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

There is an urgent need for specific antiviral treatments directed against SARS-CoV-2 to prevent the most severe forms of COVID-19. By drug repurposing, affordable therapeutics could be supplied worldwide in the present pandemic context. Targeting the nucleoprotein N of the SARS-CoV-2 coronavirus could be a strategy to impede viral replication and possibly other essential functions associated with viral N. The antiviral properties of naproxen, a non-steroidal anti-inflammatory drug (NSAID) that was previously demonstrated to be active against Influenza A virus, were evaluated against SARS-CoV-2. Intrinsic fluorescence spectroscopy, fluorescence anisotropy, and dynamic light scattering assays demonstrated naproxen binding to the nucleoprotein of SARS-Cov-2 as predicted by molecular modeling. Naproxen impeded recombinant N oligomerization and inhibited viral replication in infected cells. In VeroE6 cells and reconstituted human primary respiratory epithelium models of SARS-CoV-2 infection, naproxen specifically inhibited viral replication and protected the bronchial epithelia against SARS-CoV-2-induced damage. No inhibition of viral replication was observed with paracetamol or the COX-2 inhibitor celecoxib. Thus, among the NSAID tested, only naproxen combined antiviral and anti-inflammatory properties. Naproxen addition to the standard of care could be beneficial in a clinical setting, as tested in an ongoing clinical study.

• Klíčová slova
• SARS-CoV-2, antiviral, drug repurposing, inflammation, influenza, nucleoprotein, oligomerization, structure-based drug design,
• MeSH
• antiflogistika nesteroidní farmakologie   MeSH
• antivirové látky farmakologie   MeSH
• buněčné linie MeSH
• Cercopithecus aethiops MeSH
• farmakoterapie COVID-19 * MeSH
• lidé MeSH
• naproxen farmakologie   MeSH
• nukleoproteiny antagonisté a inhibitory   metabolismus   MeSH
• přehodnocení terapeutických indikací léčivého přípravku MeSH
• replikace viru účinky léků   MeSH
• SARS-CoV-2 účinky léků   fyziologie   MeSH
• simulace molekulového dockingu MeSH
• Vero buňky MeSH
• virové proteiny antagonisté a inhibitory   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antiflogistika nesteroidní MeSH
• antivirové látky MeSH
• naproxen MeSH
• nukleoproteiny MeSH
• virové proteiny MeSH

Influenza A virus (IAV) encodes a polymerase composed of three subunits: PA, with endonuclease activity, PB1 with polymerase activity and PB2 with host RNA five-prime cap binding site. Their cooperation and stepwise activation include a process called cap-snatching, which is a crucial step in the IAV life cycle. Reproduction of IAV can be blocked by disrupting the interaction between the PB2 domain and the five-prime cap. An inhibitor of this interaction called pimodivir (VX-787) recently entered the third phase of clinical trial; however, several mutations in PB2 that cause resistance to pimodivir were observed. First major mutation, F404Y, causing resistance was identified during preclinical testing, next the mutation M431I was identified in patients during the second phase of clinical trials. The mutation H357N was identified during testing of IAV strains at Centers for Disease Control and Prevention. We set out to provide a structural and thermodynamic analysis of the interactions between cap-binding domain of PB2 wild-type and PB2 variants bearing these mutations and pimodivir. Here we present four crystal structures of PB2-WT, PB2-F404Y, PB2-M431I and PB2-H357N in complex with pimodivir. We have thermodynamically analysed all PB2 variants and proposed the effect of these mutations on thermodynamic parameters of these interactions and pimodivir resistance development. These data will contribute to understanding the effect of these missense mutations to the resistance development and help to design next generation inhibitors.

• Klíčová slova
• VX-787, antivirals, influenza A polymerase, pimodivir, resistance,
• MeSH
• krystalografie rentgenová MeSH
• kvantová teorie MeSH
• molekulární modely MeSH
• mutace genetika   MeSH
• mutantní proteiny metabolismus   MeSH
• podjednotky proteinů antagonisté a inhibitory   chemie   metabolismus   MeSH
• proteinové domény MeSH
• pyridiny chemie   farmakologie   MeSH
• pyrimidiny chemie   farmakologie   MeSH
• pyrroly chemie   farmakologie   MeSH
• RNA-dependentní RNA-polymerasa antagonisté a inhibitory   chemie   metabolismus   MeSH
• termodynamika MeSH
• virová léková rezistence účinky léků   MeSH
• virové proteiny antagonisté a inhibitory   chemie   metabolismus   MeSH
• virus chřipky A účinky léků   enzymologie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• mutantní proteiny MeSH
• PB2 protein, Influenzavirus A MeSH Prohlížeč
• pimodivir MeSH Prohlížeč
• podjednotky proteinů MeSH
• pyridiny MeSH
• pyrimidiny MeSH
• pyrroly MeSH
• RNA-dependentní RNA-polymerasa MeSH
• virové proteiny MeSH

