yield about 106 focus-forming units of the sarcoma virus per milliliter of the culture medium. No td virus could be detected by plating of the virus at the endpoint dilution and no 35 S td virus RNA but only 38 S sarcoma virus RNA was found in virions. Hybridization kinetic studies indicated that three different clones contain about 2 virus genome equivalents, and one clone contains about 4 virus genome equivalents per diploid cell. Upon transfection the proviruses of different clones generated sarcoma viruses and no td viruses. Finally digestion with EcoRI restriction endonuclease released in all four clones a 1.9 × 106-dalton fragment characteristic of the complete src gene, while no 0.8 × 10⁶-dalton fragment characteristic of a td provirus could be detected. We concluded that the clones of RSV-transformed quail cells contain only nondefective sarcoma proviruses and produce from these proviruses nondefective focus-forming virions in the absence of any segregant td virions.     

Restitution of fibroblast-transforming ability in src deletion mutants of avian sarcoma virus during animal passage.

The Schmidt-Ruppin strain of Rous sarcoma virus subgroup D (SR-D) gives rise to transformation defective (td) mutants which have lost either all or almost all of the src gene (standard td or std viruses) or have only a partial deletion of src. These partial deletion mutants, designated ptd viruses, contain genomic RNA slightly larger than std isolates, and heteroduplex analyses suggest that ptd viruses retain approximately 25% of src from the 5′ end of that gene [Lai et al. (1977) Proc. Natl. Acad. Sci. USA74, 4781–4785]. Several ptd isolates of SR-D were injected into newly hatched chickens and after prolonged latent periods caused sarcomas in about 30% of the birds. The tumors occurred in internal organs away from the site of injection. Infectious sarcoma viruses isolated from these growths show the envelope markers of subgroup D are nondefective for replication and induce a transformation in vitro which is morphologically distinct from that of SR-D. Electrophoresis of 35 S genomic RNA from these recovered sarcoma viruses shows it to be of the size characteristic for nondefective sarcoma viruses. Fingerprint analysis of ³²P-labeled RNA from one of the new sarcoma viruses detected all oligonucleotides present in ptd viruses, the src-specific oligonucleotides of SR-D, and one new oligonucleotide not present in SR-D. This new RNase T₁-resistant oligonucleotide and the src-specific oligonucleotides identical to those of SR-D map close to the 3′ end in the genome of the recovered sarcoma virus, which is the position expected for the src gene. These studies suggest that recovered avian sarcoma viruses have acquired cellular sequences which are closely related in structure and function to the viral src gene.     

Structural and nonstructural proteins of strain Colburn cytomegalovirus

The growth of most Rous sarcoma viruses (RSV) is severely restricted on MSB-1 cells (a line of chicken T lymphoblasts) in comparison to growth on chicken embryo fibroblast (CEF). Nonconditional transformation defective mutants of RSV from which the complete src region has been deleted (td RSV) are not subject to growth restriction. We examined the formation and integration of RSV and td RSV in MSB-1 cells following high multiplicity infection. Nearly equivalent quantities of the linear form of unintegrated RSV and td RSV DNA were formed in these cells during the first 10 hr after infection. Linear RSV DNA from MSB-1 cells could not be distinguished from linear RSV from CEF by restriction endonuclease analysis and by previously described transfection assays (P. E. Neiman, C. McMillin-Helsel, and G. M. Cooper, 1978, Virology 89,360–371). Beyond 10 hr after infection, and with progressive cell growth in the MSB-1 cultures, the level of RSV linear DNA rapidly decreased. Presumptive circular RSV DNA was detected only transiently, and at very low levels, about 15 hr after infection. Association of RSV DNA with high-molecular-weight chromosomal DNA, i.e., integration, was not detected in this study. In contrast, nearly constant levels of td RSV unintegrated linear DNA and, after 20 hr, circular DNA persisted in MSB-1 cells for at least 7 days after infection. Integration of td RSV proviral DNA was inefficient, occurring in only about 5% of MSB-1 cells (even at very high multiplicities of infection) in the first round of infection, and in 25–40% of cells by 3 days after infection. Almost all MSB-1 cells containing td RSV DNA produced virus. Analysis of eight nonconditional transformation defective mutants of RSV which retain the src region to different extents showed that all of these mutants replicated to the same normal titer on MSB-1 cells as on CEF without further deletion of the src region. Two temperature sensitive src mutants that thermal inactivation of the scr gene on MSB-1 cells at both 35° and 41°, indicating that thermal inactivation of the src gene product could not abrogate the replication block. These studies clearly demonstrate that the presence of the src region in RSV impedes the formation and/or integration of provirus in some types of host cells.     

