DNA-binding region of the simian virus 40 tumor antigen.

The simian virus 40 (SV40) tumor (T) antigen was purified by immunoaffinity chromatography and cleaved with small amounts of trypsin, and the resulting fragments were subjected to SV40 DNA cellulose chromatography. A 44,000-molecular-weight fragment (44K fragment) from the left end of the molecule and a 30K fragment mapping from approximately Lys 131 to Lys 371 bound to the column and were eluted with 1 M NaCl. In a second series of experiments, T antigen was immunoprecipitated with hamster anti-T serum or various monoclonal antibodies and partially digested with trypsin. Fragments that were solubilized by this treatment were tested for DNA-binding activity by using an SV40 DNA fragment-binding assay. A 17K fragment which originated from the amino-terminal region of the polypeptide had no apparent binding activity in this assay. On the other hand, larger fragments (76K, 46K, and 30K) whose amino termini were mapped around Lys 131 did display DNA-binding activity. Finally, complexes consisting of SV40 DNA and T-antigen fragments were precipitated in the DNA-binding assay with monoclonal antibodies that recognize the central region of the protein; however, antibodies with specificities to the amino- or carboxy-terminal regions were inactive. These results strongly suggest that the DNA-binding region of T antigen lies approximately between Lys 131 and Lys 371, corresponding to 0.51 and 0.37 map units on the DNA.     

Epitopes on the major capsid protein of simian virus 40

Thirteen monoclonal antibodies which react with the major capsid protein (VP1) of simian virus 40 (SV40) have been isolated. Of these, five neutralized viral infectivity when added in sufficient concentration. Seven of the antibodies reacted with denatured VP1 and also recognized fragments generated by protease or cyanogen bromide cleavage. The region of VP1 recognized by all seven antibodies was mapped within a nine-amino-acid segment located in the carboxyl portion of the protein (from amino acid positions 312 to 321). This region is likely to protrude from the surface of the protein as judged by high hydrophilicity and low hydropathy predicted from the amino acid sequence and lack of secondary structure by contrast with the rest of the protein for which predominantly beta-sheet structure is predicted. Competition between these antibodies and synthetic peptides for binding to virus particles confirmed that the continuous epitope is contained within the nine-amino-acid sequence. Competition between the different monoclonal antibodies suggested that the continuous epitope was also part of more complex discontinuous epitopes recognized by some of the other antibodies. These results support a model in which a segment of the carboxyl-terminal portion of VP1 protrudes from the surface of the virus to form an antigenic structure.     

DNA-binding properties of simian virus 40 T-antigen mutants defective in viral DNA replication.

Three simian virus 40 (SV40)-transformed monkey cell lines, C2, C6, and C11, producing T-antigen variants that are unable to initiate viral DNA replication, were analyzed with respect to their affinity for regulatory sequences at the viral origin of replication. C2 and C11 T antigens both bound specifically to sequences at sites 1 and 2 at the viral origin region, whereas C6 T antigen showed no specific affinity for any viral DNA sequences under all conditions tested. Viral DNA sequences encoding the C6 T antigen have recently been cloned out of C6 cells and used to transform an established rat cell line. T antigen from several cloned C6-SV40-transformed rat lines failed to bind specifically to the origin. C6 DNA contains three mutations: two located close to the amino terminus of T antigen at amino acid positions 30 and 51 and a third located internally at amino acid position 153. Two recombinant SV40 DNA mutants were prepared containing either the amino-terminal mutations at positions 30 and 51 (C6-1) or the internally located mutation at position 153 (C6-2) and used to transform Rat 2 cells. Whereas T antigen from C6-2-transformed cells lacked any specific affinity for these sequences. Therefore, the single mutation at amino acid position 153 (Asn leads to Thr) is sufficient to abolish the origin-binding property of T antigen. A T antigen-specific monoclonal antibody, PAb 100, which had been previously shown to immunoprecipitate an immunologically distinct origin-binding subclass of T antigen, recognized wild-type or C6-1 antigens, but failed to react with C6 or C6-2 T antigens. These results indicate that viral replication function comprises properties of T antigen that exist in addition to its ability to bind specifically to the SV40 regulatory sequences. Furthermore, it is concluded from these data that specific viral origin binding is not a necessary feature of the transforming function of T antigen.     

