Chia Min Lee, Xuping Xie, Jing Zou, Shi-Hua Li, Michelle Yue Qi Lee, Hongping Dong, Cheng-Feng Qin, Congbao Kang, and Pei-Yong Shi J. Virol. JVI.00546-15; Accepted manuscript posted online 1 April 2015, doi:10.1128/JVI.00546-15
Abstract:
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-2 NS4A protein forms oligomers in infected cells or when expressed alone. Deletion mutagenesis mapped amino acids 50-76 (spanning the first transmembrane domain [TMD1]) of NS4A as the major determinant for oligomerization, while the N-terminal 50 residues only slightly contribute to the oligomerization. NMR analysis of NS4A amino acids 17-80 suggests that residues L31, L52, E53, G66, and G67 could participate in oligomerization. Ala-substitution of fifteen flavivirus-conserved NS4A residues revealed that these amino acids are important for viral replication. Among the fifteen mutated NS4A residues, two 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-lethal 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 mutant (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.