HVEM

HVEM Carl F. Ware* Division of Molecular Immunology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121, USA * corresponding author tel: 858-678-4660, fax: 858-558-3595, e-mail: [email protected] DOI: 10.1006/rwcy.2000.16011.

SUMMARY Herpesvirus entry mediator type A (HVEM, also known as HveA) is related to the receptors for tumor necrosis factor. HVEM is a single transmembrane glycoprotein with a canonical cysteine-rich extracellular domain that binds to two cellular ligands, LIGHT and lymphotoxin , which are related to TNF. HVEM shares ligand specificity with the LT receptor and the two receptors for TNF, which also bind LT. HVEM binds a virus-encoded ligand, the envelope glycoprotein D (gD) of Herpes simplex virus (HSV) types 1 and 2, and serves as one of several entry routes used by HSV to infect cells. Envelope gD can sterically inhibit the binding of membrane LIGHT to HVEM, acting as a virokine, potentially disrupting signal transduction. The cytoplasmic tail of HVEM binds TRAF2 and TRAF5 adapter proteins, which propagate signals leading to the activation of NFB and AP-1 transcription factors, which control many genes involved in inflammation and immune responses. HVEM is prominently expressed on cells in the immune system, particularly T cells and dendritic cells and activation of HVEM can provide a costimulator function for T cells. HVEM targets HSV to activated T cells inducing Fas ligand, which can lead to fratricide of bystander lymphocytes. HSV can also infect dendritic cells and block their maturation. Together, HVEM-mediated entry of HSV may lead to localized

immune suppression. LIGHT can interfere with HSV entry, thus potentially acting as a virus deterrent. The UL144 gene in human cytomegalovirus ( -herpesvirus) is a structural homolog of HVEM, suggesting a long evolutionary history between the TNF superfamily and herpesviruses.

BACKGROUND

Discovery Herpesvirus entry mediator (HVEM) was discovered by Spear and colleagues as a factor that allowed Herpes simplex virus (HSV-1 and -2) to infect Chinese hamster ovary cells (CHO) (Montgomery et al., 1996). CHO cells are normally resistant to infection by HSV, and thus, transfer of a cDNA library into CHO cells was used to select genes that allowed virus entry and subsequent expression of a virus-encoded marker gene ( -galactosidase). Sequencing of one such clone revealed a membrane glycoprotein with a cysteine-rich extracellular domain that showed significant homology to TNFR superfamily. This assay led to the discovery of several additional entry factors for HSV-1 (Geraghty et al., 1998). In addition, HVEM was identified in several databases as a TNFR homolog (Marsters et al., 1997; Kwon et al., 1997; Hsu et al., 1997).

1720 Carl F. Ware Table 1 Cellular entry factors for herpes simplex virus Gene

Other namesa

Gene familyb

Virus attachmentc

Tissued

HveA

HVEM, ATAR,TR2

TNF receptor

gD HSV1&2

Lymphoid

HveB

Prr2

Ig fold

gD HSV2

Epithelial

HveC

Prr1

Ig fold

gD HSV1&2

Neuronal

Pvr-HveD

Pvr

Ig fold

PRV and BHV1

Unknown

a

HVEM, herpesvirus entry mediator; ATAR, another TRAF-associated receptor; TR2, TNFR-like receptor 2; Pvr, poliovirus receptor; Prr, poliovirus receptor-related protein. b Structural relationship with: TNF receptor superfamily or immunoglobulin superfamily (Ig fold). c HSV, herpes simplex virus; PRV, pseudorabiesvirus; BHV1, bovineherpesvirus 1. d Tissue type within which this entry protein may serve as an HSV entry route.

Alternative names Herpesvirus entry mediator (HVEM) is the original designation for this protein, but TR2 and ATAR are also found in the literature. Recently, Spear suggested redesignation as HveA, based on a nomenclature scheme that groups it with other HSV entry factors (Geraghty et al., 1998) (Table 1). This scheme groups functionally similar, but structurally distinct, proteins that bind envelope glycoprotein D (gD) of HSV. Additionally, for the purposes of cataloging the large numbers of human genes, HVEM is also designated TNFRSF14 (TNF receptor superfamily 14) by the Human Gene Nomenclature Committee, HUGO (www.gene.ucl.ac.uk/nomenclature).

