Human immunodeficiency virus type 1 gp120 envelope glycoprotein regions important for association with the gp41 transmembrane glycoprotein.

Insertion of four amino acids into various locations within the amino-terminal halves of the human immunodeficiency virus type 1 gp120 or gp41 envelope glycoprotein disrupts the noncovalent association of these two envelope subunits (M. Kowalski, J. Potz, L. Basiripour, T. Dorfman, W. C. Goh, E. Terwilliger, A. Dayton, C. Rosen, W. A. Haseltine, and J. Sodroski, Science 237:1351-1355, 1987). To localize the determinants on the gp120 envelope glycoprotein important for subunit association, amino acids conserved among primate immunodeficiency viruses were changed. Substitution mutations affecting either of two highly conserved regions located at the amino (residues 36 to 45) and carboxyl (residues 491 to 501) ends of the mature gp120 molecule resulted in nearly complete dissociation of the envelope glycoprotein subunits. Partial dissociation phenotypes were observed for some changes affecting residues in the third and fourth conserved gp120 regions. These results suggest that hydrophobic regions at both ends of the gp120 glycoprotein contribute to noncovalent association with the gp41 transmembrane glycoprotein.     

Analysis of the interaction of the human immunodeficiency virus type 1 gp120 envelope glycoprotein with the gp41 transmembrane glycoprotein.

The human immunodeficiency virus type 1 (HIV-1) gp120 exterior envelope glycoprotein interacts with the viral receptor (CD4) and with the gp41 transmembrane envelope glycoprotein. To study the interaction of the gp120 and gp41 envelope glycoproteins, we compared the abilities of anti-gp120 monoclonal antibodies to bind soluble gp120 and a soluble glycoprotein, sgp140, that contains gp120 and gp41 exterior domains. The occlusion or alteration of a subset of gp120 epitopes on the latter molecule allowed the definition of a gp41 "footprint" on the gp120 antibody competition map. The occlusion of these epitopes on the sgp140 glycoprotein was decreased by the binding of soluble CD4. The gp120 epitopes implicated in the interaction with the gp41 ectodomain were disrupted by deletions of the first (C1) and fifth (C5) conserved gp120 regions. These deletions did not affect the integrity of the discontinuous binding sites for CD4 and neutralizing monoclonal antibodies. Thus, the gp41 interface on the HIV-1 gp120 glycoprotein, which elicits nonneutralizing antibodies, can be removed while retaining immunologically desirable gp120 structures.     

Antibody cross-competition analysis of the human immunodeficiency virus type 1 gp120 exterior envelope glycoprotein.

Forty-six monoclonal antibodies (MAbs) able to bind to the native, monomeric gp120 glycoprotein of the human immunodeficiency virus type 1 (HIV-1) LAI (HXBc2) strain were used to generate a competition matrix. The data suggest the existence of two faces of the gp120 glycoprotein. The binding sites for the viral receptor, CD4, and neutralizing MAbs appear to cluster on one face, which is presumably exposed on the assembled, oligomeric envelope glycoprotein complex. A second gp120 face, which is presumably inaccessible on the envelope glycoprotein complex, contains a number of epitopes for nonneutralizing antibodies. This analysis should be useful for understanding both the interaction of antibodies with the HIV-1 gp120 glycoprotein and neutralization of HIV-1.     

gp120-independent fusion mediated by the human immunodeficiency virus type 1 gp41 envelope glycoprotein: a reassessment.

In a natural context, membrane fusion mediated by the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins involves both the exterior envelope glycoprotein (gp120) and the transmembrane glycoprotein (gp41). Perez et al. (J. Virol. 66:4134-4143, 1992) reported that a mutant HIV-1 envelope glycoprotein containing only the signal peptide and carboxyl terminus of the gp120 exterior glycoprotein fused to the complete gp41 glycoprotein was properly cleaved and that the resultant gp41 glycoprotein was able to induce the fusion of even CD4-negative cells. In the studies reported herein, mutant proteins identical or similar to those studied by Perez et al. lacked detectable cell fusion activity. The proteolytic processing of these proteins was very inefficient, and one processed product identified by Perez et al. as the authentic gp41 glycoprotein was shown to contain carboxyl-terminal gp120 sequences. Furthermore, no fusion activity was observed for gp41 glycoproteins exposed after shedding of the gp120 glycoprotein by soluble CD4. Thus, evidence supporting a gp120-independent cell fusion activity for the HIV-1 gp41 glycoprotein is currently lacking.     

Membrane permeabilization by different regions of the human immunodeficiency virus type 1 transmembrane glycoprotein gp41.

The transmembrane glycoprotein (gp41) of human immunodeficiency virus type 1 (HIV-1) has been implicated in the cytopathology observed during HIV infection. The first amino acids located at the amino terminus are involved in membrane fusion and syncytium formation, while sequences located at the carboxy terminus have been predicted to interact with membranes and modify membrane permeability. The HIV-1 gp41 gene has been cloned and expressed in Escherichia coli cells by using pET vectors to analyze changes in membrane permeability produced by this protein. This system is well suited for expressing toxic genes in an inducible manner and for analyzing the function of proteins that modify membrane permeability. gp41 enhances the permeability of the bacterial membrane to hygromycin B despite the low level of expression of this protein. To localize the regions of gp41 responsible for these effects, a number of fragments spanning different portions of gp41 were inducibly expressed in E. coli. Two regions of gp41 were shown to increase membrane permeability: one located at the carboxy terminus, where two highly amphipathic helices have been predicted, and another one corresponding to the membrane-spanning domain. Expression of the central region of gp41 comprising this domain was highly lytic for E. coli cells and increased membrane permeability to a number of compounds. These findings are discussed in the light of HIV-induced cytopathology and gp41 structure.     

Association of human immunodeficiency virus type 1 envelope glycoprotein with particles depends on interactions between the third variable and conserved regions of gp120.

