Mutational analysis of the candidate internal fusion peptide of the avian leukosis and sarcoma virus subgroup A envelope glycoprotein

The transmembrane subunit (TM) of the avian leukosis and sarcoma virus (ALSV) envelope glycoprotein (Env) contains a stretch of conserved hydrophobic amino acids internal to its amino terminus (residues 21 to 42). By analogy with similar sequences in other viral envelope glycoproteins, this region has been proposed to be a fusion peptide. We investigated the role of this region by changing each of three hydrophobic residues (Ile-21, Val-30, and Ile-39) to glutamatic acid and lysine in the ALSV subgroup A Env. Like wild-type (wt) Env, all six mutant Env proteins were proteolytically processed, oligomerized, and expressed at the cell surface in a form that bound Tva, the ALSV subgroup A receptor. Like wt Env, Ile21Glu, Ile21Lys, Va30Glu, and Val30Lys changed conformation upon binding Tva, as assayed by sensitivity to thermolysin. Ile39Glu and Ile39Lys were cleaved by thermolysin in both the absence and presence of Tva. Although incorporated into virus particles at approximately equal levels, all mutant Envs were compromised in their ability to support infection. The mutants at residues 21 and 30 showed levels of infection 2 to 3 orders of magnitude lower than that of wt Env. The mutants at residue 39 were noninfectious. Furthermore, none of the mutants displayed activity in a cell-cell fusion assay. Our results support the contention that residues 21 to 42 of ALSV subgroup A Env constitute its fusion peptide.     

Three-dimensional structure-activity analysis of a series of porphyrin derivatives with anti-HIV-1 activity targeted to the V3 loop of the gp120 envelope glycoprotein of the human immunodeficiency virus type 1

Using comparative molecular field analysis (CoMFA), a 3D-QSAR model was developed for 21 porphyrin derivatives which have anti-HIV-1 activity and bind to the V3 loop of the envelope glycoprotein gp120 of the human immunodeficiency virus type 1. A significant PLS cross-validated r2cv (0.590) was obtained, indicating that the model could be used as a predictive tool for further design of porphyrin analogs. The model revealed at least three important sites for favorable electrostatic interactions and indicated favorable and unfavorable steric interaction sites. It was found that the occurrence of at least three positively charged and several hydrophobic amino acid residues is highly conserved at fixed positions of gp120 V3 loop sequences. This may support the validity of the proposed model and the hypothesis that porphyrins containing anionic and hydrophobic groups may interact with some of the highly conserved positively charged and hydrophobic sites, respectively, of the V3 loop. These interactions may induce conformational changes in the gp120 envelope glycoprotein leading to inhibition of virus entry into cells and of syncytium formation (cell-to-cell fusion) and thus to inhibition of virus replication.     

Effect of major deletions in the V1 and V2 loops of a macrophage-tropic HIV type 1 isolate on viral envelope structure, cell entry, and replication

Two HIV-1 envelope mutant proteins were generated by introducing deletions in the first and second hypervariable gp120 regions (V1 and V2 loops, respectively) of a macrophage-tropic primary HIV-1 isolate, SF162, to study the effect of the deleted sequences on envelope structure, viral entry, and replication potentials. The first mutant lacked 17 amino acids of the V1 loop and the latter 30 amino acids of the V2 loop. A comparison of the immunochemical structure of the wild-type and mutant monomeric and virion-associated gp120 molecules revealed that the V1 and V2 loop deletions differentially altered the structure of the V3 loop, the CD4-binding site, and epitopes within conserved regions of gp120. Regardless of differences in structure, both mutated envelope proteins supported viral replication into peripheral blood mononuclear cells to levels comparable to those of the wild-type SF162 virus. However, they decreased the viral replication potential in macrophages, even though they did not alter the coreceptor usage of the viruses. These studies support and extend previous observations that a complex structural interaction between the V1, V2, and V3 loops and elements of the CD4-binding site of gp120 controls entry of virus into cells. The present studies, however, suggest that the effect of the V1 and V2 loops in viral entry is cell dependent.     

