©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Vaccinia Virus B18R Gene Encodes a Type I Interferon-binding Protein That Blocks Interferon Transmembrane Signaling (*)

Oscar R. Colamonici (1)(§), Paul Domanski (1), Sharon M. Sweitzer (2), Andrew Larner (2), R. Mark L. Buller (3)

From the (1)Department of Pathology, University of Tennessee, Memphis, Tennessee 38163, the (2)Food and Drug Administration, Bethesda, Maryland 20892, and the (3)Department of Molecular Microbiology and Immunology, St. Louis University, St. Louis, Missouri 63104

ABSTRACT
INTRODUCTION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Poxviruses encode a large number of proteins that attenuate the inflammatory and immune responses to infection. In this report we demonstrate that a number of orthopoxviruses express a type I interferon (IFN)-binding protein, which is encoded by the B18R open reading frame in the WR strain of vaccinia virus. The B18R protein has significant regions of homology with the subunits of the mouse, human, and bovine type I IFN receptors, bound human IFN2 with high affinity, and inhibited transmembrane signaling as demonstrated by inhibition of Fc receptor factor 1/2 and interferon-stimulated gene factor-3 formation as well as inhibition of the IFN antiviral response. Among viral host response modifiers, the B18R protein is unique inasmuch as it exists as a soluble extracellular as well as a cell surface protein and thus should effectively block both autocrine and paracrine functions of IFN.


INTRODUCTION

The type I interferon (IFNs)()are the cornerstone of the early antiviral host response in the skin, which is the site of replication of the majority of poxviruses. The keratinocytes of the epidermis have been shown to be potent producers of both IFN and IFN(1) , which in turn induce the transcription of a large number of genes, some of which encode proteins with potent antiviral activity such as the RNA-dependent protein kinase and the 2`,5`-oligo nucleotide A systems (for review see Refs. 2 and 3). Accordingly, poxviruses have evolved effective counter measures in the form of proteins that block the activation of the RNA-dependent protein kinase (ORF E3L; Refs. 4 and 5), the phosphorylation of initiation factor eIF2 (ORF K3L; Ref. 6), and possibly the action of 2`,5`-oligo nucleotide A system (ORF D11L; Ref. 7). Despite the effectiveness of these virus genes at blocking the antiviral activity induced by the pretreatment of certain cell lineages with type I IFNs, they appear to be ineffectual in other cell types such as macrophages, which are known to support the replication of the poxvirus ectromelia in vivo(8) . This suggests that type I IFNs can block poxvirus replication in other ways and could imply existence of additional virus gene(s) for blocking type I IFN action.

In the course of using the VV/T7 polymerase system to express the different subunits of the type I interferon receptor (IFN-R) in mouse SVX.2 cells(9) , we noticed that human I-IFN2 was cross-linked to a cell surface protein with an approximate mass of 62-68 kDa (data not shown). This binding protein was initially believed to correspond to the subunit of the type I IFN-R; however, a more detailed characterization revealed that the IFNaR3 (anti- subunit) and the IFNaR1 (anti- subunit) mAbs failed to immunoprecipitate I-IFN2 cross-linked to this IFN-binding protein and to block I-IFN2 binding, respectively (data not shown). These results raised the possibility that the 62-68-kDa protein either corresponded to an IFN2-binding protein encoded in the VV genome, or that VV infection of L-929 cells induced the expression of an IFN2-binding protein. This second possibility was highly unlikely as mouse L-929 cells do not bind human IFN2(2, 10) . Fig. 1A shows that SVX.2 cells infected with VV/T7 polymerase, but not mock-infected cells, expressed the 62-68-kDa (82-88 including 20 kDa corresponding to radioiodinated IFN2) IFN-binding protein on their cell surface. Similar results were obtained with parental L-929 cells (data not shown) and confirmed the viral origin of this protein. Fig. 1A also shows that binding of radioiodinated IFN2 to the VV protein(s) was blocked by different human type I IFNs including IFN1 (data not shown), IFN2, IFN, IFN, IFN7, IFN8, but not by huIFN. Natural murine IFN at a concentration of 1 10 units/ml was less effective in blocking binding than human type I IFNs. Furthermore, the anti- subunit antibody IFNaR1 failed to block binding of radiolabeled IFN2 supporting the viral origin for this type I IFN binding activity.


