Cellular Iron Deficiency Disrupts Thyroid Hormone Regulated Gene Expression in Developing Hippocampal Neurons.

BACKGROUND: Developing neurons have high thyroid hormone and iron requirements to support their metabolically demanding growth. Early-life iron and thyroid-hormone deficiencies are prevalent and often coexist, and each independently increases risk of permanently impaired neurobehavioral function in children. Early-life dietary iron deficiency reduces thyroid-hormone concentrations and impairs thyroid hormone-responsive gene expression in the neonatal rat brain, but it is unclear whether the effect is cell-intrinsic. OBJECTIVES: This study determined whether neuronal-specific iron deficiency alters thyroid hormone-regulated gene expression in developing neurons. METHODS: Iron deficiency was induced in primary mouse embryonic hippocampal neuron cultures with the iron chelator deferoxamine (DFO) beginning at 3 d in vitro (DIV). At 11DIV and 18DIV, thyroid hormone-regulated gene messenger ribonucleic acid (mRNA)concentrations indexing thyroid hormone homeostasis (Hairless, mu-crystallin, Type II deiodinase, solute carrier family member 1c1, and solute carrier family member 16a2) and neurodevelopment (neurogranin, Parvalbumin, and Krüppel-like factor 9) were quantified. To assess the effect of iron repletion, DFO was removed at 14DIV from a subset of DFO-treated cultures, and gene expression and adenosine 5'-triphosphate (ATP) concentrations were quantified at 21DIV. RESULTS: At 11DIV and 18DIV, neuronal iron deficiency decreased neurogranin, Parvalbumin, and mu-crystallin, and by 18DIV, solute carrier family member 16a2, solute carrier family member 1c1, Type II deiodinase, and Hairless were increased, suggesting cellular sensing of a functionally abnormal thyroid hormone state. Dimensionality reduction with Principal component analysis reveals that thyroid hormone homeostatic genes strongly correlate with and predict iron status. Iron repletion from 14-21DIV did not restore ATP concentration, and Principal component analysis suggests that, after iron repletion, cultures maintain a gene expression signature indicative of previous iron deficiency. CONCLUSIONS: These novel findings suggest there is an intracellular mechanism coordinating cellular iron/thyroid hormone activities. We speculate this is a part of the homeostatic response to acutely match neuronal energy production and growth signaling. However, the adaptation to iron deficiency may cause permanent deficits in thyroid hormone-dependent neurodevelopmental processes even after recovery from iron deficiency.

Cellular Iron Deficiency Disrupts Thyroid Hormone Regulated Gene Expression in Developing Hippocampal Neurons.

Background: Developing neurons have high thyroid hormone and iron requirements to support their metabolism and growth. Early-life iron and thyroid hormone deficiencies are prevalent, often coexist, and increase the risk of permanently impaired neurobehavioral function in children. Early-life dietary iron deficiency reduces thyroid hormone levels and impairs thyroid hormone-responsive gene expression in the neonatal rat brain. Objective: This study determined whether neuronal-specific iron deficiency alters thyroid hormone-regulated gene expression in developing neurons. Methods: Iron deficiency was induced in primary mouse embryonic hippocampal neuron cultures with the iron chelator deferoxamine (DFO) beginning at 3 days in vitro (DIV). At 11DIV and 18DIV, mRNA levels for thyroid hormone-regulated genes indexing thyroid hormone homeostasis (Hr, Crym, Dio2, Slco1c1, Slc16a2) and neurodevelopment (Nrgn, Pvalb, Klf9) were quantified. To assess the effect of iron repletion, DFO was removed at 14DIV from a subset of DFO-treated cultures and gene expression and ATP levels were quantified at 21DIV. Results: At 11DIV and 18DIV, neuronal iron deficiency decreased Nrgn, Pvalb, and Crym, and by 18DIV, Slc16a2, Slco1c1, Dio2, and Hr were increased; collectively suggesting cellular sensing of a functionally abnormal thyroid hormone state. Dimensionality reduction with Principal Component Analysis (PCA) reveals that thyroid hormone homeostatic genes strongly correlate with and predict iron status (Tfr1 mRNA). Iron repletion from 14-21DIV restored neurodevelopmental genes, but not all thyroid hormone homeostatic genes, and ATP concentrations remained significantly altered. PCA clustering suggests that cultures replete with iron maintain a gene expression signature indicative of previous iron deficiency. Conclusions: These novel findings suggest there is an intracellular mechanism coordinating cellular iron/thyroid hormone activities. We speculate this is a part of homeostatic response to match neuronal energy production and growth signaling for these important metabolic regulators. However, iron deficiency may cause permanent deficits in thyroid hormone-dependent neurodevelopmental processes even after recovery from iron deficiency.