Sequence-specific interactions between a cellular DNA-binding protein and the simian virus 40 origin of DNA replication.

The core origin of simian virus 40 (SV40) DNA replication is composed of a 64-base-pair sequence encompassing T-antigen-binding site II and adjacent sequences on either side. A 7-base-pair sequence to the early side of T-antigen-binding site II which is conserved among the papovavirus genomes SV40, BK, JC, and SA12 was recently shown to be part of a 10-base-pair sequence required for origin activity (S. Deb, A.L. DeLucia, C.-P. Baur, A. Koff, and P. Tegtmeyer, Mol. Cell. Biol. 6:1663-1670, 1986), but its functional role was not defined. In the present report, we have used gel retention assays to identify a monkey cell factor that interacts specifically with double-stranded DNA carrying this sequence and also binds to single-stranded DNA. DNA-protein complexes formed with extracts from primate cells are more abundant and display electrophoretic mobilities distinct from those formed with rodent cell extracts. The binding activity of the factor on mutant templates is correlated with the replication activity of the origin. The results suggest that the monkey cell factor may be involved in SV40 DNA replication.     

Modification of simian virus 40 protein A.

The A protein of simian virus 40 is phosphorylated in both productive and transforming infection. The phosphorylated amino acid has been identified as serine and has been localized in a single tryptic peptide of the protein. Because the A protein synthesized in infection by A mutants is phosphorylated to the same extent and in the same peptide as in infection by wild-type virus, the functional defect of the A mutants is apparently unrelated to phosphorylation. At least three distinct forms of the A protein with apparent molecular weights of 85,000, 88,000, and 100,000 can be identified in extracts of cells infected by wild-type virus. After exposure of cells to Nonidet P-40, the 85,000- and 88,000-dalton proteins were found in varying amounts in extracts of permissive cells but not in extracts of transformed cells. This finding raised the question of the possible functional importance of the smaller proteins in productive infection. However, the virtual absence of the 85,000- and 88,000-dalton proteins in some extracts of the fully permissive CV-1 cell line indicates that a conversion of the larger to the smaller forms of the A protein is not required in significant quantity for productive infection. Furthermore, a study of extraction conditions shows that the smaller proteins are easily generated during extraction and provides an explanation for the appearance of these proteins in some cells after extraction under unfavorable conditions.     

Identification of simian virus 40 protein A.

A large simian virus 40 (SV40)-specific protein can be efficiently immunoprecipitated from infected cell extracts with antisera obtained from hamsters bearing SV40-induced tumors. The protein has an apparent molecular weight of 88,000 to 100,000 with respect to markers with known molecular weights, but behaves anomalously on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Cell lines infected by two different strains of SV40 synthesize immunoreactive proteins that differ slightly in mobility during SDS-polyacrylamide gel electrophoresis, evidence that the protein is coded for by the virus. These differences in protein size correlate with differences in the electrophoretic mobility of viral DNA fragments obtained by digestion with HindII and III restriction enzymes. The size of the viral capsid proteins VP2 and VP3 also varies with the strain of virus. dl-1001, a constructed deletion mutant that lacks part of the SV40A gene, directs the synthesis of a 33,000-dalton polypeptide that is not detected in cells infected with wild-type virus. The deletion fragment, like the larger protein, is phosphorylated. Maps of tryptic peptides from the 88,000- to 100,000-dalton protein and the 33,000-dalton fragment show common peptides and provide strong direct evidence that the proteins are products of the SV40 A gene. The deletion fragment reacts with antitumor sera and binds to double-stranded DNA in the presence of the complete A protein.     

Properties of the DNA-binding domain of the simian virus 40 large T antigen.

