Coordination of Zika Virus Infection and Viroplasm Organization by Microtubules and Microtubule-Organizing Centers.

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.

Zika Virus Infection Induces DNA Damage Response in Human Neural Progenitors That Enhances Viral Replication.

Zika virus (ZIKV) infection attenuates the growth of human neural progenitor cells (hNPCs). As these hNPCs generate the cortical neurons during early brain development, the ZIKV-mediated growth retardation potentially contributes to the neurodevelopmental defects of the congenital Zika syndrome. Here, we investigate the mechanism by which ZIKV manipulates the cell cycle in hNPCs and the functional consequence of cell cycle perturbation on the replication of ZIKV and related flaviviruses. We demonstrate that ZIKV, but not dengue virus (DENV), induces DNA double-strand breaks (DSBs), triggering the DNA damage response through the ATM/Chk2 signaling pathway while suppressing the ATR/Chk1 signaling pathway. Furthermore, ZIKV infection impedes the progression of cells through S phase, thereby preventing the completion of host DNA replication. Recapitulation of the S-phase arrest state with inhibitors led to an increase in ZIKV replication, but not of West Nile virus or DENV. Our data identify ZIKV's ability to induce DSBs and suppress host DNA replication, which results in a cellular environment favorable for its replication.IMPORTANCE Clinically, Zika virus (ZIKV) infection can lead to developmental defects in the cortex of the fetal brain. How ZIKV triggers this event in developing neural cells is not well understood at a molecular level and likely requires many contributing factors. ZIKV efficiently infects human neural progenitor cells (hNPCs) and leads to growth arrest of these cells, which are critical for brain development. Here, we demonstrate that infection with ZIKV, but not dengue virus, disrupts the cell cycle of hNPCs by halting DNA replication during S phase and inducing DNA damage. We further show that ZIKV infection activates the ATM/Chk2 checkpoint but prevents the activation of another checkpoint, the ATR/Chk1 pathway. These results unravel an intriguing mechanism by which an RNA virus interrupts host DNA replication. Finally, by mimicking virus-induced S-phase arrest, we show that ZIKV manipulates the cell cycle to benefit viral replication.

Molecular signatures associated with ZIKV exposure in human cortical neural progenitors.

Zika virus (ZIKV) infection causes microcephaly and has been linked to other brain abnormalities. How ZIKV impairs brain development and function is unclear. Here we systematically profiled transcriptomes of human neural progenitor cells exposed to Asian ZIKVC, African ZIKVM, and dengue virus (DENV). In contrast to the robust global transcriptome changes induced by DENV, ZIKV has a more selective and larger impact on expression of genes involved in DNA replication and repair. While overall expression profiles are similar, ZIKVC, but not ZIKVM, induces upregulation of viral response genes and TP53. P53 inhibitors can block the apoptosis induced by both ZIKVC and ZIKVM in hNPCs, with higher potency against ZIKVC-induced apoptosis. Our analyses reveal virus- and strain-specific molecular signatures associated with ZIKV infection. These datasets will help to investigate ZIKV-host interactions and identify neurovirulence determinants of ZIKV.

Identification of small-molecule inhibitors of Zika virus infection and induced neural cell death via a drug repurposing screen.

In response to the current global health emergency posed by the Zika virus (ZIKV) outbreak and its link to microcephaly and other neurological conditions, we performed a drug repurposing screen of ∼6,000 compounds that included approved drugs, clinical trial drug candidates and pharmacologically active compounds; we identified compounds that either inhibit ZIKV infection or suppress infection-induced caspase-3 activity in different neural cells. A pan-caspase inhibitor, emricasan, inhibited ZIKV-induced increases in caspase-3 activity and protected human cortical neural progenitors in both monolayer and three-dimensional organoid cultures. Ten structurally unrelated inhibitors of cyclin-dependent kinases inhibited ZIKV replication. Niclosamide, a category B anthelmintic drug approved by the US Food and Drug Administration, also inhibited ZIKV replication. Finally, combination treatments using one compound from each category (neuroprotective and antiviral) further increased protection of human neural progenitors and astrocytes from ZIKV-induced cell death. Our results demonstrate the efficacy of this screening strategy and identify lead compounds for anti-ZIKV drug development.

Brain-Region-Specific Organoids Using Mini-bioreactors for Modeling ZIKV Exposure.

Cerebral organoids, three-dimensional cultures that model organogenesis, provide a new platform to investigate human brain development. High cost, variability, and tissue heterogeneity limit their broad applications. Here, we developed a miniaturized spinning bioreactor (SpinΩ) to generate forebrain-specific organoids from human iPSCs. These organoids recapitulate key features of human cortical development, including progenitor zone organization, neurogenesis, gene expression, and, notably, a distinct human-specific outer radial glia cell layer. We also developed protocols for midbrain and hypothalamic organoids. Finally, we employed the forebrain organoid platform to model Zika virus (ZIKV) exposure. Quantitative analyses revealed preferential, productive infection of neural progenitors with either African or Asian ZIKV strains. ZIKV infection leads to increased cell death and reduced proliferation, resulting in decreased neuronal cell-layer volume resembling microcephaly. Together, our brain-region-specific organoids and SpinΩ provide an accessible and versatile platform for modeling human brain development and disease and for compound testing, including potential ZIKV antiviral drugs.

Zika Virus Infects Human Cortical Neural Progenitors and Attenuates Their Growth.

The suspected link between infection by Zika virus (ZIKV), a re-emerging flavivirus, and microcephaly is an urgent global health concern. The direct target cells of ZIKV in the developing human fetus are not clear. Here we show that a strain of the ZIKV, MR766, serially passaged in monkey and mosquito cells efficiently infects human neural progenitor cells (hNPCs) derived from induced pluripotent stem cells. Infected hNPCs further release infectious ZIKV particles. Importantly, ZIKV infection increases cell death and dysregulates cell-cycle progression, resulting in attenuated hNPC growth. Global gene expression analysis of infected hNPCs reveals transcriptional dysregulation, notably of cell-cycle-related pathways. Our results identify hNPCs as a direct ZIKV target. In addition, we establish a tractable experimental model system to investigate the impact and mechanism of ZIKV on human brain development and provide a platform to screen therapeutic compounds.

The missing pieces of the HCV entry puzzle.

The past decade has witnessed steady and rapid progress in HCV research, which has led to the recent breakthrough in therapies against this significant human pathogen. Yet a deeper understanding of the life cycle of the virus is required to develop more affordable treatments and to advance vaccine design. HCV entry presents both a challenge for scientific research and an opportunity for alternative intervention approaches, owning to its highly complex nature and the myriad of players involved. More than half a dozen cellular proteins are implicated in HCV entry; and a more definitive picture regarding the structures of the glycoproteins is emerging. A role of apolipoproteins in HCV entry has also been established. Still, major questions remain, and the answers to these, which we summarize in this review, will hopefully close the gaps in our understanding and complete the puzzle that is HCV entry.