The CD4-gp120 interaction and AIDS pathogenesis.

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Annu. Rev. Immunol. 1991. 9:649-78 Copyright © 1991 by Annual Reviews Inc. All rights reserved

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THE CD4-gp120 INTERACTION AND AIDS PATHOGENESIS

Annu. Rev. Immunol. 1991.9:649-678. Downloaded from www.annualreviews.org by York University on 11/14/12. For personal use only.

Daniel J. Capon* and Rebecca H. R. Ward

Genentech Inc., 460 Point San Bruno Boulevard, South San Francisco, California 94080 KEY

WORDS:

HIV, viral entry, AIDS therapeutics, viral attachment, cell fusion

Abstract

Infection by the human immunodeficiency virus (HIV) leads to progressive destruction of the CD4 T subset of T lymphocytes, resulting in immuno deficiency and AIDS. The selectivity of CD4+ cell destruction is due to the specific binding of gp120, the external envelope glycoprotein of HIV, to CD4, initiating viral entry. Binding of gp 120 to CD4 on the cell surface may also lead to CD4+ cell depletion by inappropriate immune targeting, and may interfere with CD4+ cell function and ontogeny by disrupting CD4-mediated cell signaling. The CD4-gp1 20 interaction is thus an ob vious target for AIDS therapeutics. INTRODUCTION

One of the hallmarks of infection by the human immunodeficiency virus (HIV) is the selective destruction of the CD4 subset of T lymphocytes. This is one of two major subsets of peripheral T lymphocytes, which can be distinguished by their mutually exclusive expression of the cell surface molecules CD4 and CD8. Both classes of T lymphocyte use T cell receptors (TcRs) to recognize peptide antigens bound to highly polymorphic major histocompatibility complex (MHC) molecules on the surface of antigen presenting cells. CD4+ cells appear to recognize antigen only when it is presented by class-II MHC, while CD8+ cells recognize antigen presented by class-I MHC. This has led to the notion that CD4 and CD8 act as coreceptors, binding to a nonpolymorphic region of the MHC molecule, * Present address: Cell Genesys, Inc.,

344 Lakeside Drive,

Foster City, California

94404.

649 0732-0582/91/0410-0649$02.00


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while the TcR binds to a highly polymorphic surface encompassing the peptide antigen. The expression of CD4 is correlated with helper cell function, whereas CD8+ cells normally have cytotoxic function. The pro gressive depletion of CD4+ cells in HIV-infected individuals thus leads to loss of the helper cell function and ultimately to acquired immune deficiency syndrome (AIDS). Since CD8+ cells are relatively unaffected by this disease, despite their broad similarity to CD4+ cells in many respects, the theory quickly arose that CD4 itself was the receptor for HIV. This has proved to be the case; gp120, the external envelope glycoprotein of HIV, binds with high affinity to CD4, initiating the process of viral entry. While alternative or additional routes of infection are now suspected to exist, the CD4-gp120 interaction is fundamental to the pathogenesis of AIDS and is therefore an obvious target for study and intervention. VIRAL ATTACHMENT: CD4-gp120 BINDING

The observation that CD4 + cells are selectively depleted in AIDS patients (1-3) was followed, after the isolation ofRIV- l , by the finding thatRIV- l infection in vivo is confined to the CD4+ and not the CD8+ cell subset, and that only CD4+ cells are infectable by HIV -1 in vitro (4). The inference drawn from these studies that CD4 might be the HIV receptor was con clusively proven by showing that: (i) antibodies to CD4 block viral infec tion and virus-mediated cell fusion (syncytium formation) (5-7); (ii) human cells lacking CD4, which are normally resistant to HIV infection, become susceptible when transfected with a CD4 expression vector (8); and (iii) gp1 20, the envelope protein of HIV-l , binds to CD4 specifically (9) and with high affinity (10, 1 1 ). CD4-dependent viral entry involves at least two distinguishable steps: attachment, and subsequent uptake of the virus into the cell. The first of these processes is relatively well understood, the second less so. We first address here what is known about the structural requirements for binding, for both CD4 and gp120; later sections review viral uptake. CD4 Structure and the gp120 Binding Site

The structure of the CD4 protein is shown schematically in Figure 1. The mature polypeptide has an extracellular region, a hydrophobic trans membrane domain, and a highly charged cytoplasmic domain of 370, 26, and 38 residues, respectively (12, 13). There are four recognized domains in the extracellular region of CD4, denoted VI-V4. The cytoplasmic domain of CD4 is strongly conserved across mammalian species, with 79% homology between human and mouse sequences (14, 15). In contrast, the extracellular and transmembrane regions show overall homologies of only �

CD4--gpl20 INTERACTIONS

Figure 1

651

Schematic outline of the CD4

protein showing the four Ig-Iike domains

(VI-V4),

disulfide

bonds

(S-S),

gly

cosylation sites (triangles), and the trans membrane (TM) and cytoplasmic (CYT)


regions.

COO"

55% between human and mouse. Mouse CD4 does not bind to gp120, and indeed only the peripheral blood mononuclear cells of higher primates can support efficient HIV infection ( 1 6). This difference has been exploited to map the residues important to gpI20 binding. The four extracellular domains of CD4 (VI-V4) have been classified as members of the immunoglobulin (Ig) superfamily (17), which are thought to share a basic structure of a stable fold of two p-pleated sheets composed of short (5-10 amino acids) antiparallel p strands (see ref. 18 for review). In immunoglobulins this fold is stabilized by a hydrophobic interior and by a conserved disulfide bond linking the two sheets. Recently, several groups have obtained crystals of soluble CD4 derivatives. Those con taining all four extracellular domains diffract poorly, although crystallo graphic (18a,b) and electron microscopic (S. Harrison, pers. comm.) studies suggest that the molecule is rod-like, about 125 A long and 25-30 A wide. However, VIV2 fragments of CD4 give good crystals which diffract to high resolution (19, 19a,b). Two groups have recently solved the structure of V lV2 crystals (19a,b); one view of this structure is shown in Figure 2a. As predicted, both VI and V2 resemble Ig domains. Each is a p-strand sandwich with Ig domain topology, VI consisting of 9 p-strands, and V2 of 7 /3-strands. The two domains are intimately associated, with the last strand of VI becoming the first strand of V2. VI is very close in structure to Ig V regions; strands B, D and E make up one P sheet, and strands A, C, C. C", F and G make up the other, with a disulfide bond linking strands B and F. The V2 domain is a truncated p barrel with strands A, Band E making up one p-sheet and strands C, C, F and G the other. The disulfide bond in this domain is between two strands in the same sheet (C and F), as previously predicted (20), which is unusual for Ig-like domains. The structures of the V3 and V4 domains are still


