Transformation by adenovirus early region 2A temperature-sensitive mutants and their revertants

H5ts107 is a temperature-sensitive mutant of adenovirus type 5 whose alteration maps in the structural gene for the viral DNA binding protein. Temperature-independent revertants of this mutant that form plaques at 39° in HeLa cells have been isolated. These revertants fall into two classes: (1) ts⁺ revertants for growth and plaque formation at 39° in both HeLa cells and 293 cells, a human cell line transformed by type 5 adenovirus; (2) ts⁺ for growth and plaque formation at 39° in HeLa cells, but temperature-sensitive (ts) for growth and plaque formation in 293 cells. The frequency of rat cell transformation by H5tsl07 was fivefold higher than that of wild-type adenovirus. A class 1 revertant, r(tsl07)127, transformed cells at the wild-type virus frequency while a class 2 revertant, r(tsl07)202, retained the ability to transform cells at the higher frequency. When the XhoI-C DNA fragment from these viruses, which contains the left end 15.5% of the viral genome, was employed to transform rat cells, then Ad5 wt, H5tsl07, r(tsl07)127, and r(ts107)202 DNAs all transformed with approximately equal frequencies.     

A temperature-sensitive mutant of simian virus 40 affecting transforming ability

In productive infection of monkey BSC-1 cells, ts 640, a temperature-sensitive (ts) mutant of simian virus 40 (SV40), was defective in its ability to synthesize infectious viral DNA and infectious viral particles at the restrictive temperature of 40 °, but not at the permissive temperature of 33 °. Growth of the mutant after infection with viral DNA was also temperature-sensitive, indicating that the block to multiplication at 40 ° is not due to failure in adsorption, penetration or uncoating. Results of mixed infections with other ts mutants showed that the gene identified in this mutant is different from those two cistrons which had been suggested to specify virion proteins.In nonproductive infection of rat 3Y1 cells, ts 640 was temperature-sensitive in its transforming ability at 40 ° but not at 33 °, providing evidence that cell transformation by SV40 is under the control of a viral gene function. The transformed cells obtained at 33 ° appeared not to revert to normal when shifted to 40 °, as judged from their colonial morphology under the standard culture conditions.     

Complementation between temperature-sensitive (ts) and host range nontransforming (hr-t) mutants of polyoma virus.

Two major classes of polyoma mutants are defective in cell transformation: early temperature-sensitive mutants of the tsA type which are defective in viral DNA synthesis and transformation at 39°, but not at 32°; and host range nontransforming (hr-t) mutants which fail to transform at either temperature. Mixed infection of mouse 3T3 cells by hr-t mutants and early tsA-type mutants results in enhanced growth of the tsA-type mutants at 39°, indicating that the hr-t mutants can supply the early viral function required for viral DNA synthesis. The hr-t mutants also complement late is mutants which fail to produce infectious progeny at 39° because of alterations in the 45,000-dalton major virion protein. Mixed infection of hamster BHK or rat Y1 cells by hr-t and tsA-type mutants results in efficient transformation at 39°, indicating that the two classes of mutants can complement for transformation. No complementation is observed in pair-wise crosses among the early tsA-type mutants alone. The tsA-type mutants are located in the distal portion of the early region of the polyoma genome [Miller, L. K., and Fried, M. (1976) J. Virol.18, 824–832]. The hr-t mutants are located in the proximal portion [Feunteun, J., Sompayrac, L., Fluck, M., and Benjamin, T. (1976) Proc. Nat. Acad. Sci. USA]. These results suggest that the early region of the polyoma genome is divided into two functional regions which can complement for transformation. The ts3 mutant of polyoma is located in the proximal portion of the late region.     

ts Transformation mutants of avian sarcoma virus PRCII: lack of strict correlation between transforming ability and properties of the P105-associated kinase

