Analysis of protein determinants of host-specific infection properties of polyomaviruses using machine learning.

BACKGROUND: The large tumor antigen (LT-Ag) and major capsid protein VP1 are known to play important roles in determining the host-specific infection properties of polyomaviruses (PyVs). OBJECTIVE: The objective of this study was to investigate the physicochemical properties of amino acids of LT-Ag and VP1 that have important effects on host specificity, as well as classification techniques used to predict PyV hosts. METHODS: We collected and used reference sequences of 86 viral species for analysis. Based on the clustering pattern of the reconstructed phylogenetic tree, the dataset was divided into three groups: mammalian, avian, and fish. We then used random forest (RF), naïve Bayes (NB), and k-nearest neighbors (kNN) algorithms for host classification. RESULTS: Among the three algorithms, classification accuracy using kNN was highest in both LT-Ag (ACC = 98.83) and VP1 (ACC = 96.51). The amino acid physicochemical property most strongly correlated with host classification was charge, followed by solvent accessibility, polarity, and hydrophobicity in LT-Ag. However, in VP1, amino acid composition showed the highest correlation with host classification, followed by charge, normalized van der Waals volume, and solvent accessibility. CONCLUSIONS: The results of the present study suggest the possibility of determining or predicting the host range and infection properties of PyVs at the molecular level by identifying the host species of active and emerging PyVs that exhibit different infection properties among diverse host species. Structural and biochemical differences of LT-Ag and VP1 proteins in host species that reflect these amino acid properties can be considered primary factors that determine the host specificity of PyV.

Photocleavable Surfactant-Enabled Extracellular Matrix Proteomics.

The extracellular matrix (ECM) provides an architectural meshwork that surrounds and supports cells. The dysregulation of heavily post-translationally modified ECM proteins directly contributes to various diseases. Mass spectrometry (MS)-based proteomics is an ideal tool to identify ECM proteins and characterize their post-translational modifications, but ECM proteomics remains challenging owing to the extremely low solubility of the ECM. Herein, enabled by effective solubilization of ECM proteins using our recently developed photocleavable surfactant, Azo, we have developed a streamlined ECM proteomic strategy that allows fast tissue decellularization, efficient extraction and enrichment of ECM proteins, and rapid digestion prior to reversed-phase liquid chromatography (RPLC)-MS analysis. A total of 173 and 225 unique ECM proteins from mouse mammary tumors have been identified using 1D and 2D RPLC-MS/MS, respectively. Moreover, 87 (from 1DLC-MS/MS) and 229 (from 2DLC-MS/MS) post-translational modifications of ECM proteins, including glycosylation, phosphorylation, and hydroxylation, were identified and localized. This Azo-enabled ECM proteomics strategy will streamline the analysis of ECM proteins and promote the study of ECM biology.

Direct single-molecule observations of DNA unwinding by SV40 large tumor antigen under a negative DNA supercoil state.

Superhelices, which are induced by the twisting and coiling of double-helical DNA in chromosomes, are thought to affect transcription, replication, and other DNA metabolic processes. In this study, we report the effects of negative supercoiling on the unwinding activity of simian virus 40 large tumor antigen (SV40 TAg) at a single-molecular level. The supercoiling density of linear DNA templates was controlled using magnetic tweezers and monitored using a fluorescent microscope in a flow cell. SV40 TAg-mediated DNA unwinding under relaxed and negative supercoil states was analyzed by the direct observation of both single- and double-stranded regions of single DNA molecules. Increased negative superhelicity stimulated SV40 TAg-mediated DNA unwinding more strongly than a relaxed state; furthermore, negative superhelicity was associated with an increased probability of SV40 TAg-mediated DNA unwinding. These results suggest that negative superhelicity helps to regulate the initiation of DNA replication.

Transformation-induced changes in the DNA-nuclear matrix interface, revealed by high-throughput analysis of DNA halos.

In higher eukaryotic nuclei, DNA is periodically anchored to an extraction-resistant protein structure, via matrix attachment regions. We describe a refined and accessible method to non-subjectively, rapidly and reproducibly measure both size and stability of the intervening chromatin loops, and use it to demonstrate that malignant transformation compromises the DNA-nuclear matrix interface.

