Viroids, small circular non-coding RNAs, act as infectious pathogens in higher plants, demonstrating high stability despite consisting solely of naked RNA. Their dependence of replication on host machinery poses the question of whether RNA modifications play a role in viroid biology. Here, we explore RNA modifications in the avocado sunblotch viroid (ASBVd) and the citrus exocortis viroid (CEVd), representative members of viroids replicating in chloroplasts and the nucleus, respectively, using LC - MS and Oxford Nanopore Technology (ONT) direct RNA sequencing. Although no modification was detected in ASBVd, CEVd contained approximately one m6A per RNA molecule. ONT sequencing predicted three m6A positions. Employing orthogonal SELECT method, we confirmed m6A in two positions A353 and A360, which are highly conserved among CEVd variants. These positions are located in the left terminal region of the CEVd rod-like structure where likely RNA Pol II and and TFIIIA-7ZF bind, thus suggesting potential biological role of methylation in viroid replication.

• Klíčová slova
• 6-methyladenosine, LC-MS, m6A SELECT, RNA modification, Viroid, direct RNA-seq,
• MeSH
• konformace nukleové kyseliny MeSH
• kruhová RNA * genetika   metabolismus   MeSH
• nemoci rostlin virologie   MeSH
• posttranskripční úpravy RNA * MeSH
• replikace viru MeSH
• RNA virová * genetika   metabolismus   chemie   MeSH
• sekvenční analýza RNA MeSH
• viroidy * genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kruhová RNA * MeSH
• RNA virová * MeSH

We explored how a simple retrovirus, Mason-Pfizer monkey virus (M-PMV) to facilitate its replication process, utilizes DHX15, a cellular RNA helicase, typically engaged in RNA processing. Through advanced genetic engineering techniques, we showed that M-PMV recruits DHX15 by mimicking cellular mechanisms, relocating it from the nucleus to the cytoplasm to aid in viral assembly. This interaction is essential for the correct packaging of the viral genome and critical for its infectivity. Our findings offer unique insights into the mechanisms of viral manipulation of host cellular processes, highlighting a sophisticated strategy that viruses employ to leverage cellular machinery for their replication. This study adds valuable knowledge to the understanding of viral-host interactions but also suggests a common evolutionary history between cellular processes and viral mechanisms. This finding opens a unique perspective on the export mechanism of intron-retaining mRNAs in the packaging of viral genetic information and potentially develop ways to stop it.

• Klíčová slova
• DEAH-box RNA helicase, DHX15, G-patch, gRNA packaging, retrovirus,
• MeSH
• buněčné jádro metabolismus   virologie   MeSH
• DEAD-box RNA-helikasy metabolismus   genetika   MeSH
• genom virový MeSH
• HEK293 buňky MeSH
• lidé MeSH
• Masonův-Pfizerův opičí virus * genetika   metabolismus   fyziologie   MeSH
• replikace viru genetika   fyziologie   MeSH
• RNA virová * metabolismus   genetika   MeSH
• RNA-helikasy metabolismus   genetika   MeSH
• sestavení viru * genetika   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DEAD-box RNA-helikasy MeSH
• DHX15 protein, human MeSH Prohlížeč
• RNA virová * MeSH
• RNA-helikasy MeSH

We currently lack antivirals for most human viruses. In a quest for new molecules, focusing on viral RNA, instead of viral proteins, can represent a promising strategy. In this study, new inhibitors were identified starting from a published crystal structure of the tertiary SARS-CoV-2 RNA involved in the -1 programmed ribosomal frameshift. The pseudoknot structure was refined, and a virtual screening was performed using the repository of binders to the nucleic acid library, taking into consideration RNA flexibility. Hit compounds were validated against the wild-type virus and with a dual-luciferase assay measuring the frameshift efficiency. Several active molecules were identified. Our study reveals new inhibitors of SARS-CoV-2 but also highlights the feasibility of targeting RNA starting from virtual screening, a strategy that could be broadly applied to drug development.

