RESUMO
How multi-segmented double-stranded RNA (dsRNA) viruses correctly incorporate their genomes into their capsids remains unclear for many viruses, including Bluetongue virus (BTV), a Reoviridae member, with a genome of 10 segments. To address this, we used an RNA-cross-linking and peptide-fingerprinting assay (RCAP) to identify RNA binding sites of the inner capsid protein VP3, the viral polymerase VP1 and the capping enzyme VP4. Using a combination of mutagenesis, reverse genetics, recombinant proteins and in vitro assembly, we validated the importance of these regions in virus infectivity. Further, to identify which RNA segments and sequences interact with these proteins, we used viral photo-activatable ribonucleoside crosslinking (vPAR-CL) which revealed that the larger RNA segments (S1-S4) and the smallest segment (S10) have more interactions with viral proteins than the other smaller segments. Additionally, using a sequence enrichment analysis we identified an RNA motif of nine bases that is shared by the larger segments. The importance of this motif for virus replication was confirmed by mutagenesis followed by virus recovery. We further demonstrated that these approaches could be applied to a related Reoviridae member, rotavirus (RV), which has human epidemic impact, offering the possibility of novel intervention strategies for a human pathogen.
Assuntos
Vírus Bluetongue , Capsídeo , RNA Viral , Proteínas Virais , Animais , Humanos , Vírus Bluetongue/química , Vírus Bluetongue/metabolismo , Capsídeo/química , Capsídeo/metabolismo , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA Viral/metabolismo , Replicação Viral , Proteínas Virais/química , Proteínas Virais/metabolismoRESUMO
A functional cure of chronic hepatitis B requires eliminating the hepatitis B virus (HBV)-encoded surface antigen (HBsAg), which can suppress immune responses. STOPS are phosphorothioated single-stranded oligonucleotides containing novel chemistries that significantly reduce HBsAgs produced by HBV-infected liver cells. The STOPS molecule ALG-10000 functions inside cells to reduce the levels of multiple HBV-encoded molecules. However, it does not bind HBV molecules. An affinity resin coupled with ALG-10000 was found to bind several proteins from liver cells harboring replicating HBV. Silencing RNAs targeting host factors SRSF1, HNRNPA2B1, GRP78 (HspA5), RPLP1, and RPLP2 reduced HBsAg levels and other HBV molecules that are concomitantly reduced by STOPS. Host proteins RPLP1/RPLP2 and GRP78 function in the translation of membrane proteins, protein folding, and degradation. ALG-10000 and the knockdowns of RPLP1/2 and GRP78 decreased the levels of HBsAg and increased their ubiquitination and proteasome degradation. GRP78, RPLP1, and RPLP2 affected HBsAg production only when HBsAg was expressed with HBV regulatory sequences, suggesting that HBV has evolved to engage with these STOPS-interacting molecules. The STOPS inhibition of HBsAg levels in HBV-infected cells occurs by sequestering cellular proteins needed for proper expression and folding of HBsAg.
RESUMO
Beta-caryophyllene is an odoriferous bicyclic sesquiterpene found in various herbs and spices. Recently, it was found that beta-caryophyllene is a ligand of the cannabinoid receptor 2 (CB2). Activation of CB2 will decrease pain, a major signal for inflammatory responses. We hypothesized that beta-caryophyllene can affect wound healing by decreasing inflammation. Here we show that cutaneous wounds of mice treated with beta-caryophyllene had enhanced re-epithelialization. The treated tissue showed increased cell proliferation and cells treated with beta-caryophyllene showed enhanced cell migration, suggesting that the higher re-epithelialization is due to enhanced cell proliferation and cell migration. The treated tissues also had up-regulated gene expression for hair follicle bulge stem cells. Olfactory receptors were not involved in the enhanced wound healing. Transient Receptor Potential channel genes were up-regulated in the injured skin exposed to beta-caryophyllene. Interestingly, there were sex differences in the impact of beta- caryophyllene as only the injured skin of female mice had enhanced re-epithelialization after exposure to beta-caryophyllene. Our study suggests that chemical compounds included in essential oils have the capability to improve wound healing, an effect generated by synergetic impacts of multiple pathways.
