A role for Z-DNA binding in vaccinia virus pathogenesis

Abstract

The N-terminal domain of the E3L protein of vaccinia virus has sequence similarity to a family of Z-DNA binding proteins of defined three-dimensional structure and it is necessary for pathogenicity in mice. When other Z-DNA-binding domains are substituted for the similar E3L domain, the virus retains its lethality after intracranial inoculation. Mutations decreasing Z-DNA binding in the chimera correlate with decreases in viral pathogenicity, as do analogous mutations in wild-type E3L. A chimeric virus incorporating a related protein that does not bind Z-DNA is not pathogenic, but a mutation that creates Z-DNA binding makes a lethal virus. The ability to bind the Z conformation is thus essential to E3L activity. This finding may allow the design of a class of antiviral agents, including agents against variola (smallpox), which has an almost identical E3L.

Fig. 1.

A family of Z-DNA-binding proteins. (a) DNA–protein contacts. A portion of the Z-DNA-binding domain of the ADAR1 editing enzyme (Zα_ADAR1) is shown complexed to a fragment of Z-DNA, as revealed in the structure of the cocrystal (10). The view is down the recognition helix of the protein (left), and a number of amino acids are shown that interact with left-handed Z-DNA stabilized by electrostatic and van der Waals interactions. The Z-DNA backbone is red and water molecules are green. There is an edge-to-face van der Waals interaction between guanine 4 and tyrosine 177. Note the van der Waals interactions between prolines 192, 193, and the Z-DNA backbone. (b) The amino acid sequences of Z-DNA binding and related proteins are shown underneath the secondary structure diagram, as revealed in the cocrystal structures of human Zα_ADAR1 (10) and mouse Zα_DLM1 (11). A number of poxvirus E3L sequences are listed, as is the related sequence of human Zβ_ADAR1. Yellow bars indicate residues important for the protein fold (red dots) and for Z-DNA recognition (blue triangles). The amino acid numbering at the beginning and the end of each sequence is indicated. The GenBank accession numbers for the various sequences are as follows: double-stranded RNA adenosine deaminase, AAB06697.1 (Homo sapiens); Z-DNA-binding protein 1; tumor stroma and activated macrophage protein DLM-1 (Mus musculus) NP_067369; the E3L proteins, AAA02759 (vaccinia virus); NP_042088 (variola virus); AAC08018 (orf virus); AAK84995 (lumpy skin disease virus); NP_570192 (Swinepox); NP_073419 (yaba-like disease virus); and CAC42100 (cowpox virus). (c) Three different families of proteins are illustrated, with domains shown as colored boxes. The Zα and Zβ domains of both ADAR1 and DLM1 are blue, as is the related domain of E3L. Vaccinia E3L protein and the editing enzyme ADAR1 both have domains that bind to double-stranded RNA, shown in red.

Fig. 2.

The N terminus of the vaccinia E3L protein can be functionally replaced by Z-DNA-binding domains from ADAR1 and DLM1. Chimeric viruses were created with either Zα_ADAR1 or Zα_DLM1 at the E3L N terminus and wild-type E3L sequences at the C terminus. In these and subsequent experiments, groups of four to six C57BL/6 mice (4–6 weeks old) were infected intracranially with the indicated number of pfu of vaccinia virus constructs in 10 μl unless otherwise specified. The method has been described (2). One hundred pfu were used in this experiment and the mice were monitored for mortality for 2 weeks. Data for wild-type vaccinia virus are a composite of four different experiments, each with four mice. Percent survival is plotted against days postinfection. WT, wild-type E3L; E3LΔ83N has 83 amino acids deleted from the N terminus, and Zα_ADAR1 and Zα_DLM1 are chimeric viruses Zα_ADAR1-E3L_C and Zα_DLM1-E3L_C, respectively.

Fig. 3.