Quantitative omics analyses of NCOA4 deficiency reveal an integral role of ferritinophagy in iron homeostasis of hippocampal neuronal HT22 cells.

Introduction: Neurons require iron to support their metabolism, growth, and differentiation, but are also susceptible to iron-induced oxidative stress and cytotoxicity. Ferritin, a cytosolic iron storage unit, mediates cellular adaptation to fluctuations in iron delivery. NCOA4 has been characterized as a selective autophagic cargo receptor facilitating the mobilization of intracellular iron from ferritin. This process named ferritinophagy results in the degradation of ferritin and the consequent release of iron into the cytosol. Methods: Here we demonstrate that NCOA4 is important for the adaptation of the HT22 mouse hippocampal neuronal cell line to cellular iron restriction. Additionally, we determined the pathophysiological implications of impaired ferritinophagy via functional analysis of the omics profile of HT22 cells deficient in NCOA4. Results: NCOA4 silencing impaired ferritin turnover and was cytotoxic when cells were restricted of iron. Quantitative proteomics identified IRP2 accumulation among the most prominent protein responses produced by NCOA4 depletion in HT22 cells, which is indicative of functional iron deficiency. Additionally, proteins of apoptotic signaling pathway were enriched by those responsive to NCOA4 deficiency. Transcriptome profiles of NCOA4 depletion revealed neuronal cell death, differentiation of neurons, and development of neurons as potential diseases and bio functions affected by impaired ferritinophagy, particularly, when iron was restricted. Discussion: These findings identify an integral role of NCOA4-mediated ferritinophagy in the maintenance of iron homeostasis by HT22 cells, and its potential implications in controlling genetic pathways of neurodevelopment and neurodegenerative diseases.

Choline Supplementation Partially Restores Dendrite Structural Complexity in Developing Iron-Deficient Mouse Hippocampal Neurons.

BACKGROUND: Fetal-neonatal iron deficiency causes learning/memory deficits that persist after iron repletion. Simplified hippocampal neuron dendrite structure is a key mechanism underlying these long-term impairments. Early life choline supplementation, with postnatal iron repletion, improves learning/memory performance in formerly iron-deficient (ID) rats. OBJECTIVES: To understand how choline improves iron deficiency-induced hippocampal dysfunction, we hypothesized that direct choline supplementation of ID hippocampal neurons may restore cellular energy production and dendrite structure. METHODS: Embryonic mouse hippocampal neuron cultures were made ID with 9 µM deferoxamine beginning at 3 d in vitro (DIV). At 11 DIV, iron repletion (i.e., deferoxamine removal) was performed on a subset of ID cultures. These neuron cultures and iron-sufficient (IS) control cultures were treated with 30 µM choline (or vehicle) between 11 and 18 DIV. At 18 DIV, the independent and combined effects of iron and choline treatments (2-factor ANOVA) on neuronal dendrite numbers, lengths, and overall complexity and mitochondrial respiration and glycolysis were analyzed. RESULTS: Choline treatment of ID neurons (ID + Cho) significantly increased overall dendrite complexity (150, 160, 180, and 210 µm from the soma) compared with untreated ID neurons to a level of complexity that was no longer significantly different from IS neurons. The average and total length of primary dendrites in ID + Cho neurons were significantly increased by ∼15% compared with ID neurons, indicating choline stimulation of dendrite growth. Measures of mitochondrial respiration, glycolysis, and ATP production rates were not significantly altered in ID + Cho neurons compared with ID neurons, remaining significantly reduced compared with IS neurons. Iron repletion significantly improved mitochondrial respiration, ATP production rates, overall dendrite complexity (100-180 µm from the soma), and dendrite and branch lengths compared with untreated ID neurons. CONCLUSIONS: Because choline partially restores dendrite structure in ID neurons without iron repletion, it may have therapeutic potential when iron treatment is not possible or advisable. Choline's mechanism in ID neurons requires further investigation.

