Early Temperature-sensitive Mutant Research Articles

Sister chromatid exchange in FR3T3 rat fibroblasts transformed by simian virus 40

The frequency of Sister Chromatid Exchange (SCE) was determined at low (33 °C) and high (40.5 °C) temperatures in cell lines derived from FR3T3 rat fibroblast cells after transformation either with Wild-Type Simian Virus 40 (SV40-WT), with an origin-defective SV40 (SV40-ori −), or with the early temperature-sensitive mutant tsA30. Of these cell lines, SV40-WT-, SV40-ori −-, and one class of tsA430-transformants (A-type) express the transformed phenotype both at 33 and 40.5 °C. The other tsA30-transformants (N-type) revert to a normal phenotype at high temperature. As compared with normal FR3T3 cells, all transformants exhibited, at 33 °C, increased numbers of metaphases with high SCE rates. At 40.5 °C, all cell lines which expressed a transformed phenotype (SV40-WT, tsA30 type A, SV40-ori −) exhibited substantially increased SCE rates. That this increase was not related to a possible induction of viral replication by BrdU, was proven by Southern blot analysis and by SCE data on SV40-ori −-transformed cells. By contrast, no such temperature-induced increase of SCE rates was observed in tsA30-transformants of type N.

Immunogenicity of herpes simplex virus glycoproteins gC and gB and their role in protective immunity.

The relative antigenicity of the individual herpes simplex virus type 1 (KOS) glycoproteins gC and gB was analyzed in BALB/c mice by using KOS mutants altered in their ability to present these antigens on cell surface membranes during infection. The mutants employed were as follows: syn LD70 , a non-temperature-sensitive mutant defective in the synthesis of cell surface membrane gC; tsF13 , a temperature-sensitive mutant defective in the processing of the precursor form of gB to the mature cell surface form at 39 degrees C; and ts606 , an immediate early temperature-sensitive mutant defective in the production of all early and late proteins including the glycoproteins. By comparing the relative susceptibility to immunolysis of mouse 3T3 cells infected at 39 degrees C with wild-type virus, presenting the full complement of the glycoprotein antigens, gC, gB, and gD, with target cells infected with mutants presenting only subsets of these antigens, we determined that a major portion of cytolytic antibody contained in hyperimmune anti-herpes simplex virus type 1 (KOS) mouse antiserum was directed against glycoproteins gC and gB. The relative immunogenicity of wild-type and mutant virus-infected cells also was compared in BALB/c mice. Immunogen lacking the mature form of gB induced a cytolytic antibody titer comparable to that of the wild-type virus, whereas that lacking the mature form of gC showed a 70% reduction in titer. The absence of the mature cell surface forms of gB and gC in immunogen preparations resulted in a 4- to 15-fold reduction in in virus neutralizing titer. Animals immunized with ts606 -infected cells (39 degrees C) induced relatively little virus-specific cytolytic and neutralizing antibody. Analysis of the glycoprotein specificities of these antisera by radioimmunoprecipitation showed that the antigens immunoprecipitated reflected the viral plasma membrane glycoprotein profiles of the immunogens. The absence of the mature forms of gC or gB in the immunizing preparation did not appreciably affect the immunoprecipitating antibody response to other antigens. Mice immunized with wild-type and mutant virus-infected cells were tested for their resistance to intracranial and intraperitoneal challenge with the highly virulent WAL strain of herpes simplex virus type 1. Despite the observed alterations in serum virus-specific antibody induced with the individual immunogens, all animals survived an intraperitoneal challenge of 10 50% lethal doses. However, differences in the survival of animals were obtained upon intracranial challenge.(ABSTRACT TRUNCATED AT 400 WORDS)

Stabilization of the large T protein in temperature-independent (type A) FR 3T3 rat cells transformed with the simian virus 40 tsA30 mutant.

