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.     

Glycosaminoglycans and sialylated glycans sequentially facilitate Merkel cell polyomavirus infectious entry

Merkel cell polyomavirus (MCV or MCPyV) appears to be a causal factor in the development of Merkel cell carcinoma, a rare but highly lethal form of skin cancer. Although recent reports indicate that MCV virions are commonly shed from apparently healthy human skin, the precise cellular tropism of the virus in healthy subjects remains unclear. To begin to explore this question, we set out to identify the cellular receptors or co-receptors required for the infectious entry of MCV. Although several previously studied polyomavirus species have been shown to bind to cell surface sialic acid residues associated with glycolipids or glycoproteins, we found that sialylated glycans are not required for initial attachment of MCV virions to cultured human cell lines. Instead, glycosaminoglycans (GAGs), such as heparan sulfate (HS) and chondroitin sulfate (CS), serve as initial attachment receptors during the MCV infectious entry process. Using cell lines deficient in GAG biosynthesis, we found that N-sulfated and/or 6-O-sulfated forms of HS mediate infectious entry of MCV reporter vectors, while CS appears to be dispensable. Intriguingly, although cell lines deficient in sialylated glycans readily bind MCV capsids, the cells are highly resistant to MCV reporter vector-mediated gene transduction. This suggests that sialylated glycans play a post-attachment role in the infectious entry process. Results observed using MCV reporter vectors were confirmed using a novel system for infectious propagation of native MCV virions. Taken together, the findings suggest a model in which MCV infectious entry occurs via initial cell binding mediated primarily by HS, followed by secondary interactions with a sialylated entry co-factor. The study should facilitate the development of inhibitors of MCV infection and help shed light on the infectious entry pathways and cellular tropism of the virus.     

Detection of Merkel cell polyomavirus in cervical squamous cell carcinomas and adenocarcinomas from Japanese patients

ABSTRACT: BACKGROUND: Merkel cell polyomavirus (MCPyV) was identified originally in Merkel cell carcinoma (MCC), a rare form of human skin neuroendocrine carcinoma. Evidence of MCPyV existence in other forms of malignancy such as cutaneous squamous cell carcinomas (SCCs) is growing. Cervical cancers became the focus of our interest in searching for potentially MCPyV-related tumors because: (i) the major histological type of cervical cancer is the SCC; (ii) the uterine cervix is a common site of neuroendocrine carcinomas histologically similar to MCCs; and (iii) MCPyV might be transmitted during sexual interaction as demonstrated for human papillomavirus (HPV). In this study, we aimed to clarify the possible presence of MCPyV in cervical SCCs from Japanese patients. Cervical adenocarcinomas (ACs) were also studied. RESULTS: Formalin-fixed paraffin-embedded tissue samples from 48 cervical SCCs and 16 cervical ACs were examined for the presence of the MCPyV genome by polymerase chain reaction (PCR) and sequencing analyses. PCR analysis revealed that 9/48 cervical SCCs (19 %) and 4/16 cervical ACs (25 %) were positive for MCPyV DNA. MCPyV-specific PCR products were sequenced to compare them with reference sequences. The nucleotide sequences in the MCPyV large-T (LT)-sequenced region were the same among MCPyV-positive cervical SCCs and AC. Conversely, in the MCPyV viral protein 1 (VP1)-sequenced, two cervical SCCs and three cervical ACs showed several nucleotide substitutions, of which three caused amino acid substitutions. These sequencing results suggested that three MCPyV variants of the VP1 were identified in our cases. Immunohistochemistry showed that the LT antigen was expressed in tumor cells in MCPyV-positive samples. Genotyping of human HPV in the MCPyV-positive samples revealed that infected HPVs were HPV types 16, 31 and 58 for SCCs and HPV types 16 and 18 for ACs. CONCLUSIONS: This study provides the first observation that MCPyV coexists in a subset of HPV-associated cervical cancers from Japanese patients. The prevalence of MCPyV in these lesions was close to that observed in the cutaneous SCCs. Further worldwide epidemiological surveys are warranted to determine the possible association of MCPyV with pathogenesis of cervical cancers.     

The ionophore monensin inhibits mouse polyomavirus DNA replication and destabilizes viral early mRNAs

Monensin is a ionophore compound with different biological activities. It raises the intralysosomal pH, it binds the plasma membranes particularly at the level of the cisternal system of the Golgi apparatus. It causes imbalance in the intramembrane ion traffic and inhibits export of secretory proteins at membrane level. Monensin blocks endocytosis and therefore impedes entry of toxic molecules. The drug also inhibits viral proliferation of RNA and DNA viruses such as vesicular stomatitis, influenza and human polyomaviruses. In this report we show that monensin effectively abolishes viral DNA replication of mouse polyomavirus. Results show that the half life of viral early mRNAs is significantly reduced in the presence of the drug. Therefore we suggest that the reduction of viral DNA synthesis is a consequence of the reduced intranuclear pool of viral early antigens.     