Figure 1: Vaccinia virus encodes a type I IFN-binding protein. A, a culture of mouse SVX.2 cells expressing constitutively the human type I IFN-R subunit was infected with 0.1 plaque-forming units/cell of VV/T7 polymerase, for 16 h at 37 °C. Radioiodination of IFN2 and affinity cross-linking methods were performed as described previously (39-41). The specific activity of the radioiodinated IFN2 was 47 µCi/µg. Affinity cross-linking was performed in the presence or absence of a 500-fold excess of unlabeled IFN2, IFN, IFN, IFN7, and IFN8. Natural murine IFN and human IFN were used at 1 10 and 6.5 10 units/ml, respectively. The IFNaR1 mAb was used at a final concentration of 100 µg/ml. Similar results were obtained in parental L-929 cells infected with VV/T7 polymerase (data not shown). B, the B18R protein has homology with the subunits of the type I IFN-R. Comparison of the protein sequences obtained from GenBank was performed using the multiple alignment program MACAW (42). The different blocks within the binding domains (BDI and BDII, corresponding to the NH- and COOH-terminal binding domains, respectively) are indicated with a B, followed by the number of the block as originally described by Bazan (15). The presence of a block in the first or second fibronectin module of the binding domain is denoted by absence or presence of a primesymbol, respectively (i.e.BD II-B6 or BD II-B6`). Those residues that are common to the B18R protein and the one or more subunits are boxed. The shaded areas correspond to regions of homology found with computer program MACAW (42). Asterisks indicate those highly conserved residues previously described in cytokine receptors (15).



Since the IFN-binding protein associated with VV infection has the ability to bind both human and mouse type I IFN (although with different affinities), we searched for homology between the VV encoded surface and secreted proteins and the known type I IFN-R subunits. Protein alignment analysis revealed that the product of the B18R gene had significant homology with the mouse(11) , human(12) , and bovine (13, 14) subunits of the type I IFN-R (Fig. 1B). Bazan (15) has proposed that the subunit of the human type I IFN-R (12) has duplicated 200-residue binding domains (Fig. 1B, BD I and BD II, for the amino- and carboxyl-terminal binding domains, respectively). Each binding domain is formed by approximately two 100-residue modules with similarity to fibronectin type III repeats and can be further subdivided in seven blocks (i.e.BDII-B4; Ref. 15). The alignment in Fig. 1B shows that the homology between B18R and the subunits begins in block 7 of the second module of the binding domain I (BD I-B7`) and extends without gaps to the third block of the first fibronectin module of the binding domain II (BD II-B3). Homology was also observed between B18R and parts of blocks 4 and 5 (BD II-B4 and BD II-B5) of the first fibronectin module of binding domain II, and blocks 2, 3, and part of 6 of the second fibronectin module (BD II-B2`/3`/6`). Some distinctive common features conserved in the B18R protein and found in binding domain II that deserve mentioning were (i) the prolines in block 1, (ii) the tryptophan residues in blocks 2 and 3 of the first module and block 3 of the second module, (iii) the characteristic pattern of aromatic residues separated by 3 residues in block 3, and (iv) the conserved tyrosine in block 6. These data suggested that these regions should play a major role in the interaction between the receptor and IFN. It is also worth mentioning that only one cysteine is conserved in the viral protein, considering that cysteine pairs are postulated to be necessary for folding of receptor proteins. The reason for this difference is unclear.