The stabilities of in vivo [35S]methionine-labeled large T and small t proteins, synthesized in temperature-sensitive (type N) and temperature-insensitive (type A) FR 3T3 rat cells transformed by an early temperature-sensitive mutant of simian virus 40 (SV40), tsA30, were analyzed at the permissive and restrictive temperatures. The two polypeptides, detected in greatly reduced amounts in cells of the N type at the restrictive temperature, were also unstable at the permissive temperature. However, both were made in similar amounts and were apparently stable in cells of the A type, irrespective of the temperature. The structures of the viral RNAs present at the permissive temperature were analyzed for transformants representative of each type, and containing a single integration of viral DNA. The two cell lines synthesized transcripts identical to the large T and small t mRNAs identified in SV40-infected monkey cells. Similar amounts of viral RNA were found in A and N transformants in active growth at the permissive and restrictive temperatures, which argued against a control at a transcriptional level. Assay of a defined function of the protein, namely, the binding of nucleotide detected by affinity labeling with periodate-oxidized [alpha-32P]ATP, clearly showed that the large T proteins from both types of transformants exhibited, at least for that particular biochemical function, the same in vitro temperature sensitivity. In transformants of the A type only could a reduced binding activity be detected in extracts from cells grown at the restrictive temperature. Thus, the temperature-independent behavior of the A transformants may result from an in vivo partial stabilization of the newly synthesized large T protein, probably through interaction with a cellular component(s).

Expression of adenovirus type 12 E1A gene in monkey cells, using a simian virus 40 vector.

Simian virus 40 (SV40) recombinants carrying the adenovirus type 12 E1A gene were constructed. The SV40 expression vector was constructed by removing most of the VP1 gene and an internal part of the intervening sequence for late 16S RNA and by joining the 5' and 3' splice sites into a small segment. The adenovirus type 12 E1A gene with or without its own promoter was inserted downstream from the SV40 late promoter and the splicing junctions. The recombinant DNA was propagated and packaged in monkey cells by cotransfection with an early temperature-sensitive mutant (tsA58) DNA as helper. Immunofluorescent staining of the monkey cells infected with the resulting virus stocks showed that up to 20% of the cells overproduced the E1A gene products in the nuclei. Two-dimensional gel electrophoresis of the products indicated that the products were very similar or identical to the authentic polypeptides synthesized in adenovirus type 12-infected human embryo kidney cells. The E1A mRNA was initiated at the SV40 late promoter irrespective of the presence of the E1A promoter and terminated at either the E1A or the SV40 polyadenylation signal. These hybrid mRNAs were correctly spliced in the E1A coding region.

Differential complementation of adenovirus type 5 temperature-sensitive early mutants by adenovirus types 3 and 12

We have assayed the ability of human adenoviruses from heterologous subgroups to complement early temperature-sensitive mutants of the C group virus Ad5 by analyzing coinfections at the restrictive temperature for serotype-specific DNA and late protein synthesis. Our results indicate that the B group virus AM, does not complement either ts125 or the N complementation group of Ad5 (ts36, ts69, and ts149). In contrast, studies with Ad12, an A group virus, and these mutants, indicate a differential complementation in that Ad12 complements all of the mutants in the Ad5 N complementation group but does not complement ts125. Failure to complement the ts125 defect in coinfected cells has been shown to be due to the inability of the heterologous wt gene product to substitute for the ts125 gene product directly at the level of DNA replication and not at some earlier event in the virus growth cycle.

Inducible permissive cells transformed by a temperature-sensitive polyoma virus: superinfection does not allow excision of the resident viral genome.