CATH (https://www.cathdb.info) identifies domains in protein structures from wwPDB and classifies these into evolutionary superfamilies, thereby providing structural and functional annotations. There are two levels: CATH-B, a daily snapshot of the latest domain structures and superfamily assignments, and CATH+, with additional derived data, such as predicted sequence domains, and functionally coherent sequence subsets (Functional Families or FunFams). The latest CATH+ release, version 4.3, significantly increases coverage of structural and sequence data, with an addition of 65,351 fully-classified domains structures (+15%), providing 500 238 structural domains, and 151 million predicted sequence domains (+59%) assigned to 5481 superfamilies. The FunFam generation pipeline has been re-engineered to cope with the increased influx of data. Three times more sequences are captured in FunFams, with a concomitant increase in functional purity, information content and structural coverage. FunFam expansion increases the structural annotations provided for experimental GO terms (+59%). We also present CATH-FunVar web-pages displaying variations in protein sequences and their proximity to known or predicted functional sites. We present two case studies (1) putative cancer drivers and (2) SARS-CoV-2 proteins. Finally, we have improved links to and from CATH including SCOP, InterPro, Aquaria and 2DProt.

• MeSH
• anotace sekvence MeSH
• COVID-19 epidemiologie   prevence a kontrola   virologie   MeSH
• databáze proteinů statistika a číselné údaje   MeSH
• epidemie MeSH
• internet MeSH
• lidé MeSH
• proteinové domény * MeSH
• proteiny chemie   genetika   metabolismus   MeSH
• SARS-CoV-2 genetika   metabolismus   fyziologie   MeSH
• sekvence aminokyselin MeSH
• sekvenční analýza proteinů metody   MeSH
• sekvenční homologie aminokyselin MeSH
• virové proteiny chemie   genetika   metabolismus   MeSH
• výpočetní biologie metody   statistika a číselné údaje   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteiny MeSH
• virové proteiny MeSH

Influenza viruses can cause severe respiratory infections in humans, leading to nearly half a million deaths worldwide each year. Improved antiviral drugs are needed to address the threat of development of novel pandemic strains. Current therapeutic interventions target three key proteins in the viral life cycle: neuraminidase, the M2 channel and RNA-dependent-RNA polymerase. Protein-protein interactions between influenza polymerase subunits are potential new targets for drug development. Using a newly developed assay based on AlphaScreen technology, we screened a peptide panel for protein-protein interaction inhibitors to identify a minimal PB1 subunit-derived peptide that retains high inhibition potential and can be further modified. Here, we present an X-ray structure of the resulting decapeptide bound to the C-terminal domain of PA polymerase subunit from pandemic isolate A/California/07/2009 H1N1 at 1.6 Å resolution and discuss its implications for the design of specific, potent influenza polymerase inhibitors.

• Klíčová slova
• AlphaScreen, Antiviral peptides, Influenza a polymerase, Protein-protein interaction,
• MeSH
• antivirové látky farmakologie   MeSH
• interakční proteinové domény a motivy účinky léků   fyziologie   MeSH
• krystalizace MeSH
• lidé MeSH
• RNA-dependentní RNA-polymerasa chemie   metabolismus   MeSH
• vazba proteinů MeSH
• virové proteiny antagonisté a inhibitory   chemie   metabolismus   MeSH
• virus chřipky A, podtyp H1N1 účinky léků   enzymologie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antivirové látky MeSH
• influenza virus polymerase basic protein 1 MeSH Prohlížeč
• RNA-dependentní RNA-polymerasa MeSH
• virové proteiny MeSH

An enigmatic localized pneumonia escalated into a worldwide COVID-19 pandemic from Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). This review aims to consolidate the extensive biological minutiae of SARS-CoV-2 which requires decipherment. Having one of the largest RNA viral genomes, the single strand contains the genes ORF1ab, S, E, M, N and ten open reading frames. Highlighting unique features such as stem-loop formation, slippery frameshifting sequences and ribosomal mimicry, SARS-CoV-2 represents a formidable cellular invader. Hijacking the hosts translational engine, it produces two polyprotein repositories (pp1a and pp1ab), armed with self-cleavage capacity for production of sixteen non-structural proteins. Novel glycosylation sites on the spike trimer reveal unique SARS-CoV-2 features for shielding and cellular internalization. Affording complexity for superior fitness and camouflage, SARS-CoV-2 challenges diagnosis and vaccine vigilance. This review serves the scientific community seeking in-depth molecular details when designing drugs to curb transmission of this biological armament.

• Klíčová slova
• 2019-nCoV, COVID-19, RNA, bats, coronavirus, pandemic, virus,
• MeSH
• COVID-19 genetika   metabolismus   virologie   MeSH
• fylogeneze MeSH
• lidé MeSH
• otevřené čtecí rámce MeSH
• pandemie MeSH
• RNA virová genetika   MeSH
• SARS-CoV-2 genetika   metabolismus   MeSH
• virové proteiny metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• RNA virová MeSH
• virové proteiny MeSH

The biological effects of flavonoids on mammal cells are diverse, ranging from scavenging free radicals and anti-cancer activity to anti-influenza activity. Despite appreciable effort to understand the anti-influenza activity of flavonoids, there is no clear consensus about their precise mode-of-action at a cellular level. Here, we report the development and validation of a screening assay based on AlphaScreen technology and illustrate its application for determination of the inhibitory potency of a large set of polyols against PA N-terminal domain (PA-Nter) of influenza RNA-dependent RNA polymerase featuring endonuclease activity. The most potent inhibitors we identified were luteolin with an IC50 of 72 ± 2 nM and its 8-C-glucoside orientin with an IC50 of 43 ± 2 nM. Submicromolar inhibitors were also evaluated by an in vitro endonuclease activity assay using single-stranded DNA, and the results were in full agreement with data from the competitive AlphaScreen assay. Using X-ray crystallography, we analyzed structures of the PA-Nter in complex with luteolin at 2.0 Å resolution and quambalarine B at 2.5 Å resolution, which clearly revealed the binding pose of these polyols coordinated to two manganese ions in the endonuclease active site. Using two distinct assays along with the structural work, we have presumably identified and characterized the molecular mode-of-action of flavonoids in influenza-infected cells.

• Klíčová slova
• Influenza, Neuraminidase, PA(N) endonuclease Inhibitors, Polyphenols, RNA polymerase,
• MeSH
• antivirové látky chemie   metabolismus   MeSH
• endonukleasy antagonisté a inhibitory   chemie   metabolismus   MeSH
• enzymatické testy metody   MeSH
• flavonoidy chemie   metabolismus   MeSH
• inhibitory enzymů chemie   metabolismus   MeSH
• krystalografie rentgenová MeSH
• mikrobiální testy citlivosti MeSH
• molekulární struktura MeSH
• preklinické hodnocení léčiv MeSH
• proteinové domény MeSH
• RNA-dependentní RNA-polymerasa antagonisté a inhibitory   chemie   metabolismus   MeSH
• vazba proteinů MeSH
• virové proteiny antagonisté a inhibitory   chemie   metabolismus   MeSH
• virus chřipky A enzymologie   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antivirové látky MeSH
• endonukleasy MeSH
• flavonoidy MeSH
• inhibitory enzymů MeSH
• RNA-dependentní RNA-polymerasa MeSH
• virové proteiny MeSH

The Epstein-Barr virus (EBV) immediate early transactivator Zta plays a key role in regulating the transition from latency to the lytic replication stages of EBV infection. Regulation of Zta is known to be controlled through a number of transcriptional and posttranscriptional events. Here, we show that Zta is targeted for ubiquitin modification and that this can occur in EBV-negative and in EBV-infected cells. Genetic studies show critical roles for both an amino-terminal region of Zta and the basic DNA binding domain of Zta in regulating Zta ubiquitination. Pulse-chase experiments demonstrate that the bulk population of Zta is relatively stable but that at least a subset of ubiquitinated Zta molecules are targeted for degradation in the cell. Mutation of four out of a total of nine lysine residues in Zta largely abrogates its ubiquitination, indicating that these are primary ubiquitination target sites. A Zta mutant carrying mutations at these four lysine residues (lysine 12, lysine 188, lysine 207, and lysine 219) cannot induce latently infected cells to produce and/or release infectious virions. Nevertheless, this mutant can induce early gene expression, suggesting a possible defect at the level of viral replication or later in the lytic cascade. As far as we know, this is the first study that has investigated the targeting of Zta by ubiquitination or its role in Zta function.IMPORTANCE Epstein-Barr virus (EBV) is a ubiquitous human pathogen and associated with various human diseases. EBV undergoes latency and lytic replication stages in its life cycle. The transition into the lytic replication stage, at which virus is produced, is mainly regulated by the viral gene product, Zta. Therefore, the regulation of Zta function becomes a central issue regarding viral biology and pathogenesis. Known modifications of Zta include phosphorylation and sumoylation. Here, we report the role of ubiquitination in regulating Zta function. We found that Zta is subjected to ubiquitination in both EBV-infected and EBV-negative cells. The ubiquitin modification targets 4 lysine residues on Zta, leading to both mono- and polyubiquitination of Zta. Ubiquitination of Zta affects the protein's stability and likely contributes to the progression of viral lytic replication. The function and fate of Zta may be determined by the specific lysine residue being modified.

