The retinoblastoma gene product regulates progression through the G1 phase of the cell cycle.

The RB gene product is a nuclear phosphoprotein that undergoes cell cycle-dependent changes in its phosphorylation status. To test whether RB regulates cell cycle progression, purified RB proteins, either full-length or a truncated form containing the T antigen-binding region, were injected into cells. Injection of either protein early in G1 inhibits progression into S phase. Co-injection of anti-RB antibodies antagonizes this effect. Injection of RB into cells arrested at G1/S or late in G1 has no effect on BrdU incorporation, suggesting that RB does not inhibit DNA synthesis in S phase. These results indicate that RB regulates cell proliferation by restricting cell cycle progression at a specific point in G1 and establish a biological assay for RB activity. Neither co-injection of RB with a T antigen peptide nor injection into cells expressing T antigen prevents cells from progressing into S phase, which supports the hypothesis that T antigen binding has functional consequences for RB.     

Biochemical activities of T-antigen proteins encoded by simian virus 40 A gene deletion mutants.

We have analyzed T antigens produced by a set of simian virus 40 (SV40) A gene deletion mutants for ATPase activity and for binding to the SV40 origin of DNA replication. Virus stocks of nonviable SV40 A gene deletion mutants were established in SV40-transformed monkey COS cells. Mutant T antigens were produced in mutant virus-infected CV1 cells. The structures of the mutant T antigens were characterized by immunoprecipitation with monoclonal antibodies directed against distinct regions of the T-antigen molecule. T antigens in crude extracts prepared from cells infected with 10 different mutants were immobilized on polyacrylamide beads with monoclonal antibodies, quantified by Coomassie blue staining, and then assayed directly for T antigen-specific ATPase activity and for binding to the SV40 origin of DNA replication. Our results indicate that the T antigen coding sequences required for origin binding map between 0.54 and 0.35 map units on the SV40 genome. In contrast, sequences closer to the C terminus of T antigen (between 0.24 and 0.20 map units) are required for ATPase activity. The presence of the ATPase activity correlated closely with the ability of the mutant viruses to replicate and to transform nonpermissive cells. The origin binding activity was retained, however, by three mutants that lacked these two functions, indicating that this activity is not sufficient to support either cellular transformation or viral replication. Neither the ATPase activity nor the origin binding activity correlated with the ability of the mutant DNA to activate silent rRNA genes or host cell DNA synthesis.     

Permeable FGF-1 Nuclear Localization Signal Peptide Stimulates DNA Synthesis in Various Cell Types but Is Cell-Density Sensitive and Unable to Support Cell Proliferation

An earlier report indicated that a 26-amino-acid peptide (SA), comprised of the nuclear localization signal (NLS) of fibroblast growth factor-1 (FGF-1) and a membrane-permeable peptide, was able to stimulate DNA synthesis after it was taken up by NIH3T3 fibroblasts. Here, we report that SA, but not a mutant with the NLS motif destroyed, induced DNA synthesis in BALB/c3T3 murine fibroblasts, human vascular endothelial (HUVE) cells, and primary cultured hepatocytes, although the activity was weaker than that of FGF-1. The kinetics of SA-induced DNA synthesis and G₁cyclin expression were similar to those elicited by FGF-1, indicating that SA induces cell cycle progression. Kinetic analysis also suggested that SA stimulates only a fraction of the DNA replication in BALB/c3T3 cells. At high cell densities, SA-induced G₁cyclin expression and DNA synthesis were more strongly inhibited than those induced by FGF-1. SA did not induce cell division in HUVE and BALB/c3T3 cells and did not interfere with FGF-1-stimulated proliferation of HUVE cells. These results indicate that SA is able to partially induce cell cycle progression through a contact-inhibition sensitive signaling pathway, but it is insufficient to support cell mitosis. We also suggest that signaling by SA does not interfere with that of FGF-1.     

