Corticosterone induced morphological changes of hippocampal and amygdaloid cell lines are dependent on 5-HT7 receptor related signal pathway.

Stress is an unavoidable life experience. It induces mood, cognitive dysfunction and plasticity changes in chronically stressed individuals. Among the various brain regions that have been studied, the hippocampus and amygdala have been observed to have different roles in controlling the limbic-hypothalamic-pituitary-adrenal axis (limbic-HPA axis). This study investigated how the stress hormone corticosterone (CORT) affects neuronal cells. The first aim is to test whether administration of CORT to hippocampal and amygdaloid cell lines induces different changes in the 5-HT receptor subtypes. The second goal is to determine whether stress induced morphological changes in these two cell lines were involved in the 5-HT receptor subtypes expression. We now show that 5-HT(7) receptor mRNA levels were significantly upregulated in HT-22 cells, but downregulated in AR-5 cells by exposure to a physiologically relevant level of CORT (50 µM) for 24 h, which was later confirmed by primary hippocampal and amygdaloid neuron cultures. Additionally, pretreatment of cells with 5-HT(7) antagonist SB-269970 or agonist LP-44 reversed CORT induced cell lesion in a dose-dependent manner. Moreover, CORT induced different changes in neurite length, number of neurites and soma size in HT-22 and AR-5 cells were also reversed by pretreatment with either SB-269970 or LP-44. The different effects of 5-HT(7) receptors on cell lines were observed in two members of the Rho family small GTPase expression: the Cdc-42 and RhoA. These observed results support the hypothesis that 5-HT may differentially modulate neuronal morphology in the hippocampus and amygdala depending on the expression levels of the 5-HT receptor subtypes during stress hormone insults.

Restructuring the neuronal stress response with anti-glucocorticoid gene delivery.

Glucocorticoids, the adrenal steroids released during stress, compromise the ability of neurons to survive neurological injury. In contrast, estrogen protects neurons against such injuries. We designed three genetic interventions to manipulate the actions of glucocorticoids, which reduced their deleterious effects in both in vitro and in vivo rat models. The most effective of these interventions created a chimeric receptor combining the ligand-binding domain of the glucocorticoid receptor and the DNA-binding domain of the estrogen receptor. Expression of this chimeric receptor reduced hippocampal lesion size after neurological damage by 63% and reversed the outcome of the stress response by rendering glucocorticoids protective rather than destructive. Our findings elucidate three principal steps in the neuronal stress-response pathway, all of which are amenable to therapeutic intervention.

Gene therapy and the aging nervous system.

In recent years, the first attempts have been made to apply gene transfer technology to protect neurons from death following neurological insults. There has been sufficient progress in this area that it becomes plausible to consider similar gene therapy approaches meant to delay aspects of aging of the nervous system. In this review, we briefly consider such progress and how it might be applied to the realm of the aging brain. Specifically, we consider: (a) the means of delivery of such therapeutic genes; (b) the choice of such genes; and (c) technical elaborations in gene delivery systems which can more tightly regulate the magnitude and duration of transgene protection.

Posttranslational processing of infected cell protein 22 mediated by viral protein kinases is sensitive to amino acid substitutions at distant sites and can be cell-type specific.

Infected cell protein 22 (ICP22) is posttranslationally phosphorylated by the viral kinases encoded by U(S)3 and U(L)13 and nucleotidylylated by casein kinase II. In rabbit and rodent cells and in primary human fibroblasts infected with mutants from which the alpha 22 gene encoding ICP22 had been deleted, a subset of late (gamma(2)) gene products exemplified by U(L)38 and U(S)11 proteins are expressed at a reduced level, as measured by the accumulation of both mRNA and protein. The same phenotype was observed in cells infected with mutants lacking the U(L)13 gene. The focus of this report is on three serine- and threonine-rich domains of ICP22. Two of these domains are homologs located between residues 38 to 66 and 300 to 328. The third domain is near the carboxyl terminus and contains the sequence T374SS. The results were as follows. (i) Alanine substitutions in the amino-terminal homolog precluded the posttranslational processing of ICP22 in rabbit skin cells and in Vero cells but had no effect on the accumulation of either U(S)11 or U(L)38 protein. (ii) Alanine substitutions in the carboxyl-terminal homolog had no effect on posttranslational processing of ICP22 accumulating in Vero cells but precluded full processing of ICP22 accumulating in rabbit skin cells. The effect on accumulation of U(L)38 and U(S)11 proteins was insignificant in Vero cells and minimal in rabbit skin cells. (iii) Substitutions of alanine for the threonine and serines in the third domain precluded full processing of ICP22 and caused a reduction of accumulation of U(S)11 and U(L)38 proteins. These results indicate the following. (i) The posttranslational processing of ICP22 is sensitive to mutations within the domains of ICP22 tested and is cell-type dependent. (ii) Posttranslational processing of ICP22 is not required for accumulation of U(L)38 and U(S)11 proteins to the same level as that seen in cells infected with the wild-type virus. (iii) The T374SS sequence shared by ICP22 and the U(S)1.5 proteins is essential for the accumulation of a subset of gamma(2) proteins exemplified by U(S)11 and U(L)38 and is the first step in mapping of the sequences necessary for optimal accumulation of U(S)11 and U(L)38 proteins.