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Figure 2

(A) Backbone representation of the VI V2 fragment of CD4. Domain I is lightly

shaded, domain 2 is heavily shaded; f3 strands are indicated by letters. Strand G of domain

1 becomes strand A of domain 2. First and last residues in each strand are indicated by single-letter code and sequence numbers. CB) Residues that affect the interaction of gpl20 with CD4, from the same angle as Figure 2A. Dark-shaded circles are exposed residues on the C'C" ridge, and dark-shaded squares are buried residues. Light-shaded circles are exposed residues that are displaced from the C'C" ridge. Light-shaded diamonds, positions in domain 2 reported to affect gpl20 interaction. Adapted from Ref. 19a by permission from Nature.

unknown. V4, like V2, is truncated and predicted to fold like an Ig constant (C) region, having jJ-sheets with 4 and 3 jJ-strands. V3 is noteworthy in lacking the conserved disulfide bond of the classical Ig domain, yet has good homology to Ig V regions. The conserved disulfide bond is no longer considered essential for admission to the Ig superfamily, since several Ig like sequences, including one functional antibody, lack it. In V3, the Cys residues are replaced with hydrophobic amino acids which may point inwards, stabilizing the Ig-like fold in place of the disulfide bond. The gp l 20-binding constant for cell-surface CD4 has been measured using recombinant gp120 from the HIV-l IlIa isolate, and found to be 4 x 10- 9 M (10). Affinity constants in the same range have been deter-


CD4-gp120 INTERACTIONS

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mined using solubilized intact CD4, and soluble secreted CD4 comprising the whole extracellular region (11). A fragment of CD4 containing only the V I domain has also been shown to bind gp120 with high affinity (21, 22). Several approaches have been taken to define the residues in VI important for gp120 binding, including random saturation mutagenesis coupled with complement-mediated selection of escape mutants (23), inser tional mutagenesis (24), and homolog scanning mutagenesis (25-28). These investigations have identified a single amino acid stretch (residues 40-55) as critical for gp120 binding. This suggested that the structure of the gp120 binding site might be relatively simple. However, a synthetic peptide based on this region (residues 25-58) only weakly inhibits HIV-1 induced cell fusion (29), and peptides from another region (residues 81-92) also block syncytium formation (30), suggesting a greater degree of complexity in the structure of the gpl 20 binding site on CD4. But since this peptide is inactive unless benzylated, the significance of these findings is unclear. Two recent detailed investigations of CD4 residues critical for gpl20 binding support the idea that the gp l20 binding site extends beyond amino acids 40-55. In one study, residues in VI predicted to be analogous to the four loop-like regions facing bound antigen in an Ig V" region were individually replaced with the analogous murine residue (or glycine if the two sequences are identical) (31); in the other, each of the hydrophilic residues in VI were systematically replaced with alanine (32). Mutations which disrupted binding to gp120, but not binding to a panel of anti-CD4 monoclonal antibodies recognizing conformationally-dependent epitopes, were considered potential contact residues for gp120. Four locations out side the CDR2-like sequence (residues 29, 59-64, 77-81 and 85) were identified as potential contact sites. Interestingly, mutations that show increased binding affinity to gp l20 can be found in four of these regions (ref. 32; A. Ashkenazi & DJC, unpublished results), supporting the con clusion that these regions are involved in the contacts CD4 makes with gpI20. The residues in VI that affect the interaction of gpI20 with CD4 are shown in Figure 2b. Interestingly, and perhaps obviously in hindsight, it is here that the largest structural differences between VI and Ig variable domains are seen (19a,b). The CC" loop in CD4 is longer than that of an Ig V/( region, while the nearby CC loop, and the FG loop, are both considerably shortened. Residues 40-55 form an exposed ridge (the C'C" ridge) on one side of the molecule, implying that gpl20 has a comple mentary groove in the CD4-binding region. Several residues that appear by mutational analysis to be involved in the interaction with gp l20 reside on adjacent {1-strands. While some of these have side chains that project towards the C'C" ridge, others are further within the body of the protein

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(see Figure 2b), and may therefore only indirectly affect gp120 binding. Using this newly available structure information, it will now be possible to design CD4 mutants to probe the structure of the CD4-binding region of gp120 more accurately.


gp120 Structure and the CD4 Binding Site

The envelope glycoproteins of HIV- 1 are initially synthesized as a single polyprotein precursor, gp160 (33), which is cleaved at a cluster of basic residues by a host cell�associated enzyme to give gp l 20 and the integral membrane protein gp41 (34). gp1 20 has the appearance of "knobs" on the virus surface; each particle is studded with 70-80 such protrusions (35). These are associated with the mature virion by a poorly understood, apparently noncovalent, interaction with gp41 and are spontaneously shed from the viral surface at a significant rate (36, 37). This may explain the relatively low infectivity of HIV preparations. If shedding also occurs in vivo, most virus-associated gpl 20 should be rcleascd into the circulation; however, no evidence for circulating soluble gp1 20 has yet been reported. The cleavage of gp 1 60 is critical to viral infectivity. Substitution of the carboxy-terminal arginine residue of gpl 20 with threonine abolishes all detectable cleavage and viral infectivity, although the mutant gp 1 60 molecule is transported to the cell surface and binds CD4 normally (38). Similarly, replacement of this tryptic-like endoproteolytic cleavage site with a chymotryptic-like site abolishes cleavage and infectivity, which can be restored by exposure to limiting concentrations of chymotrypsin (39). The primary sequence of gpI 20 predicts a 60-kd polypeptide with 24 potential asparagine-linked glycosylation sites, giving a mature gly coprotein of 1 20 kd. The sequence of different isolates of gp 1 20 has revealed an extraordinary degree of variability (up to 30%) in amino acid sequence. This variability is highly localized to five hypervariable regions, which contain deletions, insertions, and extensive amino acid substitutions (40--42). Leonard et al (43) have recently determined the disulfide bond structure and glycosylation pattern of recombinant gp 1 20 of HIV-IIIB produced in Chinese hamster ovary cells (shown schematically in Figure 3). gp 1 20 contains 5 disulfide-bonded loop structures; the first and fourth are simple loops formed by a single bond, while the others are complex, containing nested disulfide bonds. The fourth loop, which corresponds to the third hypervariable domain, contains the major type-specific neu tralizing epitope of gp1 20 (44-48). Remarkably, a 24-amino-acid peptide from this region (residues 27l�295) completely blocks the fusion inhibition activity both of antibodies raised against recombinant gp 1 20, and of serum from an infected chimp, in a type-specific manner (46). Thus, the principal epitope that elicits fusion-inhibiting antibodies is located in the central



