Zika virus (ZIKV) is a mosquito-transmitted orthoflavivirus that caused an epidemic in 2015–2016 in the Americas and raised serious global health concerns due to its association with congenital brain anomalies when infections occur during pregnancy. Various viruses can form compartments within the cell to facilitate viral replication and assembly, referred to as viroplasms, replication organelles, or virus factories depending on the type of virus. ZIKV assembles virus particles in virus-generated compartments adjacent to the nucleus, referred to here as a replication compartment (RC), which is formed by remodeling the host cell endoplasmic reticulum (ER). How the viral proteins control RC assembly remains unknown. Here we show that the ZIKV non-structural protein 3 (NS3), a dual-function protease and RNA helicase, is sufficient to drive the assembly of a replication compartment-like structure (RCLS) in human cells. While sufficient to generate the RCLS, NS3 is less efficient in several aspects compared to ZIKV-induced RC assembly. Nonetheless, the RCLS is similar to the ZIKV RC in its assembly at the nuclear periphery, its recruitment of ER, association with the Golgi and centrosome, and the arrangement of microtubules at its surface. Moreover, NS3 expression results in activation of the unfolded protein response (UPR), but attenuates expression of the downstream transcription factor CHOP, mirroring the manipulation of the different aspects of the UPR by ZIKV infection. We further show that the helicase domain and not the protease domain is required for optimal RCLS formation and organelle recruitment, yet each domain affects different control over the UPR. Overall, these findings advance our understanding of the mechanism of RC assembly by ZIKV, its involvement in hijacking the UPR, and the central role of NS3 in the process.

In many cell types, disparate non-centrosomal microtubule-organizing centers (ncMTOCs) replace functional centrosomes and serve the unique needs of the cell types in which they are formed. In Drosophila fat body cells (adipocytes), an ncMTOC is organized on the nuclear surface. This perinuclear ncMTOC is anchored by Msp300, encoded by one of two nesprin-encoding genes in Drosophila. Msp300 and the spectraplakin short stop (shot) are co-dependent for localization to the nuclear envelope to generate the ncMTOC where they recruit the microtubule minus-end stabilizer Patronin (CAMSAP). The Msp300 gene is complex, encoding at least eleven isoforms. Here we show that two Msp300 isoforms, Msp300-PE and -PG, are required and only one, Msp300-PE, appears sufficient for generation of the ncMTOC. Loss of Msp300-PE and -PG results in severe loss of localization of shot and Patronin, disruption of the MT array, nuclear mispositioning and loss of endosomal trafficking. Furthermore, upon loss of Msp300-PE and -PG, other isoforms are retained at the nuclear surface despite the loss of nuclear positioning and MT organization, indicating that they are not sufficient to generate the ncMTOC. Msp300-PE has an unusual domain structure including a lack of a KASH domain and very few spectrin repeats and appears therefore to have a highly derived function to generate an ncMTOC on the nuclear surface.