Elements of the polyomavirus replication origin required for homologous recombination mediated by large T antigen.

We introduced various elements of the polyomavirus origin of DNA replication into the genome of rat cells, and we analyzed their capacity to elicit rearrangements within the integrated sequences when exposed to large T antigen. The cis-acting sequences required for homologous recombination were those that make up a functional replication origin.     

Polyomavirus origin for DNA replication comprises multiple genetic elements.

To define the minimal cis-acting sequences required for polyomavirus DNA replication (ori), we constructed a number of polyomavirus-plasmid recombinants and measured their replicative capacity after transfection of a permissive mouse cell line capable of providing polyomavirus large T antigen in trans (MOP cells). Recombinant plasmids containing a 251-base-pair fragment of noncoding viral DNA replicate efficiently in MOP cells. Mutational analyses of these viral sequences revealed that they can be physically separated into two genetic elements. One of these elements, termed the core, contains an adenine-thymine-rich area, a 32-base-pair guanine-cytosine-rich palindrome, and a large T antigen binding site, and likely includes the site from which bidirectional DNA replication initiates. The other, termed beta, is located adjacent to the core near the late region and is devoid of outstanding sequence features. Surprisingly, another sequence element named alpha, located adjacent to beta but outside the borders of the 251-base-pair fragment, can functionally substitute for beta. This sequence too contains no readily recognized sequence features and possesses no obvious homology to the beta element. The three elements together occupy a contiguous noncoding stretch of DNA no more than 345 base pairs in length in the order alpha, beta, and core. These results indicate that the polyomavirus origin for DNA replication comprises multiple genetic elements.     

Requirements for species-specific papovavirus DNA replication.

Replication of papovavirus DNA requires a functional replication origin, a virus-encoded protein, large T antigen, and species-specific permissive factors. How these components interact to initiate and sustain viral DNA replication is not known. Toward that end, we have attempted to identify the viral target(s) of permissive factors. The functionally defined replication origins of polyomavirus and simian virus 40, two papovaviruses that replicate in different species (mice and monkeys, respectively), are composed of two functionally distinct domains: a core domain and an auxiliary domain. The origin cores of the two viruses are remarkably similar in primary structure and have common binding sites for large T antigen. By contrast, their auxiliary domains share few sequences and serve as binding sites for cellular proteins. It seemed plausible, therefore, that if cellular permissive factors interacted with the replication origin, their targets were likely to be in the auxiliary domain. To test this hypothesis we constructed hybrid origins for DNA replication that were composed of the auxiliary domain of one virus and the origin core of the other and assessed their capacity to replicate in a number of mouse and monkey cell lines, which express the large T antigen of one or the other virus. The results of this analysis showed that the auxiliary domains of the viral replication origins could substitute for one another in DNA replication, provided that the viral origin core and its cognate large T antigen were present in a permissive cellular milieu. Surprisingly, the large T antigens of the viruses could not substitute for one another, regardless of the species of origin of the host cell, even though the two large T antigens bind to the same sequence motif in vitro. These results suggest that species-specific permissive factors do not interact with the origin-auxiliary domains but, rather, with either the origin core or the large T antigen or with both components to effect DNA replication.     

Initiation of JC virus DNA replication in vitro by human and mouse DNA polymerase alpha-primase.

Host species specificity of the polyomaviruses simian virus 40 (SV40) and mouse polyomavirus (PyV) has been shown to be determined by the host DNA polymerase alpha-primase complex involved in the initiation of both viral and host DNA replication. Here we demonstrate that DNA replication of the related human pathogenic polyomavirus JC virus (JCV) can be supported in vitro by DNA polymerase alpha-primase of either human or murine origin indicating that the mechanism of its strict species specificity differs from that of SV40 and PyV. Our results indicate that this may be due to differences in the interaction of JCV and SV40 large T antigens with the DNA replication initiation complex.     

Asp-286----Asn-286 in polyomavirus large T antigen relaxes the specificity of binding to the polyomavirus origin.

We isolated revertants of a polyomavirus whose origin of DNA replication contains a point mutation in the palindrome to which large T antigen binds. Four independent second-site revertants contain an Asp-286----Asn-286 substitution in large T antigen. This mutant large T antigen activates replication of DNAs containing the mutant polyomavirus origin as well as replication of DNAs containing the wild-type origin; however, replication of DNAs with enhancer mutations is not activated by this large T antigen. The Asn-286 mutation occurs in a positively charge region of large T antigen near the location of several mutations which inactivate DNA replication. We suggest that this region of large T antigen is responsible for recognition of specific DNA sequences at the origin and that ionic forces are important for this interaction.     

