• 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; Aurelian, Laure (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.
    • 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; Aurelian, Laure (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.
    • 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.; Aurelian, Laure (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.
    • Neoplastic transformation, cellular localization and protein kinase activity of the RR1 subunit of herpes simplex virus type 2 (ICP10)

      Hunter, James Colin Ramsay; Aurelian, Laure (1996)
      The large subunit of the herpes simplex virus (HSV) Type 1 and Type 2 ribonucleotide reductase (ICP6 and ICP10, respectively) is a chimaera consisting of a protein kinase (PK) domain at the amino terminus and a ribonucleotide reductase domain at the carboxy terminus. The present work was performed in order to further characterize this PK activity and determine its biological significance. Computer-assisted analyses of ICP10 and ICP6 in comparison to other, known protein kinases suggest that they are members of a previously unidentified sub-family of serine/threonine (Ser/Thr) growth factor receptors. Using cells infected with laboratory and patient isolates of HSV, ICP10 was demonstrated to possess a Ser/Thr specific auto- and transphosphorylating kinase activity. ICP6 exhibited a similar PK activity, but was considerably weaker. Cells that constitutively express ICP10 or a library of ICP10 mutants illustrated a direct relationship between ICP10 PK activity and its ability to transform cells as determined by anchorage-independent growth. All mutations which abolish PK activity resulted in a concomitant abrogation in its transforming potential. Cells transfected to constitutively express ICP10 demonstrated its internalization from the membrane and degradation in lysosomes in a manner characteristic of a typical activated growth factor receptor. ICP10 was also found bound to cytoskeletal actin, similar to epidermal growth factor receptor. A PK-negative mutant of ICP10, deleted in the transmembrane domain was recoevered from the cytosol, but did not localize to the membrane, nor was it found in endosomes or lysosomes, nor in association with actin. In ICP10 constitutively expressing cells, Raf-1 and Erk evidenced decreased electrophoretic mobility consistent with an activated state, suggesting that ICP10 PK may cause transformation through modulation of the MAP kinase pathway. It is suggested that the PK domains of ICP6 and ICP10 were cellular genes integrated into the genome of an ancestral herpes simplex virus and subsequently conserved to promote viral replication and/or reactivation from latency.