Many regions within the envelope of human immunodeficiency virus type 1 (HIV-1) that affect its structure and function have been identified. We have previously reported that the interaction of the second conserved (C2) and third variable (V3) regions of gp120 influences the ability of HIV-1 to establish a productive infection in susceptible cells. To better understand the basis for this interaction, we have conducted structure-function analyses of envelope expressed from molecular proviral clones of HIV-1 containing defined mutations in C2 and V3 that individually and in combination differentially affect envelope function. The substitution of a glutamine for an asparagine residue (Q-267) at a potential asparagine-linked glycosylation site in C2, which severely impairs virus infectivity, reduces intracellular processing of gp160 into gp120, the association of gp120 with virions, and the ability of gp120 to bind to the HIV-1 cell surface receptor protein, CD4. The change of an arginine to an isoleucine codon in V3 (I-308), in the presence of the Q-267 mutation, restores virus infectivity to near wild-type levels by increasing the amount of gp120 associated with virions as compared with the Q-267 mutant but does not compensate for the Q-267-induced processing defect. The I-308 change in the context of the wild-type HIV-1 has no affect on processing, association, or CD4 binding. These results indicate that the impaired infectivity of the Q-267 mutant virus is due to a marked reduction in the amount of virion gp120 and suggest that the interaction of C2 and V3 stabilizes the association of gp120 with gp41.     

Changes in the transmembrane region of the human immunodeficiency virus type 1 gp41 envelope glycoprotein affect membrane fusion.

The charged amino acids near or within the membrane-spanning region of the human immunodeficiency virus type 1 gp41 envelope glycoprotein were altered. Two mutants were defective for syncytium formation and virus replication even though levels of envelope glycoproteins on the cell or virion surface and CD4 binding were comparable to those of the wild-type proteins. Thus, in addition to anchoring the envelope glycoproteins, sequences proximal to the membrane-spanning gp41 region are important for the membrane fusion process.     

Oligomeric modeling and electrostatic analysis of the gp120 envelope glycoprotein of human immunodeficiency virus

The human immunodeficiency virus envelope glycoproteins, gp120 and gp41, function in cell entry by binding to CD4 and a chemokine receptor on the cell surface and orchestrating the direct fusion of the viral and target cell membranes. On the virion surface, three gp120 molecules associate noncovalently with the ectodomain of the gp41 trimer to form the envelope oligomer. Although an atomic-level structure of a monomeric gp120 core has been determined, the structure of the oligomer is unknown. Here, the orientation of gp120 in the oligomer is modeled by using quantifiable criteria of carbohydrate exposure, occlusion of conserved residues, and steric considerations with regard to the binding of the neutralizing antibody 17b. Applying similar modeling techniques to influenza virus hemagglutinin suggests a rotational accuracy for the oriented gp120 of better than 10 degrees. The model shows that CD4 binds obliquely, such that multiple CD4 molecules bound to the same oligomer have their membrane-spanning portions separated by at least 190 A. The chemokine receptor, in contrast, binds to a sterically restricted surface close to the trimer axis. Electrostatic analyses reveal a basic region which faces away from the virus, toward the target cell membrane, and is conserved on core gp120. The electrostatic potentials of this region are strongly influenced by the overall charge, but not the precise structure, of the third variable (V3) loop. This dependence on charge and not structure may make electrostatic interactions between this basic region and the cell difficult to target therapeutically and may also provide a means of viral escape from immune system surveillance.     

Characterization of the outer domain of the gp120 glycoprotein from human immunodeficiency virus type 1

The core of the gp120 glycoprotein from human immunodeficiency virus type 1 (HIV-1) is comprised of three major structural domains: the outer domain, the inner domain, and the bridging sheet. The outer domain is exposed on the HIV-1 envelope glycoprotein trimer and contains binding surfaces for neutralizing antibodies such as 2G12, immunoglobulin G1b12, and anti-V3 antibodies. We expressed the outer domain of HIV-1(YU2) gp120 as an independent protein, termed OD1. OD1 efficiently bound 2G12 and a large number of anti-V3 antibodies, indicating its structural integrity. Immunochemical studies with OD1 indicated that antibody responses against the outer domain of the HIV-1 gp120 envelope glycoprotein are rare in HIV-1-infected human sera that potently neutralize the virus. Surprisingly, such outer-domain-directed antibody responses are commonly elicited by immunization with recombinant monomeric gp120. Immunization with soluble, stabilized HIV-1 envelope glycoprotein trimers elicited antibody responses that more closely resembled those in the sera of HIV-1-infected individuals. These results underscore the qualitatively different humoral immune responses elicited during natural infection and after gp120 vaccination and help to explain the failure of gp120 as an effective vaccine.     

Reevaluation of amino acid variability of the human immunodeficiency virus type 1 gp120 envelope glycoprotein and prediction of new discontinuous epitopes

To elucidate the evolutionary mechanisms of the human immunodeficiency virus type 1 gp120 envelope glycoprotein at the single-site level, the degree of amino acid variation and the numbers of synonymous and nonsynonymous substitutions were examined in 186 nucleotide sequences for gp120 (subtype B). Analyses of amino acid variabilities showed that the level of variability was very different from site to site in both conserved (C1 to C5) and variable (V1 to V5) regions previously assigned. To examine the relative importance of positive and negative selection for each amino acid position, the numbers of synonymous and nonsynonymous substitutions that occurred at each codon position were estimated by taking phylogenetic relationships into account. Among the 414 codon positions examined, we identified 33 positions where nonsynonymous substitutions were significantly predominant. These positions where positive selection may be operating, which we call putative positive selection (PS) sites, were found not only in the variable loops but also in the conserved regions (C1 to C4). In particular, we found seven PS sites at the surface positions of the alpha-helix (positions 335 to 347 in the C3 region) in the opposite face for CD4 binding. Furthermore, two PS sites in the C2 region and four PS sites in the C4 region were detected in the same face of the protein. The PS sites found in the C2, C3, and C4 regions were separated in the amino acid sequence but close together in the three-dimensional structure. This observation suggests the existence of discontinuous epitopes in the protein's surface including this alpha-helix, although the antigenicity of this area has not been reported yet.     

