Early Gene Products Research Articles

BDNF augmentation reverses cranial radiation therapy-induced cognitive decline and neurodegenerative consequences.

Cranial radiation therapy (RT) for brain cancers is often associated with the development of radiation-induced cognitive dysfunction (RICD). RICD significantly impacts the quality of life for cancer survivors, highlighting an unmet medical need. Previous human studies revealed a marked reduction in plasma brain-derived neurotrophic factor (BDNF) post-chronic chemotherapy, linking this decline to a substantial cognitive dysfunction among cancer survivors. Moreover, riluzole (RZ)-mediated increased BDNF in vivo in the chemotherapy-exposed mice reversed cognitive decline. RZ is an FDA-approved medication for ALS known to increase BDNF in vivo . In an effort to mitigate the detrimental effects of RT-induced BDNF decline in RICD, we tested the efficacy of RZ in a cranially irradiated (9 Gy) adult mouse model. Notably, RT-exposed mice exhibited significantly reduced hippocampal BDNF, accompanied by increased neuroinflammation, loss of neuronal plasticity-related immediate early gene product, cFos, and synaptic density. Spatial transcriptomic profiling comparing the RT+Veh with the RT+RZ group showed gene expression signatures of neuroprotection of hippocampal excitatory neurons post-RZ. RT-exposed mice performed poorly on learning and memory, and memory consolidation tasks. However, irradiated mice receiving RZ (13 mg/kg, drinking water) for 6-7 weeks showed a significant improvement in cognitive function compared to RT-exposed mice receiving vehicle. Dual-immunofluorescence staining, spatial transcriptomics, and biochemical assessment of RZ-treated irradiated brains demonstrated preservation of synaptic integrity and neuronal plasticity but not neurogenesis and reduced neuroinflammation concurrent with elevated BDNF levels and transcripts compared to vehicle-treated irradiated brains. In summary, oral administration of RZ represents a viable and translationally feasible neuroprotective approach against RICD.

MicroRNA-focused CRISPR/Cas9 screen identifies miR-142 as a key regulator of Epstein-Barr virus reactivation.

Reactivation from latency plays a significant role in maintaining persistent lifelong Epstein-Barr virus (EBV) infection. Mechanisms governing successful activation and progression of the EBV lytic phase are not fully understood. EBV expresses multiple viral microRNAs (miRNAs) and manipulates several cellular miRNAs to support viral infection. To gain insight into the host miRNAs regulating transitions from EBV latency into the lytic stage, we conducted a CRISPR/Cas9-based screen in EBV+ Burkitt lymphoma (BL) cells using anti-Ig antibodies to crosslink the B cell receptor (BCR) and induce reactivation. Using a gRNA library against >1500 annotated human miRNAs, we identified miR-142 as a key regulator of EBV reactivation. Genetic ablation of miR-142 enhanced levels of immediate early and early lytic gene products in infected BL cells. Ago2-PAR-CLIP experiments with reactivated cells revealed miR-142 targets related to Erk/MAPK signaling, including components directly downstream of the B cell receptor (BCR). Consistent with these findings, disruption of miR-142 enhanced SOS1 levels and Mek phosphorylation in response to surface Ig cross-linking. Effects could be rescued by inhibitors of Mek (cobimetinib) or Raf (dabrafenib). Taken together, these results show that miR-142 functionally regulates SOS1/Ras/Raf/Mek/Erk signaling initiated through the BCR and consequently, restricts EBV entry into the lytic cycle.

Analysis of Arc/Arg3.1 Oligomerization In Vitro and in Living Cells.

