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1.
Phytomedicine ; 110: 154650, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36649670

RESUMO

BACKGROUND: Dengue caused by dengue virus (DENV) spreads rapidly around the world. However, there are no worldwide licensed vaccines or specific antivirals to combat DENV infection. Quassinoids are the most characteristic components of Eurycoma longifolia, which have been reported to display a variety of biological activities. However, whether quassinoids exert anti-DENV activities remains unknown. PURPOSE: To test the quassinoids of E. longifolia for their activity against DENV and to clarify the potential mechanisms. METHODS: The quassinoids from E. longifolia were isolated by chromatography techniques, and their chemical structures were elucidated by spectroscopic analysis. The anti-DENV activities of quassinoids on baby hamster kidney cells BHK-21 were determined by lactate dehydrogenase (LDH) assay. The synthesis of progeny virus was measured by plaque assay. The expression levels of envelope protein (E) and non-structural protein 1 (NS1) were evaluated by qRT-PCR, Western blot and immunofluorescence assays. Molecular docking was used to screen the potential targets of the most active quassinoid against DENV-2, and surface plasmon resonance analysis was employed to confirm the direct binding between the most active quassinoid and potential target. RESULTS: Twenty-four quassinoids, including three new quassinoids (1 - 3), were isolated from the ethanol extract of E. longifolia. Quassinoids 4, 5, 9, 11, 12, 15, 16, 17, 19 and 20 significantly reduced the LDH release at the stages of viral binding and entry or intracellular replication. Among them, 19 (6α-hydroxyeurycomalactone, 6α-HEL) exhibited the best anti-DENV-2 activities with an EC50 value of 0.39 ± 0.02 µM. Further experiments suggested that 6α-HEL remarkably inhibited progeny virus synthesis and mRNA and protein expression levels of E and NS1 of DENV-2. Time-of-drug-addition assay suggested that 6α-HEL inhibited intracellular replication of DENV-2 at an early stage. Moreover, 6α-HEL was shown to interact with NS5-RdRp domain at a binding affinity of -8.15 kcal/mol. SPR assay further verified 6α-HEL bound to RdRp protein with an equilibrium dissociation constant of 1.49 × 10-7 M. CONCLUSION: Ten quassinoids from E. longifolia showed anti-DENV activities at processes of virus binding and entry or intracellular replication. The most active quassinoid 6α-HEL exerts the anti-DENV-2 activities at intracellular replication stage by directly targeting the NS5-RdRp protein. These results suggest that 6α-HEL could be a promising candidate for the treatment of DENV-2 infection.


Assuntos
Dengue , Eurycoma , Quassinas , Animais , Cricetinae , Humanos , Eurycoma/química , Simulação de Acoplamento Molecular , Antivirais/farmacologia , Antivirais/química , Quassinas/farmacologia , Replicação Viral , Dengue/tratamento farmacológico , RNA Polimerase Dependente de RNA
2.
J Org Chem ; 88(2): 838-851, 2023 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-36622749

RESUMO

In the present study, we herein report a DDQ-catalyzed new protocol for the synthesis of substituted 3-acylindoles. Being a potential system for virtual hydrogen storage, introduction of catalytic DDQ in combination with Fe(NO3)3·9H2O and molecular oxygen as co-catalysts offers a regioselective oxo-functionalization of C-3 alkyl-/aryllidine indolines even with scale-up investigations. Intermediate isolation, their spectroscopic characterization, and the density functional theory calculations indicate that the method involves dehydrogenative allylic hydroxylation and 1,3-functional group isomerization/aromatization followed by terminal oxidation to afford 3-acylindoles quantitatively with very high regioselectivity. This method is very general for a large number of substrates with varieties of functional groups tolerance emerging high-yield outcome. Moreover, molecular docking studies were performed for some selected ligands with an RNA-dependent RNA polymerase complex (RdRp complex) of SARS-CoV-2 to illustrate the binding potential of those ligands. The docking results revealed that few of the ligands possess the potential to inhibit the RdRp of SARS-Cov-2 with binding energies (-6.7 to -8.19 kcal/mol), which are comparably higher with respect to the reported binding energies of the conventional re-purposed drugs such as Remdesivir, Ribavirin, and so forth (-4 to -7 kcal/mol).