Activation of the SV40 late promoter: direct effects of T antigen in the absence of viral DNA replication

We have examined the activation of the SV40 late promoter by inserting the late promoter and the viral origin of replication into chloramphenicol acetyltransferase (CAT) transient expression vectors. Very little late promoter activity was detected in CV-1 cells, compared with high activity in COS cells, in which replication occurs due to endogenous T antigen. Nonreplicative counterparts of these plasmids, containing a mutated origin of replication, produced significantly more late promoter activity in COS cells than any of the plasmids in CV-1 cells. When plasmids were cotransfected into CV-1 cells with a plasmid that supplies T antigen, the nonreplicative plasmid displayed 30% of the activity of the replicative plasmid. Using mutant T antigens unable to replicate viral DNA, late promoter activation occurred only with mutant T antigens that retain DNA binding activity. These results demonstrate that T antigen can substantially stimulate late promoter activity directly and independent of viral DNA replication.     

Binding of Polyomavirus Small T Antigen to Protein Phosphatase 2A Is Required for Elimination of p27 and Support of S-Phase Induction in Concert with Large T Antigen

Although polyomavirus large T antigen readily transactivates S-phase-specific enzymes in serum-starved Swiss 3T3 mouse fibroblasts, it is incapable by itself to efficiently drive such cells into S phase. We describe here that this inability correlates with a weak proficiency of the viral protein to induce the synthesis of cyclin A and cyclin E and to stimulate the respective cyclin/cdk activities. Polyomavirus small T antigen, which together with the large T protein supports S-phase induction, strongly contributes to the synthesis of cyclin A. In addition, small T antigen causes a dramatic induction of cyclin A- and, together with large T antigen, of cyclin E-specific protein kinase activity. This latter function of polyomavirus small T antigen correlates with its competence to provoke the elimination of the kinase inhibitor p27(Kip1). An interaction of the small T antigen with the protein phosphatase 2A is essential for this activity. Hence, the ability to drive quiescent Swiss 3T3 cells into S phase results from the capacity of large T antigen to transactivate DNA synthesis enzymes by its interaction with retinoblastoma-type proteins and from the potential of the large and the small T antigens together to stimulate cyclin A synthesis and cyclin A- and cyclin E-dependent protein kinase activity.     

Genetic and biochemical analysis of transformation-competent, replication-defective simian virus 40 large T antigen mutants.

To study the role of the biochemical and physiological activities of simian virus 40 (SV40) large T antigen in the lytic and transformation processes, we have analyzed DNA replication-defective, transformation-competent T-antigen mutants. Here we describe two such mutants, C8/SV40 and T22/SV40, and also summarize the properties of all of the mutants in this collection. C8/SV40 and T22/SV40 were isolated from C8 and T22 cells (simian cell lines transformed with UV-irradiated SV40). Early regions encoding the defective T antigens were cloned into a plasmid vector to generate pC8 and pT22. The mutations responsible for the defects in viral DNA replication were localized by marker rescue, and subsequent DNA sequencing revealed missense and one nonsense mutation. The T22 mutation predicts a change of histidine to glutamine at residue 203. C8 has two mutations, one predicts lysine224 to glutamamic acid and the other changes the codon for glutamic acid660 to a stop codon; therefore, C8 T antigen lacks the 49 carboxy-terminal amino acids. pC8A and pC8B were constructed to contain the C8 mutations separately. Plasmids pT22, pC8, pC8A, and pC8B were able to transform primary rodent cell cultures. T22 T antigen is defective in binding to the SV40 origin. C8B (49-amino-acid truncation) is a host-range mutant defective in a late function in CV-1 but not BSC cells. Analysis of T antigens in mutant SV40-transformed mouse cells suggests that the replicative function of T antigen is important in generating SV40 DNA rearrangements that allow the expression of "100K" variant T antigens in the transformants.     

T antigen and template requirements for SV40 DNA replication in vitro.

A cell-free system for replication of SV40 DNA was used to assess the effect of mutations altering either the SV40 origin of DNA replication or the virus-encoded large tumor (T) antigen. Plasmid DNAs containing various portions of the SV40 genome that surround the origin of DNA replication support efficient DNA synthesis in vitro and in vivo. Deletion of DNA sequences adjacent to the binding sites for T antigen either reduce or prevent DNA synthesis. This analysis shows that sequences that had been previously defined by studies in vivo to constitute the minimal core origin sequences are also necessary for DNA synthesis in vitro. Five mutant T antigens containing amino acid substitutions that affect SV40 replication have been purified and their in vitro properties compared with the purified wild-type protein. One protein is completely defective in the ATPase activity of T antigen, but still binds to the origin sequences. Three altered proteins are defective in their ability to bind to origin DNA, but retain ATPase activity. Finally, one of the altered T antigens binds to origin sequences and contains ATPase activity and thus appears like wild-type for these functions. All five proteins fail to support SV40 DNA replication in vitro. Interestingly, in mixing experiments, all five proteins efficiently compete with the wild-type protein and reduce the amount of DNA replication. These data suggest that an additional function of T antigen other than origin binding or ATPase activity, is required for initiation of DNA replication.     

