The Merkel Cell Polyomavirus Minor Capsid Protein

The surface of polyomavirus virions is composed of pentameric knobs of the major capsid protein, VP1. In previously studied polyomavirus species, such as SV40, two interior capsid proteins, VP2 and VP3, emerge from the virion to play important roles during the infectious entry process. Translation of the VP3 protein initiates at a highly conserved Met-Ala-Leu motif within the VP2 open reading frame. Phylogenetic analyses indicate that Merkel cell polyomavirus (MCV or MCPyV) is a member of a divergent clade of polyomaviruses that lack the conserved VP3 N-terminal motif. Consistent with this observation, we show that VP3 is not detectable in MCV-infected cells, VP3 is not found in native MCV virions, and mutation of possible alternative VP3-initiating methionine codons did not significantly affect MCV infectivity in culture. In contrast, VP2 knockout resulted in a >100-fold decrease in native MCV infectivity, despite normal virion assembly, viral DNA packaging, and cell attachment. Although pseudovirus-based experiments confirmed that VP2 plays an essential role for infection of some cell lines, other cell lines were readily transduced by pseudovirions lacking VP2. In cell lines where VP2 was needed for efficient infectious entry, the presence of a conserved myristoyl modification on the N-terminus of VP2 was important for its function. The results show that a single minor capsid protein, VP2, facilitates a post-attachment stage of MCV infectious entry into some, but not all, cell types.     

Quantitation of Human Seroresponsiveness to Merkel Cell Polyomavirus

Merkel cell carcinoma (MCC) is a relatively uncommon but highly lethal form of skin cancer. A majority of MCC tumors carry DNA sequences derived from a newly identified virus called Merkel cell polyomavirus (MCV or MCPyV), a candidate etiologic agent underlying the development of MCC. To further investigate the role of MCV infection in the development of MCC, we developed a reporter vector-based neutralization assay to quantitate MCV-specific serum antibody responses in human subjects. Our results showed that 21 MCC patients whose tumors harbored MCV DNA all displayed vigorous MCV-specific antibody responses. Although 88% (42/48) of adult subjects without MCC were MCV seropositive, the geometric mean titer of the control group was 59-fold lower than the MCC patient group (p<0.0001). Only 4% (2/48) of control subjects displayed neutralizing titers greater than the mean titer of the MCV-positive MCC patient population. MCC tumors were found not to express detectable amounts of MCV VP1 capsid protein, suggesting that the strong humoral responses observed in MCC patients were primed by an unusually immunogenic MCV infection, and not by viral antigen expressed by the MCC tumor itself. The occurrence of highly immunogenic MCV infection in MCC patients is unlikely to reflect a failure to control polyomavirus infections in general, as seroreactivity to BK polyomavirus was similar among MCC patients and control subjects. The results support the concept that MCV infection is a causative factor in the development of most cases of MCC. Although MCC tumorigenesis can evidently proceed in the face of effective MCV-specific antibody responses, a small pilot animal immunization study revealed that a candidate vaccine based on MCV virus-like particles (VLPs) elicits antibody responses that robustly neutralize MCV reporter vectors in vitro. This suggests that a VLP-based vaccine could be effective for preventing the initial establishment of MCV infection.     

Characterization of Self-Assembled Virus-Like Particles of Merkel Cell Polyomavirus

In our recombinant baculovirus system, VP1 protein of merkel cell polyomavirus (MCPyV), which is implicated as a causative agent in Merkel cell carcinoma, was self-assembled into MCPyV-like particles (MCPyV-LP) with two different sizes in insect cells, followed by being released into the culture medium. DNA molecules of 1.5- to 5-kb, which were derived from host insect cells, were packaged in large, ~50-nm spherical particles but not in small, ~25-nm particles. Structure reconstruction using cryo-electron microscopy showed that large MCPyV-LPs are composed of 72 pentameric capsomeres arranged in a T = 7 icosahedral surface lattice and are 48 nm in diameter. The MCPyV-LPs did not share antigenic determinants with BK- and JC viruses (BKPyV and JCPyV). The VLP-based enzyme immunoassay was applied to investigate age-specific prevalence of MCPyV infection in the general Japanese population aged 1–70 years. While seroprevalence of MCPyV increased with age in children and young individuals, its seropositivity in each age group was lower compared with BKPyV and JCPyV.     

