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1.
Antimicrob Agents Chemother ; 66(11): e0095122, 2022 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-36314868

RESUMO

LHF-535 is a small-molecule antiviral currently under development as a therapeutic option to treat Lassa fever and other viral hemorrhagic fevers of arenavirus origin. The human safety and pharmacokinetics of LHF-535 were evaluated in two phase 1 trials in healthy volunteers. The first study was a double-blind, single ascending dose trial that evaluated weight-based oral doses ranging from 0.3 mg/kg in the first cohort to 40 mg/kg in the last cohort. The second study was a double-blind, multiple ascending dose trial that evaluated a 14-day oral dosing regimen, with three sequential cohorts receiving fixed doses of 450, 900, or 1,125 mg per day; the third cohort (1,125 mg/day) received a higher (loading) dose of 2,250 mg for the first dose. Each cohort in both studies consisted of eight participants randomized to either placebo (n = 2) or LHF-535 (n = 6). LHF-535 was well tolerated in both studies. Treatment-emergent adverse events were more frequent in placebo recipients than in LHF-535 recipients in both studies. LHF-535 exhibited rapid absorption, a long half-life, and exposures predicted to suppress viral replication.


Assuntos
Febres Hemorrágicas Virais , Febre Lassa , Humanos , Adulto , Febre Lassa/tratamento farmacológico , Antivirais/efeitos adversos , Antivirais/farmacocinética , Método Duplo-Cego , Voluntários Saudáveis , Relação Dose-Resposta a Droga
2.
PLoS Pathog ; 14(12): e1007439, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30576397

RESUMO

Arenaviruses are a significant cause of hemorrhagic fever, an often-fatal disease for which there is no approved antiviral therapy. Lassa fever in particular generates high morbidity and mortality in West Africa, where the disease is endemic, and a recent outbreak in Nigeria was larger and more geographically diverse than usual. We are developing LHF-535, a small-molecule viral entry inhibitor that targets the arenavirus envelope glycoprotein, as a therapeutic candidate for Lassa fever and other hemorrhagic fevers of arenavirus origin. Using a lentiviral pseudotype infectivity assay, we determined that LHF-535 had sub-nanomolar potency against the viral envelope glycoproteins from all Lassa virus lineages, with the exception of the glycoprotein from the LP strain from lineage I, which was 100-fold less sensitive than that of other strains. This reduced sensitivity was mediated by a unique amino acid substitution, V434I, in the transmembrane domain of the envelope glycoprotein GP2 subunit. This position corresponds to the attenuation determinant of Candid#1, a live-attenuated Junín virus vaccine strain used to prevent Argentine hemorrhagic fever. Using a virus-yield reduction assay, we determined that LHF-535 potently inhibited Junín virus, but not Candid#1, and the Candid#1 attenuation determinant, F427I, regulated this difference in sensitivity. We also demonstrated that a daily oral dose of LHF-535 at 10 mg/kg protected mice from a lethal dose of Tacaribe virus. Serial passage of Tacaribe virus in LHF-535-treated Vero cells yielded viruses that were resistant to LHF-535, and the majority of drug-resistant viruses exhibited attenuated pathogenesis. These findings provide a framework for the clinical development of LHF-535 as a broad-spectrum inhibitor of arenavirus entry and provide an important context for monitoring the emergence of drug-resistant viruses.


Assuntos
Antivirais/farmacologia , Febre Lassa , Vírus Lassa/genética , Virulência/efeitos dos fármacos , Virulência/genética , Animais , Chlorocebus aethiops , Farmacorresistência Viral/efeitos dos fármacos , Farmacorresistência Viral/genética , Células HEK293 , Humanos , Vírus Lassa/efeitos dos fármacos , Camundongos , Mutação , Células Vero , Proteínas do Envelope Viral/genética
3.
Antiviral Res ; 229: 105952, 2024 09.
Artigo em Inglês | MEDLINE | ID: mdl-38945484

