High-performance biochar-loaded MgAl-layered double oxide adsorbents derived from sewage sludge towards nanoplastics removal: Mechanism elucidation and QSAR modeling.

Utilization of sewage sludge for the fabrication of environmental functional materials is highly desirable to achieve pollution mitigation and resource recovery. In the present work, we introduced a novel MgAl-layered double oxide (LDO)@biochar composite adsorbent in-situ fabricated from Al-rich sewage sludge, and its excellent application in nanoplastics adsorption. Initially, fifteen model contaminants with varied conjugate structures, hydrogen bonding and ionic properties were selected for an investigation of adsorption behavior and adsorption selectivity on LDO@biochar. Structural variation of LDO@biochar suggested reconstruction of the layered double hydroxide (LDH) during the adsorption process due to the "memory effect". Under the synergy of LDH and biochar, the contaminants were adsorbed via multiple adsorbent-adsorbate interactions, including anion exchange, electrostatic interaction, hydrogen bonding and π-π conjugation. Then, a quantitative structure-activity relationship (QSAR) model was constructed by integrating the number of hydrogen bond acceptors, polarity surface area, number of aromatic rings, and Fukui index f(-)x together to reflect the affinity of each contaminant to the adsorbent. Guided by the QSAR model, the negatively charged polystyrene nanoplastics with continuously conjugated aromatic rings were predicted to be effectively adsorbed on LDO@biochar. Experimental tests confirmed a great capacity of LDO@biochar towards the polystyrene nanoplastics, given the equilibrium adsorption capacity as high as 360 mg g-1 at 30-50 °C. This work not only opened up a new avenue for sustainable utilization of sewage sludge towards high-performance environmental functional materials, but also demonstrated the potential of the QSAR analysis as a rapid and accurate approach for guiding the application of an adsorbent to new emerging containments.

SOX2 downregulation of PML increases HCMV gene expression and growth of glioma cells.

The presence of human cytomegalovirus (HCMV) in glioblastoma (GBM) and improved outcomes of GBM patients receiving therapies targeting the virus have implicated HCMV in GBM progression. However, a unifying mechanism that accounts for the contribution of HCMV to the malignant phenotype of GBM remains incompletely defined. Here we have identified SOX2, a marker of glioma stem cells (GSCs), as a key determinant of HCMV gene expression in gliomas. Our studies demonstrated that SOX2 downregulated promyelocytic leukemia (PML) and Sp100 and consequently facilitated viral gene expression by decreasing the amount of PML nuclear bodies in HCMV-infected glioma cells. Conversely, the expression of PML antagonized the effects of SOX2 on HCMV gene expression. Furthermore, this regulation of SOX2 on HCMV infection was demonstrated in a neurosphere assay of GSCs and in a murine xenograft model utilizing xenografts from patient-derived glioma tissue. In both cases, SOX2 overexpression facilitated the growth of neurospheres and xenografts implanted in immunodeficient mice. Lastly, the expression of SOX2 and HCMV immediate early 1 (IE1) protein could be correlated in tissues from glioma patients, and interestingly, elevated levels of SOX2 and IE1 were predictive of a worse clinical outcome. These studies argue that HCMV gene expression in gliomas is regulated by SOX2 through its regulation of PML expression and that targeting molecules in this SOX2-PML pathway could identify therapies for glioma treatment.

Human Cytomegalovirus IE1 Impairs Neuronal Migration by Downregulating Connexin 43.