Three mutants of avian sarcoma virus PRC-II, LA42, LA46, and LA47, have a temperature-sensitive (ts) lesion affecting cellular transformation in vitro. At the nonpermissive temperature (41.5°) they do not induce focus formation in fibroblast cultures. LA46 also fails to induce colonies in soft agar at 41.5°, while LA42 and LA47 have retained this ability. The mutations appear to be located in the transformation-specific insert of the defective sarcoma virus genomes, since association with different wild-type (wt) helper viruses does not lead to changes in the transforming phenotypes. The transformation-specific protein P105 of PRCII is detectable at the nonpermissive temperature in moderately reduced quantity in wt- and LA42-infected cells, while the amounts of P105 precipitable from LA47-infected cultures under these conditions are significantly decreased. LA46 made barely detectable quantities of P105 at 41.5°. This temperature sensitivity of LA46 in the synthesis of P105 may reflect the greatly reduced levels of transformation-specific RNA in LA46-infected cells at 41.5°. Intracellular phosphorylation of P105 was not found to be ts in the mutants or in wt PRCII at both serine and tyrosine acceptor sites. P105 extracted from wt-, ts mutant- or wt-revertant-infected cells at permissive and nonpermissive temperatures did not vary significantly in the specific activity of its associated protein kinase as assayed in vitro by phosphorylation of P105 itself. However, preincubation of P105 in vitro at 41.5° revealed greater instability of protein kinase reactions measured in P105 immunoprecipitates from mutant- as compared to wt-infected cells. Also the elevation of cellular phosphotyrosine, characteristics of PRCII-transformed cells, was greatly reduced in ts mutant-infected cells at the nonpermissive temperature but was restored to wt levels in genetic revertants derived from the ts mutants. These observations suggest that there is no direct correlation between in vivo or in vitro phosphorylation of P105 and the induction of all parameters of oncogenic transformation. The increase of total cellular phosphotyrosine appears to be correlated with focus formation, but not with the ability to induce agar colonies.     

A rat mutant cell clone showing temperature-dependent transformed phenotypes with functional expression of the src gene product

The cellular mutant B814 isolated from a Fischer rat cell line shows temperature-sensitivity of focus formation on infection with Moloney murine sarcoma virus (Mo-MSV) and Rous sarcoma virus (RSV). An RSV-transformed clone (S814-2) isolated from B814 cells shows temperature-sensitive transformed phenotypes for morphology, growth in soft agar, and glucose uptake. The expression, phosphorylation, and tyrosine kinase activity of pp60v-src in S814-2 were not affected at the nonpermissive temperature, and virus rescued from this clone had wild-type transforming ability, suggesting that a cellular factor altered in S814-2 is responsible for the cellular steps of transformation after the function of pp60v-src. In addition, the cellular 36K protein, a possible candidate as a target of pp60v-src, was phosphorylated at the nonpermissive temperature in S814-2, indicating that phosphorylation of the 36K protein is not correlated with transformed phenotypes.     

The properties of recombinant Sendai virus having the P gene of Sendai virus pi strain derived from BHK cells persistently infected with Sendai virus

We prepared the chimeric recombinant Sendai virus [rSeV(Ppi)] by replacing the P gene of the Z strain with that of pi strain for analyzing the function of Ppi, Vpi and Cpi proteins. Intriguingly, HA production by rSeV(Ppi) is significantly lower at 38°C than at 32°C, showing that virus growth of rSeV(Ppi) is slightly suppressed at 38°C. However, the main phenotypes of SeVpi, a marked temperature sensitivity as viral replication and an ability of establishing persistent infection, are not explained by the Ppi, Vpi and Cpi proteins. The V and C proteins form inclusion bodies in L929 cells infected with rSeV(Ppi) and incubated at 38°C. L929 cells infected with rSeV(Ppi) and L929 cells stably expressing the Cpi protein show resistance to interferon-β at 32 and 38°C, indicating that the Cpi protein per se is not temperature-sensitive to inhibition of IFN signaling. The complete genome sequences of Sendai virus (SeV) pi and parent Nagoya strains were determined. Fifty nucleic acid substitutions are found in the genome sequence of SeV pi strain in comparison with Nagoya strain. There are three nucleic acid substitutions in the leader sequence, while the trailer, intergenic, gene-end and gene-start sequences of both strains are completely identical. Deletions and insertions of nucleotide are not found. There are 32 amino acid substitutions in Sendai virus pi strain. The specific amino acid substitutions unique to the SeVpi are 18. Information about the complete genome sequences of SeVpi strain is important to totally understand the persistent infection and lower pathogenicity of SeV.Machiko Nishio and Ai Nagata have contributed equally.     

