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.

• Klíčová slova
• density gradient purification, mouse polyomavirus, polyomavirus, virus quantification,
• 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

The nuclear lamina is a dense network of intermediate filaments beneath the inner nuclear membrane. Composed of A-type lamins (lamin A/C) and B-type lamins (lamins B1 and B2), the nuclear lamina provides a scaffold for the nuclear envelope and chromatin, thereby maintaining the structural integrity of the nucleus. A-type lamins are also found inside the nucleus where they interact with chromatin and participate in gene regulation. Viruses replicating in the cell nucleus have to overcome the nuclear envelope during the initial phase of infection and during the nuclear egress of viral progeny. Here, we focused on the role of lamins in the replication cycle of a dsDNA virus, mouse polyomavirus. We detected accumulation of the major capsid protein VP1 at the nuclear periphery, defects in nuclear lamina staining and different lamin A/C phosphorylation patterns in the late phase of mouse polyomavirus infection, but the nuclear envelope remained intact. An absence of lamin A/C did not affect the formation of replication complexes but did slow virus propagation. Based on our findings, we propose that the nuclear lamina is a scaffold for replication complex formation and that lamin A/C has a crucial role in the early phases of infection with mouse polyomavirus.

• Klíčová slova
• VP1, lamin A/C, lamin B, mouse polyomavirus, viral replication centres,
• MeSH
• buněčné jádro metabolismus   virologie   MeSH
• fosforylace MeSH
• infekce onkogenními viry virologie   patologie   metabolismus   genetika   MeSH
• jaderná lamina * metabolismus   virologie   MeSH
• jaderný obal metabolismus   virologie   MeSH
• lamin typ A * metabolismus   genetika   MeSH
• lamin typ B metabolismus   genetika   MeSH
• myši MeSH
• polyomavirové infekce * virologie   metabolismus   genetika   patologie   MeSH
• Polyomavirus * genetika   patogenita   fyziologie   MeSH
• replikace viru * MeSH
• virové plášťové proteiny metabolismus   genetika   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• lamin typ A * MeSH
• lamin typ B MeSH
• virové plášťové proteiny MeSH
• VP1 protein, polyomavirus MeSH Prohlížeč

Murine polyomavirus mutants are frequently produced for experimental as well as therapy purposes. Commonly used methods for preparation of mutant viral genomes from recombinant vectors are laborious and give variable yields and quality. We describe an efficient and reproducible Cre/loxP-mediated recombination system that generates polyomavirus genomes from recombinant plasmid in vivo. We designed and constructed two variants of recombinant vectors containing the wild-type polyomavirus genome flanked by loxP homologous sites. The loxP sites were introduced either into the intronic region of early genes or between the two poly(A) signal sites of convergent transcriptional units. After cotransfection of the recombinant plasmids with the Cre-expressing vector into mouse 3T6 cells, we obtained infectious virus from the genome variant containing loxP site in the intronic region, but we failed to isolate any infectious virus from the viral genome containing loxP site between poly(A) signals. We show that the Cre/loxP-based method of polyomavirus production is simple, expedient, and reproducible and works with satisfactory efficiency.

• Klíčová slova
• Cre recombination, MPyV, Murine polyomavirus, PAS, Viral genome modification, Virus production, loxP, polyadenylation signal,
• MeSH
• buněčné linie MeSH
• molekulární biologie metody   MeSH
• myši MeSH
• plazmidy MeSH
• Polyomavirus genetika   růst a vývoj   MeSH
• rekombinace genetická * MeSH
• virologie metody   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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.

• Klíčová slova
• cGAS sensor, immune sensing of DNA, mouse polyomavirus, p204 sensor, pattern recognition receptors,
• 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
• Názvy látek
• cGAS protein, mouse MeSH Prohlížeč
• DNA virů MeSH
• fosfoproteiny MeSH
• Ifi16 protein, mouse MeSH Prohlížeč
• interferon beta MeSH
• jaderné proteiny MeSH
• membránové proteiny MeSH
• nukleotidyltransferasy MeSH
• Sting1 protein, mouse MeSH Prohlížeč

Microtubules, part of the cytoskeleton, are indispensable for intracellular movement, cell division, and maintaining cell shape and polarity. In addition, microtubules play an important role in viral infection. In this review, we summarize the role of the microtubules' network during polyomavirus infection. Polyomaviruses usurp microtubules and their motors to travel via early and late acidic endosomes to the endoplasmic reticulum. As shown for SV40, kinesin-1 and microtubules are engaged in the release of partially disassembled virus from the endoplasmic reticulum to the cytosol, and dynein apparently assists in the further disassembly of virions prior to their translocation to the cell nucleus-the place of their replication. Polyomavirus gene products affect the regulation of microtubule dynamics. Early T antigens destabilize microtubules and cause aberrant mitosis. The role of these activities in tumorigenesis has been documented. However, its importance for productive infection remains elusive. On the other hand, in the late phase of infection, the major capsid protein, VP1, of the mouse polyomavirus, counteracts T-antigen-induced destabilization. It physically binds microtubules and stabilizes them. The interaction results in the G2/M block of the cell cycle and prolonged S phase, which is apparently required for successful completion of the viral replication cycle.