After exposure of mouse embryo cells to the early temperature-sensitive mutant tsP155 of polyoma virus (Py), a transformed cell line (Cyp line) that can be readily induced to synthesize Py by transfer to 33 degrees C was isolated at 39 degrees C (7). Virus production and synthesis of free viral DNA occurring after temperature shiftdown or superinfection with wild-type Py or both were studied in several clonal isolates of the Cyp cell line. Measurements of virus yields indicated that, although some could be induced more effectively than others, all cell clones behaved as highly permissive when subjected to superinfection. We analyzed the origin of free viral DNA accumulating in the superinfected cultures, taking advantage of (i) the unique physical properties of the low-molecular-weight DNA which, in the case of one of the Cyp clones, accumulates during temperature shiftdown, and (ii) the differences between resident and superinfecting viral genomes in their susceptibilities towards restriction endonucleases. At 33 degrees C, both viral genomes were found to accumulate in all clones studied whereas in the case of the clones with lower inducibility, the replication of the resident genome appeared to be enhanced by superinfection. At 39 degrees C, however, accumulation of the superinfecting genome was not accompanied by that of the resident genome, unless it had already been initiated before superinfection. These findings demonstrate that, when routinely cultivated at 39 degrees C, Cyp cells contain few viral DNA molecules readily available for autonomous replication and that, upon transfer to 33 degrees C, therefore, excision must first take place before the resident genome can accumulate as free viral DNA. Our findings also suggest that, unlike the P155 gene product provided by the resident viral genome upon induction, the allelic gene product supplied by the superinfecting genome may be less effective in triggering excision than in promoting replication.

Simian virus 40 tandem repeated sequences as an element of the early promoter.

On the late side of the simian virus 40 (SV40) DNA replication origin are several sets of tandem repeated sequences, the largest of which is 72 base pairs long. The role of these sequences was examined through construction of deletion mutants of SV40. A mutant from which one of the 72-base-pair repeated units was removed is viable upon transfection of monkey kidney cells with viral DNA. Extension of this deletion into the second repeated unit, however, leads to nonviability, as recognized by the absence of early transcription and of tumor antigen production. These observations indicate that the 72-base-pair repeated sequences form an essential element in the early viral transcriptional promoter and explain the inability of such a deleted genome to complement an early temperature-sensitive mutant of SV40, tsA, as well as the failure to replicate its DNA. In a parallel experiment it was found that the extended deletion mutant was also unable to complement a late temperature-sensitive mutant of SV40, tsB. This suggests that the extended mutant is also defective in DNA replication or late transcription (or both).

Monkey cells transformed by SV40 DNA fragments: flat revertants synthesize large and small T antigens.

SV40 causes tumor formation in animals and transforms tissue-culture cells. Experiments with early temperature-sensitive mutants (tsA) indicated that synthesis of wild-type (wt) SV40 large T antigen is required for the induction and maintenance of the transformed state (Martin and Chou 1975; Tegtmeyer 1975; Brugge and Butel 1975; Osborn and Weber 1975). Also, SV40 small T antigen seems to be involved in the process of cell transformation. Early SV40 mutants with deletions between 0.54 and 0.59 map units (e.g. dl884) code for wild-type large T antigen, but for small T antigens of reduced sizes (Bouck et al. 1978; Crawford et al. 1978; Sleigh et al. 1978). These mutants have reduced transformation capacities. However, it is still uncertain which of the early SV40-specific functions attributed to the viral T antigens determine the oncogenicity of SV40.

Induction of virus multiplication in permissive cells transformed by a temperature-sensitive polyoma virus. I. Isolation and partial characterization of survivors.

A fibroblastic cell line transformed by ts-P155, an early temperature-sensitive mutant of polyoma virus, has been isolated after infection of secondary mouse embryo cells at the restrictive temperature of 39. These cells, designated Cyp, undergo a massive CPE accompanied by the production of infectious, temperature-sensitive virus when transferred to the nonrestrictive temperature of 33. Several independent clones were established from cells which survived the induction of virus multiplication occurring at 33. When routinely cultured at this temperature, these cells, termed R, displayed the growth properties of transformants and produced variable amounts of infectious, temperature-sensitive virus. This shedding of virus could not be "cured' by the addition to the culture medium of antipolyoma serum which had already been found to effectively suppress virus production in Cyp cell cultures transferred to 33. Moreover, R cells seemed to have developed a resistance to superinfection that the parent Cyp cell line did not exhibit. Therefore, virus production in R cell cultures probably reflects the occasional excision and replication of integrated viral genomes rather than a carrier state.

