β Receptor Homologs

Poxvirus IFN/ Receptor Homologs Grant McFadden1,* and Richard Moyer2 1

The John P. Robarts Research Institute and Department of Microbiology and Immunology, The University of Western Ontario, 1400 Western Road, London, Ontario, N6G 2V4, Canada 2 Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, PO Box 100266, Gainesville, FL 32610-0266, USA * corresponding author tel: (519)663-3184, fax: (519)663-3847, e-mail: [email protected] DOI: 10.1006/rwcy.2000.14015.

SUMMARY The discovery of extracellular poxvirus inhibitors of type I interferons in 1995 was made by direct inhibition studies, rather than by sequence homology analysis. In fact, the vaccinia virus (strain Western Reserve) prototype of this family, B18R, is more closely related to members of the Ig superfamily than to the cellular type I interferon receptors, at least in terms of overall similarity scores. Nevertheless, B18R binds and inhibits cellular type I interferons with a relatively broad species-specificity. The protein is expressed both as a secreted and cell surface glycoprotein and plays an important role in the pathogenesis of virus infection. In fact, the safest vaccinia strains used historically for smallpox vaccinations either did not express this gene, or the variant protein expressed by the strain was relatively inefficient in terms of type I interferon inhibition.

BACKGROUND

Discovery Poxvirus control of interferons (IFNs), like that of IL-1 , is both intracellular and extracellular. In the orthopoxviruses, intracellular control of IFNs is mediated by genes E3L and K3L (vaccinia). The E3L gene, an RNA-binding protein, functions to prevent

PKR activation by double-stranded RNA (Chang et al., 1992) whereas the K3L gene acts to prevent phosphorylation of eIF2 by PKR (Beattie et al., 1991; Davies et al., 1992). The action of extracellular type I interferons (IFN/ ) is inhibited by the secreted IFN/ receptor homolog encoded by the B18R ORF (vaccinia). The B18R ORF was initially identified by routine sequencing (Smith and Chan, 1991) and was believed to be involved in the regulation of IL-1 . The role of this ORF in IFN/ control was unanticipated because, unlike the cellular type IFN receptors which belong to the type II cytokine receptor superfamily and contain type 3 fibronectin domain repeats (Farrar and Schreiber, 1993), the B18R protein is a member of the Ig superfamily (Smith and Chan, 1991). An independent screening of proteins secreted from vaccinia virus-infected cells led to the identification of an approximately 65 kDa protein which bound to human type I interferon (Symons et al., 1995). The protein was rapidly identified as the B18R gene product. The protein is not expressed in all orthopoxviruses, and is notably absent from both the Lister and Ankara strains of vaccinia. The Wyeth strain contains the gene but expresses a protein which exhibits atypically low avidity for type I interferons (Symons et al., 1995). The gene is also present in ectromelia virus (Colamonici et al., 1995). The protein is also known as the orthopoxvirus S antigen (Morikawa and Ueda, 1993).

1856 Grant McFadden and Richard Moyer

Alternative names B18R ORF: (vaccinia WR, used as reference in this article) orthopoxvirus S antigen.

Structure The ORF, typical of poxvirus genes, is contiguous and contains no introns.

Main activities and pathophysiological roles The soluble B18R protein binds type I IFNs with high avidity to prevent IFN engagement with the appropriate cellular receptors (Symons et al., 1995), and thereby serves to block signal transduction and subsequent phosphorylation of JAK kinases (Colamonici et al., 1995). The protein binds many human IFNs (IFN1, IFN2, IFN7, IFN8, IFN ). Binding affinities are high, with Kd values of  1±4 nM (Colamonici et al., 1995; Symons et al., 1995). The B18R protein also binds a novel type of IFN (Vancova et al., 1998) but not IFN (Colamonici et al., 1995). Type I IFN binding exhibits a broad specificity that includes bovine, rabbit, and rat. Binding affinities to mouse type I IFNs are considerably lower. It is important to note that the interaction of this protein with type I IFNs is probably different from that for cellular type I IFN receptors. Binding of type I IFNs to cellular receptors is blocked by monoclonal antibodies to the N-terminal but not the C-terminal portion of the molecule. However, antibodies against both the C- and N-terminal portion of the B18R protein inhibit IFN binding (LiptaÂkova et al., 1997).

