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Human proteins repurposed as biologics for clinical use have been engineered through in vitro techniques that improve the affinity of the biologics for their ligands. However, the techniques do not select against properties, such as protease sensitivity or self-reactivity, that impair the biologics' clinical efficacy. Here we show that the B-cell receptors of primary murine B cells can be engineered to affinity mature in vivo the human CD4 domains of the HIV-1-entry inhibitor CD4 immunoadhesin (CD4-Ig). Specifically, we introduced genes encoding the CD4 domains 1 and 2 (D1D2) of a half-life-enhanced form of CD4-Ig (CD4-Ig-v0) into the heavy-chain loci of murine B cells and adoptively transferred these cells into wild-type mice. After immunization, the B cells proliferated, class switched, affinity matured and produced D1D2-presenting antibodies. Somatic hypermutations in the D1D2-encoding region of the engrafted cells improved the binding affinity of CD4-Ig-v0 for the HIV-1 envelope glycoprotein and the inhibitor's ability to neutralize a panel of HIV-1 isolates without impairing its pharmacokinetic properties. In vivo affinity maturation of non-antibody protein biologics may guide the development of more effective therapeutics., Competing Interests: Competing interests: A.P., W.H., T.O., Y.Y. and M.F. are inventors of a patent describing the in vivo affinity maturation of antibodies and biologics. C.C.B., M.D.A. and M.F. have equity stakes in Emmune, Inc., which developed CD4-Ig-v0. The other authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Mice adoptively transferred with mouse B cells edited via CRISPR to express human antibody variable chains could help evaluate candidate vaccines and develop better antibody therapies. However, current editing strategies disrupt the heavy-chain locus, resulting in inefficient somatic hypermutation without functional affinity maturation. Here we show that these key B-cell functions can be preserved by directly and simultaneously replacing recombined mouse heavy and kappa chains with those of human antibodies, using a single Cas12a-mediated cut at each locus and 5' homology arms complementary to distal V segments. Cells edited in this way to express the human immunodeficiency virus type 1 (HIV-1) broadly neutralizing antibody 10-1074 or VRC26.25-y robustly hypermutated and generated potent neutralizing plasma in vaccinated mice. The 10-1074 variants isolated from the mice neutralized a global panel of HIV-1 isolates more efficiently than wild-type 10-1074 while maintaining its low polyreactivity and long half-life. We also used the approach to improve the potency of anti-SARS-CoV-2 antibodies against recent Omicron strains. In vivo affinity maturation of B cells edited at their native loci may facilitate the development of broad, potent and bioavailable antibodies., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

V2-glycan/apex broadly neutralizing antibodies (bnAbs) recognize a closed quaternary epitope of the HIV-1 envelope glycoprotein (Env). This closed structure is necessary to elicit apex antibodies and useful to guide the maturation of other bnAb classes. To compare antigens designed to maintain this conformation, we evaluated apex-specific responses in mice engrafted with a diverse repertoire of B cells expressing the HCDR3 of the apex bnAb VRC26.25. Engineered B cells affinity matured, guiding the improvement of VRC26.25 itself. We found that soluble Env (SOSIP) variants differed significantly in their ability to raise anti-apex responses. A transmembrane SOSIP (SOSIP-TM) delivered as an mRNA-lipid nanoparticle elicited more potent neutralizing responses than multimerized SOSIP proteins. Importantly, SOSIP-TM elicited neutralizing sera from B cells engineered with the predicted VRC26.25-HCDR3 progenitor, which also affinity matured. Our data show that HCDR3-edited B cells facilitate efficient in vivo comparisons of Env antigens and highlight the potential of an HCDR3-focused vaccine approach., Competing Interests: Declaration of interests W.H., T.O., Y.Y., and M.F. are inventors on a pending patent application describing methods for editing B cells., (Copyright © 2023 Elsevier Inc. All rights reserved.)

B cells have been engineered ex vivo to express an HIV-1 broadly neutralizing antibody (bNAb). B cell reprograming may be scientifically and therapeutically useful, but current approaches limit B cell repertoire diversity and disrupt the organization of the heavy-chain locus. A more diverse and physiologic B cell repertoire targeting a key HIV-1 epitope could facilitate evaluation of vaccines designed to elicit bNAbs, help identify more potent and bioavailable bNAb variants, or directly enhance viral control in vivo. Here we address the challenges of generating such a repertoire by replacing the heavy-chain CDR3 (HCDR3) regions of primary human B cells. To do so, we identified and utilized an uncharacterized Cas12a ortholog that recognizes PAM motifs present in human JH genes. We also optimized the design of 200 nucleotide homology-directed repair templates (HDRT) by minimizing the required 3'-5' deletion of the HDRT-complementary strand. Using these techniques, we edited primary human B cells to express a hemagglutinin epitope tag and the HCDR3 regions of the bNAbs PG9 and PG16. Those edited with bNAb HCDR3 efficiently bound trimeric HIV-1 antigens, implying they could affinity mature in vivo in response to the same antigens. This approach generates diverse B cell repertoires recognizing a key HIV-1 neutralizing epitope., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Three variable 2 (V2) loops of HIV-1 envelope glycoprotein (Env) trimer converge at the Env apex to form the epitope of an important classes of HIV-1 broadly neutralizing antibodies (bNAbs). These V2-glycan/apex antibodies are exceptionally potent but less broad (∼60 to 75%) than many other bNAbs. Their CDRH3 regions are typically long, acidic, and tyrosine sulfated. Tyrosine sulfation complicates efforts to improve these antibodies through techniques such as phage or yeast display. To improve the breadth of CAP256-VRC26.25 (VRC26.25), a very potent apex antibody, we adapted and extended a B cell display approach. Specifically, we used CRISPR/Cas12a to introduce VRC26.25 heavy- and light-chain genes into their respective loci in a B cell line, ensuring that each cell expresses a single VRC26.25 variant. We then diversified these loci through activation-induced cytidine deaminase-mediated hypermutation and homology-directed repair using randomized CDRH3 sequences as templates. Iterative sorting with soluble Env trimers and further randomization selected VRC26.25 variants with successively improving affinities. Three mutations in the CDRH3 region largely accounted for this improved affinity, and VRC26.25 modified with these mutations exhibited greater breadth and potency than the original antibody. Our data describe a broader and more-potent form of VRC26.25 as well as an approach useful for improving the breadth and potency of antibodies with functionally important posttranslational modifications., Competing Interests: The authors declare no competing interest.

The engineered antibody-like entry inhibitor eCD4-Ig neutralizes every human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus isolate it has been tested against. The exceptional breadth of eCD4-Ig derives from its ability to closely and simultaneously emulate the HIV-1 receptor CD4 and coreceptors, either CCR5 or CXCR4. Here we investigated whether viral escape from eCD4-Ig is more difficult than that from CD4-Ig or the CD4-binding site antibody NIH45-46. We observed that a viral swarm selected with high concentrations of eCD4-Ig was increasingly resistant to but did not fully escape from eCD4-Ig. In contrast, viruses selected under the same conditions with CD4-Ig or NIH45-46 fully escaped from those inhibitors. eCD4-Ig-resistant viruses acquired unique changes in the V2 apex, V3, V4, and CD4-binding regions of the HIV-1 envelope glycoprotein (Env). Most of the alterations did not directly affect neutralization by eCD4-Ig or neutralizing antibodies. However, alteration of Q428 to an arginine or lysine resulted in markedly greater resistance to eCD4-Ig and CD4-Ig, with correspondingly dramatic losses in infectivity and greater sensitivity to a V3 antibody and to serum from an infected individual. Compensatory mutations in the V3 loop (N301D) and in the V2 apex (K171E) partially restored viral fitness without affecting serum or eCD4-Ig sensitivity. Collectively, these data suggest that multiple mutations will be necessary to fully escape eCD4-Ig without loss of viral fitness. IMPORTANCE HIV-1 broadly neutralizing antibodies (bNAbs) and engineered antibody-like inhibitors have been compared for their breadths, potencies, and in vivo half-lives. However, a key limitation in the use of antibodies to treat an established HIV-1 infection is the rapid emergence of fully resistant viruses. Entry inhibitors of similar breadths and potencies can differ in the ease with which viral escape variants arise. Here we show that HIV-1 escape from the potent and exceptionally broad entry inhibitor eCD4-Ig is more difficult than that from CD4-Ig or the bNAb NIH45-46. Indeed, full escape was not observed under conditions under which escape from CD4-Ig or NIH45-46 was readily detected. Moreover, viruses that were partially resistant to eCD4-Ig were markedly less infective and more sensitive to antibodies in the serum of an infected person. These data suggest that eCD4-Ig will be more difficult to escape and that even partial escape will likely extract a high fitness cost., (Copyright © 2019 American Society for Microbiology.)

