Melanopsin retinal ganglion cells mediate light-promoted brain development.

During development, melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) become light sensitive much earlier than rods and cones. IpRGCs project to many subcortical areas, whereas physiological functions of these projections are yet to be fully elucidated. Here, we found that ipRGC-mediated light sensation promotes synaptogenesis of pyramidal neurons in various cortices and the hippocampus. This phenomenon depends on activation of ipRGCs and is mediated by the release of oxytocin from the supraoptic nucleus (SON) and the paraventricular nucleus (PVN) into cerebral-spinal fluid. We further characterized a direct connection between ipRGCs and oxytocin neurons in the SON and mutual projections between oxytocin neurons in the SON and PVN. Moreover, we showed that the lack of ipRGC-mediated, light-promoted early cortical synaptogenesis compromised learning ability in adult mice. Our results highlight the importance of light sensation early in life on the development of learning ability and therefore call attention to suitable light environment for infant care.

Cytoplasmic PARP1 links the genome instability to the inhibition of antiviral immunity through PARylating cGAS.

Virus infection modulates both host immunity and host genomic stability. Poly(ADP-ribose) polymerase 1 (PARP1) is a key nuclear sensor of DNA damage, which maintains genomic integrity, and the successful application of PARP1 inhibitors for clinical anti-cancer therapy has lasted for decades. However, precisely how PARP1 gains access to cytoplasm and regulates antiviral immunity remains unknown. Here, we report that DNA virus induces a reactive nitrogen species (RNS)-dependent DNA damage and activates DNA-dependent protein kinase (DNA-PK). Activated DNA-PK phosphorylates PARP1 on Thr594, thus facilitating the cytoplasmic translocation of PARP1 to inhibit the antiviral immunity both in vitro and in vivo. Mechanistically, cytoplasmic PARP1 interacts with and directly PARylates cyclic GMP-AMP synthase (cGAS) on Asp191 to inhibit its DNA-binding ability. Together, our findings uncover an essential role of PARP1 in linking virus-induced genome instability with inhibition of host immunity, which is of relevance to cancer, autoinflammation, and other diseases.

Tumor-associated macrophage subtypes on cancer immunity along with prognostic analysis and SPP1-mediated interactions between tumor cells and macrophages.

Tumor-associated macrophages (TAM) subtypes have been shown to impact cancer prognosis and resistance to immunotherapy. However, there is still a lack of systematic investigation into their molecular characteristics and clinical relevance in different cancer types. Single-cell RNA sequencing data from three different tumor types were used to cluster and type macrophages. Functional analysis and communication of TAM subpopulations were performed by Gene Ontology-Biological Process and CellChat respectively. Differential expression of characteristic genes in subpopulations was calculated using zscore as well as edgeR and Wilcoxon rank sum tests, and subsequently gene enrichment analysis of characteristic genes and anti-PD-1 resistance was performed by the REACTOME database. We revealed the heterogeneity of TAM, and identified eleven subtypes and their impact on prognosis. These subtypes expressed different molecular functions respectively, such as being involved in T cell activation, apoptosis and differentiation, or regulating viral bioprocesses or responses to viruses. The SPP1 pathway was identified as a critical mediator of communication between TAM subpopulations, as well as between TAM and epithelial cells. Macrophages with high expression of SPP1 resulted in poorer survival. By in vitro study, we showed SPP1 mediated the interactions between TAM clusters and between TAM and tumor cells. SPP1 promoted the tumor-promoting ability of TAM, and increased PDL1 expression and stemness of tumor cells. Inhibition of SPP1 attenuated N-cadherin and ß-catenin expression and the activation of AKT and STAT3 pathway in tumor cells. Additionally, we found that several subpopulations could decrease the sensitivity of anti-PD-1 therapy in melanoma. SPP1 signal was a critical pathway of communication between macrophage subtypes. Some specific macrophage subtypes were associated with immunotherapy resistance and prognosis in some cancer types.

