• Login
    Search 
    •   UMB Digital Archive
    • School, Graduate
    • Theses and Dissertations All Schools
    • Search
    •   UMB Digital Archive
    • School, Graduate
    • Theses and Dissertations All Schools
    • Search
    JavaScript is disabled for your browser. Some features of this site may not work without it.

    Browse

    All of UMB Digital ArchiveCommunitiesPublication DateAuthorsTitlesSubjectsThis CollectionPublication DateAuthorsTitlesSubjects

    My Account

    LoginRegister

    Filter by Category

    Author
    Aurelian, Laure (4)
    Chung, Theodore Dookjong K. (1)Luo, Jianhua (1)Peng, Tao (1)Wachsman, Matthew (1)Subject
    Health Sciences, Pharmacology (4)
    Biology, Molecular (3)Herpesvirus 2, Human--genetics (3)ICP10 (3)Biology, Microbiology (2)Chemistry, Biochemistry (2)Ribonucleotide Reductases (2)Health Sciences, Immunology (1)Herpesvirus 2, Human--immunology (1)vaccinia recombinants (1)View MoreDate Issued1996 (1)1991 (3)

    Statistics

    Display statistics
     

    Search

    Show Advanced FiltersHide Advanced Filters

    Filters

    Now showing items 1-4 of 4

    • List view
    • Grid view
    • Sort Options:
    • Relevance
    • Title Asc
    • Title Desc
    • Issue Date Asc
    • Issue Date Desc
    • Results Per Page:
    • 5
    • 10
    • 20
    • 40
    • 60
    • 80
    • 100

    • 4CSV
    • 4RefMan
    • 4EndNote
    • 4BibTex
    • Selective Export
    • Select All
    • Help
    Thumbnail

    Construction and characterization of herpes simplex virus type 2 mutants deleted in the protein kinase or ribonucleotide reductase domains of the chimeric ICP10 protein

    Peng, Tao (1996)
    The large subunit of ribonucleotide reductase (RR1) encoded by Herpes Simplex Virus Type 1 and 2 (ICP6 and ICP10, respectively) is a chimeric protein consisting of a Ser/Threonine protein kinase (PK) domain at the amino terminus and a ribonucleotide reductase (RR) domain at the carboxy terminus. The findings that the PK domain is present only in HSV RR1 proteins, it is dispensable for ribonucleotide reduction and it functions as immediate-early (IE) protein during HSV infection, suggest that the PK activity plays a significant role in virus pathogenesis. The present work was initiated to confirm the intrinsic nature of ICP10 PK activity and to elucidate its role in HSV-2 infection. In stably transfected eukaryotic cells, ICP10 PK activity was eliminated by deletion of the conserved PK catalytic motifs or of the transmembrane (TM) segment and it was significantly impaired by mutation of the invariant Lys (Lys{dollar}\sp{lcub}176{rcub}{dollar}). Loss of PK activity by Lys{dollar}\sp{lcub}176{rcub}{dollar} mutation resulted in the failure to bind ATP. A truncated ICP10 PK expressed in bacteria (pp29{dollar}\rm\sp{lcub}la1{rcub}{dollar}) retained auto- and transphosphorylating activity (for calmodulin) after purification to apparent homogeneity. In cells infected with laboratory and patient isolates of HSV, RR1 had auto- and transphosphorylating activity for the small subunit of HSV ribonucleotide reductase (RR2) and Immunoglobulin G (IgG). Two HSV-2 (G) mutants deleted in the protein kinase or ribonucleotide reductase domains of ICP10 (ICP10{dollar}\Delta{dollar}PK and ICP10{dollar}\Delta{dollar}RR, respectively) were constructed by marker transfer. ICP10{dollar}\Delta{dollar}PK virus lost its intrinsic PK activity but retained its RR activity; ICP10{dollar}\Delta{dollar}RR virus retained its PK activity but lost its RR activity. ICP10{dollar}\Delta{dollar}PK virus does not replicate during the first 10 hrs postinfection (p.i.). However, its titers catch up with those of the wild type virus by 24 hrs p.i.; ICP10{dollar}\Delta{dollar}RR virus replicates as well as the wild type virus in exponentially growing cells but it is significantly impaired for growth in growth-restricted cells. HSV-2 and ICP10{dollar}\Delta{dollar}RR virus produce similar clear plaques but ICP10{dollar}\Delta{dollar}PK virus produces hazy plaques, which under magnification consist of a mixture of lysed and unlysed cells. The studies suggest that (i) ICP10 has intrinsic auto- and transphosphorylating PK activity, (ii) ICP10 PK and RR are functionally dissociable in virus infected cells, (iii) ICP10 PK is required for virus replication during the first 10 hrs p.i. and (iv) ICP10 PK may be involved in cell death in virus infected cells.
    Thumbnail

