DNA binding activity of the herpes simplex virus type 1 origin binding protein, UL9, can be modulated by sequences in the N terminus: correlation between transdominance and DNA binding

Abstract

UL9, the origin binding protein of herpes simplex virus type 1, is a member of the SF2 family of helicases. Cotransfection of cells with infectious viral DNA and plasmids expressing either full-length UL9 or the C-terminal DNA binding domain alone results in the drastic inhibition of plaque formation which can be partially relieved by an insertion mutant lacking DNA binding activity. In this work, C-terminally truncated mutants which terminate at or near residue 359 were shown to potentiate plaque formation, while other C-terminal truncations were inhibitory. Thus, residues in the N-terminal region appear to regulate the inhibitory properties of UL9. To identify which residues were involved in this regulation, a series of N-terminally truncated mutants were constructed which contain the DNA binding domain and various N-terminal extensions. Mutants whose N terminus is either at residue 494 or 535 were able to bind the origin efficiently and were inhibitory to plaque formation, whereas constructs whose N terminus is at residue 304 or 394 were defective in origin binding activity and were able to relieve inhibition. Since UL9 is required for viral infection at early but not late times and is inhibitory to infection when overexpressed, we propose that the DNA binding activities of UL9 are regulated during infection. For infection to proceed, UL9 may need to switch from a DNA binding to a non-DNA binding mode, and we suggest that sequences residing in the N terminus play a role in this switch.

FIG. 1.

Mutations ending at or around 359 are potentiating. (A) Schematic diagram of UL9 truncations. The N-terminal domain of UL9 is depicted as an open box, and the C terminus is depicted as a gray box. The N-terminal seven conserved motifs of superfamily II helicases are depicted as black boxes. An asterisk points out the position of the MV (G354A) mutation, and the position of the frame shift (FS) mutation (at 359 aa) has been marked by a pound sign (#). The size of the protein synthesized from each truncation is indicated at the right side of the figure. (B) Amino acid sequences present between residues 354 to 363 of the wild-type and mutated versions of UL9. The mutant residues are underlined. Sequencing was repeated three times to confirm the presence of these mutations. (C) The 17B antibody was used to detect truncated UL9 proteins in concentrated cell lysates from Vero cells transfected with the plasmids used in the plaque reduction assay shown in panel D. Prestained protein markers are depicted on the left. (D) Plaque reduction assays were performed by cotransfecting infectious viral DNA and the plasmid expressing the indicated protein. EP, empty plasmid. Error bars represent standard deviations calculated from the results of three independent experiments.

FIG. 2.

Inhibitory activity of the C terminus of UL9 protein in HSV-1 replication is relieved in the presence of residues 304 to 394. (A) A schematic representation of the truncated UL9 proteins is shown. The N terminus of the UL9 protein is represented by the black box. The C terminus of the protein is depicted by three black lines, and dashed lines represent the deleted amino acid sequences. The size of the protein synthesized from each truncation is indicated on the right. (B) The RH-7 antibody was used to detect UL9 truncations in concentrated cell lysates from Vero cells transfected with the plasmids used in plaque reduction assay shown in panel C. Wild-type UL9 and mutated UL9 proteins expressed in Vero cells are marked by asterisks. (C) Plaque reduction assays were performed by cotransfecting infectious viral DNA and the plasmid expressing the indicated protein. EP, empty plasmid. Error bars represent standard deviations calculated from the results of three independent experiments.

FIG. 3.

The ability to bind the origin of replication correlates with inhibition in the plaque reduction assay. (A) SDS-polyacrylamide gel electrophoresis of in vitro-synthesized wild-type UL9 and truncated proteins. Synthesis was performed in rabbit reticulocyte lysates containing 2 μg of each plasmid DNA according to the procedure described in Materials and Methods. EP, reaction was carried out using empty plasmid as a control. (B) Origin DNA binding affinity was determined by an EMSA in a 30-min reaction, using a partially double-stranded DNA (containing box I ori sequence of HSV-1 replication) as the substrate. Lane 1 represents the control reaction in the absence of proteins. For each experimental sample, two different protein concentrations were used for the binding assay. The actual binding efficiency is defined as 100 × [protein-DNA complex/(protein-DNA + free DNA)]. Squares indicate the areas of the image that have been used to calculate the binding efficiency of these proteins.

FIG. 4.

Intracellular localization of UL9 in cells transfected with wild-type and mutant versions of UL9. Vero cells were transfected with UL9-WT (A), UL9-C (B), UL9-N (C), plasmid UL9-494-851 (D), plasmid UL9-349-851 (E), and plasmid UL9-304-851 (F). Panels A, B, D, E, and F were stained with R249 antibody (recognizes the C terminus of UL9). Panel C was stained with KST-1 (directed against the whole UL9 protein), indicating that, in the absence of NLS, fragmented UL9 protein localizes in the cytoplasm. Panels G and H contain the images of Vero cells transfected with empty plasmid (pCDNA3 vector) and stained with R249 and KST-1 antibodies, respectively, which can be considered background staining.

FIG. 5.

The inhibition of plaque formation is correlated with DNA binding ability. The normalized plaque numbers (black bars) and DNA binding efficiencies (white bars) were plotted for the wild-type and truncated UL9 proteins. Error bars represent standard deviations calculated from the results of three independent experiments. In some cases, error bars are too small to see.

FIG. 6.

Model for potentiation and transdominance of HSV-1 replication. A schematic diagram of UL9 is shown at the top. The two potential regulatory regions 1 to 35 and 292 to 404 are depicted by diamond and oval shapes, respectively. The DNA binding domain (DBD) is marked by black diagonal stripes in the C terminus. For the potentiating mutants (a), we propose that the truncated protein can form a heterodimer causing a conformational change such that the DNA binding domain is masked and DNA binding is inhibited. In the case of transdominant mutants, the region required for this negative effect is either not present (b) or not able to cause this conformational change (c).