T antigen (Tag) from simian virus 40 binds specifically to two distinct sites in the viral origin of replication and to single-stranded DNA. Analysis of the protein domain responsible for these activities revealed the following. (i) The C-terminal boundary of the origin-specific and single-strand-specific DNA-binding domain is at or near amino acid 246; furthermore, the maximum of these DNA-binding activities coincides with a narrow C-terminal boundary, spanning 4 amino acids (246 to 249) and declines sharply in proteins with C termini which differ by a few (4 to 10) amino acids; (ii) a polypeptide spanning residues 132 to 246 of Tag is an independent domain responsible for origin-specific DNA binding and presumably for single-stranded DNA binding; and (iii) a comparison of identical N-terminal fragments of Tag purified from mammalian and bacterial cells revealed differential specificity and levels of activity between the two sources of protein. A role for posttranslational modification (phosphorylation) in controlling the DNA-binding activity of Tag is discussed.     

Studies on the origin-specific DNA-binding domain of simian virus 40 large T antigen.

The origin-specific DNA-binding domain of simian virus 40 large T antigen was analyzed, and its C-terminal boundary was found to be at or before amino acid 259. This does not include the zinc finger structural motif located at amino acids 302 to 320 (J. M. Berg, Science 232:485-486, 1986). Interestingly, N-terminal fragments of 266 and 272 amino acids and larger displayed dramatically reduced origin-binding activity. In addition, the specific DNA-binding properties of truncated proteins purified from both bacterial and mammalian sources were compared. Truncated T antigens from mammalian cells bound specific DNA fragments more efficiently than did their bacterial counterparts. These results implicate posttranslational modification with a role in regulating the DNA-binding activity of large T antigen.     

Helicase, DNA-binding, and immunological properties of replication-defective simian virus 40 mutant T antigens

Simian virus 40 T antigen (TAg) exhibits nonspecific and origin-specific DNA binding (ori binding) and ATPase and helicase activities, all of which are related to its roles in viral DNA replication. We have characterized some of the properties of four replication-defective but transformation-competent mutant TAgs, C6-2, T22, C11, and C8A. C6-2 and T22 TAgs were each previously determined to lack ori-binding properties, while C11 TAg was reported to lack ATPase activity. The C8A TAg did not exhibit defects in either ori-binding or ATPase functions. We have analyzed additional aspects of these mutant TAgs pertaining to their helicase, DNA-binding, and immunological properties. With the exception of the C11 TAg, all the other TAgs exhibited helicase activity. The lack of helicase activity by C11 TAg was consistent with its previously shown inability to hydrolyze ATP or to replicate viral DNA. These results therefore show that ori-binding and helicase activities are separate functions of TAg. Wild-type and mutant TAgs bound with similar efficiency to either native or denatured calf thymus DNA-cellulose, indicating no marked differences in their nonspecific DNA-binding properties. We also tested the binding of wild-type and mutant TAgs to a monoclonal antibody, PAb 100, that was previously shown to recognize an extremely small class of TAg that may represent a unique conformational form of the protein. Interestingly, while less than 10% of the wild-type, C6-2, C11, and T22 mutant TAgs were recognized by PAb 100, more than 60% of the C8A mutant TAg was bound by this antibody. Therefore, although no defect in biochemical function was observed with the C8A TAg, its deficiency in viral DNA replication may be related to an unusual conformation, as detected by its dramatically increased recognition by PAb 100. These results show that the helicase activity of TAg is not required for its transformation function.     

The DNA-binding domain of simian virus 40 tumor antigen has multiple functions.

The DNA-binding domain of simian virus 40 tumor antigen has been previously shown to participate in a number of different activities. Besides being involved in binding to sequences at the viral replication origin, this domain appears to be required for nonspecific DNA binding, for structurally distorting origin DNA (melting and untwisting), and possibly for oligomerization of the protein into hexamers and double hexamers. We now provide evidence that it also takes part in unwinding origin DNA sequences, contributes a function specifically related to in vivo DNA replication, and perhaps supports the assembly of the virus or release of the virus from the cell. This 100-amino-acid domain appears to be an excellent model system for studying how a small region of a protein could have a number of distinct activities.     

Analysis of a nuclear localization signal of simian virus 40 major capsid protein Vp1.

