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Infection control measures to prevent viral and bacterial infection spread are critical to maintaining a healthy environment. Pathogens such as viruses and pyogenic bacteria can cause infectious complications. Viruses such as SARS-CoV-2 are known to spread through the aerosol route and on fomite surfaces, lasting for a prolonged time in the environment. Developing technologies to mitigate the spread of pathogens through airborne routes and on surfaces is critical, especially for patients at high risk for infectious complications. Multifunctional coatings with a broad capacity to bind pathogens that result in inactivation can disrupt infectious spread through aerosol and inanimate surface spread. This study uses C-POLAR, a proprietary cationic, polyamine, organic polymer with a charged, dielectric property coated onto air filtration material and textiles. Using both SARS-CoV-2 live viral particles and bovine coronavirus models, C-POLAR-treated material shows a dramatic 2-log reduction in circulating viral inoculum. This reduction is consistent in a static room model, indicating simple airflow through a static C-POLAR hanging can capture significant airborne particles. Finally, Gram-positive and Gram-negative bacteria are applied to C-POLAR textiles using a viability indicator to demonstrate eradication on fomite surfaces. These data suggest that a cationic polymer surface can capture and eradicate human pathogens, potentially interrupting the infectious spread for a more resilient environment. IMPORTANCE: Infection control is critical for maintaining a healthy home, work, and hospital environment. We test a cationic polymer capable of capturing and eradicating viral and bacterial pathogens by applying the polymer to the air filtration material and textiles. The data suggest that the simple addition of cationic material can result in the improvement of an infectious resilient environment against viral and bacterial pathogens.
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Highly sulfated malto-oligomers, similar to heparin and heparan-sulfate, have good antiviral, antimetastatic, anti-inflammatory and cell growth inhibitory effects. Due to their broad biological activities and simple structure, sulfated malto-oligomer derivatives have a great therapeutic potential, therefore, the development of efficient synthesis methods for their production is of utmost importance. In this work, preparation of α-(1â4)-linked oligoglucosides containing a sulfonatomethyl moiety at position C-6 of each glucose unit was studied by different approaches. Malto-oligomeric sulfonic acid derivatives up to dodecasaccharides were prepared by polymerization using different protecting groups, and the composition of the product mixtures was analyzed by MALDI-MS methods and size-exclusion chromatography. Synthesis of lower oligomers was also accomplished by stepwise and block synthetic methods, and then the oligosaccharide products were persulfated. The antiviral, anti-inflammatory and cell growth inhibitory activity of the fully sulfated malto-oligosaccharide sulfonic acids were determined by in vitro tests. Four tested di- and trisaccharide sulfonic acids effectively inhibited the activation of the TNF-α-mediated inflammatory pathway without showing cytotoxicity.
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Oligossacarídeos , Sulfatos , Polimerização , Oligossacarídeos/farmacologia , Ácidos Sulfônicos , Anti-Inflamatórios/farmacologia , Antivirais/farmacologiaRESUMO
(-)-Cannabidiol (CBD) and (-)-cannabigerol (CBG) are two major non-psychotropic phytocannabinoids that have many beneficial biological properties. However, due to their low water solubility and prominent first-pass metabolism, their oral bioavailability is moderate, which is unfavorable for medicinal use. Therefore, there is a great need for appropriate chemical modifications to improve their physicochemical and biological properties. In this study, Mannich-type reaction was used for the synthetic modification of CBD and CBG for the first time, and thus fifteen new cannabinoid derivatives containing one or two tertiary amino groups were prepared. Thereafter the antiviral, antiproliferative and antibacterial properties of the derivatives and their effects on certain skin cells were investigated. Some modified CBD derivatives showed remarkable antiviral activity against SARS-CoV-2 without cytotoxic effect, while synthetic modifications on CBG resulted in a significant increase in antiproliferative activity in some cases compared to the parent compound.