Human cytomegalovirus (HCMV) is a leading cause of congenital birth defects. Though the underlying mechanisms remain poorly characterized, mouse models of congenital CMV infection have demonstrated that the neuronal migration process is damaged. In this study, we evaluated the effects of HCMV infection on connexin 43 (Cx43), a crucial adhesion molecule mediating neuronal migration. We show in multiple cellular models that HCMV infection downregulated Cx43 posttranslationally. Further analysis identified the immediate early protein IE1 as the viral protein responsible for the reduction of Cx43. IE1 was found to bind the Cx43 C terminus and promote Cx43 degradation through the ubiquitin-proteasome pathway. Deletion of the Cx43-binding site in IE1 rendered it incapable of inducing Cx43 degradation. We validated the IE1-induced loss of Cx43 in vivo by introducing IE1 into the fetal mouse brain. Noteworthily, ectopic IE1 expression induced cortical atrophy and neuronal migration defects. Several lines of evidence suggest that these damages result from decreased Cx43, and restoration of Cx43 levels partially rescued IE1-induced interruption of neuronal migration. Taken together, the results of our investigation reveal a novel mechanism of HCMV-induced neural maldevelopment and identify a potential intervention target. IMPORTANCE Congenital CMV (cCMV) infection causes neurological sequelae in newborns. Recent studies of cCMV pathogenesis in animal models reveal ventriculomegaly and cortical atrophy associated with impaired neural progenitor cell (NPC) proliferation and migration. In this study, we investigated the mechanisms underlying these NPC abnormalities. We show that Cx43, a critical adhesion molecule mediating NPC migration, is downregulated by HCMV infection in vitro and HCMV-IE1 in vivo. We provide evidence that IE1 interacts with the C terminus of Cx43 to promote its ubiquitination and consequent degradation through the proteasome. Moreover, we demonstrate that introducing IE1 into mouse fetal brains led to neuronal migration defects, which was associated with Cx43 reduction. Deletion of the Cx43-binding region in IE1 or ectopic expression of Cx43 rescued the IE1-induced migration defects in vivo. Our study provides insight into how cCMV infection impairs neuronal migration and reveals a target for therapeutic interventions.

Human cytomegalovirus pUL97 upregulates SOCS3 expression via transcription factor RFX7 in neural progenitor cells.

Congenital human cytomegalovirus (HCMV) infection causes severe damage to the fetal brain, and the underlying mechanisms remain elusive. Cytokine signaling is delicately controlled in the fetal central nervous system to ensure proper development. Here we show that suppressor of cytokine signaling 3 (SOCS3), a negative feedback regulator of the IL-6 cytokine family signaling, was upregulated during HCMV infection in primary neural progenitor cells (NPCs) with a biphasic expression pattern. From viral protein screening, pUL97 emerged as the viral factor responsible for prolonged SOCS3 upregulation. Further, by proteomic analysis of the pUL97-interacting host proteins, regulatory factor X 7 (RFX7) was identified as the transcription factor responsible for the regulation. Depletion of either pUL97 or RFX7 prevented the HCMV-induced SOCS3 upregulation in NPCs. With a promoter-luciferase activity assay, we demonstrated that the pUL97 kinase activity and RFX7 were required for SOCS3 upregulation. Moreover, the RFX7 phosphorylation level was increased by either UL97-expressing or HCMV-infection in NPCs, suggesting that pUL97 induces RFX7 phosphorylation to drive SOCS3 transcription. We further revealed that elevated SOCS3 expression impaired NPC proliferation and migration in vitro and caused NPCs migration defects in vivo. Taken together, these findings uncover a novel regulatory mechanism of sustained SOCS3 expression in HCMV-infected NPCs, which perturbs IL-6 cytokine family signaling, leads to NPCs proliferation and migration defects, and consequently affects fetal brain development.

MORC3 restricts human cytomegalovirus infection by suppressing the major immediate-early promoter activity.

During the long coevolution of human cytomegalovirus (HCMV) and humans, the host has formed a defense system of multiple layers to eradicate the invader, and the virus has developed various strategies to evade host surveillance programs. The intrinsic immunity primarily orchestrated by promyelocytic leukemia (PML) nuclear bodies (PML-NBs) represents the first line of defense against HCMV infection. Here, we demonstrate that microrchidia family CW-type zinc finger 3 (MORC3), a PML-NBs component, is a restriction factor targeting HCMV infection. We show that depletion of MORC3 through knockdown by RNA interference or knockout by CRISPR-Cas9 augmented immediate-early protein 1 (IE1) gene expression and subsequent viral replication, and overexpressing MORC3 inhibited HCMV replication by suppressing IE1 gene expression. To relief the restriction, HCMV induces transient reduction of MORC3 protein level via the ubiquitin-proteasome pathway during the immediate-early to early stage. However, MORC3 transcription is upregulated, and the protein level recovers in the late stages. Further analyses with temporal-controlled MORC3 expression and the major immediate-early promoter (MIEP)-based reporters show that MORC3 suppresses MIEP activity and consequent IE1 expression with the assistance of PML. Taken together, our data reveal that HCMV enforces temporary loss of MORC3 to evade its repression against the initiation of immediate-early gene expression.