Cellular factors implicated in prion replication

Prions are the unconventional infectious agents responsible for prion diseases, which are composed mainly by the misfolded prion protein (PrP^Sc) that replicates by converting the host associated cellular prion protein (PrP^C). Several lines of evidence suggest that other cellular components participate in prion conversion, however, the identity or even the chemical nature of such factors are entirely unknown. In this article we study the conversion factor activity by complementation of a PMCA procedure employing purified PrP^C and PrP^Sc. Our results show that the conversion factor is present in all major organs of diverse mammalian species, and is predominantly located in the lipid raft fraction of the cytoplasmic membrane. On the other hand, it is not present in the lower organisms tested (yeast, bacteria and flies). Surprisingly, treatments that eliminate the major classes of chemical molecules do not affect conversion activity, suggesting that various different compounds may act as conversion factor in vitro. This conclusion is further supported by experiments showing that addition of various classes of molecules have a small, but detectable effect on enhancing prion replication in vitro. More research is needed to elucidate the identity of these factors, their detailed mechanism of action and whether or not they are essential component of the infectious particle.     

Quantitative analysis of viral load per haploid genome revealed the different biological features of Merkel cell polyomavirus infection in skin tumor

Merkel cell polyomavirus (MCPyV) has recently been identified in Merkel cell carcinoma (MCC), an aggressive cancer that occurs in sun-exposed skin. Conventional technologies, such as polymerase chain reaction (PCR) and immunohistochemistry, have produced conflicting results for MCPyV infections in non-MCC tumors. Therefore, we performed quantitative analyses of the MCPyV copy number in various skin tumor tissues, including MCC (n?=?9) and other sun exposure-related skin tumors (basal cell carcinoma [BCC, n?=?45], actinic keratosis [AK, n?=?52], Bowen's disease [n?=?34], seborrheic keratosis [n?=?5], primary cutaneous anaplastic large-cell lymphoma [n?=?5], malignant melanoma [n?=?5], and melanocytic nevus [n?=?6]). In a conventional PCR analysis, MCPyV DNA was detected in MCC (9 cases; 100%), BCC (1 case; 2%), and AK (3 cases; 6%). We then used digital PCR technology to estimate the absolute viral copy number per haploid human genome in these tissues. The viral copy number per haploid genome was estimated to be around 1 in most MCC tissues, and there were marked differences between the MCC (0.119-42.8) and AK (0.02-0.07) groups. PCR-positive BCC tissue showed a similar viral load as MCC tissue (0.662). Immunohistochemistry with a monoclonal antibody against the MCPyV T antigen (CM2B4) demonstrated positive nuclear localization in most of the high-viral-load tumor groups (8 of 9 MCC and 1 BCC), but not in the low-viral-load or PCR-negative tumor groups. These results demonstrated that MCPyV infection is possibly involved in a minority of sun-exposed skin tumors, including BCC and AK, and that these tumors display different modes of infection.     

Cellular mobile genetic elements in the regulatory region of the pneumotropic mouse polyomavirus genome: structure and function in viral gene expression and DNA replication

DNA from the murine pneumotropic virus was extracted from virus in lung tissue of infected mice, and the regulatory region of the genome was amplified by PCR. The regulatory region of individual plasmid cloned DNA molecules appeared to have heterogeneous enhancer segments, whereas the protein-coding part of the genome had a uniform length. Nucleotide sequence analysis revealed that the majority of the DNA molecules had a structure differing from the standard type. A 220-bp insertion at nucleotide position 142 with a concomitant deletion of nucleotides 143 to 148 was prominent. There were two variants of the 220-bp insertion, differing at two nucleotide positions at one of the termini. Other DNA molecules had complete or partial deletions of these structures and surrounding sequences in the viral enhancer. However, the end of the insertion at nucleotide 142 was frequently preserved. The viral early and late promoter activity of the variant regulatory regions was tested in a luciferase reporter assay by using transfected NIH 3T3 cells. In relation to the standard-type DNA, all variants, including a G272T mutant, had much stronger late promoters. In contrast, the early promoter activity was influenced in a positive or negative direction by individual mutations. Also, the activity of the viral origin of DNA replication was affected by the sequence variation of the regulatory region, although the effects were smaller than for the late promoter. Analysis by Southern blotting and quantification using dot blots showed that approximately 10(3) copies of material related to the 220-bp insert in murine pneumotropic virus DNA was present in mouse and human DNA but not in Escherichia coli DNA. Moreover, analysis by PCR indicated that there were multiple copies in the mouse genome of sequences that were identical or closely related to the 220-bp viral DNA segment. These data together with the nucleotide sequence analysis suggest that the 220-bp insertion is related to a transposable element of a novel type.     