The structure of SV40 large T hexameric helicase in complex with AT-rich origin DNA.

DNA replication is a fundamental biological process. The initial step in eukaryotic DNA replication is the assembly of the pre-initiation complex, including the formation of two head-to-head hexameric helicases around the replication origin. How these hexameric helicases interact with their origin dsDNA remains unknown. Here, we report the co-crystal structure of the SV40 Large-T Antigen (LT) hexameric helicase bound to its origin dsDNA. The structure shows that the six subunits form a near-planar ring that interacts with the origin, so that each subunit makes unique contacts with the DNA. The origin dsDNA inside the narrower AAA+ domain channel shows partial melting due to the compression of the two phosphate backbones, forcing Watson-Crick base-pairs within the duplex to flip outward. This structure provides the first snapshot of a hexameric helicase binding to origin dsDNA, and suggests a possible mechanism of origin melting by LT during SV40 replication in eukaryotic cells.

SV40 T-Antigen Amino Acid Changes that Disrupt Cul-7 or Bub-1 Binding Do Not Globally Distort the T-Common Region.

BACKGROUND: Amino acids 1-107 of the SV40 T antigen constitute a functionally important and complex region. Cellular proteins, Hsc70, Bub-1, Cul-7, and Rb, each of which is involved in cell growth control or genomic stability, bind within this portion of the T antigen. Mutational analysis has mapped the J domain/Hsc70, Bub-1, and the Rb binding motifs. Two regions of the T antigen have been implicated in Cul-7 binding. Mutation of F98A diminished Cul-7 binding, and deletion of amino acids 68-83 abolished it. The authors suggest, based on T-antigen structure, that F98 is inaccessible and that the F98A change altered the configuration of the upstream region, preventing Cul-7 binding. Our objective was to determine, by using monoclonal T-antigen antibodies, whether F98 is accessible and whether F98A substitution globally distorted the T-common region. METHODS: Cell-expressing T antigens, immunoprecipitation, and immunoblot were used to determine the accessibility of amino acids. CONCLUSION: Full-length T-antigen and N-terminal fragments containing F98A were immunoprecipitated by monoclonal antibody PAb902, which recognizes a conformation-dependent epitope within the first 82 amino acids. Therefore, this alteration does not globally distort the entire T-common region. Additionally, PAb416, which displaces Cul-7 from the T antigen and immunoprecipitates bound pRb peptides, depends on F98 for binding, implying that amino acid 98 is part of the epitope and accessible in the native T antigen.

Export-deficient monoubiquitinated PEX5 triggers peroxisome removal in SV40 large T antigen-transformed mouse embryonic fibroblasts.

Peroxisomes are ubiquitous cell organelles essential for human health. To maintain a healthy cellular environment, dysfunctional and superfluous peroxisomes need to be selectively removed. Although emerging evidence suggests that peroxisomes are mainly degraded by pexophagy, little is known about the triggers and molecular mechanisms underlying this process in mammalian cells. In this study, we show that PEX5 proteins fused to a bulky C-terminal tag trigger peroxisome degradation in SV40 large T antigen-transformed mouse embryonic fibroblasts. In addition, we provide evidence that this process is autophagy-dependent and requires monoubiquitination of the N-terminal cysteine residue that marks PEX5 for recycling. As our findings also demonstrate that the addition of a bulky tag to the C terminus of PEX5 does not interfere with PEX5 monoubiquitination but strongly inhibits its export from the peroxisomal membrane, we hypothesize that such a tag mimics a cargo protein that cannot be released from PEX5, thus keeping monoubiquitinated PEX5 at the membrane for a sufficiently long time to be recognized by the autophagic machinery. This in turn suggests that monoubiquitination of the N-terminal cysteine of peroxisome-associated PEX5 not only functions to recycle the peroxin back to the cytosol, but also serves as a quality control mechanism to eliminate peroxisomes with a defective protein import machinery.

Mechanism of subunit coordination of an AAA+ hexameric molecular nanomachine.

