Allosteric Modulation of the HIV-1 gp120-gp41 Association Site by Adjacent gp120 Variable Region 1 (V1) N-Glycans Linked to Neutralization Sensitivity

The HIV-1 gp120-gp41 complex, which mediates viral fusion and cellular entry, undergoes rapid evolution within its external glycan shield to enable escape from neutralizing antibody (NAb). Understanding how conserved protein determinants retain functionality in the context of such evolution is important for their evaluation and exploitation as potential drug and/ or vaccine targets. In this study, we examined how the conserved gp120-gp41 association site, formed by the N- and Cterminal segments of gp120 and the disulfide-bonded region (DSR) of gp41, adapts to glycan changes that are linked to neutralization sensitivity. To this end, a DSR mutant virus (K601D) with defective gp120-association was sequentially passaged in peripheral blood mononuclear cells to select suppressor mutations. We reasoned that the locations of suppressors point to structural elements that are functionally linked to the gp120-gp41 association site. In culture 1, gp120 association and viral replication was restored by loss of the conserved glycan at Asn136 in V1 (T138N mutation) inconjunction with the L494I substitution in C5 within the association site. In culture 2, replication was restored with deletion of the N139INN sequence, which ablates the overlapping Asn141-Asn142-Ser-Ser potential N-linked glycosylation sequons inV1, in conjunction with D601N in the DSR. The 136 and 142 glycan mutations appeared to exert their suppressive effects by altering the dependence of gp120-gp41 interactions on the DSR residues, Leu593, Trp596 and Lys601. The 136 and/or 142glycan mutations increased the sensitivity of HIV-1 pseudovirions to the glycan-dependent NAbs 2G12 and PG16, and also pooled IgG obtained from HIV-1-infected individuals. Thus adjacent V1 glycans allosterically modulate the distal gp120-gp41 association site. We propose that this represents a mechanism for functional adaptation of the gp120-gp41 association site to an evolving glycan shield in a setting of NAb selection

The trans-ancestral genomic architecture of glycemic traits

Glycemic traits are used to diagnose and monitor type 2 diabetes and cardiometabolic health. To date, most genetic studies of glycemic traits have focused on individuals of European ancestry. Here we aggregated genome-wide association studies comprising up to 281,416 individuals without diabetes (30% non-European ancestry) for whom fasting glucose, 2-h glucose after an oral glucose challenge, glycated hemoglobin and fasting insulin data were available. Trans-ancestry and single-ancestry meta-analyses identified 242 loci (99 novel; P < 5 x 10(-8)), 80% of which had no significant evidence of between-ancestry heterogeneity. Analyses restricted to individuals of European ancestry with equivalent sample size would have led to 24 fewer new loci. Compared with single-ancestry analyses, equivalent-sized trans-ancestry fine-mapping reduced the number of estimated variants in 99% credible sets by a median of 37.5%. Genomic-feature, gene-expression and gene-set analyses revealed distinct biological signatures for each trait, highlighting different underlying biological pathways. Our results increase our understanding of diabetes pathophysiology by using trans-ancestry studies for improved power and resolution. A trans-ancestry meta-analysis of GWAS of glycemic traits in up to 281,416 individuals identifies 99 novel loci, of which one quarter was found due to the multi-ancestry approach, which also improves fine-mapping of credible variant sets

Genome-Wide Association Study of the Modified Stumvoll Insulin Sensitivity Index Identifies BCL2 and FAM19A2 as Novel Insulin Sensitivity Loci