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Canabidiol , Canabinoides , Canabidiol/farmacologia , Canabinoides/farmacologia , Disponibilidade Biológica , Antivirais/farmacologiaAssuntos
COVID-19 , Criança , Humanos , COVID-19/diagnóstico , SARS-CoV-2 , Criança Hospitalizada , MãosRESUMO
d-Arabinofuranosyl-pyrimidine and -purine nucleoside analogues containing alkylthio-, acetylthio- or 1-thiosugar substituents at the C2' position were prepared from the corresponding 3',5'-O-silylene acetal-protected nucleoside 2'-exomethylenes by photoinitiated, radical-mediated hydrothiolation reactions. Although the stereochemical outcome of the hydrothiolation depended on the structure of both the thiol and the furanoside aglycone, in general, high d-arabino selectivity was obtained. The cytotoxic effect of the arabinonucleosides was studied on tumorous SCC (mouse squamous cell) and immortalized control HaCaT (human keratinocyte) cell lines by MTT assay. Three pyrimidine nucleosides containing C2'-butylsulfanylmethyl or -acetylthiomethyl groups showed promising cytotoxicity at low micromolar concentrations with good selectivity towards tumor cells. SAR analysis using a methyl ß-d-arabinofuranoside reference compound showed that the silyl-protecting group, the nucleobase and the corresponding C2' substituent are crucial for the cell growth inhibitory activity. The effects of the three most active nucleoside analogues on parameters indicative of cytotoxicity, such as cell size, division time and cell generation time, were investigated by near-infrared live cell imaging, which showed that the 2'-acetylthiomethyluridine derivative induced the most significant functional and morphological changes. Some nucleoside analogues also exerted anti-SARS-CoV-2 and/or anti-HCoV-229E activity with low micromolar EC50 values; however, the antiviral activity was always accompanied by significant cytotoxicity.
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COVID-19 , Nucleosídeos de Pirimidina , Tioaçúcares , Humanos , Camundongos , Animais , Arabinonucleosídeos/química , Arabinonucleosídeos/farmacologia , Nucleosídeos/farmacologia , Nucleosídeos/química , Antivirais/farmacologia , Acetais , Compostos de Sulfidrila/química , Purinas , Relação Estrutura-AtividadeRESUMO
Influenza virus causes severe respiratory infection in humans. Current antivirotics target three key proteins in the viral life cycle: neuraminidase, the M2 channel and the endonuclease domain of RNA-dependent-RNA polymerase. Due to the development of novel pandemic strains, additional antiviral drugs targetting different viral proteins are still needed. The protein-protein interaction between polymerase subunits PA and PB1 is one such possible target. We recently identified a modified decapeptide derived from the N-terminus of the PB1 subunit with high affinity for the C-terminal part of the PA subunit. Here, we optimized its amino acid hotspots to maintain the inhibitory potency and greatly increase peptide solubility. This allowed thermodynamic characterization of peptide binding to PA. Solving the X-ray structure of the peptide-PA complex provided structural insights into the interaction. Additionally, we optimized intracellular delivery of the peptide using a bicyclic strategy that led to improved inhibition in cell-based assays.
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Influenza Humana , Humanos , Influenza Humana/tratamento farmacológico , Ligação Proteica , RNA Polimerase Dependente de RNA , Peptídeos/farmacologia , Peptídeos/metabolismo , TermodinâmicaRESUMO
Patients infected with SARS-CoV-2 risk co-infection with Gram-positive bacteria, which severely affects their prognosis. Antimicrobial drugs with dual antiviral and antibacterial activity would be very useful in this setting. Although glycopeptide antibiotics are well-known as strong antibacterial drugs, some of them are also active against RNA viruses like SARS-CoV-2. It has been shown that the antiviral and antibacterial efficacy can be enhanced by synthetic modifications. We here report the synthesis and biological evaluation of seven derivatives of teicoplanin bearing hydrophobic or superbasic side chain. All but one teicoplanin derivatives were effective in inhibiting SARS-CoV-2 replication in VeroE6 cells. One lipophilic and three perfluoroalkyl conjugates showed activity against SARS-CoV-2 in human Calu-3 cells and against HCoV-229E, an endemic human coronavirus, in HEL cells. Pseudovirus entry and enzyme inhibition assays established that the teicoplanin derivatives efficiently prevent the cathepsin-mediated endosomal entry of SARS-CoV-2, with some compounds inhibiting also the TMPRSS2-mediated surface entry route. The teicoplanin derivatives showed good to excellent activity against Gram-positive bacteria resistant to all approved glycopeptide antibiotics, due to their ability to dually bind to the bacterial membrane and cell-wall. To conclude, we identified three perfluoralkyl and one monoguanidine analog of teicoplanin as dual inhibitors of Gram-positive bacteria and SARS-CoV-2.