The Effects of Early-Life Iron Deficiency on Brain Energy Metabolism.

Iron deficiency (ID) is one of the most prevalent nutritional deficiencies in the world. Iron deficiency in the late fetal and newborn period causes abnormal cognitive performance and emotional regulation, which can persist into adulthood despite iron repletion. Potential mechanisms contributing to these impairments include deficits in brain energy metabolism, neurotransmission, and myelination. Here, we comprehensively review the existing data that demonstrate diminished brain energetic capacity as a mechanistic driver of impaired neurobehavioral development due to early-life (fetal-neonatal) ID. We further discuss a novel hypothesis that permanent metabolic reprogramming, which occurs during the period of ID, leads to chronically impaired neuronal energetics and mitochondrial capacity in adulthood, thus limiting adult neuroplasticity and neurobehavioral function. We conclude that early-life ID impairs energy metabolism in a brain region- and age-dependent manner, with particularly strong evidence for hippocampal neurons. Additional studies, focusing on other brain regions and cell types, are needed.

Potential Mechanisms Driving Mitochondrial Motility Impairments in Developing Iron-Deficient Neurons.

Brain development is highly demanding energetically, requiring neurons to have tightly regulated and highly dynamic metabolic machinery to achieve their ultimately complex cellular architecture. Mitochondria are the main source of neuronal adenosine 5'-triphosphate (ATP) and regulate critical neurodevelopmental processes including calcium signaling, iron homeostasis, oxidative stress, and apoptosis. Metabolic perturbations during critical neurodevelopmental windows impair neurological function not only acutely during the period of rapid growth/development, but also in adulthood long after the early-life insult has been rectified. Our laboratory uses iron deficiency (ID), the most common nutrient deficiency, as a model of early-life metabolic disruptions of neuronal metabolism because iron has a central role in mitochondrial function. Recently, we published that ID reduces hippocampal neuronal dendritic mitochondrial motility and size. In this commentary, we delve deeper into speculation about potential cellular mechanisms that drive the effects of neuronal ID on mitochondrial dynamics and quality control pathways. We propose that understanding the basic cellular biology of how mitochondria respond and adapt to ID and other metabolic perturbations during brain development may be a key factor in designing strategies to reduce the risk of later-life psychiatric, cognitive, and neurodegenerative disorders associated with early-life ID.

Chronic Energy Depletion due to Iron Deficiency Impairs Dendritic Mitochondrial Motility during Hippocampal Neuron Development.