Merkel Cell Polyomavirus Small T Antigen Induces Cancer and Embryonic Merkel Cell Proliferation in a Transgenic Mouse Model

Merkel cell polyomavirus (MCV) causes the majority of human Merkel cell carcinomas (MCC) and encodes a small T (sT) antigen that transforms immortalized rodent fibroblasts in vitro. To develop a mouse model for MCV sT-induced carcinogenesis, we generated transgenic mice with a flox-stop-flox MCV sT sequence homologously recombined at the ROSA locus (ROSA ^sT), allowing Cre-mediated, conditional MCV sT expression. Standard tamoxifen (TMX) administration to adult Ubc ^CreERT2; ROSA ^sT mice, in which Cre is ubiquitously expressed, resulted in MCV sT expression in multiple organs that was uniformly lethal within 5 days. Conversely, most adult Ubc ^CreERT2; ROSA ^sT mice survived low-dose tamoxifen administration but developed ear lobe dermal hyperkeratosis and hypergranulosis. Simultaneous MCV sT expression and conditional homozygous p53 deletion generated multi-focal, poorly-differentiated, highly anaplastic tumors in the spleens and livers of mice after 60 days of TMX treatment. Mouse embryonic fibroblasts from these mice induced to express MCV sT exhibited anchorage-independent cell growth. To examine Merkel cell pathology, MCV sT expression was also induced during mid-embryogenesis in Merkel cells of Atoh1 ^CreERT2/+; ROSA ^sT mice, which lead to significantly increased Merkel cell numbers in touch domes at late embryonic ages that normalized postnatally. Tamoxifen administration to adult Atoh1 ^CreERT2/+; ROSA ^sT and Atoh1 ^CreERT2/+; ROSA ^sT; p53f ^lox/flox mice had no effects on Merkel cell numbers and did not induce tumor formation. Taken together, these results show that MCV sT stimulates progenitor Merkel cell proliferation in embryonic mice and is a bona fide viral oncoprotein that induces full cancer cell transformation in the p53-null setting.     

Distinct Merkel Cell Polyomavirus Molecular Features in Tumour and Non Tumour Specimens from Patients with Merkel Cell Carcinoma

Merkel Cell Polyomavirus (MCPyV) is associated with Merkel Cell carcinoma (MCC), a rare, aggressive skin cancer with neuroendocrine features. The causal role of MCPyV is highly suggested by monoclonal integration of its genome and expression of the viral large T (LT) antigen in MCC cells. We investigated and characterized MCPyV molecular features in MCC, respiratory, urine and blood samples from 33 patients by quantitative PCR, sequencing and detection of integrated viral DNA. We examined associations between either MCPyV viral load in primary MCC or MCPyV DNAemia and survival. Results were interpreted with respect to the viral molecular signature in each compartment. Patients with MCC containing more than 1 viral genome copy per cell had a longer period in complete remission than patients with less than 1 copy per cell (34 vs 10 months, P = 0.037). Peripheral blood mononuclear cells (PBMC) contained MCPyV more frequently in patients sampled with disease than in patients in complete remission (60% vs 11%, P = 0.00083). Moreover, the detection of MCPyV in at least one PBMC sample during follow-up was associated with a shorter overall survival (P = 0.003). Sequencing of viral DNA from MCC and non MCC samples characterized common single nucleotide polymorphisms defining 8 patient specific strains. However, specific molecular signatures truncating MCPyV LT were observed in 8/12 MCC cases but not in respiratory and urinary samples from 15 patients. New integration sites were identified in 4 MCC cases. Finally, mutated-integrated forms of MCPyV were detected in PBMC of two patients with disseminated MCC disease, indicating circulation of metastatic cells. We conclude that MCPyV molecular features in primary MCC tumour and PBMC may help to predict the course of the disease.     

Seroprevalence of Hepatitis E Virus Infection among Swine Farmers and the General Population in Rural Taiwan

Objectives

Hepatitis E virus (HEV) is an emerging pathogen. We evaluated the seroprevalence of HEV infection among swine farmers and the general population in Taiwan, a nonendemic country.