Mutational analysis of residues in the coiled-coil domain of human immunodeficiency virus type 1 transmembrane protein gp41

The envelope glycoprotein (Env) of human immunodeficiency virus mediates virus entry into cells by undergoing conformational changes that lead to fusion between viral and cellular membranes. A six-helix bundle in gp41, consisting of an interior trimeric coiled-coil core with three exterior helices packed in the grooves (core structure), has been proposed to be part of a fusion-active structure of Env (D. C. Chan, D. Fass, J. M. Berger, and P. S. Kim, Cell 89:263-273, 1997; W. Weissenhorn, A. Dessen, S. C. Harrison, J. J. Skehel, and D. C. Wiley, Nature 387:426-430, 1997; and K. Tan, J. Liu, J. Wang, S. Shen, and M. Lu, Proc. Natl. Acad. Sci. USA 94:12303, 1997). We analyzed the effects of amino acid substitutions of arginine or glutamic acid in residues in the coiled-coil (heptad repeat) domain that line the interface between the helices in the gp41 core structure. We found that mutations of leucine to arginine or glutamic acid in position 556 and of alanine to arginine in position 558 resulted in undetectable levels of Env expression. Seven other mutations in six positions completely abolished fusion activity despite incorporation of the mutant Env into virions and normal gp160 processing. Single-residue substitutions of glutamic acid at position 570 or 577 resulted in the only viable mutants among the 16 mutants studied, although both viable mutants exhibited impaired fusion activity compared to that of the wild type. The glutamic acid 577 mutant was more sensitive than the wild type to inhibition by a gp41 coiled-coil peptide (DP-107) but not to that by another peptide corresponding to the C helix in the gp41 core structure (DP-178). These results provide insight into the gp41 fusion mechanism and suggest that the DP-107 peptide may inhibit fusion by binding to the homologous region in gp41, probably by forming a peptide-gp41 coiled-coil structure.     

Oxidative stress and interleukins in seminal plasma during leukocytospermia

Various roles for the viral receptor, CD4, have been proposed in facilitating human immunodeficiency virus type 1 (HIV-1) entry, including virion binding to the target cell and the induction of conformational changes in the viral envelope glycoproteins required for the membrane fusion reaction. Here, we compare the structural requirements in the CDR2-like loop of CD4 domain 1, the major contact site of the gp120 envelope glycoprotein, for gp120 binding and virus entry. For every CD4 mutant examined, the level of cell surface expression and the gp120 binding affinity were sufficient to explain the relative ability to function as a viral receptor. The decrease in relative infectibility associated with decreased gp120 binding affinity was more pronounced at lower cell surface CD4 concentrations. These results imply that both receptor density and affinity determine the efficiency of HIV-1 entry and that specific structures in the CD4 residues examined are probably not required for HIV-1 entry functions other than gp120 binding.     

The course of childhood bronchiectasis: a case report and considerations of hospital use

Using an inhibitory synthetic peptide (DP-178) from HIV-1 gp41, we have trapped HIV-1 envelope glycoprotein (Env) undergoing conformational changes during virus entry. Our data show that DP-178 binds gp41 and inhibits Env-mediated membrane fusion after gp120 interacts with cellular receptors, indicating that conformational changes involving the coiled coil domain of gp41 are required for entry. Capture of this fusion-active conformation of Env provides insights into the early events leading to Env-mediated membrane fusion.     

Resistance to a drug blocking human immunodeficiency virus type 1 entry (RPR103611) is conferred by mutations in gp41

A triterpene derived from betulinic acid (RPR103611) blocks human immunodeficiency virus type 1 (HIV-1) infection and fusion of CD4+ cells with cells expressing HIV-1 envelope proteins (gp120 and gp41), suggesting an effect on virus entry. This compound did not block infection by a subtype D HIV-1 strain (NDK) or cell-cell fusion mediated by the NDK envelope proteins. The genetic basis of drug resistance was therefore addressed by testing envelope chimeras derived from NDK and a drug-sensitive HIV-1 strain (LAI, subtype B). A drug-resistant phenotype was observed for all chimeras bearing the ectodomain of NDK gp41, while the origins of gp120 and of the membrane anchor and cytoplasmic domains of gp41 had no apparent role. The envelope gene of a LAI variant, fully resistant to the antiviral effect of RPR103611, was cloned and sequenced. Its product differed from the parental sequence at two positions in gp41, with changes of arginine 22 to alanine (R22A) and isoleucine 84 to serine (I84S), the gp120 being identical. In the context of LAI gp41, the I84S substitution was sufficient for drug resistance. Therefore, in two different systems, differences in gp41 were associated with sensitivity or resistance to RPR103611. Modifications of gp41 can affect the quaternary structure of gp120 and gp41 and the accessibility of gp120 to antiviral agents such as neutralizing antibodies. However, a direct effect of RPR103611 on a gp41 target must also be envisioned, in agreement with the blocking of apparently late steps of HIV-1 entry. This compound could be a valuable tool for structure-function studies of gp41.     