RESUMO

Argentine hemorrhagic fever, caused by Junín virus (JUNV), is the most common of the South American arenaviral hemorrhagic fevers. The disease has a case fatality rate of 15-30% in untreated patients. Although early intervention with immune plasma is effective, diminishing stocks and limited availability outside of Argentina underscores the need for new therapeutics. Ideally, these would be broadly active agents effective against all the pathogenic arenaviruses. The fusion inhibitor LHF-535 and the nucleoside analog favipiravir have shown promise in animal models of Lassa fever, a disease endemic in parts of Africa and the most prominent of the arenaviral hemorrhagic fevers. Against JUNV, a high dose of favipiravir is required to achieve protection in the gold-standard guinea pig infection model. Here, we demonstrate a synergistic effect by the coadministration of LHF-535 with a sub-optimal dose of favipiravir in guinea pigs challenged with JUNV. Administered individually, LHF-535 and sub-optimal favipiravir only delayed the onset of severe disease. However, combined dosing of the drugs afforded complete protection against lethal JUNV infection in guinea pigs. The benefits of the drug combination were also evident by the absence of viremia and infectious virus in tissues compared to guinea pigs treated with only the placebos. Thus, combined targeting of JUNV-endosomal membrane fusion and the viral polymerase with pan-arenaviral LHF-535 and favipiravir may expand their indication beyond Lassa fever, providing a significant barrier to drug resistance.


Assuntos
Amidas , Antivirais , Modelos Animais de Doenças , Vírus Junin , Pirazinas , Pirazinas/farmacologia , Pirazinas/administração & dosagem , Pirazinas/uso terapêutico , Animais , Cobaias , Amidas/farmacologia , Amidas/uso terapêutico , Amidas/administração & dosagem , Vírus Junin/efeitos dos fármacos , Antivirais/farmacologia , Antivirais/administração & dosagem , Antivirais/uso terapêutico , Febre Hemorrágica Americana/tratamento farmacológico , Febre Hemorrágica Americana/virologia , Quimioterapia Combinada , Feminino , Sinergismo Farmacológico , Infecções por Arenaviridae/tratamento farmacológico
4.
Bioorg Med Chem Lett ; 23(21): 5840-3, 2013 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-24064500

RESUMO

Previously we reported the optimization of antiviral scaffolds containing benzimidazole and related heterocycles possessing activity against a variety of arenaviruses. These series of compounds were discovered through an HTS campaign of a 400,000 small molecule library using lentivirus-based pseudotypes incorporated with the Lassa virus envelope glycoprotein (LASV GP). This screening also uncovered an alternate series of very potent arenavirus inhibitors based upon an acylhydrazone scaffold. Subsequent SAR analysis of this chemical series involved various substitutions throughout the chemical framework along with assessment of the preferred stereochemistry. These studies led to an optimized analog (ST-161) possessing subnanomolar activity against LASV and submicromolar activity against a number of other viruses in the Arenaviridae family.


Assuntos
Antivirais/química , Antivirais/farmacologia , Hidrazonas/química , Hidrazonas/farmacologia , Vírus Lassa/efeitos dos fármacos , Acilação , Descoberta de Drogas , Humanos , Febre Lassa/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia
5.
Bioorg Med Chem Lett ; 23(3): 750-6, 2013 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-23265900

RESUMO

A series of potent arenavirus inhibitors sharing a benzimidazole core were previously reported by our group. SAR studies were expanded beyond the previous analysis, which involved the attached phenyl rings and methylamino linker portion, to include modifications focused on the benzimidazole core. These changes included the introduction of various substituents to the bicyclic benzimidazole ring system along with alternate core heterocycles. Many of the analogs containing alternate nitrogen-based bicyclic ring systems were found to retain antiviral potency compared to the benzimidazole series from which we derived our lead compound, ST-193. In fact, 21 h, built on an imidazopyridine core, possessed a near tenfold increase in potency against Lassa virus pseudotypes compared to ST-193. As found with the benzimidazole series, broad-spectrum arenavirus activity was also observed for a number of the analogs discovered during this study.