The HIV-1 envelope glycoprotein (Env) trimer mediates cell entry and is conformationally dynamic 1-8 . Imaging by single-molecule fluorescence resonance energy transfer (smFRET) has revealed that, on the surface of intact virions, mature pre-fusion Env transitions from a pre-triggered conformation (state 1) through a default intermediate conformation (state 2) to a conformation in which it is bound to three CD4 receptor molecules (state 3) 8-10 . It is currently unclear how these states relate to known structures. Breakthroughs in the structural characterization of the HIV-1 Env trimer have previously been achieved by generating soluble and proteolytically cleaved trimers of gp140 Env that are stabilized by a disulfide bond, an isoleucine-to-proline substitution at residue 559 and a truncation at residue 664 (SOSIP.664 trimers) 5,11-18 . Cryo-electron microscopy studies have been performed with C-terminally truncated Env of the HIV-1 JR-FL strain in complex with the antibody PGT151 19 . Both approaches have revealed similar structures for Env. Although these structures have been presumed to represent the pre-triggered state 1 of HIV-1 Env, this hypothesis has never directly been tested. Here we use smFRET to compare the conformational states of Env trimers used for structural studies with native Env on intact virus. We find that the constructs upon which extant high-resolution structures are based predominantly occupy downstream conformations that represent states 2 and 3. Therefore, the structure of the pre-triggered state-1 conformation of viral Env that has been identified by smFRET and that is preferentially stabilized by many broadly neutralizing antibodies-and thus of interest for the design of immunogens-remains unknown.

Adeno-associated virus (AAV) delivery of potent and broadly neutralizing antibodies (bNAbs is a promising approach for the prevention of HIV-1 infection. The immunoglobulin G (IgG)1 subtype is usually selected for this application, because it efficiently mediates antibody effector functions and has a somewhat longer half-life. However, the use of IgG1-Fc has been associated with the generation of anti-drug antibodies (ADAs) that correlate with loss of antibody expression. In contrast, we have shown that expression of the antibody-like molecule eCD4-Ig bearing a rhesus IgG2-Fc domain showed reduced immunogenicity and completely protected rhesus macaques from simian-HIV (SHIV)-AD8 challenges. To directly compare the performance of the IgG1-Fc and the IgG2-Fc domains in a prophylactic setting, we compared AAV1 expression of rhesus IgG1 and IgG2 forms of four anti-HIV bNAbs: 3BNC117, NIH45-46, 10-1074, and PGT121. Interestingly, IgG2-isotyped bNAbs elicited significantly lower ADA than their IgG1 counterparts. We also observed significant protection from two SHIV-AD8 challenges in macaques expressing IgG2-isotyped bNAbs, but not from those expressing IgG1. Our data suggest that monoclonal antibodies isotyped with IgG2-Fc domains are less immunogenic than their IgG1 counterparts, and they highlight ADAs as a key barrier to the use of AAV1-expressed bNAbs., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Broadly neutralizing antibodies (bNAbs) target five major epitopes on the HIV-1 envelope glycoprotein (Env). The most potent bNAbs have median half-maximal inhibitory concentration (IC 50 ) values in the nanomolar range, and the broadest bNAbs neutralize up to 98% of HIV-1 strains. The engineered HIV-1 entry inhibitor eCD4-Ig has greater breadth than bNAbs and similar potency. eCD4-Ig is markedly more potent than CD4-Ig due to its C-terminal coreceptor-mimetic peptide. Here we investigated whether the coreceptor-mimetic peptide mim6 improved the potency of bNAbs with different epitopes. We observed that when mim6 was appended to the C terminus of the heavy chains of bNAbs, this sulfopeptide improved the potency of all classes of bNAbs against HIV-1 isolates that are sensitive to neutralization by the sulfopeptide alone. However, mim6 did not significantly enhance neutralization of other isolates when appended to most classes of bNAbs, with one exception. Specifically, mim6 improved the potency of bNAbs of the V3-glycan class, including PGT121, PGT122, PGT128, and 10-1074, by an average of 2-fold for all HIV-1 isolates assayed. Despite this difference, 10-1074 does not induce exposure of the coreceptor-binding site, and addition of mim6 to 10-1074 did not promote shedding of the gp120 subunit of Env. Mixtures of 10-1074 and an Fc domain fused to mim6 neutralized less efficiently than a 10-1074/mim6 fusion, indicating that mim6 enhances the avidity of this fusion. Our data show that mim6 can consistently improve the potency of V3-glycan antibodies and suggest that these antibodies bind in an orientation that facilitates mim6 association with Env. IMPORTANCE HIV-1 requires both the cellular receptor CD4 and a tyrosine-sulfated coreceptor to infect its target cells. CD4-Ig is a fusion of the HIV-1-binding domains of CD4 with an antibody Fc domain. Previous studies have demonstrated that the potency of CD4-Ig is markedly increased by appending a coreceptor-mimetic sulfopeptide to its C terminus. We investigated whether this coreceptor-mimetic peptide improves the potency of broadly neutralizing antibodies (bNAbs) targeting five major epitopes on the HIV-1 envelope glycoprotein (Env). We observed that inclusion of the sulfopeptide dramatically improved the potency of all bNAb classes against isolates with more-open Env structures, typically those that utilize the coreceptor CXCR4. In contrast, the sulfopeptide improved only V3-glycan antibodies when neutralizing primary isolates, on average by 2-fold. These studies improve the potency of one class of bNAbs, show that coreceptor-mimetic sulfopeptides enhance neutralization through distinct mechanisms, and provide insight for the design of novel multispecific entry inhibitors., (Copyright © 2019 American Society for Microbiology.)

Human Immunodeficiency Virus-1 (HIV-1) entry is dependent on the envelope glycoprotein (Env) that is present on the virion and facilitates fusion between the envelope and the cellular membrane. The protein consists of two subunits, gp120 and gp41, with the former required for binding the CD4 receptor and either the CXCR4 or CCR5 coreceptor, and the latter for mediating fusion. The requirement of fusion for infection has made Env an attractive target for HIV therapy development and led to the FDA approval of enfuvirtide, a fusion inhibitor. Continued development of entry inhibitors is warranted because enfuvirtide resistant HIV-1 strains have emerged. In this study, a novel HIV-1 fusion assay was validated using neutralizing antibodies and then used to investigate the mechanism of action of eCD4-Igmim2, an HIV-1 inhibitor proposed to cooperatively bind the CD4 binding site and the sulfotyrosine-binding pocket of gp120. Greater reduction in fusion levels was observed with eCD4-Igmim2 in the fusion assay than all of the gp120 antibodies evaluated. Lab adapted isolates, HIV-1HXB2 and HIV-1YU2, were sensitive to eCD4-Igmim2 in the fusion assay, while primary isolates, HIV-1BG505 and HIV-1ZM651 were resistant. These results correlated with greater IC50 values for primary isolates compared to the lab adapted isolates observed in a virus neutralization assay. Analysis of gp120 models identified differences in the V1 and V2 domains that are associated with eCD4-Igmim2 sensitivity. This study highlights the use of a fusion assay to identify key areas for improving the potency of eCD4-Igmim2., Competing Interests: The authors have declared that no competing interests exist.