The nuclear localization signal of the major structural protein, Vp1, of simian virus 40 was further defined by mutagenesis. The targeting activity was examined in cells microinjected with SV-Vp1 variant viral DNAs bearing either an initiation codon mutation of the agnoprotein or mutations in the Vp1 coding sequence or microinjected with pSG5-Vp1 and pSG5-Vp1 mutant DNAs in which Vp1 or mutant Vp1 is expressed from simian virus 40 early promoter. The Vp1 nuclear localization signal functioned autonomously without agno-protein once the Vp1 protein was synthesized in the cytoplasm. The targeting activity was localized to the amino-terminal 19 residues. While replacement of cysteine 10 with glycine, alanine, or serine did not affect the activity, replacement of arginine 6 with glycine caused the cytoplasmic phenotype. When multiple mutations were introduced among residue 5, 6, 7, 16, 17, or 19, the targeting activity was found to reside in two clusters of basic residues, a cluster of lysine 5, arginine 6, and lysine 7 and a cluster of lysine 16, lysine 17, and lysine 19. The clusters are independently important for nuclear localization activity.     

The finger domain of simian virus 40 large T antigen controls DNA-binding specificity.

The specificity and regulation of protein-DNA interactions play a crucial role in all aspects of communication between genotype and phenotype in a cell. The large T antigen of simian virus 40 binds to identical, yet quite differently arranged, pentanucleotide motifs in the simian virus 40 control region, sites I and II. Wild-type T antigen preferentially binds site I. We demonstrate that a bacterial peptide encoding residues 1 to 259 (T260) includes the essential amino acids required for binding to both DNA sites but predominantly binds site II. However, a longer peptide (residues 1 to 369; T370) binds almost exclusively to site I. Thus, the addition of amino acids 260 to 369 to the T260 peptide results in the loss of site II binding. This region includes a single putative metal-binding region, and mutation of T370 at either conserved cysteine of the finger results in equal but inefficient binding to both sites. While no metal binding has been shown to be directly associated with this sequence, these results suggest a novel, perhaps structural, function for such a finger motif, since this domain of T antigen appears to play a crucial role in modulating the DNA-binding behavior of T-antigen peptides.     

DNA-binding properties of the major core protein of adenovirus 2.

The major adenovirus core protein (P.VII) binds to various species of duplex and single-stranded DNA molecules as a linear function of P.VII concentration. P.VII progressively condenses 32S Ad2 DNA into rapidly sedimenting forms having an S value of around 2,280. P.VII does not coat DNA like cytochrome C, instead DNA-protein beads are visualized in the electron microscope at low protein concentration. These beads appear to interact forming larger structures and at high P.VII concentrations the DNA molecule becomes highly compacted. Analysis of DNA fragments formed after digestion of P.VII-DNA complexes and isolated cores with micrococcal nuclease suggest that the organization of the DNA in the two structures is essentially identical. The initial P.VII and DNA interaction is sensitive to both ionic and hydrophobic environments, whereas the in vitro DNA-P.VII complexes are extremely stable and are not disrupted in the presence of 3 M NaCl, 1% sarcosyl or 5% deoxycholate. Properties of these in vitro DNA-protein VII complexes share striking similarities to isolated viral core particles.     

Characterization of the DNA-binding properties of the origin-binding domain of simian virus 40 large T antigen by fluorescence anisotropy

The affinity of the origin-binding domain (OBD) of simian virus 40 large T antigen for its cognate origin was measured at equilibrium using a DNA binding assay based on fluorescence anisotropy. At a near-physiological concentration of salt, the affinities of the OBD for site II and the core origin were 31 and 50 nM, respectively. Binding to any of the four 5'-GAGGC-3' binding sites in site II was only slightly weaker, between 57 and 150 nM. Although the OBD was shown previously to assemble as a dimer on two binding sites spaced by 7 bp, we found that increasing the distance between both binding sites by 1 to 3 bp had little effect on affinity. Similar results were obtained for full-length T antigen in absence of nucleotide. Addition of ADP-Mg, which promotes hexamerization of T antigen, greatly increased the affinity of full-length T antigen for the core origin and for nonspecific DNA. The implications of these findings for the assembly of T antigen at the origin and its transition to a non-specific DNA helicase are discussed.     