Evidence for a functional interaction between the V1/V2 and C4 domains of human immunodeficiency virus type 1 envelope glycoprotein gp120.

The domains of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein that are required for envelope function have been partially characterized. Little is known, however, about the nature of the interactions between these domains. To identify regions of the HIV-1 envelope glycoprotein that are involved in interactions necessary for proper envelope function, we constructed a series of 14 envelope recombinants between the env genes of two HIV-1 isolates. The envelope chimeras were examined for their ability to induce syncytia, to be proteolytically processed, and to function during a spreading viral infection. Our results demonstrate that the exchange between the two isolates of the first and second hypervariable regions (V1/V2) of gp120 results in defects in envelope glycoprotein processing, syncytium formation, and infectivity. Long-term passage of cultures infected with virus bearing a V1/V2 chimeric envelope glycoprotein leads to the emergence of a revertant virus with replication characteristics comparable to those of the wild type. Analysis of the revertant indicated that an Ile-->Met change in the C4 region of gp120 (between hypervariable regions V4 and V5) is responsible for the revertant phenotype. This single amino acid change restores infectivity without significantly affecting gp160 processing, CD4 binding, or the levels of virion-associated gp120. While the Ile-->Met change in C4 greatly enhances the fusogenic potential of the V1/V2 chimeric envelope glycoprotein, it has a detrimental effect on syncytium formation when analyzed in the context of the wild-type envelope. These results suggest that an interaction required for proper envelope glycoprotein function occurs between the V1/V2 and C4 regions of gp120.     

Structural and functional analysis of interhelical interactions in the human immunodeficiency virus type 1 gp41 envelope glycoprotein by alanine-scanning mutagenesis

Membrane fusion by human immunodeficiency virus type 1 (HIV-1) is promoted by the refolding of the viral envelope glycoprotein into a fusion-active conformation. The structure of the gp41 ectodomain core in its fusion-active state is a trimer of hairpins in which three antiparallel carboxyl-terminal helices pack into hydrophobic grooves on the surface of an amino-terminal trimeric coiled coil. In an effort to identify amino acid residues in these grooves that are critical for gp41 activation, we have used alanine-scanning mutagenesis to investigate the importance of individual side chains in determining the biophysical properties of the gp41 core and the membrane fusion activity of the gp120-gp41 complex. Alanine substitutions at Leu-556, Leu-565, Val-570, Gly-572, and Arg-579 positions severely impaired membrane fusion activity in envelope glycoproteins that were for the most part normally expressed. Whereas alanine mutations at Leu-565 and Val-570 destabilized the trimer-of-hairpins structure, mutations at Gly-572 and Arg-579 led to the formation of a stable gp41 core. Our results suggest that the Leu-565 and Val-570 residues are important determinants of conserved packing interactions between the amino- and carboxyl-terminal helices of gp41. We propose that the high degree of sequence conservation at Gly-572 and Arg-579 may result from selective pressures imposed by prefusogenic conformations of the HIV-1 envelope glycoprotein. Further analysis of the gp41 activation process may elucidate targets for antiviral intervention.     

Envelope glycoprotein incorporation, not shedding of surface envelope glycoprotein (gp120/SU), Is the primary determinant of SU content of purified human immunodeficiency virus type 1 and simian immunodeficiency virus

Human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) particles typically contain small amounts of the surface envelope protein (SU), and this is widely believed to be due to shedding of SU from mature virions. We purified proteins from HIV-1 and SIV isolates using procedures which allow quantitative measurements of viral protein content and determination of the ratios of gag- and env-encoded proteins in virions. All of the HIV-1 and most of the SIV isolates examined contained low levels of envelope proteins, with Gag:Env ratios of approximately 60:1. Based on an estimate of 1,200 to 2,500 Gag molecules per virion, this corresponds to an average of between 21 and 42 SU molecules, or between 7 and 14 trimers, per particle. In contrast, some SIV isolates contained levels of SU at least 10-fold greater than SU from HIV-1 isolates. Quantification of relative amounts of SU and transmembrane envelope protein (TM) provides a means to assess the impact of SU shedding on virion SU content, since such shedding would be expected to result in a molar excess of TM over SU on virions that had shed SU. With one exception, viruses with sufficient SU and TM to allow quantification were found to have approximately equivalent molar amounts of SU and TM. The quantity of SU associated with virions and the SU:TM ratios were not significantly changed during multiple freeze-thaw cycles or purification through sucrose gradients. Exposure of purified HIV-1 and SIV to temperatures of 55 degrees C or greater for 1 h resulted in loss of most of the SU from the virus but retention of TM. Incubation of purified virus with soluble CD4 at 37 degrees C resulted in no appreciable loss of SU from either SIV or HIV-1. These results indicate that the association of SU and TM on the purified virions studied is quite stable. These findings suggest that incorporation of SU-TM complexes into the viral membrane may be the primary factor determining the quantity of SU associated with SIV and HIV-1 virions, rather than shedding of SU from mature virions.     

Differential loss of envelope glycoprotein gp120 from virions of human immunodeficiency virus type 1 isolates: effects on infectivity and neutralization.

Several parameters which may affect the infectivity of human immunodeficiency virus type 1 in tissue culture were analyzed. In particular, we used gel exclusion chromatography to investigate how the loss of the surface glycoprotein gp120 from virions of the HTLV-IIIB (IIIB), HTLV-IIIRF (RF), and SF-2 isolates modulates infectivity. In IIIB and RF cultures, a high proportion of the total gp120 was virion bound initially but was gradually lost from the virions over time. In contrast, most of the gp120 (and p24) in SF-2-infected cultures was soluble and the few particles present had a fivefold-lower level of virus-bound gp120. However, this reduced level of virion-bound gp120 was more resistant to shedding. Loss of a major proportion of gp120 from IIIB and RF virions resulted in reduced infectivities, and in addition, the resulting accumulation of soluble gp120 in the cultures could competitively inhibit viral infection, especially with SF-2. Increased shedding of virion gp120 also affected the neutralization of IIIB and RF particles. However, the high sensitivity to human serum neutralization characteristic of SF-2 was unaffected by soluble gp120 in cultures, suggesting that the epitopes responsible are not present on soluble gp120.     