Induction of viral DNA synthesis in clonal derivatives of a permissive cell line transformed by a temperature-sensitive polyoma virus

After exposure of mouse embryo cells to ts-P155, an early temperature-sensitive mutant of polyoma virus (Py) that transforms but replicates poorly at 39°, a transformed cell line (Cyp line) was obtained which was propagated at this temperature ( P. Bourgaux, L. Delbecchi, K. K. Y., Yu, E. Herring, and D. Bourgaux-Ramoisy, 1978 Virology 88, 348–360), Whereas viral DNA is not readily detectable in Cyp cells maintained at 39°, it is synthesized in large amounts by these cells from the 30th hour after temperature shift-down. We have thus examined the low molecular weight, covalently closed, cyclic DNA (CCC-DNA) accumulating at 33° in cultures of various clones of Cyp cells isolated in liquid medium, as well as in cells recloned in soft agar. This DNA was found to consist primarily of genome-size Py DNA (P155 DNA I), for all inducible clones analyzed except one. In cells from that clone (C12-22/a1), two discrete species of CCC-DNA molecules were consistently detected at 33°. The predominant species, referred to as Rm I, sedimented in sucrose solution, and migrated in agarose gels, as a molecule of about 4.4 × 10 6 daltons. Under these conditions, the minor species behaved as P155 DNA I. By comparison with other clones, cells from clone C12-22/a1 produced lower amounts of virus following temperature shift-down. When used to infect mouse embryo cells at 33°, the virus produced by C12-22/a1 cells directed the synthesis of DNA indistinguishable from P155 DNA I by restriction enzyme analysis. In contrast, Rm I molecules found in C12-22/a1 cells displayed a distinctive pattern after cleavage by restriction enzymes. Several enzymes were found to cleave P155 DNA and Rm I into the same number of fragments, only one fragment migrating differently in the two digests, as if the Rm I fragment was 1 × 10 6 daltons heavier than the corresponding P155 fragment. In all Rm I digests, the missing—or enlarged—viral fragment was the one overlapping Py DNA map positions 26.6 to 32.6. As compared with P155 DNA, Rm I contains one additional site for HindIII as well as one for BamHI, both located in the 1.0 × 10 6 dalton addition. By the use of the Southern transfer procedure, it was demonstrated that the sequences which are cleaved by either of these enzymes in Py DNA are not present in the addition. It thus appears that Rm I is a molecule comprising possibly the whole of the ts-P155 genome, as well as a piece of 1 × 10 6 daltons of DNA that possibly originates from the host cell.

Functional similarity between the early antigens of simian virus 40 and human papovavirus BK

The functional properties of the early antigens of simian virus 40 (SV40) and human papovavirus BK (BKV) were investigated. Infection of African green monkey kidney cells with BKV permitted the bidirectional replication of an early temperature-sensitive mutant (tsA) at a nonpermissive temperature. Conceivably, an early gene product (T-antigen) of BKV can substitute functionally for the defective SV40 T-antigen. On the other hand, SV40 DNA replication remained undetectable in human embryonic kidney cells preinfected with BKV, suggesting that BKV early antigens alone are not sufficient to provide for the replication of SV40. Preinfection of African green monkey kidney cells with BKV restored the normal pattern of late lytic SV40 transcription, suppressing the overproduction of early RNA by an SV40 tsA mutant at the nonpermissive temperature. Furthermore, preinfection of African green monkey kidney cells with BKV supported the growth of adenovirus type 2, providing a "helper function" similar to that provided by SV40 for the growth of human adenovirus in monkey kidney cells.

Autoregulation of Adenovirus Type 5 Early Gene Expression III. Transcription Studies in Isolated Nuclei

The rate of adenovirus RNA synthesis was compared in nuclei isolated from cells infected at 40.5 degrees C in the presence of 1-beta-d-arabinofuranosylcytosine with adenovirus 5 or an early temperature-sensitive mutant of adenovirus type 5, H5ts125 (ts125). In nuclei isolated at various times after infection, the maximum amount of virus RNA synthesis occurred at 6 h after infection, after which time virus RNA synthesis declined in nuclei from wild-type infections but remained high in nuclei from ts125 infections. At 12 h after infection, the amount of virus RNA synthesis was 8- to 11-fold higher in nuclei from ts125 infections than in nuclei from wild-type infections. However, the kinetics of virus RNA synthesis in nuclei isolated from both infections were similar. When a ts125-infected culture was shifted to 32 degrees C for 3 h (12 to 15 h after infection) before nucleus isolation, the amount of virus RNA synthesis in the isolated nuclei was reduced to nearly wild-type levels. A pulse-chase experiment showed little difference in degradation rates of virus RNA in isolated nuclei from wild-type and ts125 infections. Hybridization of RNA synthesized in vitro to restriction fragments of adenovirus type 5 DNA was consistent with early virus RNA. These results support the idea that the 72,000-dalton DNA-binding protein encoded by the mutant gene in ts125 can regulate early adenovirus gene expression by inhibiting initiation of transcription of the adenovirus genome.

