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.     

Strain specificity of changes in adenylate cyclase activity in cells transformed by avian sarcoma viruses.

Chick embryo fibroblasts transformed by avian sarcoma viruses, B77, the Prague strain of Rous sarcoma virus (PR-RSV), and the Schmidt-Ruppin strain of RSV (SR-RSV) had lower activity of adenylate cyclase, but the same activity of cyclic AMP phosphodiesterase as control cells. The cells infected with their temperature-sensitive mutants in the transforming capacity also had reduced cyclase activity when grown at 36°, but normal activity when grown at 41°, showing a close correlation between viral transformation and cellular adenylate cyclase activity.The characteristics, Km value for ATP, Mg²⁺ ion dependency, and sensitivity to NaF, of the adenylate cyclase activity reduced by viral transformation varied with the viral strain. It was concluded that the changes of the enzyme activity on transformation were specific to the virus strain used and were not related to the subgroup of virus.     

A temperature-sensitive lesion affecting levels of transformation-specific viral RNA in a mutant of avian sarcoma virus PRCII

LA46 is a mutant of avian sarcoma virus PRCII that is temperature sensitive (ts) in the maintenance of oncogenic transformation and in the production of infectious virus. These ts properties are independent of the helper virus. At the nonpermissive temperature LA46-infected cells also fail to produce significant quantities of the transformation-specific protein, and the amount of intracellular transformation-specific viral RNA is reduced at least 100-fold. The probable primary defect of LA46, a temperature sensitivity in transformation-specific viral RNA, can account for the phenotype of the mutant. The molecular mechanism of this defect is currently investigated.     

Characterization of transformation-sensitive membrane-proteins in chick embryo fibroblasts transformed with avian sarcoma virus

When chick embryo fibroblasts (CEF) were transformed with avian sarcoma virus (ASV), marked increases of membrane proteins with molecular weights of 90,000 daltons (P90) and 79,000 daltons (P79) and a decrease of a 50,000 dalton protein (P50) were detected. The mechanism of accumulation of the transformation-sensitive protein P90 was studied. On two-dimensional gel electrophoresis, P90 had the same isoelectric point as P91, a membrane component of nontransformed cells. From the similarity of tryptic peptide maps of ¹²⁵I-labeled P90 and ¹²⁵I-labeled-P91, and the findings that [¹⁴C]glucosamine was incorporated into P91 but not into P90 and that P90 accumulated when glycosylation was blocked with tunicamycin, it was concluded that P90 and P91 have very similar amino acid sequences, but that P91 has an oligosaccharide side chain not present in P90. However, no evidence of the interconversions of P90 and P91 was obtained in chase experiments after specific labeling of one of the two proteins. It was concluded that accumulation of P90 resulted from reduced glycosylation of growing polypeptides of P90, the synthesis of which was enhanced in transformed cells. The enhanced synthesis of P90 in transformed cells seemed to be controlled by the concentration of glucose in the medium, since large excess of glucose diminished the amount of P90 accumulated in transformed cells, and the accumulation of P90 was also observed in nontransformed cells by deprivation of glucose in the culture medium as shown by Shiu et al. (Glucose Depletion Accounts for the Induction of Two Transformation-Sensitive Membrane Proteins in Rous Sarcoma Virus-Transformed Chick Embryo Fibroblasts. Proc. Nat. Acad. Sci. USA74, 3840–3844, 1977).     

Specific RNA sequences of Rous sarcoma virus (RSV) recovered from tumors induced by transformation-defective RSV deletion mutants.

We have investigated the RNAs of two avian sarcoma viruses recovered (rASV) from tumors induced in chickens by a deletion mutant of Schmidt-Ruppin Rous sarcoma virus (SR-RSV) that had lost part, but not all, of its sarcoma gene (src). The RNAs of the rASVs had the same size as SR-RSV RNA and were larger than the predominant RNA species of the partial src deletion mutant, if measured by electrophoresis in polyacrylamide gels. Fingerprinting of RNase T,-resistant oligonucleotides indicated that the rASVs shared one src gene oligonucleotide with SR-D which was also present in the partial src deletion mutant of SR-RSV. The two rASVs shared one other, probable src oligonucleotide, that was not found in SR-RSV, and SR-RSV contained a src oligonucleotide not found in the rASVs. However, the distinctive src oligonucleotide of the rASVs was structurally closely related to that of SR-RSV. We conclude that the src genes of the rASVs and that of SR-RSV are closely related. Possible mechanisms by which a partial src deletion may recover a complete src gene are discussed in view of our results.     

A nonconditional mutant of Rous sarcoma virus containing defective polymerase.

