Autophagy, an intracellular degradation process, has emerged as a crucial innate immune response against various plant pathogens, including viruses. Tomato spotted wilt orthotospovirus (TSWV) is a highly destructive plant pathogen that infects over 1000 plant species and poses a significant threat to global food security. However, the role of autophagy in defence against the TSWV pathogen, and whether the virus counteracts this defence, remains unknown. In this study, we report that autophagy plays an important role in antiviral defence against TSWV infection; however, this autophagy-mediated defence is counteracted by the viral effector NSs. Transcriptome profiling revealed the up-regulation of autophagy-related genes (ATGs) upon TSWV infection. Blocking autophagy induction by chemical treatment or knockout/down of ATG5/ATG7 significantly enhanced TSWV accumulation. Notably, the TSWV nucleocapsid (N) protein, a major component of the viral replication unit, strongly induced autophagy. However, the TSWV nonstructural protein NSs was able to effectively suppress N-induced autophagy in a dose-dependent manner. Further investigation revealed that NSs inhibited ATG6-mediated autophagy induction. These findings provide new insights into the defence role of autophagy against TSWV, a representative segmented negative-strand RNA virus, as well as the tospoviral pathogen counterdefence mechanism.

• Klíčová slova
• TSWV, antiviral defence, autophagy, counterdefence, nonstructural protein NSs, nucleocapsid protein,
• MeSH
• autofagie * MeSH
• nemoci rostlin * virologie   imunologie   MeSH
• Solanum lycopersicum virologie   imunologie   genetika   MeSH
• tabák virologie   imunologie   genetika   MeSH
• Tospovirus * fyziologie   patogenita   MeSH
• virové nestrukturální proteiny metabolismus   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• virové nestrukturální proteiny MeSH

Since the emergence of SARS-CoV-2, mutations in all subunits of the RNA-dependent RNA polymerase (RdRp) of the virus have been repeatedly reported. Although RdRp represents a primary target for antiviral drugs, experimental studies exploring the phenotypic effect of these mutations have been limited. This study focuses on the phenotypic effects of substitutions in the three RdRp subunits: nsp7, nsp8, and nsp12, selected based on their occurrence rate and potential impact. We employed nano-differential scanning fluorimetry and microscale thermophoresis to examine the impact of these mutations on protein stability and RdRp complex assembly. We observed diverse impacts; notably, a single mutation in nsp8 significantly increased its stability as evidenced by a 13°C increase in melting temperature, whereas certain mutations in nsp7 and nsp8 reduced their binding affinity to nsp12 during RdRp complex formation. Using a fluorometric enzymatic assay, we assessed the overall effect on RNA polymerase activity. We found that most of the examined mutations altered the polymerase activity, often as a direct result of changes in stability or affinity to the other components of the RdRp complex. Intriguingly, a combination of nsp8 A21V and nsp12 P323L mutations resulted in a 50% increase in polymerase activity. To our knowledge, this is the first biochemical study to demonstrate the impact of amino acid mutations across all components constituting the RdRp complex in emerging SARS-CoV-2 subvariants.

• Klíčová slova
• RNA‐dependent RNA polymerase (RdRp), SARS‐CoV‐2, mutations, phenotypic effect,
• MeSH
• COVID-19 virologie   MeSH
• koronavirová RNA-replikasa * genetika   metabolismus   chemie   MeSH
• lidé MeSH
• mutace * MeSH
• RNA-dependentní RNA-polymerasa genetika   chemie   metabolismus   MeSH
• SARS-CoV-2 * genetika   enzymologie   MeSH
• stabilita proteinů MeSH
• vazba proteinů MeSH
• virové nestrukturální proteiny * genetika   chemie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• koronavirová RNA-replikasa * MeSH
• NS8 protein, SARS-CoV-2 MeSH Prohlížeč
• NSP12 protein, SARS-CoV-2 MeSH Prohlížeč
• NSP7 protein, SARS-CoV-2 MeSH Prohlížeč
• RNA-dependentní RNA-polymerasa MeSH
• virové nestrukturální proteiny * MeSH

Flaviviruses are single-stranded positive-sense RNA (+RNA) viruses that are responsible for several (re)emerging diseases such as yellow, dengue, or West Nile fevers. The Zika epidemic highlighted their dangerousness when a relatively benign virus known since the 1950s turned into a deadly pathogen. The central protein for their replication is NS5 (non-structural protein 5), which is composed of the N-terminal methyltransferase (MTase) domain and the C-terminal RNA-dependent RNA-polymerase (RdRp) domain. It is responsible for both RNA replication and installation of the 5' RNA cap. We structurally and biochemically analyzed the Ntaya virus MTase and RdRp domains and we compared their properties to other flaviviral NS5s. The enzymatic centers are well conserved across Flaviviridae, suggesting that the development of drugs targeting all flaviviruses is feasible. However, the enzymatic activities of the isolated proteins were significantly different for the MTase domains.

