RESUMO
Flaviviridae non-structural 3 protein (NS3) is a multifunctional enzyme, composed by a protease domain (NS3pro) and an RNA helicase domain (NS3hel). The activities present in NS3 have proved to be critical for viral replication. The replicative cycle of Flaviviridae requires coordinated regulation of all the activities present in the full-length NS3 protein, however, the exact nature of these interactions remains unclear. The present work aimed to determine common structural features between NS3 of dengue and hepatitis C viruses and to characterize residues involved in the regulation of the interdomain motions between NS3pro and NS3hel. Analysis of the root mean square (RMS) variation shows that NS3pro increases the stability of subdomain 1 of the RNA helicase. Moreover, the dynamic behaviour of the carboxy terminus of NS3hel, supports the hypothesis that, upon release of the carboxy-terminus from NS3pro, the residues involved in this interaction are folded back into the last alpha-helix. Using normal mode analysis, we characterized slow collective motions of NS3, and observed that the two lowest-frequency normal modes are enough to describe reorientations of NS3pro relative to NS3hel. These movements induced an increment in the exposure of the active site of NS3pro that can be important during the proteolytic processing of the viral polyprotein. The third low-frequency normal mode was correlated to subdomain reorientations of NS3hel, similar to those proposed during NTP hydrolysis and dsRNA unwinding. Based on these data, we support a dynamic model, in which the domain movements between NS3pro and NS3hel result in the regulation of its activities.
Assuntos
Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Sítios de Ligação , Gráficos por Computador , Simulação por Computador , Vírus da Dengue/química , Vírus da Dengue/metabolismo , Hepacivirus/química , Hepacivirus/metabolismo , Modelos Moleculares , Estrutura Terciária de Proteína , RNA Helicases/química , RNA Helicases/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , TermodinâmicaRESUMO
Splenic plasma cell response and systemic antibody response to intestinal amoebiasis were studied in C3H/HeJ mice from 5 to 60 days post-inoculation with Entamoeba histolytica. At various time intervals specific antibody-secreting cells (ASC) in the spleen were measured in infected mice and non-infected control mice by enzyme-linked immunospot (ELISPOT) assay. Serum antibodies were measured by enzyme-linked immunosorbent assay (ELISA). The infected animals showed high IgA ASC from 30 to 50 days post-inoculation as compared to IgM and IgG ASC. However, class-specific serum antibody showed high IgG titre from 30 to 60 days post-inoculation as compared to IgM and IgA serum titres. Our results suggest that E. histolytica trophozoites can induce a plasma cell response in the spleen that is different from anti-amoebic antibody response in serum.