Hepatitis B virus (HBV) core protein (HBc) plays many roles in the HBV life cycle, such as regulation of transcription, RNA encapsidation, reverse transcription, and viral release. To accomplish these functions, HBc interacts with many host proteins and undergoes different post-translational modifications (PTMs). One of the most common PTMs is ubiquitination, which was shown to change the function, stability, and intracellular localization of different viral proteins, but the role of HBc ubiquitination in the HBV life cycle remains unknown. Here, we found that HBc protein is post-translationally modified through K29-linked ubiquitination. We performed a series of co-immunoprecipitation experiments with wild-type HBc, lysine to arginine HBc mutants and wild-type ubiquitin, single lysine to arginine ubiquitin mutants, or single ubiquitin-accepting lysine constructs. We observed that HBc protein could be modified by ubiquitination in transfected as well as infected hepatoma cells. In addition, ubiquitination predominantly occurred on HBc lysine 7 and the preferred ubiquitin chain linkage was through ubiquitin-K29. Mass spectrometry (MS) analyses detected ubiquitin protein ligase E3 component N-recognin 5 (UBR5) as a potential E3 ubiquitin ligase involved in K29-linked ubiquitination. These findings emphasize that ubiquitination of HBc may play an important role in HBV life cycle.
- MeSH
- arginin genetika MeSH
- buněčné linie MeSH
- buňky Hep G2 MeSH
- HEK293 buňky MeSH
- hepatitida B genetika MeSH
- hepatocelulární karcinom genetika MeSH
- lidé MeSH
- lysin genetika MeSH
- nádorové buněčné linie MeSH
- posttranslační úpravy proteinů genetika MeSH
- ubikvitin genetika MeSH
- ubikvitinace genetika MeSH
- ubikvitinligasy genetika MeSH
- virové proteiny genetika MeSH
- virus hepatitidy B genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The envelope glycoprotein (Env) plays a crucial role in the retroviral life cycle by mediating primary interactions with the host cell. As described previously and expanded on in this paper, Env mediates the trafficking of immature Mason-Pfizer monkey virus (M-PMV) particles to the plasma membrane (PM). Using a panel of labeled RabGTPases as endosomal markers, we identified Env mostly in Rab7a- and Rab9a-positive endosomes. Based on an analysis of the transport of recombinant fluorescently labeled M-PMV Gag and Env proteins, we propose a putative mechanism of the intracellular trafficking of M-PMV Env and immature particles. According to this model, a portion of Env is targeted from the trans-Golgi network (TGN) to Rab7a-positive endosomes. It is then transported to Rab9a-positive endosomes and back to the TGN. It is at the Rab9a vesicles where the immature particles may anchor to the membranes of the Env-containing vesicles, preventing Env recycling to the TGN. These Gag-associated vesicles are then transported to the plasma membrane.
- MeSH
- AIDS opičí virologie MeSH
- buněčná membrána metabolismus virologie MeSH
- endozomy metabolismus virologie MeSH
- genové produkty env genetika metabolismus MeSH
- Masonův-Pfizerův opičí virus genetika fyziologie MeSH
- sestavení viru MeSH
- transport proteinů MeSH
- transportní vezikuly metabolismus virologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
To biochemically and structurally characterize viral intracytoplasmic particles (ICAPs), a sample of high purity and homogeneity is usually required. Production of ICAPs in the system closely related to their natural host cells is crucial for the analysis of host-cell binding proteins involved in ICAPs assembly, transport and budding. However, this approach is often hampered by problems with low yield of the ICAPs due to either low expression or fast release from the host cell. Another obstacle may be a low stability or fragility of the intracellular particles. The published methods for ICAPs isolation often involved several time-consuming centrifugation steps yielding damaged particles. Other papers describe the ICAPs production in non-natural host cells. Here, we optimized the method for purification of unstable Mason-Pfizer monkey virus (M-PMV) ICAPs from non-human primate derived cells, commonly used to study MPMV replication i.e. African green monkey kidney fibroblast cell line (COS-1). Our simple and rapid procedure involved separation of the intracytoplasmic particles from the cell debris and organelles by differential, low-speed centrifugation, their purification using sucrose velocity gradient and final concentrating by low-speed centrifugation. Importantly, the method was established for unstable and fragile M-PMV intracytoplasmic particles. Therefore, it may be suitable for isolation of ICAPs of other viruses.
