Prognostic value of antibodies to Merkel cell polyomavirus T antigens and VP1 protein in patients with Merkel cell carcinoma.

BACKGROUND: Merkel cell polyomavirus (MCPyV) is the main aetiological agent of Merkel cell carcinoma (MCC). Serum antibodies against the major MCPyV capsid protein (VP1) are detected in the general population, whereas antibodies against MCPyV oncoproteins (T antigens) have been reported specifically in patients with MCC. OBJECTIVES: The primary aim was to assess whether detection of serum antibodies against MCPyV proteins at baseline was associated with disease outcome in patients with MCC. The secondary aim was to establish whether evolution of these antibodies during follow-up was associated with the course of the disease. METHODS: Serum T-antigen and VP1 antibodies were assessed by enzyme-linked immunosorbent assay using recombinant proteins in a cohort of 143 patients with MCC, including 84 patients with serum samples available at baseline. RESULTS: Low titres of VP1 antibodies at baseline (< 10 000) were significantly and independently associated with increased risk of recurrence [hazard ratio (HR) 2·71, 95% confidence interval (CI) 1·13-6·53, P = 0·026] and death (HR 3·74, 95% CI 1·53-9·18, P = 0·004), whereas T-antigen antibodies were not found to be associated with outcome. VP1 antibodies did not differ between patients in remission and those with recurrence or progression during follow-up. However, T-antigen antibodies were more frequently detected in patients with recurrence or progression at 12 months (P = 0·020) and 24 months (P = 0·016) after diagnosis. CONCLUSIONS: VP1 antibodies constitute a prognostic marker at baseline, whereas T-antigen antibodies constitute a marker of disease recurrence or progression if detected > 12 months after diagnosis.

Vitamin D deficiency is associated with greater tumor size and poorer outcome in Merkel cell carcinoma patients.

BACKGROUND: Merkel cell polyomavirus has been recognized to be associated with Merkel cell carcinoma (MCC), but the evolution of this cancer probably depends on various factors. Vitamin D deficiency, defined by serum 25-hydroxyvitamin D levels <50 nmol/L, seems to influence cancer behavior and progression, but has never been assessed in MCC patients. OBJECTIVES: First, to evaluate whether vitamin D deficiency was associated with tumor characteristics and prognosis in a cohort of MCC patients. Second, to assess expression of the vitamin D receptor (VDR) in MCC tumors. METHODS: Clinical findings, Merkel cell polyomavirus markers and vitamin D status were assessed in a cohort of French MCC patients. The study was limited to the 89 patients for whom the serum sample had been collected within 3 years after the diagnosis of MCC. Correlation between vitamin D deficiency and MCC characteristics and outcome were determined in regression analyses. VDR expression in MCC tumours was assessed by immunohistochemistry. RESULTS: Vitamin D deficiency was noted in 65.1% of the patients and was independently associated with greater tumor size at diagnosis (P = 0.006) and with metastasis recurrence (HR, 2.89; 95% CI, 1.03 to 8.13; P = 0.043), but not with death from MCC, although there was a trend (HR, 5.28; 95% CI, 0.75 to 36.96; P = 0.093). VDR was found to be strongly expressed in all 28 MCC tumor specimens investigated. CONCLUSION: The association between vitamin D deficiency and MCC characteristics and outcome, together with detection of the VDR in MCC cells, suggest that vitamin D could influence the biology of MCC.

[Newly discovered forms of prion diseases in ruminants]. / Nouvelles formes de maladies à prions chez les ruminants.

Transmissible spongiform encephalopathies (TSEs), are fatal neurodegenerative diseases caused by unconventional agents, the prions. They are characterised by the accumulation in infected tissues of an abnormally folded form of the host-encoded prion protein (PrP). This pathological form is partially resistant to protease digestion, leading to the production of so-called PrP(res) fragments. Different isolates from the same host species may show different eletrophoretic profiles, reflecting the existence of different prion strains. The active surveillance of ruminant TSEs implemented in European countries, based on a large-scale biochemical testing of brain tissue samples from carcasses, has revealed PrP(res) profiles unnoticed so far. Experimental transmission of these atypical cases to various transgenic mouse lines has led to the recognition of a novel scrapie strain in sheep and goats, called Nor98, and of two variant strains of spongiform encephalopathy in cattle. This review is aimed at summarising the current knowledge on these newly recognised forms of ruminants TSEs, and at discussing their possible origin and potential implications in terms of animal and human health.

