RNA-dependent RNA polymerase 3Dpol is a key enzyme for the replication of picornaviruses. The viral genome is translated into a single polyprotein that is subsequently proteolytically processed into matured products. The 3Dpol enzyme arises from a stable 3CD precursor that has high proteolytic activity but no polymerase activity. Upon cleavage of the precursor the newly established N-terminus of 3Dpol is liberated and inserts itself into a pocket on the surface of the 3Dpol enzyme. The essential residue for this mechanism is the very first glycine that is conserved among almost all picornaviruses. However, kobuviruses and siciniviruses have a serine residue instead. Intrigued by this anomaly we sought to solve the crystal structure of these 3Dpol enzymes. The structures revealed a unique fold of the 3Dpol N-termini but the very first serine residues were inserted into a charged pocket in a similar manner as the glycine residue in other picornaviruses. These structures revealed a common underlying mechanism of 3Dpol activation that lies in activation of the α10 helix containing a key catalytical residue Asp238 that forms a hydrogen bond with the 2' hydroxyl group of the incoming NTP nucleotide.
- MeSH
- HeLa buňky MeSH
- Kobuvirus enzymologie MeSH
- krystalografie rentgenová MeSH
- lidé MeSH
- mutageneze cílená MeSH
- Picornaviridae enzymologie MeSH
- průtoková cytometrie MeSH
- RNA-dependentní RNA-polymerasa chemie genetika metabolismus MeSH
- virové proteiny chemie genetika metabolismus MeSH
- vodíková vazba MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Yellow fever virus (YFV) is responsible for devastating outbreaks of Yellow fever (YF) in humans and is associated with high mortality rates. Recent large epidemics and epizootics and exponential increases in the numbers of YF cases in humans and non-human primates highlight the increasing threat YFV poses, despite the availability of an effective YFV vaccine. YFV is the first human virus discovered, but the structures of several of the viral proteins remain poorly understood. Here we report the structure of the full-length NS5 protein, a key enzyme for the replication of flaviviruses that contains both a methyltransferase domain and an RNA dependent RNA polymerase domain, at 3.1 Å resolution. The viral polymerase adopts right-hand fold, demonstrating the similarities of the Yellow fever, Dengue and Zika polymerases. Together this data suggests NS5 as a prime and ideal target for the design of pan-flavivirus inhibitors.
- MeSH
- dengue MeSH
- epidemický výskyt choroby MeSH
- infekce virem zika MeSH
- konformace proteinů MeSH
- methyltransferasy chemie MeSH
- molekulární modely MeSH
- primáti MeSH
- proteinové domény MeSH
- racionální návrh léčiv MeSH
- replikace viru účinky léků MeSH
- RNA-dependentní RNA-polymerasa chemie MeSH
- vakcína proti žluté zimnici MeSH
- vazebná místa MeSH
- virové nestrukturální proteiny chemie účinky léků genetika MeSH
- virus žluté zimnice účinky léků genetika metabolismus MeSH
- zinek MeSH
- žlutá zimnice virologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Most single stranded plus RNA viruses hijack phosphatidylinositol 4-kinases (PI4Ks) to generate membranes highly enriched in phosphatidylinositol 4-phosphate (PI4P). These membranous compartments known as webs, replication factories or replication organelles are essential for viral replication because they provide protection from the innate intracellular immune response while serving as platforms for viral replication. Using purified recombinant proteins and biomimetic model membranes we show that the nonstructural viral 3A protein is sufficient to promote membrane hyper-phosphorylation given the proper intracellular cofactors (PI4KB and ACBD3). However, our bio-mimetic in vitro reconstitution assay revealed that rather than the presence of PI4P specifically, negative charge alone is sufficient for the recruitment of 3Dpol enzymes to the surface of the lipid bilayer. Additionally, we show that membrane tethered viral 3B protein (also known as Vpg) works in combination with the negative charge to increase the efficiency of membrane recruitment of 3Dpol.
- MeSH
- adaptorové proteiny signální transdukční genetika metabolismus MeSH
- buněčná membrána metabolismus MeSH
- fosfatidylinositolfosfáty metabolismus MeSH
- fosfotransferasy s alkoholovou skupinou jako akceptorem genetika metabolismus MeSH
- Kobuvirus enzymologie MeSH
- lidé MeSH
- membránové proteiny genetika metabolismus MeSH
- pikornavirové infekce metabolismus virologie MeSH
- virové nestrukturální proteiny genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
RNA viry při své replikaci manipulují s membránami hostitelských buněk, aby vytvořily tzv. replikační továrny. Tyto továrny napomáhají tvorbě replikačního komplexu a zároveň chrání před aktivací imunitního systému hostitelské buňky. Virové RNA dependentní RNA polymerasy (RdRp) jsou enzymy, které virům umožňují replikovat svůj genom a také připravit mediátorovou RNA pro translaci virových proteinů. Díky své relativní evoluční konzervovanosti jsou RdRp dobrým cílem pro design léčiv.
RNA viruses manipulate host cell membranes to create replication factories during its replication. These factories help the creation of replication complex and at the same time protect from host cell innate immunity activation. Viral RNA dependent RNA polymerases (RdRps) are enzymes which enable RNA viruses to replicate their genome and to prepare mRNA for translation of viral proteins. RdRps are good targets for drug design thanks to its relative evolutionary conservation.
- Klíčová slova
- replikační továrna, RNA dependentní RNA polymeráza,
- MeSH
- replikace viru * MeSH
- RNA-nukleotidyltransferasy * MeSH
- RNA-viry MeSH
- viry genetika klasifikace MeSH
- Publikační typ
- práce podpořená grantem MeSH
Fosfatidylinositoly jsou významné signální molekuly, společně se SNARE proteiny hrají důležitou roli ve vesikulárním transportu. V tomto textu se snažíme zdůraznit vzájemnou součinnost SNARE proteinů a fosfatidylinositolových kinas. Vesikulární transport je velmi kom‐ plikovaný dynamický proces, který není doposud plně prostudován. Jeho pochopení může být klíčové pro léčbu závažných lidských nemocnění jako je například Gaucherova choroba, rakovina prsu, rakovina močového měchýře, papilární karcinom štítné žlázy a další.
Phosphatidylinositols ale important signal molecules, and together with SNARE proteins play important role in vesicular transport; a pro‐ cess that is still not fully understood. In this paper, we try to underline the interaction of SNARE proteins and phosphatidylinositol kinases. Vesicular transport is very complicated dynamic process. The understanding can be crucial for treatment of several human diseases such as Gaucher disease, breast cancer, bladder cancer, papillary thyroid carcinoma, and others.