Simian virus 40 large tumor antigen (LT) is both a potent oncogenic protein and an efficient hexameric nanomachine that harnesses the energy from ATP binding/hydrolysis to melt origin DNA and unwind replication forks. However, how the six subunits of the helicase motor coordinate during ATP hydrolysis and DNA unwinding/translocation is unresolved. Here we investigated the subunit coordination mechanisms "binomial distribution mutant doping" experiments in the presence of various DNA substrates. For ATP hydrolysis, we observed multiple coordination modes, ranging from random and semi-random, and semi-coordinated modes, depending on which type of DNA is present. For DNA unwinding, however, the results indicated a fully-coordinated mode for the natural origin-containing duplex DNA, but a semi-coordinated mode for a pre-existing fork-DNA, providing direct evidence for LT to use potentially different mechanisms to unwind the two types of substrates. The results of this study provide insights into DNA translocation and unwinding mechanisms for LT hexameric biomotor. From the clinical editor: The study describes the subunit coordination of simian virus 40 large tumor antigen (LT) showing that multiple mechanisms exist that handle the specific needs of different stages of DNA replication.

New quantitative approaches reveal the spatial preference of nuclear compartments in mammalian fibroblasts.

The nuclei of higher eukaryotic cells display compartmentalization and certain nuclear compartments have been shown to follow a degree of spatial organization. To date, the study of nuclear organization has often involved simple quantitative procedures that struggle with both the irregularity of the nuclear boundary and the problem of handling replicate images. Such studies typically focus on inter-object distance, rather than spatial location within the nucleus. The concern of this paper is the spatial preference of nuclear compartments, for which we have developed statistical tools to quantitatively study and explore nuclear organization. These tools combine replicate images to generate 'aggregate maps' which represent the spatial preferences of nuclear compartments. We present two examples of different compartments in mammalian fibroblasts (WI-38 and MRC-5) that demonstrate new knowledge of spatial preference within the cell nucleus. Specifically, the spatial preference of RNA polymerase II is preserved across normal and immortalized cells, whereas PML nuclear bodies exhibit a change in spatial preference from avoiding the centre in normal cells to exhibiting a preference for the centre in immortalized cells. In addition, we show that SC35 splicing speckles are excluded from the nuclear boundary and localize throughout the nucleoplasm and in the interchromatin space in non-transformed WI-38 cells. This new methodology is thus able to reveal the effect of large-scale perturbation on spatial architecture and preferences that would not be obvious from single cell imaging.

Polyomavirus nephropathy: quantitative urinary polyomavirus-Haufen testing accurately predicts the degree of intrarenal viral disease.

BACKGROUND: A qualitative highly predictive urinary test for polyomavirus nephropathy (PVN) is the PV-Haufen test. This article evaluates whether a quantitative PV-Haufen analysis, that is, the number of PV-Haufen shed per milliliter urine, predicts PVN disease grades and the severity of intrarenal PV replication. METHODS: Polyomavirus-Haufen were counted in 40 urine samples from patients with biopsy-proven definitive PVN. The number of PV-Haufen was correlated with both histologic PVN disease grades 1 to 3 and the number of SV40-T-expressing cells as indicators of intrarenal PV replication in corresponding renal allograft biopsies (manual counts and automated morphometry). Findings from quantitative PV-Haufen analyses were compared to conventional laboratory test results, that is, BK viremia (quantitative polymerase chain reaction [PCR]) and BK viruria (quantitative PCR and decoy cell counts). RESULTS: Polyomavirus-Haufen counts showed excellent correlation (α0.77-0.86) with the severity of intrarenal PV replication and disease grades. In particular, low PV-Haufen numbers strongly correlated with early PVN grade 1 and minimal intrarenal expression of SV40-T antigen (P < 0.001). In comparison, BK viremia and viruria levels by PCR showed only modest correlations with histologic SV40-T expression (α0.40-0.49) and no significant correlation with disease grades or minimal intrarenal PV replication. No correlations were seen with urinary decoy cell counts. In contrast to conventional quantitative PCR assays or decoy cell counts, quantitative urinary PV-Haufen testing accurately reflects the severity of PV replication, tissue injury, and PVN disease grades. CONCLUSIONS: Quantitative PV-Haufen testing is a novel noninvasive approach to patient management for the diagnosis and prediction of PVN disease grades and monitoring of disease course during therapy.

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.

Two independent regions of simian virus 40 T antigen increase CBP/p300 levels, alter patterns of cellular histone acetylation, and immortalize primary cells.