Conservation of human immunodeficiency virus type 1 gp120 inner-domain sequences in lentivirus and type A and B retrovirus envelope surface glycoproteins

We recently described a sequence similarity between the small ruminant lentivirus surface unit glycoprotein (SU) gp135 and the second conserved region (C2) of the primate lentivirus gp120 which indicates a structural similarity between gp135 and the inner proximal domain of the human immunodeficiency virus type 1 gp120 (I. Hötzel and W. P. Cheevers, Virus Res. 69:47–54, 2000). Here we found that the seven-amino-acid sequence of the gp120 strand β25 in the C5 region, which is also part of the inner proximal domain, was conserved in the SU of all lentiviruses in similar or identical positions relative to the carboxy terminus of SU. Sequences conforming to the gp135-gp120 consensus for β-strand 5 in the C2 region, which is antiparallel to β25, were then sought in the SU of other lentiviruses and retroviruses. Except for the feline immunodeficiency virus, sequences similar to the gp120-gp135 consensus for β5 and part of the preceding strand β4 were present in the SU of all lentiviruses. This motif was highly conserved among strains of each lentivirus and included a strictly conserved cysteine residue in β4. In addition, the β4/β5 consensus motif was also present in the conserved carboxy-terminal region of all type A and B retroviral envelope surface glycoproteins analyzed. Thus, the antiparallel β-strands 5 and 25 of gp120 form an SU surface highly conserved among the lentiviruses and at least partially conserved in the type A and B retroviral envelope glycoproteins.Lentiviruses are a group of strictly exogenous retroviruses that infect a range of mammalian hosts. One characteristic of this group of retroviruses is the rapid sequence divergence observed between virus strains as well as different lentiviruses, which resulted in the evolution of viruses with large differences in genome organization and sequence (20). Most of the sequence homology between highly divergent lentiviruses is present in the gag and pol gene products (8, 21). Sequence homology between the envelope glycoproteins of different lentiviruses has previously been shown to occur only in the ectodomain of the transmembrane subunit (TM) but not in the surface unit (SU) glycoprotein (3, 8, 21–23). Due to this apparent lack of sequence conservation in lentiviral SU, it has been unclear how the SU of different lentiviruses are structurally related to each other. To address this question, we recently compared SU sequences from the gp120 from primate lentiviruses and the gp135 of small ruminant lentiviruses and found a statistically significant sequence similarity between the second conserved region (C2) of gp120 and a 99-amino-acid region from gp135 (10). Analysis of this gp120-gp135 sequence similarity in the context of the gp120 structure revealed a partial structural similarity between gp120 and gp135.The human immunodeficiency virus type 1 (HIV-1) gp120 core bound to CD4 is composed of two major domains, the inner and outer domains, and a minidomain composed of four antiparallel β-strands, the bridging sheet (13). Sequences from the C2 region form most of the β-strands of a two-helix, two-strand bundle and a five-stranded β-sandwich in the inner domain as well as some β-strands of the outer domain of gp120 (13). Most of the similarity motifs between gp135 and the C2 region of gp120 coincide with sequences corresponding to β-strands 4 through 8 in the HIV-1 gp120 inner domain and β-strands 11 and 12 in the outer domain (10). Significantly, all four cysteines that form two disulfide bonds in the proximal region of the gp120 inner domain as well as the first cysteine of the gp120 V3 loop in β12 (13, 15) are conserved in gp135, indicating a partial similarity between the tertiary structures of gp120 and gp135 (10).The most conserved sequences between gp120 and gp135 correspond to strands β4 and β5 in the five-stranded β-sandwich structure of the proximal region of the inner proximal domain of HIV-1 gp120. Two additional β-strands in this five-stranded β-sandwich are derived from C1 and C5 sequences of HIV-1 gp120 (13). We hypothesized that C1 and C5 sequences, which are part of a structurally conserved SU inner proximal domain, should also be conserved between gp120 and gp135 and possibly in the SU of other lentiviruses. Here we show that two short motifs located in the gp120 C2 and C5 regions which are part of an antiparallel β-sheet in the gp120 inner proximal domain are conserved in the lentiviruses, indicating that a surface of the inner domain of HIV-1 gp120 is conserved in the SU of other lentiviruses. In addition, the C2 motif is also present in the envelope glycoproteins encoded by A-type endogenous retroviral elements and type B retroviruses (type A and B retroviruses), suggesting a local structural similarity between the SU of lentiviruses and type A and B retroviruses.

Sequence motif of the C5 region of HIV-1 gp120 is present in the SU of all lentiviruses.