Analysis on the reproducing and cell-transforming capacities of a temperature sensitive mutant (ts 334) of avian sarcoma virus B77

Ts 334, a temperature sensitive mutant of avian sarcoma virus B77, cannot produce infectious progeny nor induce neoplastic transformation at the nonpermissive temperature. In order to clarify the relationship between these two functions of ts 334 we attempted to (1) isolate and characterize nonconditional transformation defective (td) mutants from ts 334, (2) isolate and characterize recombinants between ts 334 and RAV-1, and (3) reexamine rescue of ts 334 with RAV-1 at the nonpermissive temperature.All seven nonconditional td mutants isolated from ts 334 kept their temperature sensitive character in replication, although they had lost transforming capacity both at the permissive and at the nonpermissive temperatures. They appear to have a temperature sensitive step in virus maturation like ts 334.The helper function of these td mutants for the defective Bryan high titer strain of Rous sarcoma virus is also temperature-dependent.Two recombinants were isolated from cells coinfected with ts 334 and RAV-1. These recombinants combined the cell-transforming ability of ts 334 and of the envelope properties of RAV-1. These two recombinants were unable to induce cell-transformation but grew well at the nonpermissive temperature.In RAV-1 producing cells not only the genome of ts 334, but also the envelope property of ts 334 were rescued at the nonpermissive temperature, though cell transformation was not observed.These observations suggest that ts 334 has two mutations, one affecting reproduction and another cell-transformation capacities.     

Hydroxylamine mutagenesis of HSV DNA and DNA fragments: introduction of mutations into selected regions of the viral genome.

Procedures for the induction and isolation of temperature-sensitive (ts) mutants by in vitro mutagenesis of purified HSV DNA and DNA fragments have been developed. In order to establish conditions for the mutagenesis of viral DNA fragments with hydroxylamine (HA), virion-associated and intact DNA were mutagenized first. Under the conditions of mutagenesis employed, HA was shown to be a more powerful mutagen of purified DNA than of virion-associated DNA. Mutagenesis of intact DNA resulted in the identification of three new complementation groups. A mixture of EcoRI fragments A and B was next mutagenized. Mutations in DNA fragments were recovered by cotransfection with intact, wild-type DNA. Six ts mutants were isolated from the progeny of the mixed infection. Five of these mutants were analyzed further. One mutant, ts508, failed to complement tsB2 which had previously been shown to lie in fragment B. Like tsB2 and other members of this group, ts508 is DNA^?. The remaining four HA-induced mutants constitute three new DNA⁺ complementation groups. Marker rescue experiments indicate that mutants in two of these groups lie in fragment A.     

