Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease-19 pandemic. One of the key components of the coronavirus replication complex are the RNA methyltransferases (MTases), RNA-modifying enzymes crucial for RNA cap formation. Recently, the structure of the 2'-O MTase has become available; however, its biological characterization within the infected cells remains largely elusive. Here, we report a novel monoclonal antibody directed against the SARS-CoV-2 non-structural protein nsp10, a subunit of both the 2'-O RNA and N7 MTase protein complexes. Using this antibody, we investigated the subcellular localization of the SARS-CoV-2 MTases in cells infected with the SARS-CoV-2.

• Klíčová slova
• SARS-CoV-2, capping enzyme, coronavirus, methyltransferase, nsp10, nsp14, nsp16,
• MeSH
• COVID-19 virologie   MeSH
• lidé MeSH
• methyltransferasy analýza   genetika   metabolismus   MeSH
• monoklonální protilátky analýza   MeSH
• RNA čepičky genetika   metabolismus   MeSH
• RNA virová genetika   metabolismus   MeSH
• SARS-CoV-2 chemie   enzymologie   genetika   MeSH
• transport proteinů MeSH
• virové nestrukturální proteiny analýza   genetika   metabolismus   MeSH
• virové regulační a přídatné proteiny analýza   genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• methyltransferasy MeSH
• monoklonální protilátky MeSH
• NSP10 protein, SARS-CoV-2 MeSH Prohlížeč
• NSP16 protein, SARS-CoV-2 MeSH Prohlížeč
• RNA čepičky MeSH
• RNA virová MeSH
• virové nestrukturální proteiny MeSH
• virové regulační a přídatné proteiny MeSH

Picornaviruses infect a wide range of mammals including livestock such as cattle and swine. As with other picornavirus genera such as Aphthovirus, there is emerging evidence of a significant economic impact of livestock infections caused by members of the genera Enterovirus and Kobuvirus. While the human-infecting enteroviruses and kobuviruses have been intensively studied during the past decades in great detail, research on livestock-infecting viruses has been mostly limited to the genomic characterization of the viral strains identified worldwide. Here, we extend our previous studies of the structure and function of the complexes composed of the non-structural 3A proteins of human-infecting enteroviruses and kobuviruses and the host ACBD3 protein and present a structural and functional characterization of the complexes of the following livestock-infecting picornaviruses: bovine enteroviruses EV-E and EV-F, porcine enterovirus EV-G, and porcine kobuvirus AiV-C. We present a series of crystal structures of these complexes and demonstrate the role of these complexes in facilitation of viral replication.

• MeSH
• adaptorové proteiny signální transdukční metabolismus   MeSH
• bovinní enterovirus patogenita   MeSH
• buněčné linie MeSH
• enterovirové infekce metabolismus   veterinární   virologie   MeSH
• enteroviry prasat genetika   patogenita   MeSH
• HEK293 buňky MeSH
• Kobuvirus genetika   patogenita   MeSH
• lidé MeSH
• membránové proteiny metabolismus   MeSH
• pikornavirové infekce metabolismus   veterinární   virologie   MeSH
• prasata MeSH
• replikace viru genetika   MeSH
• skot MeSH
• virové proteiny genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• ACBD3 protein, human MeSH Prohlížeč
• adaptorové proteiny signální transdukční MeSH
• membránové proteiny MeSH
• virové proteiny MeSH

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.

• Klíčová slova
• Crystal structure, Kobuvirus, Picornavirus, Polymerase, RNA,
• 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
• Názvy látek
• RNA-dependentní RNA-polymerasa MeSH
• virové proteiny MeSH

Enteroviruses, members of the family of picornaviruses, are the most common viral infectious agents in humans causing a broad spectrum of diseases ranging from mild respiratory illnesses to life-threatening infections. To efficiently replicate within the host cell, enteroviruses hijack several host factors, such as ACBD3. ACBD3 facilitates replication of various enterovirus species, however, structural determinants of ACBD3 recruitment to the viral replication sites are poorly understood. Here, we present a structural characterization of the interaction between ACBD3 and the non-structural 3A proteins of four representative enteroviruses (poliovirus, enterovirus A71, enterovirus D68, and rhinovirus B14). In addition, we describe the details of the 3A-3A interaction causing the assembly of the ACBD3-3A heterotetramers and the interaction between the ACBD3-3A complex and the lipid bilayer. Using structure-guided identification of the point mutations disrupting these interactions, we demonstrate their roles in the intracellular localization of these proteins, recruitment of downstream effectors of ACBD3, and facilitation of enterovirus replication. These structures uncovered a striking convergence in the mechanisms of how enteroviruses and kobuviruses, members of a distinct group of picornaviruses that also rely on ACBD3, recruit ACBD3 and its downstream effectors to the sites of viral replication.