• MeSH
• antivirové látky * farmakologie   chemie   MeSH
• konformace nukleové kyseliny MeSH
• lidé MeSH
• molekulární modely MeSH
• preklinické hodnocení léčiv * metody   MeSH
• RNA virová * metabolismus   antagonisté a inhibitory   genetika   MeSH
• SARS-CoV-2 * účinky léků   MeSH
• simulace molekulového dockingu MeSH
• uživatelské rozhraní počítače MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antivirové látky * MeSH
• RNA virová * MeSH

Influenza A viruses, causing seasonal epidemics and occasional pandemics, rely on interactions with host proteins for their RNA genome transcription and replication. The viral RNA polymerase utilizes host RNA polymerase II (Pol II) and interacts with the serine 5 phosphorylated (pS5) C-terminal domain (CTD) of Pol II to initiate transcription. Our study, using single-particle electron cryomicroscopy (cryo-EM), reveals the structure of the 1918 pandemic influenza A virus polymerase bound to a synthetic pS5 CTD peptide composed of four heptad repeats mimicking the 52 heptad repeat mammalian Pol II CTD. The structure shows that the CTD peptide binds at the C-terminal domain of the PA viral polymerase subunit (PA-C) and reveals a previously unobserved position of the 627 domain of the PB2 subunit near the CTD. We identify crucial residues of the CTD peptide that mediate interactions with positively charged cavities on PA-C, explaining the preference of the viral polymerase for pS5 CTD. Functional analysis of mutants targeting the CTD-binding site within PA-C reveals reduced transcriptional function or defects in replication, highlighting the multifunctional role of PA-C in viral RNA synthesis. Our study provides insights into the structural and functional aspects of the influenza virus polymerase-host Pol II interaction and identifies a target for antiviral development.IMPORTANCEUnderstanding the intricate interactions between influenza A viruses and host proteins is crucial for developing targeted antiviral strategies. This study employs advanced imaging techniques to uncover the structural nuances of the 1918 pandemic influenza A virus polymerase bound to a specific host protein, shedding light on the vital process of viral RNA synthesis. The study identifies key amino acid residues in the influenza polymerase involved in binding host polymerase II (Pol II) and highlights their role in both viral transcription and genome replication. These findings not only deepen our understanding of the influenza virus life cycle but also pinpoint a potential target for antiviral development. By elucidating the structural and functional aspects of the influenza virus polymerase-host Pol II interaction, this research provides a foundation for designing interventions to disrupt viral replication and transcription, offering promising avenues for future antiviral therapies.

• Klíčová slova
• CTD, RNA polymerase II, RNA polymerases, influenza, transcription,
• MeSH
• chřipka lidská virologie   MeSH
• elektronová kryomikroskopie * MeSH
• fosforylace MeSH
• genetická transkripce MeSH
• lidé MeSH
• molekulární modely MeSH
• proteinové domény MeSH
• replikace viru MeSH
• RNA virová metabolismus   genetika   MeSH
• RNA-dependentní RNA-polymerasa * metabolismus   chemie   MeSH
• RNA-polymerasa II * metabolismus   chemie   MeSH
• vazba proteinů MeSH
• virové proteiny * metabolismus   chemie   genetika   MeSH
• virus chřipky A * metabolismus   genetika   enzymologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• RNA virová MeSH
• RNA-dependentní RNA-polymerasa * MeSH
• RNA-polymerasa II * MeSH
• virové proteiny * 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

SARS-CoV-2 nsp10-nsp16 complex is a 2'-O-methyltransferase (MTase) involved in viral RNA capping, enabling the virus to evade the immune system in humans. It has been considered a valuable target in the discovery of antiviral therapeutics, as the RNA cap formation is crucial for viral propagation. Through cross-screening of the inhibitors that we previously reported for SARS-CoV-2 nsp14 MTase activity against nsp10-nsp16 complex, we identified two compounds (SS148 and WZ16) that also inhibited nsp16 MTase activity. To further enable the chemical optimization of these two compounds towards more potent and selective dual nsp14/nsp16 MTase inhibitors, we determined the crystal structure of nsp10-nsp16 in complex with each of SS148 and WZ16. As expected, the structures revealed the binding of both compounds to S-adenosyl-L-methionine (SAM) binding pocket of nsp16. However, our structural data along with the biochemical mechanism of action determination revealed an RNA-dependent SAM-competitive pattern of inhibition for WZ16, clearly suggesting that binding of the RNA first may help the binding of some SAM competitive inhibitors. Both compounds also showed some degree of selectivity against human protein MTases, an indication of great potential for chemical optimization towards more potent and selective inhibitors of coronavirus MTases.

