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Advances in single-cell antibody cloning methods have led to the identification of a variety of broadly neutralizing anti-HIV-1 antibodies. We developed a computational tool (Antibody Database) to help identify critical residues on the HIV-1 envelope protein whose natural variation affects antibody activity. Our simplifying assumption was that, for a given antibody, a significant portion of the dispersion of neutralization activity across a panel of HIV-1 strains is due to the amino acid identity or glycosylation state at a small number of specific sites, each acting independently. A model of an antibody's neutralization IC50 was developed in which each site contributes a term to the logarithm of the modeled IC50. The analysis program attempts to determine the set of rules that minimizes the sum of the residuals between observed and modeled IC50 values. The predictive quality of the identified rules may be assessed in part by whether there is support for rules within individual viral clades. As a test case, we analyzed antibody 8ANC195, an anti-glycoprotein gp120 antibody of unknown specificity. The model for this antibody indicated that several glycosylation sites were critical for neutralization. We evaluated this prediction by measuring neutralization potencies of 8ANC195 against HIV-1 in vitro and in an antibody therapy experiment in humanized mice. These experiments confirmed that 8ANC195 represents a distinct class of glycan-dependent anti-HIV-1 antibody and validated the utility of computational analysis of neutralization panel data.

Defining the virus-host interactions responsible for HIV-1 transmission, including the phenotypic requirements of viruses capable of establishing de novo infections, could be important for AIDS vaccine development. Previous analyses have failed to identify phenotypic properties other than chemokine receptor 5 (CCR5) and CD4+ T-cell tropism that are preferentially associated with viral transmission. However, most of these studies were limited to examining envelope (Env) function in the context of pseudoviruses. Here, we generated infectious molecular clones of transmitted founder (TF; n = 27) and chronic control (CC; n = 14) viruses of subtypes B (n = 18) and C (n = 23) and compared their phenotypic properties in assays specifically designed to probe the earliest stages of HIV-1 infection. We found that TF virions were 1.7-fold more infectious (P = 0.049) and contained 1.9-fold more Env per particle (P = 0.048) compared with CC viruses. TF viruses were also captured by monocyte-derived dendritic cells 1.7-fold more efficiently (P = 0.035) and more readily transferred to CD4+ T cells (P = 0.025). In primary CD4+ T cells, TF and CC viruses replicated with comparable kinetics; however, when propagated in the presence of IFN-α, TF viruses replicated to higher titers than CC viruses. This difference was significant for subtype B (P = 0.000013) but not subtype C (P = 0.53) viruses, possibly reflecting demographic differences of the respective patient cohorts. Together, these data indicate that TF viruses are enriched for higher Env content, enhanced cell-free infectivity, improved dendritic cell interaction, and relative IFN-α resistance. These viral properties, which likely act in concert, should be considered in the development and testing of AIDS vaccines.

Efforts to design an effective antibody-based vaccine against HIV-1 would benefit from understanding how germ-line B-cell receptors (BCRs) recognize the HIV-1 gp120/gp41 envelope spike. Potent VRC01-like (PVL) HIV-1 antibodies derived from the VH1-2*02 germ-line allele target the conserved CD4 binding site on gp120. A bottleneck for design of immunogens capable of eliciting PVL antibodies is that VH1-2*02 germ-line BCR interactions with gp120 are uncharacterized. Here, we report the structure of a VH1-2*02 germ-line antibody alone and a germ-line heavy-chain/mature light-chain chimeric antibody complexed with HIV-1 gp120. VH1-2*02 residues make extensive contacts with the gp120 outer domain, including all PVL signature and CD4 mimicry interactions, but not critical CDRH3 contacts with the gp120 inner domain and bridging sheet that are responsible for the improved potency of NIH45-46 over closely related clonal variants, such as VRC01. Our results provide insight into initial recognition of HIV-1 by VH1-2*02 germ-line BCRs and may facilitate the design of immunogens tailored to engage and stimulate broad and potent CD4 binding site antibodies.

A large number of anti-HIV-1 antibodies targeting the CD4-binding site (CD4bs) on the envelope glycoprotein gp120 have recently been reported. These antibodies, typified by VRC01, are remarkable for both their breadth and their potency. Crystal structures have revealed a common mode of binding for several of these antibodies; however, the precise relationship among CD4bs antibodies remains to be defined. Here we analyze existing structural and sequence data, propose a set of signature features for potent VRC01-like (PVL) antibodies, and verify the importance of these features by mutagenesis. The signature features explain why PVL antibodies derive from a single germ-line human V(H) gene segment and why certain gp120 sequences are associated with antibody resistance. Our results bear on vaccine development and structure-based design to improve the potency and breadth of anti-CD4bs antibodies.