Induction of cellular DNA synthesis by a simian virus 40 mutant defective in nuclear transport of T antigen.

The simian virus 40 (SV40) (cT)-3 mutant [SV40(cT)-3], which is defective in nuclear transport of T antigen, was utilized to determine whether cellular DNA synthesis can be stimulated by SV40 in the absence of detectable nuclear T antigen. Cellular DNA synthesis was examined in the temperature-sensitive cell cycle mutants, BHK ts13 and BHK tsAF8, after microinjection of quiescent cells with plasmid DNA containing cloned copies of wild-type SV40 or SV40(cT)-3. The efficiency of induction of cellular DNA synthesis was identical for both wild-type SV40 and SV40(cT)-3 in both cell lines. The results suggest that cell surface-associated T antigen, either alone or possibly in combination with minimal amounts of nuclear T antigen below our limit of detection, is able to stimulate cellular DNA synthesis.     

Simian virus 40 small-t antigen stimulates viral DNA replication in permissive monkey cells.

The simian virus 40 (SV40) large-T antigen is essential for SV40 DNA replication and for late viral gene expression, but the role of the SV40 small-t antigen in these processes is still unclear. We have previously demonstrated that small t inhibits SV40 DNA replication in vitro. In this study, we investigated the effect of small t on SV40 replication in cultured cells. CV1 monkey cell infection experiments indicated that mutant viruses that lack small t replicate less efficiently than the wild-type virus. We next microinjected CV1 cells with SV40 DNA with and without purified small-t protein and analyzed viral DNA replication efficiency by Southern blotting. Replication of either wild-type SV40 or small-t deletion mutant DNA was increased three- to fivefold in cells coinjected with purified small t. Thus, in contrast to our in vitro observation, small t stimulated viral DNA replication in vivo. This result suggests that small t has cellular effects that are not detectable in a reconstituted in vitro replication system. We also found that small t stimulated progression of permissive monkey cells--but not of nonpermissive rodent cells--from G0-G1 to the S phase of the cell cycle, possibly leading to an optimal intracellular environment for viral replication.     

Possible role of SV40 T antigen in cell transformation]

The effect of purified SV40 T antigen on DNA synthesis in isolated nuclei from the confluent culture of CV-1 cells was studied. In the presence of T antigen the incorporation of [3H]TTP into DNA was found to be 2 to 3 times as high as in the control nuclei. The resulting labelled DNA was subjected to alkaline sucrose gradient centrifugation, which revealed the presence of 4S DNA species, corresponding to Okazaki fragments of animal cells. The latter finding suggests a replicative mode of DNA synthesis induced by T antigen. T antigen isolated from the cells infected with SV40 tsA-mutant and kept at a nonpermissive (41 degrees) temperature fails to stimulate DNA synthesis in isolated nuclei from resting cells. On storage at 4 degrees SV40 T antigen gradually loses its ability to stimulate DNA synthesis and by the 8th day even suppresses it when tested on isolated nuclei from a growing cell culture. No effect of T antigen on the endonuclease-induced reparative synthesis of DNA could be observed. The data described suggest that T antigen is directly involved in the control of DNA synthesis in the cells infected or transformed with SV40.     

A DNA replication-positive mutant of simian virus 40 that is defective for transformation and the production of infectious virions.