Simian virus 40 (SV40) large T antigen (SVT) interferes with normal cell regulation and thus has been used to identify cellular components controlling proliferation and homeostasis. We have previously shown that SVT-mediated transformation requires interaction with the histone acetyltransferases (HATs) CBP/p300 and now report that the ectopic expression of SVT in several cell types in vivo and in vitro results in a significant increase in the steady-state levels of CBP/p300. Furthermore, SVT-expressing cells contain higher levels of acetylated CBP/p300, a modification that has been linked to increased HAT activity. Concomitantly, the acetylation levels of histone residues H3K56 and H4K12 are markedly increased in SVT-expressing cells. Other polyomavirus-encoded large T antigens also increase the levels of CBP/p300 and sustain a rise in the acetylation levels of H3K56 and H4K12. SVT does not affect the transcription of CBP/p300, but rather, alters their overall levels through increasing the loading of CBP/p300 mRNAs onto polysomes. Two distinct regions within SVT, one located in the amino terminus and one in the carboxy terminus, can independently alter both the levels of CBP/p300 and the loading of CBP/p300 transcripts onto polysomes. Within the amino-terminal fragment, a functional J domain is necessary for increasing CBP/p300 and specific histone acetylation levels, as well as for immortalizing primary cells. These studies uncover the action of polyomavirus T antigens on cellular CBP/p300 and suggest that additional mechanisms are used by T antigens to induce cell immortalization and transformation.

Merkel cell polyomavirus large T antigen has growth-promoting and inhibitory activities.

Merkel cell carcinoma (MCC) is a rare and aggressive form of skin cancer. In at least 80% of all MCC, Merkel cell polyomavirus (MCPyV) DNA has undergone clonal integration into the host cell genome, and most tumors express the MCPyV large and small T antigens. In all cases of MCC reported to date, the integrated MCPyV genome has undergone mutations in the large T antigen. These mutations result in expression of a truncated large T antigen that retains the Rb binding or LXCXE motif but deletes the DNA binding and helicase domains. However, the transforming functions of full-length and truncated MCPyV large T antigen are unknown. We compared the transforming activities of full-length, truncated, and alternatively spliced 57kT forms of MCPyV large T antigen. MCPyV large T antigen could bind to Rb but was unable to bind to p53. Furthermore, MCPyV-truncated large T antigen was more effective than full-length and 57kT large T antigen in promoting the growth of human and mouse fibroblasts. In contrast, expression of the MCPyV large T antigen C-terminal 100 residues could inhibit the growth of several different cell types. These data imply that the deletion of the C terminus of MCPyV large T antigen found in MCC serves not only to disrupt viral replication but also results in the loss of a distinct growth-inhibitory function intrinsic to this region.

Analysis of the costructure of the simian virus 40 T-antigen origin binding domain with site I reveals a correlation between GAGGC spacing and spiral assembly.

Polyomavirus origins of replication contain multiple occurrences of G(A/G)GGC, the high-affinity binding element for the viral initiator T-antigen (T-ag). The site I regulatory region of simian virus 40, involved in the repression of transcription and the enhancement of DNA replication initiation, contains two GAGGC sequences arranged head to tail and separated by a 7-bp AT-rich sequence. We have solved a 3.2-Å costructure of the SV40 origin-binding domain (OBD) bound to site I. We have also established that T-ag assembly on site I is limited to the formation of a single hexamer. These observations have enabled an analysis of the role(s) of the OBDs bound to the site I pentanucleotides in hexamer formation. Of interest, they reveal a correlation between the OBDs bound to site I and a pair of OBD subunits in the previously described hexameric spiral structure. Based on these findings, we propose that spiral assembly is promoted by pentanucleotide pairs arranged in a head-to-tail manner. Finally, the possibility that spiral assembly by OBD subunits accounts for the heterogeneous distribution of pentanucleotides found in the origins of replication of polyomaviruses is discussed.

Circumventing cellular control of PP2A by methylation promotes transformation in an Akt-dependent manner.