Human Cytomegalovirus Hijacks WD Repeat Domain 11 for Virion Assembly Compartment Formation and Virion Morphogenesis.

Human cytomegalovirus (HCMV) has a large (∼235 kb) genome with more than 200 predicted open reading frames that exploits numerous cellular factors to facilitate its replication. A key feature of HCMV-infected cells is the emergence of a distinctive membranous cytoplasmic compartment termed the virion assembly compartment (vAC). Here, we report that host protein WD repeat domain 11 (WDR11) plays a key role in vAC formation and virion morphogenesis. We found that WDR11 was upregulated at both mRNA and protein levels during HCMV infection. At the late stage of HCMV replication, WDR11 relocated to the vAC and colocalized with markers of the trans-Golgi network (TGN) and vAC. Depletion of WDR11 hindered HCMV-induced membrane reorganization of the Golgi and TGN, altered vAC formation, and impaired HCMV secondary envelopment and virion morphogenesis. Further, motifs critical for the localization of WDR11 in TGN were identified by alanine-scanning mutagenesis. Mutation of these motifs led to WDR11 mislocation outside the TGN and loss of vAC formation. Taken together, these data indicate that host protein WDR11 is required for efficient viral replication at the stage of virion assembly, possibly by facilitating the remodeling of the endomembrane system for vAC formation and virion morphogenesis. IMPORTANCE During the late phase of human cytomegalovirus (HCMV) infection, the endomembrane system is dramatically reorganized, resulting in the formation of a unique structure termed the virion assembly compartment (vAC), which is critical for the assembly of infectious virions. The mechanism of HCMV-induced vAC formation is still not fully understood. In this report, we identified a host factor, WDR11, that plays an important role in vAC formation. Our findings argue that WDR11 contributes to the relocation of the Golgi and trans-Golgi network to the vAC, a membrane reorganization process that appears to be required for efficient virion maturation. The present work provides new insights into the vAC formation and HCMV virion morphogenesis and a potential novel target for antiviral treatment.

A congenital CMV infection model for follow-up studies of neurodevelopmental disorders, neuroimaging abnormalities, and treatment.

Congenital cytomegalovirus (cCMV) infection is the leading infectious cause of neurodevelopmental disorders. However, the neuropathogenesis remains largely elusive due to a lack of informative animal models. In this study, we developed a congenital murine CMV (cMCMV) infection mouse model with high survival rate and long survival period that allowed long-term follow-up study of neurodevelopmental disorders. This model involves in utero intracranial injection and mimics many reported clinical manifestations of cCMV infection in infants, including growth restriction, hearing loss, and impaired cognitive and learning-memory abilities. We observed that abnormalities in MRI/CT neuroimaging were consistent with brain hemorrhage and loss of brain parenchyma, which was confirmed by pathological analysis. Neuropathological findings included ventriculomegaly and cortical atrophy associated with impaired proliferation and migration of neural progenitor cells in the developing brain at both embryonic and postnatal stages. Robust inflammatory responses during infection were shown by elevated inflammatory cytokine levels, leukocyte infiltration, and activation of microglia and astrocytes in the brain. Pathological analyses and CT neuroimaging revealed brain calcifications induced by cMCMV infection and cell death via pyroptosis. Furthermore, antiviral treatment with ganciclovir significantly improved neurological functions and mitigated brain damage as shown by CT neuroimaging. These results demonstrate that this model is suitable for investigation of mechanisms of infection-induced brain damage and long-term studies of neurodevelopmental disorders, including the development of interventions to limit CNS damage associated with cCMV infection.

iTRAQ-Based Proteomics Analysis of Human Cytomegalovirus Latency and Reactivation in T98G Cells.