Phosphorylation of pp60src and the cycloheximide insensitive activation of the pp60src-associated kinase activity of transformation-defective temperature-sensitive mutants of Rous sarcoma virus

Chick embryo fibroblasts infected with two transformation defective temperature-sensitive mutants of Rous sarcoma virus (GI 202 and GI 251), when shifted from the nonpermissive temperature (42°) to the permissive temperature (35°) show a rapid increase in the detectable pp60^src-associated kinase activity. When such cells are shifted back to the nonpermissive temperature there is an equally rapid loss of demonstrable kinase activity. The rapid increase in detectable kinase activity is not substantially hindered by cycloheximide at concentrations inhibiting all de novo protein synthesis, and is associated with a cycloheximide-insensitive phosphorylation of the mutant pp60^src.     

Studies of septation and separation in a mutant of Salmonella typhimurium

Salmonella typhimurium strain 4a is a temperature-sensitive division mutant. At 39° or 42° in several media a septation lesion occurs since non-septate filaments form at these temperatures. At lower temperatures in some media separation is aberrant since long chains of cells are formed. Since these division abnormalities are corrected either by revertion to ts⁺ or phenotypically by growth with sucrose, it is likely that both are the result of one lesion and that the component (S) which is temperature-sensitive in strain 4a is involved in both septation and separation. At 42° S is irreversibly inactivated but at 39° inactivation appears to be reversible since division restarts rapidly when 39° filaments are returned to 25° and protein synthesis is not required for such division. Comparison of recovery times for 39° and 42° filaments suggests that the synthesis of enough S for division takes about 20 min at 25° while the septation and separation stages of division take only about 10 min at this temperature. Since these latter processes occur so quickly it seems likely that events other than septation and separation take up a substantial fraction of the D period.     

Loss of different pericellular matrix components of rat cells transformed with a T-Class ts mutant of rous sarcoma virus

Rat cells transformed by the T-class ts mutant LA339 derived from RSV B77 (2R cells) and the parental nontransformed Rat-1 cells were analyzed for the production and deposition of pericellular matrix components as a function of growth temperature. The cells were incubated at the permissive (35°) and nonpermissive (39.5°) temperatures for transformation and the collagenous and noncollagenous (fibronectin and laminin) matrix proteins were studied by polypeptide analysis of the metabolically labeled cells and their culture media. Immunofluorescence analysis of the pericellular matrix structures was performed using specific antibodies against laminin, fibronectin, and heparan sulfate proteoglycan. At 39.5° dense pericellular matrices were detected in both 211 and Rat-1 cell layers, whereas at 35° the 2R cells lost their pericellular matrix structures. The cells continued to synthesize and secrete fibronectin, laminin, and heparan sulfate proteoglycan as well as slightly reduced amounts of procollagen into the culture medium but failed to deposit them in a matrix form. Another major temperature-sensitive difference in the secreted polypeptides was the production by the 211 cells at 35° of a 56,000-dalton protein, similar to that reported in other transformed cell cultures. The present findings indicate that the transformed rat 2R cells lose, upon activity of the pp60^src, their capacity to deposit the synthesized matrix components into pericellular structures.     

Protease of adenovirus type 2: partial characterization.

An adenovirus-associated protease activity specific for the cleavage of core polypeptide PVII to polypeptide VII was identified and its properties were studied using an in vitro assay system. All temperature-sensitive (ts) mutants examined failed to induce protease activity at 39°. Activity was restored in revertants. The protease activity was completely inhibited by 1 mM tosylamide phenylethylchloromethyl ketone while phenylmethylsulfonylfluoride and tosyl-lysinechloromethyl ketone at 1 mM reduced enzyme activity to 36 and 10% of control, respectively. By contrast ethylenediamine tetraacetic acid had no effect. Optimum enzyme activity was observed at neutral pH. Enzyme activity was stable up to, but not beyond, 45°. Polypeptide PVII was cleaved whether it was in the soluble form, bound form, heat-, or acid-precipitated form. Wild-type young virions contain endogenous protease activity while the virions produced at 39° by tsl-infected cells do not. The PVII contained in these tsl-39 virions, however, may be processed by exogenous WT enzyme after the particles have been frozen-thawed several times. These results suggest that the adenovirus-associated protease is a chymotrypsin-like, nonmetalo, neutral protease.     