• Klíčová slova
• T antigens, VP1 capsid protein, cell cycle block, dynein, kinesin, microtubules, molecular motors, polyomavirus, virus, virus trafficking,
• MeSH
• buněčné jádro virologie   MeSH
• cytosol virologie   MeSH
• endoplazmatické retikulum virologie   MeSH
• endozomy virologie   MeSH
• interakce hostitele a patogenu * MeSH
• lidé MeSH
• mikrotubuly fyziologie   virologie   MeSH
• myši MeSH
• Polyomavirus genetika   patogenita   MeSH
• replikace viru MeSH
• vazba proteinů MeSH
• virové plášťové proteiny genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• virové plášťové proteiny MeSH
• VP1 protein, polyomavirus MeSH Prohlížeč

In two tumour sublines (T.wt/BL and T.wt/Bc), established from mammary adenocarcinomas caused by mouse polyoma (Py) infection of nu/nu mice, integration of polyomavirus DNA sequences into the c-myc gene locus was mapped. A complete Py genome was found to be integrated just upstream from the c-myc gene in T.wt/BL cell line, while only a part of the early Py region coding for the early proteins was inserted in the chromosomal DNA of T.wt/Bc cells. An interference of Py sequences with the regulation of c-myc gene expression gives further significance to a Py-derived tumour system that appears to be similar to some human mammary cancers in the modifications of c-myc expression. Both cell lines were found to produce truncated large T antigen and entire middle and small T antigens. In addition, production of VP1 protein was observed in the T.wt/BL cell line. The integration of polyomavirus sequences and/or expression of viral proteins caused an elevation of c-myc expression. The level of the c-myc expression was higher in both tumour cell lines in comparison with control normal murine mammary gland (NMuMG) lines, but substantially lower than in NMuMG cells infected with polyomavirus. Possible co-operation of Py proteins with c-Myc was examined. Through GST fusion protein pull-down experiments, we evidenced, that c-Myc forms a complex with the common part of the Py early antigens in the two tumour cell lines. Co-localisation of the c-myc and LT was observed in cells overexpressing c-Myc and LT.

• MeSH
• adenokarcinom genetika   virologie   MeSH
• antigeny transformující polyomavirové genetika   metabolismus   MeSH
• DNA nádorová analýza   MeSH
• DNA virů analýza   MeSH
• experimentální nádory mléčných žláz genetika   virologie   MeSH
• geny myc fyziologie   MeSH
• glutathiontransferasa metabolismus   MeSH
• integrace viru * MeSH
• lidé MeSH
• mléčné žlázy lidské fyziologie   virologie   MeSH
• myši MeSH
• Polyomavirus genetika   MeSH
• protoonkogenní proteiny c-myc genetika   metabolismus   MeSH
• virová transformace buněk MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny transformující polyomavirové MeSH
• DNA nádorová MeSH
• DNA virů MeSH
• glutathiontransferasa MeSH
• protoonkogenní proteiny c-myc 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
• Názvy látek
• mikrotubulární proteiny MeSH
• molekulární motory 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.

• Klíčová slova
• capsid proteins, importin β1, mouse polyomavirus, nuclear localization signal, trafficking into the 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
• Názvy látek
• DNA virů MeSH
• jaderné lokalizační signály MeSH
• karyoferiny MeSH
• virové plášťové proteiny 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.

• Klíčová slova
• Assembly, Encapsidation signal, Mouse polyomavirus, Packaging, Pseudovirion,
• 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

Polyomavirus mutants E, Q and H, expressing non-myristylated VP2, were generated by replacing the N-terminal glycine residue with glutamic acid, glutamine or histidine, respectively. Viruses mutated in either VP2 or VP3 translation initiation codons were also prepared. All mutated genomes, when transfected into murine host cells, gave rise to viral particles. Infectivity of VP2- and VP3- viruses, as measured by the number of cells expressing viral antigens, was dramatically diminished, indicative of defects in the early stages of infection. In contrast, the absence of a myristyl moiety on VP2 did not substantially affect the early steps of virus infection. No differences in numbers of cells expressing early or late viral antigens were observed between wild-type (wt) and E or Q myr- viruses during the course of a life cycle. Furthermore, no delay in virus DNA replication was detected. However, when cells were left for longer in culture, the number of infected cells, measured by typical virus bursts, was much lower when mutant rather than wt genomes were used. In situ, cell fractionation studies revealed differences in the interaction of viral particles with host cell structures. The infectivity of mutants was affected not only by loss of the myristyl group on VP2, but also, and to a greater extent, by alterations of the N-terminal amino acid composition.

• MeSH
• antigeny virové biosyntéza   MeSH
• buněčné linie MeSH
• DNA virů biosyntéza   MeSH
• fibroblasty virologie   MeSH
• kapsida genetika   MeSH
• kodon iniciační MeSH
• kyselina myristová metabolismus   MeSH
• mutace MeSH
• myši MeSH
• Polyomavirus genetika   imunologie   fyziologie   MeSH
• replikace DNA MeSH
• replikace viru MeSH
• transfekce MeSH
• virové plášťové proteiny MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• antigeny virové MeSH
• DNA virů MeSH
• kodon iniciační MeSH
• kyselina myristová MeSH
• virové plášťové proteiny MeSH
• VP2 protein, Polyomavirus MeSH Prohlížeč