Oligomerization and origin DNA-binding activity of simian virus 40 large T antigen

Simian virus 40 (SV40) large tumor antigen (T antigen) exists in multiple molecular forms, some of which are separable by zone velocity sedimentation of soluble extracts from infected monkey cells. Three subclasses of this antigen from SV40-infected monkey cells have been separated and characterized: the 5S, 7S, and 14S forms. Newly synthesized T antigen occurs primarily in the 5S form. Chemical cross-linking provided evidence that the 14S form is primarily a tetramer, whereas the 5S and 7S forms could not be cross-linked into oligomers. The DNA-binding properties of each subclass were investigated after immunopurification. The affinities of the three forms for SV40 DNA and for a synthetic 19-base-pair sequence from binding site I are very similar (equilibrium dissociation constant [KD], 0.3 to 0.4 nM). The specific activity of DNA binding was greatest for the 5S and 7S subclasses and least for the 14S subclass. Moreover, the specific activity of the 5S and 7S subclasses increased sharply at about 40 h after infection, whereas the activity of the 14S subclass was maintained at a constant low level throughout infection. A model relating oligomerization and DNA binding of T antigen in infected cells is presented.     

Subcellular distribution of viral structural proteins during simian virus 40 infection.

The amounts of simian virus 40 structural polypeptides Vp1, Vp2, and Vp3 in different subcellular fractions at various times after lytic infection were determined by a quantitative immunoblotting procedure. Simian virus 40-infected cells were lysed with a buffer containing Nonidet P-40 to yield a soluble fraction. The Nonidet P-40-insoluble fraction was further fractionated in the presence of deoxycholate and Tween 40 to yield a soluble fraction (cytoskeletal) and an insoluble fraction (Nuc), which is primarily cell nuclei. At 33 h postinfection, the majority of viral structural proteins was found in the cell nucleus, whereas, at 48 to 65 h postinfection, Vp1 was distributed evenly among all cell fractions and Vp2 and Vp3 were found predominantly in the cytoskeletal and Nuc fractions. Thus, not all of the viral polypeptides synthesized in the cytoplasm migrated into the cell nucleus. Throughout infection, the molar ratio (Vp3/Vp2) was rather constant in all subcellular fractions, indicating that the synthesis or processing or both of Vp2 and Vp3 are coordinately regulated. The molar ratio of Vp1/(Vp2 + Vp3) varied among the fractions. The Vp1/(Vp2 + Vp3) molar ratio in the soluble fraction varied during the course of infection; however, constant ratios were maintained in the cytoskeletal and Nuc fractions. Thus, the mechanism which controls the movement of Vp1 to different compartments of the cell appears to be different from that of Vp2 and Vp3. The Vp1/(Vp2 + Vp3) value in the Nuc fraction was similar to the ratio found in virus particles. The constant molar distribution of Vp1, Vp2, and Vp3 in the Nuc fraction throughout infection suggests that there is a specific mechanism which regulates the transport of viral structural proteins. These results support the hypothesis that the structural proteins of simian virus 40 are transported into the cell nucleus in precise proportions.     

Stimulation of non-histone chromosomal protein synthesis in simian virus 40-infected simian cells.

The pattern of synthesis of non-histone chromosomal proteins in simian virus (SV) 40-infected African green monkey kidney cells was analyzed by polyacryl-amide gel electrophoresis to see whether the changes in chromosomal protein metabolism are involved in the viral-induced synthesis of cellular DNA and mRNA. During the prereplicative phase of infection, the rate of histone synthesis was decreased until 15 h postinfection, whereas that of non-histone protein synthesis was increased after 5 h postinfection and reached a maximum at 10 to 15 h postinfection when viral-induced synthesis of cellular DNA and mRNA began to be observed. Stimulation of non-histone protein synthesis was also observed in the infected cells treated with cytosine arabinoside and was dependent on the multiplicity of infection. Stimulation occurred in almost all species of non-histone proteins. These results suggest that the stimulation of non-histone protein synthesis is caused by an early SV40 function and occurs prior to the viral-induced synthesis of cellular DNA and mRNA. During the replicative phase of infection, a marked increase in the rate of synthesis was observed in the non-histone proteins with molecular weights of about 48,000, 35,000, and 23,000, which were subsequently found to be SV40 capsid proteins.