CCR5 coreceptor utilization involves a highly conserved arginine residue of HIV type 1 gp120

The seven-transmembrane CCR5 was recently found to double as a coreceptor for a genetically diverse family of human and nonhuman primate lentiviruses. Paradoxically, the main region of the envelope protein believed to be involved in CCR5 utilization was mapped to hypervariable region 3, or V3, of the envelope glycoprotein gp120. In this study, we addressed the question of whether functional convergence in CCR5 utilization is mediated by certain V3 residues that are highly conserved among HIV type 1 (HIV-1), HIV type 2, and simian immunodeficiency virus. Site-directed mutagenesis carried out on three such V3 residues revealed that the Arg-298 of HIV-1 gp120 has an important role in CCR5 utilization. In contrast, no effect was observed for the other residues we tested. The inability of Arg-298 mutants to use CCR5 was not attributed to global alteration of gp120 conformation. Neither the expression, processing, and incorporation of mutant envelope proteins into virions, nor CD4 binding were significantly affected by the mutations. This interpretation is further supported by the finding that alanine substitutions of five residues immediately adjacent to the arginine residue had no effect on CCR5 utilization. Taken together, our data strongly suggests that the highly conserved Arg-298 residue identified in the V3 of HIV-1 has a significant role in CCR5 utilization, and may represent an unusually conserved target for future anti-viral designs.     

CD4-Induced conformational changes in the human immunodeficiency virus type 1 gp120 glycoprotein: consequences for virus entry and neutralization

Human immunodeficiency virus type 1 (HIV-1) entry into target cells involves sequential binding of the gp120 exterior envelope glycoprotein to CD4 and to specific chemokine receptors. Soluble CD4 (sCD4) is thought to mimic membrane-anchored CD4, and its binding alters the conformation of the HIV-1 envelope glycoproteins. Two cross-competing monoclonal antibodies, 17b and CG10, that recognize CD4-inducible gp120 epitopes and that block gp120-chemokine receptor binding were used to investigate the nature and functional significance of gp120 conformational changes initiated by CD4 binding. Envelope glycoproteins derived from both T-cell line-adapted and primary HIV-1 isolates exhibited increased binding of the 17b antibody in the presence of sCD4. CD4-induced exposure of the 17b epitope on the oligomeric envelope glycoprotein complex occurred over a wide range of temperatures and involved movement of the gp120 V1/V2 variable loops. Amino acid changes that reduced the efficiency of 17b epitope exposure following CD4 binding invariably compromised the ability of the HIV-1 envelope glycoproteins to form syncytia or to support virus entry. Comparison of the CD4 dependence and neutralization efficiencies of the 17b and CG10 antibodies suggested that the epitopes for these antibodies are minimally accessible following attachment of gp120 to cell surface CD4. These results underscore the functional importance of these CD4-induced changes in gp120 conformation and illustrate viral strategies for sequestering chemokine receptor-binding regions from the humoral immune response.     

Determinants of human immunodeficiency virus type 1 envelope glycoprotein activation by soluble CD4 and monoclonal antibodies