Heterotrimeric protein phosphatase 2A (PP2A) consists of catalytic C (PP2Ac), structural A, and regulatory B-type subunits, and its dysfunction has been linked to cancer. Reversible methylation of PP2Ac by leucine carboxyl methyltransferase 1 (LCMT-1) and protein phosphatase methylesterase 1 (PME-1) differentially regulates B-type subunit binding and thus PP2A function. Polyomavirus middle (PyMT) and small (PyST) tumor antigens and SV40 small tumor antigen (SVST) are oncoproteins that block PP2A function by replacing certain B-type subunits, resulting in cellular transformation. Whereas the B-type subunits replaced by these oncoproteins seem to exhibit a binding preference for methylated PP2Ac, PyMT does not. We hypothesize that circumventing the normal cellular control of PP2A by PP2Ac methylation is a general strategy for ST- and MT-mediated transformation. Two predictions of this hypothesis are (1) that PyST and SVST also bind PP2A in a methylation-insensitive manner and (2) that down-regulation of PP2Ac methylation will activate progrowth and prosurvival signaling and promote transformation. We found that SVST and PyST, like PyMT, indeed form PP2A heterotrimers independently of PP2Ac methylation. In addition, reducing PP2Ac methylation through LCMT-1 knockdown or PME-1 overexpression enhanced transformation by activating the Akt and p70/p85 S6 kinase (S6K) pathways, pathways also activated by MT and ST oncoproteins. These results support the hypothesis that MT and ST oncoproteins circumvent cellular control of PP2A by methylation to promote transformation. They also implicate LCMT-1 as a negative regulator of Akt and p70/p85 S6K. Therefore, disruption of PP2Ac methylation may contribute to cancer, and modulation of this methylation may serve as an anticancer target.

C-terminal deletions of Merkel cell polyomavirus large T-antigen, a highly specific surrogate marker for virally induced malignancy.

In 67-100% of cutaneous Merkel cell carcinomas (MCC) the Merkel cell polyomavirus (MCPyV) integrates into the host genome. Mutations and deletions truncating the C-terminal helicase domain of the T-antigen (TAg) protein have been detected in these MCCs, but not in healthy skin specimens. C-terminal deletions of the TAg nucleic acid sequences are characteristic for about 38% of these cases. While the association of MCPyV with MCC has been proven, it is unknown whether MCPyV may play a similar role in other tumor entities. We describe in detail the development and validation of a novel Merkel cell polyomavirus TAg C-terminus deletion assay (MCPyV ΔC-TAg). The triplex real-time PCR quantifies the N- and C-terminal part of the MCPyV TAg gene and the cellular ß-globin gene. By comparing the copy numbers of the N- and C-terminal part, deletions of the MCPyV TAg C-terminus are rapidly identified. MCPyV ΔC-TAg was used to assess the physical state of MCPyV TAg in a large series of 55 MCCs, 15 cutaneous lymphomas and 47 forehead smears of healthy individuals. Neither DNA positivity nor viral load was able to discriminate MCCs from the other different types of samples. However, deleted TAg C-terminus sequences were detected only in MCPyV positive MCCs (39%). Consequently, detection of deleted C-terminal TAg sequences appears to be a highly specific surrogate marker for virally induced malignancy and should be used to support novel assumed MCPyV-tumor associations. The study further supports the notion that MCPyV does not play a role in cutaneous lymphoma pathogenesis.

Binding to retinoblastoma pocket domain does not alter the inter-domain flexibility of the J domain of SV40 large T antigen.

Simian Virus 40 uses the large T antigen (Tag) to bind and inactivate retinoblastoma tumor suppressor proteins (Rb), which can result in cellular transformation. Tag is a modular protein with four domains connected by flexible linkers. The N-terminal J domain of Tag is necessary for Rb inactivation. Binding of Rb is mediated by an LXCXE consensus motif immediately C-terminal to the J domain. Nuclear magnetic resonance (NMR) and small angle X-ray scattering (SAXS) were used to study the structural dynamics and interaction of Rb with the LXCXE motif, the J domain and a construct (N(260)) extending from the J domain through the origin binding domain (OBD). NMR and SAXS data revealed substantial flexibility between the domains in N(260). Binding of pRb to a construct containing the LXCXE motif and the J domain revealed weak interactions between pRb and the J domain. Analysis of the complex of pRb and N(260) indicated that the OBD is not involved and retains its dynamic independence from the remainder of Tag. These results support a 'chaperone' model in which the J domain of Tag changes its orientation as it acts upon different protein complexes.

