Viruses from the genus Enterovirus are important human pathogens. Receptor binding or exposure to acidic pH in endosomes converts enterovirus particles to an activated state that is required for genome release. However, the mechanism of enterovirus uncoating is not well understood. Here, we use cryo-electron microscopy to visualize virions of human echovirus 18 in the process of genome release. We discover that the exit of the RNA from the particle of echovirus 18 results in a loss of one, two, or three adjacent capsid-protein pentamers. The opening in the capsid, which is more than 120 Å in diameter, enables the release of the genome without the need to unwind its putative double-stranded RNA segments. We also detect capsids lacking pentamers during genome release from echovirus 30. Thus, our findings uncover a mechanism of enterovirus genome release that could become target for antiviral drugs.
- MeSH
- Cercopithecus aethiops MeSH
- dvouvláknová RNA chemie genetika MeSH
- elektronová kryomikroskopie MeSH
- enterovirus B lidský genetika ultrastruktura MeSH
- epitelové buňky ultrastruktura virologie MeSH
- genom virový * MeSH
- kapsida chemie ultrastruktura MeSH
- koncentrace vodíkových iontů MeSH
- lidé MeSH
- RNA virová chemie genetika MeSH
- simulace molekulární dynamiky MeSH
- svlékání virového obalu genetika MeSH
- virion genetika ultrastruktura MeSH
- zvířata MeSH
- Check Tag
- lidé 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
Topical microbicides to stop sexually transmitted diseases, such as herpes simplex virus type 2 (HSV-2), are urgently needed. The emerging field of nanotechnology offers novel suitable tools for addressing this challenge. Our objective was to study, in vitro and in vivo, antiherpetic effect and antiviral mechanisms of several polyanionic carbosilane dendrimers with anti-HIV-1 activity to establish new potential microbicide candidates against sexually transmitted diseases. Plaque reduction assay on Vero cells proved that G2-S16, G1-S4, and G3-S16 are the dendrimers with the highest inhibitory response against HSV-2 infection. We also demonstrated that our dendrimers inhibit viral infection at the first steps of HSV-2 lifecycle: binding/entry-mediated events. G1-S4 and G3-S16 bind directly on the HSV-2, inactivating it, whereas G2-S16 adheres to host cell-surface proteins. Molecular modeling showed that G1-S4 binds better at binding sites on gB surface than G2-S16. Significantly better binding properties of G1-S4 than G2-S16 were found in an important position for affecting transition of gB trimer from G1-S4 prefusion to final postfusion state and in several positions where G1-S4 could interfere with gB/gH-gL interaction. We demonstrated that these polyanionic carbosilan dendrimers have a synergistic activity with acyclovir and tenofovir against HSV-2, in vitro. Topical vaginal or rectal administration of G1-S4 or G2-S16 prevents HSV-2 transmission in BALB/c mice in values close to 100%. This research represents the first demonstration that transmission of HSV-2 can be blocked by vaginal/rectal application of G1-S4 or G2-S16, providing a step forward to prevent HSV-2 transmission in humans.
- MeSH
- acyklovir farmakologie MeSH
- antiinfekční látky farmakologie MeSH
- antivirové látky farmakologie MeSH
- aplikace rektální MeSH
- Cercopithecus aethiops MeSH
- dendrimery chemie MeSH
- epitelové buňky účinky léků virologie MeSH
- herpes simplex MeSH
- koncentrace vodíkových iontů MeSH
- lidé MeSH
- lidský herpesvirus 2 účinky léků MeSH
- molekulární modely MeSH
- myši inbrední BALB C MeSH
- polymery chemie MeSH
- rektum účinky léků virologie MeSH
- silany chemie MeSH
- tenofovir farmakologie MeSH
- vagina účinky léků virologie MeSH
- Vero buňky MeSH
- virové proteiny metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Endogenous retroviruses (ERVs) were acquired during evolution of their host organisms after infection and mendelian inheritance in the germline by their exogenous counterparts. The ERVs can spread in the host genome and in some cases they affect the host phenotype. The cervid endogenous gammaretrovirus (CrERV) is one of only a few well-defined examples of evolutionarily recent invasion of mammalian genome by retroviruses. Thousands of insertionally polymorphic CrERV integration sites have been detected in wild ranging mule deer (Odocoileus hemionus) host populations. Here, we describe for the first time induction of replication competent CrERV by cocultivation of deer and human cells. We characterize the physical properties and tropism of the induced virus. The genomic sequence of the induced virus is phylogenetically related to the evolutionarily young endogenous CrERVs described so far. We also describe the level of replication block of CrERV on deer cells and its capacity to establish superinfection interference.
