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.

Fig 1

Alignment of ICP8 homolog sequences. Amino acid sequences for HSV-1 ICP8 and ICP8 homologs from representative viruses in each of the three subfamilies of herpesviruses (alphaherpesviruses, betaherpesviruses, and gammaherpesviruses) were aligned using the T-Coffee alignment algorithm (http://www.tcoffee.org [6]). The ICP8 homologs included in the analysis were from HSV-1 strain KOS (NCBI accession number P17470), HSV-2 (NP_044499), varicella-zoster virus (AEW89446), Marek's disease virus (Q9E6P0), Epstein-Barr virus (P03227), human cytomegalovirus (P17147), murine cytomegalovirus (MCMV) (P30672), human herpesvirus 7 (O56282), and Kaposi's sarcoma-associated herpesvirus (ADQ57880). Sites with similar amino acids in 4 or more ICP8 homologs are in black letters highlighted in light gray; sites with identical amino acids in 4 or more ICP8 homologs are in white letters highlighted in black; and sites with identical amino acids in all 9 ICP8 homologs are in white letters highlighted in dark gray. Two regions are shown, identifying conserved amino acids at positions 545 and 547 (based on their positions in HSV-1 ICP8) and the complete conservation of an aspartic acid residue at position 1087. A full alignment can be found in Fig. S1 in the supplemental material.

Fig 2

Transient complementation assay. HeLa cells were either mock transfected, transfected with an empty vector (pCIΔ), or transfected with plasmids expressing wild-type ICP8, the ICP8 d105 deletion mutant, or ICP8 with the codons that encode the indicated amino acids mutated to encode alanine. At 24 h posttransfection, the cells were either harvested to prepare samples for immunoblotting (A) or infected with the ICP8 mutant 8lacZ (B). Viral yield samples were harvested at 24 h postinfection, and viral yield was determined by a plaque assay on ICP8-complementing V529 cells. The reported values are percentages of the complementation by cells transfected with the plasmid encoding wild-type ICP8 and are averages for 4 independent experiments. Error bars represent standard deviations. Similar results were seen in four additional independent experiments in Vero cells.

Fig 3

Effect of ICP8 DDE mutation on DNA binding. (A) His-tagged wild-type and DDE mutant ICP8 proteins were expressed from recombinant baculoviruses and were purified from infected Sf21 cells. Purified proteins were resolved by SDS-PAGE, and the gel was stained with Coomassie blue stain. (B) The indicated concentration of each protein was incubated with radiolabeled oligo(dT)₂₅ DNA, and protein-DNA complexes were resolved on a 5% native polyacrylamide gel.

Fig 4

Effect of the ICP8 DDE mutation on recognition by the conformation-specific antibody 39S. Vero cells were either mock infected or infected with the indicated virus at an MOI of 10 PFU/cell. At 10 hpi, the cells were fixed and stained with either the α-ICP8 39S conformation-specific antibody (green; left) or the 3-83 antibody, which detects total ICP8 (red; center).

Fig 5

Effects of the ICP8 DDE mutation on viral yield and plating efficiency. (A) Vero and V529 cells were infected at an MOI of 10 PFU/cell with either wild-type HSV-1, the 8DDEm mutant virus, the 8DDEm-R rescued virus, or pm1.a virus. Viral yield samples were harvested at the times indicated, and viral yields were determined by plaque assays on V529 cells. Similar results were seen in two additional independent experiments. (B) Serial dilutions of wild-type HSV-1, the 8DDEm mutant virus, and the 8DDEm-R rescued virus were plated on Vero and V529 cells. The titer of each virus on each cell line is displayed. The values shown are averages for two independent experiments, each performed in duplicate. Error bars represent standard deviations.

Fig 6

Effect of the ICP8 DDE mutation on viral DNA synthesis. Vero cells were infected at an MOI of 10 PFU/cell with either wild-type HSV-1, the ICP8 DDEm mutant, the 8DDEm-R rescued virus, or the ICP8 mutant pm1.a, which is defective for DNA binding and replication of viral DNA. Total DNA was harvested at the times indicated, and viral DNA levels in each sample were determined by real-time PCR and were normalized to the levels of cellular DNA. Similar results were seen in two additional independent experiments.

Fig 7

Effect of the ICP8 DDE mutation on viral gene expression. Vero cells were infected with either wild-type HSV-1, the ICP8 DDEm mutant, or the 8DDEm-R rescued virus, and lysates were prepared for immunoblotting at the indicated times (hours). Polypeptides were resolved by SDS-PAGE, transferred to a PVDF membrane, and probed for representative immediate-early (ICP27), early (ICP8), and late (gC) gene products.

Fig 8

Effect of the DDE mutation on iron-dependent cleavage of ICP8. Purified His-tagged wild-type ICP8 or ICP8 DDE mutant protein, either in the presence or in the absence of oligo(dT)₂₅ DNA, was either mock treated or treated with the iron/hydroxyl radical reagents. Following the iron-dependent cleavage reaction, samples were resolved by SDS-PAGE, transferred to a PVDF membrane, and probed for ICP8.