[Polyomavirus newly discovered]. / Virus Polyoma nouvellement découverts.

Three new polyoma viruses have been recently identified; two of them, the KI et WU viruses are present in nasopharyngeal aspirates during the course of acute respiratory infections. The incidence of these viruses is low compared to other respiratory viruses and the disease has not shown a high severity of clinical signs. The physiopathology of the diseases and the mode of cultivation of these viruses remain unknown. The third virus was discovered from cutaneous biopsies of Merkel tumours with a higher incidence than in tissue from healthy patients. Its mode of transmission and its role in the cancerogenesis need more studies. However, as the virus can integrate into the cellular DNA, it signifies that the virus may have a role in various human tumors.

A cell line infectible by prion strains from different species.

It has been shown previously that ovine prion protein (PrP(C)) renders rabbit epithelial RK13 cells permissive to the multiplication of ovine prions, thus providing evidence that species barriers can be crossed in cultured cells through the expression of a relevant PrP(C). The present study significantly extended this observation by showing that mouse and bank vole prions can be propagated in RK13 cells that express the corresponding PrP(C). Importantly, the respective molecular patterns of abnormal PrP (PrP(res)) and, where examined, the neuropathological features of the infecting strains appeared to be maintained during the propagation in cell culture. These findings indicate that RK13 cells can be genetically engineered to replicate prion strains faithfully from different species. Such an approach may facilitate investigations of the molecular basis of strain identity and prion diversity.

PrPSc accumulation in myocytes from sheep incubating natural scrapie.

Because variant Creutzfeldt-Jakob disease (vCJD) in humans probably results from consumption of products contaminated with tissue from animals with bovine spongiform encephalopathy, whether infectious prion protein is present in ruminant muscles is a crucial question. Here we show that experimentally and naturally scrapie-affected sheep accumulate the prion protein PrP(Sc) in a myocyte subset. In naturally infected sheep, PrP(Sc) is detectable in muscle several months before clinical disease onset. The relative amounts of PrP(Sc) suggest a 5,000-fold lower infectivity for muscle as compared to brain.

Unexpected high testis-specific transcriptional activity of the cyclin T1 promoter in transgenic mice.

The ubiquitously expressed cyclin T1 gene encodes for a protein involved in human immunodeficiency virus type 1 (HIV-1) transcription activation. The goat gene was recently shown to share an expression pattern similar to that of its endogenous counterpart when incorporated into mice using a BAC insert. To assess if its promoter could target ubiquitous expression of the bovine Prnp in transgenic mice, two constructs carrying either 1 or 30 kb of cyclin T1 5'-flanking sequences were built and microinjected. Both constructs resulted in the unexpected high male germ cell-specific expression of the prion protein. These data re-question the suspected location of the cyclin T1 gene regulatory elements.

Efficient and specific down-regulation of prion protein expression by RNAi.

Prion diseases are fatal neurodegenerative disorders associated with an abnormal isoform of the PrPc host-encoded protein. Invalidation of the Prnp gene, that encodes PrPc, led to transgenic mice that are viable, apparently healthy, and resistant to challenge by the infectious agent. These results indicated that a down-regulation of the Prnp gene expression is a potential therapeutic approach. In the present report, we demonstrate that RNAi targeted towards the Prnp mRNA can efficiently and highly specifically reduce the level of PrPc in transfected cells. It, thus, indicates that RNAi is an attractive therapeutic approach to fight against prion diseases.

Organization of two transmissible gastroenteritis coronavirus membrane protein topologies within the virion and core.