Many broadly neutralizing antibodies (bNAbs) against human immunodeficiency virus type 1 (HIV-1) were shown effective in animal models, and are currently evaluated in clinical trials. However, use of these antibodies in humans is hampered by the rapid emergence of resistant viruses. Here we show that soft-randomization can be used to accelerate the parallel identification of viral escape pathways. As a proof of principle, we soft-randomized the epitope regions of VRC01-class bNAbs in replication-competent HIV-1 and selected for resistant variants. After only a few passages, a surprisingly diverse population of antibody-resistant viruses emerged, bearing both novel and previously described escape mutations. We observed that the escape variants resistant to some VRC01-class bNAbs are resistant to most other bNAbs in the same class, and that a subset of variants was completely resistant to every well characterized VRC01-class bNAB, including VRC01, NIH45-46, 3BNC117, VRC07, N6, VRC-CH31, and VRC-PG04. Thus, our data demonstrate that soft randomization is a suitable approach for accelerated detection of viral escape, and highlight the challenges inherent in administering or attempting to elicit VRC01-class antibodies., Competing Interests: The authors have declared that no competing interests exist.

The human immunodeficiency virus type 1 (HIV-1) entry inhibitor eCD4-Ig is a fusion of CD4-Ig and a coreceptor-mimetic peptide. eCD4-Ig is markedly more potent than CD4-Ig, with neutralization efficiencies approaching those of HIV-1 broadly neutralizing antibodies (bNAbs). However, unlike bNAbs, eCD4-Ig neutralized all HIV-1, HIV-2, and simian immunodeficiency virus (SIV) isolates that it has been tested against, suggesting that it may be useful in clinical settings, where antibody escape is a concern. Here, we characterize three new eCD4-Ig variants, each with a different architecture and each utilizing D1.22, a stabilized form of CD4 domain 1. These variants were 10- to 20-fold more potent than our original eCD4-Ig variant, with a construct bearing four D1.22 domains (eD1.22-HL-Ig) exhibiting the greatest potency. However, this variant mediated less efficient antibody-dependent cell-mediated cytotoxicity (ADCC) activity than eCD4-Ig itself or several other eCD4-Ig variants, including the smallest variant (eD1.22-Ig). A variant with the same architecture as the original eCD4-Ig (eD1.22-D2-Ig) showed modestly higher thermal stability and best prevented the promotion of infection of CCR5-positive, CD4-negative cells. All three variants, and eCD4-Ig itself, mediated more efficient shedding of the HIV-1 envelope glycoprotein gp120 than did CD4-Ig. Finally, we show that only three D1.22 mutations contributed to the potency of eD1.22-D2-Ig and that introduction of these changes into eCD4-Ig resulted in a variant 9-fold more potent than eCD4-Ig and 2-fold more potent than eD1.22-D2-Ig. These studies will assist in developing eCD4-Ig variants with properties optimized for prophylaxis, therapy, and cure applications. IMPORTANCE HIV-1 bNAbs have properties different from those of antiretroviral compounds. Specifically, antibodies can enlist immune effector cells to eliminate infected cells, whereas antiretroviral compounds simply interfere with various steps in the viral life cycle. Unfortunately, HIV-1 is adept at evading antibody recognition, limiting the utility of antibodies as a treatment for HIV-1 infection or as part of an effort to eradicate latently infected cells. eCD4-Ig is an antibody-like entry inhibitor that closely mimics HIV-1's obligate receptors. eCD4-Ig appears to be qualitatively different from antibodies, since it neutralizes all HIV-1, HIV-2, and SIV isolates. Here, we characterize three new structurally distinct eCD4-Ig variants and show that each excels in a key property useful to prevent, treat, or cure an HIV-1 infection. For example, one variant neutralized HIV-1 most efficiently, while others best enlisted natural killer cells to eliminate infected cells. These observations will help generate eCD4-Ig variants optimized for different clinical applications., (Copyright © 2018 American Society for Microbiology.)

Antibody-dependent cell-mediated cytotoxity (ADCC) can eliminate HIV-1 infected cells, and may help reduce the reservoir of latent virus in infected patients. Sera of HIV-1 positive individuals include a number of antibodies that recognize epitopes usually occluded on HIV-1 envelope glycoprotein (Env) trimers. We have recently described eCD4-Ig, a potent and exceptionally broad inhibitor of HIV-1 entry that can be used to protect rhesus macaques from multiple high-dose challenges with simian-human immunodeficiency virus AD8 (SHIV-AD8). Here we show that eCD4-Ig bearing an IgG1 Fc domain (eCD4-IgG1) can mediate efficient ADCC activity against HIV-1 isolates with differing tropisms, and that it does so at least 10-fold more efficiently than CD4-Ig, even when more CD4-Ig molecules bound cell surface-expressed Env. An ADCC-inactive IgG2 form of eCD4-Ig (eCD4-IgG2) exposes V3-loop and CD4-induced epitopes on cell-expressed trimers, and renders HIV-1-infected cells susceptible to ADCC mediated by antibodies of these classes. Moreover, eCD4-IgG2, but not IgG2 forms of the broadly neutralizing antibodies VRC01 and 10-1074, enhances the ADCC activities of serum antibodies from patients by 100-fold, and significantly enhanced killing of two latently infected T-cell lines reactivated by vorinostat or TNFα. Thus eCD4-Ig is qualitatively different from CD4-Ig or neutralizing antibodies in its ability to mediate ADCC, and it may be uniquely useful in treating HIV-1 infection or reducing the reservoir of latently infected cells.

Purpose of Review: Here we discuss recently developed HIV-1 entry inhibitors that can target multiple epitopes on the HIV-1 envelope glycoprotein (Env), with an emphasis on eCD4-Ig. Some of these inhibitors are more potent and broader than any single antibody characterized to date. We also discuss the use of recombinant adeno-associated virus (rAAV) vectors as a platform for long-term expression of these inhibitors., Recent Findings: Much of the exterior of HIV-1 Env can be targeted by broadly neutralizing antibodies (bNAbs). Recent studies combine the variable regions or Fabs from different bNAbs, often with the receptor-mimetic components, to create broad, potent, and hard-to-escape inhibitors. rAAV vectors can express these inhibitors for years in vivo, highlighting their ability to prevent or treat HIV-1 infection., Summary: By targeting multiple epitopes on Env, bispecific and antibody-like inhibitors can be broader and more potent than bNAbs. These inhibitors can provide long-term protection from, and perhaps suppression of, HIV-1 if they are administered by a delivery platform, like rAAV vectors, but only after rAAV limitations are addressed.