Assuntos
Sesquiterpenos Policíclicos/farmacologia , Receptor CB2 de Canabinoide/efeitos dos fármacos , Cicatrização/fisiologia , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Folículo Piloso/metabolismo , Inflamação/tratamento farmacológico , Queratinócitos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reepitelização , Receptor CB2 de Canabinoide/genética , Receptor CB2 de Canabinoide/metabolismo , Sesquiterpenos/metabolismo , Fatores Sexuais , Pele/metabolismo , Cicatrização/efeitos dos fármacosRESUMO
The genomes of the Reoviridae, including the animal pathogen bluetongue virus (BTV), are multisegmented double-stranded RNA (dsRNA). During replication, single-stranded (ss) positive-sense RNA segments are packaged into the assembling virus capsid, triggering genomic dsRNA synthesis. However, exactly how this packaging event occurs is not clear. A minor capsid protein, VP6, unique for the orbiviruses, has been proposed to be involved in the RNA-packaging process. In this study, we sought to characterize the RNA binding activity of VP6 and its functional relevance. A novel proteomic approach was utilized to map the ssRNA/dsRNA binding sites of a purified recombinant protein and the genomic dsRNA binding sites of the capsid-associated VP6. The data revealed that each VP6 protein has multiple distinct RNA-binding regions and that only one region is shared between recombinant and capsid-associated VP6. A combination of targeted mutagenesis and reverse genetics identified the RNA-binding region that is essential for virus replication. Using an in vitro RNA-binding competition assay, a unique cell-free assembly assay, and an in vivo single-cycle replication assay, it was possible to identify a motif within the shared binding region that binds BTV ssRNA preferentially in a manner consistent with specific RNA recruitment during capsid assembly. These data highlight the critical roles that this unique protein plays in orbivirus genome packaging and replication.IMPORTANCE Genome packaging is a critical stage during virus replication. For viruses with segmented genomes, the genome segments need to be correctly packaged into a newly formed capsid. However, the detailed mechanism of this packaging is unclear. Here we focus on VP6, a minor viral protein of bluetongue virus, which is critical for genome packaging. We used multiple approaches, including a robust RNA-protein fingerprinting assay, to map the ssRNA binding sites of recombinant VP6 and the genomic dsRNA binding sites of capsid-associated VP6. By these means, together with virological and biochemical methods, we identify the viral RNA-packaging motif of a segmented dsRNA virus for the first time.
Assuntos
Vírus Bluetongue/crescimento & desenvolvimento , Vírus Bluetongue/genética , Proteínas do Capsídeo/genética , RNA Viral/metabolismo , Montagem de Vírus/genética , Animais , Sítios de Ligação/genética , Capsídeo/metabolismo , Linhagem Celular , Cricetinae , Genoma Viral/genética , RNA Viral/genética , Motivos de Ligação ao RNA/genéticaRESUMO
Human parechoviruses (HPeV) are picornaviruses with a highly-ordered RNA genome contained within icosahedrally-symmetric capsids. Ordered RNA structures have recently been shown to interact with capsid proteins VP1 and VP3 and facilitate virus assembly in HPeV1. Using an assay that combines reversible cross-linking, RNA affinity purification and peptide mass fingerprinting (RCAP), we mapped the RNA-interacting regions of the capsid proteins from the whole HPeV1 virion in solution. The intrinsically-disordered N-termini of capsid proteins VP1 and VP3, and unexpectedly, VP0, were identified to interact with RNA. Comparing these results to those obtained using recombinantly-expressed VP0 and VP1 confirmed the virion binding regions, and revealed unique RNA binding regions in the isolated VP0 not previously observed in the crystal structure of HPeV1. We used RNA fluorescence anisotropy to confirm the RNA-binding competency of each of the capsid proteins' N-termini. These findings suggests that dynamic interactions between the viral RNA and the capsid proteins modulate virus assembly, and suggest a novel role for VP0.
