The mechanism by which DNA viruses interact with different DNA sensors and their connection with the activation of interferon (IFN) type I pathway are poorly understood. We investigated the roles of protein 204 (p204) and cyclic guanosine-adenosine synthetase (cGAS) sensors during infection with mouse polyomavirus (MPyV). The phosphorylation of IFN regulatory factor 3 (IRF3) and the stimulator of IFN genes (STING) proteins and the upregulation of IFN beta (IFN-β) and MX Dynamin Like GTPase 1 (MX-1) genes were detected at the time of replication of MPyV genomes in the nucleus. STING knockout abolished the IFN response. Infection with a mutant virus that exhibits defective nuclear entry via nucleopores and that accumulates in the cytoplasm confirmed that replication of viral genomes in the nucleus is required for IFN induction. The importance of both DNA sensors, p204 and cGAS, in MPyV-induced IFN response was demonstrated by downregulation of the IFN pathway observed in p204-knockdown and cGAS-knockout cells. Confocal microscopy revealed the colocalization of p204 with MPyV genomes in the nucleus. cGAS was found in the cytoplasm, colocalizing with viral DNA leaked from the nucleus and with DNA within micronucleus-like bodies, but also with the MPyV genomes in the nucleus. However, 2'3'-Cyclic guanosine monophosphate-adenosine monophosphate synthesized by cGAS was detected exclusively in the cytoplasm. Biochemical assays revealed no evidence of functional interaction between cGAS and p204 in the nucleus. Our results provide evidence for the complex interactions of MPyV and DNA sensors including the sensing of viral genomes in the nucleus by p204 and of leaked viral DNA and micronucleus-like bodies in the cytoplasm by cGAS.
- MeSH
- DNA virů genetika imunologie MeSH
- fosfoproteiny antagonisté a inhibitory genetika metabolismus MeSH
- fosforylace MeSH
- infekce onkogenními viry imunologie virologie MeSH
- interakce hostitele a patogenu MeSH
- interferon beta metabolismus MeSH
- jaderné proteiny antagonisté a inhibitory genetika metabolismus MeSH
- membránové proteiny antagonisté a inhibitory genetika metabolismus MeSH
- myši MeSH
- nukleotidyltransferasy antagonisté a inhibitory genetika metabolismus MeSH
- polyomavirové infekce imunologie virologie MeSH
- Polyomavirus genetika imunologie MeSH
- přirozená imunita imunologie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Human polyomavirus (HPyV)6 and HPyV7 are shed chronically from human skin. HPyV7, but not HPyV6, has been linked to a pruritic skin eruption of immunosuppression. OBJECTIVE: We determined whether biopsy specimens showing a characteristic pattern of dyskeratosis and parakeratosis might be associated with polyomavirus infection. METHODS: We screened biopsy specimens showing "peacock plumage" histology by polymerase chain reaction for HPyVs. Cases positive for HPyV6 or HPyV7 were then analyzed by immunohistochemistry, electron microscopy, immunofluorescence, quantitative polymerase chain reaction, and complete sequencing, including unbiased, next-generation sequencing. RESULTS: We identified 3 additional cases of HPyV6 or HPyV7 skin infections. Expression of T antigen and viral capsid was abundant in lesional skin. Dual immunofluorescence staining experiments confirmed that HPyV7 primarily infects keratinocytes. High viral loads in lesional skin compared with normal-appearing skin and the identification of intact virions by both electron microscopy and next-generation sequencing support a role for active viral infections in these skin diseases. LIMITATION: This was a small case series of archived materials. CONCLUSION: We have found that HPyV6 and HPyV7 are associated with rare, pruritic skin eruptions with a distinctive histologic pattern and describe this entity as "HPyV6- and HPyV7-associated pruritic and dyskeratotic dermatoses."
- MeSH
- antigeny virové nádorové analýza MeSH
- biopsie MeSH
- dospělí MeSH
- keratinocyty virologie MeSH
- keratóza patologie virologie MeSH
- kůže patologie virologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- polyomavirové infekce komplikace virologie MeSH
- Polyomavirus genetika imunologie izolace a purifikace MeSH
- pruritus patologie virologie MeSH
- retrospektivní studie MeSH
- studie případů a kontrol MeSH
- virová nálož MeSH
- virové plášťové proteiny analýza MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
The aim of this study was to develop a suitable vaccine antigen against porcine circovirus 2 (PCV2), the causative agent of post-weaning multi-systemic wasting syndrome, which causes significant economic losses in swine breeding. Chimeric antigens containing PCV2b Cap protein sequences based on the mouse polyomavirus (MPyV) nanostructures were developed. First, universal vectors for baculovirus-directed production of chimeric MPyV VLPs or pentamers of the major capsid protein, VP1, were designed for their exploitation as vaccines against other pathogens. Various strategies were employed based on: A) exposure of selected immunogenic epitopes on the surface of MPyV VLPs by insertion into a surface loop of the VP1 protein, B) insertion of foreign protein molecules inside the VLPs, or C) fusion of a foreign protein or its part with the C-terminus of VP1 protein, to form giant pentamers of a chimeric protein. We evaluated these strategies by developing a recombinant vaccine against porcine circovirus 2. All candidate vaccines induced the production of antibodies against the capsid protein of porcine circovirus after immunization of mice. The candidate vaccine, Var C, based on fusion of mouse polyomavirus and porcine circovirus capsid proteins, could induce the production of antibodies with the highest PCV2 neutralizing capacity. Its ability to induce the production of neutralization antibodies was verified after immunization of pigs. The advantage of this vaccine, apart from its efficient production in insect cells and easy purification, is that it represents a DIVA (differentiating infected from vaccinated animals) vaccine, which also induces an immune response against the mouse polyoma VP1 protein and is thus able to distinguish between vaccinated and naturally infected animals.