Figure 1 Significant features of herpesvirus entry mediator (HVEM). Herpesvirus entry mediator HVEM • aka HVEM, HveA • TNFR/NGFR superfamily • Mediates HSV entry via HSV gD • Cellular ligands are LIGHT and LTα • Chr 1p36 • TRAF2 and 5 signaling • Activates NFκB and AP1 • Co-activation of T cells tm TRAF

Structure HVEM is a type 1 single transmembrane glycoprotein of 283 amino acids that contains a cysteine-rich extracellular domain, which is homologous to members of the TNFR superfamily (Figure 1). The HVEM gene maps to chromosome 1p36. HVEM binds three known ligands: LIGHT, LT, and envelope glycoprotein D (gD) of Herpes simplex virus. The relatively short cytosolic signaling domain binds to select members of the TRAF (TNFR-associated factor) family of signaling proteins that can activate NFB and AP-1 transcription factors. The mouse

HVEM homolog is 276 amino acids long and shares 44% overall identity.

Main activities and pathophysiological roles HVEM is linked to the biology of herpesvirus as an entry factor and as a signaling receptor for the lymphotoxin-like cytokine LIGHT (Mauri et al., 1998). HVEM is considered an integral component of

HVEM 1721 the immediate TNF family because it also binds the LT homotrimer, and LIGHT binds to the LT R, the receptor for the LT1 2 heterotrimer (Figure 2). Gene deletion or transgenic systems have not yet defined the role of HVEM in immune physiology, however, it is well established by these systems that the LT R is an essential regulator of lymphoid organogenesis and splenic architecture (Futterer et al., 1998) as well as NK-T cell differentiation (Fu and Chaplin, 1999). These results, and the close structural homology and shared ligands with HVEM, entice speculation that this receptor may participate in similar physiologic processes. However, the prominent expression of HVEM on T and B lymphocytes (LT R is absent on lymphocytes), and the ability of HVEM to activate NFB and AP-1 transcription factors hints that HVEM is intimately involved in the cascade of cytokines orchestrating lymphocyte differentiation and effector activity. Tissue culture models suggest that HVEM may serve as a costimulator for lymphocyte activation and cytokine secretion (Kwon et al., 1997). HVEM is likely to play a significant role in the pathophysiology of HSV infection as an entry factor. Viral entry is a complex process involving several viral envelope glycoproteins and several cellular components (Spear et al., 1992). Virus attachment to the cell surface is mediated via the binding of virus

Figure 2 HVEM is a member of the immediate TNF/LT family. Arrows indicate the receptor± ligand binding specificity of the various members. Solid lines indicate high-affinity interactions; dashed line refers to weak interactions.

envelope glycoprotein C (gC) to cell surface sulfated proteoglycans. Virus fusion with the cell membrane (entry) requires HSV gD, gB, gH, gL acting in concert with one of several distinct cellular receptors. Of the several entry routes that HSV can utilize (Geraghty et al., 1998), HVEM may serve as a major entry route for a subset of T lymphocytes (Montgomery et al., 1996). Interestingly, contact between HSV-infected fibroblasts and CTLs or NK cells rapidly inactivates their cytolytic capacity (Posavad et al., 1994), suggesting that HSV may use the HVEM entry route as a mechanism of immune suppression. HSV-1 entry is only dependent on the ligand-binding and transmembrane domains, as deletion of the cytoplasmic tail of HVEM does not interfere with entry. Soluble HVEM inhibits HSV infection in many cell types by binding to gD in the envelope of the virion (Geraghty et al., 1998). The binding site on gD for HVEM topographically overlaps with the site recognized by HveC as they compete with each other to bind gD (Whitbeck et al., 1997). HVEM also plays a role in virus-induced cell fusion (Terry-Allison et al., 1998) and promotes cell-to-cell spread of virus (Roller and Rauch, 1998). As a possible viral deterrent, LIGHT interferes with the entry of HSV-1 into CHO cells transfected with HVEM (Mauri et al., 1998). HSV envelope gD also causes a similar interference phenomenon (Johnson and Spear, 1989), indicating that coexpression of HVEM with either ligand inhibits virus entry. This could occur by direct competition with the binding of virion gD, or by the ability of ligands to inhibit HVEM expression at the cell surface. HSV-1 gD directly and specifically competes with HVEM binding to membrane-anchored LIGHT. Thus, at the membrane contact region between the lymphocyte and virus-infected cell, gD could block the initiation of LIGHT/HVEM signaling pathways and subsequent cellular responses. An additional molecular link between herpesvirus and the LT/TNF cytokine family was revealed by the discovery of a structural homolog of HVEM that is encoded in clinical isolates of human cytomegalovirus. The UL144 open reading frame (ORF) encodes a single transmembrane glycoprotein with 46% identity to the cysteine-rich extracellular (ecto) domain of HVEM (Benedict et al., 1999). UL144 is expressed early after infection of fibroblasts, however it is retained intracellularly. A YRTL motif in the highly conserved cytoplasmic tail of UL144 contributes to its subcellular distribution consistent with a site of action in clathrin-coated vesicles. This, together with the findings that no known ligand of the TNF family binds UL144, suggests that its mechanism of action is distinct from other known virus immune-evasion

1722 Carl F. Ware genes. Specific antibodies to UL144 can be detected in the serum of a subset of HCMV seropositive individuals infected with HIV, indicating that this virus gene product is relevant to the human immune response.