• Klíčová slova
• COVID-19, SARS-CoV-2, SS148, WZ16, nsp10, nsp16,
• MeSH
• farmakoterapie COVID-19 * MeSH
• lidé MeSH
• methyltransferasy chemie   MeSH
• RNA virová metabolismus   MeSH
• SARS-CoV-2 * MeSH
• virové nestrukturální proteiny chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• methyltransferasy MeSH
• RNA virová MeSH
• virové nestrukturální proteiny MeSH

The use of high-throughput small RNA sequencing is well established as a technique to unveil the miRNAs in various tissues. The miRNA profiles are different between infected and non-infected tissues. We compare the SARS-CoV-2 positive and SARS-CoV-2 negative RNA samples extracted from human nasopharynx tissue samples to show different miRNA profiles. We explored differentially expressed miRNAs in response to SARS-CoV-2 in the RNA extracted from nasopharynx tissues of 10 SARS-CoV-2-positive and 10 SARS-CoV-2-negative patients. miRNAs were identified by small RNA sequencing, and the expression levels of selected miRNAs were validated by real-time RT-PCR. We identified 943 conserved miRNAs, likely generated through posttranscriptional modifications. The identified miRNAs were expressed in both RNA groups, NegS and PosS: miR-148a, miR-21, miR-34c, miR-34b, and miR-342. The most differentially expressed miRNA was miR-21, which is likely closely linked to the presence of SARS-CoV-2 in nasopharynx tissues. Our results contribute to further understanding the role of miRNAs in SARS-CoV-2 pathogenesis, which may be crucial for understanding disease symptom development in humans.

• Klíčová slova
• SARS-CoV-2, miRNAs, mir-21, real-time RT-PCR, small RNA sequencing,
• MeSH
• analýza hlavních komponent MeSH
• COVID-19 patologie   virologie   MeSH
• down regulace MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• lidé MeSH
• mikro RNA chemie   metabolismus   MeSH
• nazofarynx metabolismus   virologie   MeSH
• RNA virová metabolismus   MeSH
• SARS-CoV-2 genetika   izolace a purifikace   fyziologie   MeSH
• sekvenční analýza RNA MeSH
• transkriptom MeSH
• upregulace MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mikro RNA MeSH
• RNA virová MeSH

Coronaviral methyltransferases (MTases), nsp10/16 and nsp14, catalyze the last two steps of viral RNA-cap creation that takes place in cytoplasm. This cap is essential for the stability of viral RNA and, most importantly, for the evasion of innate immune system. Non-capped RNA is recognized by innate immunity which leads to its degradation and the activation of antiviral immunity. As a result, both coronaviral MTases are in the center of scientific scrutiny. Recently, X-ray and cryo-EM structures of both enzymes were solved even in complex with other parts of the viral replication complex. High-throughput screening as well as structure-guided inhibitor design have led to the discovery of their potent inhibitors. Here, we critically summarize the tremendous advancement of the coronaviral MTase field since the beginning of COVID pandemic.

• MeSH
• aminokyseliny chemie   MeSH
• Coronavirus účinky léků   enzymologie   genetika   MeSH
• lidé MeSH
• methyltransferasy antagonisté a inhibitory   chemie   metabolismus   MeSH
• metylace MeSH
• molekulární konformace MeSH
• molekulární modely MeSH
• molekulární struktura MeSH
• objevování léků MeSH
• RNA virová chemie   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aminokyseliny MeSH
• methyltransferasy MeSH
• RNA virová MeSH