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Simulação de Acoplamento Molecular , Ligantes , Antivirais/farmacologia , Antivirais/química , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Indóis/farmacologia
3.
Viruses ; 15(1)2023 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-36680185

RESUMO

A novel virus with a double-stranded RNA (dsRNA) genome was isolated from Fusarium avenaceum strain GS-WW-224, the causal agent of potato dry rot. The virus has been designated as Fusarium avenaceum alternavirus 1 (FaAV1). Its genome consists of two dsRNA segments, 3538 bp (dsRNA1) and 2477 bp (dsRNA2) in length, encoding RNA-dependent RNA polymerase (RdRp) and a hypothetical protein (HP), respectively. The virions of FaAV1 are isometric spherical and approximately 30 nm in diameter. Multiple sequence alignments and phylogenetic analyses based on the amino acid sequences of RdRp and HP indicated that FaAV1 appears to be a new member of the proposed family Alternaviridae. No significant differences in colony morphology and spore production were observed between strains GS-WW-224 and GS-WW-224-VF, the latter strain being one in which FaAV1 was eliminated from strain GS-WW-224. Notably, however, the dry weight of mycelial biomass of GS-WW-224 was higher than that of mycelial biomass of GS-WW-224-VF. The depth and the width of lesions on potato tubers caused by GS-WW-224 were significantly greater, relative to GS-WW-224-VF, suggesting that FaAV1 confers hypervirulence to its host, F. avenaceum. Moreover, FaAV1 was successfully transmitted horizontally from GS-WW-224 to ten other species of Fusarium, and purified virions of FaAV1 were capable of transfecting wounded hyphae of the ten species of Fusarium. This is the first report of an alternavirus infecting F. avenaceum and conferring hypervirulence.


Assuntos
Fusarium , Solanum tuberosum , Fusarium/genética , Filogenia , RNA Polimerase Dependente de RNA/genética
4.
Viruses ; 15(1)2023 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-36680206

RESUMO

Norovirus is the leading viral agent of gastroenteritis in humans. RNA-dependent RNA polymerase (RdRp) is essential in the replication of norovirus RNA. Here, we present a comprehensive evolutionary analysis of the norovirus GI RdRp gene. Our results show that the norovirus GI RdRp gene can be divided into three groups, and that the most recent common ancestor was 1484. The overall evolutionary rate of GI RdRp is 1.821 × 10-3 substitutions/site/year. Most of the amino acids of the GI RdRp gene were under negative selection, and only a few positively selected sites were recognized. Amino acid substitutions in the GI RdRp gene accumulated slowly over time. GI.P1, GI.P3 and GI.P6 owned the higher evolutionary rates. GI.P11 and GI.P13 had the faster accumulation rate of amino acid substitutions. GI.P2, GI.P3, GI.P4, GI.P6 and GI.P13 presented a strong linear evolution. These results reveal that the norovirus GI RdRp gene evolves conservatively, and that the molecular evolutionary characteristics of each P-genotype are diverse. Sequencing in RdRp and VP1 of norovirus should be advocated in the surveillance system to explore the effect of RdRp on norovirus activity.


Assuntos
Infecções por Caliciviridae , Norovirus , Humanos , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Norovirus/genética , Norovirus/metabolismo , Genótipo , Aminoácidos/genética , Evolução Molecular , Filogenia
5.
Arch Virol ; 168(2): 50, 2023 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-36609709

RESUMO

The whole genome sequence of mulberry crinivirus (MuCV), a novel member of the genus Crinivirus (family Closteroviridae) identified in mulberry (Morus alba L), was determined. The virus possesses a bipartite genome. RNA1 contains 8571 nucleotides (nt) with four open reading frames (ORFs). ORF1a encodes a putative polyprotein with papain-like protease, methyltransferase, and RNA helicase domains. ORF1b putatively encodes an RNA-dependent RNA polymerase (RdRp), which is probably expressed via a + 1 ribosomal frameshift. RNA2 consists of 8082 nt, containing eight ORFs that are similar in size and position to orthologous genes of other criniviruses. Phylogenetic analysis based on RdRp amino acid sequences of criniviruses placed MuCV in group 1.