A new endonuclease restriction site which is at the locus of a temperature-sensitive mutation in vaccinia virus is associated with true and pseudoreversion

A temperature-sensitive mutant of vaccinia IHD-W, designated is 9251, possesses a novel EcoRI restriction endonuclease site in the fragment D of the parental genome. Spontaneous ts⁺ revertants of ts 9251 fall into two distinct categories: the majority of revertants reacquired the parental EcoRI restriction profile, while one isolate maintained the mutant cleavage site. Using two-dimensional gel analysis of viral polypeptides induced by vaccinia virus in infected cytoplasms, it was observed that the fingerprint of mutant ts 9251 differed from the parental IHD-W in that a single viral protein of a molecular weight of 37,000 daltons migrated to a new isoelectric point. The revertants which had regained the wild-type restriction profile now encoded for a 37K polypeptide identical to that of the wild-type virus while the single revertant which maintained the novel EcoRI site possessed a 37K protein of a charge intermediate between ts 9251 and wild type. We conclude that ts 9251 can revert to the ts⁺ phenotype either by true reversion at the original mutant locus or by a second independent mutation within the same gene.     

Temperature sensitive mutants of vesicular stomatitis virus: tryptic peptide maps of the proteins modified in complementation groups II and IV

The proteins specifically modified by mutations in complementations groups II and IV of vesicular stomatitis virus were identified using ion-exchange chromatography analysis. Labeled tryptic peptides from mutants and their revertants were compared and unambiguous results were obtained. For each mutant only one peptide in one protein was eluted at a place different from that of its revertant counterpart. The modified peptide was located in the M protein for ts 023 (III), as expected from previous data (F. Lafay (1974). J. Virol.14, 1220–1228). The modified peptide was found in the NS protein for ts 052 (II) and in the N protein for ts 0111 (IV). From these results we conclude that NS protein is encoded by VSV gene II and N protein, by gene IV.     

Transformation-enhancing factor(s) released from chicken Rous sarcoma cells: effect on some transformation parameters

The medium of chick embryo fibroblasts (CEF) transformed by Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV) contains a factor(s) which complements the expression of some transformation parameters depending on the src gene. Notably, it reverses the block by puromycin of morphological transformation of cells infected with three ts-T mutants after shift-down from restrictive (41.5°) to permissive (37°) temperature. This reversal is not due to the release of inhibition of protein synthesis produced by puromycin, and is accompanied by the expression of two other src-dependent transformation parameters: disorganization of the cytoskeleton and loss of cell surface-associated fibronectin. The factor(s) able to overcome the puromycin block of morphological transformation was operationally called transformation-enhancing factor (TEF) like a previously reported factor favoring transformation by RSV (Krycève et al., Int. J. Cancer17, 370–379, 1976). It is lacking in media of untransformed cells, uninfected or infected with a nontransforming virus (RAV-1), and its production by RSV-infected cells seems to depend on the acquisition of the transformed phenotype, therefore on the expression of the src gene. Its effect was also shown to persist beyond the period of contact with the cells. It appears to be a glycoprotein which can be resolved by gel filtration into two peaks of 250K and 190K, apparently distinct from other known factors spontaneously released by transformed cells. A similar activity was also found in the medium of mammalian (rodent) cells transformed by SR-RSV and by other RNA and DNA oncogenic viruses, but not in the medium of untransformed controls.     

Location of a new endonuclease restriction site associated with a temperature-sensitive mutation of vaccinia virus

The novel EcoRI recognition site at the temperature-sensitive locus of ts 9251, derived from vaccinia IHD-W, has been mapped on the poxvirus genome. Using Southern blot hybridization, it was demonstrated that the EcoRI fragment D of vaccinia IHD-W and of rabbit poxvirus were identical in terms of sequence and presumed map position and that the novel restriction site found in ts 9251 occurred at a point 28 megadaltons from the right terminus of the molecule. This study reveals the first identification of a poxvirus mutation on the physical map and provides a potential marker for further, genetic mapping of temperature-sensitive mutations of vaccinia and related poxviruses.     

Detection and enumeration of transformation-defective strains of avian sarcoma virus with molecular hybridization.