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COVID-19 , Fluorocarbonos , Antibacterianos/química , Antivirais/química , Catepsinas/farmacologia , Fluorocarbonos/farmacologia , Glicopeptídeos/química , Bactérias Gram-Positivas , Humanos , SARS-CoV-2 , Teicoplanina/farmacologiaRESUMO
Positive-sense single-stranded RNA (+RNA) viruses have proven to be important pathogens that are able to threaten and deeply damage modern societies, as illustrated by the ongoing COVID-19 pandemic. Therefore, compounds active against most or many +RNA viruses are urgently needed. Here, we present PR673, a helquat-like compound that is able to inhibit the replication of SARS-CoV-2 and tick-borne encephalitis virus in cell culture. Using in vitro polymerase assays, we demonstrate that PR673 inhibits RNA synthesis by viral RNA-dependent RNA polymerases (RdRps). Our results illustrate that the development of broad-spectrum non-nucleoside inhibitors of RdRps is feasible.
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COVID-19 , Vírus da Encefalite Transmitidos por Carrapatos , Humanos , Pandemias , RNA Polimerase Dependente de RNA , SARS-CoV-2RESUMO
The current pandemic resulted in a rapidly increasing demand for personal protective equipment (PPE) initially leading to severe shortages of these items. Hence, during an unexpected and fast virus spread, the possibility of reusing highly efficient protective equipment could provide a viable solution for keeping both healthcare professionals and the general public equipped and protected. This requires an efficient decontamination technique that preserves functionality of the sensitive materials used for PPE production. Non-thermal plasma (NTP) is a decontamination technique with documented efficiency against select bacterial and fungal pathogens combined with low damage to exposed materials. We have investigated NTP for decontamination of high-efficiency P3 R filters from viral respiratory pathogens in comparison to other commonly used techniques. We show that NTP treatment completely inactivates SARS-CoV-2 and three other common human respiratory viruses including Influenza A, Rhinovirus and Adenovirus, revealing an efficiency comparable to 90°C dry heat or UVC light. Unlike some of the tested techniques (e.g., autoclaving), NTP neither influenced the filtering efficiency nor the microstructure of the filter. We demonstrate that NTP is a powerful and economic technology for efficient decontamination of protective filters and other sensitive materials from different respiratory pathogens.
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The newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cause life-threatening diseases in millions of people worldwide, in particular, in patients with cancer, and there is an urgent need for antiviral agents against this infection. While inâ vitro activities of artemisinins against SARS-CoV-2 and cancer have recently been demonstrated, no study of artemisinin and/or synthetic peroxide-based hybrid compounds active against both cancer and SARS-CoV-2 has been reported yet. However, the hybrid drug's properties (e. g., activity and/or selectivity) can be improved compared to its parent compounds and effective new agents can be obtained by modification/hybridization of existing drugs or bioactive natural products. In this study, a series of new artesunic acid and synthetic peroxide based new hybrids were synthesized and analyzed inâ vitro for the first time for their inhibitory activity against SARS-CoV-2 and leukemia cell lines. Several artesunic acid-derived hybrids exerted a similar or stronger potency against K562 leukemia cells (81-83 % inhibition values) than the reference drug doxorubicin (78 % inhibition value) and they were also more efficient than their parent compounds artesunic acid (49.2 % inhibition value) and quinoline derivative (5.5 % inhibition value). Interestingly, the same artesunic acid-quinoline hybrids also show inhibitory activity against SARS-CoV-2 inâ vitro (EC50 13-19â µm) and no cytotoxic effects on Vero E6 cells (CC50 up to 110â µM). These results provide a valuable basis for design of further artemisinin-derived hybrids to treat both cancer and SARS-CoV-2 infections.