Methods

We conducted a cross-sectional seroepidemiologic study in rural Taiwan in 2012 and 2013. The study included swine farmers, health examination attendees, pregnant women, and students. A commercial enzyme-linked immunosorbent assay was used to detect immunoglobulin G (IgG) and IgM against HEV. Pertinent information was collected using a questionnaire.

Results

In total, 660 participants were enrolled in the study, including 156 swine farmers, 314 health examination attendees, 100 pregnant women, and 90 students. IgG anti-HEV was detected in 29.5% of swine farmers, 11.5% of health examination attendees, 2% of pregnant women, and 1.1% of students. Two subjects were positive for IgM anti-HEV. Logistic regression analysis revealed that swine farmers had an approximately 3.5-fold increased risk (odds ratio [OR], 3.46; 95% confidence interval [CI], 1.91–6.27; p<0.0001) for being seropositive for IgG anti-HEV as compared to the general population. Age was positively associated with seropositive rate (OR, 1.07 per year; 95% CI, 1.05–1.09; p<0.0001).

Conclusion

HEV infection is prevalent in Taiwan. The seroprevalence of HEV infection is high in swine farmers and in the elderly population.     

Ganglioside GT1b Is a Putative Host Cell Receptor for the Merkel Cell Polyomavirus

The Merkel cell polyomavirus (MCPyV) was identified recently in human Merkel cell carcinomas, an aggressive neuroendocrine skin cancer. Here, we identify a putative host cell receptor for MCPyV. We found that recombinant MCPyV VP1 pentameric capsomeres both hemagglutinated sheep red blood cells and interacted with ganglioside GT1b in a sucrose gradient flotation assay. Structural differences between the analyzed gangliosides suggest that MCPyV VP1 likely interacts with sialic acids on both branches of the GT1b carbohydrate chain. Identification of a potential host cell receptor for MCPyV will aid in the elucidation of its entry mechanism and pathophysiology.Members of the polyomavirus (PyV) family, including simian virus 40 (SV40), murine PyV (mPyV), and BK virus (BKV), bind cell surface gangliosides to initiate infection (2, 8, 11, 15). PyV capsids are assembled from 72 pentamers (capsomeres) of the major coat protein VP1, with the internal proteins VP2 and VP3 buried within the capsids (7, 12). The VP1 pentamer makes direct contact with the carbohydrate portion of the ganglioside (10, 12, 13) and dictates the specificity of virus interaction with the cell. Gangliosides are glycolipids that contain a ceramide domain inserted into the plasma membrane and a carbohydrate domain that directly binds the virus. Specifically, SV40 binds to ganglioside GM1 (2, 10, 15), mPyV binds to gangliosides GD1a and GT1b (11, 15), and BKV binds to gangliosides GD1b and GT1b (8).Recently, a new human PyV designated Merkel cell PyV (MCPyV) was identified in Merkel cell carcinomas, a rare but aggressive skin cancer of neuroendocrine origin (3). It is as yet unclear whether MCPyV is the causative agent of Merkel cell carcinomas (17). A key to understanding the infectious and transforming properties of MCPyV is the elucidation of its cellular entry pathway. In this study, we identify a putative host cell receptor for MCPyV.Because an intact infectious MCPyV has not yet been isolated, we generated recombinant MCPyV VP1 pentamers in order to characterize cellular factors that bind to MCPyV. VP1 capsomeres have been previously shown to be equivalent to virus with respect to hemagglutination properties (4, 16), and the atomic structure of VP1 bound to sialyllactose has demonstrated that the capsomere is sufficient for this interaction (12, 13). The MCPyV VP1 protein (strain w162) was expressed