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.

Effects of prescribed fire timing on vigor of the invasive forb sericea lespedeza (Lespedeza cuneata), total forage biomass accumulation, plant-community composition, and native fauna on tallgrass prairie in the Kansas Flint Hills.

The predominant grazing-management practice of the Kansas Flint Hills involves annual prescribed burning in March or April with postfire grazing by yearling beef cattle at a high stocking density from April to August. There has been a dramatic increase in sericea lespedeza (Lespedeza cuneata [Dumont] G. Don) coincident with this temporally focused use of prescribed fire in the Flint Hills region. The species is an aggressive invader and a statewide noxious weed in Kansas. Control has generally been attempted using repeated herbicide applications. This approach has not limited proliferation of sericea lespedeza and resulted in collateral damage to nontarget flora and fauna. Alternative timing of prescribed fire has not been evaluated for its control. Our objectives for this 4-yr experiment were to (1) document the effects of prescribed burning during early April, early August, or early September on vigor of sericea lespedeza, standing forage biomass, and basal cover of native graminoids, forbs, and shrubs and (2) measure responses to fire regimes by grassland bird and butterfly communities. Whole-plant dry mass, basal cover, and seed production of sericea lespedeza were markedly less (P < 0.01) in areas treated with prescribed fire in August or September compared with April. Forage biomass did not differ (P ≥ 0.43) among treatments when measured during July; moreover, frequencies of bare soil, litter, and total basal plant cover were not different (P ≥ 0.29) among treatments. Combined basal covers of C4 grasses, C3 grasses, annual grasses, forbs, and shrubs also did not differ (P ≥ 0.11) between treatments. Densities of grasshopper sparrow (Ammodramus savannarum), dickcissel (Spiza americana), and eastern meadowlark (Sturnella magna) were not negatively affected (P > 0.10) by midsummer or late-summer fires relative to early-spring fires. There were no differences (P > 0.10) in densities of grassland-specialist butterfly species across fire regimes. Under the conditions of our experiment, prescribed burning during summer produced no detrimental effects on forage production, desirable nontarget plant species, grassland birds, or butterfly communities but had strong suppressive effects on sericea lespedeza. Additional research is warranted to investigate how to best incorporate late-summer prescribed fire into common grazing-management practices in the Kansas Flint Hills.

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.

Discovery of a Coregulatory Interaction between Kaposi's Sarcoma-Associated Herpesvirus ORF45 and the Viral Protein Kinase ORF36.

UNLABELLED: Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of three human malignancies. KSHV ORF36 encodes a serine/threonine viral protein kinase, which is conserved throughout all herpesviruses. Although several studies have identified the viral and cellular substrates of conserved herpesvirus protein kinases (CHPKs), the precise functions of KSHV ORF36 during lytic replication remain elusive. Here, we report that ORF36 interacts with another lytic protein, ORF45, in a manner dependent on ORF36 kinase activity. We mapped the regions of ORF36 and ORF45 involved in the binding. Their association appears to be mediated by electrostatic interactions, since deletion of either the highly basic N terminus of ORF36 or an acidic patch of ORF45 abolished the binding. In addition, the dephosphorylation of ORF45 protein dramatically reduced its association with ORF36. Importantly, ORF45 enhances both the stability and kinase activity of ORF36. Consistent with previous studies of CHPK homologs, we detected ORF36 protein in extracellular virions. To investigate the roles of ORF36 in the context of KSHV lytic replication, we used bacterial artificial chromosome mutagenesis to engineer both ORF36-null and kinase-dead mutants. We found that ORF36-null/mutant virions are moderately defective in viral particle production and are further deficient in primary infection. In summary, our results uncover a functionally important interaction between ORF36 and ORF45 and indicate a significant role of ORF36 in the production of infectious progeny virions. IMPORTANCE: Kaposi's sarcoma-associated herpesvirus (KSHV) is a human tumor virus with a significant public health burden. KSHV ORF36 encodes a serine/threonine viral protein kinase, whose functions throughout the viral life cycle have not been elucidated. Here, we report that ORF36 interacts with another KSHV protein, ORF45. We mapped the regions of ORF36 and ORF45 involved in their association and further characterized the consequences of this interaction. We engineered ORF36 mutant viruses in order to investigate the functional roles of ORF36 in the context of KSHV lytic replication, and we confirmed that ORF36 is a component of KSHV virions. Moreover, we found that ORF36 mutants are defective in virion production and primary infection. In summary, we discovered and characterized a functionally important interaction between KSHV ORF36 and ORF45, and our results suggest a significant role of ORF36 in the production of infectious progeny virions, a process critical for KSHV pathogenesis.

