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

Tick-borne encephalitis virus (TBEV) causes serious, potentially fatal neurological infections that affect humans in endemic regions of Europe and Asia. Neurons are the primary target for TBEV infection in the central nervous system. However, knowledge about this viral infection and virus-induced neuronal injury is fragmental. Here, we directly examined the pathology that occurs after TBEV infection in human primary neurons. We exploited the advantages of advanced high-pressure freezing and freeze-substitution techniques to achieve optimal preservation of infected cell architecture. Electron tomographic (ET) reconstructions elucidated high-resolution 3D images of the proliferating endoplasmic reticulum, and individual tubule-like structures of different diameters in the endoplasmic reticulum cisternae of single cells. ET revealed direct connections between the tubule-like structures and viral particles in the endoplasmic reticulum. Furthermore, ET showed connections between cellular microtubules and vacuoles that harbored the TBEV virions in neuronal extensions. This study was the first to characterize the 3D topographical organization of membranous whorls and autophagic vacuoles in TBEV-infected human neurons. The functional importance of autophagy during TBEV replication was studied in human neuroblastoma cells; stimulation of autophagy resulted in significantly increased dose-dependent TBEV production, whereas the inhibition of autophagy showed a profound, dose-dependent decrease of the yield of infectious virus.

• MeSH
• autofagie účinky léků   genetika   MeSH
• benzylaminy farmakologie   MeSH
• chinazoliny farmakologie   MeSH
• endoplazmatické retikulum účinky léků   ultrastruktura   virologie   MeSH
• lidé MeSH
• mikrotubuly účinky léků   ultrastruktura   virologie   MeSH
• nádorové buněčné linie MeSH
• neurony účinky léků   ultrastruktura   virologie   MeSH
• nokodazol farmakologie   MeSH
• primární buněčná kultura MeSH
• replikace viru účinky léků   MeSH
• sirolimus farmakologie   MeSH
• tomografie elektronová MeSH
• virion účinky léků   růst a vývoj   ultrastruktura   MeSH
• viry klíšťové encefalitidy účinky léků   růst a vývoj   ultrastruktura   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The intracellular transport of Mason-Pfizer monkey virus (M-PMV) assembled capsids from the pericentriolar region to the plasma membrane (PM) requires trafficking of envelope glycoprotein (Env) to the assembly site via the recycling endosome. However, it is unclear if Env-containing vesicles play a direct role in trafficking capsids to the PM. Using live cell microscopy, we demonstrate, for the first time, anterograde co-transport of Gag and Env. Nocodazole disruption of microtubules had differential effects on Gag and Env trafficking, with pulse-chase assays showing a delayed release of Env-deficient virions. Particle tracking demonstrated an initial loss of linear movement of GFP-tagged capsids and mCherry-tagged Env, followed by renewed movement of Gag but not Env at 4h post-treatment. Thus, while delayed capsid trafficking can occur in the absence of microtubules, efficient anterograde transport of capsids appears to be mediated by microtubule-associated Env-containing vesicles.

• MeSH
• AIDS opičí metabolismus   virologie   MeSH
• buněčná membrána virologie   MeSH
• Cercopithecus aethiops MeSH
• genové produkty env genetika   metabolismus   MeSH
• genové produkty gag genetika   metabolismus   MeSH
• Macaca mulatta MeSH
• Masonův-Pfizerův opičí virus genetika   metabolismus   MeSH
• mikrotubuly metabolismus   virologie   MeSH
• transport proteinů MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Immature capsids of the Betaretrovirus, Mason-Pfizer Monkey virus (M-PMV), are assembled in the pericentriolar region of the cell, and are then transported to the plasma membrane for budding. Although several studies, utilizing mutagenesis, biochemistry, and immunofluorescence, have defined the role of some viral and host cells factors involved in these processes, they have the disadvantage of population analysis, rather than analyzing individual capsid movement in real time. In this study, we created an M-PMV vector in which the enhanced green fluorescent protein, eGFP, was fused to the carboxyl-terminus of the M-PMV Gag polyprotein, to create a Gag-GFP fusion that could be visualized in live cells. In order to express this fusion protein in the context of an M-PMV proviral backbone, it was necessary to codon-optimize gag, optimize the Kozak sequence preceding the initiating methionine, and mutate an internal methionine codon to one for alanine (M100A) to prevent internal initiation of translation. Co-expression of this pSARM-Gag-GFP-M100A vector with a WT M-PMV provirus resulted in efficient assembly and release of capsids. Results from fixed-cell immunofluorescence and pulse-chase analyses of wild type and mutant Gag-GFP constructs demonstrated comparable intracellular localization and release of capsids to untagged counterparts. Real-time, live-cell visualization and analysis of the GFP-tagged capsids provided strong evidence for a role for microtubules in the intracellular transport of M-PMV capsids. Thus, this M-PMV Gag-GFP vector is a useful tool for identifying novel virus-cell interactions involved in intracellular M-PMV capsid transport in a dynamic, real-time system.

• MeSH
• biologický transport MeSH
• buněčná membrána metabolismus   MeSH
• fluorescenční barviva metabolismus   MeSH
• genetické vektory genetika   MeSH
• genové produkty gag genetika   metabolismus   MeSH
• HEK293 buňky MeSH
• kapsida metabolismus   MeSH
• kinetika MeSH
• lidé MeSH
• Masonův-Pfizerův opičí virus genetika   metabolismus   fyziologie   MeSH
• mikrotubuly metabolismus   virologie   MeSH
• molekulární zobrazování MeSH
• pohyb MeSH
• proviry genetika   metabolismus   fyziologie   MeSH
• rekombinantní fúzní proteiny genetika   metabolismus   MeSH
• sestavení viru MeSH
• transport proteinů MeSH
• viabilita buněk MeSH
• zelené fluorescenční proteiny genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH