West Nile virus and Zika virus infections induce aggresome formation in human neural progenitor and A549 cells

ZIKA viral replication vesicles (VRVs) were previously observed to cluster asymmetrically on one side of the nucleus in infected Huh7, A549 and human neural progenitor cells (h-NPCs). West Nile virus (WNV) infections in these cells also induce asymmetrically clustered VRVs. The sequential cell responses leading to asymmetric clustering of the WNV and ZIKV VRVs were investigated. Both infections activated the cellular unfolded protein response (UPR) as indicated by increased expression of HSPA5 (GRP78), the master regulator of endoplasmic reticular stress, and activation of the PERK branch of the UPR (p-PERK–p-eIF2α–GADD34). Increased microtubule (MT) acetylation was also detected. As the infection progressed, the level of ubiquitinated proteins increased and GRP78, HDAC6, ubiquitinated proteins and VRVs clustered at the cellular microtubule organizing center (MTOC) and became surrounded by a vimentin “cage.” The components involved in the formation of asymmetric virus replication complexes are characteristic of aggresome formation, an additional cellular response that copes with excessive misfolded proteins when the UPR is overwhelmed. During aggresome formation, HDAC6 links polyubiquitinated proteins to the dynein motor complex for transport along MTs to the MTOC where they aggregate and are surrounded by a vimentin cage. HDAC6 knockdown prevented the asymmetric clustering of the viral replication complexes and resulted in activation of the p-PERK–p-eIF2α–CHOP apoptotic pathway. The threshold for triggering aggresome formation varied with the infecting orthoflavivirus and cell type. The formation of aggresome-like, asymmetric VRVs in A549 cells and h-NPCs infected with WNV or ZIKV prolonged cell survival and increased the viral yield.