Arc (also known as Arg3.1) is an activity-dependent immediate early gene product enriched in neuronal dendrites. Arc plays essential roles in long-term potentiation, long-term depression, and synaptic scaling. Although its mechanisms of action in these forms of synaptic plasticity are not completely well established, the activities of Arc include the remodeling of the actin cytoskeleton, the facilitation of AMPA receptor (AMPAR) endocytosis, and the regulation of the transcription of AMPAR subunits. In addition, Arc has sequence and structural similarity to retroviral Gag proteins and self-associates into virus-like particles that encapsulate mRNA and perhaps other cargo for intercellular transport. Each of these activities is likely to be influenced by Arc's reversible self-association into multiple oligomeric species. Here, we used mass photometry to show that Arc exists predominantly as monomers, dimers, and trimers at approximately 20 nM concentration in vitro. Fluorescence fluctuation spectroscopy revealed that Arc is almost exclusively present as low-order (monomer to tetramer) oligomers in the cytoplasm of living cells, over a 200 nM to 5 μM concentration range. We also confirmed that an α-helical segment in the N-terminal domain contains essential determinants of Arc's self-association.

Phage protein Gp11 blocks Staphylococcus aureus cell division by inhibiting peptidoglycan biosynthesis.

Understanding the interplay between phages and their hosts is important for the development of novel therapies against pathogenic bacteria. Although phages have been used to control methicillin-resistant Staphylococcus aureus infections, our knowledge related to the processes in the early stages of phage infection is still limited. Owing to the fact that most of the phage early proteins have been classified as hypothetical proteins with uncertain functions, we screened phage early-gene products that inhibit cell growth in S. aureus, and one protein, Gp11, selectively targets essential host genes to block the synthesis of the peptidoglycan component lipid II, ultimately leading to cell growth arrest in S. aureus. Our study provides a novel insight into the strategy by which Gp11 blocks essential host cellular metabolism to influence phage-host interaction. Importantly, dissecting the interactions between phages and host cells will contribute to the development of new and effective therapies to treat bacterial infections.

HBO1/KAT7/MYST2 HAT complex regulates human adenovirus replicative cycle

Human adenoviruses (HAdV) belong to a small DNA tumor virus family that continues as valuable models in understanding the viral strategies of usurping cell growth regulation. A number of HAdV type 2/5 early viral gene products interact with a variety of cellular proteins to build a conducive environment that promotes viral replication. Here we show that HBO1 (Histone Acetyltransferase Binding to ORC1), a member of the MYST histone acetyltransferase (HAT) complex (also known as KAT7 and MYST2) that acetylates most of the histone H3 lysine 14, is essential for HAdV5 growth. HBO1/MYST2/KAT7 HAT complexes are critical for a variety of cellular processes including control of cell proliferation. In HBO1 downregulated human cells, HAdV5 infection results in reduced expression of E1A and other viral early genes, virus growth is also reduced significantly. Importantly, HBO1 downregulation reduced H3 lysine 14 acetylation at viral promoters during productive infection, likely driving reduced viral gene expression. HBO1 was also associated with viral promoters during infection and co-localized with viral replication centers in the nuclei of infected cells. In transiently transfected cells, overexpression of E1A along with HBO1 stimulated histone acetyltransferase activity of HBO1. E1A also co-immunoprecipitated with HBO1 in transiently transfected cells. In summary, our results demonstrate that HAdV recruits the HBO1 HAT complex to aid in viral replication.

A Revision of Herpes Simplex Virus Type 1 Transcription: First, Repress; Then, Express.

The herpes virus genome bears more than 80 strong transcriptional promoters. Upon entry into the host cell nucleus, these genes are transcribed in an orderly manner, producing five immediate-early (IE) gene products, including ICP0, ICP4, and ICP22, while non-IE genes are mostly silent. The IE gene products are necessary for the transcription of temporal classes following sequentially as early, leaky late, and true late. A recent analysis using precision nuclear run-on followed by deep sequencing (PRO-seq) has revealed an important step preceding all HSV-1 transcription. Specifically, the immediate-early proteins ICP4 and ICP0 enter the cell with the incoming genome to help preclude the nascent antisense, intergenic, and sense transcription of all viral genes. VP16, which is also delivered into the nucleus upon entry, almost immediately reverses this repression on IE genes. The resulting de novo expression of ICP4 and ICP22 further repress antisense, intergenic, and early and late viral gene transcription through different mechanisms before the sequential de-repression of these gene classes later in infection. This early repression, termed transient immediate-early protein-mediated repression (TIEMR), precludes unproductive, antisense, intergenic, and late gene transcription early in infection to ensure the efficient and orderly progression of the viral cascade.

