Restoration of replication-defective dengue type 1 virus bearing mutations in the N-terminal cytoplasmic portion of NS4A by additional mutations in NS4B

Flavivirus NS4A has an N-terminal hydrophilic cytoplasmic portion; however, the role of this portion remains poorly understood. In this study, we show that a recombinant dengue type 1 virus (DENV-1) in which a subportion (amino acids 27-34) of the N-terminal portion of NS4A is replaced by the corresponding region from Japanese encephalitis virus (JEV) is defective in replication. Using the defective mutant clone NS4A(27-34(JEV)), we recovered suppressor mutant viruses that carry various non-synonymous mutations. Site-directed mutational analysis indicated that a single non-synonymous mutation in NS4B that is found in the suppressor viruses is sufficient to restore NS4A(27-34(JEV)). Recombinant DENV-1 with single mutations in NS4B had increased growth properties as compared to the wild-type virus and NS4A(27-34(JEV)) virus bearing the same NS4B mutation. Collectively, our results suggest that the NS4B mutation enhanced the growth of DENV-1, irrespective of the sequence of the 27-34 subportion NS4A.