Assuntos
Proteínas do Capsídeo/metabolismo , Parechovirus/fisiologia , RNA Viral/metabolismo , Vírion/metabolismo , Montagem de Vírus , Proteínas do Capsídeo/química , Reagentes de Ligações Cruzadas/química , Células HT29 , Humanos , Modelos Moleculares , RNA Viral/químicaRESUMO
Signaling by the interleukin-36 receptor (IL-36R) is linked to inflammatory diseases such as psoriasis. However, the regulation of IL-36R signaling is poorly understood. Activation of IL-36R signaling in cultured cells results in an increased polyubiquitination of the receptor subunit, IL-1Rrp2. Treatment with deubiquitinases shows that the receptor subunit of IL-36R, IL-1Rrp2, is primarily polyubiquitinated at the K63 position, which is associated with endocytic trafficking and signal transduction. A minor amount of ubiquitination is at the K48 position that is associated with protein degradation. A focused siRNA screen identified RNF125, an E3 ubiquitin ligase, to ubiquitinate IL-1Rrp2 upon activation of IL-36R signaling while not affecting the activated IL-1 receptor. Knockdown of RNF125 decreases signal transduction by the IL-36R. Overexpression of RNF125 in HEK293T cells activates IL-36R signaling and increases the ubiquitination of IL-1Rrp2 and its subsequent turnover. RNF125 can coimmunoprecipitate with the IL-36R, and it traffics with IL-1Rrp2 from the cell surface to lysosomes. Mutations of Lys568 and Lys569 in the C-terminal tail of IL-1Rrp2 decrease ubiquitination by RNF125 and increase the steady-state levels of IL-1Rrp2. These results demonstrate that RNF125 has multiple regulatory roles in the signaling, trafficking, and turnover of the IL-36R.
Assuntos
Inflamação/imunologia , Subunidade alfa de Receptor de Interleucina-18/metabolismo , Lisossomos/metabolismo , Psoríase/imunologia , Receptores de Interleucina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Endocitose , Células HEK293 , Humanos , Subunidade alfa de Receptor de Interleucina-18/genética , Mutação/genética , Transporte Proteico , RNA Interferente Pequeno/genética , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
In the brome mosaic virus (BMV) virion, the coat protein (CP) selectively contacts the RNA motifs that regulate translation and RNA replication (Hoover et al., 2016. J. Virol. 90, 7748). We hypothesize that the unstructured N-terminal arm (NTA) of the BMV CP can specifically recognize RNA motifs. Using ion mobility spectrometry-mass spectrometry, we demonstrate that peptides containing the NTA of the CP were found to preferentially bind to an RNA hairpin motif that directs the initiation of BMV RNA synthesis. RNA binding causes the peptide to change from heterogeneous structures to a single family of structures. Fluorescence anisotropy, fluorescence quenching and size exclusion chromatography experiments all confirm that the NTA can specific recognize the RNA motif. The peptide introduced into plants along with BMV virion increased accumulation of the BMV CP and accelerated the rate of minus-strand RNA synthesis. The intrinsically disordered BMV NTA could thus specifically recognize BMV RNAs to affect viral infection.