A nonconditional mutant of Rous sarcoma virus (RSV) cd SE52dPR-C (SE52d), which is a recombinant between PR-RSV-C and RAV-0, has been isolated. It is shed from a clone of transformed quail cells, but is unable to exogenously infect or transform any other avian cells. It contains subgroup C envelope glycoproteins and the internal structural protein p27o characteristic of RAV-0. SE52d virions contain no detectable polymerase activity as measured in either an endogenous reaction or after addition of exogenous template. The genome of SE52d contains a deletion of approximately 10% of the genome as compared to PR-RSV, which can be localized between about 0.7 and 0.8 of the genome length from the 3′ terminus of the RNA. The deletion includes about 30% of the polymerase gene. The virions contain no α or β polymerase subunits (95,000 and 65,000 daltons), but do appear to have a small amount of a new protein species of about 57,000 daltons which precipitates with antibody to viral polymerase.     

Changes in membrane polypeptides that occur when chick embryo fibroblasts and NRK cells are transformed with avian sarcoma viruses

Studies were designed to detect changes in the plasma membrane polypeptide composition of chick embryo fibroblasts (CEF) and Normal rat kidney (NRK) cells following infection with avian RNA tumor viruses. A method capable of isolating large fragments of plasma cell membranes, based on fractionation in a two-phase polymer system, was developed for this purpose.Infection with the Prague (subgroup C) or SR (subgroup A) strains of RSV, or with ts 68 of SR-RSV-A or ts 339 of B77 at the permissive temperature, caused CEF to transform morphologically; simultaneously a limited number of changes occurred in their plasma cell membrane composition. They were: (a) a large increase in the rate of labeling with ¹⁴C-valine and in the total amount of a polypeptide with an apparent MW of 73,000; a similar small increase occurred with respect to a polypeptide with an MW of 95,000; neither could be labeled with ¹⁴C-glucosamine; (b) a decrease in the rate of labeling and in the total amount of high molecular weight glycopoly-peptides with MWs of about 250,000; (c) a decrease in the rate of labeling and in the total amount of a polypeptide with an MW of 39,000. Infection with ts 68 and ts 339 at the nonpermissive temperature, or with a nontransforming mutant of SR-RSV-A, or with RAV-7, did not cause alterations (a) and (b), but did cause alteration (c).Identical membrane polypeptide changes were observed in NRK cells infected with the Prague strain of RSV or with ts 339 of B77 at the permissive but not at the nonpermissive temperature. The significance of the fact that these changes, in particular the increases in the amounts of the 73,000 and 95,000 dalton polypeptides, occurred to the same extent in the membranes of both avian and mammalian cells is discussed.     

Intoxication of normal and virus-transformed cells by diphtheria toxin.

Trypsinization causes Schmidt-Ruppin Rous sarcoma virus (SR-RSV)-transformed chick embryo fibroblasts (CEF) and polyoma-transformed BHK-21 cells to be more sensitive to intoxication than their respective normal cell lines by reducing the toxin sensitivity of the normal cells but not affecting the transformed cells. Protection from trypsin is dependent on transformation but not virus production. The selective effect of trypsin on toxin sensitivity is specific. A low concentration of CRM 197 protein (0.5 μg/ml) inhibits diphtheria intoxication in trypsinized CEF but does not protect nontrypsinized CEF from intoxication.     

Mapping of nonconditional and conditional mutants in the src gene of Prague strain Rous sarcoma virus

Restriction endonuclease EcoRI digestion of the viral DNA of 12 nonconditional transformation defective (td) mutants of Prague strain Rous sarcoma virus (PR-RSV) has divided these mutants into two groups. Five mutants possess an EcoRI B (src gene-containing) fragment of the same size as that from wild type PR-RSV and thus these mutants have no detectable diminution in the transforming src gene. The other 7 mutants bear deletions of 1.0 to 1.8 kilobases in the 3.2-kilobase EcoRI B fragment. The extents of these deletions have been mapped using a number of restriction endonucleases and by comparing these results with studies on the nucleotide sequence of src(Czernilovsky et al., Nature (London)287, 198–203, 1980) we conclude that the td mutants have deleted sequences at the 5′ end of src, and in some cases also in regions between src and env, leaving intact at least some 3′ src sequences. These td mutants recombine in differing patterns with 14 temperature-sensitive (ts) src gene mutants. This enables many of the ts mutations to be localized in limited regions of src, 10 of them being clustered in the 3′ 40% of the gene, the remaining four bearing at least one mutation in the 5′ 60% of src. A nonconditional src gene mutant that transforms cells to a fusiform as opposed to round cell morphology (td SF/LO 104) also possesses a lesion that maps in the 5′ 60% of the src gene.     

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.     