During development, neurons require highly integrated metabolic machinery to meet the large energy demands of growth, differentiation, and synaptic activity within their complex cellular architecture. Dendrites/axons require anterograde trafficking of mitochondria for local ATP synthesis to support these processes. Acute energy depletion impairs mitochondrial dynamics, but how chronic energy insufficiency affects mitochondrial trafficking and quality control during neuronal development is unknown. Because iron deficiency impairs mitochondrial respiration/ATP production, we treated mixed-sex embryonic mouse hippocampal neuron cultures with the iron chelator deferoxamine (DFO) to model chronic energetic insufficiency and its effects on mitochondrial dynamics during neuronal development. At 11 days in vitro (DIV), DFO reduced average mitochondrial speed by increasing the pause frequency of individual dendritic mitochondria. Time spent in anterograde motion was reduced; retrograde motion was spared. The average size of moving mitochondria was reduced, and the expression of fusion and fission genes was altered, indicating impaired mitochondrial quality control. Mitochondrial density was not altered, suggesting that respiratory capacity and not location is the key factor for mitochondrial regulation of early dendritic growth/branching. At 18 DIV, the overall density of mitochondria within terminal dendritic branches was reduced in DFO-treated neurons, which may contribute to the long-term deficits in connectivity and synaptic function following early-life iron deficiency. The study provides new insights into the cross-regulation between energy production and dendritic mitochondrial dynamics during neuronal development and may be particularly relevant to neuropsychiatric and neurodegenerative diseases, many of which are characterized by impaired brain iron homeostasis, energy metabolism and mitochondrial trafficking.SIGNIFICANCE STATEMENT This study uses a primary neuronal culture model of iron deficiency to address a gap in understanding of how dendritic mitochondrial dynamics are regulated when energy depletion occurs during a critical period of neuronal maturation. At the beginning of peak dendritic growth/branching, iron deficiency reduces mitochondrial speed through increased pause frequency, decreases mitochondrial size, and alters fusion/fission gene expression. At this stage, mitochondrial density in terminal dendrites is not altered, suggesting that total mitochondrial oxidative capacity and not trafficking is the main mechanism underlying dendritic complexity deficits in iron-deficient neurons. Our findings provide foundational support for future studies exploring the mechanistic role of developmental mitochondrial dysfunction in neurodevelopmental, psychiatric, and neurodegenerative disorders characterized by mitochondrial energy production and trafficking deficits.

Iron Deficiency Impairs Developing Hippocampal Neuron Gene Expression, Energy Metabolism, and Dendrite Complexity.

Iron deficiency (ID), with and without anemia, affects an estimated 2 billion people worldwide. ID is particularly deleterious during early-life brain development, leading to long-term neurological impairments including deficits in hippocampus-mediated learning and memory. Neonatal rats with fetal/neonatal ID anemia (IDA) have shorter hippocampal CA1 apical dendrites with disorganized branching. ID-induced dendritic structural abnormalities persist into adulthood despite normalization of the iron status. However, the specific developmental effects of neuronal iron loss on hippocampal neuron dendrite growth and branching are unknown. Embryonic hippocampal neuron cultures were chronically treated with deferoxamine (DFO, an iron chelator) beginning at 3 days in vitro (DIV). Levels of mRNA for Tfr1 and Slc11a2, iron-responsive genes involved in iron uptake, were significantly elevated in DFO-treated cultures at 11DIV and 18DIV, indicating a degree of neuronal ID similar to that seen in rodent ID models. DFO treatment decreased mRNA levels for genes indexing dendritic and synaptic development (i.e. BdnfVI,Camk2a,Vamp1,Psd95,Cfl1, Pfn1,Pfn2, and Gda) and mitochondrial function (i.e. Ucp2,Pink1, and Cox6a1). At 18DIV, DFO reduced key aspects of energy metabolism including basal respiration, maximal respiration, spare respiratory capacity, ATP production, and glycolytic rate, capacity, and reserve. Sholl analysis revealed a significant decrease in distal dendritic complexity in DFO-treated neurons at both 11DIV and 18DIV. At 11DIV, the length of primary dendrites and the number and length of branches in DFO-treated neurons were reduced. By 18DIV, partial recovery of the dendritic branch number in DFO-treated neurons was counteracted by a significant reduction in the number and length of primary dendrites and the length of branches. Our findings suggest that early neuronal iron loss, at least partially driven through altered mitochondrial function and neuronal energy metabolism, is responsible for the effects of fetal/neonatal ID and IDA on hippocampal neuron dendritic and synaptic maturation. Impairments in these neurodevelopmental processes likely underlie the negative impact of early life ID and IDA on hippocampus-mediated learning and memory.