Assuntos
Crinivirus , Morus , Crinivirus/genética , Sequência de Bases , Filogenia , Genoma Viral , Nucleotídeos , Fases de Leitura Aberta , RNA Polimerase Dependente de RNA/genética , RNA Viral/genética
6.
Arch Virol ; 168(2): 34, 2023 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-36609790

RESUMO

A novel dsRNA mycovirus named Ilyonectria crassa alternavirus 1 (IcAV1) was found in Ilyonectria crassa isolate NW-FVA 1829. The fungus was isolated from an ash (Fraxinus excelsior L.) necrotic trunk disc infected with Hymenoscyphus fraxineus [(T. Kowalski) Baral, Queloz, Hosoya] causing ash dieback. The complete genome of IcAV1 is composed of three segments, each containing a single ORF on the positive-sense RNA. The extreme 5' UTRs of dsRNA 1 (3604 bp), dsRNA 2 (2547 bp), and dsRNA 3 (2518 bp) share a conserved hexadecamer sequence (5'-GGCTGTGTGTTTAGTT-3') and are capped. The 3' UTRs are polyadenylated. In silico analysis showed that the viral RdRP is encoded on dsRNA 1 and the capsid-protein subunits are encoded on dsRNA 3. Maximum-likelihood analysis of the aa sequence of the viral RdRP showed that IcAV1 clusters with alternaviruses from Fusarium spp., while the type member of the proposed family "Alternaviridae", Alternaria alternata virus 1 (AaV1), formed a clade together with Stemphylium lycopersici mycovirus (SlV). The function of the protein encoded on segment 2 is unknown. Based on its genome organization and its phylogenetic position, IcAV1 is suggested to be a new member of the proposed family "Alternaviridae". This is the first report of a mycovirus infecting I. crassa.


Assuntos
Micovírus , Vírus de RNA , Filogenia , Genoma Viral , RNA Polimerase Dependente de RNA/genética , RNA Viral/genética , Fases de Leitura Aberta , RNA de Cadeia Dupla/genética
7.
Eur J Med Chem ; 247: 115035, 2023 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-36603507

RESUMO

Influenza is one of the leading causes of disease-related mortalities worldwide. Several strategies have been implemented during the past decades to hinder the replication cycle of influenza viruses, all of which have resulted in the emergence of resistant virus strains. The most recent example is baloxavir marboxil, where a single mutation in the active site of the target endonuclease domain of the RNA-dependent-RNA polymerase renders the recent FDA approved compound ∼1000-fold less effective. Raltegravir is a first-in-class HIV inhibitor that shows modest activity to the endonuclease. Here, we have used structure-guided approaches to create rationally designed derivative molecules that efficiently engage the endonuclease active site. The design strategy was driven by our previously published structures of endonuclease-substrate complexes, which allowed us to target functionally conserved residues and reduce the likelihood of resistance mutations. We succeeded in developing low nanomolar equipotent inhibitors of both wild-type and baloxavir-resistant endonuclease. We also developed macrocyclic versions of these inhibitors that engage the active site in the same manner as their 'open' counterparts but with reduced affinity. Structural analyses provide clear avenues for how to increase the affinity of these cyclic compounds.