The adenine-thymine domain of the simian virus 40 core origin directs DNA bending and coordinately regulates DNA replication.

The simian virus 40 origin of replication contains a 20-base-pair adenine-thymine-rich segment with the sequence 5'-TGCATAAATAAAAAAAATTA-3'. The continuous tract of eight adenines is highly conserved among polyomaviruses. We used single-base substitutions to map structural and functional features of this DNA. Mutations in the AAA and AAAAAAAATT sequences significantly reduce DNA replication and thus identify two sequence-specific functional domains or a single domain with two parts. The AAAAAAAATT sequence also determines a DNA conformation that is characteristic of DNA bending. Single-base mutations in this domain change the degree of net bending, presumably by altering the length and location of the bending sequence. Thus, DNA bending in the correct conformation and location may be a structural signal for replication in polyomavirus origins and perhaps in other origins of replication with consecutive runs of adenines. The first five base pairs (TGCAT) of the 20-base-pair segment and the T between the AAA and AAAAAAAATT domains serve a sequence-independent function that may establish proper spacing within the core origin.     

Polyomavirus Large T Antigen Binds Cooperatively to Its Multiple Binding Sites in the Viral Origin of DNA Replication

Polyomavirus large T antigen binds to multiple 5′-G(A/G)GGC-3′ pentanucleotide sequences in sites 1/2, A, B, and C within and adjacent to the origin of viral DNA replication on the polyomavirus genome. We asked whether the binding of large T antigen to one of these sites could influence binding to other sites. We discovered that binding to origin DNA is substantially stronger at pH 6 to 7 than at pH 7.4 to 7.8, a range often used in DNA binding assays. Large T antigen-DNA complexes formed at pH 6 to 7 were stable, but a fraction of these complexes dissociated at pH 7.6 and above upon dilution or during electrophoresis. Increased binding at low pH is therefore due at least in part to increased stability of protein-DNA complexes, and binding at higher pH values is reversible. Binding to fragments of origin DNA in which one or more sites were deleted or inactivated by point mutations was measured by nitrocellulose filter binding and DNase I footprinting. The results showed that large T antigen binds cooperatively to its four binding sites in viral DNA, suggesting that the binding of this protein to one of these sites stabilizes its binding to other sites via protein-protein contacts. Sites A, B, and C may therefore augment DNA replication by facilitating the binding of large T antigen to site 1/2 at the replication origin. ATP stabilized large T antigen-DNA complexes against dissociation in the presence, but not the absence, of site 1/2, and ATP specifically enhanced protection against DNase I digestion in the central 10 to 12 bp of site 1/2, at which hexamers are believed to form and begin unwinding DNA. We propose that large T antigen molecules bound to these multiple sites on origin DNA interact with each other to form a compact protein-DNA complex and, furthermore, that ATP stimulates their assembly into hexamers at site 1/2 by a “handover” mechanism mediated by these protein-protein contacts.     

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.     

Mechanisms of simian virus 40 T-antigen activation by phosphorylation of threonine 124.

Previous studies have shown that phosphorylation of simian virus 40 (SV40) T antigen at threonine 124 enhances the binding of T antigen to the SV40 core origin of replication and the unwinding of the core origin DNA via hexamer-hexamer interactions. Here, we report that threonine 124 phosphorylation enhances the interaction of T-antigen amino acids 1 to 259 and 89 to 259 with the core origin of replication. Phosphorylation, therefore, activates the minimal DNA binding domain of T antigen even in the absence of domains required for hexamer formation. Activation is mediated by only one of three DNA binding elements in the minimal DNA binding domain of T antigen. This element, including amino acids 167, 215, and 219, enhances binding to the unique arrangement of four pentanucleotides in the core origin but not to other pentanucleotide arrangements found in ancillary regions of the SV40 origin of replication. Interestingly, the same four pentanucleotides in the core origin are necessary and sufficient for phosphorylation-enhanced DNA binding. Further, we show that phosphorylation of threonine 124 promotes the assembly of high-order complexes of the minimal DNA binding domain of T antigen with core origin DNA. We propose that phosphorylation induces conformational shifts in the minimal DNA binding domain of T antigen and thereby enhances interactions among T-antigen subunits oriented by core origin pentanucleotides. Similar subunit interactions would enhance both assembly of full-length T antigen into binary hexamer complexes and origin unwinding.     