Palmitoylation of KSHV pORF55 is required for Golgi localization and efficient progeny virion production.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a double-stranded DNA virus etiologically associated with multiple malignancies. Both latency and sporadic lytic reactivation contribute to KSHV-associated malignancies, however, the specific roles of many KSHV lytic gene products in KSHV replication remain elusive. In this study, we report that ablation of ORF55, a late gene encoding a tegument protein, does not impact KSHV lytic reactivation but significantly reduces the production of progeny virions. We found that cysteine 10 and 11 (C10 and C11) of pORF55 are palmitoylated, and the palmytoilation is essential for its Golgi localization and secondary envelope formation. Palmitoylation-defective pORF55 mutants are unstable and undergo proteasomal degradation. Notably, introduction of a putative Golgi localization sequence to these palmitoylation-defective pORF55 mutants restores Golgi localization and fully reinstates KSHV progeny virion production. Together, our study provides new insight into the critical role of pORF55 palmitoylation in KSHV progeny virion production and offers potential therapeutic targets for the treatment of related malignancies.

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.

EphA2 is a functional entry receptor for HCMV infection of glioblastoma cells.

Human cytomegalovirus (HCMV) infection is associated with human glioblastoma, the most common and aggressive primary brain tumor, but the underlying infection mechanism has not been fully demonstrated. Here, we show that EphA2 was upregulated in glioblastoma and correlated with the poor prognosis of the patients. EphA2 silencing inhibits, whereas overexpression promotes HCMV infection, establishing EphA2 as a crucial cell factor for HCMV infection of glioblastoma cells. Mechanistically, EphA2 binds to HCMV gH/gL complex to mediate membrane fusion. Importantly, the HCMV infection was inhibited by the treatment of inhibitor or antibody targeting EphA2 in glioblastoma cells. Furthermore, HCMV infection was also impaired in optimal glioblastoma organoids by EphA2 inhibitor. Taken together, we propose EphA2 as a crucial cell factor for HCMV infection in glioblastoma cells and a potential target for intervention.

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.

Targeting lipid metabolism for ferroptotic cancer therapy.

It has been 10 years since the concept of ferroptosis was put forward and research focusing on ferroptosis has been increasing continuously. Ferroptosis is driven by iron-dependent lipid peroxidation, which can be antagonized by glutathione peroxidase 4 (GPX4), ferroptosis inhibitory protein 1 (FSP1), dihydroorotate dehydrogenase (DHODH) and Fas-associated factor 1 (FAF1). Various cellular metabolic events, including lipid metabolism, can modulate ferroptosis sensitivity. It is worth noting that the reprogramming of lipid metabolism in cancer cells can promote the occurrence and development of tumors. The metabolic flexibility of cancer cells opens the possibility for the coordinated targeting of multiple lipid metabolic pathways to trigger cancer cells ferroptosis. In addition, cancer cells must obtain immortality, escape from programmed cell death including ferroptosis, to promote cancer progression, which provides new perspectives for improving cancer therapy. Targeting the vulnerability of ferroptosis has received attention as one of the significant possible strategies to treat cancer given its role in regulating tumor cell survival. We review the impact of iron and lipid metabolism on ferroptosis and the potential role of the crosstalk of lipid metabolism reprogramming and ferroptosis in antitumor immunity and sum up agents targeting lipid metabolism and ferroptosis for cancer therapy.

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.

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.

Human cytomegalovirus infection perturbs neural progenitor cell fate via the expression of viral microRNAs.

Human cytomegalovirus (HCMV) preferentially targets neural progenitor cells (NPCs) in congenitally infected fetal brains, inducing neurodevelopmental disorders. While HCMV expresses several microRNAs (miRNAs) during infection, their roles in NPC infection are unclear. Here, we characterized expression of cellular and viral miRNAs in HCMV-infected NPCs during early infection by microarray and identified seven differentially expressed cellular miRNAs and six significantly upregulated HCMV miRNAs. Deep learning approaches were used to identify potential targets of significantly upregulated HCMV miRNAs against differentially expressed cellular messenger RNA (mRNAs), and the associations with miRNA-mRNA expression changes were observed. Gene ontology enrichment analysis indicated cellular gene targets were significantly enriched in pathways involved in neurodevelopment and cell-cycle processes. Viral modulation of selected miRNAs and cellular gene targets involved in neurodevelopmental processes were further validated by real-time quantitative reverse transcription polymerase chain reaction. Finally, a predicted 3' untranslated region target site of hcmv-miR-US25-1 in Jag1, a factor important for neurogenesis, was confirmed by mutagenesis. Reduction of Jag1 RNA and protein levels in NPCs was observed in response to transient expression of hcmv-miR-US25-1. A hcmv-miR-US25-1 mutant virus (ΔmiR-US25) displayed limited ability to downregulate Jag1 mRNA levels and protein levels during the early infection stage compared with the wild type virus. Our collective experimental and computational investigation of miRNAs and cellular mRNAs expression in HCMV-infected NPCs yields new insights into the roles of viral miRNAs in regulating NPC fate and their contributions to HCMV neuropathogenesis.