Infection by some human immunodeficiency virus type 1 (HIV-1) isolates is enhanced by the binding of subneutralizing concentrations of soluble receptor, soluble CD4 (sCD4), or monoclonal antibodies directed against the viral envelope glycoproteins. In this work, we studied the abilities of different antibodies to mediate activation of the envelope glycoproteins of a primary HIV-1 isolate, YU2, and identified the regions of gp120 envelope glycoprotein contributing to activation. Binding of antibodies to a variety of epitopes on gp120, including the CD4 binding site, the third variable (V3) loop, and CD4-induced epitopes, enhanced the entry of viruses containing YU2 envelope glycoproteins. Fab fragments of antibodies directed against either the CD4 binding site or V3 loop also activated YU2 virus infection. The activation phenotype was conferred on the envelope glycoproteins of a laboratory-adapted HIV-1 isolate (HXBc2) by replacing the gp120 V3 loop or V1/V2 and V3 loops with those of the YU2 virus. Infection by the YU2 virus in the presence of activating antibodies remained inhibitable by macrophage inhibitory protein 1beta, indicating dependence on the CCR5 coreceptor on the target cells. Thus, antibody enhancement of YU2 entry involves neither Fc receptor binding nor envelope glycoprotein cross-linking, is determined by the same variable loops that dictate enhancement by sCD4, and probably proceeds by a process fundamentally similar to the receptor-activated virus entry pathway.     

Membrane fusion induced by the HIV type 1 fusion peptide: modulation by factors affecting glycoprotein 41 activity and potential anti-HIV compounds

Peptides representing a sequence of 23 amino acid residues at the N terminus of human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp41 bind and subsequently induce fusion of large unilamellar vesicles (LUV), an activity presumably related to gp41 function in viral infection. These in vitro effects can be modulated by several factors that are known to affect HIV-1 infectivity and gp41-mediated virus-cell fusion. Peptide-induced membrane fusion but not peptide binding can be inhibited by two factors known to block gp41 activity: a polar amino acid substitution V --> E in position 2 and the presence of the N-terminal hexapeptide of gp41 in addition to the parent sequence. Whereas inclusion of the alternative gp120 receptor galactosylceramide in membranes has virtually no effect, membrane cholesterol stimulates fusion activity. In view of its putative physiological relevance, we have used the fusion activity of the peptides as a tool to evaluate the inhibitory effect of antivirals that might target this sequence. We describe three dissimilar effects: Amphotericin B inhibits in a cholesterol-independent way peptide-induced fusion but not binding, human serum albumin inhibits binding and consequently fusion, and dextran sulfate (M(r) 5000) does not affect either binding or fusion.     

Determinants for sensitivity of human immunodeficiency virus coreceptor CXCR4 to the bicyclam AMD3100

The bicyclam AMD3100 is a potent and selective inhibitor of the replication of human immunodeficiency virus type 1 and type 2 (HIV-1 and HIV-2). It was recently demonstrated that the compound inhibited HIV entry through CXCR4 but not through CCR5. Selectivity of AMD3100 for CXCR4 was further indicated by its lack of effect on HIV-1 and HIV-2 infection mediated by the CCR5, CCR3, Bonzo, BOB, and US28, coreceptors. AMD3100 completely blocked HIV-1 infection mediated by a mutant CXCR4 bearing a deletion of most of the amino-terminal extracellular domain. In contrast, relative resistance to AMD3100 was conferred by different single amino acid substitutions in the second extracellular loop (ECL2) or in the adjacent membrane-spanning domain, TM4. Only substitutions of a neutral residue for aspartic acid and of a nonaromatic residue for phenylalanine (Phe) were associated with drug resistance. This suggests a direct interaction of AMD3100 with these amino acids rather than indirect effects of their mutation on the CXCR4 structure. The interaction of aspartic acids of ECL2 and TM4 with AMD3100 is consistent with the positive charge of bicyclams, which might block HIV-1 entry by preventing electrostatic interactions between CXCR4 and the HIV-1 envelope protein gp120. Other features of AMD3100 must account for its high antiviral activity, in particular the presence of an aromatic linker between the cyclam units. This aromatic group might engage in hydrophobic interactions with the Phe-X-Phe motifs of ECL2 or TM4. These results confirm the importance of ECL2 for the HIV coreceptor activity of CXCR4.     

