Dengue Virus Encapsidation and Its Interplay with the Host Cell

IGLESIAS, N.G.; BYK, L; MONDOTTE, J.A.; SAMSA, M.M.; GAMARNIK, A.V.

Cartagena

Congreso; THIRD PANAMERICAN DENGUE RESEARCH NETWORK MEETING; 2012

PANAMERICAN DENGUE RESEARCH NETWORK

Dengue and other flavivirus capsid proteins bind the viral genome to form a nucleocapsid during particle formation. Although this process is essential for viral propagation, the mechanism by which the capsid protein recruits the viral genome is largely unknown. Capsid is a highly basic protein that forms homodimers in solution and binds nucleic acids with high affinity and low specificity. In dengue virus (DENV) infected cells, the capsid protein distributes between the nucleus and the cytoplasm. In the cytoplasm, the protein associates to ER membranes and accumulates around lipid droplets (LDs). The function of this specific subcellular location of capsid during viral replication is still unclear. We are interested in understanding the multiple functions of the viral capsid protein. During DENV infection, capsid accumulates progressively around LDs. We have previously defined the requirement of hydrophobic residues (L50 and L54) as determinants for LD accumulation. Mutation of these amino acids results in the lack of capsid association to these organelles and abrogation of viral particle formation. The mechanism by which cellular proteins are transported to LDs is still unclear. Using dominant negative constructs, siRNAs, and different inhibitors, we found that a subset of cellular components of the COPI system, involved in the retrograde transport of cargo from Golgi to ER, is necessary for capsid protein accumulation on LDs during DENV infection. In order to investigate determinants in the capsid protein involved in infectious particle formation, a DENV infectious clone, in which RNA replication signals were dissociated from the capsid coding region, was used. A systematic mutational analysis indicated that a high density of basic amino acids is necessary at the N-terminus of capsid for infectious particle production. In addition, substitution of basic residues present at the center of the alpha helix-4, decreased about 1000 fold viral particle formation. The results support a model in which basic residues at both ends of the protein, the N-terminus and the alpha 4 helix, are necessary for DENV capsid function.