Unlabelled: The HIV-1 envelope glycoprotein (Env) is a trimer of gp120/gp41 heterodimers that mediates viral entry. Env binds cellular CD4, an association which stabilizes a conformation favorable to its subsequent association with a coreceptor, typically CCR5 or CXCR4. The CD4- and coreceptor-binding sites serve as epitopes for two classes of HIV-1-neutralizing antibodies: CD4-binding site (CD4bs) and CD4-induced (CD4i) antibodies, respectively. Here we observed that, at a fixed total concentration, mixtures of the CD4i antibodies (E51 or 412d) and the CD4bs antibody VRC01 neutralized the HIV-1 isolates 89.6, ADA, SG3, and SA32 more efficiently than either antibody alone. We found that E51, and to a lesser extent 412d and 17b, promoted association of four CD4bs antibodies to the Env trimer but not to monomeric gp120. We further demonstrated that the binding of the sulfotyrosine-binding pocket by CCR5mim2-Ig was sufficient for promoting CD4bs antibody binding to Env. Interestingly, the relationship is not reciprocal: CD4bs antibodies were not as efficient as CD4-Ig at promoting E51 or 412d binding to Env trimer. Consistent with these observations, CD4-Ig, but none of the CD4bs antibodies tested, substantially increased HIV-1 infection of a CD4-negative, CCR5-positive cell line. We conclude that the ability of CD4i antibodies to promote VRC01 association with Env trimers accounts for the increase potency of VRC01 and CD4i antibody mixtures. Our data further suggest that potent CD4bs antibodies avoid inducing Env conformations that bind CD4i antibodies or CCR5., Importance: Potent HIV-1-neutralizing antibodies can prevent viral transmission and suppress an ongoing infection. Here we show that CD4-induced (CD4i) antibodies, which recognize the conserved coreceptor-binding site of the HIV-1 envelope glycoprotein (Env), can increase the association of Env with potent broadly neutralizing antibodies that recognize the CD4-binding site (CD4bs antibodies). We further show that, unlike soluble forms of CD4, CD4bs antibodies poorly induce envelope glycoprotein conformations that efficiently bind CCR5. This study provides insight into the properties of potent CD4bs antibodies and suggests that, under some conditions, CD4i antibodies can improve their potency. These observations may be helpful to the development of vaccines designed to elicit specific antibody classes., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Most simian-human immunodeficiency viruses (SHIVs) bearing envelope (Env) glycoproteins from primary HIV-1 strains fail to infect rhesus macaques (RMs). We hypothesized that inefficient Env binding to rhesus CD4 (rhCD4) limits virus entry and replication and could be enhanced by substituting naturally occurring simian immunodeficiency virus Env residues at position 375, which resides at a critical location in the CD4-binding pocket and is under strong positive evolutionary pressure across the broad spectrum of primate lentiviruses. SHIVs containing primary or transmitted/founder HIV-1 subtype A, B, C, or D Envs with genotypic variants at residue 375 were constructed and analyzed in vitro and in vivo. Bulky hydrophobic or basic amino acids substituted for serine-375 enhanced Env affinity for rhCD4, virus entry into cells bearing rhCD4, and virus replication in primary rhCD4 T cells without appreciably affecting antigenicity or antibody-mediated neutralization sensitivity. Twenty-four RMs inoculated with subtype A, B, C, or D SHIVs all became productively infected with different Env375 variants-S, M, Y, H, W, or F-that were differentially selected in different Env backbones. Notably, SHIVs replicated persistently at titers comparable to HIV-1 in humans and elicited autologous neutralizing antibody responses typical of HIV-1. Seven animals succumbed to AIDS. These findings identify Env-rhCD4 binding as a critical determinant for productive SHIV infection in RMs and validate a novel and generalizable strategy for constructing SHIVs with Env glycoproteins of interest, including those that in humans elicit broadly neutralizing antibodies or bind particular Ig germ-line B-cell receptors.

Unlabelled: The cytoplasmic tails of human and simian immunodeficiency virus (HIV and SIV, respectively) envelope glycoproteins contain a highly conserved, membrane-proximal endocytosis motif that prevents the accumulation of Env on the surface of infected cells prior to virus assembly. Using an assay designed to measure the killing of virus-infected cells by antibody-dependent cell-mediated cytotoxicity (ADCC), we show that substitutions in this motif increase the susceptibility of HIV-1- and SIV-infected cells to ADCC in a manner that directly correlates with elevated Env levels on the surface of virus-infected cells. In the case of HIV-1, this effect is additive with a deletion in vpu recently shown to enhance the susceptibility of HIV-1-infected cells to ADCC as a result of tetherin-mediated retention of budding virions on the cell surface. These results reveal a previously unappreciated role for the membrane-proximal endocytosis motif of gp41 in protecting HIV-1- and SIV-infected cells from antibody responses by regulating the amount of Env present on the cell surface., Importance: This study reveals an unappreciated role for the membrane-proximal endocytosis motif of gp41 in protecting HIV-1- and SIV-infected cells from elimination by Env-specific antibodies. Thus, strategies designed to interfere with this mechanism of Env internalization may improve the efficacy of antibody-based vaccines and antiretroviral therapies designed to enhance the immunological control of HIV-1 replication in chronically infected individuals., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Unlabelled: The HIV-1 envelope glycoprotein binds cooperatively to its cellular receptor CD4 and a coreceptor, principally CXCR4 or CCR5. We have previously improved a natural amino-acid form of a scorpion toxin-derived CD4-mimetic peptide and in parallel generated sulfopeptide mimetics of the CCR5 amino terminus. Here we show that some fusions of these CCR5- and CD4-mimetic peptides, expressed as immunoadhesins, neutralize HIV-1 more efficiently than CD4-Fc or equimolar mixtures of immunoadhesin forms of each peptide. Specifically, double-mimetic peptides with linkers of 11 amino acids or greater, and with the CCR5-mimetic component preceding the CD4-mimetic component, were more efficient than constructs with shorter linkers or in a reverse orientation. The potency of these constructs derives from (i) their ability to simultaneously and cooperatively bind the CD4- and CCR5-binding sites of a single gp120 monomer of the HIV-1 envelope glycoprotein trimer and (ii) the ability of the CCR5-mimetic component to prevent the CD4-mimetic peptide from promoting infection when cellular CD4 is limiting. Thus, there is a significant advantage to simultaneously targeting both conserved regions of the HIV-1 envelope glycoprotein., Importance: This report describes a novel class of peptides that potently inhibit HIV-1 entry. These peptides simultaneously target the receptor- and coreceptor-binding sites of the HIV-1 envelope glycoprotein gp120. Peptides of this class overcome key limitations of inhibitors that target only one gp120 binding region and illustrate the utility of binding the sulfotyrosine-binding pockets of gp120.

Phage display is a key technology for the identification and maturation of high affinity peptides, antibodies, and other proteins. However, limitations of bacterial expression restrict the range and sensitivity of assays that can be used to evaluate phage-selected variants. To address this problem, selected genes are typically transferred to mammalian expression vectors, a major rate-limiting step in the iterative improvement of peptides and proteins. Here we describe a system that combines phage display and efficient mammalian expression in a single vector, pDQ1. This system permits immediate expression of phage-selected genes as IgG1-Fc fusions in mammalian cells, facilitating the rapid, sensitive characterization of a large number of library outputs for their biochemical and functional properties. We demonstrate the utility of this system by improving the ability of a CD4-mimetic peptide to bind the HIV-1 envelope glycoprotein and neutralize HIV-1 entry. We further improved the potency of the resulting peptide, CD4mim6, by limiting its ability to induce the CD4-bound conformation of the envelope glycoprotein. Thus, CD4mim6 and its variants can be used to investigate the properties of the HIV-1 envelope glycoprotein, and pDQ1 can accelerate the discovery of new peptides and proteins through phage display.

A tyrosine-sulfated CCR5-mimetic peptide, CCR5mim1, inhibits HIV-1 infection more efficiently than sulfopeptides based on the CCR5 amino terminus. Here we characterized sulfopeptide chimeras of CCR5mim1 and the heavy-chain CDR3 of the antibody PG16. Two chimeras bound a range of envelope glycoproteins and neutralized HIV-1 more efficiently than CCR5mim1. An immunoadhesin form of one of these, CCR5mim2-Ig, synergized with CD4-Ig to neutralize HIV-1. These sulfopeptides are among the broadest and most potent CCR5-mimetic peptides described to date.

Human immunodeficiency virus type 1 (HIV-1) does not replicate in primary cells of New World primates. To better understand this restriction, we expressed owl monkey (Aotus nancymaae) CD4 and CXCR4 in the owl monkey kidney cell line, OMK. An HIV-1 variant modified to evade the owl monkey restriction factor TRIM-cyp replicated efficiently in these cells but could not replicate in primary A. nancymaae CD4-positive T cells. To understand this difference, we examined APOBEC3G and tetherin orthologs from OMK cells and primary A. nancymaae cells. We observed that OMK cells expressed substantially lower levels of APOBEC3G than did A. nancymaae cells. A. nancymaae, but not marmoset (Callithrix jacchus), APOBEC3G was partially downregulated by HIV-1 vif and reduced but did not abolish HIV-1 replication when stably expressed in OMK cells. The functional difference between A. nancymaae and marmoset APOBEC3Gs mapped to residue 128, previously shown to distinguish African green monkey from human APOBEC3G. We also characterized tetherin orthologs from OMK and A. nancymaae cells. The A. nancymaae tetherin ortholog, but not OMK tetherin, prevented HIV-1 release. Alteration of threonine 181 of OMK tetherin rescued its function and its efficient N glycosylation. All alleles of Aotus lemurinus griseimembra examined, but none of A. nancymaae or Aotus vociferans, encoded this nonfunctional tetherin ortholog. Our data indicate that HIV-1 replication in owl monkeys is not restricted at entry but can be limited by APOBEC3G and tetherin. Further, A. lemurinus griseimembra does not restrict HIV-1 replication via tetherin, a property likely useful for the study of tetherin-restricted viruses.