A novel cellular protein, p60, interacting with both herpes simplex virus 1 regulatory proteins ICP22 and ICP0 is modified in a cell-type-specific manner and Is recruited to the nucleus after infection.

Herpes simplex virus 1 encodes two multifunctional regulatory proteins, infected-cell proteins 22 and 0 (ICP22 and ICP0). ICP0 is a promiscuous transactivator, whereas ICP22 is required in vivo and for efficient replication and expression of a subset of late (gamma2) genes in rodent or rabbit cell lines and in primary human cell strains (restrictive cells) but not in HEp-2 or Vero (permissive) cells. We report the identification in the yeast two-hybrid system of a cellular protein designated p60 that interacts with ICP22. This protein (apparent Mr of 60,000) has not been previously described and has no known motifs. Analyses of p60 revealed the following. (i) p60 bound fast-migrating, underprocessed wild-type ICP22 and ICP22 lacking the carboxyl-terminal 24 amino acids but not ICP22 lacking the carboxyl-terminal 40 amino acids, whereas the previously identified cellular protein p78 (R. Bruni and B. Roizman, J. Virol. 72:8525-8531, 1998) bound all forms of ICP22. The interaction of p60 with only one isoform of ICP22 supports that hypothesis that each isoform of herpes simplex virus proteins performs a specific function that may be different from that of other isoforms. (ii) p60 also bound ICP0; the binding of ICP0 was independent of that of ICP22. (iii) p60 localized in uninfected rabbit skin cells in both nuclei and cytoplasm. In rabbit skin cells infected with wild-type virus, p60 was posttranslationally processed to a higher apparent Mr but was not redistributed. Posttranslational processing required the presence of the genes encoding ICP22 and UL13 protein kinase. (iv) In uninfected HEp-2 cells, p60 localized primarily in nuclei. Soon after infection with wild-type virus, the p60 localized in discrete small nuclear structures with ICP0. Late in infection, both ICP0 and p60 tended to disperse but p60 did not change in apparent Mr. The localization of p60 was independent of ICP22, but p60 tended to be more localized in small nuclear structures and less dispersed in cells infected with mutants lacking the genes encoding the UL13 or US3 protein kinases. The results suggest that posttranslational modification of p60 is mediated either by ICP0 (permissive cells) or by ICP22 and UL13 protein kinase (restrictive rabbit skin cells) and that the restrictive phenotype of rabbit skin cells may be related to the failure to process p60 by mutants lacking the genes encoding UL13 or ICP22.

Functional anatomy of herpes simplex virus 1 overlapping genes encoding infected-cell protein 22 and US1.5 protein.

Earlier studies have shown that (i) the coding domain of the alpha22 gene encodes two proteins, the 420-amino-acid infected-cell protein 22 (ICP22) and a protein, US1.5, which is initiated from methionine 147 of ICP22 and which is colinear with the remaining portion of that protein; (ii) posttranslational processing of ICP22 mediated largely by the viral protein kinase UL13 yields several isoforms differing in electrophoretic mobility; and (iii) mutants lacking the carboxyl-terminal half of the ICP22 and therefore DeltaUS1.5 are avirulent and fail to express normal levels of subsets of both alpha (e.g., ICP0) or gamma2 (e.g., US11 and UL38) proteins. We have generated and analyzed two sets of recombinant viruses. The first lacked portions of or all of the sequences expressed solely by ICP22. The second set lacked 10 to 40 3'-terminal codons of ICP22 and US1. 5. The results were as follows. (i) In cells infected with mutants lacking amino-terminal sequences, translation initiation begins at methionine 147. The resulting protein cannot be differentiated in mobility from authentic US1.5, and its posttranslational processing is mediated by the UL13 protein kinase. (ii) Expression of US11 and UL38 genes by mutants carrying only the US1.5 gene is similar to that of wild-type parent virus. (iii) Mutants which express only US1. 5 protein are avirulent in mice. (iv) The coding sequences Met147 to Met171 are essential for posttranslational processing of the US1.5 protein. (v) ICP22 made by mutants lacking 15 or fewer of the 3'-terminal codons are posttranslationally processed whereas those lacking 18 or more codons are not processed. (vi) Wild-type and mutant ICP22 proteins localized in both nucleus and cytoplasm irrespective of posttranslational processing. We conclude that ICP22 encodes two sets of functions, one in the amino terminus unique to ICP22 and one shared by ICP22 and US1.5. These functions are required for viral replication in experimental animals. US1.5 protein must be posttranslationally modified by the UL13 protein kinase to enable expression of a subset of late genes exemplified by UL38 and US11. Posttranslational processing is determined by two sets of sequences, at the amino terminus and at the carboxyl terminus of US1.5, respectively, a finding consistent with the hypothesis that both domains interact with protein partners for specific functions.