- MeSH
- Circovirus * genetika imunologie MeSH
- myši MeSH
- nanostruktury * MeSH
- Polyomavirus * genetika imunologie MeSH
- prasata MeSH
- rekombinantní fúzní proteiny * genetika imunologie MeSH
- Sf9 buňky MeSH
- Spodoptera MeSH
- virové plášťové proteiny * genetika imunologie MeSH
- virové vakcíny * genetika imunologie farmakologie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Human polyomaviruses HPyV6, HPyV7, TSPyV, HPyV9, MWPyV, and KIPyV have been discovered between 2007 and 2012. TSPyV causes a rare skin disease trichodysplasia spinulosa in immunocompromised patients, the role of remaining polyomaviruses in human pathology is not clear. In this study, we assessed the occurrence of serum antibodies against above polyomaviruses in healthy blood donors. Serum samples were examined by enzyme-linked immunoassay (ELISA), using virus-like particles (VLPs) based on the major VP1 capsid proteins of these viruses. Overall, serum antibodies against HPyV6, HPyV7, TSPyV, HPyV9, MWPyV, and KIPyV were found in 88.2%, 65.7%, 63.2%, 31.6%, 84.4%, and 58%, respectively, of this population. The seroprevalence generally increased with age, the highest rise we observed for HPyV9 and KIPyV specific antibodies. The levels of anti-HPyV antibodies remained stable across the age-groups, except for TSPyV and HPyV9, where we saw change with age. ELISAs based on VLP and GST-VP1 gave comparable seroprevalence for HPyV6 antibodies (88.2% vs.85.3%) but not for HPyV7 antibodies (65.7% vs. 77.2%), suggesting some degree of crossreactivity between HPyV6 and HPyV7 VP1 proteins. In conclusion, these results provide evidence that human polyomaviruses HPyV6, HPyV7, TSPyV, HPyV9, MwPyV, and KIPyV circulate widely in the Czech population and their seroprevalence is comparable to other countries.
- MeSH
- dárci krve * MeSH
- dospělí MeSH
- imunokompromitovaný pacient MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- myši MeSH
- polyomavirové infekce epidemiologie imunologie virologie MeSH
- Polyomavirus klasifikace genetika imunologie MeSH
- protilátky virové krev MeSH
- senioři MeSH
- séroepidemiologické studie MeSH
- virion imunologie izolace a purifikace MeSH
- virové plášťové proteiny genetika imunologie MeSH
- zdraví dobrovolníci pro lékařské studie MeSH
- zkřížené reakce MeSH
- zvířata MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- myši MeSH
- senioři MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Československo epidemiologie MeSH
JC virus (JCPyV) has gained novel clinical importance as cause of progressive multifocal leukoencephalopathy (PML), a rare demyelinating disease recently associated to immunomodulatory drugs, such as natalizumab used in multiple sclerosis (MS) cases. Little is known about the mechanisms leading to PML, and this makes the need of PML risk stratification among natalizumab-treated patients very compelling. Clinical and laboratory-based risk-stratification markers have been proposed, one of these is represented by the JCPyV-seropositive status, which includes about 54% of MS patients. We recently proposed to investigate the possible protective role of neutralizing humoral immune response in preventing JCPyV reactivation. In this proof-of-concept study, by cloning the first human monoclonal antibody (GRE1) directed against a neutralizing epitope on JCPyV/VP1, we optimized a robust anti-JCPyV neutralization assay. This allowed us to evaluate the neutralizing activity in JCPyV-positive sera from MS patients, demonstrating the lack of correlation between the level of anti-JCPyV antibody and anti-JCPyV neutralizing activity. Relevant consequences may derive from future clinical studies induced by these findings; indeed the study of the serum anti-JCPyV neutralizing activity could allow not only a better risk stratification of the patients during natalizumab treatment, but also a better understanding of the pathophysiological mechanisms leading to PML, highlighting the contribution of peripheral versus central nervous system JCPyV reactivation. Noteworthy, the availability of GRE1 could allow the design of novel immunoprophylactic strategies during the immunomodulatory treatment.
