RESUMO
The assembly of hepatitis C virus (HCV) is not well understood. We investigated HCV nucleocapsid assembly in vitro and the role of electrostatic/hydrophobic interactions in this process. We developed a simple and rapid in vitro assay in which the progress of assembly is monitored by measuring an increase in turbidity, thereby allowing the kinetics of assembly to be determined. Assembly is performed using a truncated HCV core (C1-82), containing the minimal assembly domain, purified from Escherichia coli. The increase in turbidity is linked to the formation of nucleocapsid-like particles (NLPs) in solution, and nucleic acids are essential to initiate nucleocapsid assembly under the experimental conditions used. The sensitivity of NLP formation to salt strongly suggests that electrostatic forces govern in vitro assembly. Mutational analysis of C1-82 demonstrated that it is the global positive charge of C1-82 rather than any specific basic residue that is important for the assembly process. Our in vitro assembly assay provides an easy and efficient means of screening for assembly inhibitors, and we have identified several inhibitory peptides that could represent a starting point for drug design.
Assuntos
Hepacivirus/fisiologia , Proteínas do Core Viral/fisiologia , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA/genética , DNA Viral/genética , Avaliação Pré-Clínica de Medicamentos/métodos , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , Microscopia Eletrônica , Dados de Sequência Molecular , Mutação , Nucleocapsídeo/efeitos dos fármacos , Nucleocapsídeo/fisiologia , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Sais/farmacologia , Eletricidade Estática , Proteínas do Core Viral/antagonistas & inibidores , Proteínas do Core Viral/genética , Montagem de Vírus/efeitos dos fármacosRESUMO
The core (C) protein of hepatitis C virus (HCV) appears to be a multifunctional protein that is involved in many viral and cellular processes. Although its effects on host cells have been extensively discussed in the literature, little is known about its main function, the assembly and packaging of the viral genome. We have studied the in vitro assembly of several deleted versions of recombinant HCV C protein expressed in E. coli. We demonstrated that the 75 N-terminal residues of the C protein were sufficient to assemble and generate nucleocapsid-like particles (NLPs) in vitro. However, homogeneous particles of regular size and shape were observed only when NLPs were produced from at least the first 79 N-terminal amino acids of the C protein. This small protein unit fused to the endoplasmic reticulum-anchoring domain also generated NLPs in yeast cells. These data suggest that the N-terminal half of the C protein is important for formation of NLPs. Similarities between the HCV C protein and C proteins of other members of the Flaviviridae are discussed.
Assuntos
Hepacivirus/fisiologia , Nucleocapsídeo/fisiologia , Proteínas do Core Viral/fisiologia , Sequência de Aminoácidos , Sequência de Bases , DNA Viral/genética , Escherichia coli/genética , Hepacivirus/genética , Técnicas In Vitro , Microscopia Eletrônica , Modelos Biológicos , Dados de Sequência Molecular , Nucleocapsídeo/química , Nucleocapsídeo/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/fisiologia , Pichia/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas do Core Viral/química , Proteínas do Core Viral/genética , Montagem de VírusRESUMO
Recently, proinflammatory activities had been described for S100A8 and S100A9, two proteins found at inflammatory sites and within the neutrophil cytoplasm. In this study, we investigated the role of these proteins in neutrophil migration in vivo in response to LPS. LPS was injected into the murine air pouch, which led to the release of S100A8, S100A9, and S100A8/A9 in the pouch exudates that preceded accumulation of neutrophils. Passive immunization against S100A8 and S100A9 led to a 52% inhibition of neutrophil migration in response to LPS at 3 h postinjection. Injection of LPS was also associated with an increase in peripheral blood neutrophils and the presence in serum of S100A9 and S100A8/A9. Intravenous injection of S100A8, S100A9, or S100A8/A9 augmented the number of circulating neutrophils and diminished the number of neutrophils in the bone marrow, demonstrating that S100A8 and S100A9 induced the mobilization of neutrophils from the bone marrow to the blood. Finally, passive immunization with anti-S100A9 inhibited the neutrophilia associated with LPS injection in the air pouch. These results suggest that S100A8 and S100A9 play a role in the inflammatory response to LPS by inducing the release of neutrophils from the bone marrow and directing their migration to the inflammatory site.
Assuntos
Calgranulina A/antagonistas & inibidores , Calgranulina A/fisiologia , Calgranulina B/fisiologia , Inibição de Migração Celular , Lipopolissacarídeos/administração & dosagem , Neutrófilos/citologia , Neutrófilos/imunologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Calgranulina A/imunologia , Calgranulina A/metabolismo , Calgranulina B/imunologia , Calgranulina B/metabolismo , Agregação Celular/imunologia , Dimerização , Modelos Animais de Doenças , Espaço Extracelular/metabolismo , Imunoglobulina G/administração & dosagem , Injeções Intravenosas , Injeções Subcutâneas , Leucocitose/imunologia , Leucocitose/metabolismo , Leucocitose/patologia , Leucocitose/prevenção & controle , Camundongos , Neutrófilos/patologia , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologiaRESUMO
We investigated the proinflammatory activities of S100A12 in the context of synovial inflammation. S100A12 levels were increased in the synovial fluids and plasma of patients with gout, rheumatoid arthritis, psoriatic arthritis, and undetectable in osteoarthritis, a noninflammatory disorder. S100A12 proved to induce neutrophil adhesion to fibrinogen via Mac-1 at concentrations similar to those found in the synovial fluids. Similar concentrations induced the recruitment of large numbers of neutrophils and monocytes in the murine air pouch model. To characterize the effect of increased S100A12 plasma levels, mice were injected intravenously with S100A12. This led to the mobilization of neutrophils from the bone marrow to the peripheral blood. These results suggest that S100A12 stimulates the accumulation of neutrophil by inducing their release from the bone marrow, as well as by activating their adhesion and migration toward inflammatory sites.