GENE

Accession numbers Human HVEM cDNA: U70321

Chromosome location and linkages The HVEM gene is located at chromosome 1p36.22± 36.3, which is close to several other TNFR genes including TNFR2, 4-1BB, CD30, Ox40, and DR3/TRAMP (Kwon et al., 1997). The genomic organization has not been published at this time. Possible polymorphisms in the coding regions of HVEM cDNA between different isolates (U81232) are not apparent in corrected sequences.

PROTEIN

Description of protein HVEM is a type I single transmembrane glycoprotein of 283 amino acids (276 for mouse HVEM) encoded by a major mRNA transcript of 1.8 kb. HVEM is defined as a member of the TNF receptor superfamily by virtue of its extracellular cysteine-rich (20 residues) domain (CRD) (Figure 3). For many TNFR-related proteins, each CRD contains six cysteines that form three disulfide bonds creating loops linked around a core sequence of CxxCxxC that is highly conserved. This basic motif is repeated from two to six times depending upon the individual receptor. The crystal structure of TNFR1 reveals an elongated molecule, where the three disulfide bonds in each CRD resemble the rungs of a ladder (Banner et al., 1993). For HVEM, the first two CRDs correspond exactly to the pattern displayed by TNFR1 and can be readily modeled on the TNFR1 structure (Guex and Peitsch, 1997). However, the spacing of remaining cysteines has a complex pattern that deviates substantially from TNFR1, making the demarcation

Figure 3 Sequence and domain structure of human HVEM. The predicted protein sequence for HVEM reveals a type 1 transmembrane glycoprotein. The repeated cysteine-rich domains (CRD) are predicted by Profilescan (www.isrec.isb-sib.ch/software/PFSCAN_form.html) although the division for CRD3 and 4 is arbitrary. The disulfide bond pattern (1±2, 3±5, 4±6) for CRD1 and 2 is based on TNFR1 and is shown by connecting lines. Demarcation of transmembrane domain and cytoplasmic tail is by PSORTII (www.psort.nibb.ac.jp:8800/). The TRAF-binding region was deduced by mutation (Hsu et al., 1997).

HVEM 1723 of CRD3 and 4 in HVEM difficult. Motif profiling programs do not recognize this pattern as a TNFR motif, unless low stringent criteria are used. The HVEM ectodomain (truncated at His200) under native conditions behaves as a dimer of  40 kDa, and treatment with reducing agents does not alter the dimeric structure, indicating that all cysteines are probably involved in intramolecular disulfide bonds (Whitbeck et al., 1997). The ectodomain of HVEM has two N-linked glycosylation sites and exhibits size heterogeneity on SDS-PAGE that is reduced by treatment with endoglycosidase F and O-glycanase (Table 2).

Relevant homologies and species differences HVEM is most closely related in overall sequence homology to TNFR80, CD40, and LT R, and this homology is primarily reflected in the ectodomain (Figure 4). HVEM also shows significant homology (46%) to the UL144 ORF encoded by human cytomegalovirus (Figure 5). The species homology for mouse and human HVEM is only 41% (Figure 6) significantly less than for related receptors in this

system, which range from 63 to 68% or higher (Hsu et al., 1997) (Table 3). Given that mice do not have a natural -herpesvirus equivalent, it is tempting to speculate that HSV may have served as the strong selective pressure driving this divergence.

Affinity for ligand(s) HVEM binds three distinct ligands: LIGHT, LT (Mauri et al., 1998), and HSV-encoded gD (Whitbeck et al., 1997). LIGHT and LT are members of the superfamily of TNF-related ligands. LIGHT is a type II membrane protein, whereas LT is naturally produced as a soluble protein unless assembled as a complex with LT (Browning et al., 1993). These proteins are structurally related and both form trimers. By contrast, HSV gD is a type I membrane protein that readily forms dimers. HSV gD is found in the virion envelope and is also expressed on the surface of infected cells (Cohen et al., 1988). No structural relationship is observed between gD and members of the TNF ligand superfamily. HVEM exhibits an avidity binding constant, as a dimeric HVEM-Fc molecule, of Kd=0.5 nM with a soluble form of recombinant LIGHT; the estimated

Table 2 Some physical properties of HVEM Property

Human

Mouse

mRNA (kb)

1.8 and 3.8

nd

Amino acids

283

276

30.4

30.3

32/42

nd

2 (110; 173)

2 (184; 197)

36±37

38±39

209±225

214±230

Molecular mass (kDa)a Sequence Observed N-Glycosylation

b

Signal cleavagec Transmembrane Alternate forms

c

nd

Deletion 101±158

CRD2/3

Deletion 61±84

CRD1

Truncation 158

soluble protein

Truncation 101

soluble protein

a

Calculated from sequence; observed by SDS-PAGE for in vitro translated cDNA and estimated from a partial cDNA expressed in CHO cells.

b c

Number of sites and their amino acid position.

Determined using PSORTII analysis (www.psort.nibb.ac.jp:8800/)

1724 Carl F. Ware Figure 4 Alignment of HVEM with other TNFR superfamily members. The cysteine-rich domain 2 of several TNFR superfamily members were aligned with ClustalW (PAM250 series, MacVector). The boxed areas show identical regions.

affinity for a single site was 44 nM (Harrop et al., 1998) (Table 4). Given that HVEM forms dimers, binding to LIGHT is predicted to be of relatively high affinity on the cell surface. Competition binding assays using HVEM-Fc and membrane-bound LIGHT showed half maximum binding at 10±30 nM for HVEM-Fc, whereas LT behaved as a competitive binding ligand at 50±70 nM. This suggests that LTHVEM interaction is relatively weak compared to LT binding to TNFR (Kdˆ  0.1 nM) (Mauri et al., 1998). In this same format soluble recombinant gD exhibited half-maximal binding at  1 mM in competition with HVEM-Fc binding to membrane LIGHT. A mutant of gD, gD
290±299t, competes with LIGHT binding to HVEM-Fc at  10-fold higher affinity than wild-type gD. Interestingly, HSV-1 virions that express the gD
290±299t mutant are inactive. Hence, from the point of view of the virus, too high an affinity between gD and HVEM is deleterious to infection. The molar stoichiometry of gD binding HVEM is 2 : 1, whereas LIGHT or LT are trimers and can bind up to three HVEM molecules.

Cell types and tissues expressing the receptor HVEM exhibits relatively broad tissue distribution, but is prominent in lymphoid tissues, including spleen, peripheral blood, and thymus (Montgomery

et al., 1996) (Table 5). At the cellular level, T and B lymphocytes express HVEM prominently, a feature that distinguishes HVEM from the LT R. The level of HVEM detected by FACS is in the same range as TNFR2 (CD120b or 75±80 kDa TNFR) on activated T cells, which is on the order of 5  103 receptors per cell.

Regulation of receptor expression HVEM mRNA and protein is constitutively expressed in freshly isolated T and B lymphocytes from peripheral blood, and levels increase following activation (Kwon et al., 1997). In cell lines of myelomonocytic lineage, such as U937, mRNA levels are induced following activation with phorbol ester, or in HL60 with dimethyl sulfoxide; TNF can also induce expression in the osteosarcoma line MG63.

Release of soluble receptors In the mouse, four alternately spliced forms of HVEM have been identified that result in deletions in the ectodomain or truncate the C-terminus, creating soluble receptors (Hsu et al., 1997) (Table 2). No evidence has been reported that these forms are expressed or functional. Additionally, no evidence has been obtained indicating that HVEM is proteolytically cleaved (shed) from the cell surface.

HVEM 1725 Figure 5 Sequence homology of UL144 with the TNFR superfamily. (a) Deduced amino acid sequence of UL144-F. The predicted signal cleavage site is denoted by the arrow, N-linked glycosylation sites by (*), the transmembrane region is underlined and the putative disulfide bonds in CRD2 modeled after those in TNFR60 are connected by bars. (b) Multiple alignment of UL144 sequences (ClustalW, PAM series) from Fiala (F), Toledo (T) and two other low passages isolates, LU and ME. The outlined and shaded regions identify identical and conserved residues. The bars denote positions of CRD1 and 2, the transmembrane (tm) and cytoplasmic (cyto) domains. (c) Pairwise alignment of CRD1 and 2 of HVEM and UL144F where the boxed and shaded regions show identity and conservative substitutions. (d) Homology between UL144 and members of the TNF superfamily. Alignments between the extracellular domains (ecto) or the second cysteine-rich domains of UL144 and the indicated TNFR were made with ClustalW and the percentage of identical residues was determined.

1726 Carl F. Ware Figure 6 Human and mouse HVEM. Alignment of protein sequence of human and mouse sequences were performed using pairwise alignment (Pam 250 series Macvector) of the (a) ecto and (b) cytoplasmic domains. Shaded areas show sequence identity.

Table 4 Affinity of HVEM for cellular and viral ligands Table 3 Species homology in the TNF superfamily

Receptora

% identity human and mouse Overall

Ectodomaina

Receptors HVEM

41

51

LT R

68

69

TNFR1

63

70

LIGHT

76

83

LT

60

75

LT

72

79

Ligands

a Ectodomain for the receptors includes leader up to the transmembrane segment. The ligand ectodomain is defined as the receptor-binding region involved in trimer formation.