Highly dynamic inflammatory and excitability transcriptional profiles in hippocampal CA1 following status epilepticus

Transient brain insults including status epilepticus (SE) can initiate a process termed ‘epileptogenesis’ that results in chronic temporal lobe epilepsy. As a consequence, the entire tri-synaptic circuit of the hippocampus is fundamentally impaired. A key role in epileptogenesis has been attributed to the CA1 region as the last relay station in the hippocampal circuit and as site of aberrant plasticity, e.g. mediated by acquired channelopathies. The transcriptional profiles of the distinct hippocampal neurons are highly dynamic during epileptogenesis. Here, we aimed to elucidate the early SE-elicited mRNA signature changes and the respective upstream regulatory cascades in CA1. RNA sequencing of CA1 was performed in the mouse pilocarpine-induced SE model at multiple time points ranging from 6 to 72 h after the initial insult. Bioinformatics was used to decipher altered gene expression, signalling cascades and their corresponding cell type profiles. Robust transcriptomic changes were detected at 6 h after SE and at subsequent time points during early epileptogenesis. Major differentially expressed mRNAs encoded primarily immediate early and excitability-related gene products, as well as genes encoding immune signalling factors. Binding sites for the transcription factors Nfkb1, Spi1, Irf8, and two Runx family members, were enriched within promoters of differentially expressed genes related to major inflammatory processes, whereas the transcriptional repressors Suz12, Nfe2l2 and Rest were associated with hyperexcitability and GABA / glutamate receptor activity. CA1 quickly responds to SE by inducing transcription of genes linked to inflammation and excitation stress. Transcription factors mediating this transcriptomic switch represent targets for new highly selected, cell type and time window-specific anti-epileptogenic strategies.

Activity-regulated cytoskeletal-associated protein (Arc) in presynaptic terminals and extracellular vesicles in hippocampal synapses.

The activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) is a neuron-specific immediate early gene (IEG) product. The protein regulates synaptic strength through modulation of spine density and morphology, AMPA receptor endocytosis, and as being part of a retrovirus-like inter-cellular communication mechanism. However, little is known about the detailed subsynaptic localization of the protein, and especially its possible presynaptic localization. In the present study, we provide novel electron microscopical data of Arc localization at hippocampal Schaffer collateral synapses in the CA1 region. The protein was found in both pre-and postsynaptic cytoplasm in a majority of synapses, associated with small vesicles. We also observed multivesicular body-like structures positive for Arc. Furthermore, the protein was located over the presynaptic active zone and the postsynaptic density. The relative concentration of Arc was 25% higher in the postsynaptic spine than in the presynaptic terminal. Notably, small extracellular vesicles labeled for Arc were detected in the synaptic cleft or close to the synapse, supporting a possible transsynaptic transmission of the protein in the brain.

The Dynamics of Synthesis and Localization of Jumbo Phage RNA Polymerases inside Infected Cells.

A nucleus-like structure composed of phage-encoded proteins and containing replicating viral DNA is formed in Pseudomonas aeruginosa cells infected by jumbo bacteriophage phiKZ. The PhiKZ genes are transcribed independently from host RNA polymerase (RNAP) by two RNAPs encoded by the phage. The virion RNAP (vRNAP) transcribes early viral genes and must be injected into the cell with phage DNA. The non-virion RNAP (nvRNAP) is composed of early gene products and transcribes late viral genes. In this work, the dynamics of phage RNAPs localization during phage phiKZ infection were studied. We provide direct evidence of PhiKZ vRNAP injection in infected cells and show that it is excluded from the phage nucleus. The nvRNAP is synthesized shortly after the onset of infection and localizes in the nucleus. We propose that spatial separation of two phage RNAPs allows coordinated expression of phage genes belonging to different temporal classes.