Heterozygosity for the CCR5 Delta32 allele is associated with delayed progression to AIDS in human immunodeficiency virus type 1 (HIV-1) infection. Here we describe an unusual HIV-1 isolate from the blood of an asymptomatic individual who was heterozygous for the CCR5 Delta32 allele and had reduced levels of CCR5 expression. The primary virus used CCR5, CXCR4, and an unusually broad range of alternative coreceptors to enter transfected cells. However, only CXCR4 and CCR5 were used to enter primary T cells and monocyte-derived macrophages, respectively. Full-length Env clones had an unusually long V1/V2 region and rare amino acid variants in the V3 and C4 regions. Mutagenesis studies and structural models suggested that Y308, D321, and to a lesser extent K442 and E444, contribute to the broad coreceptor usage of these Envs, whereas I317 is likely to be a compensatory change. Furthermore, database analysis suggests that covariation can occur at positions 308/317 and 308/321 in vivo. Y308 and D321 reduced dependence on the extracellular loop 2 (ECL2) region of CCR5, while these residues along with Y330, K442, and E444 enhanced dependence on the CCR5 N-terminus compared to clade B consensus residues at these positions. These results suggest that expanded coreceptor usage of HIV-1 can occur in some individuals without rapid progression to AIDS as a consequence of changes in the V3 region that reduce dependence on the ECL2 region of CCR5 by enhancing interactions with conserved structural elements in G-protein-coupled receptors.

Sulfated tyrosines at the amino terminus of the principal HIV-1 coreceptor CCR5 play a critical role in its ability to bind the HIV-1 envelope glycoprotein gp120 and mediate HIV-1 entry. Human antibodies that recognize the CCR5-binding region of gp120 are also modified by tyrosine sulfation, which is necessary for their ability to neutralize HIV-1. Here we demonstrate that a sulfated peptide derived from the CDR3 region of one of these antibodies, E51, can efficiently bind gp120. Association of this peptide, pE51, with gp120 requires tyrosine sulfation and is enhanced by, but not dependent on, CD4. Alteration of any of four pE51 tyrosines, or alteration of gp120 residues 420, 421, or 422, critical for association with CCR5, prevents gp120 association with pE51. pE51 neutralizes HIV-1 more effectively than peptides based on the CCR5 amino terminus and may be useful as a fusion partner with other protein inhibitors of HIV-1 entry. Our data provide further insight into the association of the CCR5 amino terminus with gp120, show that a conserved, sulfate-binding region of gp120 is accessible to inhibitors in the absence of CD4, and suggest that soluble mimetics of CCR5 can be more effective than previously appreciated.

HIV-1 has maximized its utilization of syndecans. It uses them as in cis receptors to infect macrophages and as in trans receptors to infect T-lymphocytes. In this study, we investigated at a molecular level the mechanisms that control HIV-1-syndecan interactions. We found that a single conserved arginine (Arg-298) in the V3 region of gp120 governs HIV-1 binding to syndecans. We found that an amine group on the side chain of this residue is necessary for syndecan utilization by HIV-1. Furthermore, we showed that HIV-1 binds syndecans via a 6-O sulfation, demonstrating that this binding is not the result of random interactions between basic residues and negative charges, but the result of specific contacts between gp120 and a well defined sulfation in syndecans. Surprisingly, we found that Arg-298, which mediates HIV-1 binding to syndecans, also mediates HIV-1 binding to CCR5. We postulated that HIV-1 recognizes similar motifs on syndecans and CCR5. Supporting this hypothesis, we obtained several lines of evidence that suggest that the 6-O sulfation recognized by HIV-1 on syndecans mimics the sulfated tyrosines recognized by HIV-1 in the N terminus of CCR5. Our finding that CCR5 and syndecans are exploited by HIV-1 via a single determinant echoes the mechanisms by which chemokines utilize these two disparate receptors and suggests that the gp120/chemokine mimicry may represent a common strategy in microbial pathogenesis.

The human immunodeficiency virus (HIV-1) transmembrane envelope glycoprotein, gp41, which mediates virus-cell fusion, exists in at least three different conformations within the trimeric envelope glycoprotein complex. The structures of the prefusogenic and intermediate states are unknown; structures representing the postfusion state have been solved. In the postfusion conformation, three helical heptad repeat 2 (HR2) regions pack in an antiparallel fashion into the hydrophobic grooves on the surface of a triple-helical coiled coil formed by the heptad repeat 1 (HR1) regions. We studied the prefusogenic conformation of gp41 by mutagenic alteration of membrane-anchored and soluble forms of the HIV-1 envelope glycoproteins. Our results indicate that, in the HIV-1 envelope glycoprotein precursor, the gp41 HR1 region is in a conformation distinct from that of a trimeric coiled coil. Thus, the central gp41 coiled coil is formed during the transition of the HIV-1 envelope glycoproteins from the precursor state to the receptor-bound intermediate.

Interaction of the human immunodeficiency virus type 1 (HIV-1) gp120 envelope glycoprotein with the primary receptor, CD4, promotes binding to a chemokine receptor, either CCR5 or CXCR4. The chemokine receptor-binding site on gp120 elicits CD4-induced (CD4i) antibodies in some HIV-1-infected individuals. Like CCR5 itself, the CD4i antibody 412d exhibits a preference for CCR5-using HIV-1 strains and utilizes sulfated tyrosines to achieve binding to gp120. Here, we show that 412d binding requires the gp120 beta19 strand and the base of the V3 loop, elements that are important for the binding of the CCR5 N terminus. Two gp120 residues in the V3 loop base determined 412d preference for CCR5-using HIV-1 strains. A chimeric molecule in which the 412d heavy-chain third complementarity-determining loop sequence replaces the CCR5 N terminus functioned as an efficient second receptor, selectively supporting the entry of CCR5-using HIV-1 strains. Sulfation of N-terminal tyrosines contributed to the function of this chimeric receptor. These results emphasize the close mimicry of the CCR5 N terminus by the gp120-interactive region of a naturally elicited CD4i antibody.

CXCR4 is a co-receptor along with CD4 for human immunodeficiency virus type 1 (HIV-1). We investigated the role of N-linked glycosylation in the N-terminus of CXCR4 in binding to HIV-1 gp120 envelope glycoproteins. Gp120s from CXCR4 (X4) and CCR5 (R5) using HIV-1 strains bound more efficiently to non-N-glycosylated than to N-glycosylated CXCR4 proteoliposomes in a CD4-dependent manner. Similar results were observed in binding studies using non-N-glycosylated or N-glycosylated CXCR4 expressed on cells. Mutation of the N-glycosylation site N11 in CXCR4 (N11Q-CXCR4) enhanced CD4-dependent binding of X4 and R5 gp120s and allowed more efficient entry of viruses pseudotyped with X4 or R5 HIV-1 envelope glycoproteins. However, the binding of R5 gp120 to N11Q-CXCR4 and entry of R5 HIV-1 viruses into cells expressing N11Q-CXCR4 were 20- and 100- to 1000-fold less efficient, respectively, than the levels achieved using X4 gp120 or X4 HIV-1 viruses. Binding of stromal cell-derived factor (SDF)-1alpha, the natural ligand of CXCR4, and SDF-1alpha-induced signaling were reduced by the N11Q mutation. These findings demonstrate that N-glycosylation at N11 inhibits the binding of CXCR4 to X4 and R5 HIV-1 gp120, and provide a better understanding of the structural elements of CXCR4 involved in HIV-1 Env-co-receptor interactions.

