Search

Dengue virus (DV) is a flavivirus and infects mammalian cells through mosquito vectors. This study investigates the roles of domain III of DV type 2 envelope protein (EIII) in DV binding to the host cell. Recombinant EIII interferes with DV infection to BHK21 and C6/36 cells by blocking dengue virion adsorption to these cells. Inhibition of EIII on BHK21 cells was broad with no serotype specificity; however, inhibition of EIII on C6/36 cells was relatively serotype specific. Soluble heparin completely blocks binding of EIII to BHK21 cells, suggesting that domain III binds mainly to cell surface heparan sulfates. This suggestion is supported by the observation that EIII binds very weakly to gro2C and sog9 mutant mammalian cell lines that lack heparan sulfate. In contrast, heparin does not block binding of EIII to mosquito cells. Furthermore, a synthetic peptide that includes amino acids (aa) 380 to 389 of EIII, IGVEPGQLKL, inhibits binding of EIII to C6/36 but not BHK21 cells. This peptide corresponds to a lateral loop region on domain III of E protein, indicating a possible role of this loop in binding to mosquito cells. In summary, these results suggest that EIII plays an important role in binding of DV type 2 to host cells. In addition, EIII interacts with heparan sulfates when binding to BHK21 cells, and a loop region containing aa 380 to 389 of EIII may participate in DV type 2 binding to C6/36 cells. [ABSTRACT FROM AUTHOR]

Vaccinia virus does not grow in Chinese hamster ovary (CHO-K1) cells in the absence of a viral host range factor, cowpox protein CP77. In this study, CP77 was fused to the C terminus of green fluorescence protein (GFP-CP77) and a series of nested deletion mutants of GFP-CP77 was constructed for insertion into a vaccinia virus host range mutant, VV-hr, and expressed from a viral early promoter. Deletion mapping analyses demonstrated that the N-terminal 352 amino acids of CP77 were sufficient to support vaccinia virus growth in CHO-K1 cells, whereas the C-terminal residues 353 to 668 were dispensable. In yeast two-hybrid analyses, CP77 bound to a cellular protein, HMG20A, and GST pulldown analyses showed that residues 1 to 234 of CP77 were sufficient for this interaction. After VV-hr virus infection of CHO-K1 cells, HMG20A was translocated from the nucleus to viral factories and bound to the viral genome via the HMG box region. In control VV-hr-infected CHO-K1 cells, binding of HMG20A to the viral genome persisted from 2 to 8 h postinfection (h p.i.); in contrast, when CP77 was expressed, the association of HMG20A with viral genome was transient, with little HMG20A remaining bound at 8 h p.i. This indicates that dissociation of HMG20A from viral factories correlates well with CP77 host range activity in CHO-K1 cells. Finally, in cells expressing a CP77 deletion protein (amino acids 277 to 668) or a δANK5 mutant that did not support vaccinia virus growth and did not contain the HMG20A binding site, HMG20A remained bound to viral DNA, demonstrating that the binding of CP77 to HMG20A is essential for its host range function. In summary, our data revealed that a novel cellular protein, HMG20A, the dissociation of which from viral DNA is regulated by CP77, providing the first cellular target regulated by viral host range CP77 protein. [ABSTRACT FROM AUTHOR]