and purified as described previously (1, 6). Briefly, a glutathione S-transferase-MCPyV VP1 fusion protein was expressed in Escherichia coli and purified using glutathione-Sepharose affinity chromatography. The fusion protein was eluted using glutathione and cleaved in solution with thrombin. The thrombin-cleaved sample was then rechromatographed on a second glutathione-Sepharose column to remove glutathione transferase and any uncleaved protein. The unbound VP1 was then chromatographed on a P-11 phosphocellulose column, and peak fractions eluting between 400 and 450 mM NaCl were collected. The purified protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by Coomassie blue staining (Fig. ​(Fig.1A,1A, left) and immunoblotting using an antibody (I58) that generally recognizes PyV VP1 proteins (Fig. ​(Fig.1A,1A, right) (9). Transmission electron microscopy (Philips CM10) analysis confirmed that the purified recombinant MCPyV VP1 formed pentamers (capsomeres), which did not assemble further into virus-like particles (Fig. ​(Fig.1B).1B). In an initial screening of its cell binding properties, we tested whether the MCPyV VP1 pentamers hemagglutinated red blood cells (RBCs). The MCPyV VP1 pentamers were incubated with sheep RBCs and assayed as previously described (5). SV40 and mPyV recombinant VP1 pentamers served as negative and positive controls, respectively. We found that MCPyV VP1 hemagglutinated the RBCs with the same efficiency as mPyV VP1 (protein concentration/hemagglutination unit) (Fig. ​(Fig.1C,1C, compare rows B and C from wells 1 to 11), suggesting that MCPyV VP1 engages a plasma membrane receptor on the RBCs. The recombinant murine VP1 protein used for comparison was from the RA strain, a small plaque virus (4). Thus, MCPyV VP1 has the hemagglutination characteristics of a small plaque mPyV (12, 13).Open in a separate windowFIG. 1.Characterization of MCPyV VP1. Recombinant MCPyV VP1 forms pentamers and hemagglutinates sheep RBCs. (A) Coomassie blue-stained SDS-PAGE and an immunoblot of the purified recombinant MCPyV VP1 protein are shown. Molecular mass markers are indicated. (B) Electron micrograph of the purified MCPyV VP1. MCPyV VP1 (shown in panel A) was diluted to 100 μg/ml and absorbed onto Formvar/carbon-coated copper grids. Samples were washed with phosphate-buffered saline, stained with 1% uranyl acetate, and visualized by transmission electron microscopy at 80 kV. Bar = 20 nm. (C) Sheep RBCs (0.5%) were incubated with decreasing concentrations of purified recombinant SV40 VP1 (row A), mPyV VP1 (row B), and MCPyV VP1 (row C). Wells 1 to 11 contain twofold serial dilutions of protein, starting at 2 μg/ml (well 1). Well 12 contains buffer only and serves as a negative control. Well 7 (rows B and C) corresponds to 128 hemagglutination units per 2 μg/ml VP1 protein.To characterize the chemical nature of the putative receptor for MCPyV, total membranes from RBCs were purified as described previously (15). The plasma membranes (30 μg) were incubated with MCPyV VP1 (0.5 μg) and floated on a discontinuous sucrose gradient (15). After fractionation, the samples were analyzed by SDS-PAGE, followed by immunoblotting with I58. VP1 was found in the bottom of the gradient in the absence of the plasma membranes (Fig. ​(Fig.2A,2A, first panel). In the presence of plasma membranes, a fraction of the VP1 floated to the middle of the gradient (Fig. ​(Fig.2A,2A, second panel), supporting the hemagglutination results that suggested that MCPyV VP1 binds to a receptor on the plasma membrane.Open in a separate windowFIG. 