|1398-1||Interaction Studies of a Recombinant Dodecahedron Containing The Short Fiber Protein of Human Adenovirus 41 with HEK- 293 and CaCo2 cells|
|Autores:||Joselma Siqueira Silva (ICB-USP - Instituto de Ciências Biomédicas II da USP) ; Juliana Cristina Marinheiro (ICB-USP - Instituto de Ciências Biomédicas II da USP) ; Rafaella Almeida Lima Nunes (ICB-USP - Instituto de Ciências Biomédicas II da USP) ; Cibele Felisberto de Amorim (UNINOVE - Universidade Nove de Julho) ; Sarah Ribeiro Rocha (UNINOVE - Universidade Nove de Julho) ; Daphna Fenel (IBS - Institut de Biologie Structurale Jean Pièrre Ebel) ; Evelyne Gout (IBS - Institut de Biologie Structurale Jean Pièrre Ebel) ; Guy Schoehn (IBS - Institut de Biologie Structurale Jean Pièrre Ebel / UVHCI - Unit of Virus Host Cell Interactions (UVHCI), UMI 3265) ; Pascal Fender (UVHCI - Unit of Virus Host Cell Interactions (UVHCI), UMI 3265) ; Charlotte Marianna Hársi (ICB-USP - Instituto de Ciências Biomédicas II da USP) |
Human adenovirus F have attracted considerable attention in recent years as vectors for therapy or vaccination. The molecular basis of their specific pathogenesis is not known, but may be related to the capsid structures. The virions contain two different fibers: the long (LF) and the short (SF). The LF recognizes CAR but the role of SF in the infection is unknown. In order to acquire an overview of the entry of a particle containing the SF of HAdV-41 in human cells, a dodecahedron containing the SF of HAdV-41 and the penton base of HAdV-3 (DBAd3+SFAd41) and another dodecaedron with the penton base of HAdV-3 (DBAd3) were expressed using a recombinant baculovirus expression system. Cell entry kinetics assays were perfomed in CaCo2 and HEK-293. The cells were incubated with 2 g of each dodecaedron and harvested at 1, 5, 10, 20 and 30 min. Viral proteins were revealed with sera against SF and/or base Ad3 and cultures analysed with confocal microscopy. DBAd3+SFAd41 entry was more efficient than DBAd3 in both cells. In HEK-293, DBAd3+SFAd41 was observed at the membrane and already at the cytoplasm from the first min post incubation (p.i.) with intense cytoplasmic fluorescence at 5 min. In CaCo2, DBAd3+SFAd41 remain in the membrane at 1 min p.i. and the cytoplasmic fluorescence was observed only after 5 min. At 10 minutes p.i., both cells presented intense cytoplasmatic fluorescence. DBAd3 entry in cells were much slower and more discrete: In HEK-293, at 1 min p.i., fluorescence was observed only in cell membrane and at 5 min p.i. few particles at the cytoplasm. In CaCo2, very discrete fluorescence was observed at membrane and cytoplasm only from 10 min p.i. At 20 and 30 min p.i., both cells presented fluorescence but always less intense than with DBAd3+SFAd41. The SF may have a role in dodecahedron entry or in intracelular traffic. The results obtained may be reference for the development of gene-therapy vectors directed to intestinal epithelium.
(Support FAPESP – Fundação de Amparo à Pesquisa do Estado de São Paulo – Brazil; CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico)
Palavras-chave: Entry kinetics, HAdV-41, Recombinant dodecahedron, Short Fiber (SF)