The difference in membrane (M) protein compositions between the transmissible gastroenteritis coronavirus (TGEV) virion and the core has been studied. The TGEV M protein adopts two topologies in the virus envelope, a Nexo-Cendo topology (with the amino terminus exposed to the virus surface and the carboxy terminus inside the virus particle) and a Nexo-Cexo topology (with both the amino and carboxy termini exposed to the virion surface). The existence of a population of M molecules adopting a Nexo-Cexo topology in the virion envelope was demonstrated by (i) immunopurification of (35)S-labeled TGEV virions using monoclonal antibodies (MAbs) specific for the M protein carboxy terminus (this immunopurification was inhibited only by deletion mutant M proteins that maintained an intact carboxy terminus), (ii) direct binding of M-specific MAbs to the virus surface, and (iii) mass spectrometry analysis of peptides released from trypsin-treated virions. Two-thirds of the total number of M protein molecules found in the virion were associated with the cores, and one-third was lost during core purification. MAbs specific for the M protein carboxy terminus were bound to native virions through the M protein in a Nexo-Cexo conformation, and these molecules were removed when the virus envelope was disrupted with NP-40 during virus core purification. All of the M protein was susceptible to N-glycosidase F treatment of the native virions, which indicates that all the M protein molecules are exposed to the virus surface. Cores purified from glycosidase-treated virions included M protein molecules that completely or partially lost the carbohydrate moiety, which strongly suggests that the M protein found in the cores was also exposed in the virus envelope and was not present exclusively in the virus interior. A TGEV virion structure integrating all the data is proposed. According to this working model, the TGEV virion consists of an internal core, made of the nucleocapsid and the carboxy terminus of the M protein, and the envelope, containing the spike (S) protein, the envelope (E) protein, and the M protein in two conformations. The two-thirds of the molecules that are in a Nexo-Cendo conformation (with their carboxy termini embedded within the virus core) interact with the internal core, and the remaining third of the molecules, whose carboxy termini are in a Nexo-Cexo conformation, are lost during virus core purification.

Markedly increased susceptibility to natural sheep scrapie of transgenic mice expressing ovine prp.

The susceptibility of sheep to scrapie is known to involve, as a major determinant, the nature of the prion protein (PrP) allele, with the VRQ allele conferring the highest susceptibility to the disease. Transgenic mice expressing in their brains three different ovine PrP(VRQ)-encoding transgenes under an endogenous PrP-deficient genetic background were established. Nine transgenic (tgOv) lines were selected and challenged with two scrapie field isolates derived from VRQ-homozygous affected sheep. All inoculated mice developed neurological signs associated with a transmissible spongiform encephalopathy (TSE) disease and accumulated a protease-resistant form of PrP (PrPres) in their brains. The incubation duration appeared to be inversely related to the PrP steady-state level in the brain, irrespective of the transgene construct. The survival time for animals from the line expressing the highest level of PrP was reduced by at least 1 year compared to those of two groups of conventional mice. With one isolate, the duration of incubation was as short as 2 months, which is comparable to that observed for the rodent TSE models with the briefest survival times. No survival time reduction was observed upon subpassaging of either isolate, suggesting no need for adaptation of the agent to its new host. Overexpression of the transgene was found not to be required for transmission to be accelerated compared to that observed with wild-type mice. Conversely, transgenic mice overexpressing murine PrP were found to be less susceptible than tgOv lines expressing ovine PrP at physiological levels. These data argue that ovine PrP(VRQ) provided a better substrate for sheep prion replication than did mouse PrP. Altogether, these tgOv mice could be an improved model for experimental studies on natural sheep scrapie.

Ex vivo propagation of infectious sheep scrapie agent in heterologous epithelial cells expressing ovine prion protein.

Transmissible spongiform encephalopathies, or prion diseases, are fatal degenerative disorders of the central nervous system that affect humans and animals. Prions are nonconventional infectious agents whose replication depends on the host prion protein (PrP). Transmission of prions to cultured cells has proved to be a particularly difficult task, and with a few exceptions, their experimental propagation relies on inoculation to laboratory animals. Here, we report on the development of a permanent cell line supporting propagation of natural sheep scrapie. This model was obtained by stable expression of a tetracycline-regulatable ovine PrP gene in a rabbit epithelial cell line. After exposure to scrapie agent, cultures were repeatedly found to accumulate high levels of abnormal PrP (PrPres). Cell extracts induced a scrapie-like disease in transgenic mice overexpressing ovine PrP. These cultures remained healthy and stably infected upon subpassaging. Such data show that (i) cultivated cells from a nonneuronal origin can efficiently replicate prions; and (ii) species barrier can be crossed ex vivo through the expression of a relevant PrP gene. This approach led to the ex vivo propagation of a natural transmissible spongiform encephalopathy agent (i.e., without previous experimental adaptation to rodents) and might be applied to human or bovine prions.

The membrane M protein carboxy terminus binds to transmissible gastroenteritis coronavirus core and contributes to core stability.