The antigenic structure of the HIV gp120 envelope glycoprotein

The human immunodeficiency virus HIV-1 establishes persistent infections in humans which lead to acquired immunodeficiency syndrome (AIDS). The HIV-1 envelope glycoproteins, gp120 and gp41, are assembled into a trimeric complex that mediates virus entry into target cells. HIV-1 entry depends on the sequential interaction of the gp120 exterior envelope glycoprotein with the receptors on the cell, CD4 and members of the chemokine receptor family. The gp120 glycoprotein, which can be shed from the envelope complex, elicits both virus-neutralizing and non-neutralizing antibodies during natural infection. Antibodies that lack neutralizing activity are often directed against the gp120 regions that are occluded on the assembled trimer and which are exposed only upon shedding. Neutralizing antibodies, by contrast, must access the functional envelope glycoprotein complex and typically recognize conserved or variable epitopes near the receptor-binding regions. Here we describe the spatial organization of conserved neutralization epitopes on gp120, using epitope maps in conjunction with the X-ray crystal structure of a ternary complex that includes a gp120 core, CD4 and a neutralizing antibody. A large fraction of the predicted accessible surface of gp120 in the trimer is composed of variable, heavily glycosylated core and loop structures that surround the receptor-binding regions. Understanding the structural basis for the ability of HIV-1 to evade the humoral immune response should assist in the design of a vaccine.     

Regions required for CD4 binding in the external glycoprotein gp120 of simian immunodeficiency virus

The external domain of the envelope glycoprotein, gp120, of simian immunodeficiency virus (SIV) has been expressed as a mature secreted product using recombinant baculoviruses and the expressed protein, which has an observed molecular mass of 110 kDa, was purified by monoclonal antibody (MAb) affinity chromatography. N-terminal sequence analysis showed a signal sequence cleavage identity similar to that of the gp120s of both human immunodeficiency virus type 1 (HIV-1) and HIV type 2. The expressed molecule bound to soluble CD4 with an affinity that was approximately 10-fold lower than that of gp120 from HIV-1. A screening of the ability of SIV envelope MAbs to inhibit CD4 binding revealed two groups of inhibitory MAbs. One group is dependent on conformation, while the second group maps to a discrete epitope near the amino terminus. The particular role of the V3 loop region of the molecule in CD4 binding was investigated by the construction of an SIV-HIV hybrid in which the V3 loop of SIV was precisely replaced with the equivalent domain from HIV-1 MN. The hybrid glycoprotein bound HIV-1 V3 loop MAbs and not SIV V3 MAbs but continued to bind conformational SIV MAbs and soluble CD4 as well as the parent molecule.     

Mutagenesis analysis of the murine leukemia virus matrix protein: identification of regions important for membrane localization and intracellular transport

We have created two sets of substitution mutations in the Moloney murine leukemia virus (Mo-MuLV) matrix protein in order to identify domains involved in association with the plasma membrane and in incorporation of the viral envelope glycoproteins into virus particles. The first set of mutations was targeted at putative membrane-associating regions similar to those of the human immunodeficiency virus type 1 matrix protein, which include a polybasic region at the N terminus of the Mo-MuLV matrix protein and two regions predicted to form beta strands. The second set of mutations was created within hydrophobic residues to test for the production of virus particles lacking envelope proteins, with the speculation of an involvement of the membrane-spanning region of the envelope protein in incorporation into virus particles. We have found that mutation of the N-terminal polybasic region redirected virus assembly to the cytoplasm, and we show that tryptophan residues may also play a significant role in the intracellular transport of the matrix protein. In total, 21 mutants of the Mo-MuLV matrix protein were produced, but we did not observe any mutant virus particles lacking the envelope glycoproteins, suggesting that a direct interaction between the Mo-MuLV matrix protein and envelope proteins either may not exist or may occur through multiple redundant interactions.     

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.     

Human immunodeficiency virus type 1 and 2 envelope glycoproteins oligomerize through conserved sequences

Hetero-oligomerization between human immunodeficiency virus type 2 (HIV-2) envelope glycoprotein (Env) truncation mutants and epitope-tagged gp160 is dependent on the presence of gp41 transmembrane protein (TM) amino acids 552 to 589, a putative amphipathic alpha-helical sequence. HIV-2 Env truncation mutants containing this sequence were also able to form cross-type hetero-oligomers with HIV-1 Env. HIV-2/HIV-1 hetero-oligomerization was, however, more sensitive to disruption by mutagenesis or increased temperature. The conservation of the Env oligomerization function of the HIV-1 and HIV-2 alpha-helical sequences suggests that retroviral TM alpha-helical motifs may have a universal role in oligomerization.     