Simian virus 40 (SV40) mutant 5002 carries base pair substitutions of C-5109----T and C-5082----T. These mutations lie in a region of the genome that encodes amino acids common to the large and small viral tumor antigens (T and t antigens, respectively) and result in amino acid substitutions of Leu-19----Phe and Pro-28----Ser. In contrast to wild-type SV40, which produces large plaques that are clearly visible 8 days postinfection, mutant 5002 is defective for productive infection, producing tiny plaques that arise at around 21 days postinfection. However, 5002 is capable of replicating viral DNA and producing normal amounts of capsid proteins, indicating that the mutations alter an activity of T antigen that is required subsequent to DNA synthesis, such as maturation, viral assembly, or release of virions. The mutant T antigen has normal ATPase activity, is phosphorylated in a manner that is indistinguishable from that of the wild-type T antigen, and retains the ability to oligomerize. 5002 complements mutants defective in T antigen host range-adenovirus helper function for productive infection. Thus, T antigen encodes two activities that affect at least two different steps in viral infection other than DNA replication, one inactivated by mutations in the host range-adenovirus helper domain and one inactivated by the mutations present in 5002. The 5002-encoded T antigen is also defective for transformation of REF52 cells when expressed from the normal SV40 early promoter, although this defect can be partially overcome by expressing the protein from stronger promoters.     

Effective DNA vaccination against listeriosis by prime/boost inoculation with the gene gun.

Protective immunity against Listeria monocytogenes strongly depends on CD8+ T lymphocytes, and both IFN-gamma secretion and target cell killing are considered relevant to protection. We analyzed whether we could induce a protective type 1 immune response by DNA vaccination with the gene gun using plasmids encoding for two immunodominant listerial Ags, listeriolysin and p60. To induce a Th1 response, we 1) coprecipitated a plasmid encoding for GM-CSF, 2) employed a prime/boost vaccination schedule with a 45-day interval, and 3) coinjected oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs. DNA immunization of BALB/c mice with plasmids encoding for listeriolysin (pChly) and p60 (pCiap) efficiently induced MHC class I-restricted, Ag-specific CD8+ T cells that produced IFN-gamma. Coinjection of CpG-ODN significantly increased the frequency of specific IFN-gamma-secreting T cells. Although pChly induced specific CD8+ T cells expressing CTL activity, it failed to stimulate CD4+ T cells. Only pCiap induced significant CD4+ T cell and humoral responses, which were predominantly of Th2 type. Vaccination with either plasmid induced protective immunity against listerial challenge, and coinjection of CpG ODN improved vaccine efficacy in some situations. This study demonstrates the feasibility of gene gun administration of plasmid DNA for inducing immunity against an intracellular pathogen for which protection primarily depends on type 1 CD8+ T cells.     

Simian virus 40 large T-antigen point mutants that are defective in viral DNA replication but competent in oncogenic transformation.

The large T antigen of simian virus 40 (SV40) is a multifunctional protein that is essential in both the virus lytic cycle and the oncogenic transformation of cells by SV40. To investigate the role of the numerous biochemical and physiological activities of T antigen in the lytic and transformation processes, we have studied DNA replication-deficient, transformation-competent large T-antigen mutants. Here we describe the genetic and biochemical analyses of two such mutants, C2/SV40 and C11/SV40. The mutants were isolated by rescuing the integrated SV40 DNA from C2 and C11 cells (CV-1 cell lines transformed with UV-irradiated SV40). The mutant viral early regions were cloned into the plasmid vector pK1 to generate pC2 and pC11. The mutations that are responsible for the deficiency in viral DNA replication were localized by marker rescue. Subsequent DNA sequencing revealed point mutations that predict amino acid substitutions in the carboxyl third of the protein in both mutants. The pC2 mutation predicts the change of Lys----Arg at amino acid 516. pC11 has two mutations, one predicting a change of Pro----Ser at residue 522, and another predicting a Pro----Arg change at amino acid 549. The two C11 mutations were separated from each other to form two distinct viral genomes in pC11A and pC11B. pC2, pC11, pC11A, and pC11B are able to transform both primary and established rodent cell cultures. The C11 and C11A T antigens are defective in ATPase activity, suggesting that wild-type levels of ATPase activity are not necessary for the oncogenic transformation of cells by T antigen.     

Polyomavirus large and small T antigens cooperate in induction of the S phase in serum-starved 3T3 mouse fibroblasts.