Contribution of µ Opioid Receptor-expressing Dorsal Horn Interneurons to Neuropathic Pain-like Behavior in Mice.

Intersectional genetics have yielded tremendous advances in our understanding of molecularly identified subpopulations and circuits within the dorsal horn in neuropathic pain. The authors tested the hypothesis that spinal µ opioid receptor-expressing neurons (Oprm1-expressing neurons) contribute to behavioral hypersensitivity and neuronal sensitization in the spared nerve injury model in mice. The authors coupled the use of Oprm1Cre transgenic reporter mice with whole cell patch clamp electrophysiology in lumbar spinal cord slices to evaluate the neuronal activity of Oprm1-expressing neurons in the spared nerve injury model of neuropathic pain. The authors used a chemogenetic approach to activate or inhibit Oprm1-expressing neurons, followed by the assessment of behavioral signs of neuropathic pain. The authors reveal that spared nerve injury yielded a robust neuroplasticity of Oprm1-expressing neurons. Spared nerve injury reduced Oprm1 gene expression in the dorsal horn as well as the responsiveness of Oprm1-expressing neurons to the selective µ agonist (D-Ala2, N-MePhe4, Gly-ol)-enkephalin (DAMGO). Spared nerve injury sensitized Oprm1-expressing neurons, as reflected by an increase in their intrinsic excitability (rheobase, sham 38.62 ± 25.87 pA [n = 29]; spared nerve injury, 18.33 ± 10.29 pA [n = 29], P = 0.0026) and spontaneous synaptic activity (spontaneous excitatory postsynaptic current frequency in delayed firing neurons: sham, 0.81 ± 0.67 Hz [n = 14]; spared nerve injury, 1.74 ± 1.68 Hz [n = 10], P = 0.0466), and light brush-induced coexpression of the immediate early gene product, Fos in laminae I to II (%Fos/tdTomato+: sham, 0.42 ± 0.57% [n = 3]; spared nerve injury, 28.26 ± 1.92% [n = 3], P = 0.0001). Chemogenetic activation of Oprm1-expressing neurons produced mechanical hypersensitivity in uninjured mice (saline, 2.91 ± 1.08 g [n = 6]; clozapine N-oxide, 0.65 ± 0.34 g [n = 6], P = 0.0006), while chemogenetic inhibition reduced behavioral signs of mechanical hypersensitivity (saline, 0.38 ± 0.37 g [n = 6]; clozapine N-oxide, 1.05 ± 0.42 g [n = 6], P = 0.0052) and cold hypersensitivity (saline, 6.89 ± 0.88 s [n = 5] vs. clozapine N-oxide, 2.31 ± 0.52 s [n = 5], P = 0.0017). The authors conclude that nerve injury sensitizes pronociceptive µ opioid receptor-expressing neurons in mouse dorsal horn. Nonopioid strategies to inhibit these interneurons might yield new treatments for neuropathic pain.

Early zygotic gene product Dunk interacts with anillin to regulate Myosin II during Drosophila cleavage.

Drosophila melanogaster cellularization is a special form of cleavage that converts syncytial embryos into cellular blastoderms by partitioning the peripherally localized nuclei into individual cells. An early event in cellularization is the recruitment of nonmuscle myosin II ("myosin") to the leading edge of cleavage furrows, where myosin forms an interconnected basal array before reorganizing into individual cytokinetic rings. The initial recruitment and organization of basal myosin are regulated by a cellularization-specific gene, dunk, but the underlying mechanism is unclear. Through a genome-wide yeast two-hybrid screen, we identified anillin (Scraps in Drosophila), a conserved scaffolding protein in cytokinesis, as the primary binding partner of Dunk. Dunk colocalizes with anillin and regulates its cortical localization during the formation of cleavage furrows, while the localization of Dunk is independent of anillin. Furthermore, Dunk genetically interacts with anillin to regulate the basal myosin array during cellularization. Similar to Dunk, anillin colocalizes with myosin since the very early stage of cellularization and is required for myosin retention at the basal array, before the well-documented function of anillin in regulating cytokinetic ring assembly. Based on these results, we propose that Dunk regulates myosin recruitment and spatial organization during early cellularization by interacting with and regulating anillin.