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Artemisininas , Tratamento Farmacológico da COVID-19 , Leucemia , Neoplasias , Quinolinas , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Artemisininas/farmacologia , Chlorocebus aethiops , Humanos , Leucemia/tratamento farmacológico , Neoplasias/tratamento farmacológico , Peróxidos , Quinolinas/uso terapêutico , SARS-CoV-2 , Células VeroRESUMO
Hepatitis B virus uses e antigen (HBe), which is dispensable for virus infectivity, to modulate host immune responses and achieve viral persistence in human hepatocytes. The HBe precursor (p25) is directed to the endoplasmic reticulum (ER), where cleavage of the signal peptide (sp) gives rise to the first processing product, p22. P22 can be retro-translocated back to the cytosol or enter the secretory pathway and undergo a second cleavage event, resulting in secreted p17 (HBe). Here, we report that translocation of p25 to the ER is promoted by translocon-associated protein complex. We have found that p25 is not completely translocated into the ER; a fraction of p25 is phosphorylated and remains in the cytoplasm and nucleus. Within the p25 sp sequence, we have identified three cysteine residues that control the efficiency of sp cleavage and contribute to proper subcellular distribution of the precore pool.
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Antígenos E da Hepatite B , Hepatite B , Proteínas de Ligação ao Cálcio , Cisteína/metabolismo , Retículo Endoplasmático/metabolismo , Hepatite B/metabolismo , Antígenos E da Hepatite B/metabolismo , Vírus da Hepatite B/metabolismo , Humanos , Glicoproteínas de Membrana , Sinais Direcionadores de Proteínas/genética , Receptores Citoplasmáticos e Nucleares , Receptores de PeptídeosRESUMO
Chronic hepatitis caused by infection with the Hepatitis B virus is a life-threatening condition. In fact, 1 million people die annually due to liver cirrhosis or hepatocellular carcinoma. Recently, several studies demonstrated a molecular connection between the host DNA damage response (DDR) pathway and HBV replication and reactivation. Here, we investigated the role of Ataxia-telangiectasia-mutated (ATM) and Ataxia telangiectasia and Rad3-related (ATR) PI3-kinases in phosphorylation of the HBV core protein (HBc). We determined that treatment of HBc-expressing hepatocytes with genotoxic agents, e.g., etoposide or hydrogen peroxide, activated the host ATM-Chk2 pathway, as determined by increased phosphorylation of ATM at Ser1981 and Chk2 at Thr68. The activation of ATM led, in turn, to increased phosphorylation of cytoplasmic HBc at serine-glutamine (SQ) motifs located in its C-terminal domain. Conversely, down-regulation of ATM using ATM-specific siRNAs or inhibitor effectively reduced etoposide-induced HBc phosphorylation. Detailed mutation analysis of S-to-A HBc mutants revealed that S170 (S168 in a 183-aa HBc variant) is the primary site targeted by ATM-regulated phosphorylation. Interestingly, mutation of two major phosphorylation sites involving serines at positions 157 and 164 (S155 and S162 in a 183-aa HBc variant) resulted in decreased etoposide-induced phosphorylation, suggesting that the priming phosphorylation at these serine-proline (SP) sites is vital for efficient phosphorylation of SQ motifs. Notably, the mutation of S172 (S170 in a 183-aa HBc variant) had the opposite effect and resulted in massively up-regulated phosphorylation of HBc, particularly at S170. Etoposide treatment of HBV infected HepG2-NTCP cells led to increased levels of secreted HBe antigen and intracellular HBc protein. Together, our studies identified HBc as a substrate for ATM-mediated phosphorylation and mapped the phosphorylation sites. The increased expression of HBc and HBe antigens in response to genotoxic stress supports the idea that the ATM pathway may provide growth advantage to the replicating virus.
