Poxvirus TNF Receptor Homologs

Poxvirus TNF 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.2000.14011.

SUMMARY Following the cloning and sequencing of the cellular TNF receptors in the early 1990s, it was noticed that several virus genes in the database shared remarkable sequence similarity to the external cysteine-rich domain (CRD) repeat sequences that comprised the binding sites for TNF. Unlike the cellular receptors, however, the viral versions were predicted to be secreted glycoproteins that could scavenge host TNF prior to binding with its cognate cellular receptors. These viral genes, first identified in several poxviruses, were the first documented examples of `viroceptors', or virus-encoded receptor homologs that function to bind and sequester cellular ligands away from the appropriate cellular receptors.

BACKGROUND

Discovery The first known example of a virus-encoded homolog of a cellular cytokine receptor was the S-T2 gene of Shope fibroma virus (SFV) (Smith et al., 1990). In 1987, the SFV T2 open reading frame, the second gene from the terminus, was sequenced and shown to be expressed as a virus early gene (Macaulay et al., 1987; Upton et al., 1987). When the cellular type I and type II tumor necrosis factor receptors (TNFR)

were cloned in 1990, it was realized that the S-T2 gene belonged to the TNFR superfamily and we now know that this family includes not only TNFRI and TNFRII, but also other important immunomodulators such as Fas, CD40, CD30, CD27, Ox40, and 4-1BB (Smith et al., 1990, 1994). S-T2 protein was subsequently expressed from transfected COS cells and shown to bind TNF (Smith et al., 1991). A related TNFR homolog, designated M-T2, was also found in myxoma virus and was shown in 1991 to be an important virulence determinant for myxoma pathogenesis (Upton et al., 1991). Multiple TNFR homologs (designated crmB, crmC, crmD) have also been found in other poxviruses, such as cowpox virus, ectromelia virus, and variola virus (Shchelkunov et al., 1993; Hu et al., 1994; Smith et al., 1996; Loparev et al., 1998). The vaccinia virus genome contains two discontinuous, and thus nonfunctional, fragments of TNFR, but this virus is highly attenuated by passage outside a true vertebrate host (Goebel et al., 1990; Howard et al., 1991). Table 1 summarizes the different poxviral and cellular TNFR family members. All the poxvirus TNFR homologs possess 3 or 4 cysteine-rich domains (CRDs) at the N-terminus, which constitute the binding domain for TNF (see Figure 1).

Alternative names M-T2, S-T2, vTNF-R, crmB, crmC, crmD.

1634 Grant McFadden and Richard Moyer

Structure

Table 1 Accession numbers for poxvirus-encoded TNFR homologs: genes

The structural features of poxvirus TNFR homologs are reviewed elsewhere (Sedger and McFadden, 1996; McFadden et al., 1997; Xu et al., 1998). The secreted viral proteins generally have an N-terminal signal sequence followed by three or four copies of CRDs that define their membership in the TNFR superfamily, and a C-terminal extension that is unrelated to other cellular genes. The C-terminal domains of the viral proteins are generally not hydrophobic, allowing for protein secretion of at least some of the members from infected cells.

Poxvirus

Gene

GenBank

Myxoma virus

M-T2

M95181

S-T2

A23727

Main activities and pathophysiological roles The viral TNFR homologs are believed to be secreted scavengers of host TNF, and function as viroceptors to block the biological activities of host TNF and lymphotoxin prior to receptor engagement (Sedger and McFadden, 1996; McFadden et al., 1997; Xu et al., 1998). In the case of the M-T2 protein from myxoma, a second activity has been described of inhibiting apoptosis in virus-infected lymphocytes in a TNF-independent fashion (Macen et al., 1996; Schreiber et al., 1997).

GENE

Shope fibroma virus Variola virus Bangladesh 1975

a

G2R

L22579

Cowpox virus

crmB

L08906

Cowpox virus

crmC

Cowpox virus Monkeypox virus

crmD c

Y15035 b

crmB

U87234 U87842

a G2R/crmB homologs have been sequenced from other variola virus strains, including Garcia 1966 (Brazilian alastrim, minor), Harvey 1944, Butler 1952, Congo 1970, Somalia 1977, Sierra Leone 1968, Chimp 9-2, and Chimp 9-4.

bFour strains of ectromelia virus also encode crmD homologs. cCrmB genes from monkeypox virus strains Zaire 1996, Sierra Leone 1970, Nigeria 1971, Benin 1978, Zaire 1979, CW-N1, CV1, Zaire 1970, WMP, and UTC have been sequenced and reported in GenBank. Truncated and presumably nonfunctional homologs of crmB are found in rabbitpox, buffalopox, taterapox, and most strains of vaccinia virus.