Assuntos
Antivirais/síntese química , Antivirais/farmacologia , Arenavirus/efeitos dos fármacos , Benzimidazóis/química , Descoberta de Drogas , Compostos Heterocíclicos/síntese química , Antivirais/química , Benzimidazóis/síntese química , Benzimidazóis/farmacologia , Compostos Heterocíclicos/química , Compostos Heterocíclicos/farmacologia , Vírus Lassa/efeitos dos fármacos , Relação Estrutura-Atividade
6.
Bioorg Med Chem Lett ; 23(3): 744-9, 2013 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-23265895

RESUMO

A chemically diverse library of about 400,000 small molecules was screened for antiviral activity against lentiviral pseudotypes with the Lassa virus envelope glycoprotein (LASV GP) gene incorporated. High-throughput screening resulted in discovery of a hit compound (ST-37) possessing a benzimidazole core which led to a potent compound series. Herein, we report SAR studies which involved structural modifications to the phenyl rings and methylamino linker portion attached to the benzimidazole core. Many analogs in this study possessed single digit nanomolar potency against LASV pseudotypes. Compounds in this benzimidazole series also exhibited nanomolar antiviral activity against pseudotypes generated from other arenavirus envelopes indicating the potential for development of a broad-spectrum inhibitor. Ultimately, lead compound ST-193 was identified and later found to be efficacious in a lethal LASV guinea pig model showing superior protection compared to ribavirin treatment.


Assuntos
Antivirais/síntese química , Antivirais/farmacologia , Arenavirus/efeitos dos fármacos , Benzimidazóis/química , Descoberta de Drogas , Animais , Antivirais/química , Benzimidazóis/síntese química , Benzimidazóis/farmacologia , Modelos Animais de Doenças , Cobaias , Bibliotecas de Moléculas Pequenas
7.
J Biol Chem ; 286(8): 6192-200, 2011 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-21159779

RESUMO

Arenaviruses are responsible for acute hemorrhagic fevers worldwide and are recognized to pose significant threats to public health and biodefense. Small molecule compounds have recently been discovered that inhibit arenavirus entry and protect against lethal infection in animal models. These chemically distinct inhibitors act on the tripartite envelope glycoprotein (GPC) through its unusual stable signal peptide subunit to stabilize the complex against pH-induced activation of membrane fusion in the endosome. Here, we report the production and characterization of the intact transmembrane GPC complex of Junín arenavirus and its interaction with these inhibitors. The solubilized GPC is antigenically indistinguishable from the native protein and forms a homogeneous trimer in solution. When reconstituted into a lipid bilayer, the purified complex interacts specifically with its cell-surface receptor transferrin receptor-1. We show that small molecule entry inhibitors specific to New World or Old World arenaviruses bind to the membrane-associated GPC complex in accordance with their respective species selectivities and with dissociation constants comparable with concentrations that inhibit GPC-mediated membrane fusion. Furthermore, competitive binding studies reveal that these chemically distinct inhibitors share a common binding pocket on GPC. In conjunction with previous genetic studies, these findings identify the pH-sensing interface of GPC as a highly vulnerable target for antiviral intervention. This work expands our mechanistic understanding of arenavirus entry and provides a foundation to guide the development of small molecule compounds for the treatment of arenavirus hemorrhagic fevers.


Assuntos
Antígenos CD/metabolismo , Antivirais/farmacologia , Infecções por Arenaviridae/tratamento farmacológico , Vírus Junin/metabolismo , Receptores da Transferrina/metabolismo , Proteínas do Envelope Viral/antagonistas & inibidores , Proteínas do Envelope Viral/metabolismo , Animais , Antígenos CD/genética , Antivirais/química , Infecções por Arenaviridae/genética , Infecções por Arenaviridae/metabolismo , Chlorocebus aethiops , Humanos , Vírus Junin/genética , Receptores da Transferrina/genética , Células Vero , Proteínas do Envelope Viral/genética , Internalização do Vírus/efeitos dos fármacos
8.
Bioorg Med Chem Lett ; 22(13): 4263-72, 2012 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-22664128