Ligand affinity ( Kd nM) LIGHTb

LTc

gDd

Super gDe

HVEM-Fc (FACS)

1

50

1000

200

HVEM-Fc (PRS)

0.4

nd

nd

nd

HVEM (206t)

nd

nd

1000

200

a Different forms of HVEM used in affinity measurements: as a Fc fusion protein of the ectodomain or as a soluble ectodomain truncated at residue 206. Plasmon resonance (BIACORE) or flow cytometry was used to calculate half-maximum binding concentrations. b FACS analysis used stably transfected lines expressing membrane LIGHT. c LT-binding affinity was determined by competition with HVEM-Fc binding to membrane LIGHT. d Recombinant soluble form of gD. e Recombinant soluble form of the deletion mutant of gD
290±299t.

HVEM 1727 Table 5 Tissue and cellular expression patterns of HVEM Tissue

Humana

Mouseb

Adult

Strong: spleen, PBL, thymus, heart, lung, liver, kidney

Strong: bone marrow, thymus, spleen, small intestine, lung, heart, cecum, colon

Weak: prostate, small intestine, colon, brain, prostate, skeletal muscle

Weak: kidney, skin, heart, liver, stomach

Lung, liver, kidney

Lung, liver, kidney

Fetal Cell lines

a,c

Lymphocytes

II-23 CD4+ T cell hybridoma Jurkat CD4+ Raji B lymphoblastoid

Peripheral blood:

CD4+ resting T cells CD8+ resting T cells B cells (CD19+)

Monocytic

U937 HL60 THP1

Nonlymphoid

HT29 colonc HeLa carcinomac

a

Determined by northern blots. Determined by RNase protection assay. c Determined by flow cytometry. b

SIGNAL TRANSDUCTION

Associated or intrinsic kinases The binding of a trivalent ligand, which induces an ordered aggregation or `clustering' of receptors, initiates signal transduction by TNFR family members. Antireceptor antibodies, or mere overexpression, can induce receptor clustering and lead to activation of signal transduction pathways. HVEM, as well as all the other related TNF receptors, have no intrinsic kinase or other enzymatic activities encoded by their cytosolic domain (Figure 3). Rather, signal transduction occurs through the recruitment of cytosolic adapters, which directly or indirectly confer enzymatic activity to the receptor. Initial characterization of HVEM signaling pathways indicates that the cytoplasmic domain interacts directly with members of the TNFR-associated factor (TRAF) family of zinc RING finger proteins (Arch et al., 1998) (Figure 7).

Our understanding of the mechanisms and pathways involved in HVEM signaling is at an early stage, however, reasonable predictions can be drawn from the fact that HVEM interacts with a subset of the TRAF adapter molecules (Hsu et al., 1997; Marsters et al., 1997). TRAF2 and 5 have been shown to bind HVEM directly using yeast two-hybrid analysis or direct binding assays with GST fusion proteins. The binding of TRAF1 and 3 to HVEM is equivocal as two groups differ in their results. Yeast two-hybrid analysis showed strong interactions between HVEM with TRAF2 and 5, but not 1 or 3 (Hsu et al., 1997). In contrast, GST fusion protein binding analysis using extracts from TRAFs overexpressed in HEK293 cells showed TRAF1 and 3, as well as 2 and 5, but not TRAF6 bound to HVEM (Marsters et al., 1997). TRAF1 binding could occur indirectly in the latter model because of the propensity of TRAF1 to form heterocomplexes with TRAF2. Regardless, TRAF3 binding to HVEM is weak when compared to that to LT R. Mutation of glutamic acid at position 271

1728 Carl F. Ware Figure 7 Schematic diagram of the HVEM-signaling pathway. HVEM utilizes TRAF2 and TRAF5 in initiating signaling pathways. NFB induces gene expression resulting in proinflammatory and antiapoptotic activities. The JNK/AP-1 pathway induces stress responses, including cytokine secretion.