Monoclonal antibodies b12 and 4E10 are broadly neutralizing against a variety of strains of the human immunodeficiency virus type 1 (HIV-1). The epitope for b12 maps to the CD4-binding site in the gp120 subunit of HIV-1's trimeric gp120-gp41 envelope spike, whereas 4E10 recognizes the membrane-proximal external region (MPER) of gp41. Here, we constructed and compared a series of architectures for the b12 and 4E10 combining sites that differed in size, valency, and flexibility. In a comparative analysis of the ability of the b12 and 4E10 constructs to neutralize a panel of clade B HIV-1 strains, we observed that the ability of bivalent constructs to cross-link envelope spikes on the virion surface made a greater contribution to neutralization by b12 than by 4E10. Increased distance and flexibility between antibody combining sites correlated with enhanced neutralization for both antibodies, suggesting restricted mobility for the trimeric spikes embedded in the virion surface. The size of a construct did not appear to be correlated with neutralization potency for b12, but larger 4E10 constructs exhibited a steric occlusion effect, which we interpret as evidence for restricted access to its gp41 epitope. The combination of limited avidity and steric occlusion suggests a mechanism for evading neutralization by antibodies that target epitopes in the highly conserved MPER of gp41.

Huntington's disease and several other neurological diseases are caused by expanded polyglutamine [poly(Gln)] tracts in different proteins. Mechanisms for expanded (>36 Gln residues) poly(Gln) toxicity include the formation of aggregates that recruit and sequester essential cellular proteins [Preisinger, E., Jordan, B. M., Kazantsev, A. & Housman, D. (1999) Phil. Trans. R. Soc. London B 354, 1029-1034; Chen, S., Berthelier, V., Yang, W. & Wetzel, R. (2001) J. Mol. Biol. 311, 173-182] and functional alterations, such as improper interactions with other proteins [Cummings, C. J. & Zoghbi, H. Y. (2000) Hum. Mol. Genet. 9, 909-916]. Expansion above the "pathologic threshold" ( approximately 36 Gln) has been proposed to induce a conformational transition in poly(Gln) tracts, which has been suggested as a target for therapeutic intervention. Here we show that structural analyses of soluble huntingtin exon 1 fusion proteins with 16 to 46 glutamine residues reveal extended structures with random coil characteristics and no evidence for a global conformational change above 36 glutamines. An antibody (MW1) Fab fragment, which recognizes full-length huntingtin in mouse brain sections, binds specifically to exon 1 constructs containing normal and expanded poly(Gln) tracts, with affinity and stoichiometry that increase with poly(Gln) length. These data support a "linear lattice" model for poly(Gln), in which expanded poly(Gln) tracts have an increased number of ligand-binding sites as compared with normal poly(Gln). The linear lattice model provides a rationale for pathogenicity of expanded poly(Gln) tracts and a structural framework for drug design.

The Drosophila mutant methuselah (mth) was identified from a screen for single gene mutations that extended average lifespan. Mth mutants have a 35% increase in average lifespan and increased resistance to several forms of stress, including heat, starvation, and oxidative damage. The protein affected by this mutation is related to G protein-coupled receptors of the secretin receptor family. Mth, like secretin receptor family members, has a large N-terminal ectodomain, which may constitute the ligand binding site. Here we report the 2.3-A resolution crystal structure of the Mth extracellular region, revealing a folding topology in which three primarily beta-structure-containing domains meet to form a shallow interdomain groove containing a solvent-exposed tryptophan that may represent a ligand binding site. The Mth structure is analyzed in relation to predicted Mth homologs and potential ligand binding features.

The cation-pi interaction is an important, general force for molecular recognition in biological receptors. Through the sidechains of aromatic amino acids, novel binding sites for cationic ligands such as acetylcholine can be constructed. We report here a number of calculations on prototypical cation-pi systems, emphasizing structures of relevance to biological receptors and prototypical heterocycles of the type often of importance in medicinal chemistry. Trends in the data can be rationalized using a relatively simple model that emphasizes the electrostatic component of the cation-pi interaction. In particular, plots of the electrostatic potential surfaces of the relevant aromatics provide useful guidelines for predicting cation-pi interactions in new systems.