RESUMO

A series of acylthiourea derivatives were designed, synthesized, and evaluated for broad-spectrum antiviral activity with selected viruses from Poxviridae (vaccinia virus) and two different genera of the family Bunyaviridae (Rift Valley fever and La Crosse viruses). A compound selected from a library screen, compound 1, displayed submicromolar antiviral activity against both vaccinia virus (EC(50)=0.25 µM) and La Crosse virus (EC(50)=0.27 µM) in cytopathic effect (CPE) assays. SAR analysis was performed to further improve antiviral potency and to optimize drug-like properties of the initial hits. During our analysis, we identified 26, which was found to be nearly fourfold more potent than 1 against both vaccinia and La Crosse viruses. Selected compounds were further tested to more fully characterize the spectrum of antiviral activity. Many of these possessed single digit micromolar and sub-micromolar antiviral activity against a diverse array of targets, including influenza virus (Orthomyxoviridae), Tacaribe virus (Arenaviridae), and dengue virus (Flaviviridae).


Assuntos
Antivirais/química , Tioureia/química , Antivirais/síntese química , Antivirais/farmacologia , Arenavirus/efeitos dos fármacos , Vírus da Dengue/efeitos dos fármacos , Vírus La Crosse/efeitos dos fármacos , Orthomyxoviridae/efeitos dos fármacos , Relação Estrutura-Atividade , Tioureia/síntese química , Tioureia/farmacologia , Vaccinia virus/efeitos dos fármacos
9.
Sci Rep ; 12(1): 19911, 2022 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-36402782

RESUMO

LHF-535 is a small molecule antiviral currently in development for the treatment of Lassa fever, a zoonotic disease endemic in West Africa that generates significant morbidity and mortality. Current treatment options are inadequate, and there are no approved therapeutics or vaccines for Lassa fever. LHF-535 was evaluated in a lethal guinea pig model of Lassa pathogenesis, using once-daily administration of a fixed dose (50 mg/kg/day) initiating either 1 or 3 days after inoculation with a lethal dose of Lassa virus. LHF-535 reduced viremia and clinical signs and protected all animals from lethality. A subset of surviving animals was rechallenged four months later with a second lethal challenge of Lassa virus and were found to be protected from disease. LHF-535 pharmacokinetics at the protective dose in guinea pigs showed plasma concentrations well within the range observed in clinical trials in healthy volunteers, supporting the continued development of LHF-535 as a Lassa therapeutic.


Assuntos
Febre Lassa , Cobaias , Animais , Febre Lassa/tratamento farmacológico , Febre Lassa/prevenção & controle , Antivirais/farmacologia , Antivirais/uso terapêutico , Vírus Lassa , Viremia/tratamento farmacológico , Vacinação
10.
J Virol ; 82(21): 10932-9, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18768973

RESUMO

The arenavirus envelope glycoprotein (GPC) mediates viral entry through pH-induced membrane fusion in the endosome. This crucial process in the viral life cycle can be specifically inhibited in the New World arenaviruses by the small-molecule compound ST-294. Here, we show that ST-294 interferes with GPC-mediated membrane fusion by targeting the interaction of the G2 fusion subunit with the stable signal peptide (SSP). We demonstrate that amino acid substitutions at lysine-33 of the Junín virus SSP confer resistance to ST-294 and engender de novo sensitivity to ST-161, a chemically distinct inhibitor of the Old World Lassa fever virus. These compounds, as well as a broadly active inhibitor, ST-193, likely share a molecular target at the SSP-G2 interface. We also show that both ST-294 and ST-193 inhibit pH-induced dissociation of the G1 receptor-binding subunit from GPC, a process concomitant with fusion activation. Interestingly, the inhibitory activity of these molecules can in some cases be overcome by further lowering the pH used for activation. Our results suggest that these small molecules act to stabilize the prefusion GPC complex against acidic pH. The pH-sensitive interaction between SSP and G2 in GPC represents a robust molecular target for the development of antiviral compounds for the treatment of arenavirus hemorrhagic fevers.


Assuntos
Arenavirus/efeitos dos fármacos , Arenavirus/fisiologia , Fusão de Membrana/efeitos dos fármacos , Fusão de Membrana/fisiologia , Sulfonamidas/farmacologia , Ureia/análogos & derivados , Proteínas do Envelope Viral/metabolismo , Internalização do Vírus/efeitos dos fármacos , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Antivirais/farmacologia , Farmacorresistência Viral , Concentração de Íons de Hidrogênio , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Alinhamento de Sequência , Ureia/farmacologia
11.
J Virol ; 82(21): 10768-75, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18715909

RESUMO

Several arenaviruses, including Lassa virus (LASV), are causative agents of hemorrhagic fever, for which effective therapeutic options are lacking. The LASV envelope glycoprotein (GP) gene was used to generate lentiviral pseudotypes to identify small-molecule inhibitors of viral entry. A benzimidazole derivative with potent antiviral activity was identified from a high-throughput screen utilizing this strategy. Subsequent lead optimization for antiviral activity identified a modified structure, ST-193, with a 50% inhibitory concentration (IC(50)) of 1.6 nM against LASV pseudotypes. ST-193 inhibited pseudotypes generated with other arenavirus envelopes as well, including the remaining four commonly associated with hemorrhagic fever (IC(50)s for Junín, Machupo, Guanarito, and Sabiá were in the 0.2 to 12 nM range) but exhibited no antiviral activity against pseudotypes incorporating either the GP from the LASV-related arenavirus lymphocytic choriomeningitis virus (LCMV) or the unrelated G protein from vesicular stomatitis virus, at concentrations of up to 10 microM. Determinants of ST-193 sensitivity were mapped through a combination of LASV-LCMV domain-swapping experiments, genetic selection of viral variants, and site-directed mutagenesis. Taken together, these studies demonstrate that sensitivity to ST-193 is dictated by a segment of about 30 amino acids within the GP2 subunit. This region includes the carboxy-terminal region of the ectodomain and the predicted transmembrane domain of the envelope protein, revealing a novel antiviral target within the arenavirus envelope GP.


Assuntos
Antivirais/isolamento & purificação , Antivirais/farmacologia , Arenavirus/efeitos dos fármacos , Benzimidazóis/isolamento & purificação , Benzimidazóis/farmacologia , Internalização do Vírus/efeitos dos fármacos , Sequência de Aminoácidos , Sítios de Ligação , Concentração Inibidora 50 , Estrutura Molecular , Mutagênese Sítio-Dirigida , Alinhamento de Sequência , Proteínas do Envelope Viral/antagonistas & inibidores , Proteínas do Envelope Viral/genética
12.
Antiviral Res ; 92(2): 187-94, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21820471

RESUMO

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) emerged as the causal agent of an endemic atypical pneumonia, infecting thousands of people worldwide. Although a number of promising potential vaccines and therapeutic agents for SARS-CoV have been described, no effective antiviral drug against SARS-CoV is currently available. The intricate, sequential nature of the viral entry process provides multiple valid targets for drug development. Here, we describe a rapid and safe cell-based high-throughput screening system, dual envelope pseudovirion (DEP) assay, for specifically screening inhibitors of viral entry. The assay system employs a novel dual envelope strategy, using lentiviral pseudovirions as targets whose entry is driven by the SARS-CoV Spike glycoprotein. A second, unrelated viral envelope is used as an internal control to reduce the number of false positives. As an example of the power of this assay a class of inhibitors is reported with the potential to inhibit SARS-CoV at two steps of the replication cycle, viral entry and particle assembly. This assay system can be easily adapted to screen entry inhibitors against other viruses with the careful selection of matching partner virus envelopes.


