The zinc finger region of simian virus 40 large T antigen is needed for hexamer assembly and origin melting.

Simian virus 40 large T antigen contains a single sequence element with an arrangement of cysteines and histidines that is characteristic of a zinc finger motif. The finger region maps from amino acids 302 through 320 and has the sequence C-302 L K C-305 I K K E Q P S H Y K Y H-317 E K H-320. Previous genetic analysis has shown that the cysteine and histidine sequences and the contiguous S H Y K Y region in the finger are important for DNA replication in vivo. We show here that representative mutations in either of these elements of the finger prevent the assembly of large T antigen into stable hexamers in vitro. These same mutations have a characteristic effect on the interaction of T antigen with the simian virus 40 core origin of replication. The mutant T antigens bind to the central pentanucleotide domain of the core origin but fail to melt the adjacent inverted repeat domain and to untwist the adenine-thymine domain. These defects would prevent the formation of a replication bubble and the initiation of DNA replication. Finger mutations have lesser effects on the helicase function of T antigen and no observable effect on binding of T antigen to the mouse p53 protein. We propose that the zinc finger region contributes to protein-protein interactions essential for the assembly of stable T-antigen hexamers at the origin of replication and that hexamers are needed for subsequent alterations in the structure of origin DNA. We cannot exclude the possibility that the zinc finger region also makes specific contacts with components of origin DNA.     

The simian virus 40 T antigen double hexamer assembles around the DNA at the replication origin.

An initial step in the replication of simian virus (SV40) DNA is the ATP-dependent formation of a double hexamer of the SV40 large tumor (T) antigen at the SV40 DNA replication origin. In the absence of DNA, T antigen assembled into hexamers in the presence of magnesium and ATP. Hexameric T antigen was stable and could be isolated by glycerol gradient centrifugation. The ATPase activities of hexameric and monomeric T antigen isolated from parallel glycerol gradients were identical. However, while monomeric T antigen was active in the ATP-dependent binding, untwisting, unwinding, and replication of SV40 origin-containing DNA, hexameric T antigen was inactive in these reactions. Isolated hexamers incubated at 37 degrees C in the presence of ATP remained intact, but dissociated into monomers when incubated at 37 degrees C in the absence of ATP. This dissociation restored the activity of these preparations in the DNA replication reaction, indicating that hexameric T antigen is not permanently inactivated but merely assembled into a nonproductive structure. We propose that the two hexamers of T antigen at the SV40 origin assemble around the DNA from monomer T antigen in solution. This complex untwists the DNA at the origin, melting specific DNA sequences. The resulting single-stranded regions may be utilized by the T antigen helicase activity to initiate DNA unwinding bidirectionally from the origin.     

Mapping of helicase and helicase substrate-binding domains on simian virus 40 large T antigen.

We generated fragments of simian virus 40 large tumor antigen (T antigen) by tryptic digestion and assayed them for helicase activity and helicase substrate (mostly single-stranded DNA)-binding activity in order to map the domain sites on the protein. The N-terminal 130 amino acids were not required for either activity, since a 76-kilodalton (kDa) fragment (amino acids 131 to 708) was just as active as intact T antigen. To map the helicase domain further, smaller tryptic fragments were generated. A 66-kDa fragment (131 to about 616) retained some activity, whereas a slightly smaller 62-kDa fragment (137 or 155 to 616) had none. This suggests that the minimal helicase domain maps from residue 131 to approximately residue 616. To map the helicase substrate-binding domain, we tested various fragments in a substrate-binding assay. The smallest fragment for which we could clearly demonstrate activity was a 46-kDa fragment (131 to 517). To determine the relationship between the helicase substrate domain and the origin-binding domain (131 to 257, minimal core region; 131 to 371, optimal region), we performed binding experiments with competitor DNAs present. We found that origin-containing double-stranded DNA was an excellent competitor of the binding of the helicase substrate to T antigen, suggesting that the two domains overlap. Therefore, full helicase activity requires at least a partial origin-binding domain as well as an active ATPase domain. Additionally, we found that the helicase substrate was a poor competitor of origin-binding activity, indicating that T antigen has a much higher affinity to origin sequences than to the helicase substrate.     

Two separable functional domains of simian virus 40 large T antigen: carboxyl-terminal region of simian virus 40 large T antigen is required for efficient capsid protein synthesis.

