Membrane topology and function of dengue virus NS2A protein

Flavivirus non-structural protein 2A (NS2A) is a component of virus replication complex, functions in virion assembly, and antagonizes host immune response. Although flavivirus NS2A is known to associate with endoplasmic reticulum (ER) membrane, the detailed topology of this protein has not been determined. Here we report the first topology model of flavivirus NS2A on ER membrane. Using dengue virus (DENV) NS2A as a model, we show that (i) the N-terminal 68 amino acids are located in ER lumen with one segment (amino acids 30-52) that peripherally interacts with ER membrane without traversing the lipid bilayer; (ii) amino acids 69 to 209 form five trans-membrane segments (TMS), each of which integrally spans the ER membrane; (iii) the C-terminal tail (amino acids 210-218) is located in cytosol. NMR structural analysis showed that the first TMS (amino acids 69-93) consists of two helices separated by a helix “breaker”. The helix “breaker” structure is formed by Pro85 and two positively charged residues (Lys82 and Arg84). Functional analysis using replicon and genome-length RNAs of DENV-2 indicates that Pro85 is not important for viral replication. However, when Arg84 was substituted with Glu, the mutation attenuated both viral RNA synthesis and virus production. Remarkably, when R84 was changed to Ser or Ala, the mutations (R84S or R84A) did not affect viral RNA synthesis, but significantly reduced virus particle formation. Collectively, the mutagenesis results demonstrate that NS2A functions in both viral RNA synthesis and virion assembly/release. The topology model of DENV NS2A provides a good starting point to study how flavivirus NS2A modulates viral replication and evasion of host immune response.