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.

Differential Roles of Phospholipase D Proteins in FcεRI-Mediated Signaling and Mast Cell Function.

Phospholipase D (PLD) proteins are enzymes that catalyze the hydrolysis of phosphatidylcholine to generate an important signaling lipid, phosphatidic acid. Phosphatidic acid is a putative second messenger implicated in the regulation of vesicular trafficking and cytoskeletal reorganization. Previous studies using inhibitors and overexpression of PLD proteins indicate that PLD1 and PLD2 play positive roles in FcεRI-mediated signaling and mast cell function. We used mice deficient in PLD1, PLD2, or both to study the function of these enzymes in mast cells. In contrast to published studies, we found that PLD1 deficiency impaired FcεRI-mediated mast cell degranulation; however, PLD2 deficiency enhanced it. Biochemical analysis showed that PLD deficiency affected activation of the PI3K pathway and RhoA. Furthermore, our data indicated that, although PLD1 deficiency impaired F-actin disassembly, PLD2 deficiency enhanced microtubule formation. Together, our results suggested that PLD1 and PLD2, two proteins that catalyze the same enzymatic reaction, regulate different steps in mast cell degranulation.

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.

Role of LAT in the granule-mediated cytotoxicity of CD8 T cells.

Linker for activation of T cells (LAT) is a transmembrane adaptor protein that is essential to bridge T cell receptor (TCR) engagement to downstream signaling events. The indispensable role of LAT in thymocyte development and T cell activation has been well characterized; however, the function of LAT in cytotoxic-T-lymphocyte (CTL) cytotoxicity remains unknown. We show here that LAT-deficient CTLs failed to upregulate FasL and produce gamma interferon after engagement with target cells and had impaired granule-mediated killing. We further dissected the effect of the LAT deletion on each step of granule exocytosis. LAT deficiency led to altered synapse formation, subsequently causing unstable T cell-antigen-presenting cell (APC) conjugates. Microtubule organizing center polarization and granule reorientation were also impaired by LAT deficiency, leading to reduced granule delivery. Despite these defects, granule release was still observed in LAT-deficient CTLs due to residual calcium flux and phospholipase C (PLC) activity. Our data demonstrated that LAT-mediated signaling intricately regulates CTL cytotoxicity at multiple steps.

The incidence of visual perceptual impairment in patients with severe traumatic brain injury.

PRIMARY OBJECTIVES: To investigate the incidence of visual perceptual impairments in a sample of patients with traumatic brain injury (TBI) using the Occupational Therapy Adult Perceptual Screening Test (OT-APST), compare incidence rates to a normative sample and explore the relationship between the presence of visual perceptual impairment and the severity of cognitive and functional impairment following TBI. RESEARCH DESIGN: Cohort study using a convenience sample of patients with TBI and a normative sample. METHODS AND PROCEDURES: Thirty-one patients with severe TBI and 195 healthy people were compared on the OT-APST and measures of cognition and function. MAIN OUTCOMES AND RESULTS: The most common impairments in the TBI sample were unilateral neglect (45.2%) and impairments of body scheme (25.8%) and constructional skills (25.8%). Significantly higher incidences of agnosia, apraxia, unilateral neglect and impairments in body scheme and constructional skills were found in the TBI sample compared to the normative sample. No significant relationship was found between the presence of visual perceptual impairments and the level of cognitive and functional impairment after TBI. CONCLUSIONS: Visual perceptual changes are evident in patients with severe TBI when compared to a normative sample. Routine use of a screening tool such as the OT-APST may help identify visual perceptual impairments in these patients and the need for more detailed assessment.