Zika virus (ZIKV) became a global health concern in 2016 due to its links to congenital microcephaly and other birth defects. Flaviviruses, including ZIKV, reorganize the endoplasmic reticulum (ER) to form a viroplasm, a compartment where virus particles are assembled. Microtubules (MTs) and microtubule-organizing centers (MTOCs) coordinate structural and trafficking functions in the cell, and MTs also support replication of flaviviruses. Here we investigated the roles of MTs and the cell’s MTOCs on ZIKV viroplasm organization and virus production. We show that a toroidal-shaped viroplasm forms upon ZIKV infection, and MTs are organized at the viroplasm core and surrounding the viroplasm. We show that MTs are necessary for viroplasm organization and impact infectious virus production. In addition, the centrosome and the Golgi MTOC are closely associated with the viroplasm, and the centrosome coordinates the organization of the ZIKV viroplasm toroidal structure. Surprisingly, viroplasm formation and virus production are not significantly impaired when infected cells have no centrosomes and impaired Golgi MTOC, and we show that MTs are anchored to the viroplasm surface in these cells. We propose that the viroplasm is a site of MT organization, and the MTs organized at the viroplasm are sufficient for efficient virus production.

The Drosophila melanogaster cell line 1182-4, which constitutively lacks centrioles, was established many years ago from haploid embryos laid by females homozygous for the maternal haploid (mh) mutation. This was the first clear example of animal cells regularly dividing in the absence of this organelle. However, the cause of the acentriolar nature of the 1182-4 cell line remained unclear and could not be clearly assigned to a particular genetic event. Here, we detail historically the longstanding mystery of the lack of centrioles in this Drosophila cell line. Recent advances, such as the characterization of the mh gene and the genomic analysis of 1182-4 cells, allow now a better understanding of the physiology of these cells. By combining these new data, we propose three reasonable hypotheses of the genesis of this remarkable phenotype.

Non-centrosomal microtubule-organizing centers (ncMTOCs) have a variety of roles presumed to serve the diverse functions of the range of cell types in which they are found. ncMTOCs are diverse in their composition, subcellular localization, and function. Here we report a novel perinuclear MTOC inDrosophilafat body cells that is anchored by Msp300/Nesprin at the cytoplasmic surface of the nucleus. Msp300 recruits the MT minus-end protein Patronin/CAMSAP, which functions redundantly with Ninein to further recruit the MT polymerase Msps/XMAP215 to assemble non-centrosomal MTs and does so independently of the widespread MT nucleation factorγ-tubulin. Functionally, the fat body ncMTOC and the radial MT arrays it organizes is essential for nuclear positioning and for secretion of basement membrane components via retrograde dynein-dependent endosomal trafficking that restricts plasma membrane growth. Together, this study identifies a perinuclear ncMTOC with unique architecture and MT regulation properties that serves vital functions.Highlights: A novel perinuclear MTOC in differentiated fat body cells The predominant nucleator,γ-tubulin, is not required at the fat body ncMTOC Msp300/Nesprin organizes the ncMTOC at the nuclear surface by recruiting Patronin/CAMSAP and the spectraplakin Shot Patronin cooperates with Ninein to control MT assembly at the fat body ncMTOC by recruiting Msps Msps, a MT polymerase, is essential for radial MT elongation from the fat body ncMTOC Patronin and Msps associate The ncMTOC and radial MTs, but not actin, control nuclear positioning in the fat body The fat body MTOC controls retrograde endocytic trafficking to regulate plasma membrane growth and secretion of basement membrane proteins

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.