New analytical techniques that overcome major drawbacks of current routinely used viral infection diagnosis methods, i.e., the long analysis time and laboriousness of real-time reverse-transcription polymerase chain reaction (qRT-PCR) and the insufficient sensitivity of "antigen tests", are urgently needed in the context of SARS-CoV-2 and other highly contagious viruses. Here, we report on an antifouling terpolymer-brush biointerface that enables the rapid and sensitive detection of SARS-CoV-2 in untreated clinical samples. The developed biointerface carries a tailored composition of zwitterionic and non-ionic moieties and allows for the significant improvement of antifouling capabilities when postmodified with biorecognition elements and exposed to complex media. When deployed on a surface of piezoelectric sensor and postmodified with human-cell-expressed antibodies specific to the nucleocapsid (N) protein of SARS-CoV-2, it made possible the quantitative analysis of untreated samples by a direct detection assay format without the need of additional amplification steps. Natively occurring N-protein-vRNA complexes, usually disrupted during the sample pre-treatment steps, were detected in the untreated clinical samples. This biosensor design improved the bioassay sensitivity to a clinically relevant limit of detection of 1.3 × 104 PFU/mL within a detection time of only 20 min. The high specificity toward N-protein-vRNA complexes was validated both by mass spectrometry and qRT-PCR. The performance characteristics were confirmed by qRT-PCR through a comparative study using a set of clinical nasopharyngeal swab samples. We further demonstrate the extraordinary fouling resistance of this biointerface through exposure to other commonly used crude biological samples (including blood plasma, oropharyngeal, stool, and nasopharyngeal swabs), measured via both the surface plasmon resonance and piezoelectric measurements, which highlights the potential to serve as a generic platform for a wide range of biosensing applications.

• Klíčová slova
• SARS-CoV-2, functional coatings, piezoelectric biosensor, polymer brush, rapid detection, zwitterionic materials,
• MeSH
• biosenzitivní techniky MeSH
• biotest MeSH
• bioznečištění MeSH
• COVID-19 diagnóza   MeSH
• fosfoproteiny chemie   MeSH
• hmotnostní spektrometrie MeSH
• ionty MeSH
• koronavirové nukleokapsidové proteiny chemie   MeSH
• lidé MeSH
• limita detekce MeSH
• nazofarynx virologie   MeSH
• nosní sliznice virologie   MeSH
• odběr biologického vzorku MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• polymery chemie   MeSH
• reprodukovatelnost výsledků MeSH
• RNA virová metabolismus   MeSH
• SARS-CoV-2 * MeSH
• senzitivita a specificita MeSH
• testování na COVID-19 * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fosfoproteiny MeSH
• ionty MeSH
• koronavirové nukleokapsidové proteiny MeSH
• nucleocapsid phosphoprotein, SARS-CoV-2 MeSH Prohlížeč
• polymery MeSH
• RNA virová MeSH

Fullerene derivatives with hydrophilic substituents have been shown to exhibit a range of biological activities, including antiviral ones. For a long time, the anti-HIV activity of fullerene derivatives was believed to be due to their binding into the hydrophobic pocket of HIV-1 protease, thereby blocking its activity. Recent work, however, brought new evidence of a novel, protease-independent mechanism of fullerene derivatives' action. We studied in more detail the mechanism of the anti-HIV-1 activity of N,N-dimethyl[70]fulleropyrrolidinium iodide fullerene derivatives. By using a combination of in vitro and cell-based approaches, we showed that these C70 derivatives inhibited neither HIV-1 protease nor HIV-1 maturation. Instead, our data indicate effects of fullerene C70 derivatives on viral genomic RNA packaging and HIV-1 cDNA synthesis during reverse transcription-without impairing reverse transcriptase activity though. Molecularly, this could be explained by a strong binding affinity of these fullerene derivatives to HIV-1 nucleocapsid domain, preventing its proper interaction with viral genomic RNA, thereby blocking reverse transcription and HIV-1 infectivity. Moreover, the fullerene derivatives' oxidative activity and fluorescence quenching, which could be one of the reasons for the inconsistency among reported anti-HIV-1 mechanisms, are discussed herein.

• Klíčová slova
• HIV-1, RNA packaging, fullerene, inhibition, nucleocapsid,
• MeSH
• fullereny metabolismus   farmakologie   MeSH
• genom virový účinky léků   MeSH
• genové produkty gag - virus lidské imunodeficience metabolismus   MeSH
• HEK293 buňky MeSH
• HIV-1 účinky léků   genetika   metabolismus   fyziologie   MeSH
• látky proti HIV metabolismus   farmakologie   MeSH
• lidé MeSH
• nukleokapsida - proteiny metabolismus   MeSH
• reverzní transkripce MeSH
• RNA virová metabolismus   MeSH
• svlékání virového obalu účinky léků   MeSH
• vazba proteinů MeSH
• virion metabolismus   MeSH
• zabalení virového genomu účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fullereny MeSH
• genové produkty gag - virus lidské imunodeficience MeSH
• látky proti HIV MeSH
• nukleokapsida - proteiny MeSH
• RNA virová MeSH