LIGHT/LTα HVEM

traf2 JNK/SAPK

traf5 NIKinase

AP-1

Stress & inflammation

NFκB

Anti-apoptosis

Inmmune response

(E271) to alanine in HVEM specifically ablates TRAF2 and 5 binding in yeast two-hybrid analysis (Hsu et al., 1997). The V269EET region is homologous to the TRAF2 and 5-binding region in CD40 (Figure 3). Recent results indicate that HVEM is located in a distinct subcellular compartment separate from LT R and cannot recruit TRAF3, which may account for the inability of HVEM to signal death. The interaction of HVEM with TRAF2 or 5 is expected to initiate activation of both NFB and cjun N-terminal kinase (JNK)/AP-1 pathways. HVEM overexpression in HEK caused activation of the p65/ p50 heterocomplex of B as measured by gel shift and reporter (pELAM-luciferase) assays, especially when cotransfected with TRAF5 (Hsu et al., 1997). TRAF2 signaling was less robust in this model. Overexpression of HVEM in 293 cells activated JNK as measured by phosphorylation of c-Jun-GST fusion protein on serine 63 (Marsters et al., 1997). Electrophoretic mobility shift assays showed that AP-1 nucleotide binding activity induced by HVEM was supershifted by an antibody to JunD. Overexpression of TRAF2 or TRAF5 has similar effect on AP-1. TRAF2 is known to be essential for TNFR1 activation of the JNK/AP-1 pathway in mice, suggesting that TRAF2 may be utilized by HVEM to activate this pathway (Yeh and Ohashi, 1997; Lee et al., 1997).

Cytoplasmic signaling cascades The TNFR superfamily is divided into two major groups based on the functional properties of their cytoplasmic tails: the death domain receptors, such as Fas and TNFR1, and the TRAF-binding receptors, such as LT R, CD40, and HVEM among others. The TRAF family of signal transducers consists of six members. TRAF2, 5, and 6 are involved directly in activating NFB and JNK pathways, which are prominent for the induction of genes involved in inflammation and stress responses (Arch et al., 1998). TRAF3 is involved in regulating cell death for LT R (Force et al., 1997), CD40, and CD30, whereas roles for TRAF1 and 4 have not clearly emerged. Three distinct structural motifs are found in the TRAF molecule. Located at the N-terminus, most TRAF proteins have a zinc RING finger and multiple zinc fingers (TRAF1 lacks a RING motif), a centrally positioned coiled coil region, and a C-terminal (TRAF) domain that binds directly to the receptor's cytoplasmic domain. TRAF2, 5, and 6 act as adapter molecules that recruit kinases and other components to their receptors. As exemplified by TRAF2, several distinct proteins interact with this adapter including NIK and ASK1. The NFB-inducing kinase (NIK) is a serine kinase involved in activating IKK-1 and 2, which in turn phosphorylate IB (inhibitor of B) causing its degradation and the release the transcriptionally active form of NFB. The interaction of TRAF2 with ASK1, a MAP3 kinase, activates the JNK-dependent cascade that in turn activates the AP1 transcription factor. TRAF5 and 6 exhibit several redundant activities with TRAF2, including NIK and ASK1 activation. Mice deleted of TRAF2 reveal absolute dependence of this adapter for JNK activation, but not NFB activation; these mice show a perinatal demise indicating the importance of this protein in development. TRAF5 knockout mice show no deficit in signaling, suggesting it may function redundantly with TRAF2. For a comprehensive review of these signaling pathways see the chapter on The TNF Ligand and TNF/NGF Receptor Families.

DOWNSTREAM GENE ACTIVATION

Transcription factors activated Genes specifically induced by HVEM signaling have not been identified. The ability of HVEM to activate both NFB and AP-1 transcription factors suggests

HVEM 1729 that HVEM signaling will elicit components involved in inflammation and cellular stress responses. However, the plethora of genes activated via these transcription factors makes it difficult to predict responses specific to HVEM. Cellular responses will also be controlled in part by the strength and duration of HVEM signals, as well as by the responding tissue and cell types. Here, the fact that naõÈ ve T cells express HVEM but lack TNFR suggests that HVEM signaling could activate genes involved during the early phases of T cell activation, such as chemokines or adhesion molecules.

Genes induced Because NFB and AP-1 transcription factors are activated by HVEM, it is expected that a subset of the genes regulated by these factors will be induced (Whitmarsh and Davis, 1996).

Promoter regions involved Genetic elements controlling HVEM gene expression have not been analyzed.

BIOLOGICAL CONSEQUENCES OF ACTIVATING OR INHIBITING RECEPTOR AND PATHOPHYSIOLOGY

Unique biological effects of activating the receptors The unique feature of HVEM is its ability to serve as an entry factor for HSV-1 and 2.

Human abnormalities No known human genetic disease is linked to the HVEM gene. However, the ability of HVEM to serve as an entry factor for Herpes simplex virus argues strongly that this molecule will play some role in the initial or recurrent infections associated with this herpesvirus. There are two major types of HSV that differ genetically and immunologically. HSV-1 is associated with primary infection of oropharyngeal

mucosal surfaces. HSV-2 is associated with infection of genitalia. Primary infection typically resolves in 2± 3 weeks and stimulates strong cellular and humoral immunity, which is specific for subtypes and strains of HSV. HSV-1 is widespread in the human population and is typically acquired in childhood by a large portion of the population. However, both types persist in a latent phase in sensory ganglion that innervate the area of the primary site of infection. Sporadically, the virus re-emerges to cause a vesicular lesion supplied by the neuron harboring latent virus. The reactivation of HSV-1 is associated with exposure to UV light, stress, menstruation, or chemotherapy, processes that may compromise local and systemic immunity. Recurrent infections typically resolve quickly (< 1 week), however in immune compromised individuals control of recurrent HSV lesions is poor. The lesions can engross a large area, and virus can disseminate to other tissues.