• MeSH
• Flavivirus enzymologie   metabolismus   MeSH
• krystalografie rentgenová MeSH
• methyltransferasy * metabolismus   chemie   MeSH
• molekulární modely * MeSH
• proteinové domény MeSH
• RNA-dependentní RNA-polymerasa * metabolismus   chemie   MeSH
• sekvence aminokyselin MeSH
• vazba proteinů MeSH
• virové nestrukturální proteiny * metabolismus   chemie   genetika   MeSH
• viry klíšťové encefalitidy metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• methyltransferasy * MeSH
• NS5 protein, flavivirus MeSH Prohlížeč
• RNA-dependentní RNA-polymerasa * MeSH
• virové nestrukturální proteiny * MeSH

Aim: Discovery of novel SARS-CoV-2 main protease (Mpro) inhibitors using a structure-based drug discovery strategy. Materials & methods: Virtual screening employing covalent and noncovalent docking was performed to discover Mpro inhibitors, which were subsequently evaluated in biochemical and cellular assays. Results: 91 virtual hits were selected for biochemical assays, and four were confirmed as reversible inhibitors of SARS CoV-2 Mpro with IC50 values of 0.4-3 μM. They were also shown to inhibit SARS-CoV-1 Mpro and human cathepsin L. Molecular dynamics simulations indicated the stability of the Mpro inhibitor complexes and the interaction of ligands at the subsites. Conclusion: This approach led to the discovery of novel thiosemicarbazones as potent SARS-CoV-2 Mpro inhibitors.

• Klíčová slova
• Mpro, SARS-CoV-2, computer-aided drug design, coronavirus, molecular docking, molecular dynamics simulations, protease inhibitors, virtual screening,
• MeSH
• antivirové látky farmakologie   chemie   MeSH
• COVID-19 * MeSH
• inhibitory proteas farmakologie   chemie   MeSH
• lidé MeSH
• SARS-CoV-2 MeSH
• simulace molekulového dockingu MeSH
• thiosemikarbazony * farmakologie   MeSH
• virové nestrukturální proteiny MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 3C-like proteinase, SARS-CoV-2 MeSH Prohlížeč
• antivirové látky MeSH
• inhibitory proteas MeSH
• thiosemikarbazony * MeSH
• virové nestrukturální proteiny MeSH

Monkeypox is a disease with pandemic potential. It is caused by the monkeypox virus (MPXV), a double-stranded DNA virus from the Poxviridae family, that replicates in the cytoplasm and must encode for its own RNA processing machinery including the capping machinery. Here, we present crystal structures of its 2'-O-RNA methyltransferase (MTase) VP39 in complex with the pan-MTase inhibitor sinefungin and a series of inhibitors that were discovered based on it. A comparison of this 2'-O-RNA MTase with enzymes from unrelated single-stranded RNA viruses (SARS-CoV-2 and Zika) reveals a conserved sinefungin binding mode, implicating that a single inhibitor could be used against unrelated viral families. Indeed, several of our inhibitors such as TO507 also inhibit the coronaviral nsp14 MTase.

• MeSH
• COVID-19 * MeSH
• infekce virem zika * MeSH
• lidé MeSH
• methyltransferasy metabolismus   MeSH
• RNA virová genetika   MeSH
• RNA MeSH
• SARS-CoV-2 genetika   MeSH
• virové nestrukturální proteiny chemie   MeSH
• virus opičích neštovic genetika   metabolismus   MeSH
• virus zika * genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• methyltransferasy MeSH
• RNA virová MeSH
• RNA MeSH
• virové nestrukturální proteiny MeSH

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) represent cornerstones of current regimens for treatment of human immunodeficiency virus type 1 (HIV-1) infections. However, NNRTIs usually suffer from low aqueous solubility and the emergence of resistant viral strains. In the present work, novel bicyclic NNRTIs derived from etravirine (ETV) and rilpivirine (RPV), bearing modified purine, tetrahydropteridine, and pyrimidodiazepine cores, were designed and prepared. Compounds 2, 4, and 6 carrying the acrylonitrile moiety displayed single-digit nanomolar activities against the wild-type (WT) virus (EC50 = 2.5, 2.7, and 3.0 nM, respectively), where the low nanomolar activity was retained against HXB2 (EC50 = 2.2-2.8 nM) and the K103N and Y181C mutated strains (fold change, 1.2-6.7×). Most importantly, compound 2 exhibited significantly improved phosphate-buffered saline solubility (10.4 μM) compared to ETV and RPV (≪1 μM). Additionally, the binding modes of compounds 2, 4, and 6 to the reverse transcriptase were studied by X-ray crystallography.

