RESUMO
The respiratory syncytial virus (RSV) fusion (F) protein is synthesized as an inactive precursor (F0), which subsequently undergoes post-translational cleavage to give the disulphide bond-linked F1 and F2 subunits. The methodology detailing the use of two-dimensional electrophoresis, endoglycosidases, and alpha-mannosidase inhibitors, as applied to investigating F protein glycan maturation, is given. Examples are used to show how this methodology was used to provide evidence for glycan heterogeneity within the mature F protein.
Assuntos
Polissacarídeos/análise , Processamento de Proteína Pós-Traducional , Subunidades Proteicas/análise , Vírus Sinciciais Respiratórios/química , Proteínas Virais de Fusão/análise , Animais , Chlorocebus aethiops , Eletroforese em Gel Bidimensional , Glicosilação , Processamento de Proteína Pós-Traducional/fisiologia , Subunidades Proteicas/metabolismo , Vírus Sinciciais Respiratórios/metabolismo , Células VeroRESUMO
Glycan heterogeneity of the respiratory syncytial virus (RSV) fusion (F) protein was demonstrated by proteomics. The effect of maturation of the virus glycoproteins-associated glycans on virus infectivity was therefore examined using the alpha-mannosidase inhibitors deoxymannojirimycin (DMJ) and swainsonine (SW). In the presence of SW the N-linked glycans on the F protein appeared in a partially mature form, whereas in the presence of DMJ no maturation of the glycans was observed. Neither inhibitor had a significant effect on G protein processing or on the formation of progeny virus. Although the level of infectious virus and syncytia formation was not significantly affected by SW-treatment, DMJ-treatment correlated with a one hundred-fold reduction in virus infectivity. Our data suggest that glycan maturation of the RSV glycoproteins, in particular those on the F protein, is an important step in virus maturation and is required for virus infectivity.
Assuntos
Inibidores Enzimáticos/farmacologia , Glicoproteínas/metabolismo , Polissacarídeos/metabolismo , Vírus Sincicial Respiratório Humano/fisiologia , Proteínas Virais/metabolismo , alfa-Manosidase/antagonistas & inibidores , Fusão Celular , Linhagem Celular Tumoral , Eletroforese em Gel Bidimensional , Glicosídeo Hidrolases , Humanos , Microscopia Eletrônica de Varredura , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Vírus Sincicial Respiratório Humano/patogenicidade , Proteínas Virais/genética , Proteínas Virais/isolamento & purificaçãoRESUMO
In this report, the interaction between respiratory syncytial virus (RSV) and heat shock protein 70 (HSP70) was examined. Although no significant increase in total HSP70 protein levels was observed during virus infection, analysis of the HSP70 content in lipid-raft membranes from mock- and virus-infected cells revealed an increase in the levels of raft-associated HSP70 during virus infection. Fluorescence microscopy demonstrated that this transport of HSP70 into lipid-raft membranes correlated with the appearance of HSP70 within virus-induced inclusion bodies. Furthermore, co-localisation of HSP70 with the virus N protein and the raft lipid GM1 was observed within these structures. Immunoprecipitation experiments demonstrated the ability of HSP70 to interact with the virus polymerase complex in lipid-rafts in an ATP-dependent manner. Collectively, these data suggest that RSV may induce cellular changes which allow the recruitment of specific host-cell factors, via lipid-raft membranes, to the polymerase complex.
Assuntos
Proteínas de Choque Térmico HSP70/fisiologia , Microdomínios da Membrana/fisiologia , Microdomínios da Membrana/virologia , Vírus Sinciciais Respiratórios/fisiologia , Vírus Sinciciais Respiratórios/patogenicidade , Proteínas Virais/fisiologia , Sequência de Aminoácidos , Linhagem Celular , Proteínas de Choque Térmico HSP70/genética , Humanos , Corpos de Inclusão/fisiologia , Corpos de Inclusão/virologia , Microscopia de Fluorescência , Dados de Sequência Molecular , Complexos Multiproteicos , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/isolamento & purificação , Vírus Sinciciais Respiratórios/genética , Proteínas Virais/genética , Replicação ViralRESUMO
The interaction between the respiratory syncytial virus (RSV) polymerase complex and lipid rafts was examined in HEp2 cells. Lipid-raft membranes were prepared from virus-infected cells and their protein content was analysed by Western blotting and mass spectrometry. This analysis revealed the presence of the N, P, L, M2-1 and M proteins. However, these proteins appeared to differ from one another in their association with these structures, with the M2-1 protein showing a greater partitioning into raft membranes compared to that of the N, P or M proteins. Determination of the polymerase activity profile of the gradient fractions revealed that 95% of the detectable viral enzyme activity was associated with lipid-raft membranes. Furthermore, analysis of virus-infected cells by confocal microscopy suggested an association between these proteins and the raft-lipid, GM1. Together, these results provide evidence that the RSV polymerase complex is able to associate with lipid rafts in virus-infected cells.