Temperature-independent revertants of adenovirus H5ts125 and H5ts107 mutants in the DNA binding protein: isolation of a new class of host range temperature conditional revertants

H5ts125 and H5ts107 are temperature sensitive mutants of type 5 adenovirus in the structural gene for the DNA binding protein. Spontaneous, temperature-independent revertants of these mutants were isolated by growth in HeLa cells at 39°. Out of 30 independently isolated temperature-independent revertants grown in HeLa cells, two isolates were found to retain their temperature sensitive phenotype when grown or plaqued in 293 cells, which are a human cell line transformed by type 5 adenovirus. These two revertants represent a new class of host range temperature conditional mutants. It is suggested that these revertants arise by either a viral-encoded extragenic suppressor mutation or a second site intragenic mutation that recognizes cellular proteins in HeLa and 293 cells differently.     

Isolation of cellular revertants from a rat cell line transformed by the E6 and E7 genes of human papillomavirus type 16.

Three revertants defective in the ability to form colonies in semisolid medium were isolated from a rat cell line transformed by the E6 and E7 genes of human papillomavirus type 16 (HPV16). These revertants appeared to be defective in a cellular factor(s) necessary for transformation by HPV16-E6E7 genes since they still expressed a comparable amount of HPV16-E6E7 mRNA and E7 protein to the parental cells, harbored rescuable transforming virus, and were resistant to retransformation by HPV16-E6E7 genes. All these reverted phenotypes of the three mutants were recessive on somatic cell hybridization with normal cells, because all the hybrids showed transformed phenotypes.     

The effect of temperature on the amplitude distributions of miniature endplate potentials in the mouse diaphragm

The effect of temperature (11 to 45°C) on miniature endplate potential (MEPP) distributions was examined at the mouse diaphragm. MEPP distributions were composed of two populations ('skew-MEPPS' and ‘bell-MEPPs’) and the mode of the skew-MEPP population (subminiature endplate potential, sub-MEPP) had an amplitude equal to 1/10 to 1/15 the mean of the normally distributed bell-MEPP class. The overall MEPP frequency increased with a Q₁₀ of 2.2 between 11 and 30°C, and an Arrhenius plot indicated two temperature-sensitive reactions between 11 and 45°C. Below 30°C, the activation energy was 10.4 kcal/mole K and between 30 and 45°C the activation energy was 38.7 kcal/ mole K. The proportion of skew-MEPPs between different fibers was independent of the overall MEPP frequency and varied between 1% and 31% at temperatures of 11–34°C. However, as the temperature was lowered below 24°C, the decrease in frequency of skew-MEPPs was more than that of bell-MEPPs. Conversely, an increase in temperature from 24 to 34°C increased the bell-MEPP frequency but either reduced or had little effect on the frequency of skew-MEPPs. Following heat challenges (T > 40°C), MEPP distributions contained a large percentage of skew-MEPPs and the profile of the MEPP distribution became uniform. Before complete cessation of spontaneous activity with multiple heat challenges, MEPP amplitude distributions were either uniform or were composed primarily of sub-MEPPs. MEPP time courses were slower after heat challenges, and in some preparations, inflections were observed on many MEPP rising phases. Amplitude histograms of these inflections yielded distributions similar to control distributions of skew-MEPPs.The presence of inflections on MEPPs following heat challenges supports the hypothesis that skew-MEPPs and bell-MEPPs are composed of subunits. These results suggest that skew- and bell-MEPPs are caused by the release of transmitter by different temperature-sensitive mechanisms.     

Partial characterization of a Moloney murine sarcoma virus 85,000-dalton polypeptide whose expression correlates with the transformed phenotype in cells infected with a temperature-sensitive mutant virus

The viral proteins specified by a Moloney murine sarcoma virus (Mo-MuSV) with a temperature-sensitive mutation in its transforming gene were examined. Normal rat kidney cells infected with this replication-defective virus have transformed cell characteristics at 33° but revert to a normal phenotype at 40°. At the temperature permissive for transformation, the cells contained an 85,000-dalton protein (P85) which had antigenic determinants of p15, pp12, and p30, and also tryptic peptides characteristic of p15 and p30 as well as additional unidentified tryptic peptides. P85 was only detectable at the permissive temperature. A 58,000-dalton protein (P58) was also detected. It had both antigenic determinants and tryptic peptides of p15, pp12, and p30. P58 was seen at both temperatures. Phosphorylation experiments indicated that P58 is a phosphoprotein whereas ³²P-labeled P85 was not observed. Temperature shift experiments showed that newly synthesized P85 was first detected between 2 and 3 hr following transfer of cultures to 33°. Morphological and biochemical changes indicative of transformation occurred 8 or more hr after temperature shift. These results are consistent with the interpretation that P85 contains peptide sequences derived from both the gag gene and the MuSV-acquired or src gene sequences.     