Fetal and neonatal iron deficiency but not copper deficiency increases vascular complexity in the developing rat brain.

OBJECTIVES: Anemia caused by nutritional deficiencies, such as iron and copper deficiencies, is a global health problem. Iron and copper deficiencies have their most profound effect on the developing fetus/infant, leading to brain development deficits and poor cognitive outcomes. Tissue iron depletion or chronic anemia can induce cellular hypoxic signaling. In mice, chronic hypoxia induces a compensatory increase in brain blood vessel outgrowth. We hypothesized that developmental anemia, due to iron or copper deficiencies, induces angiogenesis/vasculogenesis in the neonatal brain. METHODS: To test our hypothesis, three independent experiments were performed where pregnant rats were fed iron- or copper-deficient diets from gestational day 2 through mid-lactation. Effects on the neonatal brain vasculature were determined using quantitative real-time polymerase chain reaction to assess mRNA levels of angiogenesis/vasculogenesis-associated genes and GLUT1 immunohistochemistry to assess brain blood vessel density and complexity. RESULTS: Iron deficiency, but not copper deficiency, increased mRNA expression of brain endothelial cell- and angiogenesis/vasculogenesis-associated genes (i.e. Glut1, Vwf, Vegfa, Ang2, Cxcl12, and Flk1) in the neonatal brain, suggesting increased cerebrovascular density. Iron deficiency also increased hippocampal and cerebral cortical blood vessel branching by 62 and 78%, respectively. DISCUSSION: This study demonstrates increased blood vessel complexity in the neonatal iron-deficient brain, which is likely due to elevated angiogenic/vasculogenic signaling. At least initially, this is probably an adaptive response to maintain metabolic substrate homeostasis in the developing iron-deficient brain. However, this may also contribute to long-term neurodevelopmental deficits.

Fetal and neonatal iron deficiency exacerbates mild thyroid hormone insufficiency effects on male thyroid hormone levels and brain thyroid hormone-responsive gene expression.

Fetal/neonatal iron (Fe) and iodine/TH deficiencies lead to similar brain developmental abnormalities and often coexist in developing countries. We recently demonstrated that fetal/neonatal Fe deficiency results in a mild neonatal thyroidal impairment, suggesting that TH insufficiency contributes to the neurodevelopmental abnormalities associated with Fe deficiency. We hypothesized that combining Fe deficiency with an additional mild thyroidal perturbation (6-propyl-2-thiouracil [PTU]) during development would more severely impair neonatal thyroidal status and brain TH-responsive gene expression than either deficiency alone. Early gestation pregnant rats were assigned to 7 different treatment groups: control, Fe deficient (FeD), mild TH deficient (1 ppm PTU), moderate TH deficient (3 ppm PTU), severe TH deficient (10 ppm PTU), FeD/1 ppm PTU, or FeD/3 ppm PTU. FeD or 1 ppm PTU treatment alone reduced postnatal day 15 serum total T4 concentrations by 64% and 74%, respectively, without significantly altering serum total T3 concentrations. Neither treatment alone significantly altered postnatal day 16 cortical or hippocampal T3 concentrations. FeD combined with 1 ppm PTU treatment produced a more severe effect, reducing serum total T4 by 95%, and lowering hippocampal and cortical T3 concentrations by 24% and 31%, respectively. Combined FeD/PTU had a more severe effect on brain TH-responsive gene expression than either treatment alone, significantly altering Pvalb, Dio2, Mbp, and Hairless hippocampal and/or cortical mRNA levels. FeD/PTU treatment more severely impacted cortical and hippocampal parvalbumin protein expression compared with either individual treatment. These data suggest that combining 2 mild thyroidal insults during development significantly disrupts thyroid function and impairs TH-regulated brain gene expression.