Conditional lethal mutants of avian sarcoma viruses. I. Physiology of ts 75 and ts 149

Ts 75 and ts 149 are two temperature-sensitive mutants of avian sarcoma virus B77 which fail to reproduce and to induce neoplastic transformation at 41°. Both mutants are indistinguishable from wild type in somatic properties of the virion: host range, type-specific antigenicity, and rate of inactivation at 41°. The temperature-sensitive step of ts 75 occurs late in the infectious cycle allowing the synthesis of group-specific antigen in increased amounts and of viral RNA under nonpermissive conditions. Ts 149 has an early temperature sensitive phase and does not produce group-specific antigen at 41°. The maintenance of some neoplastic properties in cells transformed by either ts 75 or ts 149 is continuously dependent on a temperature sensitive viral function: Shift of transformed cells to 41° results in disappearance of neoplastic traits. Double infection of cells at 41° with wild-type avian sarcoma virus and ts 75 or ts 149 results in the rescue of 3 markers derived from the mutant virus: temperature sensitivity, host range, and morphology of the transformed cell. Rescue of ts 75 or ts 149 with wild-type avian leukosis viruses at 41° has not been accomplished.     

RNA synthesis by temperature-sensitive mutants of vesicular stomatitis virus, New Jersey serotype.

We report the results of studies using temperature-sensitive mutants of Rous sarcoma virus (ts RSV) to study the alterations in surface proteins occurring on transformation. A large external transformation-sensitive (LETS) protein is detected by lactoperoxidase-catalyzed iodination on normal chicken embryo fibroblasts (CEF) but is reduced on RSV-transformed cells. The LETS protein is temperature sensitive in ts RSV-infected CEF and the kinetics of the alterations occurring on shifts between permissive and restrictive temperature are reported. During reversion to normality on shift-up, the LETS protein appears at the surface quite rapidly and in parallel with the morphological changes, although the change is less rapid than that in glucose transport. On transformation during shift-down, the disappearance of the LETS protein lags behind the morphological change. Cycloheximide does not inhibit reappearance of iodinatable LETS protein on shift-up, suggesting that the LETS protein is synthesized in transformed cells, although it is not present at the surface. Hypotheses to explain the absence of the LETS protein at the surface are discussed and evidence is presented for an increased rate of turnover after transformation.     

Differential expression of transformation in rat and chicken cells infected with an avian sarcoma virus ts mutant

Cells of the NRK line were infected with ts 339, a mutant of avian sarcoma virus B77, and clones were isolated which displayed the characteristics of transformed cells at 33 C. These cells reverted to a normal phenotype at 37 C. In comparison, chicken cells infected with the same mutant remained transformed at 37 C and acquired properties of normal cells only after temperature shift to 41 C.     

Synthesis of virus-specific polypaptides by temperature-sensitive mutants of herpes simplex virus type 1.

The polypeptide phenotypes of 22 temperature-sensitive (ts) mutants of herpes simplex virus type 1 were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis of mutant-infected cells at permissive and nonpermissive temperatures. Following analysis of isotopically labeled polypeptides synthesized from 4–24 hr postinfection, the mutants were divided into four major phenotypic groups which include: (1) DNA^?ts mutants which share several common polypeptide defects, (2) DNA^±ts mutants which exhibit polypeptide profiles resembling the DNA^?ts mutants, (3) DNA⁺ts mutants which exhibit polypeptide phenotypes differing only slightly from that observed in wild-type virus-infected cells grown at 39°, and (4) DNA⁺ts mutants which exhibit no detectable alterations in their polypeptide profiles when compared with that of the wildtype virus. When the polypeptide phenotypes of the mutants were compared with previously determined mutant characteristics, including synthesis of viral DNA, thymidine kinase, DNA polymerase, and physical virus particles, a correlation was consistently observed between mutant polypeptide and viral DNA phenotypes.     

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.     

Use of an aberrant polypeptide as a marker in three-factor crosses: Further evidence for independent reassortment as the mechanism of recombination between temperature-sensitive mutants of reovirus type 3

The aberrant μ1 and μ2 viral polypeptide phenotype (designated μ^??) of certain groups of temperature-sensitive mutants of reovirus type 3 has been utilized as a third unselected genetic marker in three-factor crosses. Approximately 50% of the ts⁺ recombinant clones were found to express the wild type μ⁺ phenotype of one of the parents, while the remainder expressed the aberrant μ^?? phenotype of the other. The approximately random distribution of the μ phenotype in two crosses of this type supports the conclusion that recombinants which result from mixed infection with pairs of temperature-sensitive mutants of reovirus type 3 are generated by random assortment of RNA segments. The results of three-factor crosses, furthermore, suggest that the ts mutation of the group A mutant (ts201) is genetically linked to the determinant of the aberrant μ1 and μ2 polypeptide.In addition, ts⁺ revertants of groups B, D, and G retain the μ phenotype of the mutant strain from which they are derived, while the group A (ts201) revertants display a variety of phenotypes.     