African great apes are naturally infected with polyomaviruses closely related to Merkel cell polyomavirus

The oncogenic Merkel cell polyomavirus (MCPyV) infects humans worldwide, but little is known about the occurrence of viruses related to MCPyV in the closest phylogenetic relatives of humans, great apes. We analyzed samples from 30 wild chimpanzees and one captive gorilla and identified two new groups of polyomaviruses (PyVs). These new viruses are by far the closest relatives to MCPyV described to date, providing the first evidence of the natural occurrence of PyVs related to MCPyV in wild great apes. Similar to MCPyV, the prevalence of these viruses is relatively high (>30%). This, together with the fact that humans in West and Central Africa frequently hunt and butcher primates, may point toward further MCPyV-like strains spreading to, or already existing in, our species.     

Cellular factors required for papillomavirus DNA replication.

In vitro replication of papillomavirus DNA has been carried out with a combination of purified proteins and partially purified extracts made from human cells. DNA synthesis requires the viral E1 protein and the papillomavirus origin of replication. The E2 protein stimulates DNA synthesis in a binding site-independent manner. Papillomavirus DNA replication is also dependent on the cellular factors replication protein A, replication factor C, and proliferating-cell nuclear antigen as well as a phosphocellulose column fraction (IIA). Fraction IIA contains DNA polymerase alpha-primase and DNA polymerase delta. Both of these polymerases are essential for papillomavirus DNA replication in vitro. However, unlike the case with T-antigen-dependent replication from the simian virus 40 origin, purified DNA polymerase alpha-primase and delta cannot efficiently replace fraction IIA in the replication reaction. Hence, additional cellular factors seem to be required for papillomavirus DNA replication. Interestingly, replication factor C and proliferating-cell nuclear antigen are more stringently required for DNA synthesis in the papillomavirus system than in the simian virus 40 in vitro system. These distinctions indicate that there must be mechanistic differences between the DNA replication systems of papillomavirus and simian virus 40.     

Growth factors and beta cell replication

Recent studies have demonstrated that human islet allograft transplantation can be a successful therapeutic option in the treatment of patients with Type I diabetes. However, this impressive recent advance is accompanied by a very important constraint. There is a critical paucity of pancreatic islets or pancreatic beta cells for islet transplantation to become a large-scale therapeutic option in patients with diabetes. This has prompted many laboratories around the world to invigorate their efforts in finding ways for increasing the availability of beta cells or beta cell surrogates that potentially could be transplanted into patients with diabetes. The number of studies analyzing the mechanisms that govern beta cell proliferation and growth in physiological and pathological conditions has increased exponentially during the last decade. These studies exploring the role of growth factors, intracellular signaling molecules and cell cycle regulators constitute the substrate for future strategies aimed at expanding human beta cells in vitro and/or in vivo after transplantation. In this review, we describe the current knowledge on the effects of several beta cell growth factors that have been shown to increase beta cell proliferation and expand beta cell mass in vitro and/or in vivo and that they could be potentially deployed in an effort to increase the number of patients transplanted with islets. Furthermore, we also analyze in this review recent studies deciphering the relevance of these specific islet growth factors as physiological and pathophysiological regulators of beta cell proliferation and islet growth.     

Droplet-digital PCR assay to detect Merkel cell polyomavirus sequences in chorionic villi from spontaneous abortion affected females

Droplet-digital polymerase chain reaction (ddPCR) technique was set up to detect/quantify Merkel cell polyomavirus (MCPyV) DNA in clinical specimens, including chorionic villi and peripheral blood mononuclear cells (PBMCs) from spontaneous abortion (SA)-affected females. This ddPCR assay showed high accuracy, sensitivity, and specificity in detecting MCPyV DNA cloned in a recombinant plasmid vector, the control. ddPCR was extended to MCPyV DNA to investigate/quantify its sequences in clinical samples. Overall, 400 samples were analyzed, that is, 100 chorionic villi and 100 PBMCs, from SA females (n = 100), the cases, and 100 chorionic villi and 100 PBMCs from females who underwent voluntary pregnancy interruption (VI, n = 100), the control. MCPyV DNA was detected in 4/100 (4%) and 5/100 (5%) of SA and VI chorionic villi, respectively. The mean viral DNA load was 1.99 ( ± 0.94 standard mean deviation [SD]) copy/10⁴ cells in SA and 3.02 ( ± 1.86 [SD]) copy/10⁴ cells in VI. In PBMCs, MCPyV DNA was revealed in 9/100 (9%) and 14/100 (14%) of SA and VI, with a mean of 2.09 ( ± 1.17 [SD]) copy/10⁴ cells and 4.09 ( ± 4.26 [SD]) copy/10⁴ cells in SA and VI, respectively. MCPyV gene expression analysis by quantitative PCR for the large T antigen (LT) and viral capsid protein 1 (VP1) showed their mRNAs in 2/4 (50%) SA- and 2/5 (40%) VI-MCPyV-positive samples. MCPyV DNA was detected/quantified using the ddPCR technique, in chorionic villi and PBMCs from SA and VI. In our experimental conditions, ddPCR provided a powerful tool to detect/quantify MCPyV DNA sequences in clinical samples.