Role of Two Clusters of Basic Residues at the Alpha-4 Helix of Dengue Virus Capsid Protein

BYK LA; SAMSA MM; GAMARNIK AV

Boston

Congreso; Keystone Symposia - Plrus Strand RNA Viruses; 2013

Dengue virus (DENV) is the most significant mosquito-borne human viral pathogen worldwide. It is an enveloped virus with a single stranded positive sense RNA genome. The single ORF encodes a polyprotein that is processed into three structural and seven nonstructural proteins. After translation, the viral genome is first used as template for RNA amplification, and then as substrate for RNA encapsidation. During new particle formation, the capsid protein (C) recruits the viral genome to form the nucleocapsid, which buds into the ER gaining the envelope and prM proteins. The mechanism of DENV encapsidation is still unclear and the functions of C are little understood. C is a highly basic protein of 12 kDa that forms homodimers in solution. The monomer contains four alpha helices: alpha-1 to alpha-4, while the first 20 amino acids are unstructured in solution. We studied the subcellular localization of C during dengue virus infection and found that the protein distributes between the nucleus and the cytoplasm. In the cytoplasm, C associates to ER membranes and accumulates around lipid droplets (LDs). We have previously shown that residues within alpha-2 are necessary for LD association, and that the unstructured basic region at the N-terminus is crucial for viral particle formation. Here, we studied the requirement of basic residues in alpha-4 using recombinant dengue viruses and reporter systems. A mutagenesis analysis indicated that substitution of 3 residues in each of two clusters of basic amino acids found at the N-terminus and the center of alpha-4 impaired viral particle formation. We identified K73 as a critical determinant for viral particle formation, while viruses with substitutions of K74 and K76 replicated similar to the parental virus. Interestingly, substitution of K73 abolished nuclear localization of C during infection. These results indicate a function of specific amino acids of alpha 4 both in particle formation and subcellular localization of capsid in DENV infected cells.