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Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Dano ao DNA , Vírus da Hepatite B/fisiologia , Hepatócitos/virologia , Proteínas do Core Viral/metabolismo , Motivos de Aminoácidos , Quinase do Ponto de Checagem 2/metabolismo , Citoplasma/metabolismo , Citoplasma/virologia , Etoposídeo/farmacologia , Células Hep G2 , Antígenos E da Hepatite B/metabolismo , Vírus da Hepatite B/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/farmacologia , Fosforilação , Serina/metabolismo , Transativadores/genética , Transativadores/metabolismo , Proteínas do Core Viral/química , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
The protracted global COVID-19 pandemic urges the development of new drugs against the causative agent SARS-CoV-2. The clinically used glycopeptide antibiotic, teicoplanin, emerged as a potential antiviral, and its efficacy was improved with lipophilic modifications. This prompted us to prepare new lipophilic apocarotenoid conjugates of teicoplanin, its pseudoaglycone and the related ristocetin aglycone. Their antiviral effect was tested against SARS-CoV-2 in Vero E6 cells, using a cell viability assay and quantitative PCR of the viral RNA, confirming their micromolar inhibitory activity against viral replication. Interestingly, two of the parent apocarotenoids, bixin and ß-apo-8'carotenoic acid, exerted remarkable anti-SARS-CoV-2 activity. Mechanistic studies involved cathepsin L and B, as well as the main protease 3CLPro, and the results were rationalized by computational studies. Glycopeptide conjugates show dual inhibitory action, while apocarotenoids have mostly cathepsin B and L affinity. Since teicoplanin is a marketed antibiotic and the natural bixin is an approved, cheap and widely used red colorant food additive, these readily available compounds and their conjugates as potential antivirals are worthy of further exploration.
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This study describes the discovery of novel prodrugs bearing tyrosine derivatives instead of the phenol moiety present in FDA-approved tenofovir alafenamide fumarate (TAF). The synthesis was optimized to afford diastereomeric mixtures of novel prodrugs in one pot (yields up to 86%), and the epimers were resolved using a chiral HPLC column into fast-eluting and slow-eluting epimers. In human lymphocytes, the most efficient tyrosine-based prodrug reached a single-digit picomolar EC50 value against HIV-1 and nearly 300-fold higher selectivity index (SI) compared to TAF. In human hepatocytes, the most efficient prodrugs exhibited subnanomolar EC50 values for HBV and up to 26-fold higher SI compared to TAF. Metabolic studies demonstrated markedly higher cellular uptake of the prodrugs and substantially higher levels of released tenofovir inside the cells compared to TAF. These promising results provide a strong foundation for further evaluation of the reported prodrugs and their potential utility in the development of highly potent antivirals.
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Amidas/química , Antivirais/farmacologia , Descoberta de Drogas , Ácidos Fosfóricos/química , Pró-Fármacos/farmacologia , Tenofovir/farmacologia , Antivirais/química , HIV-1/efeitos dos fármacos , Vírus da Hepatite B/efeitos dos fármacos , Hepatócitos/virologia , Humanos , Testes de Sensibilidade Microbiana , Fenol/química , Pró-Fármacos/química , Estereoisomerismo , Tenofovir/química , Tirosina/químicaRESUMO
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.
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COVID-19/virologia , Metiltransferases/metabolismo , Capuzes de RNA/genética , RNA Viral/genética , SARS-CoV-2/enzimologia , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Anticorpos Monoclonais/análise , Humanos , Metiltransferases/análise , Metiltransferases/genética , Transporte Proteico , Capuzes de RNA/metabolismo , RNA Viral/metabolismo , SARS-CoV-2/química , SARS-CoV-2/genética , Proteínas não Estruturais Virais/análise , Proteínas não Estruturais Virais/genética , Proteínas Virais Reguladoras e Acessórias/análise , Proteínas Virais Reguladoras e Acessórias/genéticaRESUMO
A series of 7-deazaadenine ribonucleosides bearing alkyl, alkenyl, alkynyl, aryl, or hetaryl groups at position 7 as well as their 5'-O-triphosphates and two types of monophosphate prodrugs (phosphoramidates and S-acylthioethanol esters) were prepared and tested for antiviral activity against selected RNA viruses (Dengue, Zika, tick-borne encephalitis, West Nile, and SARS-CoV-2). The modified triphosphates inhibited the viral RNA-dependent RNA polymerases at micromolar concentrations through the incorporation of the modified nucleotide and stopping a further extension of the RNA chain. 7-Deazaadenosine nucleosides bearing ethynyl or small hetaryl groups at position 7 showed (sub)micromolar antiviral activities but significant cytotoxicity, whereas the nucleosides bearing bulkier heterocycles were still active but less toxic. Unexpectedly, the monophosphate prodrugs were similarly or less active than the corresponding nucleosides in the in vitro antiviral assays, although the bis(S-acylthioethanol) prodrug 14h was transported to the Huh7 cells and efficiently released the nucleoside monophosphate.