The carboxyl-terminal portion of simian virus 40 large T antigen is essential for productive infection of CV-1 and CV-1p green monkey kidney cells. Mutant dlA2459, lacking 14 base pairs at 0.193 map units, was positive for viral DNA replication, but unable to form plaques in CV-1p cells (J. Tornow and C.N. Cole, J. Virol. 47:487-494, 1983). In this report, the defect of dlA2459 is further defined. Simian virus 40 late mRNAs were transcribed, polyadenylated, spliced, and transported in dlA2459-infected cells, but the level of capsid proteins produced in infected CV-1 green monkey kidney cells was extremely low. dlA2459 large T antigen lacks those residues known to be required for adenovirus helper function, and the block to productive infection by dlA2459 occurs at the same stage of infection as the block to productive adenovirus infection of CV-1 cells. These results suggest that the adenovirus helper function is required for productive infection by simian virus 40. Mutant dlA2459 was able to grow on the Vero and BSC-1 lines of African green monkey kidney cells. Additional mutants affecting the carboxyl-terminal portion of large T were prepared. Mutant inv2408 contains an inversion of the DNA between the BamHI and BclI sites (0.144 to 0.189 map units). This inversion causes transposition of the carboxyl-terminal 26 amino acids of large T antigen and the carboxyl-terminal 18 amino acids of VP1. This mutant was viable, even though the essential information absent from dlA2459 large T antigen has been transferred to the carboxyl terminus of VP1 of inv2408. The VP1 polypeptide carrying this carboxyl-terminal portion of large T could overcome the defect of dlA2459. This indicates that the carboxyl terminus of large T antigen is a separate and separable functional domain.     

Association of simian virus 40 T antigen with replicating nucleoprotein complexes of simian virus 40.

An immunoprecipitation assay was established for simian virus 40 T-antigen-bound nucleoprotein complexes by means of precipitation with sera from hamsters bearing simian virus 40-induced tumors. About 80% of simian virus 40 replicating nucleoprotein complexes in various stages of replication were immunoprecipitated. In contrast, less than 21% of mature nucleoprotein complexes were immunoprecipitated. Pulse-chase experiments showed that T antigen was lost from most of the nucleoprotein complexes concurrently with completion of DNA replication. T antigen induced by dl-940, a mutant with a deletion in the region coding for small T antigen, was also associated with most of the replicating nucleoprotein complexes. Once bound with replicating nucleoprotein complexes at the permissive temperature, thermolabile T antigen induced by tsA900 remained associated with the complexes during elongation of the replicating DNA chain at the restrictive temperature. These results suggest that simian virus 40 T antigen (probably large T antigen) associates with nucleoprotein complexes at or before initiation of DNA replication and that the majority of the T antigen dissociates from the nucleoprotein complexes simultaneously with completion of DNA replication.     

New properties of simian virus 40 large T antigen

An 8,000-molecular-weight (8K) T antigen was found in all cells transformed by simian virus 40. The 8K T antigen was weakly labeled in vivo with [35S]methionine or 32Pi. A deletion in the human papovavirus BK genome, in the region coding for the carboxy-terminal end of the large T antigen, reduced the size of the 8K T antigen. The last 80 amino acids of the large T antigen include the sequence Asp-Asp-Asp-Asp unique to the activation peptide of trypsinogen. Large T antigen bound diisopropyl fluorophosphate and was retained by D-phenylalanine coupled to Sepharose beads, an affinity adsorbent that can retain chymotrypsin. The large T antigen and the recA protein of Escherichia coli, a known protease, have several properties in common as well as several similar sequences. Antibodies against large T antigen interacted with native recA protein.     

Salt-resistant association of simian virus 40 T antigen with simian virus 40 DNA in nucleoprotein complexes.

Treatment of nucleoprotein complexes (NPCs) from simian virus 40 (SV40)-infected TC7 cells with NaCl (1 or 2 M) or guanidine-hydrochloride (1 or 2 M) resulted in a significant fraction of T antigen still associated with SV40 (I) DNA. Immunoprecipitation of the salt-treated NPCs with SV40 anti-T serum indicated that T antigen is preferentially associated with SV40 (I) DNA rather than with SV40 (II) DNA. Treatment of the NPCs with 4 M guanidine-hydrochloride, however, resulted in a substantial decrease in the amount of SV40 (I) and (II) DNA associated with T antigen. As the temperature was increased to 37 degrees C during incubation of NPCs with NaCl or guanidine-hydrochloride, there was a decrease in the amount of SV40 (I) and (II) DNA immunoprecipitated with SV40 anti-T serum. In the absence of salt, temperature had no effect on the association of T antigen with the SV40 DNA in the NPCs. Treatment of NPCs from SV40 wildtype or tsA58-infected cells grown at the permissive temperature with 1 or 2 M NaCl indicated that tsA T antigen has the same sensitivities as wild-type T antigen to high salt treatment when bound to DNA in NPCs. Characterization of the proteins associated with SV40 (I) DNA after high salt treatment revealed that, in addition to T antigen, a certain amount of viral capsid proteins VP1 and VP3 remained associated with the DNA. Complexes containing SV40 (I) DNA had a sedimentation value of 53S after 1 M NaCl treatment and 43S after 2 M NaCl treatment.     

trans-dominant defective mutants of simian virus 40 T antigen.

