Proteomics in the Identification of Cellular Proteins Expressed in Response to Dengue Virus (serotype 2) Infection of Vero Cells.
Chung, J.1; de Meneses, M.D.F.2; Junqueira, M.R.1; Santos. M.F.1; Ferreira, D.F.2; Rebello, M.A.2; Domont, G.B.1
1Departamento de Bioquímica, Instituto de Química-UFRJ; 2Departamento de Virologia, Instituto de Microbiologia-UFRJ
Dengue Virus is a mosquito borne disease transmitted by Äedes aegypti and A. albopictus. It is member of the Flaviviridae family, flavivirus genus, with four serotype DEN-1 to DEN-4. The ~ 11 kb genome of DEN-2 consists of a single-stranded RNA molecule of positive polarity, enveloped, encoding the polyprotein 5'-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3', which co- and post- translationally gives rise to three structural and seven nonstructural protein. The mechanism of virus entry in the host cell and the cleavages of the polyprotein to generate individual protein is mediated by the host cell which changes its gene expression as a consequence of virus infection. In this work, we used proteomics techniques to investigate the differential protein profile of Vero cells infected by DEN-2. Cells infected with DEN-2 at multiplicity of infection (MOI=1) were recovered 72h post-infection and lysed in a solution of 9.6 M urea, 4% CHAPS and 1% Pharmalyte. Samples were analyzed by 2-DE using IPG strips, pH range 4-7 (GE Health Care) for IEF and standard vertical SDS-PAGE for the second dimension. 2D Vero cell maps of infected and non-infected (control) cells were visualized by Coomassie G-250 staining. Sixteen differential spots and over-expressed cellular proteins were identify by PMF ans MS/MS using a 4700 MALDI-TOF-TO (ABI). MASCOT platform and NCBInr database allowed, to the present, identification of cytoskeleton (e.g. actins, tubulin, vimentin) and heat-shock proteins (HSP-70 isoforms). These results indicate actin reorganization in the infected cells and support the evidence that HSP-70 participates as a receptor in dengue virus entry. Suporte: Capes, CNPq, FAPERJ.
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