RESUMO
Based on the structural similarity of viral fusion proteins within the family Paramyxoviridae, we tested recently described and newly synthesized acetanilide derivatives for their capacity to inhibit measles virus (MV)-, canine distemper virus (CDV)- and Nipah virus (NiV)-induced membrane fusion. We found that N-(3-cyanophenyl)-2-phenylacetamide (compound 1) has a high capacity to inhibit MV- and CDV-induced (IC(50) µM), but not NiV-induced, membrane fusion. This compound is of outstanding interest because it can be easily synthesized and its cytotoxicity is low [50â% cytotoxic concentration (CC(50)) ≥ 300 µM], leading to a CC(50)/IC(50) ratio of approximately 100. In addition, primary human peripheral blood lymphocytes and primary dog brain cell cultures (DBC) also tolerate high concentrations of compound 1. Infection of human PBMC with recombinant wild-type MV is inhibited by an IC(50) of approximately 20 µM. The cell-to-cell spread of recombinant wild-type CDV in persistently infected DBC can be nearly completely inhibited by compound 1 at 50 µM, indicating that the virus spread between brain cells is dependent on the activity of the viral fusion protein. Our findings demonstrate that this compound is a most applicable inhibitor of morbillivirus-induced membrane fusion in tissue culture experiments including highly sensitive primary cells.
Assuntos
Antivirais/farmacologia , Benzenoacetamidas/farmacologia , Vírus do Sarampo/efeitos dos fármacos , Vírus do Sarampo/fisiologia , Fusão de Membrana/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Animais , Antivirais/química , Antivirais/toxicidade , Benzenoacetamidas/química , Benzenoacetamidas/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Chlorocebus aethiops , Vírus da Cinomose Canina/efeitos dos fármacos , Vírus da Cinomose Canina/fisiologia , Cães , Humanos , Concentração Inibidora 50 , Linfócitos/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Vírus Nipah/efeitos dos fármacos , Vírus Nipah/fisiologiaRESUMO
Antibodies to CD9, a member of the tetraspan transmembrane-protein family, selectively inhibit Canine distemper virus (CDV)-induced cell-cell fusion. Neither CDV-induced virus-cell fusion nor cell-cell fusion induced by the closely related morbillivirus Measles virus (MV) is affected by anti-CD9 antibodies. As CDV does not bind CD9, an unknown, indirect mechanism is responsible for the observed inhibition of cell-cell fusion. It was investigated whether this effect was restricted to only one viral glycoprotein, either the haemagglutinin (H) or the fusion (F) protein, which form a fusion complex on the surface of virions and infected cells, or whether it is dependent on both in transient co-transfection assays. The susceptibility to CD9 antibodies segregates with the H protein of CDV. By exchanging portions of the H proteins of CDV and MV, it was determined that the complete extracellular domain, including the predicted stem structure (stem 1, barrel strand 1 and stem 2) and globular head domain, of the CDV-H protein mediates the effect. This suggests that interaction of the CDV-H protein with an unknown cellular receptor(s) is regulated by CD9, rather than F protein-mediated membrane fusion.
Assuntos
Antígenos CD/imunologia , Fusão Celular , Vírus da Cinomose Canina/patogenicidade , Glicoproteínas de Membrana/imunologia , Proteínas Virais/química , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Antígenos CD/metabolismo , Chlorocebus aethiops , Vírus da Cinomose Canina/genética , Cães , Células Gigantes/fisiologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Tetraspanina 29 , Transfecção , Células Vero , Proteínas Virais/genéticaRESUMO
Measles virus (MV) nucleocapsids are present abundantly in brain cells of patients with subacute sclerosing panencephalitis (SSPE). This invariably lethal brain disease develops years after acute measles as result of a persistent MV infection. Various rodent models for MV infection of the central nervous system (CNS) have been described in the past, in which the detection of viral antigens is based on histological staining procedures of paraffin embedded brains. Here, the usage of a recombinant MV (MV-EGFP-CAMH) expressing the haemagglutinin (H) of the rodent-adapted MV-strain CAM/RB and the enhanced green fluorescent protein (EGFP) is described. In newborn rodents the virus infects neurons and causes an acute lethal encephalitis. From 2 weeks on, when the immune system of the genetically unmodified animal is maturating, intracerebral (i.c.) infection is overcome subclinically, however, a focal persistent infection in groups of neurons remains. The complete brain can be analysed in 50 or 100 microm slices, and infected autofluorescent cells are readily detected. Seven and 28 days post-infection (p.i.) 86 and 81% of mice are infected, respectively, and virus persists for more than 50 days p.i. Intraperitoneal immunization with MV 1 week before infection, but not after infection, protects and prevents persistence. The high percentage of persistence demonstrates that this is a reliable and useful model of a persistent CNS infection in fully immunocompetent mice, which allows the investigation of determinants of the immune system.