We constructed a collection of linker insertion mutants in the simian virus 40 (SV40) genome and studied several of these with changes limited to a part of the large T antigen gene corresponding to an amino acid sequence shared with other ATPases. Two of these mutants were found to have a novel phenotype in that they could not be complemented for plaque formation by a late-region deletion mutant. These two mutants, in contrast to other mutants in this region, were able to transform rat cells in culture at a frequency close to that of the wild-type gene. The noncomplementing mutants were found to be potent inhibitors of SV40 DNA replication despite the presence of wild-type T antigen in the transfected cells. This inhibition was shown to be the result of the introduced mutations in the large T antigen gene. We conclude that the large T antigens of the noncomplementing mutants can act as inhibitors of SV40 DNA replication.     

Specific association of simian virus 40 tumor antigen with simian virus 40 chromatin

Simian virus 40 tumor antigen (SV40 T antigen) was bound to both replicating and fully replicated SV40 chromatin extracted with a low-salt buffer from the nuclei of infected cells, and at least a part of the association was tight specific. T antigen cosedimented on sucrose gradients with SV40 chromatin, and T antigen-chromatin complexes could be precipitated from the nuclear extract specifically with anti-T serum. From 10 to 20% of viral DNA labeled to steady state with [3H]thymidine for 12 h late in infection or 40 to 50% of replicating viral DNA pulse-labeled for 5 min was associated with T antigen in such immunoprecipitates. After reaction with antibody, most of the T antigen-chromatin complex was stable to washing with 0.5 M NaCl, but only about 20% of the DNA label remained in the precipitate after washing with 0.5 M NaCl-0.4% Sarkosyl. This tightly bound class of T antigen was associated preferentially with a subfraction of pulse-labeled replicating DNA which comigrated with an SV40 form I marker. A tight binding site for T antigen was identified tentatively by removing the histones with dextran sulfate and heparin from immunoprecipitated chromatin labeled with [32P]phosphate to steady state and then digesting the DNA with restriction endonucleases HinfI and HpaII. The site was within the fragment spanning the origin of replication, 0.641 to 0.725 on the SV40 map.     

SV40 large T antigen hexamer structure: domain organization and DNA-induced conformational changes

Simian Virus 40 replication requires only one viral protein, the Large T antigen (T-ag), which acts as both an initiator of replication and as a replicative helicase (reviewed in ). We used electron microscopy to generate a three-dimensional reconstruction of the T-ag hexameric ring in the presence and absence of a synthetic replication fork to locate the T-ag domains, to examine structural changes in the T-ag hexamer associated with DNA binding, and to analyze the formation of double hexamers on and off DNA. We found that binding DNA to the T-ag hexamer induces large conformational changes in the N- and C-terminal domains of T-ag. Additionally, we observed a significant increase in density throughout the central channel of the hexameric ring upon DNA binding. We conclude that conformational changes in the T-ag hexamer are required to accommodate DNA and that the mode of DNA binding may be similar to that suggested for some other ring helicases. We also identified two conformations of T-ag double hexamers formed in the presence of forked DNA: with N-terminal hexamer-hexamer contacts, similar to those formed on origin DNA, or with C-terminal contacts, which are unlike any T-ag double hexamers reported previously.     

