The minor structural protein VP2 and its shorter variant, VP3, of mouse polyomavirus (MPyV) are essential for virus exit from the endoplasmic reticulum (ER) during viral trafficking to the nucleus. Here, we followed the role of putative hydrophobic domains (HD) of the minor proteins in membrane affinity and viral infectivity. We prepared variants of VP2, each mutated to decrease hydrophobicity of one of three predicted hydrophobic domains: VP2-mHD1, VP2-mHD2 or VP2-mHD3 mutated in HD1 (amino acids (aa) 60-101), HD2 (aa 125-165) or HD3 (aa 287-307), respectively. Transient production of the mutated proteins revealed that only VP2-mHD2 lost the affinity for intracellular membranes. Cytotoxicity connected with the ability of VP2/VP3 to perforate membranes decreased markedly for VP2-mHD2, but only slightly for VP2-mHD1. The mutant VP2-mHD3 exhibited properties similar to the wild-type protein. MPyV genomes, each carrying one of the mutations, were prepared for virus production. MPyV-mHD1 and MPyV-mHD2 viruses could be isolated, while the HD3 mutation in VP2/VP3 prevented virus assembly. We found that both MPyV-mHD1 and MPyV-mHD2 viruses arrived at the ER without delay and were processed by ER residential enzymes. However, the ability to associate with ER membranes was decreased in the case of MPyV-mHD1 and practically abolished in the case of MPyV-mHD2. Interestingly, while MPyV-mHD2 was not infectious, infection of MPyV-mHD1 virus was delayed. These findings reveal that HD2, common to both VP2 and VP3, is responsible for the membrane binding properties of the minor proteins, while HD1 of VP2 is likely required to stabilize VP2-membrane association and to enhance viral exit from the ER.

• MeSH
• buněčné jádro metabolismus   MeSH
• endoplazmatické retikulum genetika   metabolismus   MeSH
• hydrofobní a hydrofilní interakce MeSH
• intracelulární membrány metabolismus   MeSH
• lidé MeSH
• myši MeSH
• Polyomavirus genetika   metabolismus   patogenita   MeSH
• proteinové domény MeSH
• sekvence aminokyselin MeSH
• vazba proteinů MeSH
• virové plášťové proteiny genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• úvodníky MeSH

The tumorigenic potential of mouse polyomavirus (MPyV) has been studied for decades in cell culture models and has been mainly attributed to nonstructural middle T antigen (MT), which acts as a scaffold signal adaptor, activates Src tyrosine kinases, and possesses transforming ability. We hypothesized that MPyV could also transform mouse cells independent of MT via a Toll-like receptor 4 (TLR4)-mediated inflammatory mechanism. To this end, we investigated the interaction of MPyV with TLR4 in mouse embryonic fibroblasts (MEFs) and 3T6 cells, resulting in secretion of interleukin 6 (IL-6), independent of active viral replication. TLR4 colocalized with MPyV capsid protein VP1 in MEFs. Neither TLR4 activation nor recombinant IL-6 inhibited MPyV replication in MEFs and 3T6 cells. MPyV induced STAT3 phosphorylation through both direct and MT-dependent and indirect and TLR4/IL-6-dependent mechanisms. We demonstrate that uninfected mouse fibroblasts exposed to the cytokine environment from MPyV-infected fibroblasts upregulated the expressions of MCP-1, CCL-5, and α-SMA. Moreover, the cytokine microenvironment increased the invasiveness of MEFs and CT26 carcinoma cells. Collectively, TLR4 recognition of MPyV induces a cytokine environment that promotes the cancer-associated fibroblast (CAF)-like phenotype in noninfected fibroblasts and increases cell invasiveness.

• Publikační typ
• časopisecké články MeSH

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

Infection of non-enveloped polyomaviruses depends on an intact microtubular network. Here we focus on mouse polyomavirus (MPyV). We show that the dynamics of MPyV cytoplasmic transport reflects the characteristics of microtubular motor-driven transport with bi-directional saltatory movements. In cells treated with microtubule-disrupting agents, localization of MPyV was significantly perturbed, the virus was retained at the cell periphery, mostly within membrane structures resembling multicaveolar complexes, and at later times post-infection, only a fraction of the virus was found in Rab7-positive endosomes and multivesicular bodies. Inhibition of cytoplasmic dynein-based motility by overexpression of dynamitin affected perinuclear translocation of the virus, delivery of virions to the ER and substantially reduced the numbers of infected cells, while overexpression of dominant-negative form of kinesin-1 or kinesin-2 had no significant impact on virus localization and infectivity. We also found that transport along microtubules was important for MPyV-containing endosome sequential acquisition of Rab5, Rab7 and Rab11 GTPases. However, in contrast to dominant-negative mutant of Rab7 (T22N), overexpression of dominant-negative mutant Rab11 (S25N) did not affect the virus infectivity. Altogether, our study revealed that MPyV cytoplasmic trafficking leading to productive infection bypasses recycling endosomes, does not require the function of kinesin-1 and kinesin-2, but depends on functional dynein-mediated transport along microtubules for translocation of the virions from peripheral, often caveolin-positive compartments to late endosomes and ER - a prerequisite for efficient delivery of the viral genome to the nucleus.