Human cytomegalovirus (HCMV) establishes a persistent/latent infection after primary infection, and the host factor(s) plays a key role in regulating HCMV infection status. The spread of reactivated HCMV via the hematogenous or neural route usually results in severe diseases in newborns and immunocompromised individuals. As the primary reservoirs in vivo, cells of myeloid lineage have been utilized extensively to study HCMV infection. However, the molecular mechanism of HCMV latency/reactivation in neural cells is still poorly understood. We previously showed that HCMV-infected T98G cells maintain a large number of viral genomes and support HCMV reactivation from latency upon cAMP/IBMX treatment. Here, we employed an isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomics to characterize cellular protein changes during HCMV latency and reactivation in T98G cells. A total of 168 differentially expressed proteins (DEPs) were identified, including 89 proteins in latency and 85 proteins in reactivation. Bioinformatics analysis showed that a few biological pathways were associated with HCMV latency or reactivation. Moreover, we validated 16 DEPs by both mRNA and protein expression profiles and further evaluated the effects of ApoE and the phosphatidylinositol 3-kinase (PI3K) pathway on HCMV infection. ApoE knockdown reduced HCMV loads and virus release, whereas overexpressing ApoE hampered HCMV latent infection, indicating a role in HCMV latency establishment/maintenance. Blocking the PI3K pathway by LY294002, a PI3K inhibitor, induced HCMV reactivation from latency in T98G cells. Overall, this comparative proteomics analysis delineates the cellular protein changes during HCMV latency and reactivation and provides a road map to advance our understanding of the mechanism(s) in the context of neural cells. IMPORTANCE Human cytomegalovirus (HCMV) is a highly transmissible betaherpesvirus that has a prevalence of 60% to 90% worldwide. This opportunist pathogen poses a significant threat to newborns and immunosuppressed individuals. One major obstacle for developing effective therapeutics is a poor understanding of HCMV latency/reactivation mechanisms. This study presents, for the first time, a systemic analysis of host cell protein expression changes during HCMV latency establishment and reactivation processes in neural cells. We showed that ApoE was downregulated by HCMV to facilitate latent infection. Also, the proteomics analysis has associated a few PI3K pathway-related proteins with HCMV reactivation. Altogether, this study highlights multiple host proteins and signaling pathways that can be further investigated as potential druggable targets for HCMV-related diseases, especially brain disorders.

Localization of the WD repeat-containing protein 5 to the Virion Assembly Compartment Facilitates Human Cytomegalovirus Assembly.

We previously reported that human cytomegalovirus (HCMV) utilizes the cellular protein WD repeat-containing protein 5 (WDR5) to facilitate capsid nuclear egress. Here, we further show that HCMV infection results in WDR5 localization in a juxtanuclear region, and that its localization to this cellular site is associated with viral replication and late viral gene expression. Furthermore, WDR5 accumulated in the virion assembly compartment (vAC) and co-localized with vAC markers of gamma-tubulin (γ-tubulin), early endosomes, and viral vAC marker proteins pp65, pp28, and glycoprotein B (gB). WDR5 co-immunoprecipitated with multiple virion proteins, including MCP, pp150, pp65, pIRS1, and pTRS1, which may explain WDR5 accumulation in the vAC during infection. WDR5 fractionated with virions either in the presence or absence of Triton X-100 and was present in purified viral particles, suggesting that WDR5 was incorporated into HCMV virions. Thus, WDR5 localized to the vAC and was incorporated into virions, raising the possibility that in addition to capsid nuclear egress, WDR5 could also participate in cytoplasmic HCMV virion morphogenesis.Importance Human cytomegalovirus (HCMV) has a large (∼235-kb) genome that contains over 170 ORFs and exploits numerous cellular factors to facilitate its replication. In the late phase of HCMV infection cytoplasmic membranes are reorganized to establish the virion assembly compartment (vAC), which has been shown to necessary for efficient assembly of progeny virions. We previously reported that WDR5 facilitates HCMV nuclear egress. Here, we show that WDR5 is localized to the vAC and incorporated into virions, perhaps contributing to efficient virion maturation. Thus, findings in this study identified a potential role for WDR5 in HCMV assembly in the cytoplasmic phase of virion morphogenesis.

Human Cytomegalovirus Immediate Early 1 Protein Causes Loss of SOX2 from Neural Progenitor Cells by Trapping Unphosphorylated STAT3 in the Nucleus.