    Vaccinia recombinants as probes of relevant HSV-2-specific immunity

    Wachsman, Matthew (1991)
    Vaccinia recombinants expressing the glycoprotein D (gD) of HSV-1 (VP176) or HSV-2 (VP221) under control of an early vaccinia virus promoter or of HSV-1 under the control of a late promoter (VP254) were studied as probes for relevant immunity. The recombinants expressed comparable amounts of gD and grew equally well in vitro and in vivo VP176 immunization protected guinea pigs against primary (p {dollar}<{dollar} 0.005) and recurrent (p {dollar}<{dollar} 0.005) cutaneous HSV-2 disease, VP221 protected against recurrent disease and VP254 immunization afforded no protection. All vaccines protected mice at 10 days post infection, but VP176 gave superior protection on day 50 post immunization (p {dollar}<{dollar} 0.0001). Although neutralizing anti-HSV antibody and immunity to vaccinia antigens induced were identical, immunization with VP176 as compared to VP254 induced significantly higher levels of T-cell mediated immunity to HSV antigen as defined by lymphocyte transformation or delayed type hypersensitivity (P {dollar}<{dollar} 0.01). L3T4+ LNC from VP176 immunized animals mediated these effects and could transfer protection but required a second non-HSV immune, radiosensitive cell. Immunity induced by prior HSV-2 immunization was less effective than that induced by VP176 in terms of preventing primary disease (p {dollar}<{dollar} 0.01) and local viral replication (100 fold higher virus titers). This was associated with lower HSV-specific blastogenic and DTH response in HSV but not VP176 immune animals following HSV reexposure. VP254 but not VP176 infected antigen presenting cells failed to produce fully glycosylated gD. This defect correlated with the failure of VP254 infected epidermal or spleen cells to express gD on the cell surface or to present antigen.
    Thumbnail

    Identification and characterization of the protein kinase activity associated with the large subunit of the herpes simplex virus type 2 ribonucleotide reductase and domain mapping of the multi-functional protein

    Chung, Theodore Dookjong K. (1991)
    The transforming region of the herpes simplex virus type 2 (HSV-2) genome encodes a 140 kDa protein, designated ICP10, which was previously demonstrated to be the large subunit of the viral ribonucleotide reductase (RR1) and to share identity with the cervical cancer associated antigen AG-4. The present work was initiated to further characterize the biochemical and functional nature of ICP10 and to elucidate its role, if any, in the induction of neoplastic transformation. Combining computer assisted sequence analyses with conventional biochemical and molecular techniques, ICP10 is demonstrated to possess multiple functionally distinct domains. Most significantly, the amino terminal one-third of ICP10, previously shown to be unique to HSV, possesses protein kinase (PK) activity, a transmembrane helical segment and an extracellular modulatory domain analogous to growth factor receptor kinases. Analysis of the RR activity reveals that leucine-rich stretch of amino acids representing the junction between the PK and RR domains is critical for association of the two virally encoded RR subunits and a previously uncharacterized cellular 180 kDa protein that functions to stabilize the RR activity. It is proposed that the ICP10 protein represents a chimera between a cellular receptor kinase-related oncoprotein and the HSV-2 RR1. Stabilization of the RR active complex may be one factor that has favored conservation of this union.
    Thumbnail