Assuntos
Dibenzotiepinas , Inibidores de Integrase de HIV , Influenza Humana , Orthomyxoviridae , Humanos , RNA Polimerase Dependente de RNA , Piridonas/farmacologia , Piridonas/uso terapêutico , Influenza Humana/tratamento farmacológico , Dibenzotiepinas/farmacologia , Dibenzotiepinas/uso terapêutico , Endonucleases , Triazinas/farmacologia , Antivirais/farmacologia
8.
Chem Commun (Camb) ; 59(7): 872-875, 2023 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-36594508

RESUMO

Replication of RNA viruses is catalysed by virus-specific polymerases, which can be targets of therapeutic strategies. In this study, we used a selection strategy to identify endogenous RNAs from a transcriptome library derived from lung cells that interact with the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2. Some of the selected RNAs weakened the activity of RdRp by forming G-quadruplexes. These results suggest that certain endogenous RNAs, which potentially form G-quadruplexes, can reduce the replication of viral RNAs.


Assuntos
COVID-19 , Quadruplex G , Humanos , SARS-CoV-2/genética , RNA Viral/genética , RNA Polimerase Dependente de RNA/metabolismo , RNA Polimerases Dirigidas por DNA/genética , Antivirais/farmacologia
9.
Arch Virol ; 168(2): 66, 2023 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-36653596

RESUMO

The complete genome sequence of a novel single-stranded [+ ssRNA] positive-sense (+) RNA mycovirus, designated as "Pleurotus citrinopileatus ourmiavirus 1" (PcOV1), isolated from Pleurotus citrinopileatus strain CCMJ2141, was determined. The complete genome of PcOV1 is composed of 2,535 nucleotides. It contains a single open reading frame (ORF), which encodes a protein of 657 amino acids (aa) containing conserved domains of an RNA-dependent RNA polymerase (RdRp). Phylogenetic analysis based on RdRp sequences revealed that PcOV1 is a new member of the genus Ourmiavirus in the family Botourmiaviridae. This is the first virus from P. citrinopileatus to be characterized.


Assuntos
Micovírus , Vírus de RNA , RNA Viral/genética , Filogenia , Genoma Viral , RNA Polimerase Dependente de RNA/genética , Fases de Leitura Aberta , RNA de Cadeia Dupla
10.
Genes (Basel) ; 14(1)2023 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-36672862

RESUMO

In the growth and development of plants, some non-coding small RNAs (sRNAs) not only mediate RNA interference at the post-transcriptional level, but also play an important regulatory role in chromatin modification at the transcriptional level. In these processes, the protein factors Argonaute (AGO), Dicer-like (DCL), and RNA-dependent RNA polymerase (RDR) play very important roles in the synthesis of sRNAs respectively. Though they have been identified in many plants, the information about these gene families in strawberry was poorly understood. In this study, using a genome-wide analysis and a phylogenetic approach, 13 AGO, six DCL, and nine RDR genes were identified in diploid strawberry Fragaria vesca. We also identified 33 AGO, 18 DCL, and 28 RDR genes in octoploid strawberry Fragaria × ananassa, studied the expression patterns of these genes in various tissues and developmental stages of strawberry, and researched the response of these genes to some hormones, finding that almost all genes respond to the five hormone stresses. This study is the first report of a genome-wide analysis of AGO, DCL, and RDR gene families in Fragaria spp., in which we provide basic genomic information and expression patterns for these genes. Additionally, this study provides a basis for further research on the functions of these genes and some evidence for the evolution between diploid and octoploid strawberries.


Assuntos
Fragaria , RNA Polimerase Dependente de RNA , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Fragaria/metabolismo , Filogenia , Genes de Plantas
11.
Proc Natl Acad Sci U S A ; 120(1): e2211425120, 2023 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-36577062

RESUMO

De novo viral RNA-dependent RNA polymerases (RdRPs) utilize their priming element (PE) to facilitate accurate initiation. Upon transition to elongation, the PE has to retreat from the active site to give room to the template-product RNA duplex. However, PE conformational change upon this transition and the role of PE at elongation both remain elusive. Here, we report crystal structures of RdRP elongation complex (EC) from dengue virus serotype 2 (DENV2), demonstrating a dramatic refolding of PE that allows establishment of interactions with the RNA duplex backbone approved to be essential for EC stability. Enzymology data from both DENV2 and hepatitis C virus (HCV) RdRPs suggest that critical transition of the refolding likely occurs after synthesis of a 4- to 5-nucleotide (nt) product together providing a key basis in understanding viral RdRP transition from initiation to elongation.