• MeSH
• buněčné linie MeSH
• endocytóza * MeSH
• endoplazmatické retikulum metabolismus   virologie   MeSH
• endozomy metabolismus   virologie   MeSH
• mikrotubulární proteiny metabolismus   MeSH
• mikrotubuly metabolismus   MeSH
• molekulární motory metabolismus   MeSH
• myši MeSH
• Polyomavirus metabolismus   MeSH
• viabilita buněk MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

VP1, the major structural protein of the mouse polyomavirus (MPyV), is the major architectural component of the viral capsid. Its pentamers are able to self-assemble into capsid-like particles and to non-specifically bind DNA. Surface loops of the protein interact with sialic acid of ganglioside receptors. Although the replication cycle of the virus, including virion morphogenesis, proceeds in the cell nucleus, a substantial fraction of the protein is detected in the cytoplasm of late-phase MPyV-infected cells. In this work, we detected VP1 mainly in the cytoplasm of mammalian cells transfected with plasmid expressing VP1. In the cytoplasm, VP1-bound microtubules, including the mitotic spindle, and the interaction of VP1 with microtubules resulted in cell cycle block at the G2/M phase. Furthermore, in the late phase of MPyV infection and in cells expressing VP1, microtubules were found to be hyperacetylated. We then sought to understand how VP1 interacts with microtubules. Dynein is not responsible for the VP1-microtubule association, as neither overexpression of p53/dynamitin nor treatment with ciliobrevin-D (an inhibitor of dynein activity) prevented binding of VP1 to microtubules. A pull-down assay for VP1-interacting proteins identified the heat shock protein 90 (Hsp90) chaperone, and Hsp90 was also detected in the VP1-microtubule complexes. Although Hsp90 is known to be associated with acetylated microtubules, it does not mediate the interaction between VP1 and microtubules. Our study provides insight into the role of the major structural protein in MPyV replication, indicating that VP1 is a multifunctional protein that participates in the regulation of cell cycle progression in MPyV-infected cells.

• MeSH
• acetylace MeSH
• buněčné jádro metabolismus   virologie   MeSH
• buňky NIH 3T3 MeSH
• cytoplazma metabolismus   virologie   MeSH
• epitelové buňky metabolismus   virologie   MeSH
• exprese genu MeSH
• HEK293 buňky MeSH
• HeLa buňky MeSH
• interakce hostitele a patogenu MeSH
• kontrolní body fáze G2 buněčného cyklu MeSH
• lidé MeSH
• mikrotubuly metabolismus   virologie   MeSH
• mléčné žlázy zvířat metabolismus   virologie   MeSH
• myši MeSH
• plazmidy chemie   metabolismus   MeSH
• Polyomavirus genetika   metabolismus   MeSH
• proteiny tepelného šoku HSP90 genetika   metabolismus   MeSH
• transfekce MeSH
• vazba proteinů MeSH
• virion genetika   metabolismus   MeSH
• virové plášťové proteiny genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Mouse polyomavirus (MPyV) is considered a potential tool for the application of gene therapy; however, the current knowledge of the encapsulation of DNA into virions is vague. We used a series of assays based on the encapsidation of a reporter vector into MPyV pseudovirions to identify putative cis-acting elements that are involved in DNA encapsidation. None of the sequences that were derived from MPyV have been shown to solely enhance the encapsidation of a reporter vector in the assay. The frequency of encapsidation strongly correlated with the total intracellular amount of the vector after transfection. The encapsidation of target DNA into the pseudovirions was shown to be non-specific, and the packaging of non-replicated DNA was observed. We propose that the actual concentration of target DNA at the sites of virion formation is the primary factor that determines its selection for encapsidation.