The mechanisms underlying neurodevelopmental damage caused by virus infections remain poorly defined. Congenital human cytomegalovirus (HCMV) infection is the leading cause of fetal brain development disorders. Previous work has linked HCMV infection to perturbations of neural cell fate, including premature differentiation of neural progenitor cells (NPCs). Here, we show that HCMV infection of NPCs results in loss of the SOX2 protein, a key pluripotency-associated transcription factor. SOX2 depletion maps to the HCMV major immediate early (IE) transcription unit and is individually mediated by the IE1 and IE2 proteins. IE1 causes SOX2 downregulation by promoting the nuclear accumulation and inhibiting the phosphorylation of STAT3, a transcriptional activator of SOX2 expression. Deranged signaling resulting in depletion of a critical stem cell protein is an unanticipated mechanism by which the viral major IE proteins may contribute to brain development disorders caused by congenital HCMV infection.IMPORTANCE Human cytomegalovirus (HCMV) infections are a leading cause of brain damage, hearing loss, and other neurological disabilities in children. We report that the HCMV proteins known as IE1 and IE2 target expression of human SOX2, a central pluripotency-associated transcription factor that governs neural progenitor cell (NPC) fate and is required for normal brain development. Both during HCMV infection and when expressed alone, IE1 causes the loss of SOX2 from NPCs. IE1 mediates SOX2 depletion by targeting STAT3, a critical upstream regulator of SOX2 expression. Our findings reveal an unanticipated mechanism by which a common virus may cause damage to the developing nervous system and suggest novel targets for medical intervention.

WDR5 Facilitates Human Cytomegalovirus Replication by Promoting Capsid Nuclear Egress.

WD repeat-containing protein 5 (WDR5) is essential for assembling the VISA-associated complex to induce a type I interferon antiviral response to Sendai virus infection. However, the roles of WDR5 in DNA virus infections are not well described. Here, we report that human cytomegalovirus exploits WDR5 to facilitate capsid nuclear egress. Overexpression of WDR5 in fibroblasts slightly enhanced the infectious virus yield. However, WDR5 knockdown dramatically reduced infectious virus titers with only a small decrease in viral genome replication or gene expression. Further investigation of late steps of viral replication found that WDR5 knockdown significantly impaired formation of the viral nuclear egress complex and induced substantially fewer infoldings of the inner nuclear membrane. In addition, fewer capsids were associated with these infoldings, and there were fewer capsids in the cytoplasm. Restoration of WDR5 partially reversed these effects. These results suggest that WDR5 knockdown impairs the nuclear egress of capsids, which in turn decreases virus titers. These findings reveal an important role for a host factor whose function(s) is usurped by a viral pathogen to promote efficient replication. Thus, WDR5 represents an interesting regulatory mechanism and a potential antiviral target.IMPORTANCE Human cytomegalovirus (HCMV) has a large (∼235-kb) genome with over 170 open reading frames and exploits numerous cellular factors to facilitate its replication. HCMV infection increases protein levels of WD repeat-containing protein 5 (WDR5) during infection, overexpression of WDR5 enhances viral replication, and knockdown of WDR5 dramatically attenuates viral replication. Our results indicate that WDR5 promotes the nuclear egress of viral capsids, the depletion of WDR5 resulting in a significant decrease in production of infectious virions. This is the first report that WDR5 favors HCMV, a DNA virus, replication and highlights a novel target for antiviral therapy.

Ex vivo non-viral vector-mediated neurotrophin-3 gene transfer to olfactory ensheathing glia: effects on axonal regeneration and functional recovery after implantation in rats with spinal cord injury.

OBJECTIVE: Combine olfactory ensheathing glia (OEG) implantation with ex vivo non-viral vector-based neurotrophin-3 (NT-3) gene therapy in attempting to enhance regeneration after thoracic spinal cord injury (SCI). METHODS: Primary OEG were transfected with cationic liposome-mediated recombinant plasmid pcDNA3.1(+)-NT3 and subsequently implanted into adult Wistar rats directly after the thoracic spinal cord (T9) contusion by the New York University impactor. The animals in 3 different groups received 4x10(5) OEG transfected with pcDNA3.1(+)-NT3 or pcDNA3.1(+) plasmids, or the OEGs without any plasmid transfection, respectively; the fourth group was untreated group, in which no OEG was implanted. RESULTS: NT-3 production was seen increased both ex vivo and in vivo in pcDNA3.1(+)-NT3 transfected OEGs. Three months after implantation of NT-3-transfected OEGs, behavioral analysis revealed that the hindlimb function of SCI rats was improved. All spinal cords were filled with regenerated neurofilament-positive axons. Retrograde tracing revealed enhanced regenerative axonal sprouting. CONCLUSION: Non-viral vector-mediated genetic engineering of OEG was safe and more effective in producing NT-3 and promoting axonal outgrowth followed by enhancing SCI recovery in rats.