Genome-wide association studies (GWAS) have found few common variants that influence fasting measures of insulin sensitivity. We hypothesized that a GWAS of an integrated assessment of fasting and dynamic measures of insulin sensitivity would detect novel common variants. We performed a GWAS of the modified Stumvoll Insulin Sensitivity Index (ISI) within the Meta-Analyses of Glucose and Insulin-Related Traits Consortium. Discovery for genetic association was performed in 16,753 individuals, and replication was attempted for the 23 most significant novel loci in 13,354 independent individuals. Association with ISI was tested in models adjusted for age, sex, and BMI and in a model analyzing the combined influence of the genotype effect adjusted for BMI and the interaction effect between the genotype and BMI on ISI (model 3). In model 3, three variants reached genome-wide significance: Rs13422522 (NYAP2; P = 8.87 × 10-11), rs12454712 (BCL2; P = 2.7 × 10-8), and rs10506418 (FAM19A2; P = 1.9 × 10-8). The association at NYAP2 was eliminated by conditioning on the known IRS1 insulin sensitivity locus; the BCL2 and FAM19A2 associations were independent of known cardiometabolic loci. In conclusion, we identified two novel loci and replicated known variants associated with insulin sensitivity. Further studies are needed to clarify the causal variant and function at the BCL2 and FAM19A2 loci

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Sex-stratified Genome-wide Association Studies Including 270,000 Individuals Show Sexual Dimorphism in Genetic Loci for Anthropometric Traits

Peer reviewe

A Conserved Gly(436)-Trp-Leu-Ala-Gly-Leu-Phe-Tyr Motif in Hepatitis C Virus Glycoprotein E2 Is a Determinant of CD81 Binding and Viral Entry

The hepatitis C virus (HCV) glycoproteins E1 and E2 form a heterodimer that mediates CD81 receptor binding and viral entry. In this study, we used site-directed mutagenesis to examine the functional role of a conserved G(436)WLAGLFY motif of E2. The mutants could be placed into two groups based on the ability of mature virion-incorporated E1E2 to bind the large extracellular loop (LEL) of CD81 versus the ability to mediate cellular entry of pseudotyped retroviral particles. Group 1 comprised E2 mutants where LEL binding ability largely correlated with viral entry ability, with conservative and nonconservative substitutions (W437 L/A, L438A, L441V/F, and F442A) inhibiting both functions. These data suggest that Trp-437, Leu-438, Leu-441, and Phe-442 directly interact with the LEL. Group 2 comprised E2 glycoproteins with more conservative substitutions that lacked LEL binding but retained between 20% and 60% of wild-type viral entry competence. The viral entry competence displayed by group 2 mutants was explained by residual binding by the E2 receptor binding domain to cellular full-length CD81. A subset of mutants maintained LEL binding ability in the context of intracellular E1E2 forms, but this function was largely lost in virion-incorporated glycoproteins. These data suggest that the CD81 binding site undergoes a conformational transition during glycoprotein maturation through the secretory pathway. The G436P mutant was an outlier, retaining near-wild-type levels of CD81 binding but lacking significant viral entry ability. These findings indicate that the G(436)WLAGLFY motif of E2 functions in CD81 binding and in pre- or post-CD81-dependent stages of viral entry

The Conserved Glycine-Rich Segment Linking the N-Terminal Fusion Peptide to the Coiled Coil of Human T-Cell Leukemia Virus Type 1 Transmembrane Glycoprotein gp21 Is a Determinant of Membrane Fusion Function

Retroviral transmembrane proteins (TMs) contain an N-terminal fusion peptide that initiates virus-cell membrane fusion. The fusion peptide is linked to the coiled-coil core through a conserved sequence that is often rich in glycines. We investigated the functional role of the glycine-rich segment, Met-326 to Ser-337, of the human T-cell leukemia virus type 1 (HTLV-1) TM, gp21, by alanine and proline scanning mutagenesis. Alanine substitution for the hydrophobic residue Ile-334 caused an ∼90% reduction in cell-cell fusion activity without detectable effects on the lipid-mixing and pore formation phases of fusion. Alanine substitutions at other positions had smaller effects (Gly-329, Val-330, and Gly-332) or no effect on fusion function. Proline substitution for glycine residues inhibited cell-cell fusion function with position-dependent effects on the three phases of fusion. Retroviral glycoprotein fusion function thus appears to require flexibility within the glycine-rich segment and hydrophobic contacts mediated by this segment

Sensitivity of T138N and ΔN¹³⁹INN mutant pseudovirions to NAbs.