Serial propagation of avian sarcoma viruses generates deletions in the viral gene responsible for cellular transformation (src). We have devised an assay for these deletion mutants which utilizes molecular hybridization and exploits the availability of DNA (cDNA_sarc) complementary to the nucleotide sequences affected by the deletion in src. Our procedure is also applicable to deletions in other viral genes and offers several advantages over conventional bioassays for the deletion mutants; moreover, it can be used to detect deletions in virus-specific intracellular nucleic acids. In order to illustrate the utility of the assay, we demonstrate that all 20 copies of the proviral DNA for avian sarcoma viruses in XC cells contain src, and we show that single avian cells can contain functioning proviruses for both avian sarcoma virus and a congenic deletion mutant. It should now be possible to use molecular hybridization to study the mechanism by which deletions in src are generated.     

Temperature-sensitive mutants of influenza virus: Identification of the loci of the two ts lesions in the Udorn-ts-1A2 donor virus and the correlation of the presence of these two ts lesions with a predictable level of attenuation

The influenza A/Udorn/72-ts-1A2 virus is currently being studied as a donor of its ts lesions to produce recombinants for use in a live virus vaccine. The Udorn/72-ts-1A2 virus, which possesses two ts lesions, was produced by mating two complementing, single-lesion ts mutants. One parent, the Udorn/72-ts-1 [A] clone 189 virus, bears a is lesion that belongs to our complementation-recombination group 1 and is located on RNA segment 1(P. Palese and M. B. Ritchey, 1977, Virology78, 183–191). The second parent, the Hong Kong/68-ts-315 virus, has a is mutation that belongs to our complementation-recombination group 5. The present study sought to determine the location of the complementation group 5 ts mutation. In addition, the RNAs of 6 is-1A2 recombinants bearing the surface antigens of influenza A/Vic/75 (H3N2) virus were analyzed by polyacrylamide gel electrophoresis in order to determine the parental origin of each gene. Finally, by correlating genotype with level of viral replication in the hamster it was possible to identify the genes of the ts-1A2 donor virus responsible for attenuation.     

A nonconditional replication-defective mutant of the Schmidt-Ruppin strain of Rous sarcoma virus.

A nonconditional mutant of the Schmidt-Ruppin strain of Rous sarcoma virus (subgroup A) that fails to synthesize infectious progeny virus is described. Nonproducing cells transformed by this mutant LA7365 have functional viral src and env genes. The gag gene of LA7365 is defective. There is no complementation with a gag ts mutant, and pulse-chase experiments show that the proteolytic processing of the gag precursor pr76 is strongly inhibited in mutant-infected cells. Whether there is an additional lesion in the pol gene of LA 7365 could not be determined from the available data.     

Molecular cloning of African swine fever virus DNA

African swine fever virus DNA (about 170 kbp) was cleaved with the restriction endonuclease EcoRI and most of the resulting 31 fragments were cloned in either the phage vector λWES.λB or the plasmid pBR325. Three fragments were not cloned in those vectors, the largest fragment EcoRI-A (21.2 kbp) and the two crosslinked terminal fragments, EcoRI-K′ and D′. Endonuclease SalI cut fragment EcoRI-A into three pieces which were cloned in plasmid pBR322. The two terminal EcoRI fragments were cloned after removal of the crosslinks with nuclease S1 and addition of EcoRI linkers to the fragment ends. The complete library of the cloned fragments accounted for about 98% of ASF virus genome, the missing sequences being those removed by the nuclease S1 in the process of cloning the terminal fragments.     

Mutants of Rous sarcoma virus with extensive deletions of the viral genome.