Aged mice are less susceptible to motion sickness and show decreased efferent vestibular activity compared to young adults.

The efferent vestibular system (EVS) is a feedback circuit thought to modulate vestibular afferent activity by inhibiting type II hair cells and exciting calyx-bearing afferents in the peripheral vestibular organs. In a previous study, we suggested EVS activity may contribute to the effects of motion sickness. To determine an association between motion sickness and EVS activity, we examined the effects of provocative motion (PM) on c-Fos expression in brainstem efferent vestibular nucleus (EVN) neurons that are the source of efferent innervation in the peripheral vestibular organs. c-Fos is an immediate early gene product expressed in stimulated neurons and is a well-established marker of neuronal activation. To study the effects of PM, young adult C57/BL6 wild-type (WT), aged WT, and young adult transgenic Chat-gCaMP6f mice were exposed to PM, and tail temperature (Ttail ) was monitored using infrared imaging. After PM, we used immunohistochemistry to label EVN neurons to determine any changes in c-Fos expression. All tissue was imaged using laser scanning confocal microscopy. Infrared recording of Ttail during PM indicated that young adult WT and transgenic mice displayed a typical motion sickness response (tail warming), but not in aged WT mice. Similarly, brainstem EVN neurons showed increased expression of c-Fos protein after PM in young adult WT and transgenic mice but not in aged cohorts. We present evidence that motion sickness symptoms and increased activation of EVN neurons occur in young adult WT and transgenic mice in response to PM. In contrast, aged WT mice showed no signs of motion sickness and no change in c-Fos expression when exposed to the same provocative stimulus.

Viral DNA Accumulation Regulates Replication Efficiency of Chlorovirus OSy-NE5 in Two Closely Related Chlorella variabilis Strains.

Many chloroviruses replicate in Chlorella variabilis algal strains that are ex-endosymbionts isolated from the protozoan Paramecium bursaria, including the NC64A and Syngen 2-3 strains. We noticed that indigenous water samples produced a higher number of plaque-forming viruses on C. variabilis Syngen 2-3 lawns than on C. variabilis NC64A lawns. These observed differences led to the discovery of viruses that replicate exclusively in Syngen 2-3 cells, named Only Syngen (OSy) viruses. Here, we demonstrate that OSy viruses initiate infection in the restricted host NC64A by synthesizing some early virus gene products and that approximately 20% of the cells produce a small number of empty virus capsids. However, the infected cells did not produce infectious viruses because the cells were unable to replicate the viral genome. This is interesting because all previous attempts to isolate host cells resistant to chlorovirus infection were due to changes in the host receptor for the virus.

Detection of Arc/Arg3.1 oligomers in rat brain: constitutive and synaptic activity-evoked dimer expression in vivo.

The immediate early gene product activity-regulated cytoskeleton-associated protein (Arc or Arg3.1) is a major regulator of long-term synaptic plasticity with critical roles in postnatal cortical development and memory formation. However, the molecular basis of Arc function is undefined. Arc is a hub protein with interaction partners in the postsynaptic neuronal compartment and nucleus. Previous in vitro biochemical and biophysical analysis of purified recombinant Arc showed formation of low-order oligomers and larger particles including retrovirus-like capsids. Here, we provide evidence for naturally occurring Arc oligomers in the mammalian brain. Using in situ protein crosslinking to trap weak Arc-Arc interactions, we identified in various preparations a prominent Arc immunoreactive band on SDS-PAGE of molecular mass corresponding to a dimer. While putative trimers, tetramers and heavier Arc species were detected, they were of lower abundance. Stimulus-evoked induction of Arc expression and dimer formation was first demonstrated in SH-SY5Y neuroblastoma cells treated with the muscarinic cholinergic agonist, carbachol, and in primary cortical neuronal cultures treated with brain-derived neurotrophic factor (BDNF). In the dentate gyrus (DG) of adult anesthetized rats, induction of long-term potentiation (LTP) by high-frequency stimulation (HFS) of medial perforant synapses or by brief intrahippocampal infusion of BDNF led to a massive increase in Arc dimer expression. Arc immunoprecipitation of crosslinked DG tissue showed enhanced dimer expression during 4 h of LTP maintenance. Mass spectrometric proteomic analysis of immunoprecipitated, gel-excised bands corroborated detection of Arc dimer. Furthermore, Arc dimer was constitutively expressed in naïve cortical, hippocampal and DG tissue, with the lowest levels in the DG. Taken together the results implicate Arc dimer as the predominant low-oligomeric form in mammalian brain, exhibiting regional differences in its constitutive expression and enhanced synaptic activity-evoked expression in LTP.