As the sequences of three of the five β-strands of the gp120 inner proximal domain β-sandwich are conserved in gp135, we first tried to determine whether the gp120-gp135 sequence similarity extends to the other two β-strands which are part of this structure. One of these strands is β1, located in the C1 region of gp120 (13). Although the sequence of β1 is relatively well conserved among the primate lentiviruses, it is only 3 amino acids long, and a reliable assignation of similar sequences in gp135 could not be done. The other strand of this β-sandwich structure is the 7-amino-acid long β25. This strand is antiparallel to β5, which is the most conserved sequence between gp120 and gp135 (10, 13). Strand β25 is located about 20 amino acid residues upstream from the carboxy terminus of HIV-1 gp120 in the C5 region, and its sequence is highly conserved among strains of primate lentiviruses (sequence KYKVVKI in HIV-1_HXB2; residues conserved between HIV-1 strains are underlined) (12, 24). The last residue of this motif has been shown to be important for anchoring of gp120 on gp41 (9), suggesting that β25 is a functionally important structure of the inner proximal domain of gp120 likely to be conserved in other lentiviral glycoproteins. Sequences similar to the HIV-1 gp120 β25 motif (C5 motif) were visually sought in the gp135 carboxy-terminal region. A similar sequence was found in the caprine arthritis-encephalitis virus (CAEV) and visna virus gp135 between 33 and 34 amino acid residues upstream from the carboxy terminus of gp135 (Fig. ​(Fig.1B).1B). Similar to the C5 motif sequence of primate lentiviruses, the gp135 C5 motif is highly conserved in the gp135 of small ruminant lentiviruses (4, 27, 31, 35, 36). The sequence similarity also included the strictly conserved residue L483 of HIV-1 gp120 in the preceding α-helix 5, which is part of the two-helix, two-strand bundle of the inner domain. Flanking regions of gp120 and gp135 did not show any sequence similarity (not shown). Due to its short length, the significance of the conservation of the C5 motif in gp120 and gp135 was unclear. If this motif is indeed part of a structurally or functionally important domain of SU and not due only to chance, it should also be conserved in the SU of other lentiviruses. Therefore, to establish the relevance of this sequence similarity, we determined whether the C5 motif was also present in the carboxy terminus of the SU of other lentiviruses. Open in a separate windowFIG. 1Alignment of the C2 (A) and C5 (B) motifs of the SU from lentiviruses and type A and B retroviruses. Numbers at the right of the alignments indicate the position of the last residue of the motif from the initiation codon. Letters above the alignment indicate residue positions within each motif. Black backgrounds represent identical amino acids or conservative variations between the lentiviruses and type A and B retroviruses for each position of the motifs. Gray backgrounds represent identical amino acids or conservative variations between the lentiviruses and type A and B retroviruses (but which are nonconservative with the residues in black background) for each position. Numbers in parentheses indicate the number of amino acids between the last position of the C5 motif and the carboxy terminus of SU for each lentivirus. Thick lines indicate sequences which are part of HIV-1 gp120 strands β4, β5, and β25 and helix α5 (13). HIV-1 and HIV-2, human immunodeficiency virus types 1 (strain HXB2, GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"K03455","term_id":"1906382"}}K03455) and 2 (strain ROD, {"type":"entrez-nucleotide","attrs":{"text":"X05291","term_id":"60146"}}X05291); CAEV, caprine arthritis-encephalitis virus ({"type":"entrez-nucleotide","attrs":{"text":"M33677","term_id":"323294"}}M33677); Visna, visna virus ({"type":"entrez-nucleotide","attrs":{"text":"M10608","term_id":"470313"}}M10608); JSRV, jaagsiekte sheep retrovirus ({"type":"entrez-nucleotide","attrs":{"text":"M80216","term_id":"331338"}}M80216); EIAV, equine infectious anemia virus (AF033820); FIV, feline immunodeficiency virus ({"type":"entrez-nucleotide","attrs":{"text":"M73965","term_id":"323946"}}M73965); BIV, bovine immunodeficiency virus ({"type":"entrez-nucleotide","attrs":{"text":"M32690","term_id":"210706"}}M32690); JDV, jembrana disease lentivirus ({"type":"entrez-nucleotide","attrs":{"text":"U21603","term_id":"733067"}}U21603); HERV-K, human endogenous retrovirus K, type 2 genome ({"type":"entrez-nucleotide","attrs":{"text":"X82272","term_id":"757871"}}X82272); MMTV, mouse mammary tumor virus ({"type":"entrez-nucleotide","attrs":{"text":"X01811","term_id":"61630"}}X01811); MIAE, mouse intracisternal A-type element ({"type":"entrez-nucleotide","attrs":{"text":"M73818","term_id":"194061"}}M73818).Sequences conforming to the C5 motif consensus were also found in the SU of the equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and the bovine jembrana disease lentivirus (JDV), 19 to 23 amino acid residues from the carboxy terminus of SU, the same relative position as the C5 motif from the carboxy terminus of gp120 in primate lentiviruses (Fig. ​(Fig.1B).1B). This sequence similarity was clear when considering the chemical similarities of amino acid side chains (Gln/Glu, Tyr/Trp, Lys/Arg/Gln, Val/Leu, or Val/Ile). A survey of lentiviral SU sequences present in GenBank revealed that the C5 motif was also highly conserved between EIAV, FIV, and BIV strains. For example, the C5 motif was found to be strictly conserved in 64 of 69 EIAV gp90 sequences in GenBank and is also stable during in vivo persistent infection (16, 39). However, the little variation that is observed between strains of a given lentivirus follows the same pattern as variation between different lentiviruses, suggesting a common constraint on sequence variation in different lentiviruses. For example, position h of the C5 motif of HIV-1 gp120 can be either of the conservative variations Lys/Arg or Gln/Glu, the same amino acids present at position h in other lentiviruses. Similarly, position h of the C5 motif of CAEV in different strains is either Lys or Arg (35), two of the residues allowed at position h in HIV-1 gp120. In addition, position b in the C5 motif of most FIV gp100 sequences in GenBank is the conservative variation Gln or Glu, the same amino acids present at position b of the C5 motif in EIAV and the small ruminant lentiviruses, respectively. Although the C5 motif is present in all lentiviruses, the flanking sequences were not consistently conserved except for a few amino acids in some pairwise alignments (not shown). Therefore, although conservation of the C5 motif may not be statistically significant in some SU pairwise alignments, the presence of this motif in the same position relative to the carboxy terminus of SU in all lentiviruses indicates that strand β25 of gp120 is an important structural or functional domain conserved in all lentiviruses.

Sequences similar to an HIV-1 gp120 C2 motif are present in the SU of most lentiviruses.