    Characterization of the intrinsic protein kinase activity and the kinase catalytic motifs of the large subunit of the herpes simplex virus type 2 ribonucleotide reductase

    Luo, Jianhua (1991)
    The large subunit of Herpes Simplex Virus type 2 (HSV-2) ribonucleotide reductase (ICP10) is a chimera protein consisting of a serine/threonine protein kinase (PK) domain at the amino-terminus and a ribonucleotide reductase (RR) domain at the carboxyl-terminus. Like growth factor receptor PKs, ICP10 is myristylated, it has features of a signal peptide and putative transmembrane (TM) segment, and its PK activity is modulated by basic proteins and by antibodies to amino acid residues upstream of the TM. To further characterize this PK domain, we constructed a bacterial expression vector (pJL11) containing DNA sequences encoding ICP10 amino acid residues 1-445. Bacteria containing pJL11 were induced to express a 29 KDa protein (designated pp29{dollar}\sp{lcub}\rm la1{rcub}){dollar} that represents a truncated portion of the ICP10 PK domain as demonstrated by immunoprecipitation with antibodies that recognize different antigenic domains, competition studies with extracts of ICP10 positive eukaryotic cells, and peptide mapping. pp29{dollar}\sp{lcub}\rm la1{rcub}{dollar} has autophosphorylating and transphosphorylating activity for calmodulin. The enzyme is activated by Mn{dollar}\sp{lcub}2+{rcub}{dollar} but not by Mg{dollar}\sp{lcub}\rm 2+{rcub}{dollar} ions, and autophosphorylation is inhibited by histone. It differs from the authentic ICP10-PK in that phosphorylation is specific only for threonine. To determine the significance of ICP10 PK catalytic motifs, site-directed and deletion mutants in PK motifs I and II, the putative signal peptide and the TM segment were used to determine the role of these elements in ICP10-PK activity. PK activity was lost by deletion of the putative TM segment (amino acid residues 85-106). However, mutation of the central Gly in PK catalytic motif I (Gly{dollar}\sp{lcub}106{rcub}{dollar}) or of the invariant Lys in PK catalytic motif II (Lys{dollar}\sp{lcub}176{rcub}{dollar}) or deletion of both of these catalytic motifs (amino acid residues 106-178) did not abolish the kinase activity as determined both in auto- and transphosphorylation assays. PK activity of the mutant deleted in domains I and II was 4-fold lower than that of the wild type ICP10 and it was insensitive to Mn{dollar}\sp{lcub}2+{rcub}{dollar}, suggesting that these motifs are involved in Mn{dollar}\sp{lcub}2+{rcub}{dollar} activation of kinase activity. The result of immunoblotting demonstrated that ICP10 complexes with GTPase activating protein (GAP). Ras GTPase activity is significantly inhibited in ICP10 transformed (JHLa1) cells. These results suggested that ICP10 may constitutively activate ras activity by blocking its down-regulation process, implying a potential signal transduction mechanism for ICP10 induced transformation.
    DSpace software (copyright © 2002 - 2019)  DuraSpace
    Quick Guide | Policies | Contact Us
    Open Repository is a service operated by 
    Atmire NV
     

    Export search results

    The export option will allow you to export the current search results of the entered query to a file. Different formats are available for download. To export the items, click on the button corresponding with the preferred download format.

    By default, clicking on the export buttons will result in a download of the allowed maximum amount of items.

    To select a subset of the search results, click "Selective Export" button and make a selection of the items you want to export. The amount of items that can be exported at once is similarly restricted as the full export.

    After making a selection, click one of the export format buttons. The amount of items that will be exported is indicated in the bubble next to export format.