2.MCPyV VP1 binds to a protease-resistant, sialic acid-containing receptor on the plasma membrane. (A) Purified recombinant MCPyV VP1 was incubated with or without the indicated plasma membranes. The samples were floated in a discontinuous sucrose gradient, and the fractions were collected from the top of the gradient, subjected to SDS-PAGE, and immunoblotted with the anti-VP1 antibody I58. (B) Control and proteinase K-treated plasma membranes were subjected to SDS-PAGE, followed by Coomassie blue staining. (C) HeLa cells treated with proteinase K (4 μg/ml) were incubated with MCPyV at 4°C, and the resulting cell lysate was probed for the presence of MCPyV VP1. (D) As described in the legend to panel C, except 293T cells were used. (E) Purified MCPyV VP1 was incubated with plasma membranes pretreated with or without α2-3,6,8 neuraminidase and analyzed as described in the legend to panel A.To determine whether the receptor is a protein or a lipid, plasma membrane preparations (30 μg) were incubated with proteinase K (Sigma), followed by analysis with SDS-PAGE and Coomassie blue staining. Under these conditions, the majority of the proteins in the plasma membranes were degraded by the protease (Fig. ​(Fig.2B,2B, compare lanes 1 and 2). Despite the lack of proteins, the proteinase K-treated plasma membranes bound MCPyV VP1 as efficiently as control plasma membranes (Fig. ​(Fig.2A,2A, compare the second and third panels), demonstrating that MCPyV VP1 interacts with a protease-resistant receptor. The absence of VP1 in the bottom fraction in Fig. ​Fig.2A2A (third panel) is consistent with the fact that the buoyant density of the membranes is lowered by proteolysis. Of note, a similar result was seen with binding of the mPyV to the plasma membrane (15). Binding of MCPyV to the cell surface of two human tissue culture cells (i.e., HeLa and 293T) was also largely unaffected by pretreatment of the cells with proteinase K (Fig. 2C and D, compare lanes 1 and 2), further indicating that a nonproteinaceous molecule on the plasma membrane engages the virus.We next determined whether the protease-resistant receptor contains a sialic acid modification. Plasma membranes (10 μg) were incubated with a neuraminidase (α2-3,6,8 neuraminidase; Calbiochem) to remove the sialic acid groups. In contrast to the control plasma membranes, the neuraminidase-treated membranes did not bind MCPyV VP1 (Fig. ​(Fig.2E,2E, compare first and second panels), indicating that the MCPyV receptor includes a sialic acid modification.Gangliosides are lipids that contain sialic acid modifications. We asked if MCPyV VP1 binds to gangliosides similar to other PyV family members. The structures of the gangliosides used in this analysis (gangliosides GM1, GD1a, GD1b, and GT1b) are depicted in Fig. ​Fig.3A.3A. To assess a possible ganglioside-VP1 interaction, we employed a liposome flotation assay established previously (15). When liposomes (consisting of phosphatidyl-choline [19 μl of 10 mg/ml], -ethanolamine [5 μl of 10 mg/ml], -serine [1 μl of 10 mg/ml], and -inositol [3 μl of 10 mg/ml]) were incubated with MCPyV VP1 and subjected to the sucrose flotation assay, the VP1 remained in the bottom fraction (Fig. ​(Fig.3B,3B, first panel), indicating that VP1 does not interact with these phospholipids. However, when liposomes containing GT1b (1 μl of 1 mM), but not GM1 (1 μl of 1 mM) or GD1a (1 μl of 1 mM), were incubated with MCPyV VP1, the vesicles bound this VP1 (Fig. ​(Fig.3B).3B). A low level of virus floated partially when incubated with liposomes containing GD1b (Fig. ​(Fig.3B),3B), perhaps reflecting a weak affinity between MCPyV and GD1b. Importantly, MCPyV binds less efficiently to neuraminidase-treated GT1b-containing liposomes than to GT1b-containing liposomes (Fig. ​(Fig.3B,3B, sixth panel), suggesting that the GT1b sialic acids are involved in virus binding. This finding is consistent with the ability of neuraminidase to block MCPyV binding to the plasma membrane (Fig. ​(Fig.2E).2E). The level of virus flotation observed in the neuraminidase-treated GT1b-containing liposomes is likely due to the inefficiency of the neuraminidase reaction with a high concentration of GT1b used to prepare the vesicles.Open in a separate windowFIG. 