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

BK polyomavirus (BKPyV) infection in humans is usually asymptomatic but ultimately results in viral persistence. In immunocompromised hosts, virus reactivation can lead to nephropathy or hemorrhagic cystitis. The urinary tract serves as a silent reservoir for the virus. Recently, it has been demonstrated that human bladder microvascular endothelial cells (HBMVECs) serve as viral reservoirs, given their unique response to infection, which involves interferon (IFN) production. The aim of the present study was to better understand the life cycle of BKPyV in HBMVECs, uncover the molecular pathway leading to IFN production, and to identify the connection between the viral life cycle and the activation of the IFN response. Here, in the early stage of infection, BKPyV virions were found in internalized monopinocytic vesicles, while later they were detected in late endosomes, lysosomes, tubuloreticular structures, and vacuole-like vesicles. The production of viral progeny in these cells started at 36 h postinfection. Increased cell membrane permeability and peaks of virion release coincided with the leakage of viral and cellular DNA into the cytosol at approximately 60 h postinfection. Leaked DNA colocalized with and activated cGAS, leading to the activation of STING and the consequent transcription of IFNB and IFN-related genes; in contrast, the IFN response was attenuated by exposure to the cGAS inhibitor, G140. These findings highlight the importance of the cGAS-STING pathway in the innate immune response of HBMVECs to BKPyV.

• Klíčová slova
• BK polyomavirus, BKPyV reservoir cells, STING, cGAS, interferon response,
• MeSH
• endoteliální buňky * virologie   MeSH
• interferony metabolismus   MeSH
• kultivované buňky MeSH
• lidé MeSH
• membránové proteiny metabolismus   genetika   MeSH
• močový měchýř * virologie   MeSH
• nukleotidyltransferasy metabolismus   genetika   MeSH
• polyomavirové infekce virologie   imunologie   MeSH
• replikace viru MeSH
• signální transdukce * MeSH
• virion MeSH
• virus BK * fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cGAS protein, human MeSH Prohlížeč
• interferony MeSH
• membránové proteiny MeSH
• nukleotidyltransferasy MeSH
• STING1 protein, human MeSH Prohlížeč

Herein, we performed a simple virus capture and photoinactivation procedure using visible light on phosphatidylcholine vesicles. l-α-Phosphatidylcholine vesicles were enriched by viral receptors, GT1b gangliosides, and the nonpolar photosensitizer 5,10,15,20-tetraphenylporphyrin. These vesicles absorb in the blue region of visible light with a high quantum yield of antiviral singlet oxygen, O2 (1Δg). Through the successful incorporation of gangliosides into the structure of vesicles and the encapsulation of photosensitizers in their photoactive and monomeric state, the photogeneration of O2(1Δg) was achieved with high efficiency on demand; this process was triggered by light, and specifically targeting/inactivating viruses were captured on ganglioside receptors due to the short lifetime (3.3 μs) and diffusion pathway (approximately 100 nm) of O2(1Δg). Time-resolved and steady-state luminescence as well as absorption spectroscopy were used to monitor the photoactivity of the photosensitizer and the photogeneration of O2(1Δg) on the surface of the vesicles. The capture of model mouse polyomavirus and its inactivation were achieved using immunofluorescence methods, and loss of infectivity toward mouse fibroblast 3T6 cells was detected.

• 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.

• 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

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.

• Klíčová slova
• VP1, histone deacetylase 6, microtubule acetylation, microtubule stabilization, microtubules, mouse polyomavirus, α-tubulin acetyltransferase 1,
• 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
• Názvy látek
• acetyltransferasy MeSH
• Hdac6 protein, mouse MeSH Prohlížeč
• histondeacetylasa 6 MeSH
• tubulin MeSH
• virové plášťové proteiny MeSH
• VP1 protein, polyomavirus MeSH Prohlížeč

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