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portion of gpI20. The positions of the cysteine residues are highly con served in the different gp120 isolates sequenced. By contrast, only l3 of the 24 glycosylation sites are conserved in position among the 20-odd HIV isolates sequenced to date (49). Although many of the remaining glycosylation sites are found in the hypervariable regions, the total number of sites appears to remain essentially constant among isolates, implying that the degree of glycosylation of the molecule is functionally important. Thus, every known gp120 isolate may be surrounded by a cloud of host derived carbohydrate residues which envelope the protein sequence, pro tecting it from immune recognition. However, enzymatic deglycosylation of gp 1 20 does not significantly diminish CD4 binding, indicating that only protein determinants are critical (50). Interestingly, a gp120 mutated at an N-linked glycosylation site (Asn 232 to GIn) is totally defective for infection although it retains CD4 binding (5 1 ). Studies with second-site revertants (Ser 98 to Asn) as well as third-site revertants (Arg 274 to lIe) to second site mutants have revealed that at least three discrete regions of gp l20 are critical to infectivity at a step subsequent to CD4 binding (52). Since HIV-2, like HIV- l , uses CD4 as its receptor (53-56), and there is limited amino acid identity (�40%) between the gp1 20s of these viruses (57), one might expect that the residues responsible for CD4 binding would be found in small conserved regions; so far this appears to be the case. A proteolytic fragment of gp1 20 containing most of the third, the fourth, and the fifth conserved domains (residue 322 to near the C-terminus) retains at least partial ability to bind CD4 (58). Consistent with this, monoclonal antibodies to gp 1 20 that block CD4 binding map to the fourth conserved domain (residues 392--402 and 396--407) ( 1 0, 59), while antisera to peptides derived from the fourth (residues 389--41 5) and fifth (residues 452--474) conserved domains substantially block CD4 binding (60). Indeed, studies with linker-insertion mutations have revealed lesions in the third (residues 333-334), fourth (residues 388-390) and fifth (residues 442--443) conserved domains which abolish CD4 binding, whereas deletion of part of the fourth hypervariable domain (residues 362-369) had no effect (61 ). Perhaps the most convincing evidence for contact residues on gp120 comes from studies with substitution mutants at Ala 403, which diminishes CD4 binding (10), and Trp 397, which abrogates CD4 binding (62). Interest ingly, a point substitution at a nearby residue, lIe 390, shows normal CD4 binding but an altered tropism; viruses carrying this mutation lose the ability to infect monocytes but retain the ability to infect T cells (62). The requirements for entry after CD4 binding may therefore be different in the two cell types (see below). Although the three-dimensional structure of gp l 20 and its receptor binding site is unknown, and likely to remain so for some time, an interest-


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'?' high man nose Figure 3

lJ.

complex

Disulfide bond and carbohydrate structure of recombinant gp120 produced by

Chinese hamster ovary cells. (Adapted from Leonard et al. (42).) Regions conserved between HIV-l and HIV-2 (CI-C5) and hypervariable regions (VI-V5) are indicated. Amino acid numbering in this figure and elsewhere in the review i� ba�ed on the mature sequence of the gpl20 ofHIV-l IIIB•

ing analogy has recently emerged between HIV and the (quite unrelated) rhinovirus family. As for HIV, cross-neutralizing antibodies that recognize all forms of rhinovirus are difficult if not impossible to raise, and like HIV, rhinoviruses use a member of the Ig superfamily (ICAM- I ) as their cellular receptor (63, 64). The three-dimensional structure of the rhinovirus capsid suggests that receptor recognition occurs within a canyon lined with highly conserved amino acids, which has a narrow rim consisting of variable residues (65). It has been suggested that by restricting the size of the


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receptor binding pocket and varying the residues exposed at its surface, the virus protects the receptor-binding site from antibody recognition, allowing it to evade the immune response (65). It is possible that a similar structure, serving a similar purpose, has evolved in the receptor-binding domain of the HIV envelope (21 ).


VIRAL ENTRY AND CELL FUSION

As for other enveloped viruses, the entry of HIV following CD4 binding is presumed to occur by membrane fusion, either directly with the plasma membrane or within an acidic endocytic vesicle following receptor mediated endocytosis of the virus-receptor complex. The observation that CD4 is internalized and recycled to the cell surface following phorbol ester treatment (66) initially led to the idea that HIV could enter by endocytosis, in a pH-dependent process. While early studies suggested that HIV entry was pH-dependent (8), subsequent studies with lysosomatropic agents that raise the pH of acidic endosomal compartments and block the entry of other viruses by the endocytic pathway have shown HIV entry to be pH independent (67, 68). Consistent with this, direct visualization by electron microscopy has revealed fusion of HIV particles with the plasma mem brane but not viral endocytosis or intracellular compartmentalization of intact virus (67). Perhaps the clearest support for the idea that HIV fusion with the plasma membrane is sufficient for infection comes from studies with CD4 mutants lacking the cytoplasmic domain (69, 70). Human cell lines transfected with such mutants are as susceptible to infection as cell expressing wild-type CD4, despite having severely impaired ability to carry out phorbol ester-induced endocytosis. The ability of HIV to induce cell fusion led to examination of the envelope sequence for homology to other fusogenic viruses; indeed gp41 contains a conserved N-terminal sequence with strong homology to the fusion glycoprotein of paramyxoviruses (71, 72). Mutants within this region are markedly impaired in their ability to induce syncytia (61); and in the analogous protein (p32) of simian immunodeficiency virus (SIVmac) , mutations that increase the overall hydrophobicity of the N-terminus increase fusogenic activity (73). Thus, it appears likely that, after CD4 binding, the next step in HIV infection of a CD4 + T cell is membrane fusion initiated by gp41. The structure of gp41 may allow the formation of antiparallel amphipathic a-helical structures, which could aggregate and after insertion destabilize the cell membrane (74). Consistent with this, gp41 is found in virions chiefly as a tetramer, with some trimers (75). Fusion appears to be dependent on an unidentified human cellular com ponent, in that transfected mouse cells expressing human CD4 are resistant