Assuntos
RNA Polimerase Dependente de RNA , RNA , RNA Polimerase Dependente de RNA/metabolismo , Hepacivirus/metabolismo , Domínio Catalítico , Nucleotídeos , RNA Viral/genética
12.
Biochem Biophys Res Commun ; 641: 50-56, 2023 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-36521285

RESUMO

Kyasanur forest disease is a neglected zoonotic disease caused by a single-stranded RNA-based flavivirus, the incidence of which was first recorded in 1957 in the Southern part of India. Kyasanur forest disease virus is transmitted to monkeys and humans through the infected tick bite of Haemophysalis spinigera. Kyasanur forest disease is a febrile illness, which in severe cases, results in neurological complications leading to mortality. The current treatment regimens are symptomatic and supportive, and no targeted therapies are available for this disease. In this study, we evaluated the ability of FDA-approved drugs sofosbuvir (and its active metabolite) and Dasabuvir to inhibit the RNA-dependent RNA polymerase activity of NS5 protein from the Kyasanur forest disease virus. NS5 protein containing the N-terminal methyl transferase domain and C-terminal RNA-dependent RNA polymerase domain was expressed in Escherichia coli, and RNA-dependent RNA polymerase activity was demonstrated with the purified protein. The RNA-dependent RNA polymerase assay conditions were optimized, followed by the determination of apparent Km,ATP to validate the enzyme preparation. Half maximal-inhibitory concentrations against RNA-dependent RNA polymerase activity were determined for Sofosbuvir and its active metabolite. Dasabuvir did not show detectable inhibition with the tested conditions. This is the first demonstration of the inhibition of RNA-dependent RNA polymerase activity of NS5 protein from the Kyasanur forest disease virus with small molecule inhibitors. These initial findings can potentially facilitate the discovery and development of targeted therapies for treating Kyasanur forest disease.


Assuntos
Vírus da Encefalite Transmitidos por Carrapatos , Doença da Floresta de Kyasanur , Animais , Humanos , Vírus da Encefalite Transmitidos por Carrapatos/genética , Haplorrinos , Índia/epidemiologia , Doença da Floresta de Kyasanur/epidemiologia , Fosfatos , Sofosbuvir/farmacologia , RNA Polimerase Dependente de RNA/metabolismo , Proteínas não Estruturais Virais/metabolismo
13.
Biomater Sci ; 11(2): 509-517, 2023 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-36533394

RESUMO

Unimolecular micelles (UIMs) exhibit promising potential in the precise diagnosis and accurate treatment of tumor tissues, a pressing problem in the field of medical treatment, because of their perfect stability in the complex and variable microenvironment. In this study, porphyrin-based four-armed star-shaped block polymers with narrow molar mass dispersity (D = 1.34) were facilely prepared by photocontrolled bromine-iodine transformation reversible-deactivation radical polymerization (BIT-RDRP). A photothermal conversion dye, ketocyanine, was covalently linked onto the PEG and then introduced into the polymers through a "grafting onto" strategy to obtain polymeric nanomaterial, THPP-4PMMA-b-4P(PEGMA-co-APMA)@NIR-800, with dual PTT/PDT function. The resulting polymers could form monodispersed UIMs in the water below critical aggregation concentration, meanwhile maintaining the capacities of singlet oxygen release and photothermal conversion. Importantly, the UIMs displayed excellent biocompatibility while exerting superior PTT and/or PDT therapeutic effects under the irradiation of specific wavelengths of light, according to in vitro cellular experiments, which is expected to become a new hot spot for cancer therapy and anti-tumor research. Overall, stable and powerful UIMs with dual PTT/PDT function is provided, which are expected to be competitive candidates in cancer therapy.