Simian virus 40 A gene function: DNA content analysis of Chinese hamster cells transformed by an early temperature-sensitive virus mutant.

Replication of two Chinese hamster embryo cell lines transformed by an early temperature-sensitive mutant of simian virus 40, tsA58, was examined by flow microfluorometry and autoradiography of [3H]thymidine-labeled cells in order to determine whether transformed cell DNA synthesis is initiated by the virus A gene. At the permissive temperature (37 degrees), cells transformed by the mutant were like the wild-type virus transformants in appearance, colony-forming ability, high saturation density, and rapid replication. At the nonpermissive temperature (40.5 degrees), the tsA58 transformed cells resembled normal embryo fibroblasts and seem to return to normal growth patterns. Although both mutant transformed cell lines at 40.5 degrees appeared to cease growth at low saturation density, the cells did not enter a resting state, but continued to replicate. The cultures were maintained at low densities by a balance among cell replication, cell death, and sloughing of dead cells into the supernatant. These results suggest that the simian virus 40 A gene function effected by the tsA58 mutation does not prevent Chinese hamster embryo transformed cells from entering a resting state, although the gene may control other phenotypic characteristics of transformation.

A mouse embryo cell line carrying an inducible, temperature-sensitive, polyoma virus genome

Secondary monolayer cultures from whole mouse embryos were infected at 39° with ts-P155, an early temperature-sensitive mutant of polyoma virus that transforms but replicates poorly at this temperature ( W. Eckhart, 1969, Virology 38, 120–125; W. Eckhart, 1974, Cold Spring Harbor Symp. Quant. Biol. 39, 37–40). The cultures were then passaged at 39° in the presence of polyoma virus antiserum until sufficient cell degeneration occurred to allow detection of foci of cells exhibiting lack of contact-inhibition. From one such focus, a cell strain—designated Cyp—was derived and cloned at 39°. Whether cloned or uncloned, Cyp cells all seem to exhibit essentially the same phenotype. At 39°, they display several properties of transformed cells and, as indicated by immunofluorescence, synthesize polyoma T antigen. They do not produce any readily detectable amounts of viral DNA or V antigen and only low amounts of viral hemagglutinin at 39°, although they are still fully permissive for polyoma virus replication at this temperature. Upon transfer to 33°, Cyp cells undergo a massive cytopathic effect detectable within 30 to 34 hr and produce large amounts of infectious, temperature-sensitive polyoma virus. Viral DNA accumulating in Cyp cells after temperature shift-down consists primarily of covalently closed monomers and, upon cleavage by HpaII, yields fragments characteristic of the polyoma virus strain from which the ts-P155 mutant had been derived. When present in the medium during temperature shift-down, polyoma virus antiserum neither curtails the cytopathic effect nor depresses viral DNA synthesis, although it suppresses virus production. These observations are in agreement with the results of infectious center assays, suggesting that most Cyp cells produce virus following transfer to 33°.