Fetal and neonatal iron deficiency reduces thyroid hormone-responsive gene mRNA levels in the neonatal rat hippocampus and cerebral cortex.

Copper (Cu), iron (Fe), and thyroid hormone (TH) deficiencies produce similar defects in late brain development including hypomyelination of axons and impaired synapse formation and function, suggesting that these micronutrient deficiencies share a common mechanism contributing to these derangements. We previously demonstrated that fetal/neonatal Cu and Fe deficiencies lower circulating TH concentrations in neonatal rats. Fe deficiency also reduces whole-brain T(3) content, suggesting impaired TH action in the developing Fe-deficient brain. We hypothesized that fetal/neonatal Cu and Fe deficiencies will produce mild or moderate TH deficiencies and will impair TH-responsive gene expression in the neonatal cerebral cortex and hippocampus. To test this hypothesis, we rendered pregnant Sprague Dawley rats Cu-, Fe-, or TH-deficient from early gestation through postnatal d 10 (P10). Mild and moderate TH deficiencies were induced by 1 and 3 ppm propylthiouracil treatment, respectively. Cu deficiency did not significantly alter serum or tissue TH concentrations or TH-responsive brain mRNA expression. Fe deficiency significantly lowered P10 serum total T(3) (45%), serum total T(4) (52%), whole brain T(3) (14%), and hippocampal T(3) (18%) concentrations, producing a mild TH deficiency similar to 1 ppm propylthiouracil treatment. Fe deficiency lowered Pvalb, Enpp6, and Mbp mRNA levels in the P10 hippocampus. Fe deficiency also altered Hairless, Dbm, and Dio2 mRNA levels in the P10 cerebral cortex. These results suggest that some of the brain defects associated with Fe deficiency may be mediated through altered thyroidal status and the concomitant alterations in TH-responsive gene transcription.

Maternal iron supplementation attenuates the impact of perinatal copper deficiency but does not eliminate hypotriiodothyroninemia nor impaired sensorimotor development.

Copper, iron and iodine/thyroid hormone (TH) deficiencies disrupt brain development. Neonatal Cu deficiency causes Fe deficiency and may impact thyroidal status. One purpose of these studies was to determine the impact of improved iron status following Cu deficiency by supplementing the diet with iron. Cu deficiency was produced in pregnant Holtzman [Experiment 1 (Exp. 1)] or Sprague-Dawley [Experiment 2 (Exp. 2)] rats using two different diets. In Exp. 2, dietary Fe content was increased from 35 to 75 mg/kg according to NRC guidelines for reproduction. Cu-deficient (CuD) Postnatal Day 24 (P24) rats from both experiments demonstrated lower hemoglobin, serum Fe and serum triiodothyronine (T3) concentrations. However, brain Fe was lower only in CuD P24 rats in Exp. 1. Hemoglobin and serum Fe were higher in Cu adequate (CuA) P24 rats from Exp. 2 compared to Exp. 1. Cu- and TH-deficient rats from Exp. 2 exhibited a similar sensorimotor functional deficit following 3 months of repletion. Results suggest that Cu deficiency may impact TH status independent of its impact on iron biology. Further research is needed to clarify the individual roles for Cu, Fe and TH in brain development.

Perinatal iron and copper deficiencies alter neonatal rat circulating and brain thyroid hormone concentrations.