THERAPEUTIC UTILITY

Effect of treatment with soluble receptor domain In tissue culture systems soluble HVEM can inhibit infection of cells by HSV-1 (Whitbeck et al., 1997). This effect is completely dependent on the type of gD protein expressed by the virion. Soluble HVEM is thought to act by steric hindrance of gD in the virion membrane, resulting in reduced attachment of gD to the cellular entry factors HveA or HveC that are expressed on the surface of the target cell. It is not known whether HVEM-Fc administered in vivo can block primary or recurrent HSV infections. HVEM-Fc inhibits the action of its ligands, LIGHT and LT, irrespective of the receptors that these ligands engage, and thus could be used to modulate immune responses that are dependent upon these cytokines. In tissue culture models, HVEM-Fc blocks LIGHT-induced growth arrest of HT29 cells and shows modest inhibition of cellular proliferation induced in mixed lymphocyte cultures (Harrop et al., 1998).

Effects of inhibitors (antibodies) to receptors Antibodies to HVEM inhibit entry of HSV-1 in a subset of T cells (Montgomery et al., 1996). No

1730 Carl F. Ware evidence is available on whether anti-HVEM antibodies suppress infection in vivo. Recent studies (Sarrias et al., 1999) have isolated peptides that bind HVEM. These were identified by affinity selection from phage-display libraries. One peptide inhibits the binding of gD to HVEM and can block infection of HVEM-transfected CHO cells by HSV-1. This exciting observation suggests that small molecules may prove useful in blocking virus entry pathways.

References Arch, R., Gedrich, R., and Thompson, C. (1998). Tumor necrosis factor receptor-associated factors (TRAFs)±a family of adaptor proteins that regulates life and death. Genes Dev. 12, 2821±2830. Banner, D. W., D'Arcy, A., Janes, W., Gentz, R., Schoenfeld, H. J., Broger, C., Loetscher, H., and Lesslauer, W. (1993). Crystal structure of the soluble human 55 kd TNF receptor-human TNF beta complex: implications for TNF receptor activation. Cell 73, 431±445. Benedict, C., Butrovich, K., Lurain, N., Corbeil, J., Rooney, I., Schneider, P., Tschopp, J., and Ware, C. (1999). Cutting edge: A novel viral TNF receptor superfamily member in virulent strains. J. Immunol. 162, 6967±6970. Browning, J. L., Ngam-ek, A., Lawton, P., DeMarinis, J., Tizard, R., Chow, E. P., Hession, C., O'Brine-Greco, B., Foley, S. F., and Ware, C. F. (1993). Lymphotoxin , a novel member of the TNF family that forms a heteromeric complex with lymphotoxin on the cell surface. Cell 72, 847±856. Cohen, G. H., Wilcox, W. C., Sodora, D. L., Long, D., Levin, J. Z., and Eisenberg, R. J. (1988). Expression of herpes simplex virus type 1 glycoprotein D deletion mutants in mammalian cells. J. Virol. 62, 1932±1940. Force, W. R., Cheung, T. C., and Ware, C. F. (1997). Dominant negative mutants of TRAF3 reveal an important role for the coiled coil domains in cell death signaling by the lymphotoxin- receptor (LT R). J. Biol. Chem. 272, 30835± 30840. Fu, Y.-X., and Chaplin, D. (1999). Development and maturation of secondary lymphoid tissues. Annu. Rev. Immunol. 17, 399± 433. Futterer, A., Mink, K., Luz, A., Kosco-Vilbois, M. H., and Pfeffer, K. (1998). The lymphotoxin beta receptor controls organigenesis and affinity maturation in peripheral lymphoid tissues. Immunity 9, 59±70. Geraghty, R. J., Krummenacher, C., Cohen, G. H., Eisenberg, R. J., and Spear, P. G. (1998). Entry of alphaherpesviruses mediated by poliovirus receptor-related protein 1 and poliovirus receptor. Science 280, 1618±1620. Guex, N., and Peitsch, M. C. (1997). SWISS-MODEL and the Swiss-Pdb Viewer: an environment for comparative protein modelling. Electrophoresis 18, 2714±2723. Harrop, J. A., McDonnell, P. C., Brigham-Burke, M., Lyn, S. D., Minton, J., Tan, K. B., Dede, K., Spampanato, J., Silverman, C., Hensley, P., DiPrinzio, R., Emery, J. G., Deen, K., Eichman, C., Chabot-Fletcher, M., Truneh, A., and Young, P. R. (1998).