Using computer-assisted searches, we were previously unable to find in EIAV, BIV, or FIV the same extensive region of similarity that is observed between the C2 region of gp120 and gp135 (10). However, the presence of the C5 (β25) motif in all lentiviruses suggests that sequences similar to gp120 β5, which is antiparallel to β25 and conserved between gp120 and gp135, are also present in degenerate form in other lentiviruses. Visual examination of SU sequences from different lentiviruses revealed the presence of a similar motif (C2 motif) in EIAV, BIV, and JDV although not in FIV (Fig. ​(Fig.1A).1A). This 12-amino-acid C2 motif encompasses most of gp120 β-strands 4 and 5 and includes a strictly conserved cysteine residue in the β4 region. The C2 motif is highly conserved between strains of EIAV and BIV. In EIAV, the C2 motif is stable during persistent infection, with few conservative changes observed (16, 39). In addition, the C2 motif was found to be strictly conserved in 176 of 179 EIAV gp90 sequences present in GenBank, despite considerable sequence variation in other regions.Although some positions of the C2 motif were not absolutely conserved, we found a common pattern of variation between distantly related lentiviruses. For example, position f of the C2 motif is either Pro in EIAV, HIV-2, and simian immunodeficiency virus or aromatic (Tyr or Trp) in the small ruminant lentiviruses BIV and JDV. Also, position h can be either Phe or Tyr even in closely related lentiviruses (HIV-1/HIV-2, visna virus/CAEV, or BIV/JDV), and position l can be either Arg or Lys in the primate and small ruminant lentiviruses or Gln, which is a common conservative substitution for Arg and Lys, in EIAV, BIV, and JDV. Therefore, the C2 motifs of different lentiviruses appear to have a common constraint on sequence variation, suggesting a structural or functional similarity between the HIV-1 gp120 C2 domain and the SU of EIAV, BIV, and JDV.The other previously described gp120-gp135 conserved motifs outside the β4/β5 region could not be identified in the SU of other lentiviruses, including the sequence of gp120 β8, which has a cysteine forming a disulfide bond with the conserved β4 cysteine. Although the C2 motif was not present in FIV gp100, a similar motif was identified in a location upstream from the FIV gp100 V3 region (sequence SYCTDPLQIPLI, amino acids 318 to 329; conserved residues are underlined), in a similar relative position from gp100 V3 as the C2 motif from the V3 region of HIV-1 gp120. However, some of the highly conserved positions of the motif (positions g, h, and j) were not conserved in FIV gp100, and the significance of this FIV gp100 motif is unclear.

C2 motif is present in type A and B retroviral envelope surface glycoproteins.