Chronic stress in solid tumor development: from mechanisms to interventions.

Chronic stress results in disturbances of body hormones through the neuroendocrine system. Cancer patients often experience recurrent anxiety and restlessness during disease progression and treatment, which aggravates disease progression and hinders treatment effects. Recent studies have shown that chronic stress-regulated neuroendocrine systems secret hormones to activate many signaling pathways related to tumor development in tumor cells. The activated neuroendocrine system acts not only on tumor cells but also modulates the survival and metabolic changes of surrounding non-cancerous cells. Current clinical evidences also suggest that chronic stress affects the outcome of cancer treatment. However, in clinic, there is lack of effective treatment for chronic stress in cancer patients. In this review, we discuss the main mechanisms by which chronic stress regulates the tumor microenvironment, including functional regulation of tumor cells by stress hormones (stem cell-like properties, metastasis, angiogenesis, DNA damage accumulation, and apoptotic resistance), metabolic reprogramming and immune escape, and peritumor neuromodulation. Based on the current clinical treatment framework for cancer and chronic stress, we also summarize pharmacological and non-pharmacological therapeutic approaches to provide some directions for cancer therapy.

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.

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.

Direct auditory cortical input to the lateral periaqueductal gray controls sound-driven defensive behavior.

Threatening sounds can elicit a series of defensive behavioral reactions in animals for survival, but the underlying neural substrates are not fully understood. Here, we demonstrate a previously unexplored neural pathway in mice that projects directly from the auditory cortex (ACx) to the lateral periaqueductal gray (lPAG) and controls noise-evoked defensive behaviors. Electrophysiological recordings showed that the lPAG could be excited by a loud noise that induced an escape-like behavior. Trans-synaptic viral tracing showed that a great number of glutamatergic neurons, rather than GABAergic neurons, in the lPAG were directly innervated by those in layer V of the ACx. Activation of this pathway by optogenetic manipulations produced a behavior in mice that mimicked the noise-evoked escape, whereas inhibition of the pathway reduced this behavior. Therefore, our newly identified descending pathway is a novel neural substrate for noise-evoked escape and is involved in controlling the threat-related behavior.

Basolateral amygdala input to the medial prefrontal cortex controls obsessive-compulsive disorder-like checking behavior.

Obsessive-compulsive disorder (OCD) affects ∼1 to 3% of the world's population. However, the neural mechanisms underlying the excessive checking symptoms in OCD are not fully understood. Using viral neuronal tracing in mice, we found that glutamatergic neurons from the basolateral amygdala (BLAGlu) project onto both medial prefrontal cortex glutamate (mPFCGlu) and GABA (mPFCGABA) neurons that locally innervate mPFCGlu neurons. Next, we developed an OCD checking mouse model with quinpirole-induced repetitive checking behaviors. This model demonstrated decreased glutamatergic mPFC microcircuit activity regulated by enhanced BLAGlu inputs. Optical or chemogenetic manipulations of this maladaptive circuitry restored the behavioral response. These findings were verified in a mouse functional magnetic resonance imaging (fMRI) study, in which the BLA-mPFC functional connectivity was increased in OCD mice. Together, these findings define a unique BLAGlu→mPFCGABA→Glu circuit that controls the checking symptoms of OCD.

Human Cytomegalovirus Protein UL94 Targets MITA to Evade the Antiviral Immune Response.

Cyclic GMP-AMP synthase (cGAS) senses double-stranded DNA and synthesizes the second messenger cyclic GMP-AMP (cGAMP), which binds to mediator of IRF3 activation (MITA) and initiates MITA-mediated signaling, leading to induction of type I interferons (IFNs) and other antiviral effectors. Human cytomegalovirus (HCMV), a widespread and opportunistic pathogen, antagonizes the host antiviral immune response to establish latent infection. Here, we identified HCMV tegument protein UL94 as an inhibitor of the cGAS-MITA-mediated antiviral response. Ectopic expression of UL94 impaired cytosolic double-stranded DNA (dsDNA)- and DNA virus-triggered induction of type I IFNs and enhanced viral replication. Conversely, UL94 deficiency potentiated HCMV-induced transcription of type I IFNs and downstream antiviral effectors and impaired viral replication. UL94 interacted with MITA, disrupted the dimerization and translocation of MITA, and impaired the recruitment of TBK1 to the MITA signalsome. These results suggest that UL94 plays an important role in the immune evasion of HCMV.IMPORTANCE Human cytomegalovirus (HCMV), a large double-stranded DNA (dsDNA) virus, encodes more than 200 viral proteins. HCMV infection causes irreversible abnormalities of the central nervous system in newborns and severe syndromes in organ transplantation patients or AIDS patients. It has been demonstrated that HCMV has evolved multiple immune evasion strategies to establish latent infection. Previous studies pay more attention to the mechanism by which HCMV evades immune response in the early phase of infection. In this study, we identified UL94 as a negative regulator of the innate immune response, which functions in the late phase of HCMV infection.