Assuntos
Neoplasias , Fotoquimioterapia , Humanos , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes , Micelas , Polímeros/uso terapêutico , Bromo/uso terapêutico , Polimerização , Neoplasias/tratamento farmacológico , RNA Polimerase Dependente de RNA/uso terapêutico , Microambiente Tumoral
14.
J Infect Public Health ; 16(1): 117-124, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36512968

RESUMO

BACKGROUND: Mucormycosis is an infection caused by fungi belonging to the order Mucorales. Rhizopus oryzae is one of the most prevalent organisms identified in mucormycosis patients. Because it spreads quickly through the blood vessels, this opportunistic illness has an exceptionally high fatality rate, even when vigorous treatment is administered. Nonetheless, it has a high tolerance to antifungal medicines, limiting treatment options. As a result, improved methods for preventing and treating mucormycosis are desperately needed. Hence, this study was aimed at assessing the effect of lupeol, quercetin, and solasodine against mucormycosis based on computational approaches. METHODS: The Rhizopus oryzae RNA-dependent RNA polymerase (RdRp) was the target for the design of drugs against the deadly mucormycosis. The three-dimensional structure of the RdRp was modelled with a Swiss model and validated using PROCHECK, VERIFY 3D, and QMEAN. Using the Schrodinger maestro module, a molecular docking study was performed between RdRp and the antimicrobial phytochemicals lupeol, quercetin, and solasodine. A molecular dynamics (MD) simulation study was used to assess the stability and interaction of the RdRp with these phytochemicals. RESULTS: The RdRp protein binds strongly to lupeol (-7.2 kcal/mol), quercetin (-9.1 kcal/mol), and solasodine (-9.6 kcal/mol), according to molecular docking assessment based on the lowest binding energy, confirmation, and bond interaction. Simulations suggest that lupeol, quercetin, and solasodine complexes with RdRp and showed stable confirmation with minimal fluctuation throughout the 200 nanoseconds based on the RMSD and RMSF trajectory assessments. CONCLUSION: The molecular docking and MD simulation investigation improved our understanding of phytochemical-RdRp interactions. Due to its high affinity for RdRp, solasodine may be a better treatment option for mucormycosis.


Assuntos
Mucormicose , Humanos , Mucormicose/tratamento farmacológico , Rhizopus/genética , Rhizopus oryzae , Simulação de Acoplamento Molecular , Quercetina/farmacologia , Quercetina/uso terapêutico , RNA Polimerase Dependente de RNA
15.
Int J Biol Macromol ; 226: 946-955, 2023 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-36528144

RESUMO

The coronavirus disease 2019 has been ravaging throughout the world for three years and has severely impaired both human health and the economy. The causative agent, severe acute respiratory syndrome coronavirus 2 employs the viral RNA dependent RNA polymerase (RdRp) complex for genome replication and transcription, making RdRp an appealing target for antiviral drug development. Systematic characterization of RdRp will undoubtedly aid in the development of antiviral drugs targeting RdRp. Here, our research reveals that RdRp can recognize and utilize nucleoside diphosphates as a substrate to synthesize RNA with an efficiency of about two thirds of using nucleoside triphosphates as a substrate. Nucleoside diphosphates incorporation is also template-specific and has high fidelity. Moreover, RdRp can incorporate ß-d-N4-hydroxycytidine into RNA while using diphosphate form molnupiravir as a substrate. This incorporation results in genome mutation and virus death. It is also observed that diphosphate form molnupiravir is a better substrate for RdRp than the triphosphate form molnupiravir, presenting a new strategy for drug design.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , RNA , Difosfatos , Nucleosídeos , RNA Polimerase Dependente de RNA/metabolismo , Antivirais/química , Nucleotídeos , RNA Viral/genética , Proteínas do Olho , Proteínas do Tecido Nervoso
16.
J Cell Biochem ; 124(1): 127-145, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36502494