• Klíčová slova
• Epstein-Barr virus, Zta, lytic replication, reactivation, ubiquitination,
• MeSH
• buněčné linie MeSH
• infekce virem Epsteina-Barrové virologie   MeSH
• lidé MeSH
• mutace MeSH
• promotorové oblasti (genetika) MeSH
• proteinové domény MeSH
• regulace exprese virových genů MeSH
• replikace viru MeSH
• trans-aktivátory genetika   metabolismus   MeSH
• ubikvitin metabolismus   MeSH
• vazba proteinů MeSH
• virové proteiny genetika   metabolismus   MeSH
• virus Epsteinův-Barrové genetika   fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• BZLF1 protein, Herpesvirus 4, Human MeSH Prohlížeč
• trans-aktivátory MeSH
• ubikvitin MeSH
• virové proteiny MeSH

Altering amounts of a protein in a cell has become a crucial tool for understanding its function. In many organisms, including the protozoan parasite Trypanosoma brucei, protein overexpression has been achieved by inserting a protein-coding sequence into an overexpression vector. Here, we have adapted the PCR only based system for tagging trypanosome proteins at their endogenous loci such that it in addition enables a tetracycline-inducible T7 RNA polymerase-mediated protein overexpression. Hence, this approach bypasses the need for molecular cloning, making it rapid and cost effective. We validated the approach for ten flagellum-associated proteins with molecular weights ranging from 40 to over 500 kDa. For a majority of the recombinant proteins a significant (3-50 fold) increase in the cellular amount was achieved upon induction of overexpression. Two of the largest proteins studied, the dynein heavy chains, were significantly overexpressed, while two were not. Our data suggest that this may reflect the extent of the T7 RNA polymerase processivity on the trypanosome genomic DNA. We further show that the overexpression is informative as to cellular functions of the studied proteins, and that these cultures can serve as an excellent source for purification of the overexpressed proteins. We believe that this rapid in locus overexpression system will become a valuable tool to interrogate cellular functions and biochemical activities of trypanosome proteins.

• Klíčová slova
• Overexpressing endogenous proteins, Protein purification, T7 RNA polymerase-mediated transcription, Trypanosomes,
• MeSH
• DNA řízené RNA-polymerasy metabolismus   MeSH
• dyneiny biosyntéza   MeSH
• exprese genu MeSH
• protozoální geny MeSH
• protozoální proteiny biosyntéza   izolace a purifikace   MeSH
• rekombinantní proteiny biosyntéza   izolace a purifikace   MeSH
• Trypanosoma brucei brucei * genetika   metabolismus   MeSH
• virové proteiny metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bacteriophage T7 RNA polymerase MeSH Prohlížeč
• DNA řízené RNA-polymerasy MeSH
• dyneiny MeSH
• protozoální proteiny MeSH
• rekombinantní proteiny MeSH
• virové proteiny MeSH

Dinucleoside polyphosphates (NpnNs) were discovered 50 years ago in all cells. They are often called alarmones, even though the molecular target of the alarm has not yet been identified. Recently, we showed that they serve as noncanonical initiating nucleotides (NCINs) and fulfill the role of 5' RNA caps in Escherichia coli. Here, we present molecular insight into their ability to be used as NCINs by T7 RNA polymerase in the initiation phase of transcription. In general, we observed NpnNs to be equally good substrates as canonical nucleotides for T7 RNA polymerase. Surprisingly, the incorporation of ApnGs boosts the production of RNA 10-fold. This behavior is due to the pairing ability of both purine moieties with the -1 and +1 positions of the antisense DNA strand. Molecular dynamic simulations revealed noncanonical pairing of adenosine with the thymine of the DNA.

• MeSH
• bakteriofág T7 enzymologie   MeSH
• dinukleosidfosfáty genetika   metabolismus   MeSH
• DNA řízené RNA-polymerasy genetika   metabolismus   MeSH
• DNA metabolismus   MeSH
• iniciace genetické transkripce * MeSH
• párování bází MeSH
• RNA čepičky genetika   MeSH
• RNA genetika   metabolismus   MeSH
• simulace molekulární dynamiky MeSH
• vazba proteinů MeSH
• virové proteiny genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bacteriophage T7 RNA polymerase MeSH Prohlížeč
• dinukleosidfosfáty MeSH
• DNA řízené RNA-polymerasy MeSH
• DNA MeSH
• RNA čepičky MeSH
• RNA MeSH
• virové proteiny MeSH