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to infection, although binding of HIV to these cells appears normal (8). Thus, the infectivity block in these cells is probably at the stage of penetration. Evidence is accumulating that CD4 sequences distinct from those required for binding may be important in the fusion process. Thus, an antibody to the V3 domain of CD4, which does not inhibit HIV binding, nevertheless inhibits infection (76). Similarly, mutants derived by chemical mutagenesis of a CD4 + T-cell line and selected for altered CD4 expression bind soluble gpl20 with normal affinity yet are markedly deficient in their ability to form syncytia; however, it has not yet been clearly shown that this difference resides in the CD4 gene (77). The situation is further com plicated by the fact that the requirements for viral infection and virus mediated syncytium induction may be distinct. Thus, chimpanzee and rhesus CD4 + T cells permit HIV entry but not syncytium formation (78). This may partially explain the observation that chimpanzees, while infectable by HIV, do not develop the profound immunodeficiency charac teristic of HIV infection in humans (79). Currently it is unclear why cell cell fusion is affected whereas virus-cell fusion is not. It has been suggested that the viral surface may have a higher density of gpl20 and/or gp41 than an infected cell surface, and therefore viral infection is less dependent on the precise sequence of CD4 (78). The difference in ability to support syncytium induction appears to be intrinsic to the CD4 molecule itself, in that human B cells transfected with a human CD4 cDNA undergo syncytium formation whereas cells transfected with a chimpanzee CD4 cDNA do not (78). However, the two molecules, which differ by only five amino acids, have identical affinities for gp l20 and appear to support infection comparably. The sequences responsible for the difference between chimpanzee and human CD4 in promoting syncytium formation are thus distinct from those involved in gp 1 20 binding. Residues affecting syncytium formation are clustered around amino acid 87; remarkably, chimpanzee CD4 containing human residue 87 (Glu) supports syncytium formation, whereas human CD4 bearing chimpanzee residue 87 (Gly) does not. The manner in which CD4 sequences participate in syncytium formation is unclear. The interaction of gp l20 with CD4 may activate or expose a fusion domain in gp41 via a change in the conformation of gp 120. Indeed, soluble recombinant CD4 (rCD4) can actually enhance the formation of syncytia in cells infected with HIV-like viruses such as SIVagm (80) and HIV-2 (81), although it blocks syncytium formation induced by HIV-l (82, 83). In contrast, a chimeric molecule consisting of the VIV2 domains of CD4 joined to the Fc domain of human IgG (rCD4-IgG) (84) blocks syncytium formation in HIV-2-infected cells, whereas a VIV2 fragment of CD4, like soluble rCD4, enhances (81 ). Two questions arise from these



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findings: why does the V1V2 fragment, but not the chimeric rCD4-IgG molecule, enhance syncytium formation; and why does soluble rCD4 enhance syncytium induction by HlV-2 or SIVagm but inhibit that induced by HIV-l? It is possible that when gp120 binds to CD4, the conformational change in gp120 that allows fusion may also require a conformational change in CD4. Perhaps the rCD4-IgG molecule is unable to support such a change due to structural or steric constraints, and this prevents the exposure of the gp41 fusion domain. The difference between the viruses is a matter of speculation at this point; while the fusion domains of HIV-I and HIV-2 are well conserved (57), there may be fundamental differences in the mode of entry of the two viruses. CD4-DEPENDENT PATHOGENESIS IN AIDS

A major pathogenetic mechanism in AIDS is the depletion of CD4 + T cells. In addition, the functions of the surviving CD4+ cells are also impaired (3). Because CD4 is important in the maturation of T cells, it is reasonable to predict that T-cell development would also be impaired as a result of the interaction of gp120 with CD4. To understand the patho genesis of AIDS, it is therefore important to understand the functions of CD4, both in the activation of mature T cells and in the development of peripheral T cells. CD4-Dependent Mechanisms of Cell Killing

Although the direct infection of CD4+ cells by virus appears to be an obvious pathway of cellular destruction in AIDS, its quantitative sig nificance is not clear. Recent work has shown that the frequency of CD4+ cells containing HIV-l DNA in AIDS patients is 1/100, although the frequency of cells actively expressing viral mRNA may be as low as 1/1000 (85). In earlier work the frequency of total mononuclear cells expressing HIV RNA was found to be 1/100,000 (86). These results prompted a search for indirect mechanisms to explain HIV -mediated killing of very high percentages of CD4+ cells when only a small number are actively infected. One possible mechanism for the killing of uninfected CD4+ cells is their recruitment by HIV-infected cells into multinucleated giant cells, or syn cytia (87). Syncytium formation is clearly dependent on CD4-gp1 20 bind ing, as it can be completely blocked by anti-CD4 antibodies (88) and soluble rCD4 (82, 83). Furthermore, studies with transfected cells or cells infected with vaccinia virus recombinants have revealed that expression of the env proteins in the absence of other HIV- l proteins is sufficient for fusion of such cells with uninfected CD4 + cells (88, 89). Syncytium for-