UL13 protein kinase of herpes simplex virus 1 complexes with glycoprotein E and mediates the phosphorylation of the viral Fc receptor: glycoproteins E and I.

Herpes simplex virus 1 encodes a Fc receptor consisting of glycoproteins E (gE) and I (gI) and two protein kinases specified by UL13 and US3, respectively. We report the following: (i) Antibody to UL13 formed immune complexes containing gE and gI in addition to UL13 protein. Immune complexes formed by monoclonal antibody to gE, but not those formed by monoclonal antibody to gI, also contained the UL13 protein. This association may reflect direct interaction between gE and UL13 inasmuch as IgG in preimmune rabbit serum and an antiserum made against another viral protein which does not react with the UL13 protein directly also bound gE and UL13. (ii) In cells infected with the wild-type virus, gE formed two sharp bands and a diffuse, slower migrating band. The slower sharp band was undetectable, and the diffuse slower migrating forms of gE were diminished in lysates of cells infected with a mutant virus lacking the UL13 gene (DeltaUL13). (iii) Both gE and gI were labeled with 32Pi in cells infected with wild-type or the DeltaUL13 virus, but the labeling was significantly stronger in cells infected with the wild-type virus than in those infected with the DeltaUL13 virus. (iv) In an in vitro protein kinase assay, UL13 immunoprecipitated from cells infected with wild-type virus labeled gE in the presence of [gamma-32P]ATP. This activity was absent in precipitates from cells infected with DeltaUL13 virus. The labeled gE comigrated with the slower, sharp band of gE. (v) gI present in the UL13 immune complex was also phosphorylated in the in vitro kinase assay. (vi) The cytoplasmic domain of gE contains recognition sequences for phosphorylation by casein kinase II (CKII). Exogenous CKII phosphorylated gE in immune complexes from lysates of cells infected with the DeltaUL13 mutant or in immune complexes from lysates of cells infected with wild-type virus that had been heated to inactivate all endogenous kinase activity including that of UL13. In both instances, CKII phosphorylated gE in both the slow and fast migrating sharp bands. We conclude that UL13 physically associates with gE and mediates the phosphorylation of gE and gI. UL13 may also be a determinant in posttranslational processing of gE.

The UL13 protein kinase and the infected cell type are determinants of posttranslational modification of ICP0.

The herpes simplex virus infected-cell protein 0 (ICP0) acts as a promiscuous transactivator of genes introduced into eukaryotic cells by transfection or infection. The protein is highly posttranslationally modified by phosphorylation and nucleotidylylation. We have examined the electrophoretic mobility and phosphorylation of ICP0 in Vero and rabbit skin cells infected with wild-type virus or viruses from which the UL13 gene (DeltaUL13) encoding a protein kinase or the alpha22/US1.5 genes (Deltaalpha22/DeltaUS1.5) encoding putative transcriptional factors has been deleted. We report the following: (i) The accumulation of ICP0 and the electrophoretic mobility of ICP0 were dependent on the nature of the infected cell type and the presence of UL13. ICP0 encoded by wild-type virus accumulated to maximum levels earlier in infected Vero cells and its electrophoretic mobility was slower than that made in rabbit skin cells. In both Vero and rabbit skin cells infected with the DeltaUL13 virus, the prevailing ICP0 form migrated faster than that accumulating in the corresponding cells infected with wild-type virus. (ii) The alteration in electrophoretic mobility of ICP0 made in cells infected with DeltaUL13 virus was due to the absence of the UL13 protein and not to failure of posttranslational modification of Deltaalpha22/DeltaUS1.5 proteins inasmuch as the mobility of ICP0 in cells infected with Deltaalpha22/DeltaUS1.5 virus could not be differentiated from that of wild-type infected cells. (iii) ICP0 is extensively phosphorylated in infected cells even in the absence of UL13 protein. ICP0 is, however, a substrate for the UL13 kinase inasmuch as ICP0 was phosphorylated in mixtures of immune complexes of ICP0 and UL13. Complexes containing ICP0 only or infected cell lysate proteins reacting with preimmune serum from the rabbit immunized with UL13 protein failed to phosphorylate ICP0. (iv) In the absence of UL13, ICP22 is overproduced-an imbalance attributed to UL13. Thus, ICP22 regulates both the utilization of splice acceptor sites and the longevity of ICP0 mRNA (K. L. Carter and B. Roizman, 1996, Proc. Natl. Acad. Sci. USA 93, 12535-12540); UL13 is involved in the posttranslational modification of ICP0 and is required for both posttranslational processing and control of abundance of ICP22.