We next designed experiments to determine if the B18R protein was indeed a type I IFN-binding protein, whether other poxviruses also encoded an analogous activity, and its cellular location. Fig. 2A shows that L-929 cells infected with VV recombinant in which the B18R gene has been inactivated (VV B18RKO), but not with deletion of the B15R gene (VV B15RKO), have lost the ability to bind radioiodinated IFN2. These results confirmed that the B18R gene product was a type I IFN-binding protein and that orthopoxviruses cowpox and ectromelia also encoded a similar binding activity in their genomes (Fig. 2A). The Lister strain, which has a naturally occurring deletion of the B18R gene(16) , also failed to show type I IFN binding activity. The B18R protein was originally identified as an early surface antigen (S antigen) on the surface of poxvirus-infected cells(17) , and recently shown to be a secreted protein(18) . The deduced amino acid sequence of the B18R protein has an amino-terminal hydrophobic region consistent with a signal sequence, but lacked an obvious membrane anchor, and thus a means to localize to the plasma membrane. We sought to determine the relative amounts of the B18R IFN binding activity in the culture supernatant and on the surface of virus-infected cells. Fig. 2B shows that very high levels of IFN2 binding activity were present in conditioned medium of cells infected with the VV WR strain, cowpox virus, ectromelia virus, VV B15RKO, but not in the Lister strain or VV B18RKO. For all viruses, the binding activity observed in conditioned medium in this experiment corresponded to approximately 12 times as much as that associated with the cell surface of L-929 cells (data not shown). Binding studies with L-929 cells revealed the B18R protein to have a single binding site with a K of 440 pM and to be present in an excess of 16,800 binding sites/cell (data not shown). This compares with 1,200 high (36 pM) and 8,200 low (700 pM) affinity receptors/cell on the surface of U-266 cells and a few hundred receptors/cell observed in most normal tissues(2) . Thus, the high affinity of the B18R protein for type I IFNs and its abundance both on the cell surface and in the extracellular milieu indicated that the B18R protein could be an extremely powerful blocker of type I IFN autocrine and paracrine functions.


Figure 2: The B18R gene encodes a type I IFN-binding protein as do genomes of other poxviruses. Cultures of L-929 cells (2 10 cells/lane) were infected for 16 h with 0.1 plaque-forming units/cell of the indicated viruses. Cells were harvested, and affinity cross-linking was performed as described in Fig. 1A. The B15RKO, B18RKO, and B15/18RKO correspond to WR recombinant viruses in which the B15R, B18R, and both B15R and B18R genes have been knockout by insertional inactivation (34). The 82-88-kDa band corresponds to the B18R protein cross-linked to one molecule of I-IFN2 (62-68 kDa corresponding to the B18R protein (18) and 20 kDa of I-IFN2). Two additional bands also detected by affinity cross-linking (51 and 100 kDa) are indicated by arrows. B, the B18R protein is present in the conditioned medium of cells infected with various poxviruses. Five hundred microliters of conditioned media from various infection conditions were incubated with 5 nMI-IFN2, cross-linked, and immunoprecipitated with an anti-IFN serum. Immunoprecipitates were analyzed as in Fig. 1A. Quantitation of the B18R protein was carried out using a PhosphorImager (Molecular Dynamics, Sunnyville, CA). C, VV B18R protein blocks binding to the normal human type I IFN-R. The indicated amounts of conditioned medium from HeLa cells infected with VV B15RKO or VV B18RKO was preincubated in a final volume of 200 µl with 5 nM radioiodinated IFN2 for 30 min at 4 °C. Then, the mixture was added to human U-266 cells and the cross-linking procedure performed as described above. The IFNaR1 mAb was used as specificity control to block binding of I-IFN2 to the human receptor (no conditioned medium was added). Arrows indicate the position of the different subunits of the type I IFN-R and the B18R cross-linked to IFN2. The high molecular mass complex contains the and subunits, and probably other receptor-associated proteins.