The architecture of transmissible gastroenteritis coronavirus includes three different structural levels, the envelope, an internal core, and the nucleocapsid that is released when the core is disrupted. Starting from purified virions, core structures have been reproducibly isolated as independent entities. The cores were stabilized at basic pH and by the presence of divalent cations, with Mg(2+) ions more effectively contributing to core stability. Core structures showed high resistance to different concentrations of detergents, reducing agents, and urea and low concentrations of monovalent ions (<200 mM). Cores were composed of the nucleoprotein, RNA, and the C domain of the membrane (M) protein. At high salt concentrations (200 to 300 mM), the M protein was no longer associated with the nucleocapsid, which resulted in destruction of the core structure. A specific ionic interaction between the M protein carboxy terminus and the nucleocapsid was demonstrated using three complementary approaches: (i) a binding assay performed between a collection of M protein amino acid substitution or deletion mutants and purified nucleocapsids that led to the identification of a 16-amino-acid (aa) domain (aa 237 to 252) as being responsible for binding the M protein to the nucleocapsid; (ii) the specific inhibition of this binding by monoclonal antibodies (MAbs) binding to a carboxy-terminal M protein domain close to the indicated peptide but not by MAbs specific for the M protein amino terminus; and (iii) a 26-residue peptide, including the predicted sequence (aa 237 to 252), which specifically inhibited the binding. Direct binding of the M protein to the nucleoprotein was predicted, since degradation of the exposed RNA by RNase treatment did not affect the binding. It is proposed that the M protein is embedded within the virus membrane and that the C region, exposed to the interior face of the virion in a population of these molecules, interacts with the nucleocapsid to which it is anchored, forming the core. Only the C region of the M protein is part of the core.

Establishment of astrocyte cell lines from sheep genetically susceptible to scrapie.

Primary cultures of the brain from sheep embryos were used to establish cell lines after transfection by the simian virus 40 (SV40) large T gene. Two of the lines (A15 and 4A6) displayed astroglial properties. They expressed the glial fibrillary acidic protein (GFAP), intermediate filament protein vimentin, and S-100 (beta-subunit) protein. While numerous rodent and human glial cell lines are available, this is to our knowledge the first description of ovine cell lines with astrocyte features. In addition, these cell lines were derived from sheep embryos chosen for their genetic susceptibility to scrapie (PrP genotype: VV136, QQ171). Therefore, they could be attractive tissue culture models for the study of propagation and pathogenesis of the scrapie agent ex vivo.

Successful transmission of three mouse-adapted scrapie strains to murine neuroblastoma cell lines overexpressing wild-type mouse prion protein.

Propagation of the agents responsible for transmissible spongiform encephalopathies (TSEs) in cultured cells has been achieved for only a few cell lines. To establish efficient and versatile models for transmission, we developed neuroblastoma cell lines overexpressing type A mouse prion protein, MoPrP(C)-A, and then tested the susceptibility of the cells to several different mouse-adapted scrapie strains. The transfected cell clones expressed up to sixfold-higher levels of PrP(C) than the untransfected cells. Even after 30 passages, we were able to detect an abnormal proteinase K-resistant form of prion protein, PrP(Sc), in the agent-inoculated PrP-overexpressing cells, while no PrP(Sc) was detectable in the untransfected cells after 3 passages. Production of PrP(Sc) in these cells was also higher and more stable than that seen in scrapie-infected neuroblastoma cells (ScN2a). The transfected cells were susceptible to PrP(Sc)-A strains Chandler, 139A, and 22L but not to PrP(Sc)-B strains 87V and 22A. We further demonstrate the successful transmission of PrP(Sc) from infected cells to other uninfected cells. Our results corroborate the hypothesis that the successful transmission of agents ex vivo depends on both expression levels of host PrP(C) and the sequence of PrP(Sc). This new ex vivo transmission model will facilitate research into the mechanism of host-agent interactions, such as the species barrier and strain diversity, and provides a basis for the development of highly susceptible cell lines that could be used in diagnostic and therapeutic approaches to the TSEs.

Obtention of porcine aminopeptidase-n transgenic mice and analysis of their susceptibility to transmissible gastroenteritis virus.