- MeSH
- centrifugace metody MeSH
- Cercopithecus aethiops MeSH
- COS buňky MeSH
- cytoplazma virologie MeSH
- lidé MeSH
- Masonův-Pfizerův opičí virus izolace a purifikace fyziologie MeSH
- replikace viru MeSH
- sestavení viru MeSH
- virologie metody MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Matrix proteins (MAs) play a key role in the transport of retroviral proteins inside infected cells and in the interaction with cellular membranes. In most retroviruses, retroviral MAs are N-terminally myristoylated. This modification serves as a membrane targeting signal and also as an anchor for membrane interaction. The aim of this work was to characterize the interactions anchoring retroviral MA at the plasma membrane of infected cell. To address this issue, we compared the structures and membrane affinity of the Mason-Pfizer monkey virus (M-PMV) wild-type MA with its two budding deficient double mutants, that is, T41I/T78I and Y28F/Y67F. The structures of the mutants were determined using solution NMR spectroscopy, and their interactions with water-soluble phospholipids were studied. Water-soluble phospholipids are widely used models for studying membrane interactions by solution NMR spectroscopy. However, this approach might lead to artificial results due to unnatural hydrophobic interactions. Therefore, we used a new approach based on the measurement of the loss of the (1)H NMR signal intensity of the protein sample induced by the addition of the liposomes containing phospholipids with naturally long fatty acids. HIV-1 MA was used as a positive control because its ability to interact with liposomes has already been described. We found that in contrast to HIV-1, the M-PMV MA interacted with the liposomes differently and much weaker. In our invivo experiments, the M-PMV MA did not co-localize with lipid rafts. Therefore, we concluded that M-PMV might adopt a different membrane binding mechanism than HIV-1.
- MeSH
- buněčná membrána metabolismus MeSH
- fosfolipidy metabolismus MeSH
- liposomy metabolismus MeSH
- magnetická rezonanční spektroskopie MeSH
- Masonův-Pfizerův opičí virus fyziologie MeSH
- mastné kyseliny metabolismus MeSH
- missense mutace MeSH
- mutantní proteiny chemie metabolismus MeSH
- proteiny virové matrix chemie metabolismus MeSH
- uvolnění viru z buňky * MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
The assembly of immature retroviral particles is initiated in the cytoplasm by the binding of the structural polyprotein precursor Gag with viral genomic RNA. The protein interactions necessary for assembly are mediated predominantly by the capsid (CA) and nucleocapsid (NC) domains, which have conserved structures. In contrast, the structural arrangement of the CA-NC connecting region differs between retroviral species. In HIV-1 and Rous sarcoma virus, this region forms a rod-like structure that separates the CA and NC domains, whereas in Mason-Pfizer monkey virus, this region is densely packed, thus holding the CA and NC domains in close proximity. Interestingly, the sequence connecting the CA and NC domains in gammaretroviruses, such as murine leukemia virus (MLV), is unique. The sequence is called a charged assembly helix (CAH) due to a high number of positively and negatively charged residues. Although both computational and deletion analyses suggested that the MLV CAH forms a helical conformation, no structural or biochemical data supporting this hypothesis have been published. Using an in vitro assembly assay, alanine scanning mutagenesis, and biophysical techniques (circular dichroism, NMR, microcalorimetry, and electrophoretic mobility shift assay), we have characterized the structure and function of the MLV CAH. We provide experimental evidence that the MLV CAH belongs to a group of charged, E(R/K)-rich, single α-helices. This is the first single α-helix motif identified in viral proteins.
- MeSH
- mutageneze MeSH
- myši MeSH
- proteinové domény MeSH
- sekundární struktura proteinů MeSH
- virové plášťové proteiny chemie genetika MeSH
- virus myší leukemie chemie genetika MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Očkování je proces, při kterém je organismu podáván antigen s cílem navodit jeho imunizaci. Tento antigen může být podán v různých podobách a formách a dle toho lze rozdělit očkovací látky – vakcíny, na několik typů. Objev první vakcíny, proti pravým neštovicím, byl jedním z nejvýznamnějších výsledků lékařské vědy. V současné době existuje mnoho typů vakcín proti různým infekčním onemocněním a každá země má svůj harmonogram, kdy jsou jaké očkovací látky podávány, proti kterým infekčním onemocněním se očkuje plošně a která očkování jsou naopak pouze doporučována.
Vaccination is the process by which the antigen is administered to an organism in order to induce its immunization. This antigen can be delivered and presented in a variety of forms and types and thus we can categorize several types of vaccines. The discovery of the first vaccine against smallpox was one of the main achievements of medical science. Nowadays, there are many types of vaccines against various infectious diseases, and each country has its own schedule, that defines when the certain vaccine is delivered, which vaccines are necessary and which are only recommended.
- MeSH
- aktivní imunoterapie MeSH
- atenuované vakcíny aplikace a dávkování imunologie MeSH
- cestovní lékařství metody MeSH
- dítě MeSH
- DNA vakcíny aplikace a dávkování imunologie MeSH
- hromadná vakcinace * organizace a řízení zákonodárství a právo MeSH
- inaktivované vakcíny aplikace a dávkování imunologie MeSH
- kojenec MeSH
- kombinované vakcíny aplikace a dávkování imunologie MeSH
- lidé středního věku MeSH
- mladiství MeSH
- novorozenec MeSH
- očkovací schéma * MeSH
- předškolní dítě MeSH
- senioři MeSH
- vakcinace * dějiny klasifikace metody trendy MeSH
- vakcíny konjugované aplikace a dávkování imunologie MeSH
- vakcíny * klasifikace terapeutické užití MeSH
- Check Tag
- dítě MeSH
- kojenec MeSH
- lidé středního věku MeSH
- mladiství MeSH
- mužské pohlaví MeSH
- novorozenec MeSH
- předškolní dítě MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Geografické názvy
- Česká republika MeSH