Assuntos
Proteínas do Capsídeo/química , Proteínas Intrinsicamente Desordenadas/química , Motivos de Nucleotídeos , RNA Viral/química , Replicação Viral , Sequência de Bases , Bromovirus/genética , Bromovirus/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Regulação Viral da Expressão Gênica , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , RNA Viral/genética , RNA Viral/metabolismo , Vírion/química , Vírion/genética , Vírion/metabolismoRESUMO
Coronaviruses are positive-sense RNA viruses that generate double-stranded RNA (dsRNA) intermediates during replication, yet evade detection by host innate immune sensors. Here we report that coronavirus nonstructural protein 15 (nsp15), an endoribonuclease, is required for evasion of dsRNA sensors. We evaluated two independent nsp15 mutant mouse coronaviruses, designated N15m1 and N15m3, and found that these viruses replicated poorly and induced rapid cell death in mouse bone marrow-derived macrophages. Infection of macrophages with N15m1, which expresses an unstable nsp15, or N15m3, which expresses a catalysis-deficient nsp15, activated MDA5, PKR, and the OAS/RNase L system, resulting in an early, robust induction of type I IFN, PKR-mediated apoptosis, and RNA degradation. Immunofluorescence imaging of nsp15 mutant virus-infected macrophages revealed significant dispersal of dsRNA early during infection, whereas in WT virus-infected cells, the majority of the dsRNA was associated with replication complexes. The loss of nsp15 activity also resulted in greatly attenuated disease in mice and stimulated a protective immune response. Taken together, our findings demonstrate that coronavirus nsp15 is critical for evasion of host dsRNA sensors in macrophages and reveal that modulating nsp15 stability and activity is a strategy for generating live-attenuated vaccines.
Assuntos
Coronavirus/genética , Coronavirus/imunologia , Macrófagos/imunologia , RNA de Cadeia Dupla/genética , Proteínas não Estruturais Virais/genética , Animais , Apoptose/genética , Apoptose/imunologia , Linhagem Celular , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Cricetinae , Endorribonucleases/metabolismo , Ativação Enzimática/genética , Imunidade Inata/imunologia , Interferon Tipo I/genética , Interferon Tipo I/imunologia , Helicase IFIH1 Induzida por Interferon/metabolismo , Macrófagos/virologia , Camundongos , Proteínas não Estruturais Virais/imunologiaRESUMO
The recent outbreak of Zika virus (ZIKV) has infected over 1 million people in over 30 countries. ZIKV replicates its RNA genome using virally encoded replication proteins. Nonstructural protein 5 (NS5) contains a methyltransferase for RNA capping and a polymerase for viral RNA synthesis. Here we report the crystal structures of full-length NS5 and its polymerase domain at 3.0 Å resolution. The NS5 structure has striking similarities to the NS5 protein of the related Japanese encephalitis virus. The methyltransferase contains in-line pockets for substrate binding and the active site. Key residues in the polymerase are located in similar positions to those of the initiation complex for the hepatitis C virus polymerase. The polymerase conformation is affected by the methyltransferase, which enables a more efficiently elongation of RNA synthesis in vitro. Overall, our results will contribute to future studies on ZIKV infection and the development of inhibitors of ZIKV replication.
Assuntos
Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Zika virus/metabolismo , Cristalografia por Raios X , Metiltransferases/metabolismo , Conformação Proteica , Capuzes de RNA , RNA Polimerase Dependente de RNA/metabolismo , Especificidade por Substrato , Replicação Viral , Zika virus/fisiologiaRESUMO
Noroviruses belong to the Caliciviridae family of single-stranded positive-sense RNA viruses. The genus Norovirus includes seven genogroups (designated GI-GVII), of which GI, GII and GIV infect humans. Human noroviruses are responsible for widespread outbreaks of acute gastroenteritis and represent one of the most common causes of foodborne illness. No vaccine or antiviral treatment options are available for norovirus infection. The RNA-dependent RNA polymerase (RdRp) of noroviruses is a key enzyme responsible for transcription and replication of the viral genome. Here, we review the progress made in understanding the structures and functions of norovirus RdRp and its use as a target for small molecule inhibitors. Crystal structures of the RdRp at different stages of substrate interaction have been determined, which shed light on its multi-step catalytic cycle. The in vitro assays and in vivo animal models that have been developed to identify and characterize inhibitors of norovirus RdRp are also summarized, followed by an update on the current antiviral research targeting different regions of norovirus RdRp. In the future, structure-based drug design and rational optimization of known nucleoside and non-nucleoside inhibitors of norovirus RdRp may pave the way towards the next generation of direct-acting antivirals.