Cellular and viral factors regulating Merkel cell polyomavirus replication.

Merkel cell polyomavirus (MCV), a previously unrecognized component of the human viral skin flora, was discovered as a mutated and clonally-integrated virus inserted into Merkel cell carcinoma (MCC) genomes. We reconstructed a replicating MCV clone (MCV-HF), and then mutated viral sites required for replication or interaction with cellular proteins to examine replication efficiency and viral gene expression. Three days after MCV-HF transfection into 293 cells, although replication is not robust, encapsidated viral DNA and protein can be readily isolated by density gradient centrifugation and typical ∼40 nm diameter polyomavirus virions are identified by electron microscopy. The virus has an orderly gene expression cascade during replication in which large T (LT) and 57kT proteins are first expressed by day 2, followed by expression of small T (sT) and VP1 proteins. VP1 and sT proteins are not detected, and spliced 57kT is markedly diminished, in the replication-defective virus suggesting that early gene splicing and late gene transcription may be dependent on viral DNA replication. MCV replication and encapsidation is increased by overexpression of MCV sT, consistent with sT being a limiting factor during virus replication. Mutation of the MCV LT vacuolar sorting protein hVam6p (Vps39) binding site also enhances MCV replication while exogenous hVam6p overexpression reduces MCV virion production by >90%. Although MCV-HF generates encapsidated wild-type MCV virions, we did not find conditions for persistent transmission to recipient cell lines suggesting that MCV has a highly restricted tropism. These studies identify and highlight the role of polyomavirus DNA replication in viral gene expression and show that viral sT and cellular hVam6p are important factors regulating MCV replication. MCV-HF is a molecular clone that can be readily manipulated to investigate factors affecting MCV replication.

Merkel cell polyomavirus large T antigen disrupts lysosome clustering by translocating human Vam6p from the cytoplasm to the nucleus.

Merkel cell polyomavirus (MCV) has been recently described as the cause for most human Merkel cell carcinomas. MCV is similar to simian virus 40 (SV40) and encodes a nuclear large T (LT) oncoprotein that is usually mutated to eliminate viral replication among tumor-derived MCV. We identified the hVam6p cytoplasmic protein involved in lysosomal processing as a novel interactor with MCV LT but not SV40 LT. hVam6p binds through its clathrin heavy chain homology domain to a unique region of MCV LT adjacent to the retinoblastoma binding site. MCV LT translocates hVam6p to the nucleus, sequestering it from involvement in lysosomal trafficking. A naturally occurring, tumor-derived mutant LT (MCV350) lacking a nuclear localization signal binds hVam6p but fails to inhibit hVam6p-induced lysosomal clustering. MCV has evolved a novel mechanism to target hVam6p that may contribute to viral uncoating or egress through lysosomal processing during virus replication.

Asymmetric assembly of Merkel cell polyomavirus large T-antigen origin binding domains at the viral origin.

The double-stranded DNA polyomavirus Merkel cell polyomavirus (MCV) causes Merkel cell carcinoma, an aggressive but rare human skin cancer that most often affects immunosuppressed and elderly persons. As in other polyomaviruses, the large T-antigen of MCV recognizes the viral origin of replication by binding repeating G(A/G)GGC pentamers. The spacing, number, orientation, and necessity of repeats for viral replication differ, however, from other family members such as SV40 and murine polyomavirus. We report here the 2.9 Å crystal structure of the MCV large T-antigen origin binding domain (OBD) in complex with a DNA fragment from the MCV origin of replication. Consistent with replication data showing that three of the G(A/G)GGC-like binding sites near the center of the origin are required for replication, the crystal structure contains three copies of the OBD. This stoichiometry was verified using isothermal titration calorimetry. The affinity for G(A/G)GGC-containing double-stranded DNA was found to be ~740 nM, approximately 8-fold weaker than the equivalent domain in SV40 for the analogous region of the SV40 origin. The difference in affinity is partially attributable to DNA-binding residue Lys331 (Arg154 in SV40). In contrast to SV40, a small protein-protein interface is observed between MCV OBDs when bound to the central region of the origin. This protein-protein interface is reminiscent of that seen in bovine papilloma virus E1 protein. Mutational analysis indicates, however, that this interface contributes little to DNA binding energy.