Sex-Dependent Attentional Impairments in a Subchronic Ketamine Mouse Model for Schizophrenia

BackgroundThe development of more effective treatments for schizophrenia targeting cognitive and negative symptoms has been limited, partly due to a disconnect between rodent models and human illness. Ketamine administration is widely used to model symptoms of schizophrenia in both humans and rodents. In mice, subchronic ketamine treatment reproduces key dopamine and glutamate dysfunction; however, it is unclear how this translates into behavioral changes reflecting positive, negative, and cognitive symptoms. MethodsIn male and female mice treated with either subchronic ketamine or saline, we assessed spontaneous and amphetamine-induced locomotor activity to measure behaviors relevant to positive symptoms, and used a touchscreen-based progressive ratio task of motivation and the rodent continuous performance test of attention to capture specific negative and cognitive symptoms, respectively. To explore neuronal changes underlying the behavioral effects of subchronic ketamine treatment, we quantified expression of the immediate early gene product, c-Fos, in key corticostriatal regions using immunofluorescence. ResultsWe showed that spontaneous locomotor activity was unchanged in male and female subchronic ketamine–treated animals, and amphetamine-induced locomotor response was reduced. Subchronic ketamine treatment did not alter motivation in either male or female mice. In contrast, we identified a sex-specific effect of subchronic ketamine on attentional processing wherein female mice performed worse than control mice due to increased nonselective responding. Finally, we showed that subchronic ketamine treatment increased c-Fos expression in prefrontal cortical and striatal regions, consistent with a mechanism of widespread disinhibition of neuronal activity. ConclusionsOur results highlight that the subchronic ketamine mouse model reproduces a subset of behavioral symptoms that are relevant for schizophrenia.

834 Inhibition of the Epileptic Engram Prevents Seizures in a Mouse Model of Temporal Lobe Epilepsy

INTRODUCTION: The cellular basis of temporal lobe epilepsy (TLE) remains elusive and consequently has hindered efforts to develop novel therapies. Similar challenges have plagued the field of learning and memory until recent work identified plasticity of a subset of neurons in a multi-focal network as the cellular source for memory (the memory engram). Building on these results, we ask if an engram exists for TLE and if it can be targeted to modulate the epileptic phenotype? METHODS: We used the intra-amygdala kainic acid status-epilepticus (SE) model in which a brief episode of SE results in spontaneous seizures days later. To identify cells active during epileptogenesis and thus possibly comprising the engram, we performed immunohistochemistry for the immediate early gene product c-Fos (a marker of cellular activity) at various time points after SE (1 to 96 hours). To modulate the persistently active cells, we used a chemical-genetic approach to first label these cells with an inhibitory DREADD (designer receptors exclusively activated by designer drugs) and then inhibit their activity with clozapine-N-oxide (a small molecular activator of DREADDs). RESULTS: We found a pattern of c-Fos expression that first appears after SE in the dentate gyrus, begins to localize in subsets of CA3 and CA1 pyramidal neurons by 12 hours, and then persists in these cells for approximately 72 hours. With our chemical-genetic approach, we successfully labeled the cells active after SE with an inhibitory DREADD and then silenced their activity once the animals were epileptic. Such silencing proved effective in significantly reducing the occurrence of subsequent seizure activity. CONCLUSIONS: Following an epilepsy-inducing insult, a subset of hippocampal neurons remains persistently active for several days. Subsequent inhibition of these neurons in epileptic animals prevents seizures, consistent with them comprising the epileptic engram.

