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.
- 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
DNA virus infections are often lifelong and can cause serious diseases in their hosts. Their recognition by the sensors of the innate immune system represents the front line of host defence. Understanding the molecular mechanisms of innate immunity responses is an important prerequisite for the design of effective antivirotics. This review focuses on the present state of knowledge surrounding the mechanisms of viral DNA genome sensing and the main induced pathways of innate immunity responses. The studies that have been performed to date indicate that herpesviruses, adenoviruses, and polyomaviruses are sensed by various DNA sensors. In non-immune cells, STING pathways have been shown to be activated by cGAS, IFI16, DDX41, or DNA-PK. The activation of TLR9 has mainly been described in pDCs and in other immune cells. Importantly, studies on herpesviruses have unveiled novel participants (BRCA1, H2B, or DNA-PK) in the IFI16 sensing pathway. Polyomavirus studies have revealed that, in addition to viral DNA, micronuclei are released into the cytosol due to genotoxic stress. Papillomaviruses, HBV, and HIV have been shown to evade DNA sensing by sophisticated intracellular trafficking, unique cell tropism, and viral or cellular protein actions that prevent or block DNA sensing. Further research is required to fully understand the interplay between viruses and DNA sensors.
- MeSH
- DNA virů metabolismus MeSH
- Herpesviridae * genetika metabolismus MeSH
- infekce DNA virem * MeSH
- lidé MeSH
- Polyomavirus * genetika MeSH
- přirozená imunita MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
The nuclear lamina is the main component of the nuclear cytoskeleton that maintains the integrity of the nucleus. However, it represents a natural barrier for viruses replicating in the cell nucleus. The lamina blocks viruses from being trafficked to the nucleus for replication, but it also impedes the nuclear egress of the progeny of viral particles. Thus, viruses have evolved mechanisms to overcome this obstacle. Large viruses induce the assembly of multiprotein complexes that are anchored to the inner nuclear membrane. Important components of these complexes are the viral and cellular kinases phosphorylating the lamina and promoting its disaggregation, therefore allowing virus egress. Small viruses also use cellular kinases to induce lamina phosphorylation and the subsequent disruption in order to facilitate the import of viral particles during the early stages of infection or during their nuclear egress. Another component of the nuclear cytoskeleton, nuclear actin, is exploited by viruses for the intranuclear movement of their particles from the replication sites to the nuclear periphery. This study focuses on exploitation of the nuclear cytoskeleton by viruses, although this is just the beginning for many viruses, and promises to reveal the mechanisms and dynamic of physiological and pathological processes in the nucleus.
- MeSH
- aktiny metabolismus MeSH
- buněčné jádro metabolismus MeSH
- cytoskelet genetika metabolismus MeSH
- druhová specificita MeSH
- interakce hostitele a patogenu * MeSH
- jaderná lamina metabolismus MeSH
- jaderný obal metabolismus MeSH
- laminy metabolismus MeSH
- lidé MeSH
- náchylnost k nemoci * MeSH
- regulace exprese virových genů MeSH
- replikace viru MeSH
- virové nemoci etiologie metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy 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
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.
- 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
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 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.
Morphogenesis of the mouse polyomavirus virion is a complex and not yet well understood process. Nuclear lysates of infected cells and cells transiently producing the major capsid protein (VP1) of the mouse polyomavirus and whole-cell lysates were separated by blue native polyacrylamide gel electrophoresis (BN-PAGE) to characterize the participation of cellular proteins in virion precursor complexes. Several VP1-specific complexes were found by immunostaining with the anti-VP1 antibody. Some of these complexes contained proteins from the heat shock protein 70 family. The BN-PAGE was found to be a useful tool for the identification of protein complexes by immunostaining of separated cell lysates. However, whole-cell lysates and lysates of isolated nuclei of cells infected with polyomavirus appeared to be too complex for BN-PAGE separation followed by mass spectrometry. No distinct bands specific for cells infected with polyomavirus were detected by Coomassie blue stained gels, hence this method is not suitable for the discovery of new cellular proteins participating in virion assembly. Nevertheless, BN-PAGE can be valuable for the analyses of different types of complexes formed by proteins after their enrichment or isolation by affinity chromatography.
- MeSH
- chromatografie afinitní MeSH
- elektroforéza v polyakrylamidovém gelu metody MeSH
- hmotnostní spektrometrie MeSH
- interakce hostitele a patogenu MeSH
- mapování interakce mezi proteiny MeSH
- myši MeSH
- Polyomavirus chemie patogenita MeSH
- proteom analýza MeSH
- vazba proteinů MeSH
- virové plášťové proteiny metabolismus MeSH
- virové proteiny analýza MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
Mouse polyomavirus (PyV) virions enter cells by internalization into smooth monopinocytic vesicles, which fuse under the cell membrane with larger endosomes. Caveolin-1 was detected on monopinocytic vesicles carrying PyV particles in mouse fibroblasts and epithelial cells (33). Here, we show that PyV can be efficiently internalized by Jurkat cells, which do not express caveolin-1 and lack caveolae, and that overexpression of a caveolin-1 dominant-negative mutant in mouse epithelial cells does not prevent their productive infection. Strong colocalization of VP1 with early endosome antigen 1 (EEA1) and of EEA1 with caveolin-1 in mouse fibroblasts and epithelial cells suggests that the monopinocytic vesicles carrying the virus (and vesicles containing caveolin-1) fuse with EEA1-positive early endosomes. In contrast to SV40, PyV infection is dependent on the acidic pH of endosomes. Bafilomycin A1 abolished PyV infection, and an increase in endosomal pH by NH4Cl markedly reduced its efficiency when drugs were applied during virion transport towards the cell nucleus. The block of acidification resulted in the retention of a fraction of virions in early endosomes. To monitor further trafficking of PyV, we used fluorescent resonance energy transfer (FRET) to determine mutual localization of PyV VP1 with transferrin and Rab11 GTPase at a 2- to 10-nm resolution. Positive FRET between PyV VP1 and transferrin cargo and between PyV VP1 and Rab11 suggests that during later times postinfection (1.5 to 3 h), the virus meets up with transferrin in the Rab11-positive recycling endosome. These results point to a convergence of the virus and the cargo internalized by different pathways in common transitional compartments.
- MeSH
- buněčné linie MeSH
- časové faktory MeSH
- elektronová mikroskopie MeSH
- endozomy metabolismus virologie MeSH
- financování organizované MeSH
- fúze membrán MeSH
- kaveolin 1 genetika metabolismus MeSH
- koncentrace vodíkových iontů MeSH
- myši MeSH
- Polyomavirus fyziologie MeSH
- rab proteiny vázající GTP metabolismus MeSH
- rezonanční přenos fluorescenční energie MeSH
- transferin metabolismus MeSH
- transport proteinů MeSH
- vazba proteinů MeSH
- virion metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