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mation is a characteristic feature of the cytopathology induced by HIV in infected T-Iymphoblastoid cell cultures, and there is increasing evidence that the ability of a virus to induce syncytia correlates with its patho genicity (see below). Tn vivo evidence for syncytium formation in AIDS is limited to reports of giant cell formation in the brains of patients with AIDS-related dementia (90-93); SIV-infected monkeys also show syncytium formation in brain (94) as well as in lymphoid tissues (95). Other mechanisms suggested to explain the substantial depletion of CD4 + cells characteristic of AIDS involve the targeting of uninfected CD4+ cells by killer cells after they bind the soluble gp 1 20 shed by the virus and by infected cells (36, 37). In vitro evidence suggests that gp 1 20 binding to CD4 can make an uninfected CD4 + cell a target for antibody dependent cell-mediated cytotoxicity (ADCC) mediated by anti-gp 1 20 antibodies present in the sera of infected individuals (96, 97). In addition, uninfected CD4 + cells that have bound, internalized, and processed gp 1 20 can also present gpl20 fragments on MHC on their surface and thus become targets for killing by unusual CD4 + cytotoxic T cells that recognize gp 120 sequences presented in the context of class-II MHC (98- 1 0 1 ). Envelope Variation and Pathogenesis

Many studies of HIV pathogenicity have relied on the ability of the virus to induce syncytium formation in vitro as a measure of its cytopathic effects in vivo. Numerous studies show that different isolates of HIV-I and HIV-2 differ in their degree of in vitro cytopathicity. Thus, while some primary HIV -I isolates ind uce marked syncytium formation and cell death, others show no such effect; moreover, such phenotypic differences are stable upon in vitro passage (1 02, 103). Increases in syncytium-inducing potential have been correlated with increased risk of progression to AIDS and to decreased survival rate following diagnosis of AIDS (104). Sequen tial isolations from individual patients have revealed transitions from non

syncytium-inducing to syncytium-inducing HIV isolates ( lOS). Further more, disease progression is often accompanied by the emergence of variants with increased cytopathicity (1 06). Thus, the ability of HIV to induce syncytium formation may well reflect pathogenic potential in VIVO.

There is an increasing appreciation that several HIV gene products, in particular those controlling replication, can affect vi rus cytopathicity. However, the high variability of the envelope proteins, their constant selection by the immune system, and their direct role in attachment and cell fusion make env-related changes in cytopathicity particularly relevant. Indeed, differences in cytopathicity have been mapped to the env gene using replication competent proviral clones (1 07). Engineered mutations


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in the HIV envelope decrease in vitro cytopathicity by several mechanisms (effects on CD4 binding, fusion ability, efficiency of expression, proces sing or transport, or gp120-gp41 association; Ref. 6 1 ); however, there is as yet little information on which of these mechanisms are important in


VIVO.

While HIV-2 infection is associated with AIDS, epidemiological studies suggest that HIV-2 may be less pathogenic than HIV-1 (reviewed in Ref. 108). HIV-2 isolates which clearly have reduced cytopathicity in vitro, yet replicate actively, have been described (109, 1 1 0). Studies with vaccinia recombinants expressing the env proteins of HIV-2Roo, which induces syncytia, and HIV-2sT' which does not, show that env determines the syncytium-inducing phenotype ( 1 1 1 ). Since no differences in env expression, processing, or transport were observed, it is likely that the interaction between the envelope protein and the target cell was altered. Indeed, when HIV-2sT was selected for increased cytopathicity in culture, the resultant highly cytopathic variant exhibits a three- to four-fold increase in affinity for CD4, as well as an increased surface expression of envelope ( 1 12). It is interesting to note that HIV-2Roo is less sensitive to inhibition by soluble CD4 than HIV-I (55, 56) and that the affinity of HIV-2RoD gp1 20 for CD4 has been reported to be 25-fold lower than that of HIV-l IIIB (11 3). The affinity of HIV-2sT gp120 is not known. It remains to be seen whether a change in CD4-binding affinity is a common mechanism for increases in cytopathicity, or whether other mechanisms apply in natural infections. Whatever the mechanism by which syncytium-inducing variants arise during late-stage disease, one might expect that those who acquire HIV from late-stage patients would have a more severe clinical course, and this does not appear to be the case. It has been proposed that such highly cytopathic variants are at a selective disadvantage in the immuno competent host, since their high rate of replication makes them immediate targets for the immune system ( 1 14). Thus, although cytoplasmic variants might arise periodically throughout the course of HIV infection, con tributing to CD4 + cell depletion, they would not become apparent until the immune system is severely compromised. CD4-MHC Interaction, Signaling and T-Cell Activation

In addition to the depletion of CD4 + cells, HIV infection is associated with functional abnormalities in this T-cell subset, as reflected in the suppression of antigen and mitogen-induced proliferative responses in T cells derived from patients (3). Once again the question arises as to whether this cellular dysfunction is a direct consequence of viral infection or the result of the interaction of extracellular soluble gp120 with the CD4 mol-


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ecule. Evidence for both mechanisms has been obtained. In HIV-infected cells, the downregulation of CD4 surface expression (7), which occurs at the level of transcription (1 1 5, 1 1 6) and by complex formation between CD4 and internally produced gp1 20 (1 1 5-1 1 8), is expected to impair T cell function. For uninfected cells, purified gp 120 has been shown to inhibit a variety of lymphocyte responses in vitro, for example, the proliferative responses to mitogens (1 19) and soluble antigens ( 1 20- 1 23). To explain the immunosuppressive properties of gp120, numerous investigators have proposed that the binding sites for class-II MHC and gp 1 20 on CD4 are situated so that gpl 20 blocks MHC binding. Before we consider the evidence that supports this hypothesis, we briefly review the state of under standing of the role of CD4 as a coreceptor in T-cell activation. The finding that CD4 + and CD8 + T cells are restricted to the recognition of antigen presented by class-II and class-I MHC-expressing target cells, respectively, led to the notion that CD4 and CD8 function as accessory molecules in T-cell activation, promoting adhesion between target and effector cells by binding to nonpolymorphic determinants on the appro priate MHC molecules (reviewed in 1 24, 1 25). Direct evidence for CD4 binding to class-II MHC comes from experiments showing that fibroblasts transfected with a CD4 expression vector adhere specifically to cells expressing class-II MHC (1 26); comparable results have been obtained for CD8 and class-I MHC ( 1 27). More recently, the view has arisen that the role of CD4 and CD8 in T-cell activation is not confined to improving adhesion between effector and target cell. In the coreceptor model, CD4 and CD8 are envisioned as an integral part of the TcRjCD3 complex on activated T cells, contributing to the signaling function of the complex (128). In the case ofCD4, this model predicts that CD4 and the TcRjCD3 complex recognize the same class-II MHC molecule and are physically associated. Evidence for an interaction between CD4 and TcR/CD3 comes from studies showing the comodulation of CD4 and CD3 molecules on the cell surface (1 29, 1 30) and the coclustering of CD4 and CD3 to the cell-cell interface upon activation of a T-cell clone by antigen-presenting cells (131). In addition, studies with bispecific antibody dimers or aggre gates show that cross-linking of these molecules is significantly more potent in activating T cells than cross-linking the TcR/CD3 complex alone ( 1 32, 1 33). Fluorescence resonance energy transfer experiments have shown that activation of the TcR by anti-CD3 antibody causes association between CD4 and the TcRjCD3 complex ( 1 34). Interestingly, this association did not occur when a truncated CD4 protein lacking the cytoplasmic domain was used instead of wild type. As the cytoplasmic domain has been impli cated in signaling (see below), this would imply that complex formation and signaling may be linked.