The synthetic peptide T-20, which corresponds to a sequence within the C-terminal heptad repeat region (HR2) of the human immunodeficiency virus type 1 (HIV-1) gp41 envelope glycoprotein, potently inhibits viral membrane fusion and entry. Although T-20 is thought to bind the N-terminal heptad repeat region (HR1) of gp41 and interfere with gp41 conformational changes required for membrane fusion, coreceptor specificity determined by the V3 loop of gp120 strongly influences the sensitivity of HIV-1 variants to T-20. Here, we show that T-20 binds to the gp120 glycoproteins of HIV-1 isolates that utilize CXCR4 as a coreceptor in a manner determined by the sequences of the gp120 V3 loop. T-20 binding to gp120 was enhanced in the presence of soluble CD4. Analysis of T-20 binding to gp120 mutants with variable loop deletions and the reciprocal competition of T-20 and particular anti-gp120 antibodies suggested that T-20 interacts with a gp120 region near the base of the V3 loop. Consistent with the involvement of this region in coreceptor binding, T-20 was able to block the interaction of gp120-CD4 complexes with the CXCR4 coreceptor. These results help to explain the increased sensitivity of CXCR4-specific HIV-1 isolates to the T-20 peptide. Interactions between the gp41 HR2 region and coreceptor-binding regions of gp120 may also play a role in the function of the HIV-1 envelope glycoproteins.

The conserved surface of the HIV-1 gp120 envelope glycoprotein that binds to the HIV-1 coreceptor is protected from humoral recognition by multiple layers of camouflage. Here we present sequence and genomic analyses for 12 antibodies that pierce these defenses and determine the crystal structures of 5. The data reveal mechanisms and atomic-level details for three unusual immune features: posttranslational mimicry of coreceptor by tyrosine sulfation of antibody, an alternative molecular mechanism controlling such sulfation, and highly selective V(H)-gene usage. When confronted by extraordinary viral defenses, the immune system unveils novel adaptive capabilities, with tyrosine sulfation enhancing the vocabulary of antigen recognition.

Sulfated tyrosines at the amino terminus of the principal HIV-1 coreceptor CCR5 play a critical role in its ability to bind the HIV-1 envelope glycoprotein gp120 and mediate HIV-1 infection. Here, we show that a number of human antibodies directed against gp120 are tyrosine sulfated at their antigen binding sites. Like that of CCR5, antibody association with gp120 is dependent on sulfate moieties, enhanced by CD4, and inhibited by sulfated CCR5-derived peptides. Most of these antibodies preferentially associate with gp120 molecules of CCR5-utilizing (R5) isolates and neutralize primary R5 isolates more efficiently than laboratory-adapted isolates. These studies identify a distinct subset of CD4-induced HIV-1 neutralizing antibodies that closely emulate CCR5 and demonstrate that tyrosine sulfation can contribute to the potency and diversity of the human humoral response.

CXCR4, a member of the G protein-coupled receptor family of proteins, is the receptor for stromal cell-derived factor (SDF-1 alpha) and is a principal coreceptor for human immunodeficiency virus type 1 (HIV-1). CXCR4 has also been implicated in breast cancer metastasis. We examined the ability of CXCR4 to homomultimerize in detergent-solubilized cell lysates and in the membranes of intact cells. CXCR4 was found to multimerize in cell lysates containing the detergents CHAPSO or Cymal-7 but not other detergents that have been shown to disrupt the native conformation of CXCR4. CXCR4 expression levels did not affect the observed multimerization and differentially tagged CXCR4 molecules associated only when coexpressed in the same cell. CXCR4 did not interact with CCR5, the other principal HIV-1 coreceptor, when the two proteins were coexpressed. Using bioluminescence resonance energy transfer (BRET(2)), we demonstrated that CXCR4 multimers are found naturally in the intact cell membrane, in both the presence and absence of multiple CXCR4 ligands. Ligand binding did not significantly alter the observed BRET(2) signal, suggesting that CXCR4 exists as a constitutive oligomer. In cell lysates prepared with non-denaturing detergents, CXCR4 sedimented in a manner consistent with a dimer, whereas CCR5 sedimented as a monomer under these conditions. The stable, constitutive dimerization of CXCR4 may contribute to its biological functions in chemokine binding, signaling, and HIV-1 entry.

Alzheimer's disease is characterized by deposition of beta-amyloid peptide (Abeta) into plaques in the brain, leading to neuronal toxicity and dementia. Human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system can also cause a dementia, and amyloid deposition in the central nervous system is significantly higher in HIV-1-infected individuals compared with uninfected controls. Here we report that Abeta fibrils stimulated, by 5-20-fold, infection of target cells expressing CD4 and an appropriate coreceptor by multiple HIV-1 isolates but did not permit infection of cells lacking these receptors. Abeta enhanced infection at the stage of virus attachment or entry into the cell. Abeta fibrils also stimulated infection by amphotrophic Moloney leukemia virus, herpes simplex virus, and viruses pseudotyped with the envelope glycoprotein of vesicular stomatitis virus. Other synthetic fibril-forming peptides similarly enhanced viral infection and may be useful in gene delivery applications utilizing retroviral vectors. These data suggest that Abeta deposition may increase the vulnerability of the central nervous system to enveloped viral infection and that amyloidogenic peptides could be useful in enhancing gene transfer by enveloped viral vectors.

The chemokine receptor CXCR4 plays critical roles in development, immune function, and human immunodeficiency virus type 1 (HIV-1) entry. Here we demonstrate that, like the CC-chemokine receptors CCR5 and CCR2b, CXCR4 is posttranslationally modified by sulfation of its amino-terminal tyrosines. The sulfate group at tyrosine 21 contributes substantially to the ability of CXCR4 to bind its ligand, stromal derived factor 1 alpha. Tyrosine sulfation plays a less significant role in CXCR4-dependent HIV-1 entry than in CCR5-dependent HIV-1 entry. In some cell lines, CXCR4 is efficiently modified by a chondroitin sulfate chain at serine 18, but neither HIV-1 entry nor stromal derived factor 1 alpha binding was affected by loss of this glycosaminoglycan. These data demonstrate a functional role for tyrosine sulfate in the CXC-chemokine receptor family and underscore a general difference in HIV-1 utilization of CCR5 and CXCR4.

Most human immunodeficiency virus type 1 (HIV-1) viruses in the brain use CCR5 as the principal coreceptor for entry into a cell. However, additional phenotypic characteristics are necessary for HIV-1 neurotropism. Furthermore, neurotropic strains are not necessarily neurovirulent. To better understand the determinants of HIV-1 neurovirulence, we isolated viruses from brain tissue samples from three AIDS patients with dementia and HIV-1 encephalitis and analyzed their ability to induce syncytia in monocyte-derived macrophages (MDM) and neuronal apoptosis in primary brain cultures. Two R5X4 viruses (MACS1-br and MACS1-spln) were highly fusogenic in MDM and induced neuronal apoptosis. The R5 viruses UK1-br and MACS2-br are both neurotropic. However, only UK1-br induced high levels of fusion in MDM and neuronal apoptosis. Full-length Env clones from UK1-br required lower CCR5 and CD4 levels than Env clones from MACS2-br to function efficiently in cell-to-cell fusion and single-round infection assays. UK1-br Envs also had a greater affinity for CCR5 than MACS2-br Envs in binding assays. Relatively high levels of UK1-br and MACS2-br Envs bound to CCR5 in the absence of soluble CD4. However, these Envs could not mediate CD4-independent infection, and MACS2-br Envs were unable to mediate fusion or infection in cells expressing low levels of CD4. The UK1-br virus was more resistant than MACS2-br to inhibition by the CCR5-targeted inhibitors TAK-779 and Sch-C. UK1-br was more sensitive than MACS2-br to neutralization by monoclonal antibodies (2F5 and immunoglobulin G1b12 [IgG1b12]) and CD4-IgG2. These results predict the presence of HIV-1 variants with increased CCR5 affinity and reduced dependence on CCR5 and CD4 in the brains of some AIDS patients with central nervous system disease and suggest that R5 variants with increased CCR5 affinity may represent a pathogenic viral phenotype contributing to the neurodegenerative manifestations of AIDS.

