Requirements of Dengue Virus Capsid in Encapsidation and Uncoating

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

Buenos Aires

Congreso; Ubiquitin & UBLs. At the crossroads from chromatin to protein; 2014

EMBO

Dengue virus (DENV) is the most significant mosquito-borne human viral pathogen worldwide, infecting more than 400 million people each year. It is an enveloped virus with a single stranded positive sense RNA genome of approximately 11kb. The single ORF encodes a polyprotein that is processed into three structural proteins, the highly basic Capsid, prM, and E, and seven nonstructural (NS) proteins. DENV enters host cells by receptor mediated endocytosis, which involves binding of E to cellular receptors. Upon internalization, fusion of viral and vesicular membranes allows the release of the nucleocapsid to the cytoplasm. Genome uncoating, which involves dissociation of the capsid protein from the RNA, takes place by an unknown process. The viral genome is recognized as mRNA and is translated by the host?s ribosomes. Once the NS proteins accumulate, the viral genome is used as a template for its amplification. The newly synthesized genome can be used for translation of more viral proteins or associate with capsid to generate new viral particles. The mechanism by which the genome is recruited by capsid during particle morphogenesis is still unknown. DENV 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 and the first 20 amino acids are unstructured in solution. We examined determinants in the dengue virus capsid protein necessary for encapsidation and uncoating. A systematic mutational analysis using a reporter virus system indicated that a high density of basic residues at the N-terminus and at the center of the alpha-4 helix, rather than specific amino acids in defined positions, were required for infectious particle production. In contrast, specific amino acids were found to reduce slightly the amount of infectious particles but completely abrogated viral infectivity. These viruses were able to enter the susceptible cell, but translation and replication were impaired, indicating defects in genome uncoating. To continue characterizing the process, we tested whether proteasome activity was required for DENV uncoating. We found that DENV C protein degradation upon entry is proteasome dependent. We are currently studying this observation in DENV uncoating.