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

Zika virus is a global health threat due to significantly elevated risk of fetus malformations in infected pregnant women. Currently, neither an effective therapy nor a prophylactic vaccination is available for clinical use, desperately necessitating novel therapeutics and approaches to obtain them. Here, we present a structural model of the Zika virus RNA-dependent RNA polymerase (ZIKV RdRp) in complex with template and nascent RNAs, Mg2+ ions and accessing nucleoside triphosphate. The model allowed for docking studies aimed at effective pre-screening of potential inhibitors of ZIKV RdRp. Applicability of the structural model for docking studies was illustrated with the NITD008 artificial nucleotide that is known to effectively inhibit the function of the ZIKV RdRp. The ZIKV RdRp - RNA structural model is provided for all possible variations of the nascent RNA bases pairs to enhance its general utility in docking and modelling experiments. The developed model makes the rational design of novel nucleosides and nucleotide analogues feasible and thus provides a solid platform for the development of advanced antiviral therapy.

• MeSH
• adenosin analogy a deriváty   chemie   farmakologie   MeSH
• hořčík chemie   MeSH
• infekce virem zika genetika   virologie   MeSH
• konformace proteinů účinky léků   MeSH
• lidé MeSH
• molekulární modely MeSH
• nukleosidy chemie   MeSH
• nukleotidy chemie   MeSH
• polyfosfáty chemie   MeSH
• replikace viru genetika   MeSH
• RNA-dependentní RNA-polymerasa chemie   genetika   MeSH
• RNA chemie   genetika   MeSH
• simulace molekulového dockingu MeSH
• virové nestrukturální proteiny chemie   genetika   MeSH
• virus zika chemie   genetika   patogenita   MeSH
• Check Tag
• lidé 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

Phage T4 lysozyme is a well folded and highly soluble protein that is widely used as an insertion tag to improve solubility and crystallization properties of poorly behaved recombinant proteins. It has been used in the fusion protein strategy to facilitate crystallization of various proteins including multiple G protein-coupled receptors, lipid kinases, or sterol binding proteins. Here, we present a structural and biochemical characterization of its novel, metal ions-binding mutant (mbT4L). We demonstrate that mbT4L can be used as a purification tag in the immobilized-metal affinity chromatography and that, in many respects, it is superior to the conventional hexahistidine tag. In addition, structural characterization of mbT4L suggests that mbT4L can be used as a purification tag compatible with X-ray crystallography.

• MeSH
• bakteriofág T4 * enzymologie   genetika   MeSH
• chromatografie afinitní metody   MeSH
• krystalografie rentgenová metody   MeSH
• muramidasa * chemie   genetika   izolace a purifikace   MeSH
• mutace * MeSH
• Publikační typ
• časopisecké články 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

Picornaviruses are small positive-sense single-stranded RNA viruses that include many important human pathogens. Within the host cell, they replicate at specific replication sites called replication organelles. To create this membrane platform, they hijack several host factors including the acyl-CoA-binding domain-containing protein-3 (ACBD3). Here, we present a structural characterization of the molecular complexes formed by the non-structural 3A proteins from two species of the Kobuvirus genus of the Picornaviridae family and the 3A-binding domain of the host ACBD3 protein. Specifically, we present a series of crystal structures as well as a molecular dynamics simulation of the 3A:ACBD3 complex at the membrane, which reveals that the viral 3A proteins act as molecular harnesses to enslave the ACBD3 protein leading to its stabilization at target membranes. Our data provide a structural rationale for understanding how these viral-host protein complexes assemble at the atomic level and identify new potential targets for antiviral therapies.

• MeSH
• adaptorové proteiny signální transdukční chemie   genetika   metabolismus   MeSH
• aminokyselinové motivy MeSH
• buněčné linie MeSH
• exprese genu MeSH
• interakce hostitele a patogenu * MeSH
• interakční proteinové domény a motivy MeSH
• klonování DNA MeSH
• Kobuvirus genetika   metabolismus   MeSH
• konformace proteinů, alfa-helix MeSH
• konformace proteinů, beta-řetězec MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• membránové proteiny chemie   genetika   metabolismus   MeSH
• rekombinantní proteiny chemie   genetika   metabolismus   MeSH
• replikace viru genetika   MeSH
• simulace molekulární dynamiky MeSH
• stabilita proteinů MeSH
• unilamelární lipozómy chemie   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• virové nestrukturální proteiny chemie   genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• 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.

• MeSH
• fosfatidylinositol-4-fosfát-3-kinasa fyziologie   MeSH
• fosfatidylinositoly * fyziologie   MeSH
• intracelulární prostor fyziologie   metabolismus   MeSH
• lidé MeSH
• vezikulární transportní proteiny * fyziologie   metabolismus   MeSH
• zdraví MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

Phosphatidylinositol 4-kinase beta (PI4KB) is one of four human PI4K enzymes that generate phosphatidylinositol 4-phosphate (PI4P), a minor but essential regulatory lipid found in all eukaryotic cells. To convert their lipid substrates, PI4Ks must be recruited to the correct membrane compartment. PI4KB is critical for the maintenance of the Golgi and trans Golgi network (TGN) PI4P pools, however, the actual targeting mechanism of PI4KB to the Golgi and TGN membranes is unknown. Here, we present an NMR structure of the complex of PI4KB and its interacting partner, Golgi adaptor protein acyl-coenzyme A binding domain containing protein 3 (ACBD3). We show that ACBD3 is capable of recruiting PI4KB to membranes both in vitro and in vivo, and that membrane recruitment of PI4KB by ACBD3 increases its enzymatic activity and that the ACBD3:PI4KB complex formation is essential for proper function of the Golgi.

• MeSH
• adaptorové proteiny signální transdukční chemie   metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• Cercopithecus aethiops MeSH
• COS buňky MeSH
• fosfatidylinositolfosfáty metabolismus   MeSH
• fosfotransferasy s alkoholovou skupinou jako akceptorem chemie   metabolismus   MeSH
• Golgiho aparát metabolismus   MeSH
• lidé MeSH
• membránové proteiny chemie   metabolismus   MeSH
• molekulární modely MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• sekundární struktura proteinů MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Intramural MeSH

Phosphoinositides are a class of phospholipids generated by the action of phosphoinositide kinases with key regulatory functions in eukaryotic cells. Here, we present the atomic structure of phosphatidylinositol 4-kinase type IIα (PI4K IIα), in complex with ATP solved by X-ray crystallography at 2.8 Å resolution. The structure revealed a non-typical kinase fold that could be divided into N- and C-lobes with the ATP binding groove located in between. Surprisingly, a second ATP was found in a lateral hydrophobic pocket of the C-lobe. Molecular simulations and mutagenesis analysis revealed the membrane binding mode and the putative function of the hydrophobic pocket. Taken together, our results suggest a mechanism of PI4K IIα recruitment, regulation, and function at the membrane.

• MeSH
• fosfatidylinositoly chemie   metabolismus   MeSH
• fosfotransferasy s alkoholovou skupinou jako akceptorem chemie   metabolismus   ultrastruktura   MeSH
• inositol chemie   MeSH
• konformace proteinů * MeSH
• krystalografie rentgenová * MeSH
• lidé MeSH
• membrány chemie   MeSH
• metoda Monte Carlo MeSH
• signální transdukce MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH