RESUMO
Canine distemper virus (CDV), a close relative of the human pathogen measles virus (MeV), is an enveloped, negative sense RNA virus that belongs to the genus Morbillivirus and causes severe diseases in dogs and other carnivores. Although the vaccination is available as a preventive measure against the disease, the occasional vaccination failure highlights the importance of therapeutic alternatives such as antivirals against CDV. The morbilliviral cell entry system relies on two interacting envelope glycoproteins: the attachment (H) and fusion (F) proteins. Here, to potentially discover novel entry inhibitors targeting CDV H, F and/or the cognate receptor: signaling lymphocyte activation molecule (SLAM) proteins, we designed a quantitative cell-based fusion assay that matched high-throughput screening (HTS) settings. By screening two libraries of small molecule compounds, we successfully identified two membrane fusion inhibitors (F2736-3056 and F2261-0043). Although both inhibitors exhibited similarities in structure and potency with the small molecule compound 3G (an AS-48 class morbilliviral F-protein inhibitor), F2736-3056 displayed improved efficacy in blocking fusion activity when a 3G-escape variant was employed. Altogether, we present a cell-based fusion assay that can be utilized not only to discover antiviral agents against CDV but also to dissect the mechanism of morbilliviral-mediated cell-binding and cell-to-cell fusion activity.
Assuntos
Antivirais/farmacologia , Vírus da Cinomose Canina/efeitos dos fármacos , Vírus da Cinomose Canina/fisiologia , Cinomose/virologia , Avaliação Pré-Clínica de Medicamentos , Internalização do Vírus , Animais , Antivirais/química , Sítios de Ligação , Células Cultivadas , Chlorocebus aethiops , Cinomose/tratamento farmacológico , Cinomose/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Interações Hospedeiro-Patógeno , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Receptores Virais/metabolismo , Bibliotecas de Moléculas Pequenas , Células Vero , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismoRESUMO
Morbilliviruses (e.g. measles virus [MeV] or canine distemper virus [CDV]) employ the attachment (H) and fusion (F) envelope glycoproteins for cell entry. H protein engagement to a cognate receptor eventually leads to F-triggering. Upon activation, F proteins transit from a prefusion to a postfusion conformation; a refolding process that is associated with membrane merging. Small-molecule morbilliviral fusion inhibitors such as the compound 3G (a chemical analog in the AS-48 class) were previously generated and mechanistic studies revealed a stabilizing effect on morbilliviral prefusion F trimers. Here, we aimed at designing 3G-resistant CDV F mutants by introducing single cysteine residues at hydrophobic core positions of the helical stalk region. Covalently-linked F dimers were generated, which highlighted substantial conformational flexibility within the stalk to achieve those irregular F conformations. Our findings demonstrate that "top-stalk" CDV F cysteine mutants (F-V571C and F-L575C) remained functional and gained resistance to 3G. Conversely, although not all "bottom-stalk" F cysteine variants preserved proper bioactivity, those that remained functional exhibited 3G-sensitivity. According to the recently determined prefusion MeV F trimer/AS-48 co-crystal structure, CDV residues F-V571 and F-L575 may directly interact with 3G. A combination of conformation-specific anti-F antibodies and low-resolution electron microscopy structural analyses confirmed that 3G lost its stabilizing effect on "top-stalk" F cysteine mutants thus suggesting a primary resistance mechanism. Overall, our data suggest that the fusion inhibitor 3G stabilizes prefusion CDV F trimers by docking at the top of the stalk domain.
Assuntos
Antivirais/farmacologia , Vírus da Cinomose Canina/efeitos dos fármacos , Vírus da Cinomose Canina/fisiologia , Farmacorresistência Viral , Proteínas Virais de Fusão/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Linhagem Celular , Chlorocebus aethiops , Cinomose , Modelos Moleculares , Mutação , Conformação Proteica , Células Vero , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismoRESUMO
We evaluated the antiviral activity of a chlorine dioxide gas solution (CD) and sodium hypochlorite (SH) against feline calicivirus, human influenza virus, measles virus, canine distemper virus, human herpesvirus, human adenovirus, canine adenovirus and canine parvovirus. CD at concentrations ranging from 1 to 100 ppm produced potent antiviral activity, inactivating >or= 99.9% of the viruses with a 15 sec treatment for sensitization. The antiviral activity of CD was approximately 10 times higher than that of SH.