The Oncogenic Small Tumor Antigen of Merkel Cell Polyomavirus Is an Iron-Sulfur Cluster Protein That Enhances Viral DNA Replication.

UNLABELLED: Merkel cell polyomavirus (MCPyV) plays an important role in Merkel cell carcinoma (MCC). MCPyV small T (sT) antigen has emerged as the key oncogenic driver in MCC carcinogenesis. It has also been shown to promote MCPyV LT-mediated replication by stabilizing LT. The importance of MCPyV sT led us to investigate sT functions and to identify potential ways to target this protein. We discovered that MCPyV sT purified from bacteria contains iron-sulfur (Fe/S) clusters. Electron paramagnetic resonance analysis showed that MCPyV sT coordinates a [2Fe-2S] and a [4Fe-4S] cluster. We also observed phenotypic conservation of Fe/S coordination in the sTs of other polyomaviruses. Since Fe/S clusters are critical cofactors in many nucleic acid processing enzymes involved in DNA unwinding and polymerization, our results suggested the hypothesis that MCPyV sT might be directly involved in viral replication. Indeed, we demonstrated that MCPyV sT enhances LT-mediated replication in a manner that is independent of its previously reported ability to stabilize LT. MCPyV sT translocates to nuclear foci containing actively replicating viral DNA, supporting a direct role for sT in promoting viral replication. Mutations of Fe/S cluster-coordinating cysteines in MCPyV sT abolish its ability to stimulate viral replication. Moreover, treatment with cidofovir, a potent antiviral agent, robustly inhibits the sT-mediated enhancement of MCPyV replication but has little effect on the basal viral replication driven by LT alone. This finding further indicates that MCPyV sT plays a direct role in stimulating viral DNA replication and introduces cidofovir as a possible drug for controlling MCPyV infection. IMPORTANCE: MCPyV is associated with a highly aggressive form of skin cancer in humans. Epidemiological surveys for MCPyV seropositivity and sequencing analyses of healthy human skin suggest that MCPyV may represent a common component of the human skin microbial flora. However, much of the biology of the virus and its oncogenic ability remain to be investigated. In this report, we identify MCPyV sT as a novel Fe/S cluster protein and show that conserved cysteine clusters are important for sT's ability to enhance viral replication. Moreover, we show that sT sensitizes MCPyV replication to cidofovir inhibition. The discovery of Fe/S clusters in MCPyV sT opens new avenues to the study of the structure and functionality of this protein. Moreover, this study supports the notion that sT is a potential drug target for dampening MCPyV infection.

High-affinity Rb binding, p53 inhibition, subcellular localization, and transformation by wild-type or tumor-derived shortened Merkel cell polyomavirus large T antigens.

UNLABELLED: Interference with tumor suppressor pathways by polyomavirus-encoded tumor antigens (T-Ags) can result in transformation. Consequently, it is thought that T-Ags encoded by Merkel cell polyomavirus (MCPyV), a virus integrated in ∼90% of all Merkel cell carcinoma (MCC) cases, are major contributors to tumorigenesis. The MCPyV large T-Ag (LT-Ag) has preserved the key functional domains present in all family members but has also acquired unique regions that flank the LxCxE motif. As these regions may mediate unique functions, or may modulate those shared with T-Ags of other polyomaviruses, functional studies of MCPyV T-Ags are required. Here, we have performed a comparative study of full-length or MCC-derived truncated LT-Ags with regard to their biochemical characteristics, their ability to bind to retinoblastoma (Rb) and p53 proteins, and their transforming potential. We provide evidence that full-length MCPyV LT-Ag may not directly bind to p53 but nevertheless can significantly reduce p53-dependent transcription in reporter assays. Although early region expression constructs harboring either full-length or MCC-derived truncated LT-Ag genes can transform primary baby rat kidney cells, truncated LT-Ags do not bind to p53 or reduce p53-dependent transcription. Interestingly, shortened LT-Ags exhibit a very high binding affinity for Rb, as shown by coimmunoprecipitation and in vitro binding studies. Additionally, we show that truncated MCPyV LT-Ag proteins are expressed at higher levels than those for the wild-type protein and are able to partially relocalize Rb to the cytoplasm, indicating that truncated LT proteins may have gained additional features that distinguish them from the full-length protein. IMPORTANCE: MCPyV is one of the 12 known polyomaviruses that naturally infect humans. Among these, it is of particular interest since it is the only human polyomavirus known to be involved in tumorigenesis. MCPyV is thought to be causally linked to MCC, a rare skin tumor. In these tumors, viral DNA is monoclonally integrated into the genome of the tumor cells in up to 90% of all MCC cases, and the integrated MCV genomes, furthermore, harbor signature mutations in the so-called early region that selectively abrogate viral replication while preserving cell cycle deregulating functions of the virus. This study describes comparative studies of early region T-Ag protein characteristics, their ability to bind to Rb and p53, and their transforming potential.

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.

Structures of Merkel cell polyomavirus VP1 complexes define a sialic acid binding site required for infection.

The recently discovered human Merkel cell polyomavirus (MCPyV or MCV) causes the aggressive Merkel cell carcinoma (MCC) in the skin of immunocompromised individuals. Conflicting reports suggest that cellular glycans containing sialic acid (Neu5Ac) may play a role in MCPyV infectious entry. To address this question, we solved X-ray structures of the MCPyV major capsid protein VP1 both alone and in complex with several sialylated oligosaccharides. A shallow binding site on the apical surface of the VP1 capsomer recognizes the disaccharide Neu5Ac-α2,3-Gal through a complex network of interactions. MCPyV engages Neu5Ac in an orientation and with contacts that differ markedly from those observed in other polyomavirus complexes with sialylated receptors. Mutations in the Neu5Ac binding site abolish MCPyV infection, highlighting the relevance of the Neu5Ac interaction for MCPyV entry. Our study thus provides a powerful platform for the development of MCPyV-specific vaccines and antivirals. Interestingly, engagement of sialic acid does not interfere with initial attachment of MCPyV to cells, consistent with a previous proposal that attachment is mediated by a class of non-sialylated carbohydrates called glycosaminoglycans. Our results therefore suggest a model in which sialylated glycans serve as secondary, post-attachment co-receptors during MCPyV infectious entry. Since cell-surface glycans typically serve as primary attachment receptors for many viruses, we identify here a new role for glycans in mediating, and perhaps even modulating, post-attachment entry processes.

Detection of Merkel cell polyomavirus and human papillomaviruses in Merkel cell carcinoma combined with squamous cell carcinoma in immunocompetent European patients.

BACKGROUND: About 10% of patients with Merkel cell carcinoma (MCC) suffer from an associated squamous cell carcinoma (SCC). In European patients, Merkel cell polyomavirus (MCPyV) is detectable in 60%-88% of the MCC tumors. In combined lesions, MCPyV was not detectable so far. METHODS: We investigated 2 combined tumors of MCC and SCC for the presence of MCPyV and human papillomavirus (HPV) by polymerase chain reaction and immunohistochemistry. RESULTS: In both lesions, MCPyV DNA was found, and in 1 case, HPV DNA was also detected. This is the first report of a coinfection with HPV and MCPyV in combined MCC-SCC tumors. CONCLUSIONS: The results underline the hypothesis of co-cancerogenesis of 2 oncogenic viruses in nonmelanoma skin cancer. Technical reasons and a low viral copy number of MCPyV hampering immunohistochemical detection may be responsible for the negative results in the literature.