Association of herpes simplex virus regulatory protein ICP22 with transcriptional complexes containing EAP, ICP4, RNA polymerase II, and viral DNA requires posttranslational modification by the U(L)13 proteinkinase.

The expression of herpes simplex virus 1 gamma (late) genes requires functional alpha proteins (gamma1 genes) and the onset of viral DNA synthesis (gamma2 genes). We report that late in infection after the onset of viral DNA synthesis, cell nuclei exhibit defined structures which contain two viral regulatory proteins (infected cell proteins 4 and 22) required for gamma gene expression, RNA polymerase II, a host nucleolar protein (EAP or L22) known to be associated with ribosomes and to bind small RNAs, including the Epstein-Barr virus small nuclear RNAs, and newly synthesized progeny DNA. The formation of these complexes required the onset of viral DNA synthesis. The association of infected cell protein 22, a highly posttranslationally processed protein, with these structures did not occur in cells infected with a viral mutant deleted in the genes U(L)13 and U(S)3, each of which specifies a protein kinase known to phosphorylate the protein.

Assemblons: nuclear structures defined by aggregation of immature capsids and some tegument proteins of herpes simplex virus 1.

In cells infected with herpes simplex virus 1 (HSV-1), the viral proteins ICP5 (infected-cell protein 5) and VP19c (the product of UL38) are associated with mature capsids, whereas the same proteins, along with ICP35, are components of immature capsids. Here we report that ICP35, ICP5, and UL38 (VP19c) coalesce at late times postinfection and form antigenically dense structures located at the periphery of nuclei, close to but not abutting nuclear membranes. These structures were formed in cells infected with a virus carrying a temperature-sensitive mutation in the UL15 gene at nonpermissive temperatures. Since at these temperatures viral DNA is made but not packaged, these structures must contain the proteins for immature-capsid assembly and were therefore designated assemblons. These assemblons are located at the periphery of a diffuse structure composed of proteins involved in DNA synthesis. This structure overlaps only minimally with the assemblons. In contrast, tegument proteins were located in asymmetrically distributed structures also partially overlapping with assemblons but frequently located nearer to nuclear membranes. Of particular interest is the finding that the UL15 protein colocalized with the proteins associated with viral DNA synthesis rather than with assemblons, suggesting that the association with DNA may take place during its synthesis and precedes the involvement of this protein in packaging of the viral DNA into capsids. The formation of three different compartments consisting of proteins involved in viral DNA synthesis, the capsid proteins, and tegument proteins suggests that there exists a viral machinery which enables aggregation and coalescence of specific viral protein groups on the basis of their function.

Processing of the herpes simplex virus regulatory protein alpha 22 mediated by the UL13 protein kinase determines the accumulation of a subset of alpha and gamma mRNAs and proteins in infected cells.

We reported previously that the posttranslational processing associated with phosphorylation of the herpes simplex virus 1 infected-cell protein 22 (ICP22), a regulatory protein, is encoded by UL13, a gene encoding a structural protein of the virion. We now report the following. (i) In cells infected with a mutant lacking UL13 (delta UL13), restricted infected cells accumulate reduced levels of the regulatory protein ICP0 and several late viral proteins. Identical reductions have been observed in the same cell lines infected with a mutant from which the alpha 22 gene, encoding ICP22, had been deleted (delta alpha 22). We conclude that the UL13-mediated processing of ICP22 is essential for its gene-regulatory function. (ii) The reduced accumulations of specific viral protein in cells infected with either delta UL13 or delta alpha 22 viruses correlate with reduced levels of specific mRNAs for both ICP0 and the affected late genes. (iii) ICP22 is not modified by the UL13 protein introduced into cells during infection. (iv) ICP22 is also modified by the protein kinase encoded by US3, but this modification is different from that of the UL13 protein kinase. These results predict that UL13 encodes a protein kinase or phosphotransferase which is expressed late in the replicative life cycle and which directly or indirectly phosphorylates ICP22. This modification is essential for stabilization or increased transcription of a specific subset of viral RNAs and, ultimately, for the accumulation of corresponding viral proteins.