<p>U87.CD4.CCR5 cells were incubated with pseudovirus-IgG mixtures for 2 days prior to lysis and assay for luciferase activity. Neutralizing activities were measured in triplicate and reported as the average percent luciferase activity. The data are representative of 2–4 independent experiments. WT: blue squares, T138N: red triangles, ΔN¹³⁹INN: green “X”s.</p

Structural models.

<p><b><i>A</i></b>, Homology model of oligomannose-glycosylated AD8 V1V2 based on the crystal structure of CAP45 V1V2 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003218#ppat.1003218-McLellan1" target="_blank">[7]</a>. The V1V2 model, generated using the Modeller algorithm <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003218#ppat.1003218-Sali1" target="_blank">[113]</a> within Discovery Studio 3.0, was glycosylated <i>in silico</i> with oligomannose side chains using the glycosciences.de server (<a href="http://www.glycosciences.de/modeling/glyprot/php/main.php" target="_blank">http://www.glycosciences.de/modeling/glyprot/php/main.php</a>) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003218#ppat.1003218-BohneLang1" target="_blank">[114]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003218#ppat.1003218-Lutteke1" target="_blank">[115]</a>. The β2–β3 hairpin that forms the V1V2 base is colored green, V1 in yellow, V2 in orange. PNGSs (Asn residues shown in CPK) and oligomannose side chains are colored according to the V1V2 subdomain to which they are attached. <b><i>B</i></b>, Model of oligomannose-glycosylated gp120 monomer. The model, prepared with the UCSF Chimera package <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003218#ppat.1003218-Pettersen1" target="_blank">[117]</a>, is based on the crystal structures of the complex formed between HXBc2 gp120 with gp41-interactive region (gp120 residues 31–284, 334–501), sCD4 and 48 d Fab (PDB ID 3JWD) and the YU2 gp120 (residues 285–333)-418d Fab-sCD4 complex (PDB ID 2QAD) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003218#ppat.1003218-Huang2" target="_blank">[5]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003218#ppat.1003218-Pancera1" target="_blank">[8]</a>. Oligomannose addition was performed <i>in silico</i> as for <b><i>A</i></b>. The gp41 association site formed by the N- and C-terminal segments is colored green, the 7 stranded β-sandwich in purple, layer 2 and the V1V2 β2–β3 hairpin base in green, layer 1 in pink, the outer domain in red. Asn residues representing PNGSs are shown in CPK. Oligomannose glycans implicated in 2G12 recognition are colored crimson. The homology models were drawn using Pymol. <b><i>C</i></b>, Alignment of V1V2 amino acid sequences. Selected V1V2 sequences were initially aligned using clustalx and then adjusted manually. PNGSs are highlighted in green. Residue numbering is according to HXB2. sens: neutralization-sensitive; res: neutralization-resistant. <i>^a</i>, Neutralization susceptibility of Envs derived from subtype B HIV-1 reference strains as determined using macaque antisera raised to SF162 gp140 immunogen <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003218#ppat.1003218-Ching1" target="_blank">[36]</a>; <i>^b</i>, Neutralization susceptibility phenotype associated with primary (M1) and chronic (M47, M46, M32 and M31) phase subtype A V1V2 sequences as determined with autologous plasma <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003218#ppat.1003218-Sagar1" target="_blank">[42]</a>; <i>^c</i>, Consensus V1V2 sequences derived from neutralization-sensitive (VP-1CON) and neutralization-resistant (VP-2CON) isolates obtained from a patient with cross-reactive neutralizing activity as determined with autologous plasma <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003218#ppat.1003218-vanGils2" target="_blank">[44]</a>.</p