Assuntos
Antivirais/metabolismo , Inibidores Enzimáticos/metabolismo , Norovirus/enzimologia , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , Transcrição Gênica , Replicação Viral , Animais , Infecções por Caliciviridae/tratamento farmacológico , Infecções por Caliciviridae/virologia , Cristalografia por Raios X , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Testes de Sensibilidade Microbiana , Norovirus/efeitos dos fármacos , Conformação Proteica , RNA Polimerase Dependente de RNA/antagonistas & inibidoresRESUMO
UNLABELLED: The four brome mosaic virus (BMV) RNAs (RNA1 to RNA4) are encapsidated in three distinct virions that have different disassembly rates in infection. The mechanism for the differential release of BMV RNAs from virions is unknown, since 180 copies of the same coat protein (CP) encapsidate each of the BMV genomic RNAs. Using mass spectrometry, we found that the BMV CP contains a complex pattern of posttranslational modifications. Treatment with phosphatase was found to not significantly affect the stability of the virions containing RNA1 but significantly impacted the stability of the virions that encapsidated BMV RNA2 and RNA3/4. Cryo-electron microscopy reconstruction revealed dramatic structural changes in the capsid and the encapsidated RNA. A phosphomimetic mutation in the flexible N-terminal arm of the CP increased BMV RNA replication and virion production. The degree of phosphorylation modulated the interaction of CP with the encapsidated RNA and the release of three of the BMV RNAs. UV cross-linking and immunoprecipitation methods coupled to high-throughput sequencing experiments showed that phosphorylation of the BMV CP can impact binding to RNAs in the virions, including sequences that contain regulatory motifs for BMV RNA gene expression and replication. Phosphatase-treated virions affected the timing of CP expression and viral RNA replication in plants. The degree of phosphorylation decreased when the plant hosts were grown at an elevated temperature. These results show that phosphorylation of the capsid modulates BMV infection. IMPORTANCE: How icosahedral viruses regulate the release of viral RNA into the host is not well understood. The selective release of viral RNA can regulate the timing of replication and gene expression. Brome mosaic virus (BMV) is an RNA virus, and its three genomic RNAs are encapsidated in separate virions. Through proteomic, structural, and biochemical analyses, this work shows that posttranslational modifications, specifically, phosphorylation, on the capsid protein regulate the capsid-RNA interaction and the stability of the virions and affect viral gene expression. Mutational analysis confirmed that changes in modification affected virion stability and the timing of viral infection. The mechanism for modification of the virion has striking parallels to the mechanism of regulation of chromatin packaging by nucleosomes.
Assuntos
Bromovirus/fisiologia , Proteínas do Capsídeo/metabolismo , Processamento de Proteína Pós-Traducional , Bromovirus/ultraestrutura , Proteínas do Capsídeo/química , Microscopia Crioeletrônica , Espectrometria de Massas , Fosforilação , Plantas , Vírion/ultraestrutura , Viroses , Replicação ViralRESUMO
Signal transduction by the IL-36 receptor (IL-36R) is linked to several human diseases. However, the structure and function of the IL-36R is not well understood. A molecular model of the IL-36R complex was generated and a cell-based reporter assay was established to assess the signal transduction of recombinant subunits of the IL-36R. Mutational analyses and functional assays have identified residues of the receptor subunit IL-1Rrp2 needed for cytokine recognition, stable protein expression, disulfide bond formation and glycosylation that are critical for signal transduction. We also observed that, overexpression of ectodomain (ECD) of Il-1Rrp2 or IL-1RAcP exhibited dominant-negative effect on IL-36R signaling. The presence of IL-36 cytokine significantly increased the interaction of IL-1Rrp2 ECD with the co-receptor IL-1RAcP. Finally, we found that single nucleotide polymorphism A471T in the Toll-interleukin 1 receptor domain (TIR) of the IL-1Rrp2 that is present in â¼2% of the human population, down-regulated IL-36R signaling by a decrease of interaction with IL-1RAcP.