Mast cells are critical components of innate and adaptive immunity that differentiate in tissues in situ from circulating committed progenitor cells. We now demonstrate that human cord blood-derived mast cell progenitors are susceptible to infection with macrophagetropic (M-tropic) and dualtropic human immunodeficiency virus type 1 (HIV-1) isolates but not with T-cell-tropic (T-tropic) strains. Mast cell progenitors (c-kit(+) CD13(+) cells with chloroacetate esterase activity) were purified from 4-week-old cultures of cord blood mononuclear cells maintained in stem cell factor, interleukin-6 (IL-6), and IL-10 using a CD14 depletion column. These progenitors expressed CCR3, CCR5, and CXCR4, as well as low levels of CD4. When infected in vitro with viruses pseudotyped with different HIV and simian immunodeficiency virus envelope glycoproteins, only M-tropic and dualtropic, but not T-tropic, viruses were able to enter mast cell progenitors. Both the CCR5-specific monoclonal antibody 2D7 and TAK-779, a nonpeptide inhibitor of CCR5-mediated viral entry, blocked HIV-1 strain ADA infection by >80%. Cultures infected with replication-competent virus produced progressively increasing amounts of virus for 21 days as indicated by p24 antigen detection. Mast cell progenitors that were exposed to an M-tropic, green fluorescent protein-expressing HIV-1 strain exhibited fluorescence indicative of viral entry and replication on a single-cell level and retained virus production during differentiation. The trafficking of mast cell progenitors to multiple tissues, combined with the long life span of mature mast cells, suggests that they could provide a widespread and persistent HIV reservoir in AIDS.

In addition to the CCR5 and CXCR4 chemokine receptors, a subset of primary human immunodeficiency virus type 1 (HIV-1) isolates can also use the seven-transmembrane-domain receptor APJ as a coreceptor. A previously identified ligand of APJ, apelin, specifically inhibited the entry of primary T-tropic and dualtropic HIV-1 isolates from different clades into cells expressing CD4 and APJ. Analysis of apelin analogues demonstrated that potent and specific antiviral activity was retained by a 13-residue, arginine-rich peptide. Antiviral potency was influenced by the integrity of methionine 75, which contributes to APJ-binding affinity, and by the retention of apelin residues 63 to 65. These studies demonstrate the ability of a small peptide ligand to block the function of APJ as an HIV-1 coreceptor, identify apelin sequences important for the inhibition, and provide new reagents for the investigation of the significance of APJ to HIV-1 infection and pathogenesis.

The sequential association of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 with CD4 and a seven-transmembrane segment coreceptor such as CCR5 or CXCR4 initiates entry of the virus into its target cell. The N terminus of CCR5, which contains several sulfated tyrosines, plays a critical role in the CD4-dependent association of gp120 with CCR5 and in viral entry. Here we demonstrate that a tyrosine-sulfated peptide based on the N terminus of CCR5, but not its unsulfated analogue, inhibits infection of macrophages and peripheral blood mononuclear cells by CCR5-dependent, but not CXCR4-dependent, HIV-1 isolates. The sulfated peptide also inhibited the association of CCR5-expressing cells with gp120-soluble CD4 complexes and, less efficiently, with MIP-1alpha. Moreover, this peptide inhibited the precipitation of gp120 by 48d and 23e antibodies, which recognize CD4-inducible gp120 epitopes, but not by several other antibodies that recognize proximal epitopes. The ability of the sulfated peptide to block 48d association with gp120 was dependent in part on seven tropism-determining residues in the third variable (V3) and fourth conserved (C4) domains of gp120. These data underscore the important role of the N-terminal sulfate moieties of CCR5 in the entry of R5 HIV-1 isolates and localize a critical contact between gp120 and CCR5.

The human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins function as a membrane-anchored trimer of three gp120 exterior glycoproteins and three gp41 transmembrane glycoproteins. Previously, we reported three approaches to stabilize soluble trimers containing parts of the gp41 ectodomains: addition of GCN4 trimeric helices, disruption of the cleavage site between gp120 and gp41, and introduction of cysteines in the gp41 coiled coil to form intersubunit disulfide bonds. Here, we applied similar approaches to stabilize soluble gp140 trimers including the complete gp120 and gp41 ectodomains. A combination of fusion with the GCN4 trimeric sequences and disruption of the gp120-gp41 cleavage site resulted in relatively homogeneous gp140 trimers with exceptional stability. The gp120 epitopes recognized by neutralizing antibodies are intact and exposed on these gp140 trimers. By contrast, the nonneutralizing antibody epitopes on the gp120 subunits of the soluble trimers are relatively occluded compared with those on monomeric gp120 preparations. This antigenic similarity to the functional HIV-1 envelope glycoproteins and the presence of the complete gp41 ectodomain should make the soluble gp140 trimers useful tools for structural and immunologic studies.

The functional unit of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins is a trimer composed of three gp120 exterior glycoproteins and three gp41 transmembrane glycoproteins. The lability of intersubunit interactions has hindered the production and characterization of soluble, homogeneous envelope glycoprotein trimers. Here we report three modifications that stabilize soluble forms of HIV-1 envelope glycoprotein trimers: disruption of the proteolytic cleavage site between gp120 and gp41, introduction of cysteines that form intersubunit disulfide bonds, and addition of GCN4 trimeric helices. Characterization of these secreted glycoproteins by immunologic and biophysical methods indicates that these stable trimers retain structural integrity. The efficacy of the GCN4 sequences in stabilizing the trimers, the formation of intersubunit disulfide bonds between appropriately placed cysteines, and the ability of the trimers to interact with a helical, C-terminal gp41 peptide (DP178) support a model in which the N-terminal gp41 coiled coil exists in the envelope glycoprotein precursor and contributes to intersubunit interactions within the trimer. The availability of stable, soluble HIV-1 envelope glycoprotein trimers should expedite progress in understanding the structure and function of the virion envelope glycoprotein spikes.

The gp120 envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1) promotes virus entry by sequentially binding CD4 and chemokine receptors on the target cell. Primary, clinical HIV-1 isolates require interaction with CD4 to allow gp120 to bind the CCR5 chemokine receptor efficiently. We adapted a primary HIV-1 isolate, ADA, to replicate in CD4-negative canine cells expressing human CCR5. The gp120 changes responsible for the adaptation were limited to alteration of glycosylation addition sites in the V2 loop-V1-V2 stem. The gp120 glycoproteins of the adapted viruses bound CCR5 directly, without prior interaction with CD4. Thus, a major function of CD4 binding in the entry of primary HIV-1 isolates can be bypassed by changes in the gp120 V1-V2 elements, which allow the envelope glycoproteins to assume a conformation competent for CCR5 binding.

Chemokine receptors and related seven-transmembrane-segment (7TMS) receptors serve as coreceptors for entry of human and simian immunodeficiency viruses (HIV-1, HIV-2, and SIV) into target cells. Each of these otherwise diverse coreceptors contains an N-terminal region that is acidic and tyrosine rich. Here, we show that the chemokine receptor CCR5, a principal HIV-1 coreceptor, is posttranslationally modified by O-linked glycosylation and by sulfation of its N-terminal tyrosines. Sulfated tyrosines contribute to the binding of CCR5 to MIP-1 alpha, MIP-1 beta, and HIV-1 gp120/CD4 complexes and to the ability of HIV-1 to enter cells expressing CCR5 and CD4. CXCR4, another important HIV-1 coreceptor, is also sulfated. Tyrosine sulfation may contribute to the natural function of many 7TMS receptors and may be a modification common to primate immunodeficiency virus coreceptors.

Biochemical and structural studies of fragments of the ectodomain of the human immunodeficiency virus type 1 (HIV-1) gp41 transmembrane envelope glycoprotein have demonstrated that the molecular contacts between alpha helices allow the formation of a trimeric coiled coil. By introducing cysteine residues into specific locations along these alpha helices, the normally labile HIV-1 gp160 envelope glycoprotein was converted into a stable disulfide-linked oligomer. Although proteolytic cleavage into gp120 and gp41 glycoproteins was largely blocked, the disulfide-linked oligomer was efficiently transported to the cell surface and was recognized by a series of conformationally dependent antibodies. The pattern of hetero-oligomer formation between this construct and an analogous construct lacking portions of the gp120 variable loops and of the gp41 cytoplasmic tail demonstrates that these oligomers are trimers. These results support the relevance of the proposed gp41 structure and intersubunit contacts to the native, complete HIV-1 envelope glycoprotein. Disulfide-mediated stabilization of the labile HIV-1 envelope glycoprotein oligomer, which has been suggested to possess advantages as an immunogen, may assist attempts to develop vaccines.