3.MCPyV VP1 binds to ganglioside GT1b. (A) Structures of gangliosides GM1, GD1a, GD1b, and GT1b. The nature of the glycosidic linkages is indicated. (B) Purified MCPyV VP1 protein was incubated with liposomes only or with liposomes containing the indicated gangliosides. The samples were analyzed as described in the legend to Fig. ​Fig.2A.2A. Where indicated, GT1b-containing liposomes were pretreated with α2-3,6,8 neuraminidase and analyzed subsequently for virus binding. (C to E) The indicated viruses were incubated with liposomes and analyzed as described in the legend to panel B.As controls, GM1-containing liposomes bound SV40 (Fig. ​(Fig.3C),3C), GD1a-containing liposomes bound mPyV (Fig. ​(Fig.3D),3D), and GD1b-containing liposomes bound BKV (Fig. ​(Fig.3E),3E), demonstrating that the liposomes were functionally intact. We note that, while all of the MCPyV VP1 floated when incubated with liposomes containing GT1b (Fig. ​(Fig.3B,3B, sixth panel), a significant fraction of SV40, mPyV, and BKV VP1 remained in the bottom fraction despite being incubated with liposomes containing their respective ganglioside receptors (Fig. 3C to E, second panels). This result is likely due to the fact that in contrast to MCPyV, which are assembled as pentamers (Fig. ​(Fig.1B),1B), the SV40, mPyV, and BKV used in these experiments are fully assembled particles: their larger and denser nature prevents efficient flotation. Nonetheless, we conclude that MCPyV VP1 binds to ganglioside GT1b efficiently.The observation that GD1a does not bind to MCPyV VP1 suggests that the monosialic acid modification on the right branch of GT1b (Fig. ​(Fig.3A)3A) is insufficient for binding. Similarly, the failure of GD1b to bind MCPyV VP1 suggests that the sialic acid on the left arm of GT1b is necessary for binding. Together, these observations suggest that MCPyV VP1 interacts with sialic acids on both branches of GT1b (Fig. ​(Fig.4).4). A recent structure of SV40 VP1 in complex with the sugar portion of GM1 (10) demonstrated that although SV40 VP1 binds both branches of GM1 (Fig. ​(Fig.4),4), only a single sialic acid in GM1 is involved in this interaction. In the case of mPyV, structures of mPyV VP1 in complex with different carbohydrates (12, 13) revealed that the sialic acid-galactose moiety on the left branch of GD1a (and GT1b) is sufficient for mPyV VP1 binding (Fig. ​(Fig.4).4). Although no structure of BKV in complex with the sugar portion of GD1b (or GT1b) is available, in vitro binding studies (8) have suggested that the disialic acid modification on the right branch of GD1b (and GT1b) is responsible for binding BKV VP1 (Fig. ​(Fig.4).4). Thus, it appears that the unique feature of the MCPyV VP1-GT1b interaction is that the sialic acids on both branches of this ganglioside are likely involved in capsid binding.Open in a separate windowFIG. 4.A potential model of the different VP1-ganglioside interactions (see the text for discussion).The identification of a potential cellular receptor for MCPyV will facilitate the study of its entry mechanism. An important issue for further study is to determine whether MCPyV targets Merkel cells preferentially, and if so, whether GT1b is found in higher levels in these cells to increase susceptibility.     