Copper (Cu), iron (Fe), and iodine/thyroid hormone (TH) deficiencies lead to similar defects in late brain development, suggesting that these micronutrient deficiencies share a common mechanism contributing to the observed derangements. Previous studies in rodents (postweanling and adult) and humans (adolescent and adult) indicate that Cu and Fe deficiencies affect the hypothalamic-pituitary-thyroid axis, leading to altered TH status. Importantly, however, relationships between Fe and Cu deficiencies and thyroidal status have not been assessed in the most vulnerable population, the developing fetus/neonate. We hypothesized that Cu and Fe deficiencies reduce circulating and brain TH levels during development, contributing to the defects in brain development associated with these deficiencies. To test this hypothesis, pregnant rat dams were rendered Cu deficient (CuD), FeD, or TH deficient from early gestation through weaning. Serum thyroxine (T(4)) and triiodothyronine (T(3)), and brain T(3) levels, were subsequently measured in postnatal d 12 (P12) pups. Cu deficiency reduced serum total T(3) by 48%, serum total T(4) by 21%, and whole-brain T(3) by 10% at P12. Fe deficiency reduced serum total T(3) by 43%, serum total T(4) by 67%, and whole-brain T(3) by 25% at P12. Brain mRNA analysis revealed that expression of several TH-responsive genes were altered in CuD or FeD neonates, suggesting that reduced TH concentrations were sensed by the FeD and CuD neonatal brain. These results indicate that at least some of the brain defects associated with neonatal Fe and Cu deficiencies are mediated through reductions in circulating and brain TH levels.

Herpes simplex virus type 1 immediate-early protein ICP22 is required for VICE domain formation during productive viral infection.

During productive infection, herpes simplex virus type 1 (HSV-1) induces the formation of discrete nuclear foci containing cellular chaperone proteins, proteasomal components, and ubiquitinated proteins. These structures are known as VICE domains and are hypothesized to play an important role in protein turnover and nuclear remodeling in HSV-1-infected cells. Here we show that VICE domain formation in Vero and other cells requires the HSV-1 immediate-early protein ICP22. Since ICP22 null mutants replicate efficiently in Vero cells despite being unable to induce VICE domain formation, it can be concluded that VICE domain formation is not essential for HSV-1 productive infection. However, our findings do not exclude the possibility that VICE domain formation is required for viral replication in cells that are nonpermissive for ICP22 mutants. Our studies also show that ICP22 itself localizes to VICE domains, suggesting that it could play a role in forming these structures. Consistent with this, we found that ICP22 expression in transfected cells is sufficient to reorganize the VICE domain component Hsc70 into nuclear inclusion bodies that resemble VICE domains. An N-terminal segment of ICP22, corresponding to residues 1 to 146, is critical for VICE domain formation in infected cells and Hsc70 reorganization in transfected cells. We previously found that this portion of the protein is dispensable for ICP22's effects on RNA polymerase II phosphorylation. Thus, ICP22 mediates two distinct regulatory activities that both modify important components of the host cell nucleus.

Identification of sequences in herpes simplex virus type 1 ICP22 that influence RNA polymerase II modification and viral late gene expression.

Previous studies have shown that the herpes simplex virus type 1 (HSV-1) immediate-early protein ICP22 alters the phosphorylation of the host cell RNA polymerase II (Pol II) during viral infection. In this study, we have engineered several ICP22 plasmid and virus mutants in order to map the ICP22 sequences that are involved in this function. We identify a region in the C-terminal half of ICP22 (residues 240 to 340) that is critical for Pol II modification and further show that the N-terminal half of the protein (residues 1 to 239) is not required. However, immunofluorescence analysis indicates that the N-terminal half of ICP22 is needed for its localization to nuclear body structures. These results demonstrate that ICP22's effects on Pol II do not require that it accumulate in nuclear bodies. As ICP22 is known to enhance viral late gene expression during infection of certain cultured cells, including human embryonic lung (HEL) cells, we used our engineered viral mutants to map this function of ICP22. It was found that mutations in both the N- and C-terminal halves of ICP22 result in similar defects in viral late gene expression and growth in HEL cells, despite having distinctly different effects on Pol II. Thus, our results genetically uncouple ICP22's effects on Pol II from its effects on viral late gene expression. This suggests that these two functions of ICP22 may be due to distinct activities of the protein.