title: Identification of a divalent metal cation binding site in herpes simplex virus 1 (HSV-1) ICP8 required for HSV replication.
abstract: Herpes simplex virus 1 (HSV-1) ICP8 is a single-stranded DNA-binding protein that is necessary for viral DNA replication and exhibits recombinase activity in vitro. Alignment of the HSV-1 ICP8 amino acid sequence with ICP8 homologs from other herpesviruses revealed conserved aspartic acid (D) and glutamic acid (E) residues. Amino acid residue D1087 was conserved in every ICP8 homolog analyzed, indicating that it is likely critical for ICP8 function. We took a genetic approach to investigate the functions of the conserved ICP8 D and E residues in HSV-1 replication. The E1086A D1087A mutant form of ICP8 failed to support the replication of an ICP8 mutant virus in a complementation assay. E1086A D1087A mutant ICP8 bound DNA, albeit with reduced affinity, demonstrating that the protein is not globally misfolded. This mutant form of ICP8 was also recognized by a conformation-specific antibody, further indicating that its overall structure was intact. A recombinant virus expressing E1086A D1087A mutant ICP8 was defective in viral replication, viral DNA synthesis, and late gene expression in Vero cells. A class of enzymes called DDE recombinases utilize conserved D and E residues to coordinate divalent metal cations in their active sites. We investigated whether the conserved D and E residues in ICP8 were also required for binding metal cations and found that the E1086A D1087A mutant form of ICP8 exhibited altered divalent metal binding in an in vitro iron-induced cleavage assay. These results identify a novel divalent metal cation-binding site in ICP8 that is required for ICP8 functions during viral replication.
authors.affiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Kevin F
authors.initials: KF
authors.lastname: Bryant
authors.forename: Zhipeng
authors.initials: Z
authors.lastname: Yan
authors.forename: David H
authors.initials: DH
authors.lastname: Dreyfus
authors.forename: David M
authors.initials: DM
authors.lastname: Knipe
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002413:Cations, Divalent / Q000378:metabolism*; D002460:Cell Line; D002522:Chlorocebus aethiops; D017124:Conserved Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006561:Herpes Simplex / Q000821:virology*; D018259:Herpesvirus 1, Human / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D016415:Sequence Alignment; D014709:Vero Cells; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D002413:Cations, Divalent; D004268:DNA-Binding Proteins; C045462:ICP8 protein, Simplexvirus; D014764:Viral Proteins