• MeSH
• buněčné linie MeSH
• genetická terapie přístrojové vybavení   MeSH
• genetické vektory genetika   fyziologie   MeSH
• kapsida metabolismus   MeSH
• lidé MeSH
• myši MeSH
• Polyomavirus genetika   fyziologie   MeSH
• reportérové geny MeSH
• sestavení viru * MeSH
• virion genetika   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Mouse polyomavirus (MPyV) is a member of the Polyomaviridae family, which comprises non-enveloped tumorigenic viruses infecting various vertebrates including humans and causing different pathogenic responses in the infected organisms. Despite the variations in host tropism and pathogenicity, the structure of the virions of these viruses is similar. The capsid, with icosahedral symmetry (ø, 45 nm, T = 7d), is composed of a shell of 72 capsomeres of structural proteins, arranged around the nucleocore containing approximately 5-kbp-long circular dsDNA in complex with cellular histones. MPyV has been one of the most studied polyomaviruses and serves as a model virus for studies of the mechanisms of cell transformation and virus trafficking, and for use in nanotechnology. It can be propagated in primary mouse cells (e.g., in whole mouse embryo cells) or in mouse epithelial or fibroblast cell lines. In this unit, propagation, purification, quantification, and storage of MPyV virions are presented.

• MeSH
• kultivace virů metody   MeSH
• kultivované buňky MeSH
• myši MeSH
• ochrana biologická metody   MeSH
• Polyomavirus růst a vývoj   izolace a purifikace   MeSH
• virová nálož metody   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Viruses have evolved mechanisms to manipulate microtubules (MTs) for the efficient realization of their replication programs. Studying the mechanisms of replication of mouse polyomavirus (MPyV), we observed previously that in the late phase of infection, a considerable amount of the main structural protein, VP1, remains in the cytoplasm associated with hyperacetylated microtubules. VP1-microtubule interactions resulted in blocking the cell cycle in the G2/M phase. We are interested in the mechanism leading to microtubule hyperacetylation and stabilization and the roles of tubulin acetyltransferase 1 (αTAT1) and deacetylase histone deacetylase 6 (HDAC6) and VP1 in this mechanism. Therefore, HDAC6 inhibition assays, αTAT1 knock out cell infections, in situ cell fractionation, and confocal and TIRF microscopy were used. The experiments revealed that the direct interaction of isolated microtubules and VP1 results in MT stabilization and a restriction of their dynamics. VP1 leads to an increase in polymerized tubulin in cells, thus favoring αTAT1 activity. The acetylation status of MTs did not affect MPyV infection. However, the stabilization of MTs by VP1 in the late phase of infection may compensate for the previously described cytoskeleton destabilization by MPyV early gene products and is important for the observed inhibition of the G2→M transition of infected cells to prolong the S phase.

• MeSH
• acetylace MeSH
• acetyltransferasy genetika   metabolismus   MeSH
• buněčné linie MeSH
• buněčný cyklus MeSH
• cytoplazma metabolismus   MeSH
• fibroblasty virologie   MeSH
• histondeacetylasa 6 genetika   metabolismus   MeSH
• interakce mikroorganismu a hostitele * MeSH
• mikrotubuly metabolismus   virologie   MeSH
• myši MeSH
• Polyomavirus genetika   metabolismus   MeSH
• posttranslační úpravy proteinů MeSH
• tubulin metabolismus   MeSH
• virové plášťové proteiny genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The mechanism used by mouse polyomavirus (MPyV) overcomes the crowded cytosol to reach the nucleus has not been fully elucidated. Here, we investigated the involvement of importin α/β1 mediated transport in the delivery of MPyV genomes into the nucleus. Interactions of the virus with importin β1 were studied by co-immunoprecipitation and proximity ligation assay. For infectivity and nucleus delivery assays, the virus and its capsid proteins mutated in the nuclear localization signals (NLSs) were prepared and produced. We found that at early times post infection, virions bound importin β1 in a time dependent manner with a peak of interactions at 6 h post infection. Mutation analysis revealed that only when the NLSs of both VP1 and VP2/3 were disrupted, virus did not bind efficiently to importin β1 and its infectivity remarkably decreased (by 80%). Nuclear targeting of capsid proteins was improved when VP1 and VP2 were co-expressed. VP1 and VP2 were effectively delivered into the nucleus, even when one of the NLS, either VP1 or VP2, was disrupted. Altogether, our results showed that MPyV virions can use VP1 and/or VP2/VP3 NLSs in concert or individually to bind importins to deliver their genomes into the cell nucleus.

• MeSH
• biologický transport MeSH
• buněčné jádro MeSH
• buněčné linie MeSH
• DNA virů metabolismus   MeSH
• fluorescenční protilátková technika MeSH
• jaderné lokalizační signály genetika   MeSH
• karyoferiny metabolismus   MeSH
• mutace MeSH
• myši MeSH
• polyomavirové infekce metabolismus   virologie   MeSH
• Polyomavirus fyziologie   ultrastruktura   MeSH
• sestavení viru MeSH
• substituce aminokyselin MeSH
• vazba proteinů MeSH
• virové plášťové proteiny genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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