Truncation of the human immunodeficiency virus type 1 envelope glycoprotein allows efficient pseudotyping of Moloney murine leukemia virus particles and gene transfer into CD4+ cells

Human immunodeficiency virus type 1 (HIV-1) can readily accept envelope (Env) glycoproteins from distantly related retroviruses. However, we previously showed that the HIV-1 Env glycoprotein complex is excluded even from particles formed by the Gag proteins of another lentivirus, visna virus, unless the matrix domain of the visna virus Gag polyprotein is replaced by that of HIV-1. We also showed that the integrity of the HIV-1 matrix domain is critical for the incorporation of wild-type HIV-1 Env protein but not for the incorporation of a truncated form which lacks the 144 C-terminal amino acids of the cytoplasmic domain of the transmembrane glycoprotein. We report here that the C-terminal truncation of the transmembrane glycoprotein also allows the efficient incorporation of HIV-1 Env proteins into viral particles formed by the Gag proteins of the widely divergent Moloney murine leukemia virus (Mo-MLV). Additionally, pseudotyping of a Mo-MLV-based vector with the truncated rather than the full-length HIV-1 Env allowed efficient transduction of human CD4+ cells. These results establish that Mo-MLV-based vectors can be used to target cells susceptible to infection by HIV-1.     

Specificity of antibodies produced against native or desialylated human immunodeficiency virus type 1 recombinant gp160

In a previous report we have shown that, in contrast to antibodies produced against native or fully deglycosylated human immunodeficiency virus type 1 (HIV-1) gp160 in rabbits, antibodies raised against desialylated HIV-1 gp160 also recognize gp140 from HIV-2 at high titers. Here, we characterize the fine specificity of these cross-reactive antibodies. Inhibition assays with a panel of synthetic peptides as competitors showed that cross-reactivity to gp140 was due to antibodies that were specific for the region encompassing HIV-1 gp41 immunodominant epitope, mimicked by peptide P39 (residues 583 to 609), the latter being able to totally inhibit the formation of complexes between radiolabeled HIV-2 gp140 and antibodies elicited by desialylated HIV-1 gp160. In addition, anti-desialylated gp160 antibodies retained on a P39 affinity column still bound HIV-2 gp140. Fine mapping has enabled us to localize the cross-reactive epitope within the N-terminal extremity of the gp41 immunodominant region. Interestingly, this cross-reactive antibody population did not recognize glycosylated or totally deglycosylated simian immunodeficiency virus gp140 despite an amino acid homology with HIV-1 within this region that is comparable to that of HIV-2. This cross-reactivity between HIV-1 and HIV-2 did not correlate with cross-neutralization. These results illustrate the influence of carbohydrate moieties on the specificity of the antibodies produced and clearly indicate that such procedures may be an efficient way to raise specific immune responses that are not type specific. Moreover, this cross-reactivity might explain the double-positive reactivity observed, in some human sera, against both HIV-1 and HIV-2 envelope antigens.     

Sensitivity of human immunodeficiency virus to bicyclam derivatives is influenced by the three-dimensional structure of gp120

The bicyclams are a new class of anti-human immunodeficiency virus (anti-HIV) compounds targeted at viral entry. From marker rescue experiments, it appears that the envelope gp120 glycoprotein plays an important role in the anti-HIV activity of the bicyclams. Bicyclam-resistant strains contain a number of amino acid changes scattered over the V2 to V5 region of gp120. Experiments aimed at estimating the relative importance of particular amino acid changes with regard to the overall resistance pattern are described. The sequences of some partially bicyclam-resistant virus strains, obtained during the resistance development process, were analyzed, and the corresponding 50% effective concentrations were determined. Selected mutations observed in bicyclam-resistant strains were introduced in the wild-type background by site-directed mutagenesis. In addition, some amino acids were back-mutated to their wild-type counterparts in an otherwise JM3100-resistant strain. The sensitivities of these mutant viruses to bicyclams were determined. Construction of chimeric viruses, carrying the V3 loop of JM3100-resistant virus in a wild-type HIV type 1 HXB2 background, enabled us to investigate the importance of the mutations in the V3 loop of JM3100-resistant virus. From the results described in the report, it can be concluded that single amino acid substitutions do not influence the observed resistance to JM3100. Also, the mutations in the V3 loop are not sufficient to engender even a partially resistant phenotype. We postulate that the overall conformation of gp120 determines the degree of sensitivity or resistance of HIV strains to bicyclams.