- MeSH
- epitopy genetika imunologie MeSH
- humanizované monoklonální protilátky škodlivé účinky terapeutické užití MeSH
- klonování DNA MeSH
- lidé MeSH
- monoklonální protilátky genetika imunologie MeSH
- neutralizační testy metody MeSH
- neutralizující protilátky genetika imunologie MeSH
- Polyomavirus imunologie MeSH
- progresivní multifokální leukoencefalopatie imunologie prevence a kontrola MeSH
- protilátky virové genetika imunologie MeSH
- roztroušená skleróza terapie MeSH
- virové plášťové proteiny antagonisté a inhibitory imunologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Mouse polyomavirus-like particles (MPyV-VLPs) carrying inside a fragment of the Bcr-Abl hybrid protein containing the epitope of chronic myeloid leukemia fusion region were prepared. A sequence encoding 171 amino acids covering Bcr-Abl breakpoint was fused to the C-terminal part of VP3 minor protein connecting it to the VP1 capsomeres. Chimeric particles, the Bcr-Abl VLPs, were tested for their ability to induce Bcr-Abl specific immune response in mice after their intranasal (i.n.) or intraperitoneal (i.p.) administration without any other adjuvants. Bcr-Abl VLPs induced strong anti-VP1 immune response in both i.n. and i.p. immunized mice. As expected, neither IgG nor IgM anti-Bcr-Abl specific antibodies were detected in the sera of immunized animals. Surprisingly, no specific CTL (cytotoxic T-lymphocyte) activity was proved using two different methods (in vitro cytotoxicity assay with CFSE-labeled target cells and highly sensitive cytotoxicity assay using MHC class I Bcr-Abl specific pentamers). In addition, no proliferative response of T-cells of i.n. immunized mice after in vitro restimulation with antigen-pulsed bone marrow-derived dendritic cells was observed. Taken together, Bcr-Abl breakpoint epitopes appeared to be weak immunogens and even MPyV-VLPs did not provide sufficient adjuvant ability to support induction of immune responses specific to Bcr-Abl fusion zone epitope.
- MeSH
- antigeny virové imunologie MeSH
- bcr-abl fúzní proteiny imunologie MeSH
- chronická myeloidní leukemie imunologie MeSH
- cytotoxicita imunologická MeSH
- epitopy imunologie MeSH
- fluorescenční protilátková technika MeSH
- imunoelektronová mikroskopie MeSH
- lidé MeSH
- myši MeSH
- Polyomavirus imunologie MeSH
- rekombinantní proteiny imunologie MeSH
- western blotting MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
Mouse polyomavirus (MPyV) VP1-pseudocapsids carrying enhanced green fluorescent protein (EGFP-VLPs) were used for intranasal immunization of mice. EGFP-VLPs induced strong anti-VP1 but not anti-EGFP antibody production. In vitro restimulation with antigen-pulsed bone marrow-derived dendritic cells (BMDCs) induced remarkable T-cell proliferative response specific for both VP1 and EGFP antigen and IL-2 and IFN-gamma production. Surprisingly, no specific cytotoxic activities against VP1 and EGFP proteins were detected. After intranasal administration of EGFP-VLPs, as well as after polyomavirus infection, a moderate reduction of CD4(+)CD25(+)Foxp3(+) T cells was observed in spleens but not in lymph nodes and peripheral blood, suggesting that both MPyV virions and pseudocapsids are able to induce changes in distribution of regulatory T cells. Treatment of EGFP-VLPs pulsed BMDCs with inhibitors of endosomal acidification proved that presentation of peptides on MHCgp class II is dependent on acidic endosomal environment. Substantial decrease of CD4-specific T-cell proliferation in the presence of proteasome inhibitor suggests that MHCgp class II might load VPL-derived peptides processed by proteasomes. Thus, polyomavirus derived VLPs appear to be promising delivery and adjuvant vehicles for therapeutic proteins.
- MeSH
- aplikace intranazální MeSH
- cytotoxické T-lymfocyty imunologie MeSH
- cytotoxické testy imunologické MeSH
- financování organizované MeSH
- interferon gama biosyntéza MeSH
- interleukin-2 biosyntéza MeSH
- krev imunologie MeSH
- lymfatické uzliny imunologie MeSH
- myši MeSH
- Polyomavirus imunologie MeSH
- proliferace buněk MeSH
- protilátky virové krev MeSH
- rekombinantní fúzní proteiny aplikace a dávkování imunologie MeSH
- slezina imunologie MeSH
- T-lymfocyty - podskupiny imunologie MeSH
- T-lymfocyty imunologie MeSH
- vakcíny VLP aplikace a dávkování imunologie MeSH
- virové plášťové proteiny aplikace a dávkování imunologie MeSH
- zelené fluorescenční proteiny imunologie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
