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.

Synthetic α-Hydroxytropolones Inhibit Replication of Wild-Type and Acyclovir-Resistant Herpes Simplex Viruses

“…Polymerase incorporation of the nucleoside analog ACV prevents chain elongation (69); however, the ␣-hydroxytropolone mechanism of action remains to be determined. Possible viral targets with NTS enzyme activity or homology include the polymerase (3=-to-5= and 5=-to-3= exonuclease activities), pUL12 nuclease, pUL15 terminase, and ICP8 single-stranded DNA binding protein (70)(71)(72)76). Our observation that the ␣-hydroxytropolones suppress ␤-galactosidase expression under the control of an HSV immediate early gene promoter suggests that the compounds target a relatively early event in HSV replication, consistent with early effects previously seen in time-of-addition experiments (33).…”

Inhibitors of Nucleotidyltransferase Superfamily Enzymes Suppress Herpes Simplex Virus Replication

“…Candidate HSV gene products with NTS enzyme activities include the predicted RNase H activity of the ICP8 single-stranded DNA-binding protein (25), the RNase H activity of the pUL30 DNA polymerase (67), the 3=-5= exonuclease activity of pUL30 (68), and the 5=-3= exonuclease activity of the pUL12 polymerase accessory protein (27) that are directly involved in virus DNA replication (21). The pUL15 terminase that cleaves the viral DNA into monomers is known to be an NTS enzyme and is also a possible target (28).…”

“…DNA replication initiates at one or more of three viral origins of DNA replication and is primed by the viral helicase-primase complex (HSV-1 proteins pUL5, pUL8, and pUL52). DNA replication requires the single-stranded DNAbinding protein pUL29 (ICP8), which is predicted to contain an RNase H-like fold (25). The viral DNA polymerase holoenzyme complex (pUL30 DNA polymerase plus pUL42) catalyzes DNA elongation by a presumed double-stranded rolling-circle mechanism.…”

Inhibition of O-Linked N -Acetylglucosamine Transferase Reduces Replication of Herpes Simplex Virus and Human Cytomegalovirus

“…After viral adsorption, the cells were washed with PBS and overlaid with DMEM supplemented with 1% BCS and containing dimethyl sulfoxide (DMSO) (0.5%) or OSMI-1 at the desired concentrations. Progeny virus titers were determined by a plaque assay as described previously (21). To assess HCMV replication in the presence of OSMI-1, HFFs were infected with HCMV at the desired multiplicities of infection (MOIs).…”