Effects of footshock stress on social behavior and neuronal activation in the medial prefrontal cortex and amygdala of male and female mice.

Social behavior is complex and fundamental, and its deficits are common pathological features for several psychiatric disorders including anxiety, depression, and posttraumatic stress disorder. Acute stress may have a negative impact on social behavior, and these effects can vary based on sex. The aim of this study was to explore the effect of acute footshock stress, using analogous parameters to those commonly used in fear conditioning assays, on the sociability of male and female C57BL/6J mice in a standard social approach test. Animals were divided into two main groups of footshock stress (22 male, 24 female) and context exposed control (23 male and 22 female). Each group had mice that were treated intraperitoneally with either the benzodiazepine-alprazolam (control: 10 male, 10 female; stress: 11 male, 11 female), or vehicle (control: 13 male, 12 female; stress: 11 male, 13 female). In all groups, neuronal activation during social approach was assessed using immunohistochemistry against the immediate early gene product cFos. Although footshock stress did not significantly alter sociability or latency to approach a social stimulus, it did increase defensive tail-rattling behavior specifically in males (p = 0.0022). This stress-induced increase in tail-rattling was alleviated by alprazolam (p = 0.03), yet alprazolam had no effect on female tail-rattling behavior in the stress group. Alprazolam lowered cFos expression in the medial prefrontal cortex (p = 0.001 infralimbic area, p = 0.02 prelimbic area), and social approach induced sex-dependent differences in cFos activation in the ventromedial intercalated cell clusters (p = 0.04). Social approach following stress-induced cFos expression was positively correlated with latency to approach and negatively correlated with sociability in the prelimbic area and multiple amygdala subregions (all p < 0.05). Collectively, our results suggest that acute footshock stress induces sex-dependent alterations in defensiveness and differential patterns of cFos activation during social approach.

Retinoblastoma Protein Is Required for Epstein-Barr Virus Replication in Differentiated Epithelia.

Coinfection of human papillomavirus (HPV) and Epstein-Barr virus (EBV) has been detected in oropharyngeal squamous cell carcinoma. Although HPV and EBV replicate in differentiated epithelial cells, we previously reported that HPV epithelial immortalization reduces EBV replication within organotypic raft culture and that the HPV16 oncoprotein E7 was sufficient to inhibit EBV replication. A well-established function of HPV E7 is the degradation of the retinoblastoma (Rb) family of pocket proteins (pRb, p107, and p130). Here, we show that pRb knockdown in differentiated epithelia and EBV-positive Burkitt lymphoma (BL) reduces EBV lytic replication following de novo infection and reactivation, respectively. In differentiated epithelia, EBV immediate early (IE) transactivators were expressed, but loss of pRb blocked expression of the early gene product, EA-D. Although no alterations were observed in markers of epithelial differentiation, DNA damage, and p16, increased markers of S-phase progression and altered p107 and p130 levels were observed in suprabasal keratinocytes after pRb knockdown. In contrast, pRb interference in Akata BX1 Burkitt lymphoma cells showed a distinct phenotype from differentiated epithelia with no significant effect on EBV IE or EA-D expression. Instead, pRb knockdown reduced the levels of the plasmablast differentiation marker PRDM1/Blimp1 and increased the abundance of c-Myc protein in reactivated Akata BL with pRb knockdown. c-Myc RNA levels also increased following the loss of pRb in epithelial rafts. These results suggest that pRb is required to suppress c-Myc for efficient EBV replication in BL cells and identifies a mechanism for how HPV immortalization, through degradation of the retinoblastoma pocket proteins, interferes with EBV replication in coinfected epithelia. IMPORTANCE Terminally differentiated epithelium is known to support EBV genome amplification and virion morphogenesis following infection. The contribution of the cell cycle in differentiated tissues to efficient EBV replication is not understood. Using organotypic epithelial raft cultures and genetic interference, we can identify factors required for EBV replication in quiescent cells. Here, we phenocopied HPV16 E7 inhibition of EBV replication through knockdown of pRb. Loss of pRb was found to reduce EBV early gene expression and viral replication. Interruption of the viral life cycle was accompanied by increased S-phase gene expression in postmitotic keratinocytes, a process also observed in E7-positive epithelia, and deregulation of other pocket proteins. Together, these findings provide evidence of a global requirement for pRb in EBV lytic replication and provide a mechanistic framework for how HPV E7 may facilitate a latent EBV infection through its mediated degradation of pRb in copositive epithelia.