Evidence for a function of the adenovirus DNA-binding protein in initiation of DNA synthesis as well as in elongation of nascent DNA chains

Early in lytic infection of KB cells with adenovirus type 5 (Ad5) a viral-coded single-strand-specific DNA-binding protein with a molecular weight of 72,000 is synthesized. The function of this protein in viral DNA synthesis was studied using two different methods for specific inactivation. First, DNA synthesis was examined in KB cells infected with an early temperature-sensitive mutant, H5ts125, which produces a thermolabile DNA-binding protein. At the nonpermissive temperature (40°) the start of the first and subsequent rounds of DNA replication was inhibited. However, viral DNA synthesis in vitro using a system of isolated nuclei obtained from H5ts125-infected KB cells was not thermosensitive compared to wild type. In this in vitro system, replicating molecules were normally converted to mature DNA at 40° without reinitiation of new replication rounds. These results show that thermal inactivation of the H5ts125 DNA-binding protein destroys its capacity to function in the initiation of new replication rounds but does not impair chain growth. As a second method of inactivation we studied viral DNA synthesis in vitro in the presence of antibody against the purified DNA-binding protein. A 50–60% inhibition of the rate of viral DNA synthesis was observed while cellular DNA synthesis was not affected. Density-labeling experiments showed that the antibody inhibits the duplication of both complementary strands, presumably by a decrease of fork movement in replicating adenovirus DNA. The results suggest that the Ad5 DNA-binding protein functions in initiation of DNA replication as well as in elongation of nascent viral DNA chains.

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.

Isolation, propagation and characterization of replication requirements of reiteration mutants of simian virus 40

We have identified five reiteration mutants from serially-propagated, defective stocks of Simian Virus 40 and DAR virus (an SV40 ‡ variant of human origin). The genomes of these mutants contain tandem repeats of specific segments of the SV40 genome. In order to propagate individual reiteration mutants, the monomer DNA segments from the mutant genomes are separated from wild-type SV40 DNA after cleavage by certain bacterial restriction endonucleases which produce short cohesive termini at their cleavage sites. These monomer segments, which are one-third, one-fourth, or one-fifth the size of wild-type SV40 DNA, are then circularized in vitro using bacteriophage T4 polynucleotide ligase and used to infect African green monkey kidney cells in the presence of wild-type or temperature-sensitive mutant DNAs as helpers. While wild-type SV40 and late temperature-sensitive mutants can serve as helpers in the replication and amplification of these minicircular DNAs, early temperature-sensitive mutant genomes are unable to do so at the nonpermissive temperature. The minicircular DNAs are amplified in vivo through an arithmetic series of oligomers. Encapsidation of reiterated molecules between 70 and 100% the size of wild-type SV40 DNA is observed, although reiterated viral DNA molecules much larger than unit size are formed in vivo.

Integration of SV40 DNA and induction of cellular DNA synthesis after a ts SV40 infection

The early temperature-sensitive mutant of simian virus 40 (SV40), ts∗101, was characterized in nonpermissive Chinese hamster (ChH) and permissive monkey CV-1 cells for integration of viral DNA and induction of cellular DNA synthesis. At 42° in CV-1 cells, this mutant did not induce the synthesis of T antigen or cellular and viral DNA. The viral DNA did not integrate into the CV-1 DNA, although nuclear penetration occurred. In ChH cells the mutant induced T antigen at 40° and 42°, and the viral DNA integrated into ChH DNA, but ChH DNA synthesis was not induced. These findings suggest that the integration of SV40 DNA does not depend on the replication of cellular DNA and that, at least functionally, integration precedes the induction of cellular DNA synthesis.

Simian virus 40-host cell interactions. I. Temperature-sensitive regulation of SV40 T antigen in 3T3 mouse cells transformed by the ts*101 temperature-sensitive early mutant of SV40.

BALB/3T3 and Swiss/3T3 mouse cells transformed at permissive temperature (33 C) by the early temperature-sensitive mutant of simian virus 40 (SV40), ts(*)101, exhibited a temperature-dependent modulation of SV40 tumor (T) antigen as assayed by immunofluorescence. The percentage of T antigen-positive nuclei in ts(*)101 transformed cells was reduced at restrictive temperature (39 C) when compared to 33 C and to wild-type SV40 transformed cells at either 33 C or 39 C. The percentage of T antigen-positive nuclei in ts(*)101 transformed cells returned to the 33 C control level when the cells were shifted from 39 to 33 C. The ts(*)101 transformed cells could be superinfected with wild-type, but not ts(*)101, virions at 39 C as assayed by an increase in T antigen-positive nuclei.