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 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 GRP78, the master regulator of UPR, and activation of the PERK branch of the UPR (p-PERK-p-eIF2α-GADD34). Increased microtubule (MT) acetylation was also induced. As the infection progressed, the level of ubiquitinated proteins increased and GRP78, ubiquitinated proteins, the deacetylase HDAC6, VRVs, and modified endoplasmic reticulum clustered at the cellular microtubule-organizing center (MTOC) and became surrounded by a vimentin "cage." The components involved in the formation of asymmetric VRVs 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 asymmetric clustering of viral replication complexes, resulted in activation of the p-PERK-p-eIF2α-CHOP apoptotic pathway, and decreased viral yield. 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 delayed activation of the cellular death pathways and increased viral yield.<h4>Importance</h4>Zika virus and West Nile virus replication complexes, which are typically symmetrically distributed around the nucleus in the perinuclear region of an infected cell, cluster asymmetrically on one side of the nucleus in A549 and human neural progenitor cells as the infections progress. Analysis of the sequential cellular responses involved showed that microtubule acetylation increased and the unfolded protein response was activated; then, the levels of ubiquitinated protein and HDAC6 increased, followed by asymmetric concentration of the viral replication complexes, ubiquitinated proteins, and HDAC6 at the cellular microtubule-organizing center, where they became surrounded by a vimentin cage. These sequential steps are characteristic of aggresome formation, an additional protective cellular response that copes with the accumulation of toxic misfolded proteins. This response delayed activation of cell death pathways and extended virus production. Both cell type-specific and orthoflavivirus-specific characteristics determine whether a particular infection induces aggresome formation.