How Do Flaviviruses Establish Replication Organelles and Assemble Viral Particles?

Zika virus (ZIKV) is an emerging mosquito-borne pathogen of tremendous public health concern with 2.2 billion people at risk of infection every year. Although historically a benign infectious agent, recent introduction of ZIKV into the Western hemisphere resulted in an explosive epidemic in South America associated with novel pathogenicity, including Guillain-Barré syndrome and fetal microcephaly (Rajah et al. ACS Infect Dis 2016; Barnard et al. Fut Virol 2017).

ZIKV is a positive-sense RNA virus with a single open reading frame flanked by highly-structured 5’ and 3’ untranslated regions (UTRs). The genome encodes a polyprotein that is cleaved by host and viral proteases into the structural [capsid (C), pre-membrane (prM), and envelope (E)], and nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The structural proteins form the virion particle with E facilitating receptor binding and cell entry, and prM mediating virion maturation during viral egress. NS1 is a glycoprotein involved in replication and host immune evasion. NS2A, NS2B and NS4A are small hydrophobic proteins implicated in viral replication organelle biogenesis, with NS2B acting as the NS3 cofactor. NS3 has protease, nucleotide triphosphatase, and helicase activities that mediate viral polyprotein processing and aid in viral replication. NS4B has unknown function, but is implicated in alteration of cellular membranes, viral replication, pathogenesis, and evasion of antiviral signaling. Finally, NS5 has both RNA-dependent RNA polymerase (RdRp) and methyltransferase (MT) activities required for viral RNA replication and is also implicated in evasion of antiviral signaling.

Interestingly, while all the NS proteins are required for replication, the NS2A and NS3 proteins are unique in that they are also central players in virion assembly. Notably, while we know that these proteins are present at both sites, little is known about how they interact with other viral proteins, their stoichiometry, and the precise role(s) they play in replication organelle biogenesis and virion assembly. We hypothesize that NS2A and NS3 are the central scaffold that orchestrate replication organelle biogenesis and virion assembly. To investigate this, we are using molecular docking and computational modeling to explore the protein-protein and protein-RNA interactions important for replication complex formation. We also use co-immunoprecipitation assays as well as cis- and trans-complementation systems to provide insight into the mechanisms of replication organelle biogenesis and virion assembly in the ZIKV life cycle.