Assuntos
Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala/métodos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/normas , Ensaios de Triagem em Larga Escala/normas , Humanos , Testes de Sensibilidade Microbiana/métodos , Testes de Sensibilidade Microbiana/normas , Cultura de Vírus/métodos , Cultura de Vírus/normas
13.
Antiviral Res ; 90(1): 70-9, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21371508

RESUMO

Lassa virus (LASV), a member of the Arenaviridae family, causes a viral hemorrhagic fever endemic to West Africa, where as many as 300,000 infections occur per year. Presently, there are no FDA-approved LASV-specific vaccines or antiviral agents, although the antiviral drug ribavirin has shown some efficacy. A recently identified small-molecule inhibitor of arenavirus entry, ST-193, exhibits submicromolar antiviral activity in vitro. To determine the antiviral utility of ST-193 in vivo, we tested the efficacy of this compound in the LASV guinea pig model. Four groups of strain 13 guinea pigs were administered 25 or 80 mg/kg ST-193, 25 mg/kg of ribavirin, or the vehicle by the intraperitoneal (i.p.) route before infection with a lethal dose of LASV, strain Josiah, and continuing once daily for 14 days. Control animals exhibited severe disease, becoming moribund between days 10 and 15 postinfection. ST-193-treated animals exhibited fewer signs of disease and enhanced survival when compared to the ribavirin or vehicle groups. Body temperatures in all groups were elevated by day 9, but returned to normal by day 19 postinfection in the majority of ST-193-treated animals. ST-193 treatment mediated a 2-3-log reduction in viremia relative to vehicle-treated controls. The overall survival rate for the ST-193-treated guinea pigs was 62.5% (10/16) compared with 0% in the ribavirin (0/8) and vehicle (0/7) groups. These data suggest that ST-193 may serve as an improved candidate for the treatment of Lassa fever.


Assuntos
Antivirais/administração & dosagem , Febre Lassa/tratamento farmacológico , Animais , Temperatura Corporal , Modelos Animais de Doenças , Feminino , Cobaias , Injeções Intraperitoneais , Febre Lassa/mortalidade , Febre Lassa/patologia , Análise de Sobrevida , Viremia/prevenção & controle
14.
J Virol ; 80(1): 353-9, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16352560

RESUMO

The envelope of class I viruses can be a target for potent viral inhibitors, such as the human immunodeficiency virus type 1 (HIV-1) inhibitor enfuvirtide, which are derived from the C-terminal heptad repeat (HR2) of the transmembrane (TM) subunit. Resistance to an HR2-based peptide inhibitor of a model retrovirus, subgroup A of the Avian Sarcoma and Leukosis Virus genus (ASLV-A), was studied by examining mutants derived by viral passage in the presence of inhibitor. Variants with reduced sensitivity to inhibitor were readily selected in vitro. Sensitivity determinants were identified for 13 different isolates, all of which mapped to the TM subunit. These determinants were identified in two regions: (i) the N-terminal heptad repeat (HR1) and (ii) the N-terminal segment of TM, between the subunit cleavage site and the fusion peptide. The latter class of mutants identified a region outside of the predicted HR2-binding site that can significantly alter sensitivity to inhibitor. A subset of the HR1 mutants displayed the unanticipated ability to infect nonavian cells. This expanded tropism was associated with increased efficiency of envelope triggering by soluble receptor at low temperatures, as measured by protease sensitivity of the surface subunit (SU) of envelope. In addition, expanded tropism was linked for the most readily triggered mutants with increased sensitivity to neutralization by SU-specific antiserum. These observations depict a class of HR2 peptide-selected mutations with a reduced activation threshold, thereby allowing the utilization of alternative receptors for viral entry.


Assuntos
Vírus da Leucose Aviária/efeitos dos fármacos , Vírus do Sarcoma Aviário/efeitos dos fármacos , Peptídeos/farmacologia , Tropismo/efeitos dos fármacos , Proteínas do Envelope Viral/fisiologia , Vírus da Leucose Aviária/patogenicidade , Vírus do Sarcoma Aviário/patogenicidade , Linhagem Celular , Sequências Repetitivas de Aminoácidos , Tropismo/fisiologia , Proteínas do Envelope Viral/genética , Proteínas Virais de Fusão/química
15.
Proc Natl Acad Sci U S A ; 101(12): 4240-5, 2004 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-15010527