The induction of an S phase in the host cell is a prerequisite for the lytic replication cycle of polyomavirus. This function was attributed to proteins coded for by the early region of the viral DNA, the T antigens. A consideration of the role of the T antigens in the initiation of a mitogenic response of the host cell has to take into account the recent discovery that virus adsorption is sufficient to induce the synthesis of proteins which are known to appear early after quiescent cells are stimulated by the addition of serum, namely fos, jun, and myc (J. Zullo, C.D. Stiles, and R.L. Garcea, Proc. Natl. Acad. Sci. USA 84:1210-1214, 1987; G. M. Glenn and W. Eckhart, J. Virol. 64:2193-2201, 1990). This induction is followed by an initiation of DNA synthesis. It is therefore important to dissociate the effects of the T antigens on the host cell from those of virus adsorption. To do so, we used dexamethasone-regulated versions of the large and small T antigens of polyomavirus stably integrated into the genome of Swiss 3T3 cells to study their function in S-phase induction. When the production of the large or small T antigen in serum-starved 3T3 mouse fibroblasts was activated, only a small fraction of cells was able to leave G0/G1 despite the synthesis of considerable amounts of the respective T antigen. Activation of both T antigens within the same cell, on the other hand, resulted in S-phase induction in a notable percentage of cells, suggesting that the two proteins cooperate in this activity. Polyomavirus T antigens appear to bypass the pathway of growth regulation involving the activation of c-fos. These results are discussed in relation to other known functions of the two virally coded proteins.     

Mutation causing premature termination of the polyoma virus medium T antigen blocks cell transformation

We used site-specific mutagenesis to introduce a termination codon, TGA, into the reading frame for the polyoma virus medium T antigen. We induced this mutation in a region of the polyoma genome in which the overlapping coding regions for the large and medium TE antigens are translated in different reading frames. Therefore, the mutation terminated translation of the medium T antigen, but it caused only a single amino acid substitution in the large T antigen and did not affect the small T antigen. Cells infected by the mutant virus produced normal-size small and large T antigens. The infected cells produced a 28,000-dalton fragment of the 48,000-dalton medium T antigen, whose size and tryptic peptide map were consistent with its being a truncated N-terminal fragment terminating at the new termination codon of the mutant. Immunoprecipitates of mutant-infected cell extracts did not show medium-T-antigen-associated protein kinase activity. The mutant virus replicated normally in mouse 3T6 cells and induced cellular DNA synthesis in resting mouse 3T3 cells, but it failed to transform rat or hamster cells, as judged by focus formation and growth in agar. The mutant complemented a tsA mutant which affects the large T antigen for transformation, implying that the mutant defect for transformation was in the medium T antigen. These results imply that the small T antigen and the large T antigen together are insufficient to cause transformation and support the conclusion that the medium T antigen is essential for cell transformation by polyoma virus.     

Mutations near the carboxyl terminus of the simian virus 40 large tumor antigen alter viral host range.

We report the characterization of three mutants of simian virus 40 with mutations that delete sequences near the 3' end of the gene encoding large tumor antigen (T antigen). Two of these mutants, dl1066 and dl1140, exhibit an altered viral host range. Wild-type simian virus 40 is capable of undergoing a complete productive infection on several types of established African green monkey kidney lines, including BSC40 and CV1P. dl1066 and dl1140 grow on BSC40 cells at 37 degrees C. However, both mutants fail to form plaques on BSC40 cells at 32 degrees C or on CV1P cells at any temperature. These mutants are capable of replicating viral DNA in the nonpermissive cell type, indicating a defect in an activity of T antigen not related to its replication function. Furthermore this defect can be complemented in trans by the wild type or by a variety of DNA replication-negative T antigen mutants, so long as they produce a normal carboxyl-terminal region of the molecule. Our data are consistent with the hypothesis that the C-terminal region of T antigen constitutes a functional domain. We propose that this domain encodes an activity that is required for simian virus 40 productive infection on the CV1P cell line, but not on BSC40.     

Mutational analysis of polyomavirus small-T-antigen functions in productive infection and in transformation.

The function of polyomavirus small T antigen in productive infection and in transformation was studied. Transfection of permissive mouse cells with mixtures of mutants that express only one type of T antigen showed that small T antigen increased large-T-antigen-dependent viral DNA synthesis approximately 10-fold. Under the same conditions, small T antigen was also essential for the formation of infectious virus particles. To analyze these activities of small T antigen, mutants producing protein with single amino acid replacements were constructed. Two mutants, bc1073 and bc1075, were characterized. Although both mutations led to the substitution of amino acid residues of more than one T antigen, the phenotype of both mutants was associated with alterations of the small T antigen. Both mutant proteins had lost their activity in the maturation of infectious virus particles. The bc1075 but not the bc1073 small T antigen had also lost its ability to stimulate viral DNA synthesis in mouse 3T6 cells. Finally, both mutants retained a third activity of small T antigen: to confer on rat cells also expressing middle T antigen the ability to grow efficiently in semisolid medium. The phenotypes of the mutants in these three assays suggest that small T antigen has at least three separate functions.     