Use of temperature-sensitive mutants to study the morphogenesis of poliovirus

Three temperature-sensitive (ts) mutants of poliovirus (type 1 Mahoney) were isolated after nitrous acid treatment and characterized as phenotypically RNA⁺. When cells were infected at 37° with two of the three RNA⁺ts mutants (ts109 and ts739), reduced levels of 14 S particles were synthesized. One RNA⁺ mutant (ts520) synthesized significant amount of viral 14 S particle subunits. All of the mutants synthesized reduced amounts of procapsids and virions at 37°. At 39.5°, with all three ts mutants, the production of all virus-related particles in infected cells was markedly suppressed. Isoelectric focusing of the viral-related particles produced at 37° by the ts mutants and electrophoretic analysis of their structural polypeptides revealed the following: (i) ts739 synthesized an altered VP0 polypeptide and produced 14 S particles with an altered isoelectric point; (ii) ts109 produced 14 S particles with a normal pI but containing what appeared to be an altered VP1; (iii) ts520 produced normal 14 S particles as demonstrated by their pI, the electrophoretic behavior of their constituent structural polypeptides in SDS-PAGE, their ability to self-assemble, and their ability to form procapsid-like structures when incubated in extracts from wild-type (wt) virus-infected cells. However, ts520-infected cells contained few, if any, procapsids and extracts made therefrom were unable to assemble ts520 or wt 14 S particles into detectable amounts of pI 6.8 empty capsids. These and other findings are consistent with ts739 (and probably ts109) possessing an altered structural protein and ts520 being mutant in its morphopoietic factor.     

Genetic factors associated with loss of the temperature-sensitive phenotype of the influenza A/Alaska/77-ts-1A2 recombinant during growth in vivo

The influenza A/Alaska/77-ts-1A2 vaccine candidate strain, which has a 37° shutoff temperature for plaque formation and temperature-sensitive (ts) lesions on the P1 and P3 genes, was administered intranasally to a seronegative child who subsequently remained well. However, starting 7 days after inoculation, A/Alaska/77 virus with a 40° shutoff temperature (ts⁺) was shed. To determine whether an extragenic suppressor mutation was responsible for this loss of ts phenotype of the vaccine candidate virus, the ts⁺ isolate was mated with wild-type virus, and ts recombinant progeny were sought. Approximately 25% of the progeny were ts, suggesting that a suppressor mutation was present in the child's ts⁺ isolate. Each of 62 ts progeny viruses had the ts P3 gene. The extragenic suppressor of this ts gene was localized to the P2 gene. The ts P3 gene of the A/Alaska/77-ts-1A2 recombinant specified a 37–38° shutoff temperature for plaque formation, whereas the ts P3 gene in the ts segregants derived from the ts⁺ isolate had a 39° shutoff temperature. Hence, the ts P3 gene in the ts⁺ isolate developed an intragenic mutation affecting its level of temperature sensitivity. In addition, the P3 RNA of the ts⁺ virus migrated more slowly in polyacrylamide gel than the P3 RNA of its A/Alaska/77-ts-1A2 parent. We failed to isolate ts P1 segregants from the ts⁺ isolate, indicating that the P1 ts mutation had reverted or undergone intragenic suppression. The ability of ts mutants to escape from their ts phenotype by suppression and other mutations may limit their usefulness in immunoprophylaxis since attenuation of ts mutants of influenza virus appears to be a function of the ts phenotype. Additionally, suppressor mutations provide new opportunities for studying the nature of protein-protein interactions.     

Characterization of two conditional early mutants of Rous sarcoma virus

Some of the biological and physical properties of two independently isolated mutants of Rous sarcoma virus have been investigated. Both ts 335 and ts 337 are unable either to transform or replicate at 41 °. The lesions in the mutants occur very early after infection (prior to 2 hr); however, the lesions appear to be expressed after adsorption to and penetration into the host cells. Both mutants contain temperature sensitive structural components, and ts 337 is also antigenically different from the wild type. The virions contain a polymerase activity which appears to be temperature sensitive in both the endogenous reaction and with poly rA:d(pT)₁₀ as the exogenous template. Upon coinfection of chick fibroblasts with ts 335 or ts 337 and Rous-associated virus (RAV), genetically stable wild-type sarcoma virus is produced at a high frequency. Therefore, the information for the function(s) which is mutant in these viruses is also present in RAV. All the parameters measured are consistent with a mutation affecting the function of the avian tumor virus RNA-dependent DNA polymerase.