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The coreceptor model also predicts that the CD4 molecule is actively involved in the transduction of signals accompanying T-cell activation. Two major pathways of signal transduction appear to be activated after TcR occupancy or cross-linking (reviewed in Ref. 1 35). In the first, acti vation of phospholipase C (PLC) leads to the hydrolysis of phos phatidylinositol 4,5-bisphosphate (PIP2), yielding the second messengers diacylglycerol, which activates protein kinase C, and inositol 1 ,4,5-tris phosphate, which triggers the release of Ca2+ from intracellular stores (reviewed in Ref. 1 36). Indeed, gp 120 affects signaling in both activated and resting T cells, although these effects are opposite in nature. Thus, gp 120 blocks PIPz hydrolysis and Ca2+ mobilization elicited by soluble antigen or by anti-CD3 antibody in tetanus toxoid-reactive T-cell clones ( 123). A combination of gp1 20 and anti-gp l20 antibody also inhibits Ca 2+ mobilization in peripheral T cells stimulated with anti-TcR antibody ( 1 37). On the other hand, gp l20 activates these same responses in resting T cells and mononuclear cells (1 38, 1 39). These findings, while apparently paradoxical, indicate that soluble gp 1 20, if present at high enough con centrations, may interfere with the normal process of T-cell activation. A second pathway suspected to be important in T-cell activation involves a tyrosine kinase encoded by the lck protooncogene, p56Jck ( 1 40). p561ck, which is expressed to high levels only in T cells, has been found to be associated noncovalently with CD4 or CD8 ( 1 4 1 , 1 42). The structure of p56lck is homologous to that of the protooncogene p6Wsrc in its C terminal kinase domain, but has a unique N-terminal domain ( 1 43). p56Jck is myristoylated at its N-terminus and associates with the inner leaflet of the plasma membrane (1 43). Chimeric p56iCk/p60c-src proteins have been used to show that the unique N-terminal domain of p56lck is responsible for its specific association with CD4 or the a chain of CD8 (1 44, 1 45). This interaction is independent of other lymphoid factors, as it is observed in transfected nonlymphoid cells ( 145, 1 46). The p56lck-binding domain of CD4 has been shown by mutational analysis to comprise the membrane proximal 28 residues of the cytoplasmic tail; the two cysteine residues in this region are critical for p56lck association ( 1 45). Cross-linking of CD4 by anti-CD4 antibodies induces a rapid increase in p56lck tyrosine kinase activity ( 1 47). Very recently it has been shown functional T-cell clones containing CD4 mutants that lack the ability to interact with p561ck also fail to undergo antigenic activation (D. Littman, pers. comm.), implying that activation of p56lck is a crucial signal in T-cell activation. Thus, evidence is accumulating that CD4 is intimately involved in T-cell signal transduction, and that inhibition of CD4 function is likely to inhibit CD4+ T-cell activation. While some of the immunosuppressive effects of gp 1 20 may result


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directly from the interaction with CD4, there is also evidence that gp1 20 can inhibit the interaction of CD4 with class-II MHC. Thus, soluble gp120 specifically blocks the formation of conjugates between CD4 + cells and cell-size lipid vesicles bearing MHC class-II proteins ( 1 4S). Similarly, murine T-cell hybridomas expressing human CD4, which specifically rosette with targets expressing human class-II MHC, are blocked from rosette formation and subsequent IL-2 generation by gp1 20 ( 1 49, 1 50). Such systems have been useful in mapping the MHC-binding domain of CD4 by homolog-scanning mutagenesis. In these studies, segments of the murine CD4 sequence (which does not bind gp120 or human class-II MHC) were used to replace the corresponding regions of nonconserved sequences in human CD4 (27, 2S). The resultant mutants were tested for their ability to bind soluble gp1 20, their ability to trigger a class-II MHC dependent IL-2 response in effector cells, and their ability to allow rosette formation with a class-II MHC-expressing target cell. Three classes of mutants were obtained: those lacking the ability to bind MHC, but that bind to gp l20 with high affinity; those lacking the ability to bind gp 1 20, yet that interact normally with class-II MHC; and mutants that are impaired in both functions. These findings indicate that the gp1 20 and class-II MHC binding sites on CD4 are distinct and separable, but overlap to a signifi cant extent. Thus, gp l20 can apparently block the coreceptor function of CD4 by sterically hindering the interaction of CD4 with class-II MHC. Thymic Development of CD4+ Cells