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Antivirais/farmacologia , Pró-Fármacos/farmacologia , Purinas/farmacologia , Vírus de RNA/efeitos dos fármacos , Ribonucleosídeos/farmacologia , COVID-19/virologia , Linhagem Celular Tumoral , Vírus da Dengue/efeitos dos fármacos , Vírus da Encefalite Transmitidos por Carrapatos/efeitos dos fármacos , Humanos , Fosfatos/farmacologia , Nucleosídeos de Purina , RNA Polimerase Dependente de RNA/metabolismo , SARS-CoV-2/efeitos dos fármacos , Vírus do Nilo Ocidental/efeitos dos fármacos , Zika virus/efeitos dos fármacos , Tratamento Farmacológico da COVID-19RESUMO
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.
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Vírus da Influenza A Subtipo H1N1/enzimologia , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Antivirais/farmacologia , Cristalização , Humanos , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Domínios e Motivos de Interação entre Proteínas/fisiologia , Proteínas Virais/antagonistas & inibidoresRESUMO
Hepatitis B virus (HBV) core protein (HBc) plays many roles in the HBV life cycle, such as regulation of transcription, RNA encapsidation, reverse transcription, and viral release. To accomplish these functions, HBc interacts with many host proteins and undergoes different post-translational modifications (PTMs). One of the most common PTMs is ubiquitination, which was shown to change the function, stability, and intracellular localization of different viral proteins, but the role of HBc ubiquitination in the HBV life cycle remains unknown. Here, we found that HBc protein is post-translationally modified through K29-linked ubiquitination. We performed a series of co-immunoprecipitation experiments with wild-type HBc, lysine to arginine HBc mutants and wild-type ubiquitin, single lysine to arginine ubiquitin mutants, or single ubiquitin-accepting lysine constructs. We observed that HBc protein could be modified by ubiquitination in transfected as well as infected hepatoma cells. In addition, ubiquitination predominantly occurred on HBc lysine 7 and the preferred ubiquitin chain linkage was through ubiquitin-K29. Mass spectrometry (MS) analyses detected ubiquitin protein ligase E3 component N-recognin 5 (UBR5) as a potential E3 ubiquitin ligase involved in K29-linked ubiquitination. These findings emphasize that ubiquitination of HBc may play an important role in HBV life cycle.
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Vírus da Hepatite B/genética , Processamento de Proteína Pós-Traducional/genética , Ubiquitinação/genética , Proteínas Virais/genética , Arginina/genética , Carcinoma Hepatocelular/genética , Linhagem Celular , Linhagem Celular Tumoral , Células HEK293 , Células Hep G2 , Hepatite B/genética , Humanos , Lisina/genética , Ubiquitina/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
A critical lack of personal protective equipment has occurred during the COVID-19 pandemic. Polylactic acid (PLA), a polyester made from renewable natural resources, can be exploited for 3D printing of protective face masks using the Fused Deposition Modelling technique. Since the possible high porosity of this material raised questions regarding its suitability for protection against viruses, we have investigated its microstructure using scanning electron microscopy and aerosol generator and photometer certified as the test system according to the standards EN 143 and EN 149. Moreover, the efficiency of decontaminating PLA surfaces by conventional chemical disinfectants including 96% ethanol, 70% isopropanol, and a commercial disinfectant containing 0.85% sodium hypochlorite has been determined. We confirmed that the structure of PLA protective masks is compact and can be considered a sufficient barrier protection against particles of a size corresponding to microorganisms including viruses. Complete decontamination of PLA surfaces from externally applied Staphylococcus epidermidis, Escherichia coli, Candida albicans and SARS-CoV-2 was achieved using all disinfectants tested, and human adenovirus was completely inactivated by sodium hypochlorite-containing disinfectant. Natural contamination of PLA masks worn by test persons was decontaminated easily and efficiently by ethanol. No disinfectant caused major changes to the PLA surface properties, and the pore size did not change despite severe mechanical damage of the surface. Therefore, PLA may be regarded as a suitable material for 3D printing of protective masks during the current or future pandemic crises.
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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.