• MeSH
• adaptorové proteiny signální transdukční chemie   genetika   metabolismus   MeSH
• fosfotransferasy s alkoholovou skupinou jako akceptorem genetika   metabolismus   MeSH
• HEK293 buňky MeSH
• interakce hostitele a patogenu * MeSH
• konformace proteinů MeSH
• krystalizace MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• membránové proteiny chemie   genetika   metabolismus   MeSH
• molekulární modely MeSH
• mutace MeSH
• Picornaviridae fyziologie   MeSH
• replikace viru * MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie MeSH
• vazba proteinů MeSH
• virové proteiny chemie   genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ACBD3 protein, human MeSH Prohlížeč
• adaptorové proteiny signální transdukční MeSH
• fosfotransferasy s alkoholovou skupinou jako akceptorem MeSH
• membránové proteiny MeSH
• virové proteiny MeSH

Tumour necrosis factor (TNF) related apoptosis inducing ligand (TRAIL), a membrane-bound ligand from the TNF family, has attracted significant attention due to its rather specific and effective ability to induce apoptotic death in various types of cancer cells via binding to and activating its pro-apoptotic death receptors. However, a significant number of primary cancer cells often develop resistance to TRAIL treatment, and the signalling platform behind this phenomenon is not fully understood. Upon blocking endosomal acidification by the vacuolar ATPase (V-ATPase) inhibitors bafilomycin A1 (BafA1) or concanamycin A, we observed a significantly reduced initial sensitivity of several, mainly colorectal, tumour cell lines to TRAIL-induced apoptosis. In cells pretreated with these inhibitors, the TRAIL-induced processing of caspase-8 and the aggregation and trafficking of the TRAIL receptor complexes were temporarily attenuated. Nuclear factor κB or mitogen activated protein/stress kinase signalling from the activated TRAIL receptors remained unchanged, and neither possible lysosomal permeabilization nor acid sphingomyelinase was involved in this process. The cell surface expression of TRAIL receptors and their TRAIL-induced internalization were not affected by V-ATPase inhibitors. The inhibitory effect of BafA1, however, was blunted by knockdown of the caspase-8 inhibitor cFLIP. Altogether, the data obtained provide the first evidence that endosomal acidification could represent an important regulatory node in the proximal part of TRAIL-induced pro-apoptotic signalling.

• Klíčová slova
• TRAIL, V-ATPase, acidification, apoptosis, caspase-8,
• MeSH
• aktivace enzymů MeSH
• apoptóza MeSH
• down regulace MeSH
• endozomy metabolismus   MeSH
• FLIP (buněčný) metabolismus   MeSH
• kaspasa 8 metabolismus   MeSH
• koncentrace vodíkových iontů MeSH
• lidé MeSH
• makrolidy farmakologie   MeSH
• nádorové buněčné linie MeSH
• protein TRAIL farmakologie   MeSH
• protinádorové látky farmakologie   MeSH
• sfingolipidy fyziologie   MeSH
• sfingomyelinfosfodiesterasa metabolismus   MeSH
• signální adaptorové proteiny receptorové domény smrti metabolismus   MeSH
• signální transdukce účinky léků   MeSH
• TRAIL receptory metabolismus   MeSH
• transport proteinů MeSH
• vakuolární protonové ATPasy antagonisté a inhibitory   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bafilomycin A1 MeSH Prohlížeč
• CASP8 protein, human MeSH Prohlížeč
• concanamycin A MeSH Prohlížeč
• FLIP (buněčný) MeSH
• kaspasa 8 MeSH
• makrolidy MeSH
• protein TRAIL MeSH
• protinádorové látky MeSH
• sfingolipidy MeSH
• sfingomyelinfosfodiesterasa MeSH
• signální adaptorové proteiny receptorové domény smrti MeSH
• TNFSF10 protein, human MeSH Prohlížeč
• TRAIL receptory MeSH
• vakuolární protonové ATPasy MeSH