The conservation of two short motifs in distant regions of SU that are located close to each other in the tertiary structure of HIV-1 gp120 suggests that this region represents a domain of SU that is of structural or functional importance. The TM ectodomains from lentiviruses and type B retroviruses have been shown to have some sequence similarity (19, 34, 38). Therefore, we asked whether sequence similarity between the Env of lentiviruses and type B retroviruses extends to the C2 and C5 motifs of SU.The type A and B retroviruses have some sequence homology in SU, and most of the sequence homology is located in the carboxy-terminal region of SU (18, 38). Visual examination of SU sequences from the human endogenous retrovirus K (18), mouse intracisternal A-type element (26), the exogenous/endogenous mouse mammary tumor virus (25), and the exogenous/endogenous type B/D jaagsiekte sheep retrovirus (JSRV) and the closely related ovine enzootic nasal tumor virus (which encode type B retroviral envelopes) (6, 38) revealed a sequence closely related to the C2 motif in their conserved carboxy-terminal region (Fig. ​(Fig.1A).1A). This sequence represents one of the most conserved sequences in the SU of this group of retroviruses and is also conserved among different strains or members of endogenous families (not shown). Some positions of the C2 motif, such as positions c, d, and g, are strictly or almost completely conserved between the lentiviruses and type A and B retroviruses. However, more informative than the sequence similarity between lentiviruses and type A and B retroviruses is the lack of distinction between the patterns of sequence variation for each position of the motif within and between retrovirus groups, even between closely related viruses. For example, position e of the C2 motif within both the lentiviruses and type A and B retroviruses can be either Pro or basic/Gln; the “dimorphic” position f encodes only Tyr/Trp or Pro (except in HIV-1); position h encodes either Phe or Tyr in all sequences; position i encodes either Ala or a hydrophobic residue in most sequences; position j encodes either Ile, Leu, or Phe in all sequences; position k encodes either Leu, Ile, or Val in all sequences; and position l is preferentially Lys, Arg, or Gln in the lentiviruses and JSRV. Most of these degenerate positions represent very conservative variations (positions a and h through l) or a restricted number of nonconservative variations (positions e and f, in the turn between β4 and β5). The sequence conservation and common pattern of variation between the C2 motifs of lentiviruses and type A and B retroviruses indicate a similar structural or functional constraint on sequence variation in the SU of these two groups of viruses.In contrast to the type A and B retroviruses, sequences similar to the C2 or C5 motifs could not be found in the SU of the Moloney murine leukemia virus, bovine leukemia virus, human T-cell leukemia virus types 1 and 2 (HTLV-1 and HTLV-2), Rous sarcoma virus, feline RD114 endogenous retrovirus, baboon endogenous retrovirus, feline leukemia virus type A, the Mason-Pfizer monkey retrovirus, or any spumaretrovirus even when using the Findpatterns program of the GCG package (7).Here we show that two short SU motifs are highly conserved in the lentiviruses and that one of these motifs is also conserved in the type A and B retroviruses. Many of the pairwise alignments were not statistically significant when tested by the Monte Carlo simulation of the Bestfit program of the GCG package and could therefore be attributed to chance. However, when all lentiviral sequences are included in the analysis and the multiple alignment is interpreted in the context of the X-ray structure of HIV-1 gp120, the conserved C2 and C5 motifs have a clear structural significance. The conservation of these motifs indicates that the region of the HIV-1 gp120 inner proximal domain centered on the antiparallel β-strands 5 and 25 forms a highly conserved lentiviral SU surface and suggests a possible structural similarity between the SU of lentiviruses and type A and B retroviruses in that domain. Although the C2 motif is too short to rule out convergent evolution between the SU of lentiviruses and type A and B retroviruses, their sequence similarity in TM (19, 34, 38) supports a common origin for most or the entire env genes of these two retroviral groups.The reason for the disagreement between the different degrees of sequence similarity in the SU of lentiviruses and the phylogenetic analyses of the pol gene products is unclear but probably reflects differences in evolutionary rates in different lentiviruses or recombination events (19, 20). Precedents for recombination events between env genes of closely or distantly related retroviruses, deduced from phylogenetic analyses, have been described. An exchange of env sequences probably occurred between HTLV-1 and HTLV-2 (19) and between a type C retrovirus closely related to the avian reticuloendotheliosis virus and a type B retrovirus which originated the type D retroviruses (19, 38).Modeling of the trimeric SU complex on the virion surface indicates that strands β5 and β25 form part of the most virion-proximal surface of the gp120 core (14, 37). While none of the residues of the C2 motif was directly tested for interactions with TM, at least one of the residues of β25 in the C5 region of HIV-1 gp120, I491, is important for stable SU-TM interactions (9). Therefore, the conserved lentiviral SU surface may represent a common structure among lentiviruses and possibly type A and B retroviruses for anchoring SU on TM in the envelope glycoprotein complex. It is interesting that the C5 motif region, which forms a β-strand in the CD4-bound gp120 core, is included in a computer-modeled pocket structure postulated to be important in SU-TM interactions (28), suggesting a structural basis for SU shedding upon receptor-induced conformational change.The sequence of the HIV-1 gp120 outer domain, shown as a cross-hatched box in Fig. ​Fig.2,2, is included entirely between the C2 and C5 motifs (13). Our previous sequence analysis indicates that the gp135s of small ruminant lentiviruses have a similar inner/outer domain organization: most strands of the inner domain β-sandwich as well as β12, located in the outer domain immediately upstream from the gp120 V3 loop, are conserved between gp135 and the gp120 of primate lentiviruses (10). The identification of a homologue of gp120 β25 in gp135 about 290 amino acid residues downstream from the C2 motif provides further support for a similar domain organization in the SU of primate and small ruminant lentiviruses. Consistent with this interpretation, the putative outer domain of gp135, located between the C2 and C5 motifs, is highly glycosylated and contains more than 80% of the potential N-linked glycosylation sites of gp135 (11), similar to the heavy glycosylation of the gp120 outer domain (37). In this gp135 domain model, the distance between the C2 and C5 motifs in the primary structure of SU would indicate a larger relative size of the putative outer domain of gp135 than gp120 outer domain. The presence of the C2 and C5 motifs in EIAV, BIV, and JDV would also suggest an analogous inner/outer domain organization for the SU of these lentiviruses. However, the shorter sequence between the C2 and C5 motifs in EIAV, BIV, and JDV may indicate either a much smaller or absent outer domain in the SU of these viruses (Fig. ​(Fig.2).2). The conserved C2 motif of EIAV gp90 was shown to be part of a minor neutralization epitope recognized by a murine monoclonal antibody (1), suggesting that the EIAV C2 motif is better exposed on the virion surface than the C2 motif of gp120, compatible with a smaller or absent outer domain in gp90. Interestingly, the C2 motif of type A and B retroviruses is located in the carboxy terminus of SU (Fig. ​(Fig.2)2) and C5 appears to be absent, indicating that the surface glycoproteins of type A and B retroviruses, although possibly structurally related to the SU of lentiviruses, probably lack an outer domain homologue and have a different domain organization than the SU of lentiviruses. Open in a separate windowFIG. 2Location of the C2 and C5 motifs in retrovirus envelope glycoproteins. The Env glycoproteins (excluding the amino-terminal leader peptide) are drawn to scale and aligned by the SU-TM cleavage sites conserved in all retroviruses (dotted line). The SU and TM domains of Env are indicated by double arrows. The boundaries of the C2, V3, and C5 regions of HIV-1 gp120 are indicated by thick lines above the alignment, and the location of the HIV-1 gp120 outer domain sequence is shown by a cross-hatched box. The black and gray boxes in the SU domain indicate the positions of the C2 and C5 motifs, respectively. Asterisks represent the described PNDs of EIAV (1), visna virus (29), FIV (17), and T-cell-adapted strains of HIV-1 (33) and HIV-2 (2).The two conserved colinear motifs of lentivirus SU could be useful as structural points of reference for comparative structural studies of SU from different lentiviruses. Variable domains of SU are important in the mechanisms of host cell invasion, tropism determination, and immune evasion. In HIV-1, the third variable loop V3 of gp120 is the main target of neutralizing antibodies in tissue culture-adapted strains and also determines coreceptor usage and tropism (5, 30, 32, 33). Whether sequences in variable regions of SU in other lentiviruses that are functionally equivalent to the gp120 V3 loop are also structurally related to the gp120 V3 loop is not clear. The position of variable domains relative to the C2 and C5 motifs could therefore indicate their structural relationship. For example, the principal neutralization domain (PND) of EIAV gp90, postulated to be functionally equivalent to the gp120 V3 loop (1, 16), is located upstream from the C2 motif instead of downstream, as the V3 loop in gp120 is (Fig. ​(Fig.2),2), suggesting that the gp90 PND and the gp120 V3 loop, while having similar roles in evasion of humoral immune responses, may not be structurally related to each other. A similar situation also occurs in visna virus, whose PND, located in the carboxy-terminal region of gp135 (29), was previously shown to be structurally unrelated to the HIV-1 gp120 V3 loop (10). This would indicate that different lentiviruses may have evolved different regions of a primordial lentivirus surface glycoprotein to perform similar functions important in virus-host interactions.     