Multiple effects of the 72-kDa, adenovirus-specified DNA binding protein on the efficiency of cellular transformation

The early region 2A gene (E2A) of adenovirus types 2 and 5 encodes a 72-kDa DNA binding protein (DBP) which contains two physical domains comprising approximately the amino-terminal one-third and carboxyl-terminal two-thirds of the protein, respectively. Previous work has shown that some Ad5 mutants containing temperature-sensitive (ts) mutations in the carboxyl-terminal domain of DBP, such as Ad5ts125, show a 3- to 8-fold enhanced ability to transform rat cells. We have examined the transformation characteristics of a series of Ad5 E2A deletion mutants, Ad5dl801-5, which encode either no functional DBP or encode truncated, defective DBPs. The E2A deletion mutants transformed rat embryo cells at frequencies similar to wild-type (wt) Ad5. These results suggest that the high transformation phenotype of carboxyl-terminal E2A mutants like Ad5ts125 is not due to the simple inactivation of DBP function, but rather results from an activity possessed by an altered DBP. This hypothesis is supported by the fact that the transformation phenotype of Adsts125 and similar mutants is dominant over the wild-type phenotype. A number of additional Ad2 and Ad5 E2A mutants were examined with respect to their ability to transform primary rat embryo cells. It was found that a carboxyl-terminal E2A mutant, Ad2+ND1ts23, also showed the enhanced transformation phenotype. In contrast, several amino-terminal E2A host-range (hr) mutants, originally isolated on the basis of their ability to replicate in monkey cells, transformed rat embryo cells at a frequency similar to wild-type virus. Ad2ts400, and E2A mutant with alterations in both DBP domains, showed a wild-type frequency of transformation, while two similar mutants, Ad5ts125 X 405 and Ad5ts125 X 404, showed an enhanced frequency. Last, it was found that coinfection of primary rat embryo cells with the hr mutants plus Ad5ts125 or Ad2+ND1ts23 resulted in a wild-type frequency of transformation, demonstrating that the hr mutants are dominant to the ts mutants with regard to transformation phenotype. Thus, DBP can both positively and negatively affect viral transformation in this system.     

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.     

Kirsten murine sarcoma virus-coded p21ras may act on multiple targets to effect pleiotropic changes in transformed cells

Kirsten murine sarcoma virus (Ki-MSV) is a replication-defective recombinant retrovirus capable of transforming cells in culture. A Ki-MSV coded, 21,000-dalton protein (p21^ras) is required for the maintenance of cellular transformation. It is unknown whether the p2l^ras of Ki-MSV induces transformation by acting on multiple targets, as has been suggested for Rous sarcoma virus transformed cells, or by a single target mechanism. In order to resolve this question, we have used a normal rat kidney cell line transformed by a temperature-sensitive mutant strain of Ki-MSV (tsKNRK), which codes for a thermolabile p21^ras to investigate the correlative aspects of the expression of several transformation-related cellular properties upon shifting from the permissive (32°) to nonpermissive (39°) temperature. Except for an altered morphology, an organized cytoskeleton, and increased adhesion to substratum, tsKNRK cells at 32° displayed similar properties to those of wild-type Ki-MSV transformants at either temperature. Upon shifting to 39°, anchorage- and density-dependent growth were restored, although the growth rate and glucose uptake were unaffected. The cells assumed a more flattened morphology, although adhesiveness did not increase significantly. Increased levels of cell surface fibronectin were observed within 48 hr post-temperature shift, although fibronectin levels comparable to that of normal rat kidney cells (NRK) were not observed until later. Epidermal growth factor (EGF) binding increased only slightly at 48 hr post-temperature shift, but did not approach EGF-binding levels of NRK cells. Cytoskeletal organization was invariant between the two temperatures. Although our results suggest a multiple target model for Ki-MSV-mediated transformation, a single target mechanism cannot totally be ruled out.