Dengue and Zika virus NS4B proteins differ in topology and in determinants of ER membrane protein complex dependency

Flaviviruses utilize the cellular endoplasmic reticulum (ER) for all aspects of their lifecycle. Genome replication and other viral activities take place in structures called replication organelles (ROs), which are invaginations induced in the ER membrane. Among the required elements for RO formation is the biogenesis of viral nonstructural proteins NS4A and NS4B. We have previously shown that NS4A and NS4B from Dengue virus (DENV) and Zika virus (ZIKV) depend on the cellular ER membrane protein complex (EMC) for biogenesis. Here, we find that this dependency extends to the NS4A and NS4B proteins of Yellow Fever virus (YFV) and West Nile virus (WNV), which share similar computationally predicted membrane topologies. However, we demonstrate that ZIKV NS4B has different determinants of its dependency on the EMC than those for DENV NS4B, as well as a different membrane topology. Furthermore, we characterize mutant isolates of DENV and ZIKV that were serially passaged in EMC knockout cells and find that none are completely independent of the EMC for infection, and that mutant NS4B proteins remain sensitive to EMC depletion, suggesting a high genetic barrier to EMC depletion. Collectively, our findings are consistent with a model in which the EMC recognizes multiple determinants in the NS4B protein to support infection in several flaviviruses of critical public health importance.IMPORTANCEThe NS4A and NS4B proteins of flaviviruses are critically important to replication, but little is known about their function. It has been previously reported that the cellular EMC supports the biogenesis of NS4A and NS4B from Dengue and Zika virus. In this work, we demonstrate that this dependency on the EMC for NS4A and NS4B biogenesis extends to the West Nile and Yellow Fever viruses. Furthermore, we examine the features of ZIKV NS4B and find that its membrane topology of ZIKV NS4B and its determinants of dependency on the EMC are different from those previously described in DENV NS4B. Finally, we present evidence that there is a high genetic barrier for Dengue and Zika viruses to overcome EMC depletion.