One difficulty in understanding how HIV infection ultimately brings about the complete destruction of the CD4 + cell subset is related to the belief that mature CD4 + cells are replenished from a bone marrow stem cell that lacks CD4 expression and therefore is thought to be resistant to HIV infection. This paradox may be more apparent than real, since the pro duction of mature CD4 + cells itself depends upon the function of CD4 and therefore may be abrogated if that function is disturbed. To illustrate this, we have provided a brief review of the role of CD4 in thymic devel opment, but the reader is referred to recent excellent reviews (151, 152) for more detailed discussion of these topics. The thymus is essential for the development of mature T cells. In a process which is not fully understood, hematopoietic cells that do not express CD4, CDS, or T-cell receptors enter the thymus and emerge as TCR-positive, CD4+CDS- or CD4- CDS+ (single-positive) T cells. It is also here that the range of permissible TcR specificities is determined, by deleting cells expressing receptors that recognize self-antigen. The events within the thymus can be summarized as follows. Precursor cells, expressing neither CD4, CDS, nor TcR, initially give rise to



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CD4+CD8+ double-positive thymocytes, some of which express a func tionally arranged r:t.{3 TCR. It is currently thought that a selected subset of these cells go on to become single-positive, TcR-bearing mature thymo cytes, while the rest die within the thymus. The selection of immature thymocytes is thought to be a combination of two processes-(a) positive selection, allowing maturation of thymocytes bearing T-cell receptors that bind to self-MHC molecules, thus selecting for their ability to recognize foreign antigen bound to MHC; and (b) negative selection, arresting the development of thymocytes bearing T-cell receptors that bind to MHC plus self-antigens. The mature T cell expresses only one of CD4 and CD8, depending on the specificity of the TcR it expresses, i.e. whether it binds to class-II or class-I MHC. Thus, transgenic mice expressing a TcR specific for class II have a greatly increased number of peripheral CD4+CD8cells (153), whereas mice expressing TcRs specific for class I generate peripheral T cells that are largely CD4-CD8+ (154). These experiments imply that the specificity of the TcR expressed on an immature thymocyte ultimately determines whether the mature T cell expresses CD4 or CD8. It appears that CD4 and CD8 are themselves involved in these selective events; when antibodies to CD4 or CD8 are given to young mice early in development, the development of the corresponding subpopulation of T cells is inhibited (155); similar results can be obtained with anti-MHC antibodies (156). Two explanations have been offered for the apparent ability of a TcR to choose which accessory molecule should be retained by a T cell. In the "instruction" model, coengagement of a CD4 molecule and a TcR with a class-II molecule instructs the T cell to delete expression of CD8. Conversely, when CD8 and a class I-reactive TcR co engage a c1ass-1 molecule, deletion of CD4 expression results. In the "selection" model, T cells lose either CD4 or CD8 randomly, and only the cells with matched pairs of accessory molecules and TcRs survive (157). Given the ability of gpl 20 to block CD4-MHC binding, one might expect that the maturation of CD4 + T cells could be blocked in the presence of gp120, if present in high enough concentration, as it is in the presence of anti-CD4 antibody. Inhibition of CD4+ T-cell replenishment may be a significant factor in the gradual depletion of these cells in HIV infection. However, no evidence has yet been presented that supports this hypothesis. While there is currently no information on the ability of HIV to infect lymphoid progenitor cells, it should be noted that CD34+ myeloid progenitor cells, which eventually give rise in culture to CD4 + cells with mature monocytoid morphology, can be infected in vitro (158). It is pos sible that lymphocyte progenitors can also be infected at an early stage, decreasing the rate of replenishment independent of effects on T-cell development.

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In addition to its effects on thymic development of CD4 + T cells, antibody to CD4 can induce tolerance to certain antigens when given concurrently (159-161). Antigen-specific tolerance induced by this method is long-lasting and independent of the abiility of such antibodies to deplete CD4 + cells (162). Similar tolerance induction has recently been observed using gp l 20 (Ref. 163, J. Greenstein, M. Gefter, personal communication).


CD4-BASED THERAPEUTIC STRATEGIES

Given that the CD4-gp120 interaction is so critical, not only to new rounds of HIV infection but also to most postulated mechanisms of CD4+ cell depletion, one approach to controlling HIV disease progression would be to inhibit the CD4-gp120 interaction. Indeed, antibodies to CD4 can block viral infection and syncytium induction, but since these may also inhibit CD4-mediated immune function and CD4+ cell development, efforts have focussed on directing such a blocking activity to gp120. Monoclonal anti bodies to gp120, an obvious choice, suffer from the limitation that they will generally be type-specific. Thus, most attention in this area has centered on the development of antagonists that bind to the CD4-binding site on gp120, using the rationale that the only part of gp120 that the virus cannot afford to change is its CD4-binding site. The first generation of such efforts used soluble forms of the CD4 molecule itself (II, 82, 83, 164--166). These molecules are normally made as truncated forms of the CD4 molecule, lacking the transmembrane and cytoplasmic domains, and are efficiently secreted by mammalian cells. Such soluble rCD4 molecules bind to gp l 20 with an affinity comparable to that of cell-surface CD4 and block both viral infection and virus-induced cell fusion in several different in vitro systems. Moreover, diminished viremia was seen in SIVmac-infected rhesus monkeys treated with soluble rCD4 (167), although the experience with soluble rCD4 in human clinical trials has been equivocal to date (168170). Unexpectedly, soluble derivatives of CD4 have proved not to inhibit CD4+ cell function (83, 121, 171) nor to block CD4--class II MHC adhesion (149, 172); such derivatives can in fact prevent the inhibition of CD4+ cell function by gp120 (172). While the molecular basis for this is incompletely understood, it would appear that the interaction between monovalent CD4 and class-II MHC is of low affinity. Smaller versions of CD4, containing approximately the first 110 amino acids (the VI domain), appear to bind gp120 and block virus infection and HIV-mediated cell fusion comparably to soluble rCD4 (19, 20). This is the smallest molecule so far shown to bind to gp120 with high affinity; although small peptides that can block HIV infectivity and HIV -induced cell fusion