The murine p53 protein blocks replication of SV40 DNA in vitro by inhibiting the initiation functions of SV40 large T antigen

We have characterized the effect of murine p53 on SV40 DNA replication in vitro. Purified wild-type murine p53 dramatically inhibited the ability of SV40 T antigen to mediate the replication of a plasmid bearing the viral origin (ori-DNA) in vitro. In contrast, polyoma ori-DNA replication in vitro was unaffected by p53. Surprisingly, both unbound p53 and SV40 T antigen-bound p53 were equally detrimental to SV40 ori-DNA replication. Thus, p53 interferes with interactions between T antigen molecules that are required for DNA synthesis. p53 inhibited the binding to and subsequent unwinding of the SV40 origin by T antigen and thus selectively blocked the initial stages of ori-DNA replication. In contrast to the nononcogenic wild-type murine p53, high concentrations of a mutant transforming p53 failed to block SV40 ori-DNA replication in vitro. These observations may provide insight into a possible role for p53 in the cell.     

Mechanism of activation of simian virus 40 DNA replication by protein phosphatase 2A.

The catalytic subunit of protein phosphatase 2A (PP2Ac) stimulates the initiation of replication of simian virus 40 DNA in vitro by dephosphorylating T antigen at specific phosphoserine residues (K. H. Scheidtmann, D. M. Virshup, and T. J. Kelly, J. Virol. 65:2098-2101, 1991). To better define the biochemical mechanism responsible for this stimulation, we investigated the effect of PP2Ac on the interaction of T antigen with wild-type and mutant origins of replication. Analysis of the binding of T antigen to the wild-type origin as a function of protein concentration revealed that binding occurs in two relatively discrete steps: the assembly of a T-antigen hexamer on one half-site of the origin, followed by the assembly of the second hexamer on the other half-site. The major effect of PP2Ac was to stimulate binding of the second hexamer, so that the binding reaction became much more cooperative. This observation suggests that dephosphorylation of T antigen by PP2Ac primarily affects interactions between the two hexamers bound to the origin. Pretreatment with PP2Ac increased the ability of the bound T antigen to unwind the origin of replication but had no effect on the intrinsic helicase activity of the protein. Thus, dephosphorylation of PP2Ac appears to increase the efficiency of the initial opening of the origin by T antigen. An insertion mutation at the dyad axis in the simian virus 40 origin, which altered the structural relationship of the two halves of the origin, abolished the effect of the phosphatase on the cooperativity of binding and completely prevented origin unwinding. These findings suggest that the ability of T antigen to open the viral origin of DNA replication is critically dependent on the appropriate functional interactions between T-antigen hexamers and that these interactions are regulated by the phosphorylation state of the viral initiator protein.     

Polyomavirus Large T Antigen Binds Symmetrical Repeats at the Viral Origin in an Asymmetrical Manner

Polyomaviruses have repeating sequences at their origins of replication that bind the origin-binding domain of virus-encoded large T antigen. In murine polyomavirus, the central region of the origin contains four copies (P1 to P4) of the sequence G(A/G)GGC. They are arranged as a pair of inverted repeats with a 2-bp overlap between the repeats at the center. In contrast to simian virus 40 (SV40), where the repeats are nonoverlapping and all four repeats can be simultaneously occupied, the crystal structure of the four central murine polyomavirus sequence repeats in complex with the polyomavirus origin-binding domain reveals that only three of the four repeats (P1, P2, and P4) are occupied. Isothermal titration calorimetry confirms that the stoichiometry is the same in solution as in the crystal structure. Consistent with these results, mutation of the third repeat has little effect on DNA replication in vivo. Thus, the apparent 2-fold symmetry within the DNA repeats is not carried over to the protein-DNA complex. Flanking sequences, such as the AT-rich region, are known to be important for DNA replication. When the orientation of the central region was reversed with respect to these flanking regions, the origin was still able to replicate and the P3 sequence (now located at the P2 position with respect to the flanking regions) was again dispensable. This highlights the critical importance of the precise sequence of the region containing the pentamers in replication.     

Immortalization of rat embryo fibroblasts by mutant polyomavirus large T antigens deficient in DNA binding.

We have identified a putative DNA-binding domain in polyomavirus large T antigen. Mutations introduced into the gene between amino acids 290 and 310 resulted in proteins that no longer bound to the high-affinity binding sites on the polyomavirus genome, showed no detectable nonspecific DNA binding, and were not able to initiate DNA replication from the viral origin. These mutant T antigen genes were introduced into rat embryo fibroblasts together with the neomycin resistance gene to allow selection for growth in the presence of G418. All the mutations tested facilitated the establishment of these cells in long-term culture at an efficiency indistinguishable from that of the wild-type protein.