Herpesvirus entry mediator ligand (HVEM-L), a novel ligand for HVEM/TR2, stimulates proliferation of T cells and inhibits HT29 cell growth. J. Biol. Chem. 273, 27548± 27556. Hsu, H., Solovyev, I., Colobero, A., Elliott, R., Kelley, M., and Boyle, W. J. (1997). ATAR, a novel tumor necrosis factor receptor family member, signals through TRAF2 and TRAF5. J. Biol. Chem. 272, 13471±13474. Johnson, R. M., and Spear, P. G. (1989). Herpes simplex virus glycoprotein D mediates interference with herpes simplex virus infection. J. Virol. 63, 819±827. Kwon, B. S., Tan, K. B., Ni, J., Lee, K. O., Kim, K. K., Kim, Y. J., Wang, S., Gentz, R., Yu, G. L., Harrop, J., Lyn, S. D., Silverman, C., Porter, T. G., Truneh, A., and Young, P. R. (1997). A newly identified member of the tumor necrosis factor receptor superfamily with a wide tissue distribution and involvement in lymphocyte activation. J. Biol. Chem. 272, 14272± 14276. Lee, S., Reichlin, A., Santana, A., Sokol, K., Nussenzweig, M., and Choi Y (1997). TRAF2 is essential for JNK but not NFkappaB activation and regulates lymphocyte proliferation and survival. Immunity 7, 703±713. Marsters, S. A., Ayres, T. M., Skuatch, M., Gray, C. L., Rothe, M. L., and Ashkenazi, A. (1997). Herpesvirus entry mediator, a member of the tumor necrosis factor receptor family (TNFR), interacts with members of the TNFR-associated factor family and activates the transcription factors NF-B and AP-1. J. Biol. Chem. 272, 14029±14032. Mauri, D. N., Ebner, R., Montgomery, R. I., Kochel, K. D., Cheung, T. C., Yu, G.-L., Ruben, S., Murphy, M., Eisenbery, R. J., Cohen, G. H., Spear, P. G., and Ware, C. F. (1998). LIGHT, a new member of the TNF superfamily and lymphotoxin  are ligands for herpesvirus entry mediator. Immunity 8, 21±30. Montgomery, R. I., Warner, M. S., Lum, B., and Spear, P. G. (1996). Herpes simplex virus 1 entry into cells mediated by a novel member of the TNF/NGF receptor family. Cell 87, 427± 436. Posavad, C. M., Newton, J. J., and Rosenthal, K. L. (1994). Infection and inhibition of human cytotoxic T lymphocytes by herpes simplex virus. J. Virol. 68, 4072±4074. Roller, R., and Rauch, D. (1998). Herpesvirus entry mediator HVEM mediates cell-cell spread in BHK(TK-) cell clones. J.Virol. 72, 1411±1417. Sarrias, M., Whitbeck, J., Rooney, I., Spruce, L., Kay, B., Montgomery, R., Spear, P., Ware, C., Eisenberg, R., Cohen, G., and Lambris, J. (1999). Inhibition of herpes simplex virus gD and lymphotoxin-alpha binding to HveA by peptide antagonists. J. Virol. 73, 5681±5687. Spear, P., Shieh, M., Herold, B., WuDunn, D., and Koshy, T. (1992). Heparan sulfate glycosaminoglycans as primary cell surface receptors for herpes simplex virus. Adv. Exp. Med. Biol. 313, 341±353. Terry-Allison, T., Montgomery, R., Whitbeck, J., Xu, R., Cohen, G., Eisenberg, R., and Spear, P. (1998). HveA (herpesvirus entry mediator A), a co-receptor for herpes simplex virus entry, also participates in virus-induced cell fusion. J. Virol. 72, 5802±5810. Whitbeck, J. C., Peng, C., Lou, H., Xu, R., Willis, S. H., Ponce de Leon, M., Peng, T., Nicola, A. V., Montgomery, R. I., Warner, M. S., Soulika, A., Spruce, L., Moor, W. T., Lambris, J. D., Spear, P. G., Cohen, G. H., and Eisenberg, R. J. (1997). Glycoprotein D of herpes simplex

HVEM 1731 virus binds directly to HVEM, a mediator of HSV entry. J. Virol. 71, 6083±6093. Whitmarsh, A., and Davis, R. (1996). Transcription factor AP-1 regulation by mitogen-activated protein kinase signal transduction pathways. J. Mol. Med. 74, 589±607.

Yeh, W. C., and Ohashi, P. (1997). Early lethality, functional NFkappaB activation, and increased sensitivity to TNF-induced cell death in TRAF2-deficient mice. Immunity 7, 715±725.