Table 2 Accession numbers for poxvirus-encoded TNFR homologs: proteins

Accession numbers

Poxvirus

Amino acids

PID

Myxoma virus

326

332310

See Table 1.

Shope fibroma virus

325

139627

Variola virus Bangladesh 1975

349

439102

PROTEIN

Cowpox virus (CrmB)

355

333519

Cowpox virus (CrmC)

186

3097018

Cowpox virus (CrmD)

320

2738033

Monkeypox virus

349

2738063

Accession numbers See Table 2.

Sequence See Figure 1.

Description of protein In early studies examining the biological activity of S-T2, the secreted viral protein was independently expressed and found to bind TNF in ligand blot

assays. Characterization of the binding properties of M-T2 to TNF have also revealed that the cytotoxicity of rabbit TNF, but not the human or murine ligands, could be completely inhibited by addition of purified M-T2 protein. Hence M-T2 binds and inhibits TNF in a distinctly species-specific manner. On the other hand, the cowpox TNF-R homolog, crmC, inhibits murine TNF and lymphotoxin while the expressed variola version inhibits the human ligands.

Poxvirus TNF Receptor Homologs 1635 Figure 1 Amino acid alignment of representative members of the poxvirus TNF receptor superfamily.

Relevant homologies and species differences Although sequence identity of the extracellular ligand-binding domain of the different TNF receptor family members is not high, amino acid sequence

alignments reveal striking conservation in the position of conserved cysteine residues (Figure 1). Similarly, the ligands that engage these receptors can also be described in a superfamily of structurally related proteins, whose predicted secondary structures are virtually superimposable. These include TNF, lymphotoxin  and , CD40L, CD30L,

1636 Grant McFadden and Richard Moyer CD27L, FasL, and TRAIL. Following binding to their respective ligands, cellular TNF receptor family members oligomerize and mediate important immunomodulatory functions, including lymphocyte activation and proliferation, B cell costimulation and, under certain circumstances, cell death or apoptosis.

Affinity for ligand(s) Scatchard analysis revealed that M-T2 binds rabbit TNF with an affinity in the range of Kd 170-195 pM. This is comparable to that measured for TNF binding to cellular receptors. The Scatchard assays also indicated that binding of M-T2 to murine TNF occurs with an affinity that is an order of magnitude lower (Kd 1.7 nM), whereas binding to human TNF could not be demonstrated at all in these assays. The crmC protein from cowpox was shown to bind TNF, but not lymphotoxin , suggesting that ligand specificities might vary substantially between the poxvirus proteins (Smith et al., 1996). The crmD protein of cowpox and ectromelia binds both TNF and lymphotoxin  (Loparev et al., 1998).

Regulation of receptor expression All of the poxvirus TNFR homologs are early genes, except for crmC of cowpox virus, which is expressed at late times post infection.

Release of soluble receptors Most poxvirus TNFR homologs are secreted as soluble glycoproteins in monomeric, dimeric, or higher-ordered oligomeric forms (Schreiber et al., 1996; Loparev et al., 1998). The dimer of M-T2 is a more potent inhibitor of TNF cytolysis than is the monomeric form, presumably by blocking ligandinduced dimerization of the receptor (Schreiber et al., 1996).

BIOLOGICAL CONSEQUENCES OF ACTIVATING OR INHIBITING RECEPTOR AND PATHOPHYSIOLOGY

Phenotypes of receptor knockouts and receptor overexpression mice In order to evaluate how M-T2 contributes to viral virulence, a recombinant myxoma virus was

constructed in which both copies of the M-T2 ORF present in the virus genome were disrupted by insertion of a dominant selectable marker. This myxoma T2-minus virus (vMyxT2ÿ) does not express detectable T2 protein, but replicates normally within infected fibroblasts in vitro. When the vMyxT2ÿ virus was used to infect susceptible European rabbits, virus disease was found to be significantly attenuated (Upton et al., 1991). An important aspect of Leporipoxvirus infection and pathogenesis is the ability of these viruses to productively infect lymphocytes and spread to secondary sites via the lymphatics. Although both myxoma and the vMyxT2ÿ viruses can replicate normally in infected fibroblasts in vitro the M-T2 knockout virus cannot productively infect CD4+ rabbit T cells. Replication of vMyxT2ÿ in these cells is abortive, due to a rapid induction of DNA fragmentation and morphological alterations of cell death by apoptosis (Macen et al., 1996). Deletion analysis revealed that only the first two CRDs of M-T2 are required to prevent apoptosis of infected lymphocytes (Schreiber et al., 1997), whereas the first three CRDs are necessary for M-T2 to bind and inhibit TNF (Schreiber and McFadden, 1996).

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