Asymmetric Assembly of Merkel Cell Polyomavirus Large T-Antigen Origin Binding Domains at the Viral Origin

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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. P olyomaviruses are small DNA tumor viruses implicated in human diseases, particularly among the immunocompromised and the elderly (1). For example, the polyomavirus JC causes progressive multifocal leukoencephalopathy (PML) (2), BK virus is correlated with renal dysfunction in kidney transplant patients (2), and Merkel cell polyomavirus (MCV) is implicated in a rare but aggressive form of skin cancer (3, 4). The recent identification of several new human polyomaviruses (e.g., KI and WU polyomaviruses [5]) have provided additional incentives to establish fundamental aspects of the polyomavirus life cycle.The best-studied member of the polyomavirus family is simian virus 40 (SV40). Advances made with SV40 include the identification of several of the tumor suppressor proteins targeted during viral transformation (reviewed in reference 6). It has also been used to identify many of the proteins required for eukaryotic DNA replication (reviewed in references 7 to 9). In related studies, it was used to establish basic mechanisms involved in the replication process (reviewed in references 10 to 12). A current focus of research in this field is providing a molecular understanding of initiation events (reviewed in reference 13). In the long term, it is anticipated that progress made in terms of understanding SV40 replication will lead to therapies for treating polyomavirus infections.Among the highly conserved features of the circular, doublestranded DNA genomes of polyomavirus family members are the origins of DNA replication. The minimal or "core" origin of SV40 is a 64-bp region that is necessary and sufficient for DNA replication (reference 14 and references therein) (Fig. 1A). The central region of the core origin (termed site II) contains a palindromic arrangement of four GAGGC pentanucleotides (termed P1 to P4). The pentanucleotides are the hi...

Section: Resultsmentioning

confidence: 63%

Section: Discussionmentioning

confidence: 96%

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

Meinke

Phelan

Harrison

et al. 2013

Self Cite

“…To date, all of the MCPyV large T antigens isolated from tumors are truncated due to nonsense mutations or deletions. At least to date, missense mutations that render the full-length large T antigen deficient in DNA binding or helicase activity have not been identified (25,45). Similarly, mutations in the MCPyV viral origin of replication predicted to disrupt the binding of wild-type MCPyV large T antigen DNA binding domain have not been identified.…”

Section: Discussionmentioning

confidence: 99%

Merkel Cell Polyomavirus Large T Antigen Has Growth-Promoting and Inhibitory Activities

Cheng

Rozenblatt-Rosen

Paulson

et al. 2013

109

145

cMerkel 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. Merkel cell carcinoma (MCC) is an aggressive skin cancer with an annual incidence of 3 per million in the United States (1). Risk factors for developing MCC include advanced age, prolonged UV exposure, and immunosuppression due to HIV, hematologic malignancy, or solid-organ transplantation (2, 3). Recently, Merkel cell polyomavirus (MCPyV) was discovered to be clonally integrated in at least 80% of MCC, raising the possibility that this pathogen contributes to carcinogenesis (4, 5).MCPyV is a typical polyomavirus with a circular, doublestranded DNA genome containing an early region that expresses large and small T antigens, a late region that encodes 3 viral coat proteins, VP1, VP2, and VP3, and a regulatory region that contains the origin of replication and a bidirectional promoter for the early and late genes. MCPyV was the fifth polyomavirus identified in humans, preceded by BKPyV, JCPyV, KIPyV, and WUPyV (6-9). Since then, 6 additional human polyomaviruses have been discovered, including HPyV6, HPyV7, TSPyV, HPyV9, MWPyV, and STLPyV (10-15). Although JCPyV and BKPyV have been detected in a variety of human cancers (16, 17), Merkel cell polyomavirus is the only polyomavirus DNA clonally integrated in human cancer. Expression of Merkel large and small T antigens can be detected in most MCC specimens (5, 18, 19).The T antigens from several polyomaviruses have oncogenic activity. Notably, the simian virus 40 (SV40) large and small T antigens can transform a variety of rodent and human cells. Expression of SV40 large and small T antigens, together with human telomerase reverse transcriptase and an oncogenic form of H-RAS, can fully transform normal human fibroblasts (20,21)...

“…A noncoding regulatory region (NCRR) divides the genome into early and late coding regions. The NCRR contains the viral origin of replication (Ori) and regulatory elements/promoters for viral gene transcription (8,9). The early region encodes three proteins, namely, large T antigen (LT), small T antigen (sT), and the 57kT antigen (7).…”

Section: Erkel Cell Polyomavirus (Mcpyv) Was Discovered In 2008 Inmentioning

confidence: 99%

“…It contains a number of domains that are conserved among polyomaviruses, including a retinoblastoma (Rb)-binding domain, DnaJ domain, and CR1 domain (15). LT also has an origin-binding domain (OBD) and a C-terminal helicase domain, both of which are required for initiating viral replication (8,9,16).…”

Section: Erkel Cell Polyomavirus (Mcpyv) Was Discovered In 2008 Inmentioning

confidence: 99%

Host DNA Damage Response Factors Localize to Merkel Cell Polyomavirus DNA Replication Sites To Support Efficient Viral DNA Replication

Tsang

Wang

et al. 2014

IMPORTANCEMCPyV is the first polyomavirus to be clearly associated with human cancer. However, the MCPyV life cycle and its oncogenic mechanism remain poorly understood. In this report, we show that, in cells infected with native MCPyV virions, components of the ATM-and ATR-mediated DDR pathways accumulate in MCPyV LT-positive nuclear foci. Such a phenotype was recapitulated using our previously established system for visualizing MCPyV replication complexes in cells. By combining immunofluorescent staining, fluorescence in situ hybridization, and BrdU incorporation analysis, we demonstrate that DDR proteins are important for maintaining robust MCPyV DNA replication. This study not only provides the first look into the microscopic details of DDR factor/LT replication complexes at the MCPyV origin but also provides a platform for further studying the mechanistic role of host DDR factors in the MCPyV life cycle and virus-associated oncogenesis.