EBV dUTPase: A Novel Modulator of Inflammation and the Tumor Microenvironment in EBV-Associated Malignancies.

There is increasing evidence that put into question the classical dogma that the Epstein-Barr virus (EBV) exists in cells as either a lytic virus in which new progeny is produced or in a latent state in which no progeny is produced. Notably, a third state has now been described, known as the abortive-lytic phase, which is characterized by the expression of some immediate early (IE) and early (E) genes, but no new virus progeny is produced. While the function of these IE and E gene products is not well understood, several recent studies support the concept they may contribute to tumor promotion by altering the tumor microenvironment (TME). The mechanisms by which these viral gene products may contribute to tumorigenesis remain unclear; however, it has been proposed that some of them promote cellular growth, immune evasion, and/or inhibit apoptosis. One of these EBV early gene products is the deoxyuridine triphosphate nucleotidohydrolase (dUTPase) encoded by BLLF3, which not only contributes to the establishment of latency through the production of activin A and IL-21, but it may also alter the TME, thus promoting oncogenesis.

ADAM9: A regulator between HCMV infection and function of smooth muscle cells.

Lots of epidemiological and clinical studies have shown that human cytomegalovirus (HCMV) is related to the pathogenesis of atherosclerosis. Released by inflammatory cells and vascular smooth muscle cell (VSMCs), metalloproteinases are observed in many pathological vessel conditions, including atherosclerosis and restenosis.This study was designed to investigate the effect of HCMV infection on the expression of metalloproteinases and their involvements in the HCMV-induced functional changes of VSMCs.Differential metalloproteinase after HCMV infection was assayed using reverse transcription-polymerase chain reaction (RT-PCR) microarray. The most significant increased a disintegrin and metalloprotease 9 (ADAM9) was chosen to investigate the mechanism of its specific increase after infection using the treatment of UV-irradiated replication-deficient HCMV, HCMV-infected cell lysate filters or Foscarnet. The function of proliferation, migration, production of inflammatoty factors and phenotypic transformation were determined by using cell counting kit-8, transwell, Enzyme-linked immunosorbent assay, RT-quantitative PCR(qPCR) and Western blot, respectively. Moreover, the effect of ADAM9 deficiency on HCMV replication was also determined using RT-qPCR and immunofluorescence.The expression levels of 6 genes were upregulated and 14 genes were downregulated at different time points after HCMV infection. Among these, the expression level of ADAM9 increased most significantly at each time point and the abnormal expression of ADAM9 might be induced by the early gene products of HCMV. Further studies found that ADAM9 promoted the proliferation, the migration, the production of inflammatory factors and the transit from the contractile phenotype (decreased ACTA2 expression) to the synthetic phenotype (increased osteopontin [OPN] expression). Knockdown theADAM9expression could rescue the decreased ACTA2 expression, but has no effect on OPN expression. ADAM-9 deficiency didn't affect the replication of HCMV.The findings of our study suggest that HCMV infection changed VSMC function through ADAM9 expression, which may contribute to the understanding of the underlying pathological mechanisms of HCMV-induced atherosclerosis.