Anterograde Viral Tracer Herpes Simplex Virus 1 Strain H129 Transports Primarily as Capsids in Cortical Neuron Axons.

The features of herpes simplex virus 1 (HSV-1) strain 129 (H129), including natural neurotropism and anterograde transneuronal trafficking, make it a potential tool for anterograde neural circuitry tracing. Recently anterograde polysynaptic and monosynaptic tracers were developed from H129 and have been applied for the identification of novel connections and functions of different neural circuitries. However, how H129 viral particles are transported in neurons, especially those of the central nervous system, remains unclear. In this study, we constructed recombinant H129 variants with mCherry-labeled capsids and/or green fluorescent protein (GFP)-labeled envelopes and infected the cortical neurons to study axonal transport of H129 viral particles. We found that different types of viral particles were unevenly distributed in the nucleus, cytoplasm of the cell body, and axon. Most H129 progeny particles were unenveloped capsids and were transported as capsids rather than virions in the axon. Notably, capsids acquired envelopes at axonal varicosities and terminals where the sites forming synapses are connected with other neurons. Moreover, viral capsids moved more frequently in the anterograde direction in axons, with an average velocity of 0.62 ± 0.18 µm/s and maximal velocity of 1.80 ± 0.15 µm/s. We also provided evidence that axonal transport of capsids requires the kinesin-1 molecular motor. These findings support that H129-derived tracers map the neural circuit anterogradely and possibly transsynaptically. These data will guide future modifications and improvements of H129-based anterograde viral tracers.IMPORTANCE Anterograde transneuronal tracers derived from herpes simplex virus 1 (HSV-1) strain 129 (H129) are important tools for mapping neural circuit anatomic and functional connections. It is, therefore, critical to elucidate the transport pattern of H129 within neurons and between neurons. We constructed recombinant H129 variants with genetically encoded fluorescence-labeled capsid protein and/or glycoprotein to visualize viral particle movement in neurons. Both electron microscopy and light microscopy data show that H129 capsids and envelopes move separately, and notably, capsids are enveloped at axonal varicosity and terminals, which are the sites forming synapses to connect with other neurons. Superresolution microscopy-based colocalization analysis and inhibition of H129 particle movement by inhibitors of molecular motors support that kinesin-1 contributes to the anterograde transport of capsids. These results shed light into the mechanisms for anterograde transport of H129-derived tracer in axons and transmission between neurons via synapses, explaining the anterograde labeling of neural circuits by H129-derived tracers.

Human cytomegalovirus protein UL42 antagonizes cGAS/MITA-mediated innate antiviral response.

Cyclic GMP-AMP synthase (cGAS) senses viral DNA in the cytosol and then catalyzes synthesis of the second messenger cGAMP, which activates the ER-localized adaptor protein Mediator of IRF3 Activator (MITA) to initiate innate antiviral response. Human cytomegalovirus (HCMV) proteins can antagonize host immune responses to promote latent infection. Here, we identified HCMV UL42 as a negative regulator of cGAS/MITA-dependent antiviral response. UL42-deficiency enhances HCMV-induced production of type I interferons (IFNs) and downstream antiviral genes. Consistently, wild-type HCMV replicates more efficiently than UL42-deficient HCMV. UL42 interacts with both cGAS and MITA. UL42 inhibits DNA binding, oligomerization and enzymatic activity of cGAS. UL42 also impairs translocation of MITA from the ER to perinuclear punctate structures, which is required for MITA activation, by facilitating p62/LC3B-mediated degradation of translocon-associated protein ß (TRAPß). These results suggest that UL42 can antagonize innate immune response to HCMV by targeting the core components of viral DNA-triggered signaling pathways.