Origin-specific unwinding of herpes simplex virus 1 DNA by the viral UL9 and ICP8 proteins: visualization of a specific preunwinding complex

Abstract

Herpes simplex virus 1 contains three origins of replication; two copies of oriS and one of a similar sequence, oriL. Here, the combined action of multiple factors known or thought to influence the opening of oriS are examined. These include the viral origin-binding protein, UL9, and single-strand binding protein ICP8, host cell topoisomerase I, and superhelicity of the DNA template. By using electron microscopy, it was observed that when ICP8 and UL9 proteins were added together to oriS-containing supertwisted DNA, a discrete preunwinding complex was formed at oriS on 40% of the molecules, which was shown by double immunolabeling electron microscopy to contain both proteins. This complex was relatively stable to extreme dilution. Addition of ATP led to the efficient unwinding of approximately 50% of the DNA templates. Unwinding proceeded until the acquisition of a high level of positive supertwists in the remaining duplex DNA inhibited further unwinding. Addition of topoisomerase I allowed further unwinding, opening >1 kb of DNA around oriS.

Figure 1

Visualization of preunwinding complexes containing UL9 protein and ICP8 assembled on a supertwisted plasmid containing oriS. UL9 protein and ICP8 were incubated with a supertwisted plasmid containing oriS in the presence of magnesium. The samples were prepared for EM by fixing the proteins in place, removing free protein by gel filtration, adsorption to carbon foils, and rotary shadowcasting with tungsten. (Lower) Enlargements of individual preunwinding complexes (see Materials and Methods). Images are shown in reverse contrast. (Scale bars: Upper, 250 nm; Lower, 25 nm.)

Figure 2

Immunoelectron microscopic demonstration that the preunwinding complex contains both ICP8 and UL9 protein. Preunwinding complexes were prepared as described for Fig. 1 and then labeled with rabbit polyclonal IgG against UL9 protein and mouse monoclonal IgG against ICP8. This was followed with addition of goat anti-rabbit IgG and goat anti-mouse IgG conjugated with 5- and 10-nm gold particles, respectively (Materials and Methods). Hence, the large gold particles denote the presence of ICP8 and the small ones denote the presence of UL9. (Insets) Complexes at higher magnification. Images are shown in reverse contrast. (Scale bar, 500 nm.)

Figure 3

Visualization of the unwinding of supertwisted DNA by UL9 protein and ICP8. Preunwinding complexes were assembled on supertwisted plasmid containing oriS for 15 min (see text) followed by the addition of 5 mM ATP for 2 min (A), 6 min (B), 10 min (C), and 30 min (D). (E) Incubation as described for D was followed by the addition of topoisomerase I for 30 min. The samples were prepared for EM as described for Fig. 1. Images are shown in reverse contrast. (Scale bar, 250 nm.) (F) In fields of molecules from an unwinding time course as described for A–D, the number of supertwists (strand crossovers) in each DNA containing an unwinding complex was scored by EM. The maximum that can be reliably counted by this method is 10–15 crossovers for this DNA.

Figure 4

Visualization of UL9 protein and ICP8 unwinding linear oriS-containing DNA. Linear pGEM822 DNA was incubated with UL9 protein and ICP8 in the presence of ATP for 15 min. The samples were then prepared for EM as described for Fig. 1. The two thick loops in A are typical of ssDNA bound by ICP8. The location of the large complexes is consistent with the location of oriS, which is ≈2,000 bp from one end of the DNA and ≈2,700 bp from the other. Images are shown in reverse contrast. (Scale bar, 100 nm.)