GENE

Accession numbers Nucleic acid accession numbers: Vaccinia virus WR: X56122 Vaccinia virus Copenhagen: D01019 Cowpox virus: Y15035 Variola virus Garcia: X72086 Variola virus Somalia: U18341 Variola virus Bangladesh: L22579

Sequence In vaccinia virus, the gene is located approximately 176 kb from the left end of the  190 kb genome

within the HindIII B fragment, the rightmost terminal HindIII fragment of the viral genome. Transcription is in the rightward direction. The protein is expressed early, prior to viral DNA synthesis.

PROTEIN

Accession numbers Vaccinia virus WR: 62238, 514205 Vaccinia virus Copenhagen: 222699 Cowpox virus: 3097038 Variola virus Garcia: 1150677 Variola virus Somalia: 439095 Variola virus India: 516438, 457079

Sequence See Figure 1.

Description of protein The complete vaccinia virus open reading frame predicts a product of 351 amino acids (see Figure 1). The mature protein is formed following cleavage of the N-terminal secretory signal sequence. Although the protein exhibits significant homology to the IgSF repeats as indicated, these repeats themselves have homology to the fibronectin repeats typical of cellular type I IFN receptors. Thus, the B18R ORF exhibits significant homology to the mouse, human, and bovine  subunits of the type I IFN receptor (Colamonici et al., 1995), particularly within the binding domains. Although classified as a secreted protein, a certain amount of the active protein is also found bound to the cell surface (Colamonici et al., 1995).

Relevant homologies and species differences The vaccinia virus protein is 93% identical to the corresponding ORF in cowpox virus, and 88±89% identical to the variola virus ORFs. The protein binds to type I IFNs in solution, which serves to prevent IFN binding to appropriate cellular receptors (Symons et al., 1995), subsequent signal transduction and phosphorylation of the JAK kinases (Colamonici et al., 1995). The protein can rebind to cells and under certain conditions be expressed at the surface of

Poxvirus IFN/ Receptor Homologs 1857 Figure 1 The sequence of the B18R ORF precursor protein from vaccinia virus WR. A predicted cleavage site is present between amino acids 19 and 20 of the N-terminal secretory signal to generate the mature, secreted protein. The predicted molecular weight ( 41 kDa), differs from the observed molecular weight of  65 kDa because of glycosylation. Putative glycosylation sites are located at amino acids 117±119, 261±263, 269±271, 321±324. Shown in boldface are the three IgSF repeat domains, which comprise most of the mature protein (Smith and Chan, 1991).

infected cells via proposed type II membrane topology (Morikawa and Ueda, 1993). Whether free in solution, or bound to the surface of cells, the protein is believed to afford protection against extracellular type I IFNs.

BIOLOGICAL CONSEQUENCES OF ACTIVATING OR INHIBITING RECEPTOR AND PATHOPHYSIOLOGY Both type I and type II interferons are known to be important in controlling poxvirus infections. Indeed as pointed out in the Discovery section, IFN is controlled by poxviruses in both an intracellular as well as extracellular fashion. Treatment of ectromelia virus-infected mice with neutralizing antibodies to either type I or type II IFN prevents normal clearance of the virus in infected mice (Karupiah et al., 1993). The role of the B18R ORF in virulence was tested by deleting the gene from vaccinia virus and performing intranasal infections of mice. An attenuation was noted and signs of clinical illness were reduced in animals infected with the mutant virus. The lethal dose of virus was some 100-fold less than for wildtype virus. Growth and viremia was measured by the assay for virus in lungs and brains respectively of infected animals. At selected doses, titres of virus in the lungs were drastically reduced in mice (undetectable in some animals) lacking the B18R gene and absent from the brains. In contrast, for animals infected with wild-type virus, virus was readily detected in both lungs and brain (Symons et al., 1995). It is noteworthy that these effects are observed despite the fact that affinity of murine type I IFNs for this protein is poor compared with the binding of human, rabbit, and rat IFNs. These results clearly

implicate a role of the type I IFNs in controlling orthopoxvirus infections.

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