Determinants of Dengue Virus NS4A Protein Oligomerization

Flavivirus NS4A protein induces host membrane rearrangement and functions as a replication complex component. The molecular details of how flavivirus NS4A exerts these functions remain elusive. Here, we used dengue virus (DENV) as a model to characterize and demonstrate the biological relevance of flavivirus NS4A oligomerization. DENV type 2 (DENV-2) NS4A protein forms oligomers in infected cells or when expressed alone. Deletion mutagenesis mapped amino acids 50 to 76 (spanning the first transmembrane domain [TMD1]) of NS4A as the major determinant for oligomerization, while the N-terminal 50 residues contribute only slightly to the oligomerization. Nuclear magnetic resonance (NMR) analysis of NS4A amino acids 17 to 80 suggests that residues L31, L52, E53, G66, and G67 could participate in oligomerization. Ala substitution for 15 flavivirus conserved NS4A residues revealed that these amino acids are important for viral replication. Among the 15 mutated NS4A residues, 2 amino acids (E50A and G67A) are located within TMD1. Both E50A and G67A attenuated viral replication, decreased NS4A oligomerization, and reduced NS4A protein stability. In contrast, NS4A oligomerization was not affected by the replication-defective mutations (R12A, P49A, and K80A) located outside TMD1. trans complementation experiments showed that expression of wild-type NS4A alone was not sufficient to rescue the replication-lethal NS4A mutants. However, the presence of DENV-2 replicons could partially restore the replication defect of some lethal NS4A mutants (L26A and K80A), but not others (L60A and E122A), suggesting an unidentified mechanism governing the outcome of complementation in a mutant-dependent manner. Collectively, the results have demonstrated the importance of TMD1-mediated NS4A oligomerization in flavivirus replication.

Importance: We report that DENV NS4A forms oligomers. Such NS4A oligomerization is mediated mainly through amino acids 50 to 76 (spanning the first transmembrane domain [TMD1]). The biological importance of NS4A oligomerization is demonstrated by results showing that mutations of flavivirus conserved residues (E50A and G67A located within TMD1) reduced the oligomerization and stability of the NS4A protein, leading to attenuated viral replication. A systematic mutagenesis analysis demonstrated that flavivirus conserved NS4A residues are important for DENV replication. A successful trans complementation of replication-lethal NS4A mutant virus requires wild-type NS4A in the context of the viral replication complex. The wild-type NS4A protein alone is not sufficient to rescue the replication defect of NS4A mutants. Intriguingly, distinct NS4A mutants yielded different complementation outcomes in the replicon-containing cells. Overall, the study has enhanced our understanding of flavivirus NS4A at the molecular level. The results also suggest that inhibitor blocking of NS4A oligomerization could be explored for antiviral drug discovery.