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have been identified from two different regions of the CD4 molecule (amino acids 25-58 and 81-92), very high concentrations are required (> 100 11M) (29, 30). One of these peptide sequences (residues 25-58) contains a major critical determinant involved in gp120 binding revealed by studies with CD4 mutants (residues 40-55; see above). It is notable that the other peptide (residues 81-92) must be derivatized to have blocking activity, although it does appear to block the CD4-gp120 interaction (173). Recently, Finberg et al (174) have exploited the observation that a Phe residue (position 43) is present in CD4 at the site of gp120 binding to develop a series of small phenylalanine-containing molecules as HIV antagonists. Certain derivatives of the dipeptide prolylphenylalanine, with an N-terminal carbomethoxycarbonyl moiety and a C-terminal benzyl ester (termed CPFs) blocked gp120 binding to CD4, inhibited HIV infec tion, and reversed the inhibition by gp120 of CD4-class II MHC binding (174). CPFs, like CD4 derivatives, appear to act by binding to gp120, in contrast to other small-molecule inhibitors such as aurin tricarboxylic acid, which inhibits gpl20 binding by binding to CD4 (175). However, their affinity for gp l20 appears quite low, with an IDso for blocking HIV infectivity of '" 100 11M. Thus, while CPFs provide an attractive avenue for further research, it is likely that their potency will need to be substantially improved. Since gpl20 is expressed on the surface of productively infected cells, the gp l 20-binding activity of CD4 can in principle be used to target toxins or liposomes containing antiviral compounds to infected cells, resulting in their destruction. Molecular fusions of CD4 with a variant of pseudomonas exotoxin (CD4-PE40) kill infected cells at least 100-fold more efficiently than uninfected cells (176). Similarly, rCD4 chemically coupled to degly cosylated ricin A chain kills HIV-infected cells with 103-fold specificity (177). However, as such preparations do not affect latently infected cells, they will probably need to be administered chronically, and since their toxin moieties are immunogenic, they may have limited utility. It is possible that linking smaller, less antigenic toxic molecules or radionuclides to CD4 derivatives may have greater potential. Another approach to adding active antiviral capabilities to CD4 is exemplified by CD4·immunoglobulin hybrids. These were initially con structed in an attempt to improve the plasma half-life of soluble rCD4, which is cleared efficiently (t 1/2 1 hour in humans; 170), probably by kidney filtration. Molecules were designed that combine the gpl20-binding portion of CD4 with the Fc portion of IgG, which confers a long plasma half-life on IgG molecules (14-21 days). Such CD4-IgG hybrids, called CD4 immunoadhesins, have been shown to possess a terminal half-life up �


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to 100-fold longer than soluble rCD4, as well as the gp l 20-binding and HIV-blocking properties of soluble rCD4 (84). Interestingly, a CD4-IgM hybrid blocks HIV-l infectivity with considerably greater efficiency than soluble CD4 itself, perhaps due to its decavalent structure ( 1 78). CD4 immunoadhesins also have a number of the active properties determined by the Fc region of immunoglobulin, including the ability to bind to the first component of complement, C l q, and to bind to Fc receptors (84, 1 78, 1 79). As predicted for a molecule that can bind to Fc receptors, CD4 immunoadhesin has recently been shown to mediate the specific killing of infected but not un infected CD4+ cells via ADCC ( 1 80). Unlike natural anti-gp 1 20 antibodies, CD4 immunoadhesin does not allow ADCC of un infected CD4-expressing "bystander" cells which have bound gp1 20 to CD4 on their surface. This is probably because gp1 20 has only one CD4-binding site; CD4 immunoadhesin, unlike natural anti-gp1 20 antibodies, cannot bind gp l 20 already bound to cell-surface CD4. In addition, CD4 immunoadhesin, like natural IgG molecules, is efficiently transferred across the placenta of a primate, whereas soluble rCD4 is not (180). This observation may have relevance for the use of CD4 immuno adhesin for the prevention of perinatally transmitted HIV infection. An underlying assumption of the use of CD4 derivatives is that to avoid binding to such molecules, the virus would need to change its envelope protein to such a degree that specific infection of CD4+ cells would no longer be possible; even if HIV were able to change so much and remain infective, the resulting virus would be unlikely to cause the CD4+ depletion and profound immunodeficiency characteristic of AIDS. Indeed, the blocking effects of soluble rCD4 are observed with a broad range of HIV laboratory isolates, as predicted (55, 56). However, this assumption appears to be only partly true, in that the virus, while dependent on binding to CD4 to retain its infectivity, nonetheless appears to retain infectivity over a range of CD4-gp120 affinities. The evidence for this is twofold. First, recent studies show that primary, unpassaged HIV isolates are much less sensitive to soluble rCD4 inhibition than are laboratory isolates that have been extensively passaged on human T-cell lines ( 1 8 1 ). An obvious interpretation of this finding is that the affinity of unpassaged strains for CD4 is lower than that of laboratory strains, although this has not yet been directly examined. The high affinity for CD4 of laboratory strains may therefore be due to constant selective pressure for virus replication in vitro. Second, an HIV- I laboratory isolate selected for growth' in the presence of high concentrations of soluble rCD4 has yielded variants that are less sensitive to rCD4, and whose gp120s have decreased affinity for CD4 (182). Interestingly, one of these variants is different in its amino acid sequence from the parent in a region identified as critical for CD4 binding


CD4--gp120 INTERACTIONS

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(residue Met 404 becomes Thr). While this finding suggests that CD4based therapies may allow the emergence of drug resistance, as do those based on inhibition of the viral reverse transcriptase, such as AZT (183), it is also possible that the strains of HIV found in vivo are already at the limit of the affinity required to infect CD4 + cells successfully and therefore may not so easily give rise to CD4-resistant strains. Soluble receptor-based agents represent a novel concept in receptor blocking therapy, and their potential for use as antivirals and in other diseases has attracted wide attention. Due to their mode of action and likely synergy with other anti-HIV agents, CD4-based therapeutics remain an attractive area for further investigation. However, the success of this approach may well require the development of analogs with higher potency, such as CD4-immunoadhesin. In addition to further improve ments in pharmacological properties and active antiviral capabilities it may also be possible to enhance CD4-based therapeutics by improving their affinity, particularly for in vivo strains of HIV. ACKNOWLEDGMENTS

We thank Drs. Daniel Littman, Randal Byrn, and Avi Ashkenazi for helpful discussions and critical review of the manuscript, Dr. Steven Har rison for providing Figure 2, Michael Spellman for allowing us to adapt Figure 3, Anne Stone, Carol Morita, and Scooter Morris for assistance with illustrations, and Cathy Hollenbach for manuscript preparation. Literature Cited

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