RESUMO

Numerous pathogens affecting human is present in the flavivirus family namely west nile, dengue, yellow fever, and zika which involves in development of global burden and distressing the environment economically. Till date, no approved drugs are available for targeting these viruses. The threat which urged the identification of small molecules for the inhibition of these viruses is the spreading of serious viral diseases. The recent outbreak of zika and dengue infections postured a solemn risk to worldwide public well-being. RNA-dependent RNA polymerase (RdRp) is the supreme adaptable enzymes of all the RNA viruses which is responsible for the replication and transcription of genome among the structural and nonstructural proteins of flaviviruses. It is understood that the RdRp of the flaviviruses are similar stating that the japanese encephalitis and west nile shares 70% identity with zika whereas the dengue serotype 2 and 3 shares the identity of 76% and 81%, respectively. In this study, we investigated the binding site of four flaviviral RdRp and provided insights into various interaction of the molecules using the computational approach. Our study helps in recognizing the potent compounds that could inhibit the viral protein as a common inhibitor. Additionally, with the conformational stability analysis, we proposed the possible mechanism of inhibition of the identified common small molecule toward RdRp of flavivirus. Finally, this study could be an initiative for the identification of common inhibitors and can be explored further for understanding the mechanism of action through in vitro studies for the study on efficacy.


Assuntos
Dengue , Flavivirus , Infecção por Zika virus , Zika virus , Humanos , Flavivirus/genética , Flavivirus/metabolismo , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Reposicionamento de Medicamentos , Proteínas Virais/metabolismo
17.
Proc Natl Acad Sci U S A ; 119(50): e2203054119, 2022 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-36469786

RESUMO

Mammalian reovirus (reovirus) is a multilayered, turreted member of Reoviridae characterized by transcription of dsRNA genome within the innermost capsid shell. Here, we present high-resolution in situ structures of reovirus transcriptase complex in an intact double-layered virion, and in the uncoated single-layered core particles in the unloaded, reloaded, pre-elongation, and elongation states, respectively, obtained by cryo-electron microscopy and sub-particle reconstructions. At the template entry of RNA-dependent RNA polymerase (RdRp), the RNA-loading region gets flexible after uncoating resulting in the unloading of terminal genomic RNA and inactivity of transcription. However, upon adding transcriptional substrates, the RNA-loading region is recovered leading the RNAs loaded again. The priming loop in RdRp was found to play a critical role in regulating transcription, which hinders the elongation of transcript in virion and triggers the rearrangement of RdRp C-terminal domain (CTD) during elongation, resulting in splitting of template-transcript hybrid and opening of transcript exit. With the integration of these structures, a transcriptional model of reovirus with five states is proposed. Our structures illuminate the RdRp activation and regulation of the multilayered turreted reovirus.


Assuntos
RNA Viral , Reoviridae , Animais , Microscopia Crioeletrônica , RNA Viral/genética , Reoviridae/genética , RNA Polimerase Dependente de RNA/genética , Capsídeo , Mamíferos/genética
18.
Viruses ; 14(12)2022 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-36560736

RESUMO

The genogroup II genotype 4 (GII.4) noroviruses are a major cause of viral gastroenteritis. Since the emergence of the Sydney_2012 variant, no novel norovirus GII.4 variants have been reported. The high diversity of noroviruses and periodic emergence of novel strains necessitates continuous global surveillance. The aim of this study was to assess the diversity of noroviruses in selected wastewater samples from Pretoria, South Africa (SA) using amplicon-based next-generation sequencing (NGS). Between June 2018 and August 2020, 200 raw sewage and final effluent samples were collected fortnightly from two wastewater treatment plants in Pretoria. Viruses were recovered using skimmed milk flocculation and glass wool adsorption-elution virus recovery methods and screened for noroviruses using a one-step real-time reverse-transcription PCR (RT-PCR). The norovirus BC genotyping region (570-579 bp) was amplified from detected norovirus strains and subjected to Illumina MiSeq NGS. Noroviruses were detected in 81% (162/200) of samples. The majority (89%, 89/100) of raw sewage samples were positive for at least one norovirus, compared with 73% (73/100) of final effluent samples. Overall, a total of 89 different GI and GII RdRp-capsid combinations were identified, including 51 putative novel recombinants, 34 previously reported RdRp-capsid combinations, one emerging novel recombinant and three Sanger-sequencing confirmed novel recombinants.