- MeSH
- biologická evoluce MeSH
- endogenní retroviry klasifikace genetika izolace a purifikace ultrastruktura MeSH
- epitelové buňky ultrastruktura virologie MeSH
- fylogeneze MeSH
- Gammaretrovirus klasifikace genetika izolace a purifikace ultrastruktura MeSH
- genom virový * MeSH
- HEK293 buňky MeSH
- kokultivační techniky MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- replikace viru MeSH
- virion genetika izolace a purifikace ultrastruktura MeSH
- vysoká zvěř virologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
UNLABELLED: The HIV-1 envelope protein (Env) is heavily glycosylated, with approximately 50% of the Env molecular mass being contributed by N-glycans. HIV-1 Env N-glycans shield the protein backbone and have been shown to play key roles in determining Env structure, surface exposure, and, consequently, antigenicity, infectivity, antibody neutralization, and carbohydrate and receptor binding. Studies of HIV-1 glycosylation have focused mainly on the position of glycosylation, rather than the types of glycans. Also, the role of Env glycan moieties on HIV-1 transmission has not been systematically defined. Using viruses with modified Env glycan content and heterogeneity, we examined the effects of Env glycan moieties on the major events of HIV-1 transmission. Compared to viruses with less oligomannose and more complex Env glycans, viruses with more oligomannose and less complex glycans more efficiently (i) transcytosed across an epithelial cell monolayer, (ii) attached to monocyte-derived macrophages (MDMs), (iii) bound monocyte-derived dendritic cells (MoDCs), and (iv) trans-infected primary lymphocytes via MoDCs. However, viruses with more oligomannose and less complex glycans displayed impaired infectivity in TZMbl cells, MDMs, primary lymphocytes, and fresh human intestinal tissue. Thus, N-linked Env glycans display discordant effects on the major events of HIV-1 transmission, with mature oligosaccharide structures on Env playing a crucial role in HIV-1 infection. Env glycosylation should be taken into consideration in the development of vaccine strategies to interdict HIV-1 transmission. IMPORTANCE: HIV-1 Env N-glycans shield the protein backbone and play key roles in determining Env structure and surface exposure, thereby impacting Env antigenicity, infectivity, antibody neutralization, and carbohydrate and receptor binding. Studies of HIV-1 glycosylation have focused mainly on the position of glycosylation, rather than the types of glycans. In the study described in this report, we investigated systematically the role of Env glycan moieties on HIV-1 transmission. We show that N-linked Env glycans display discordant effects on the major events of HIV-1 transmission. These data indicate that Env glycan moieties impact HIV-1 transmission and that modulation of Env glycan moieties offers a potential strategy for the development of therapeutic or prophylactic vaccines against HIV-1.
- MeSH
- dendritické buňky virologie MeSH
- epitelové buňky virologie MeSH
- genové produkty env - virus lidské imunodeficience chemie metabolismus MeSH
- HIV-1 fyziologie MeSH
- kultivované buňky MeSH
- lidé MeSH
- lymfocyty virologie MeSH
- makrofágy virologie MeSH
- polysacharidy analýza metabolismus MeSH
- přichycení viru * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
In most cells, vaccinia virus (VACV) infection is considered to cause a lytic cell death, an equivalent of necrosis. However, upon infection of the epithelial cell lines HeLa G and BSC-40 with VACV strain Western Reserve (WR), we have previously observed an increased activation of and activity attributable to caspases, a typical sign of apoptosis. In this paper, we have further analyzed the type of cell death in VACV-infected cells HeLa G and BSC-40. In a cell-based flow cytometric assay, we showed a specific activation of caspase-2 and 4 in HeLa G and BSC-40 cells infected with VACV, strain WR, while we did not find any effects of inhibitors of calpain and cathepsin D and E. The actual activity of the two caspases, but also of caspase-3, was then confirmed in lysates of infected HeLa G, but not in BSC-40 cells. Accordingly, poly(ADP)-ribose polymerase (PARP) cleavage was found increased only in infected HeLa G cells. Consequently, we have determined morphological features of apoptosis and/or activity of the executioner caspase-3 in infected HeLa G cells in situ, while only a background apoptosis was observed in infected BSC-40 cells. Finally, vaccination strains Dryvax and Praha were found to induce apoptosis in both HeLa G and BSC-40 cells, as characterized morphologically and by PARP cleavage. These findings may be important for understanding the differences in VACV-host interactions and post-vaccination complications in different individuals.
- MeSH
- buněčná smrt MeSH
- buněčné linie MeSH
- Cercopithecus aethiops MeSH
- epitelové buňky virologie MeSH
- kaspasy metabolismus MeSH
- lidé MeSH
- poly(ADP-ribosa)-polymerasy metabolismus MeSH
- průtoková cytometrie MeSH
- virus vakcinie patogenita MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Vaccinia virus (VV) is considered to cause lytic infection of most cells, with lysis being regarded equivalent to necrosis. Activation of caspases has not been associated with necrosis. However, we observed the activation and activity of caspases in epithelial cells HeLa G and BSC-40 lytically infected with VV. Using three different flow-cytometric approaches, we characterized the distinct stages of caspase cascade in VV-infected cells: a cleaved, activated form of caspases detected using a fluorescent pan-caspase inhibitor; caspase activity assayed by cleavage of a non-fluorescent substrate into a fluorescent product; caspase-specific cleavage of death substrates characterized by a fluorescent antibody detecting a neo-epitope in cytokeratin-18. All of these approaches yielded an increased fluorescent signal in VV-infected cells compared to mock-infected controls. Additionally, the signal was decreased by the expression of Bcl-2. The cleavage of cytokeratin-18 was confirmed by western blotting, but another key protein involved in apoptosis, PARP, was not cleaved in VV-infected lytic cells. The necrotic phenotype of the cells was confirmed by increased cell membrane permeability and/or decreased mitochondrial membrane potential. In conclusion, our data suggest that VV infection of the epithelial cells HeLa G and BSC-40 initiates the apoptotic program, however, apoptosis is not completed and switches into necrosis.
- MeSH
- aktiny genetika metabolismus MeSH
- aktivace enzymů MeSH
- apoptóza MeSH
- Cercopithecus aethiops MeSH
- cytopatogenní efekt virový MeSH
- epitelové buňky cytologie fyziologie virologie MeSH
- fenotyp MeSH
- financování organizované MeSH
- HeLa buňky MeSH
- kaspasy genetika metabolismus MeSH
- keratin-18 genetika metabolismus MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- nekróza MeSH
- permeabilita buněčné membrány MeSH
- poly(ADP-ribosa)-polymerasy genetika metabolismus MeSH
- protoonkogenní proteiny c-bcl-2 genetika metabolismus MeSH
- vakcínie patofyziologie virologie MeSH
- virus vakcinie fyziologie růst a vývoj MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