The zinc finger region of simian virus 40 large T antigen

Simian virus 40 large T antigen contains a single sequence element with an arrangement of cysteines and histidines that is characteristic of a zinc finger motif. The finger region maps from amino acids 302 through 320 and has the sequence Cys-302LeuLysCys-305IleLysLysGluGlnProSerHisTyrLysTyrHis- 317GluLysHis-320. In a conventional representation, the binding of zinc to the cysteines and histidines at positions 302, 305, 317, and 320 would form two minor loops and one major loop from the intervening amino acids. We made single amino acid substitutions at every position in the finger to identify possible functional elements within the putative metal-binding domain. Amino acids in the zinc finger could be divided into three classes characterized by distinct roles in DNA replication and transformation. Class 1 consisted of amino acids in the two minor loops of the finger and in the amino-terminal part of the major loop. Mutations here did not affect either replication or transformation. Class 2 consisted of the SerHisTyrLysTyr amino acids located in the carboxy terminus of the major loop of the finger. Mutations in this contiguous region reduced replication of the mutant viruses to different degrees. This clustering suggested that the region is an active site important for a specific function in DNA replication. With the exception of a mutation in the histidine at position 313, these mutations had no effect on transformation. Class 3 consisted of the proposed zinc-binding amino acids at positions 302, 305, 317, and 320 and the histidine at position 313 in the major loop of the finger. Mutations in these amino acids abolished the viability of the virus completely and had a distinctive effect on the transforming functions of the protein. Thus, the five cysteines and histidines of class 3 may play an important role in determining the overall structure of the protein. The histidine at position 313 may function both in the active site where it is located and in cooperation with the proposed zinc-binding ligands.     

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.     

Rat embryo fibroblasts immortalized with simian virus 40 large T antigen undergo senescence upon its inactivation.

Introduction of simian virus 40 T antigen into rodent fibroblasts gives rise to cells that can proliferate indefinitely but are dependent upon it for maintenance of their growth once the normal mitotic life span has elapsed. Inactivation of T antigen in these immortalized cells causes rapid and irreversible cessation of growth. To determine whether this growth arrest is associated with entry into senescence, we have undertaken a genetic and biological analysis of conditionally immortal (tsa) cell lines derived by immortalizing rat embryo fibroblasts with the thermolabile tsA58 T antigen. This analysis has identified the following parallels between the tsa cells after inactivation of T antigen and senescent rat embryo fibroblasts: (i) growth arrest is irreversible; (ii) it occurs in G1 as well as G2; (iii) the G1 block can be partially overcome by stimulation with 20% fetal calf serum, but the G2 block cannot be overcome; (iv) 20% fetal calf serum induces c-fos, but c-myc is unaltered; and (v) fibronectin and p21(Waf1/Cip1/Sdi1) are upregulated upon growth arrest. These results suggest that T-antigen-immortalized fibroblasts are committed to undergo senescence but are prevented from undergoing this process by T antigen. Inactivation of T antigen removes this block and results in senescence of the cells. Thus, these cell lines may represent a powerful system for study of the molecular basis of entry into senescence.     

Biochemical characterization of phosphorylation site mutants of simian virus 40 large T antigen: evidence for interaction between amino- and carboxy-terminal domains.

The simian virus 40 large T antigen is phosphorylated at eight or more sites that are clustered in an amino-terminal region and a carboxy-terminal region of the protein. Mutants carrying exchanges at these phosphorylation sites have been generated in vitro by bisulfite or oligonucleotide-directed mutagenesis and analyzed for their phosphorylation patterns. Two-dimensional phosphopeptide analyses of the mutant large T antigens confirmed most of the previously identified phosphorylation sites, namely, serine residues 106, 112, 123, 639, 677, and 679 and threonine residues 124 and 701. In addition, serine residue 120 was identified as a new site, whereas serines residues 111 and 676 were excluded. Interestingly, several of the mutants exhibited secondary effects in that a mutation in the amino-terminal region affected phosphorylation at distant and even carboxy-terminal sites and vice versa. Thus, the amino- and carboxy-terminal domains appear to be in close proximity in the three-dimensional structure of large T antigen. The possible consequences of the above findings and the role of phosphorylation are discussed.     

Homologous cellular proteins associated with simian virus 40 small T antigen and polyomavirus medium T antigen.

The simian virus 40 small T-associated 56,000-Mr (56K) and 32K cellular proteins were shown to be closely related to the polyomavirus medium T-associated 61K and 37K cellular proteins as demonstrated by two-dimensional polyacrylamide gel electrophoresis and V8 protease peptide mapping.     

Use of simian virus 40 large T-beta-galactosidase fusion proteins in an immunochemical analysis of simian virus 40 large T antigen.

Simian virus 40 large T antigen is a multifunctional protein that is encoded by the early region of the viral genome. We constructed fusion proteins between simian virus 40 large T antigen and beta-galactosidase by cloning HindIII fragments A and D of the virus into the HindIII sites of expression vectors pUR290, pUR291, and pUR292. Large amounts of the fusion protein were synthesized when the DNA fragment encoding part of simian virus 40 large T antigen was in frame with the lacZ gene of the expression vector. Using Western blotting and a competition radioimmunoassay, we assessed the binding of existing anti-T monoclonal and polyclonal antibodies to the two fusion proteins. Several monoclonal antibodies reacted with the protein encoded by the fragment A construction, but none reacted with the protein encoded by the fragment D construction. However, mice immunized with pure beta-galactosidase-HindIII fragment D fusion protein produced good levels of anti-T antibodies, which immunoprecipitated simian virus 40 large T antigen from lytically infected cells, enabling derivation of monoclonal antibodies to this region of large T antigen. Therefore, the fusion proteins allowed novel epitopes to be discovered on large T antigen and permitted the precise localization of epitopes recognized by existing antibodies. The same approach can also be used to produce antibodies against defined regions of any gene.     

Phosphorylation downregulates the DNA-binding activity of simian virus 40 T antigen.

Proteolytic fragments of simian virus 40 tumor (T) antigen and T antigen that was dephosphorylated with alkaline phosphatase bound between 1.5 to 2 times more origin-containing simian virus 40 DNA than did intact T antigen in DNA saturation experiments. Kinetic experiments showed that these treatments also enhanced the rate at which T antigen bound to the DNA. The enhanced binding of T-antigen fragments correlated with the generation of DNA-binding fragments that lacked the NH2-terminal region. Dephosphorylation of T antigen in vitro resulted in the removal of phosphate groups from the NH2-terminal region as well as from the COOH-terminal region. To test the effects of dephosphorylation on the size of the protein, immunoaffinity-purified T antigen was subjected to sedimentation with and without prior treatment with alkaline phosphatase. Most of the purified protein sedimented as a monomer and no significant effect was observed after dephosphorylation, indicating that the enhanced DNA-binding activity was probably not due to the uncovering of additional binding sites buried specifically in oligomerized T antigen. Taken together, these results indicate that in vivo phosphorylation of the NH2-terminal region (residues 106 to 124) decreases the binding of the protein to the DNA origin. The effect is reversed by in vitro dephosphorylation or by proteolysis which removes the highly phosphorylated NH2-terminal arm of the polypeptide. We suggest that phosphorylation inactivates one of two distinct DNA-binding activities on the polypeptide chain perhaps corresponding to two separate regions in T antigen.     

The distribution of large T antigen in simian virus 40 nucleoprotein complexes

Simian virus 40 large T antigen binds to two types of nucleoprotein complexes from lytically infected cells: those containing replicating virus DNA (100S complexes) and those containing nonreplicating virus DNA (70S complexes). Analysis by agarose gel electrophoresis showed that replicating DNA was found exclusively in 100S complexes, although these complexes also contained large amounts of form I and form II DNA. In contrast, no replicating DNA was found in 70S complexes, and pulse-labeled DNA in these complexes migrated as form I and form II DNA that presumably had recently completed replication. Immunoprecipitation and gel electrophoresis showed that large T antigen was associated with both types of complexes. From 21 to 62% of replicating DNA in 100S complexes was bound to T antigen. Our estimates indicated, however, that more than three-fourths of the DNA molecules in 100S complexes were nonreplicating and unassociated with T antigen. In 70S complexes, 12 to 31% of pulse-labeled DNA was bound to T antigen, but because there were more DNA molecules in the 70S complexes, they contained a greater absolute amount of T antigen.     

Differential induction of structural changes in the simian virus 40 origin of replication by T antigen.

The ATP-dependent binding of the simian virus 40 (SV40) large tumor antigen (T antigen) to the SV40 origin of replication (ori) results in the structural distortion of two critical elements within flanking regions of ori and the untwisting of the DNA helix. We examined the effect of changes in temperature, ATP concentration, and other reaction parameters on the generation of these DNA structural changes. We found that induction of the two localized structural transitions were highly and differentially sensitive to reaction conditions. Significant distortion of the early palindrome element, shown previously to result from DNA melting, required low levels of ATP (10 to 30 microM) but temperatures above 25 degrees C. Distortion of the AT tract occurred at low temperatures (5 degrees C) but required relatively high concentrations of ATP (greater than 300 microM). Thus, T antigen can induce structural changes within one critical element of ori without generating significant structural distortion within the second element. The response of ori untwisting to reaction conditions generally increased in parallel with or fell intermediate between the inductions of localized structural transitions. We suggest that ori untwisting and localized structural distortions are interdependent consequences of T-antigen binding to ori. These results suggest a model for the structural events occurring during the initial steps of SV40 DNA replication.