• MeSH
• HIV infekce * farmakoterapie   MeSH
• HIV reverzní transkriptasa metabolismus   MeSH
• HIV-1 * metabolismus   MeSH
• inhibitory reverzní transkriptasy MeSH
• látky proti HIV * chemie   MeSH
• lidé MeSH
• racionální návrh léčiv MeSH
• rilpivirin terapeutické užití   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• etravirine MeSH Prohlížeč
• HIV reverzní transkriptasa MeSH
• inhibitory reverzní transkriptasy MeSH
• látky proti HIV * MeSH
• rilpivirin MeSH

The worldwide COVID-19 pandemic caused by the coronavirus SARS-CoV-2 urgently demands novel direct antiviral treatments. The main protease (Mpro) and papain-like protease (PLpro) are attractive drug targets among coronaviruses due to their essential role in processing the polyproteins translated from the viral RNA. In this study, we virtually screened 688 naphthoquinoidal compounds and derivatives against Mpro of SARS-CoV-2. Twenty-four derivatives were selected and evaluated in biochemical assays against Mpro using a novel fluorogenic substrate. In parallel, these compounds were also assayed with SARS-CoV-2 PLpro. Four compounds inhibited Mpro with half-maximal inhibitory concentration (IC50) values between 0.41 μM and 9.0 μM. In addition, three compounds inhibited PLpro with IC50 ranging from 1.9 μM to 3.3 μM. To verify the specificity of Mpro and PLpro inhibitors, our experiments included an assessment of common causes of false positives such as aggregation, high compound fluorescence, and inhibition by enzyme oxidation. Altogether, we confirmed novel classes of specific Mpro and PLpro inhibitors. Molecular dynamics simulations suggest stable binding modes for Mpro inhibitors with frequent interactions with residues in the S1 and S2 pockets of the active site. For two PLpro inhibitors, interactions occur in the S3 and S4 pockets. In summary, our structure-based computational and biochemical approach identified novel naphthoquinonal scaffolds that can be further explored as SARS-CoV-2 antivirals.

• MeSH
• antivirové látky * farmakologie   chemie   MeSH
• COVID-19 MeSH
• inhibitory proteas * farmakologie   chemie   MeSH
• koronavirové proteasy 3C * antagonisté a inhibitory   MeSH
• koronavirové proteasy podobné papainu * antagonisté a inhibitory   MeSH
• lidé MeSH
• naftochinony * chemie   farmakologie   MeSH
• papain MeSH
• SARS-CoV-2 * účinky léků   enzymologie   MeSH
• simulace molekulového dockingu MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• 3C-like proteinase, SARS-CoV-2 MeSH Prohlížeč
• antivirové látky * MeSH
• inhibitory proteas * MeSH
• koronavirové proteasy 3C * MeSH
• koronavirové proteasy podobné papainu * MeSH
• naftochinony * MeSH
• papain-like protease, SARS-CoV-2 MeSH Prohlížeč
• papain MeSH

Some medically important viruses-including retroviruses, flaviviruses, coronaviruses, and herpesviruses-code for a protease, which is indispensable for viral maturation and pathogenesis. Viral protease inhibitors have become an important class of antiviral drugs. Development of the first-in-class viral protease inhibitor saquinavir, which targets HIV protease, started a new era in the treatment of chronic viral diseases. Combining several drugs that target different steps of the viral life cycle enables use of lower doses of individual drugs (and thereby reduction of potential side effects, which frequently occur during long term therapy) and reduces drug-resistance development. Currently, several HIV and HCV protease inhibitors are routinely used in clinical practice. In addition, a drug including an inhibitor of SARS-CoV-2 main protease, nirmatrelvir (co-administered with a pharmacokinetic booster ritonavir as Paxlovid®), was recently authorized for emergency use. This review summarizes the basic features of the proteases of human immunodeficiency virus (HIV), hepatitis C virus (HCV), and SARS-CoV-2 and discusses the properties of their inhibitors in clinical use, as well as development of compounds in the pipeline.