Z-DNA binding by mutants of Zα_ADAR1 protein correlates with pathogenicity of vaccinia viruses containing the corresponding mutations in chimeric Zα_ADAR1-E3L_C genes. (a) Mice were infected with vaccinia viruses containing Zα_ADAR1-E3L_C chimeric genes, including the indicated mutations, as described in Fig. 2. The mice were monitored for mortality for 2 weeks. (Left) Dose–response curve in which percent survival is plotted against dose of viruses administered. (Right) The ability of Zα_ADAR1 protein and two of its mutant proteins to convert d(CG)₆ from the right-handed B-form to the left-handed Z-form is shown. The ellipticity at 255 nm is monitored as a function of time, using 90 μM (base pair) d(CG)₆ and 30 μM protein, as described (26). Zα_ADAR1 protein converts the oligonucleotide to the Z-DNA conformation quite rapidly, and the mutants Y177F and Y177A affect the conversion at increasingly slower rates, corresponding to the weakened pathogenicity of the relevant viral constructs. (b) The lethality after intracranial inoculation of 100 pfu is shown for wild-type and a number of chimeric E3L vaccinia viruses, with the indicated mutations. Wild-type virus, chimeric Zα_ADAR1-E3L_C virus, and chimeric virus with Q186A or E171A mutations are all equally lethal. In contrast, the K169A mutant shows decreased virulence with considerable survival. The CD is shown (Right). Zα_ADAR1 protein and the Q186A and E171A mutants rapidly convert d(CG)₆ to the Z-DNA form. The K169A mutant achieves only partial conversion and at a slower rate.

Fig. 4.

A comparison of the in vivo pathogenic effects of analogous mutations in the Z-DNA-binding domains of Zα_ADAR1-E3L_C chimeric and wild-type E3L vaccinia virus. (a) Experiments analogous to Fig. 3a were carried out on the E3L gene of wild-type vaccinia virus. Dose–response curves are shown of the lethality after intracranial inoculation, monitored for 2 weeks, for different doses of the virus. Results are shown for virus containing wild-type E3L and for virus containing Y48F or Y48A mutations. The results are similar to those shown in Fig. 3a for the Zα_ADAR1-E3L_c chimeric virus. (b) Comparison of the effect of mutations in analogous proline residues of the E3L gene in wild-type vaccinia virus, as well as the Zα_ADAR1-E3L_C chimeric virus. (Left) Mice were infected intracranially with the indicated doses of wild-type vaccinia virus [WT (Z_E3L)], or with vaccinia virus containing P63A or P64A mutations. (Right) Mice were infected with virus containing chimeric (Zα_ADAR1-E3L_C) or the chimeric virus containing P192A or P193A mutations. The P64A mutation in the wild-type E3L gene produces decreased virulence in a manner similar to that seen with the P193A mutation of Zα_ADAR1-E3L_C chimeric virus, with an LD₅₀ of ≈10² pfu. The P63A mutation in wild-type E3L or the P192A mutations in Zα_ADAR1-E3L_C chimeric virus results in further weakening of the virus, with an LD₅₀ of 10⁴ to 10⁵ pfu.

Fig. 5.

Gain of biological function. Mutation of Zβ_ADAR1-E3L_C chimeric virus leads to Z-DNA binding and to pathogenesis in mice. Mice were infected intracranially with 10⁶ pfu of either a Zβ_ADAR1-E3L_C chimeric virus or the chimeric virus with a I335Y mutation. The Zβ_ADAR1-E3L_C chimeric virus has no lethality after intracerebral inoculation of 10⁶ pfu, and the Zβ_ADAR1 protein is unable to change the CD of d(CG)₆, as shown on the right. Substituting tyrosine for isoleucine at position 335 of Zβ_ADAR1 in Zβ_ADAR1-E3L_C chimeric virus leads to significant mortality. The same mutation in Zβ_ADAR1 protein converts d(CG)₆ from the B-DNA to the Z-DNA form.