Multimerization potential of the cytoplasmic domain of the human immunodeficiency virus type 1 transmembrane glycoprotein gp41

We previously demonstrated that an envelope mutant of human immunodeficiency virus type 1 lacking the entire cytoplasmic domain interferes in trans with the production of infectious virus by inclusion of the mutant envelope into the wild-type envelope complex. We also showed that the envelope incorporation into virions is not affected when the wild-type envelope is coexpressed with the mutant envelope. These results suggest that an oligomeric structure of the cytoplasmic domain is functionally required for viral infectivity. To understand whether the cytoplasmic domain of human immunodeficiency virus type 1 transmembrane protein gp41 has the potential to self-assemble as an oligomer, in the present study we fused the coding sequence of the entire cytoplasmic domain at 3' to the Escherichia coli malE gene, which encodes a monomeric maltose-binding protein. The expressed fusion protein was examined by chemical cross-linking, sucrose gradient centrifugation, and gel filtration. The results showed that the cytoplasmic domain of gp41 assembles into a high-ordered structural complex. The intersubunit interaction of the cytoplasmic domain was also confirmed by a mammalian two-hybrid system that detects protein-protein interactions in eucaryotic cells. A cytoplasmic domain fragment expressed in eucaryotic cells was pulled down by glutathione-Sepharose 4B beads via its association with another cytoplasmic domain fragment fused to the C terminus of the glutathione S-transferase moiety. We also found that sequences encompassing the lentiviral lytic peptide-1 and lentiviral lytic peptide-2, which are located within residues 828-856 and 770-795, respectively, play a critical role in cytoplasmic domain self-assembly. Taken together, the results from the present study indicate that the cytoplasmic domain of gp41 by itself is sufficient to assemble into a multimeric structure. This finding supports the hypothesis that a multimeric form of the gp41 cytoplasmic domain plays a crucial role in virus infectivity.     

Challenge of chimpanzees (Pan troglodytes) immunized with human immunodeficiency virus envelope glycoprotein gp120.

The human immunodeficiency virus type 1 (HIV-1), the causative agent of acquired immunodeficiency syndrome, infects humans and chimpanzees. To determine the efficacy of immunization for preventing infection, chimpanzees were immunized with gp120 purified from human T-cell lymphotrophic virus type IIIB (HTLV-IIIB)-infected cell membranes and challenged with the homologous virus, HTLV-IIIB. A challenge stock of HTLV-IIIB was prepared by using unconcentrated HTLV-IIIB produced in H9 cells. The titer of the virus from this stock on human and chimpanzee peripheral blood mononuclear cells and in human lymphoid cell lines was determined; a cell culture infectivity of 10(4) was assigned. All chimpanzees inoculated intravenously with 40 cell culture infectious units or more became infected, as demonstrated by virus isolation and seroconversion. One of two chimpanzees inoculated with 4 cell culture infectious units became infected. Chimpanzees immunized with gp120 formulated in alum developed antibodies which precipitated gp120 and neutralized HTLV-IIIB. Peripheral blood mononuclear cells from gp120-vaccinated and HIV-infected animals showed a significantly greater response in proliferation assays with HIV proteins than did peripheral blood mononuclear cells from nonvaccinated and non-HIV-infected chimpanzees. Two of the gp120-alum-immunized chimpanzees were challenged with virus from the HTLV-IIIB stock. One animal received 400 cell culture infectious units, and one received 40 infectious units. Both animals became infected with HIV, indicating that the immune response elicited by immunization with gp120 formulated in alum was not effective in preventing infection with HIV-1.     

Discontinuous, conserved neutralization epitopes overlapping the CD4-binding region of human immunodeficiency virus type 1 gp120 envelope glycoprotein.

Monoclonal antibodies have been isolated from human immunodeficiency virus type 1 (HIV-1)-infected patients that recognize discontinuous epitopes on the gp120 envelope glycoprotein, that block gp120 interaction with the CD4 receptor, and that neutralize a variety of HIV-1 isolates. Using a panel of HIV-1 gp120 mutants, we identified amino acids important for precipitation of the gp120 glycoprotein by three different monoclonal antibodies with these properties. These amino acids are located within seven discontinuous, conserved regions of the gp120 glycoprotein, four of which overlap those regions previously shown to be important for CD4 recognition. The pattern of sensitivity to amino acid change in these seven regions differed for each antibody and also differed from that of the CD4 glycoprotein. These results indicate that the CD4 receptor and this group of broadly neutralizing antibodies recognize distinct but overlapping gp120 determinants.     

CD4 binding site antibodies inhibit human immunodeficiency virus gp120 envelope glycoprotein interaction with CCR5

The human immunodeficiency virus type 1 (HIV-1) gp120 exterior glycoprotein is conformationally flexible. Upon binding the host cell receptor, CD4, gp120 assumes a conformation that is able to bind the chemokine receptors CCR5 or CXCR4, which act as coreceptors for the virus. CD4-binding-site (CD4BS) antibodies are neutralizing antibodies elicited during natural infection that are directed against gp120 epitopes that overlap the binding site for CD4. Recent studies (S. H. Xiang et al., J. Virol. 76:9888-9899, 2002) suggest that CD4BS antibodies recognize conformations of gp120 distinct from the CD4-bound conformation. This predicts that the binding of CD4BS antibodies will inhibit chemokine receptor binding. Here, we show that Fab fragments and complete immunoglobulin molecules of CD4BS antibodies inhibit CD4-independent gp120 binding to CCR5 and cell-cell fusion mediated by CD4-independent HIV-1 envelope glycoproteins. These results are consistent with a model in which the binding of CD4BS antibodies limits the ability of gp120 to assume a conformation required for coreceptor binding.     

Replication and neutralization of human immunodeficiency virus type 1 lacking the V1 and V2 variable loops of the gp120 envelope glycoprotein.

A human immunodeficiency virus type 1 (HIV-1) mutant lacking the V1 and V2 variable loops in the gp120 exterior envelope glycoprotein replicated in Jurkat lymphocytes with only modest delays compared with the wild-type virus. Revertants that replicated with wild-type efficiency rapidly emerged and contained only a few amino acid changes in the envelope glycoproteins compared with the parent virus. Both the parent and revertant viruses exhibited increased sensitivity to neutralization by antibodies directed against the V3 loop or a CD4-induced epitope on gp120 but not by soluble CD4 or an antibody against the CD4 binding site. This result demonstrates the role of the gp120 V1 and V2 loops in protecting HIV-1 from some subsets of neutralizing antibodies.