Human immunodeficiency virus type 1 (HIV-1) enters target cells by sequential binding to CD4 and specific seven-transmembrane-segment (7TMS) coreceptors. Viruses use the chemokine receptor CCR5 as a coreceptor in the early, asymptomatic stages of HIV-1 infection but can adapt to the use of other receptors such as CXCR4 and CCR3 as the infection proceeds. Here we identify one such coreceptor, Apj, which supported the efficient entry of several primary T-cell-line tropic (T-tropic) and dualtropic HIV-1 isolates and the simian immunodeficiency virus SIVmac316. Another 7TMS protein, CCR9, supported the less efficient entry of one primary T-tropic isolate. mRNAs for both receptors were present in phytohemagglutinin- and interleukin-2-activated peripheral blood mononuclear cells. Apj and CCR9 share with other coreceptors for HIV-1 and SIV an N-terminal region rich in aromatic and acidic residues. These results highlight properties common to 7TMS proteins that can function as HIV-1 coreceptors, and they may contribute to an understanding of viral evolution in infected individuals.

Seven transmembrane segment (7TMS) receptors for chemokines and related molecules have been demonstrated to be essential, in addition to CD4, for HIV and SIV infection. The beta-chemokine receptor CCR5 is the primary, perhaps sole, co-receptor for HIV-1 during the early and chronic phases of infection and supports infection by most primary HIV-1 and many SIV isolates. Late-stage primary and laboratory-adapted HIV-1, HIV-2, and SIV isolates can use other 7TMS receptors. CXCR4 appears especially important in late-stage HIV infection; several related receptors can also be used. The specificity of SIV viruses is similar. Commonalities among these receptors, combined with analyses of mutated molecules, indicate that discrete, conformationally-dependent sites on the chemokine receptors determine their association with the third variable and conserved regions of viral envelope glycoproteins. These studies are useful for elucidating the mechanism and molecular determinants of HIV-1 entry, and of inhibitors to that entry., (Copyright 1998 Academic Press.)

The bis-azo compound FP-21399 inhibits HIV-1 infection. We now show that FP-21399 acts on the HIV-1 envelope glycoprotein to prevent viral replication. This compound targets the entry step of the HIV-1 replication cycle as demonstrated by time-of-addition and single cycle viral entry assays. The entry of SIVmac239, which uses the same coreceptors (CD4/CCR5) as HIV-1, was not inhibited by FP-21399, indicating that the antiviral effect of FP-21399 is specific for the HIV-1 envelope glycoprotein and is not dependent upon the cellular receptors CD4 and CCR5. FP-21399 inhibits neither the activity of HIV-1 reverse transcriptase nor the expression of HIV-1 early mRNA. Finally, this compound inhibits gp120 shedding of the T-tropic virus. Our results suggest that the anti-HIV activity of FP-21399 is due to its interaction with HIV-1 gp120/41 complex during viral entry.

The human CXCR-4 molecule serves as a second receptor for primary, T-cell-tropic, and laboratory-adapted human immunodeficiency virus type 1 (HIV-1) isolates. Here we show that murine CXCR-4 can support the entry of some of these HIV-1 isolates. Differences between mouse and human CXCR-4 in the ability to function as an HIV-1 receptor are determined by sequences in the second extracellular loop of the CXCR-4 protein.

Human immunodeficiency virus type 1 (HIV-1) requires the presence of specific chemokine receptors in addition to CD4 to enter target cells. The chemokine receptor CCR5 is used by the macrophage-tropic strains of HIV-1 that predominate during the asymptomatic stages of infection. Here we identify a small tyrosine-rich region of CCR5 proximal to the N-terminal cysteine that is critical for entry of macrophage-tropic and dual-tropic variants of HIV-1. HIV-1 infection of cells expressing CCR5 mutants with changes in this region was substantially reduced compared with the infection of cells bearing wild-type CCR5. Simian immunodeficiency virus (SIVmac239) entry was also ablated on a subset of these mutants but enhanced on others. These differences in virus entry were correlated with the relative ability of soluble, monomeric HIV-1 and SIVmac239 gp120 glycoproteins to bind the CCR5 mutants. These results identify a region of CCR5 that is necessary for the physical association of the gp120 envelope glycoprotein with CCR5 and for HIV-1 infection.

Two different crystal structures of the human immunodeficiency virus type 1 (HIV-1) integrase (IN) catalytic domain were analyzed for interactions at the enzyme active site. Gln-62 and Glu-92 interact with active-site residue Asp-64, and Lys-136 interacts with active-site residue Asp-116 across a dimer interface. Conservative and nonconservative substitutions were introduced at these positions to probe the roles of these interactions in HIV-1 integration. Purified mutant proteins were assayed for in vitro 3' processing, DNA strand transfer, and disintegration activities, and HIV-1 mutants were assayed for virion protein composition, reverse transcription, and infectivities in human cell lines. Each of the mutant IN proteins displayed wild-type disintegration activity, indicating that none of the interactions is essential for catalysis. Mutants carrying Gln or Ala for Glu-92 displayed wild-type activities, but substituting Lys for Glu-92 reduced in vitro 3' processing and DNA strand transfer activities 5- to 10-fold and yielded a replication-defective IN active-site mutant viral phenotype. Substituting Glu for Gln-62 reduced in vitro 3' processing and DNA strand transfer activities 5- to 10-fold without grossly affecting viral replication kinetics, suggesting that HIV-1 can replicate in T-cell lines with less than the wild-type level of IN activity. The relationship between IN solubility and HIV-1 replication was also investigated. We previously showed that substituting Lys for Phe-185 dramatically increased the solubility of recombinant IN but caused an HIV-1 particle assembly defect. Mutants carrying His at this position displayed increased solubility and wild-type replication kinetics, showing that increased IN solubility per se is not detrimental to virus growth.

The human immunodeficiency virus type 1 (HIV-1) requires the presence of specific chemokine receptors in addition to CD4 to enter its target cell. The chemokine receptor CCR5 is used by macrophage-tropic strains of HIV-1, which predominate during the asymptomatic stages of infection. Here we investigate whether the ability of CCR5 to signal in response to its beta-chemokine ligands is necessary or sufficient for viral entry. Three CCR5 mutants with little or no ability to mobilize calcium in response to macrophage inflammatory protein-1beta could nonetheless support HIV-1 entry and the early steps in the virus life cycle with efficiencies comparable with those of wild-type CCR5. Conversely, a chimeric receptor with the N terminus of CCR2 replacing that of CCR5 responded to macrophage inflammatory protein-1beta and MCP-1 but did not efficiently support viral entry. These results demonstrate that chemokine signaling and HIV-1 entry are separable functions of CCR5 and that only viral entry requires the N-terminal domain of CCR5.

Several members of the chemokine receptor family are used together with CD4 for HIV-1 entry into target cells. T cell line-tropic (T-tropic) HIV-1 viruses use the chemokine receptor CXCR4 as a co-receptor, whereas macrophage-tropic (M-tropic) primary viruses use CCR5 (refs 2-6). Individuals with defective CCR5 alleles exhibit resistance to HIV-1 infection, suggesting that CCR5 has an important role in vivo in HIV-1 replication. A subset of primary viruses can use CCR3 as well as CCR5 as a co-receptor, but the in vivo contribution of CCR3 to HIV-1 infection and pathogenesis is unknown. HIV-1 infects the central nervous system (CNS) and causes the dementia associated with AIDS. Here we report that the major target cells for HIV-1 infection in the CNS, the microglia, express both CCR3 and CCR5. The CCR3 ligand, eotaxin, and an anti-CCR3 antibody inhibited HIV-1 infection of microglia, as did MIP-1beta, which is a CCR5 ligand. Our results suggest that both CCR3 and CCR5 promote efficient infection of the CNS by HIV-1.