To obtain a laboratory animal model for transmissible gastroenteritis virus (TGEV) infection, transgenic mice (Tg) were produced by introducing two porcine aminopeptidase-n (APN) cDNA-derived constructs into the mouse genome. In the first construct, the APN cDNA was fused in 5' with the 1 kb upstream region of the APN gene and in 3' with the SV40 small intron and polyadenylation site. In the second construct, the 5' end of the APN cDNA was replaced by the corresponding domain of the APN gene comprising the three first introns, an additional intron (the rabbit beta-like globine intron 2) was inserted at the 3' extremity of the construct and the resulting DNA stretch was placed under the control of the rat intestinal fatty acid-binding protein (I-FABP) gene promoter. Transgenes were obtained with these two constructs, and RNA expression was evidenced by RT-PCR with the second construct in a transgene lineage. Using two different immunoassays, expression of the porcine APN protein was not detected in the transgenic intestines of animals of the RT-PCR positive lineage. Northern blot analyses did not revealed TGEV replication in infected adult mice. Additional assays will be carried out on young animals to detect potential TGEV susceptibility.

Interferon alpha inducing property of coronavirus particles and pseudoparticles.

Previous work in our laboratory have provided evidence that the membrane glycoprotein M of TGEV is centrally involved in efficient induction of alpha interferon (IFN-alpha) synthesis by non-immune peripheral blood mononuclear cells incubated with fixed, TGEV-infected cells or inactivated virions. Here we report recent completion of studies initiated to get a better understanding of the nature of the interferogenic determinant(s). Transfected cells expressing TGEV M together with the minor structural component E (formerly called sM) were found to trigger IFN-alpha synthesis. Co-expression of these two proteins was shown to be necessary and sufficient for assembly and release of pseudoparticles resembling TGEV virions. Purified pseudoparticles exhibited an interferogenic activity close to that of authentic virions. Chimeric recombinant particles expressing BCV M ectodomain also induced IFN. Examination of cell cultures infected by viruses representative of the three Nidovirales genera revealed that the capacity to act as an efficient IFN-alpha inducer is a common feature of viral particles of the coronavirus genus. Altogether these data bring new insights regarding the putative nature of the viral structure involved in IFN-alpha induction.

Is the sialic acid binding activity of the S protein involved in the enteropathogenicity of transmissible gastroenteritis virus?

Transmissible gastroenteritis virus (TGEV) is able to recognize sialic acid on sialo-glycoconjugates. Analysis of mutants indicated that single point mutations in the S protein (around amino acids 145-155) of TGEV may result both in the loss of the sialic acid binding activity and in a drastic reduction of the enteropathogenicity. From this observation we conclude that the sialic acid binding activity is involved in the enteropathogenicity of TGEV. On the basis of our recent results we propose that binding of sialylated macromolecules to the virions surface may increase virus stability. This in turn would explain how TGEV as an enveloped virus can survive the gastrointestinal passage and cause intestinal infections.

Coronavirus pseudoparticles formed with recombinant M and E proteins induce alpha interferon synthesis by leukocytes.

Transmissible gastroenteritis virus (TGEV), an enteric coronavirus of swine, is a potent inducer of alpha interferon (IFN-alpha) both in vivo and in vitro. Incubation of peripheral blood mononuclear cells with noninfectious viral material such as inactivated virions or fixed, infected cells leads to early and strong IFN-alpha synthesis. Previous studies have shown that antibodies against the virus membrane glycoprotein M blocked the IFN induction and that two viruses with a mutated protein exhibited a decreased interferogenic activity, thus arguing for a direct involvement of M protein in this phenomenon. In this study, the IFN-alpha-inducing activity of recombinant M protein expressed in the absence or presence of other TGEV structural proteins was examined. Fixed cells coexpressing M together with at least the minor structural protein E were found to induce IFN-alpha almost as efficiently as TGEV-infected cells. Pseudoparticles resembling authentic virions were released in the culture medium of cells coexpressing M and E proteins. The interferogenic activity of purified pseudoparticles was shown to be comparable to that of TGEV virions, thus establishing that neither ribonucleoprotein nor spikes are required for IFN induction. The replacement of the externally exposed, N-terminal domain of M with that of bovine coronavirus (BCV) led to the production of chimeric particles with no major change in interferogenicity, although the structures of the TGEV and BCV ectodomains markedly differ. Moreover, BCV pseudoparticles also exhibited interferogenic activity. Together these observations suggest that the ability of coronavirus particles to induce IFN-alpha is more likely to involve a specific, multimeric structure than a definite sequence motif.