RESUMO

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is a rapidly emerging pathogen with potentially serious consequences for public health. Here we describe conditions that result not only in the efficient expression of the SARS-CoV spike (S) protein on the surface of cells, but in its incorporation into lentiviral particles that can be used to transduce cells in an S glycoprotein-dependent manner. We found that although some primate cell lines, including Vero E6, 293T and Huh-7 cells, could be efficiently transduced by SARS-CoV S glycoprotein pseudoviruses, other cells lines were either resistant or very poorly permissive to virus entry. Infection by pseudovirions could be inhibited by several lysosomotropic agents, suggesting a requirement for acidification of endosomes for efficient S-mediated viral entry. In addition, we were able to develop a cell-cell fusion assay that could be used to monitor S glycoprotein-dependent membrane fusion. Although proteolysis did not enhance the infectivity of cell-free pseudovirions, trypsin activation is required for cell-cell fusion. Additionally, there was no apparent pH requirement for S glycoprotein-mediated cell-cell fusion. Together, these studies describe important tools that can be used to study SARS-CoV S glycoprotein structure and function, including approaches that can be used to identify inhibitors of the entry of SARS-CoV into target cells.


Assuntos
Proteínas do Capsídeo/metabolismo , Glicoproteínas de Membrana/metabolismo , Síndrome Respiratória Aguda Grave/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/metabolismo , Proteínas do Envelope Viral/metabolismo , Proteínas do Capsídeo/genética , Fusão Celular , Humanos , Glicoproteínas de Membrana/genética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Glicoproteína da Espícula de Coronavírus , Proteínas do Envelope Viral/genética
16.
J Virol ; 78(24): 13430-9, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15564453

RESUMO

Fusion proteins of enveloped viruses categorized as class I are typified by two distinct heptad repeat domains within the transmembrane subunit. These repeats are important structural elements that assemble into the six-helix bundles characteristic of the fusion-activated envelope trimer. Peptides derived from these domains can be potent and specific inhibitors of membrane fusion and virus infection. To facilitate our understanding of retroviral entry, peptides corresponding to the two heptad repeat domains of the avian sarcoma and leukosis virus subgroup A (ASLV-A) TM subunit of the envelope protein were characterized. Two peptides corresponding to the C-terminal heptad repeat (HR2), offset from one another by three residues, were effective inhibitors of infection, while two overlapping peptides derived from the N-terminal heptad repeat (HR1) were not. Analysis of envelope mutants containing substitutions within the HR1 domain revealed that a single amino acid change, L62A, significantly reduced sensitivity to peptide inhibition. Virus bound to cells at 4 degrees C became sensitive to peptide within the first 5 min of elevating the temperature to 37 degrees C and lost sensitivity to peptide after 15 to 30 min, consistent with a transient intermediate in which the peptide binding site is exposed. In cell-cell fusion experiments, peptide inhibitor sensitivity occurred prior to a fusion-enhancing low-pH pulse. Soluble receptor for ASLV-A induces a lipophilic character in the envelope which can be measured by stable liposome binding, and this activation was found to be unaffected by inhibitory HR2 peptide. Finally, receptor-triggered conformational changes in the TM subunit were also found to be unaffected by inhibitory peptide. These changes are marked by a dramatic shift in mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, from a subunit of 37 kDa to a complex of about 80 kDa. Biotinylated HR2 peptide bound specifically to the 80-kDa complex, demonstrating a surprisingly stable envelope conformation in which the HR2 binding site is exposed. These experiments support a model in which receptor interaction promotes formation of an envelope conformation in which the TM subunit is stably associated with its target membrane and is able to bind a C-terminal peptide.


Assuntos
Vírus da Leucose Aviária/efeitos dos fármacos , Vírus do Sarcoma Aviário/efeitos dos fármacos , Fusão de Membrana/efeitos dos fármacos , Peptídeos/farmacologia , Proteínas Virais de Fusão/química , Sequência de Aminoácidos , Animais , Vírus da Leucose Aviária/patogenicidade , Vírus do Sarcoma Aviário/patogenicidade , Fusão Celular , Linhagem Celular , Dados de Sequência Molecular , Mutação , Peptídeos/síntese química , Peptídeos/química , Sequências Repetitivas de Aminoácidos
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