Assuntos
Proteína Acessória do Receptor de Interleucina-1 , Subunidade alfa de Receptor de Interleucina-18 , Polimorfismo Genético , Células HEK293 , Humanos , Proteína Acessória do Receptor de Interleucina-1/química , Proteína Acessória do Receptor de Interleucina-1/genética , Proteína Acessória do Receptor de Interleucina-1/metabolismo , Subunidade alfa de Receptor de Interleucina-18/química , Subunidade alfa de Receptor de Interleucina-18/genética , Subunidade alfa de Receptor de Interleucina-18/metabolismo , Domínios Proteicos , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
Capsid proteins form protective shells around viral genomes and mediate viral entry. However, many capsid proteins have additional and important roles for virus infection and in modulating cellular response to infection, with important consequences on pathogenesis. Infection by the Hepatitis C virus (HCV) can lead to liver steatosis, cirrhosis, and hepatocellular carcinoma. Herein, we focus on the role in pathogenesis of Core, the capsid protein of the HCV.
Assuntos
Proteínas do Capsídeo/metabolismo , Hepacivirus/patogenicidade , Hepatite C/virologia , Proteínas do Core Viral/metabolismo , Animais , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Carcinoma Hepatocelular/virologia , Genoma Viral , Hepacivirus/genética , Hepacivirus/imunologia , Humanos , Cirrose Hepática/virologia , Neoplasias Hepáticas/virologia , Camundongos , Proteínas do Core Viral/química , Proteínas do Core Viral/genéticaRESUMO
UNLABELLED: The cyclic dinucleotide 2',3'-cGAMP can bind the adaptor protein STING (stimulator of interferon [IFN] genes) to activate the production of type I IFNs and proinflammatory cytokines. We found that cGAMP added to the culture medium could suppress the replication of the hepatitis C virus (HCV) genotype 1b strain Con1 subgenomic replicon in human hepatoma cells. Knockdown of STING expression diminished the inhibitory effect on replicon replication, while overexpression of STING enhanced the inhibitory effects of cGAMP. The addition of cGAMP into 1b/Con1 replicon cells significantly increased the expression of type I IFNs and antiviral interferon-stimulated genes. Unexpectedly, replication of the genotype 2a JFH1 replicon and infectious JFH1 virus was less sensitive to the inhibitory effect of cGAMP than was that of 1b/Con1 replicon. Using chimeric replicons, 2a NS4B was identified to confer resistance to cGAMP. Transient expression of 2a NS4B resulted in a pronounced inhibitory effect on STING-mediated beta IFN (IFN-ß) reporter activation compared to that of 1b NS4B. 2a NS4B was found to suppress STING accumulation in a dose-dependent manner. The predicted transmembrane domain of 2a NS4B was required to inhibit STING accumulation. These results demonstrate a novel genotype-specific inhibition of the STING-mediated host antiviral immune response. IMPORTANCE: The cyclic dinucleotide cGAMP was found to potently inhibit the replication of HCV genotype 1b Con1 replicon but was less effective for the 2a/JFH1 replicon and infectious JFH1 virus. The predicted transmembrane domain in 2a NS4B was shown to be responsible for the decreased sensitivity to cGAMP. The N terminus of NS4B has been reported to suppress STING-mediated signaling by disrupting the interaction of STING and TBK1 and/or MAVS. We show that 2a/JFH1 NS4B has an additional mechanism to evade STING signaling through suppressing STING accumulation.