Deletion mutants of Rous sarcoma virus (RSV) have been isolated from a stock of Prague RSV which had been irradiated with ultraviolet light. Quail fibroblasts were infected with irradiated virus and transformed clones isolated by agar suspension culture. Three clones were obtained which did not release any virus particles. Analysis of DNA from these non-producer clones with restriction endonucleases and the Southern DNA transfer technique indicated that the clones carry defective proviruses with deletions of approximately 4 × 10⁶ daltons of proviral DNA. The defective proviruses, which retain the viral transformation (src) gene, contain only 1.7–2.0 × 10⁶ daltons of DNA. Multiple species of viral RNA containing the sequences of the src gene were detected in these clones; some of these RNAs may contain both viral and cellular sequences. The protein product of the src gene, p60^src (Brugge and Erikson, 1977), was also synthesized in the nonproducer clones. However these clones did not contain the products of the group-specific antigen (gag), DNA polymerase (pol), or envelope glycoprotein (env) genes, nor did they contain the 35 and 28 S RNA species which are believed to represent the messengers for these viral gene-products. The properties of these mutants indicate that expression of the src gene is sufficient to induce transformation. These clones may represent useful tools for the study of the expression of this region of the genome.     

Activation of mouse retrovirus by herpes simplex virus type 1 cloned DNA fragments

Cloned DNA fragments from herpes simplex virus (HSV) type 1 (strain Patton) were tested for activation of endogenous mouse retrovirus in BALB/3T3 cells. Activation within the L region of HSV-1 DNA was observed with the ～3.4-kilobase pair (kbp) BamHI fragment which contains the virus thymidine kinase (TK) gene, and the ～5.3-kbp EcoRI L fragment. Activation by the TK-containing BamHI fragment was abrogated by digestion with EcoRI. Activation within the S region of HSV-1 DNA was observed with the ～15.2-kbp EcoRI H ragment and the ～8.4-kbp Eco RI/HindIII H/G fragment. Assaying for retrovirus activation serves as an additional parameter for mapping biological functions within the HSV genome.     

The physical locations of structural genes in adenovirus DNA.

Interserotypic recombinants are formed in crosses between temperature-sensitive (ts) mutants of adenovirus type 5 (Ad5) and the nondefective adenovirus 2-SV40 hybrid virus Ad2⁺ND1. The positions of Ad2⁺ND1 and Ad5 DNA sequences in recombinant genomes can be determined by analysis of their DNA with restriction endonucleases and the sites of the mutation in many ts mutants have been mapped [Grodzicker, T., Williams, J. F., Sharp, P. A., and Sambrook, J. (1974), Cold Spring Harbor Zymp. Quant. Biol.39, 439–446; Sambrook, J., Williams, J., Sharp, P. A., and Grodzicker, T. (1975), J. Mol. Biol.97, 369–390]. We show here that several Ad5 polypeptides can be distinguished from their Ad2⁺ND1 counterparts by SDS-polyacrylamide gel electrophoresis. Analysis of the polypeptides encoded by recombinants of known genetic constitution has allowed us to locate several structural genes on the adenovirus genetic and physical maps and to assign ts mutants to specific genes. Furthermore, we have physically mapped the early Ad5 mutant ts 125 which has a defect in the gene for the adenovirus-specific single-stranded binding protein. In addition, we show that the virus-associated RNAs of Ad2 and Ad5 can be separated electrophoretically, permitting rapid and simple determination of the origin of the DNA surrounding position 0.30 in interserotypic recombinants.     

Restriction endonuclease mapping of the DNA of Rous-associated virus O reveals extensive homology in structure and sequence with avian sarcoma virus DNA

We have prepared a physical map of the DNA of Rous-associated virus O (RAV-O), an endogenous virus released by certain chicken lines, in order to examine the relationship between the genome of this virus and closely related proviruses endogenous to chickens. Nineteen recognition sties for 11 restriction endonucleases have been mapped on the unintegrated linear and circular forms of RAV-O DNA isolated from acutely infected quail cells.PvuI is the only enzyme tested which does not cleave RAV-O DNA. Most of the sites (18 of 19) occur at similar or identical positions in the DNA of avian sarcoma virus (ASV). Significant differences between the maps of corrseponding regions of RAV-O and ASV DNA are observed only with those endonucleases which recognize sites encoded near the 3′ terminus of ASV RNA (PvuI andEcoRI). Both ends of RAV-O linear DNA contain sequences copied from both the 3′ and the 5′ ends of viral RNA. Two species of closed circular DNA were found: one the same size as the terminally redundant linear DNA (5.0 × 10⁶M_r and the other lacking ca. 0.35 kb from an end of linear DNA. Thus the unintegrated forms of RAV-O DNA appear structurally similar to those of ASV DNA[Shank, P. R.,et al. (1978b).Cell15, 1383–1395;Hsu, W.,et al. (1978).J. Virol.28, 810–818]; presumably one copy of a ca. 0.35-kb terminal repeat unit is lost during formation of the smaller circle from linear DNA. The following paper illustrates the utility of the physical map for differentiating between endogenous proviruses which might or might not have sequence identity with the RAV-O genome.     