To test whether the B18R protein could ``sequester'' IFN from the type I IFN-R, I-IFN2 was preincubated with increasing concentrations of conditioned medium obtained from cells infected with VV B15RKO or VV B18RKO prior to the addition to uninfected U-266 cells and cross-linking analysis. Fig. 2C shows that there was a progressive decrease in I-IFN2 binding to the human receptor (arrow, and subunits) expressed in human U-266 cells in the presence of increasing concentrations of conditioned medium containing the B18R protein (VV B15RKO). The decrease in IFN2 binding to the human receptor was paralleled by an increase in binding to the B18R protein. One hundred microliters of conditioned medium (50% of the final reaction volume) blocked binding to the human receptor in a way comparable as the anti- subunit antibody IFNaR1 (data not shown). No effect on binding was observed when U-266 cells were incubated with conditioned medium obtained from cells infected with the VV in which the B18R gene has been deleted (data not shown). These data demonstrated that the B18R protein was highly effective in blocking binding of type I IFN to the normal cell receptor, even when present at levels higher than observed under physiological conditions. Furthermore, this experiment also shows that the B18R protein was detected on the surface of uninfected U-266 cells, indicating that this IFN-binding protein is first secreted into the supernatant and then binds back to the cell membrane. Similarly, experiments with uninfected L-929 cells and supernatant containing the B18R protein demonstrated that B18R binds to a cell surface component with saturable kinetics (data not shown).

We next determined if blocking of binding to the human receptor by the B18R protein was sufficient to inhibit the IFN signaling pathway. One of the earliest events in IFN signaling is tyrosine phosphorylation of the Jak kinases (19, 20, 21, 22) and the activation of the transcription factors Stat1, Stat1, and Stat2(23) . To determine the effect of the B18R protein on IFN signaling, whole cell extracts were prepared from U-266 cells treated with IFN2, and the IFN-dependent activation of the Stat1 and Stat2 transcriptional regulators was assessed by electrophoretic mobility shift assay (EMSA) with probes encoding IFN response region (GRR) present in the Fc1 receptor gene (Fc receptor for IgG) (24, 25) and interferon-stimulated response element (ISRE)(23, 26, 27) . Fig. 3A shows that low levels of basal FcRF2 were observed in cells treated with conditioned medium from cells infected with VV B15RKO and VV B18RKO, but not from mock-infected cells (lanes1, 7, and 13). Preincubation of 1,000 units/ml IFN2 with conditioned medium obtained from B15RKO-infected cells (lane10), but not from mock-infected (lane4) or B18RKO-infected cells (lane16), completely blocked the activation of FcRF1, FcRF2, and ISGF3 (Fig. 3B) in U-266 cells. FcRF1/2 DNA binding activity was blocked by an excess of unlabeled GRR oligonucleotide and was supershifted by an anti-Stat1 serum indicating that Stat1 was present in these complexes (Fig. 3A). Interestingly, the basal level of activation of FcRF1/2 detected after treatment with conditioned medium obtained from either VV B15RKO- or VV B18RKO-infected cells (in the absence of IFN) was specific as it was blocked by an excess of unlabeled GRR probe and was supershifted by the anti-Stat1 antibody. The nature of this basal FcRF1/2 activation is under investigation. Fig. 3C shows that IFN-dependent activation of FcRF2 is completely blocked by conditioned medium obtained from either VV B18RKO- or VV B15RKO-infected cells, but not by medium from mock-infected cells. Presumably it was the B8R protein(28) , and not the B15R or B18R proteins, that was responsible for inhibition of the IFN activation of the Jak/Stat pathway.


Figure 3: The B18R protein inhibits activation of Stat1 and Stat2. A, whole cell extracts were obtained from U-266 cells treated with 1,000 units/ml IFN2 previously incubated for 30 min in the presence of conditioned medium from VV B15RKO-, VV B18RKO-, or mock-infected cells. EMSA was performed using a GRR probe (24) in the presence of a 100-fold excess of unlabeled GRR or 0.5 µl of anti-Stat1 serum for supershifts. The positions of FcRF1, FcRF2, and free GRR are indicated. B, the same whole cell extracts from A were used for EMSA with and ISRE probe. The position of ISGF3 and free ISRE are indicated. C, whole cell extracts were prepared in the same form as in A except that cells were treated with 10 ng/ml IFN. A GRR probe was used for EMSA.