Assuntos
Norovirus , Esgotos , Humanos , Infecções por Caliciviridae , Gastroenterite/virologia , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Epidemiologia Molecular , Norovirus/genética , Norovirus/isolamento & purificação , Filogenia , RNA Polimerase Dependente de RNA/genética , Esgotos/virologia , África do Sul/epidemiologia , Vírus Reordenados/genética , Vírus Reordenados/isolamento & purificação
19.
Arch Virol ; 168(1): 7, 2022 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-36542124

RESUMO

A number of viruses have recently been discovered in all major fungal phyla using high-throughput sequencing. However, basal fungi remain among the least-explored organisms with respect to the presence of mycoviruses. In this study, we characterized two mycoviruses coinfecting the basal fungus Conidiobolus adiaeretus, which we have named "Conidiobolus adiaeretus totivirus 1" (CaTV1) and "Conidiobolus adiaeretus totivirus 2" (CaTV2). Due to their similar sizes, the genomic RNAs of these two viruses comigrated as a single band in 1.5% agarose gel electrophoresis but could be distinguished and characterized by next-generation sequencing and RT-PCR. Like those of other totiviruses, the genomes of both CaTV1 and CaTV2 have two discontinuous open reading frames: ORF1 and ORF2, encoding a putative capsid protein and a putative RNA-dependent RNA polymerase (RdRp), respectively. The RdRps of CaTV1 and CaTV2 have 62.73% and 63.76% amino acid sequence identity, respectively, to Wuhan insect virus 26 and have 62.15% amino acid sequence identity to each other. A maximum-likelihood phylogenetic tree based on RdRp amino acid sequences showed that both CaTV1 and CaTV2 clustered in a clade with members of the genus Totivirus. Therefore, we propose that CaTV1 and CaTV2 are two new members of the genus Totivirus in the family Totiviridae.


Assuntos
Conidiobolus , Micovírus , Totivirus , Totivirus/genética , Filogenia , Conidiobolus/genética , RNA Polimerase Dependente de RNA/genética , Fases de Leitura Aberta , Genoma Viral , RNA Viral/genética , RNA de Cadeia Dupla , Micovírus/genética
20.
Signal Transduct Target Ther ; 7(1): 400, 2022 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-36575184

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic has devastated global health. Identifying key host factors essential for SARS-CoV-2 RNA replication is expected to unravel cellular targets for the development of broad-spectrum antiviral drugs which have been quested for the preparedness of future viral outbreaks. Here, we have identified host proteins that associate with nonstructural protein 12 (nsp12), the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 using a mass spectrometry (MS)-based proteomic approach. Among the candidate factors, CDK2 (Cyclin-dependent kinase 2), a member of cyclin-dependent kinases, interacts with nsp12 and causes its phosphorylation at T20, thus facilitating the assembly of the RdRp complex consisting of nsp12, nsp7 and nsp8 and promoting efficient synthesis of viral RNA. The crucial role of CDK2 in viral RdRp function is further supported by our observation that CDK2 inhibitors potently impair viral RNA synthesis and SARS-CoV-2 infection. Taken together, we have discovered CDK2 as a key host factor of SARS-CoV-2 RdRp complex, thus serving a promising target for the development of SARS-CoV-2 RdRp inhibitors.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Quinase 2 Dependente de Ciclina/genética , Proteômica , COVID-19/genética , Proteínas não Estruturais Virais/genética , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo
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