Assuntos
Hepacivirus/imunologia , Hepacivirus/fisiologia , Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Imunidade Inata , Proteínas de Membrana/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo , Linhagem Celular Tumoral , Genótipo , Hepacivirus/classificação , Hepacivirus/genética , Hepatócitos/imunologia , Hepatócitos/virologia , HumanosRESUMO
Using cross-linking coupled to matrix-assisted laser desorption/ionization mass spectrometry and CLIP-Seq sequencing, we determined the peptide and oligonucleotide sequences at the interfaces between the capsid proteins and the genomic RNA of bacteriophage MS2. The results suggest that the same coat protein (CP)-RNA and maturation protein (MP)-RNA interfaces are used in every viral particle. The portions of the viral RNA in contact with CP subunits span the genome, consistent with a large number of discrete and similar contacts within each particle. Many of these sites match previous predictions of the locations of multiple, dispersed and degenerate RNA sites with cognate CP affinity termed packaging signals (PSs). Chemical RNA footprinting was used to compare the secondary structures of protein-free genomic fragments and the RNA in the virion. Some PSs are partially present in protein-free RNA but others would need to refold from their dominant solution conformations to form the contacts identified in the virion. The RNA-binding peptides within the MP map to two sections of the N-terminal half of the protein. Comparison of MP sequences from related phages suggests a similar arrangement of RNA-binding sites, although these N-terminal regions have only limited sequence conservation. In contrast, the sequences of the C-termini are highly conserved, consistent with them encompassing pilin-binding domains required for initial contact with host cells. These results provide independent and unambiguous support for the assembly of MS2 virions via a PS-mediated mechanism involving a series of induced-fit viral protein interactions with RNA.
Assuntos
Levivirus/fisiologia , Montagem de Vírus , Sítios de Ligação , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Levivirus/genética , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , RNA Viral/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Hepatitis C virus (HCV) poses a major health threat to the world. The recent development of direct-acting antivirals (DAAs) against HCV has markedly improved the response rate of HCV and reduced the side effects in comparison to the interferon-based therapy. Despite this therapeutic advance, there is still a need to develop new inhibitors that target different stages of the HCV life cycle because of various limitations of the current regimens. In this study, we performed a quantitative high throughput screening of the Molecular Libraries Small Molecule Repository (MLSMR) of â¼350,000 chemicals for novel HCV inhibitors using our previously developed cell-based HCV infection assay. Following confirmation and structural clustering analysis, we narrowed down to 158 compounds from the initial â¼3000 molecules that showed inhibitory activity for further structural and functional analyses. We were able to assign the majority of these compounds to specific stage(s) in the HCV life cycle. Three of them are direct inhibitors of NS3/4A protease. Most of the compounds appear to act on novel targets in HCV life cycle. Four compounds with novel structure and excellent drug-like properties, three targeting HCV entry and one targeting HCV assembly/secretion, were advanced for further development as lead hits. These compounds represent diverse chemotypes that are potential lead compounds for further optimization and may offer promising candidates for the development of novel therapeutics against HCV infection. In addition, they represent novel molecular probes to explore the complex interactions between HCV and the cells.
Assuntos
Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Hepatite C Crônica/tratamento farmacológico , Antivirais/química , Linhagem Celular Tumoral , Hepacivirus/fisiologia , Ensaios de Triagem em Larga Escala/métodos , Humanos , Simulação de Acoplamento Molecular , Inibidores de Proteases/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/química , Replicação Viral/efeitos dos fármacosRESUMO
Heterogeneous ribonucleoprotein K (hnRNP K) binds to the 5' untranslated region of the hepatitis C virus (HCV) and is required for HCV RNA replication. The hnRNP K binding site on HCV RNA overlaps with the sequence recognized by the liver-specific microRNA, miR-122. A proteome chip containing â¼17,000 unique human proteins probed with miR-122 identified hnRNP K as one of the strong binding proteins. In vitro kinetic study showed hnRNP K binds miR-122 with a nanomolar dissociation constant, in which the short pyrimidine-rich residues in the central and 3' portion of the miR-122 were required for hnRNP K binding. In liver hepatocytes, miR-122 formed a coprecipitable complex with hnRNP K. High throughput Illumina DNA sequencing of the RNAs precipitated with hnRNP K was enriched for mature miR-122. SiRNA knockdown of hnRNP K in human hepatocytes reduced the levels of miR-122. These results show that hnRNP K is a cellular protein that binds and affects the accumulation of miR-122. Its ability to also bind HCV RNA near the miR-122 binding site suggests a role for miR-122 recognition of HCV RNA.