• MeSH
• antivirové látky farmakologie   terapeutické užití   MeSH
• COVID-19 * MeSH
• HIV infekce * farmakoterapie   MeSH
• lidé MeSH
• SARS-CoV-2 MeSH
• virové proteasy MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• antivirové látky MeSH
• nirmatrelvir and ritonavir drug combination MeSH Prohlížeč
• virové proteasy MeSH

Tick-borne encephalitis virus (TBEV) is the most medically relevant tick-transmitted Flavivirus in Eurasia, targeting the host central nervous system and frequently causing severe encephalitis. The primary function of its capsid protein (TBEVC) is to recruit the viral RNA and form a nucleocapsid. Additional functionality of Flavivirus capsid proteins has been documented, but further investigation is needed for TBEVC. Here, we show the first capsid protein 3D structure of a member of the tick-borne flaviviruses group. The structure of monomeric Δ16-TBEVC was determined using high-resolution multidimensional NMR spectroscopy. Based on natural in vitro TBEVC homodimerization, the dimeric interfaces were identified by hydrogen deuterium exchange mass spectrometry (MS). Although the assembly of flaviviruses occurs in endoplasmic reticulum-derived vesicles, we observed that TBEVC protein also accumulated in the nuclei and nucleoli of infected cells. In addition, the predicted bipartite nuclear localization sequence in the TBEVC C-terminal part was confirmed experimentally, and we described the interface between TBEVC bipartite nuclear localization sequence and import adapter protein importin-alpha using X-ray crystallography. Furthermore, our coimmunoprecipitation coupled with MS identification revealed 214 interaction partners of TBEVC, including viral envelope and nonstructural NS5 proteins and a wide variety of host proteins involved mainly in rRNA processing and translation initiation. Metabolic labeling experiments further confirmed that TBEVC and other flaviviral capsid proteins are able to induce translational shutoff and decrease of 18S rRNA. These findings may substantially help to design a targeted therapy against TBEV.

• Klíčová slova
• capsid, nucleolus, nucleus, protein structure, tick-borne flaviviruses, translational shutoff,
• MeSH
• kapsida metabolismus   MeSH
• RNA virová metabolismus   MeSH
• virové nestrukturální proteiny metabolismus   MeSH
• virové plášťové proteiny genetika   metabolismus   MeSH
• viry klíšťové encefalitidy * genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• RNA virová MeSH
• virové nestrukturální proteiny MeSH
• virové plášťové proteiny MeSH

The helicase domain of nonstructural protein 3 (NS3H) unwinds the double-stranded RNA replication intermediate in an ATP-dependent manner during the flavivirus life cycle. While the ATP hydrolysis mechanism of Dengue and Zika viruses NS3H has been extensively studied, little is known in the case of the tick-borne encephalitis virus NS3H. We demonstrate that ssRNA binds with nanomolar affinity to NS3H and strongly stimulates the ATP hydrolysis cycle, whereas ssDNA binds only weakly and inhibits ATPase activity in a noncompetitive manner. Thus, NS3H is an RNA-specific helicase, whereas DNA might act as an allosteric inhibitor. Using modeling, we explored plausible allosteric mechanisms by which ssDNA inhibits the ATPase via nonspecific binding in the vicinity of the active site and ATP repositioning. We captured several structural snapshots of key ATP hydrolysis stages using X-ray crystallography. One intermediate, in which the inorganic phosphate and ADP remained trapped inside the ATPase site after hydrolysis, suggests that inorganic phosphate release is the rate-limiting step. Using structure-guided modeling and molecular dynamics simulation, we identified putative RNA-binding residues and observed that the opening and closing of the ATP-binding site modulates RNA affinity. Site-directed mutagenesis of the conserved RNA-binding residues revealed that the allosteric activation of ATPase activity is primarily communicated via an arginine residue in domain 1. In summary, we characterized conformational changes associated with modulating RNA affinity and mapped allosteric communication between RNA-binding groove and ATPase site of tick-borne encephalitis virus helicase.

• Klíčová slova
• ATPase, RNA helicase, crystal structure, enzyme kinetics, flavivirus, molecular dynamics, nonstructural protein 3, tick-borne encephalitis virus, viral protein,
• MeSH
• adenosintrifosfát metabolismus   MeSH
• adenosintrifosfatasy * metabolismus   MeSH
• dvouvláknová RNA metabolismus   MeSH
• fosfáty metabolismus   MeSH
• jednovláknová DNA * metabolismus   MeSH
• lidé MeSH
• RNA-helikasy * metabolismus   MeSH
• virové nestrukturální proteiny * metabolismus   MeSH
• viry klíšťové encefalitidy * enzymologie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfát MeSH
• adenosintrifosfatasy * MeSH
• dvouvláknová RNA MeSH
• fosfáty MeSH
• jednovláknová DNA * MeSH
• NS3 protein, flavivirus MeSH Prohlížeč
• RNA-helikasy * MeSH
• virové nestrukturální proteiny * MeSH