Cloning of the marmoset herpesvirus thymidine kinase gene and analyses of the boundaries of the coding region

In order to delimit the approximate boundaries of the marmoset herpesvirus (MarHV) thymidine kinase (TK) gene, HindIII and BamHI digests of MarHV DNA were cloned in plasmid pBR322. Several recombinant plasmids which transformed E. coli K12 strain RR1 to ampicillin resistance were isolated. The MarHV DNA inserts in these plasmids accounted for about half of the MarHV genome. One of the plasmids, pMAR4, contained a 9.1-kbp fragment of MarHV DNA (HindIII-G), transformed LM(TK^?) cells to TK⁺, and hybridized to the BamHI-I fragment of MarHV DNA, which had previously been shown to have TK-transforming activity. pMAR4 DNA had little or no homology to the 2-kbp PuvII fragment of HSV-1 DNA, which contains the HSV-1 TK gene. Cleavage with PvuII, SacI, SmaI, and KpnI inactivated the TK-transforming activity of pMAR4, but cleavage with HindIII, PstI, EcoRI, XhoI, XbaI, and BamHI did not. Deletion mutants pMAR401 and pMAR420, which lacked the 2.6-kbp KpnI and the 2.75-kbp EcoRI fragments, respectively, of pMAR4, lost transforming activity, whereas pMAR410, which lacked a 2.9kbp XhoI fragment of pMAR4 did not. Recombinant plasmid pMAR430, which contained a 3-kbp PstI fragment of pMAR4, also transformed LM(TK^?) cells to TK⁺. The results strongly suggest that the coding region of the MarHV TK gene was within a 2.4-kbp pMAR4 sequence extending from the PstI (0.33 kbp) to the EcoRI (2.7 kbp) cleavage sites.     

Alterations in membrane polypeptides of chick embryo fibroblasts induced by transformation with avian sarcoma viruses.

Membrane proteins of chick embryo fibroblasts (CEF) transformed with various strains of avian sarcoma viruses were analyzed by electrophoresis in SDS-polyacrylamide gels and compared with those of untransformed cells. The following differences were consistently detected in CEF transformed with B77, the Prague strain of Rous sarcoma virus (PR-RSV) or the Schmidt-Ruppin strain of RSV (SR-RSV): (1) The appearance of a polypeptide band with an apparent molecular weight of 90,000, (2) increase in amount of a polypeptide of 79,000 daltons, (3) significant decrease in amount of a polypeptide of 50,000 daltons and (4) marked decrease in amount of a protein of 200,000 daltons. CEF infected with the temperature-sensitive (ts) mutants of these strains, LA334 (of B77), LA31 (of PR-RSV) or OS122 (of SR-RSV) showed similar changes at 36°, but at 41°, except for alteration (4), the profiles of the membrane proteins were similar to those of uninfected cells. Changes (1) and (3) were reversible and clearly observable within a few hours after a temperature shift of CEF infected with ts mutants. Fusiform transformation induced by a variant of B77 was also shown to induce alterations (1) and (3).From these and other results, the appearance of the polypeptide band of 90,000 daltons, which could not be detected in untransformed cells, and the marked decrease in amount of a protein of 50,000 daltons in cell membranes were concluded to be closely correlated with transformation of CEF.