Serological Cross-Reactivity between Merkel Cell Polyomavirus and Two Closely Related Chimpanzee Polyomaviruses

Phylogenetic analyses based on the major capsid protein sequence indicate that Merkel cell polyomavirus (MCPyV) and chimpanzee polyomaviruses (PtvPyV1, PtvPyV2), and similarly Trichodysplasia spinulosa-associated polyomavirus (TSPyV) and the orangutan polyomavirus (OraPyV1) are closely related. The existence of cross-reactivity between these polyomaviruses was therefore investigated. The findings indicated serological identity between the two chimpanzee polyomaviruses investigated and a high level of cross-reactivity with Merkel cell polyomavirus. In contrast, cross-reactivity was not observed between TSPyV and OraPyV1. Furthermore, specific antibodies to chimpanzee polyomaviruses were detected in chimpanzee sera by pre-incubation of sera with the different antigens, but not in human sera.     

The T Antigen Locus of Merkel Cell Polyomavirus Downregulates Human Toll-Like Receptor 9 Expression

Establishment of a chronic infection is a key event in virus-mediated carcinogenesis. Several cancer-associated, double-stranded DNA (dsDNA) viruses act via their oncoproteins to downregulate Toll-like receptor 9 (TLR9), a key receptor in the host innate immune response that senses viral or bacterial dsDNA. A novel oncogenic virus, Merkel cell polyomavirus (MCPyV), has been recently identified that causes up to 80% of Merkel cell carcinomas (MCCs). However, it is not yet known whether this oncogenic virus also disrupts immune-related pathways. We find that MCPyV large T antigen (LT) expression downregulates TLR9 expression in epithelial and MCC-derived cells. Accordingly, silencing of LT expression results in upregulation of mRNA TLR9 levels. In addition, small T antigen (sT) also appears to inhibit TLR9 expression, since inhibition of its expression also resulted in an increase of TLR9 mRNA levels. LT inhibits TLR9 expression by decreasing the mRNA levels of the C/EBPβ transactivator, a positive regulator of the TLR9 promoter. Chromatin immunoprecipitation reveals that C/EBPβ binding at a C/EBPβ response element (RE) in the TLR9 promoter is strongly inhibited by expression of MCPyV early genes and that mutation of the C/EBP RE prevents MCPyV downregulation of TLR9. A survey of BK polyomavirus (BKPyV), JC polyomavirus (JCPyV), KI polyomavirus (KIPyV), MCPyV, simian virus 40 (SV40), and WU polyomavirus (WUPyV) early genes revealed that only BKPyV and MCPyV are potent inhibitors of TLR9 gene expression. MCPyV LT targeting of C/EBP transactivators is likely to play an important role in viral persistence and potentially inhibit host cell immune responses during MCPyV tumorigenesis.     

湖北地区普通人群卡波氏肉瘤相关疱疹病毒感染的血清学分析

卡波氏肉瘤相关疱疹病毒(KSHV)可导致人类产生卡波氏肉瘤(KS),即AIDS病人最为常见的肿瘤.广泛的流行病学研究显示,KSHV的流行与KS相似并呈现明显的地域分布型.为调查KSHV在汉族普通人群中的感染情况,我们以KSHV ORF65编码的小衣壳蛋白(small capsid protein)为抗原,采用酶联免疫(ELISA)分析方法,对湖北地区560例汉族普通人群血清样品进行了KSHV抗体检测.在检测的560份血样中,KSHV抗体总阳性率为5.2%,其中,男性阳性率为5.7%,女性为4.5%.统计学分析显示,KSHV感染率在男女性别上无差异(P=0.542),但与年龄有一定的相关性10岁以下儿童群体较之10岁以上人群KSHV感染率具有显著的统计学差异(P=0.006,OR=6.692,95%CI=1.710-26.198);60岁以上的老年人群KSHV感染率有上升趋势,但无统计学明显差异(P=0.052).上述结果表明,KSHV在这一地区的流行与西方成年人群的感染率相似,但在儿童群体中的相对较高的感染率与一些非洲地区的接近.由此提示在该群体可能存在特殊的传播模式.     

Merkel Cell Polyomavirus Large T Antigen Disrupts Host Genomic Integrity and Inhibits Cellular Proliferation

Clonal integration of Merkel cell polyomavirus (MCV) DNA into the host genome has been observed in at least 80% of Merkel cell carcinoma (MCC). The integrated viral genome typically carries mutations that truncate the C-terminal DNA binding and helicase domains of the MCV large T antigen (LT), suggesting a selective pressure to remove this MCV LT region during tumor development. In this study, we show that MCV infection leads to the activation of host DNA damage responses (DDR). This activity was mapped to the C-terminal helicase-containing region of the MCV LT. The MCV LT-activated DNA damage kinases, in turn, led to enhanced p53 phosphorylation, upregulation of p53 downstream target genes, and cell cycle arrest. Compared to the N-terminal MCV LT fragment that is usually preserved in mutants isolated from MCC tumors, full-length MCV LT shows a decreased potential to support cellular proliferation, focus formation, and anchorage-independent cell growth. These apparently antitumorigenic effects can be reversed by a dominant-negative p53 inhibitor. Our results demonstrate that MCV LT-induced DDR activates p53 pathway, leading to the inhibition of cellular proliferation. This study reveals a key difference between MCV LT and simian vacuolating virus 40 LT, which activates a DDR but inhibits p53 function. This study also explains, in part, why truncation mutations that remove the MCV LT C-terminal region are necessary for the oncogenic progression of MCV-associated cancers.