Finally, we tested the ability of the B18R protein to reverse the IFN2 mediated inhibition of vesicular stomatitis virus (VSV) replication in U-266 cells. As shown in , as little as 1 µl of conditioned culture supernatant from VV B15RKO infection completely reversed the inhibition of VSV replication induced by 10 units/ml IFN2, whereas identical and greater amounts (50 µl, data not shown) of mock or VV B18RKO culture supernatant had no effect. In the absence of IFN2, VSV replicated equally well in cultures treated with supernatants from VV B15RKO and VV B18RKO infections, and to a level similar to that observed in cultures treated with IFN2 and VV B15RKO culture supernatant.

Type I IFNs are the first tier of host defenses against virus infection in the skin. Poxviruses have been shown to encode at least three genes that effectively block individual IFN-induced antiviral mechanisms in L-929 and other cells(6, 29, 30) ; however, it is not known if these genes are effective in vivo in keratinocytes or other cells of the dermis that are productively infected at least in the ectromelia virus infection of the mouse(31) . Based on in vitro studies, which documented on abortive ectromelia virus infection of the peritoneal macrophages pretreated with type I IFN(32) , it is likely that certain cells in the skin may not support poxvirus replication if previously exposed to type I IFN. If this is the case, then the production of the type I IFN-binding protein early in poxvirus infection and the ability of the protein to bind IFNs in the extracellular space as well as attached to the cell surface of infected and uninfected neighboring cells may be critical for the establishment of the virus in the skin. Interestingly, recent DNA sequence analysis of variola virus strain Bangladesh-1975 shows an ORF homologous to B18R (33). Although poxviruses have recently been shown to encode other proteins that bind cytokines (interleukin-1 (Refs. 18 and 34), tumor necrosis factor (Refs. 35-38), and IFN (Ref. 28)), only the type I IFN-binding protein appears to be able to exist in both a soluble and surface form. The importance of the poxvirus type I IFN-binding protein in the pathogenesis of the natural infection is not known. We have determined that inactivation of the B18R gene increases the mouse intracranial LD value only by 7-fold over VV wild type.()This value was lower than expected, but may be due to the lower affinity that the VV B18R protein has for mouse IFN (Fig. 1) or to the fact that VV is not a natural pathogen of the mouse. A more precise understanding of the role of the B18R gene in poxvirus pathogenesis will come from the study of a B18R poxvirus in its natural host. We are currently initiating such studies in ectromelia virus, the causative agent of mousepox.

  
Table: B18R protein blocks the anti-viral effects of IFN2

Yield of VSV from U-266 cells pretreated with 10 units/ml IFN2 mixed with 1 µl of the indicated culture supernatant for 24 h prior to challenge with VSV. Data are presented as the mean of triplicate values ± standard error of the mean.



FOOTNOTES

*
This work has been supported by National Institutes of Health Grant CA55079 (to O. R. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Pathology, University of Tennessee, 899 Madison Ave. M-576, Memphis, TN 38163. Tel.: 901-448-6173; Fax: 901-448-6979.

The abbreviations used are: IFN, interferon; VV, vaccinia virus; IFN-R, interferon receptor; ORF, open reading frame; mAb, monoclonal antibody; EMSA, electrophoretic mobility shift assay; VSV, vesicular stomatitis virus; GRR, IFN response region; ISRE, interferon-stimulated response element.

R. M. L. Buller, D. E. Hruby, and M. K. Spriggs, unpublished data.


ACKNOWLEDGEMENTS

We are especially grateful to Drs. Dennis Hruby (Oregon State University, Corvallis, OR) and Melanie Spriggs (Immunex, Seattle, WA) for the VV knockouts. We also thank Drs. M. Brunda (Hoffman-LaRoche), Paul Trotta (Schering-Plough), G. Adolf (Ernst Boehringer Institut für Arzneimittelforschung, Vienna, Austria), L. Ling (Biogen), H. Hochkeppel (Ciba-Geigy, Basel, Switzerland), and D. Gangemi (Clemson University, Clemson, SC) for providing us with the different IFNs.


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