An insertional mutant of epidermal growth factor receptor allows dissection of diverse receptor functions.

Cultured NIH-3T3 cells devoid of endogenous EGF-receptors were transfected with cDNA constructs encoding normal human EGF-receptor and with a construct encoding an insertional mutant of the EGF-receptor containing four additional amino acids in the kinase domain after residue 708. Unlike the wild-type receptor expressed in these cells which exhibits EGF-stimulatable protein tyrosine kinase activity, the mutant receptor lacks protein tyrosine kinase activity both in vitro and in vivo. Despite this deficiency the mutant receptor is properly processed, it binds EGF and it exhibits both high and low affinity binding sites. Moreover, it undergoes efficient EGF-mediated endocytosis. However, EGF fails to stimulate DNA synthesis and is unable to stimulate the phosphorylation of S6 ribosomal protein in cells expressing this receptor mutant. Hence, it is proposed that the protein tyrosine kinase activity of EGF-receptor is essential for the initiation of S6 phosphorylation and for DNA synthesis induced by EGF. However, EGF-receptor processing, the expression of high and low affinity surface receptors and receptor internalization, require neither kinase activity nor receptor autophosphorylation. Interestingly, phorbol ester (TPA) fails to abolish the high affinity state and is also unable to stimulate the phosphorylation of this receptor mutant. This result is consistent with the notion that kinase-C phosphorylation of EGF-receptor is essential for the loss of high affinity EGF-receptors caused by TPA.     

A deletion in the simian virus 40 large T antigen impairs lytic replication in monkey cells in vivo but enhances DNA replication in vitro: new complementation function of T antigen.

We describe a new complementation function within the simian virus 40 (SV40) A gene. This function is required for viral DNA replication and virus production in vivo but, surprisingly, does not affect any of the intrinsic enzymatic functions of T antigen directly required for in vitro DNA replication. Other well-characterized SV40 T-antigen mutants, whether expressed stably from integrated genomes or in cotransfection experiments, complement these mutants for in vivo DNA replication and plaque formation. These new SV40 mutants were isolated and cloned from human cells which stably carry the viral DNA. The alteration in the large-T-antigen gene was shown by marker rescue and nucleotide sequence analysis to be a deletion of 322 bp spanning the splice-donor site of the first exon, creating a 14-amino-acid deletion in the large T antigen. The mutant gene was expressed in H293 human cells from an adenovirus vector, and the protein was purified by immunoaffinity chromatography. The mutant protein directs greater levels of DNA replication in vitro than does the wild-type protein. Moreover, the mutant protein reduces the lag time for in vitro DNA synthesis and can be diluted to lower levels than wild-type T antigen and still promote good replication, which is in clear contrast to the in vivo situation. These biochemical features of the protein are independent of the source of the cellular replication factors (i.e., HeLa, H293, COS 7, or CV1 cells) and the cells from which the T antigens were purified. The mutant T antigen does not transform Rat-2 cells. Several different models which might reconcile the differences observed in vivo and in vitro are outlined. We propose that the function of T antigen affected prepares cells for SV40 replication by activation of a limiting cellular replication factor. Furthermore, a link between the induction of a cellular replication factor and transformation by SV40 is discussed.     

Temperature-Sensitive Mutants of Simian Virus 40: Infection of Permissive Cells

Ten temperature-sensitive mutants of simian virus 40 have been isolated and characterized in permissive cells. The mutants could be divided into three functional groups and two complementation groups. Seven mutants produced T antigen, infectious viral deoxyribonucleic acid (DNA), and structural viral antigen but predominantly the empty shell type of viral particles. Two mutants produced T antigen and infectious viral DNA, but, although viral structural protein(s) could be detected immunologically, no V antigen or viral particles were found. These two functional groups of mutants did not complement each other. A single mutant was defective in the synthesis of viral DNA, viral structural antigens, and viral particles. T antigen could be detected in infected cells by fluorescent antibody but was reduced by complement fixation assay. This mutant stimulated cell DNA synthesis at the restrictive temperature and complemented the other two functional groups of mutants.