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. 2009 Mar 15;385(2):505-20.
doi: 10.1016/j.virol.2008.12.032. Epub 2009 Jan 23.

Highly complex neutralization determinants on a monophyletic lineage of newly transmitted subtype C HIV-1 Env clones from India

Smita S Kulkarni  1 , Alan Lapedes, Haili Tang, S Gnanakaran, Marcus G Daniels, Ming Zhang, Tanmoy Bhattacharya, Ming Li, Victoria R Polonis, Francine E McCutchan, Lynn Morris, Dennis Ellenberger, Salvatore T Butera, Robert C Bollinger, Bette T Korber, Ramesh S Paranjape, David C Montefiori
Affiliations

Highly complex neutralization determinants on a monophyletic lineage of newly transmitted subtype C HIV-1 Env clones from India

Smita S Kulkarni et al. Virology. .

Abstract

Little is known about the neutralization properties of HIV-1 in India to optimally design and test vaccines. For this reason, a functional Env clone was obtained from each of ten newly acquired, heterosexually transmitted HIV-1 infections in Pune, Maharashtra. These clones formed a phylogenetically distinct genetic lineage within subtype C. As Env-pseudotyped viruses the clones were mostly resistant to IgG1b12, 2G12 and 2F5 but all were sensitive to 4E10. When compared to a large multi-subtype panel of Env-pseudotyped viruses (subtypes B, C and CRF02_AG) in neutralization assays with a multi-subtype panel of HIV-1-positive plasma samples, the Indian Envs were remarkably complex. With the exception of the Indian Envs, results of a hierarchical clustering analysis showed a strong subtype association with the patterns of neutralization susceptibility. From these patterns we were able to identify 19 neutralization cluster-associated amino acid signatures in gp120 and 14 signatures in the ectodomain and cytoplasmic tail of gp41. We conclude that newly transmitted Indian Envs are antigenically complex in spite of close genetic similarity. Delineation of neutralization-associated amino acid signatures provides a deeper understanding of the antigenic structure of HIV-1 Env.

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Figures

FIG. 1
FIG. 1
Maximum likelihood tree indicating the phylogenetic relationships of the gp160 sequences used in the reference panel of sequences used to study neutralization phenotype. The newly characterized Indian gp160 genes are clustered together within the more diverse group of subtype C Envs from Africa, represented here by twelve gp160 genes that are recommended as standard reference reagents from South Africa and Zambia. The African sequences did not cluster by country, but there was a distinctive South African sub-cluster. The CRF 02_AG sequences 271 and T33 are highly related. Values at nodes indicate the percentage of bootstraps in which the cluster to the right was found; only values of 78% or greater are shown. The M group ancestor from the Los Alamos database was used as an outgroup.
FIG. 2
FIG. 2
Alignment of deduced amino acid sequences for subtype C HIV-1 gp160 genes from India. Nucleotide sequences were translated, aligned, and compared with a consensus sequence generated by MASE. Numbering of amino acid residues begins with the first residue of gp120 and does not include the signal peptide. Dashes denote sequence identity, while dots represent gaps introduced to optimize alignments. Small letters in the consensus sequence indicate sites at which fewer than 50% of the viruses share the same amino acid residue. Triangles above the consensus sequence denote cysteine residues (solid triangles indicated sequence identity, while open triangles indicate sequence variation). V1, V2, V3, V4, and V5 regions designate hypervariable HIV-1 gp120 domains. The signal peptide and Env precursor cleavage sites are indicated; “msd” denotes the membrane-spanning domain in gp41; asterisks mark in-frame stop codons. Open circles highlight altered cysteine residues. Potential N-linked glycosylation sites (NXYX motif, where X is any amino acid other than proline and Y is either serine or threonine) are bolded and underlined. Potential N-linked glycosylation sites that are conserved in at least 9 of the 10 clones are shaded.
FIG. 2
FIG. 2
Alignment of deduced amino acid sequences for subtype C HIV-1 gp160 genes from India. Nucleotide sequences were translated, aligned, and compared with a consensus sequence generated by MASE. Numbering of amino acid residues begins with the first residue of gp120 and does not include the signal peptide. Dashes denote sequence identity, while dots represent gaps introduced to optimize alignments. Small letters in the consensus sequence indicate sites at which fewer than 50% of the viruses share the same amino acid residue. Triangles above the consensus sequence denote cysteine residues (solid triangles indicated sequence identity, while open triangles indicate sequence variation). V1, V2, V3, V4, and V5 regions designate hypervariable HIV-1 gp120 domains. The signal peptide and Env precursor cleavage sites are indicated; “msd” denotes the membrane-spanning domain in gp41; asterisks mark in-frame stop codons. Open circles highlight altered cysteine residues. Potential N-linked glycosylation sites (NXYX motif, where X is any amino acid other than proline and Y is either serine or threonine) are bolded and underlined. Potential N-linked glycosylation sites that are conserved in at least 9 of the 10 clones are shaded.
FIG. 3
FIG. 3
Neutralization-sensitivity of the Indian Envs as determined with plasma samples from chronically infected subjects in India and South Africa. Bar heights represent the geometric mean titer (GMT) of neutralizing Abs against the indicated Env-pseudotyped viruses. Black bars, plasma samples from 18 subjects in India. Gray bars, plasma samples from 6 subjects in South Africa. Paired sets were compared by two-tailed Mann Witney tests. Differences that were significant (p<0.05) are designated with an asterisk.
FIG. 4
FIG. 4
Heatmap 1. Hierarchical clustering analysis of neutralization results generated with HIV-1-positive plasma samples from South Africa and subtype B, C and D plasma pools. Plasma samples from 6 HIV-1 subtype C-infected blood bank donors in Johannesburg, South Africa, and plasma pools from individuals infected with either HIV-1 subtype B, C or D, were assayed against a multi-subtype panel of 57 Env-pseudotyped viruses, including viruses pseudotyped with each of the 10 Indian Env clones. The dendogram for pseudovirus clustering is displayed on the left of the Heatmap, while the dendogram for the plasma clustering is displayed on the top. Env clones and plasma names are displayed at the tips of the respective dendograms. The magnitude of neutralization (log IC50 values) are denoted by color, and the numbers that correspond to the colors in the key are the natural log of the IC50 value. A “Brewer” color palette (www.ColorBrewer.org) was used to map neutralization values to colors: lower values are represented by less saturated light colors (e.g. light yellows), while higher values of neutralization are represented by more saturated dark colors (e.g. dark reds). Bootstrap values (the percentage of time a cluster was found among 1000 random with replacement resamplings of the data) of the major Env clusters are shown proximal to branch points; only high bootstrap values associated with the major groupings are shown. Some of the pairs and triplets also had high boostrap values. The upper (U) cluster referred to in the text is marked; it is more neutralization-sensitive and dominated by African subtype C sequences. The lower (L) clusters is less sensitive to neutralization and can be broken down into L1, dominated by the CRF02 AG envelopes, and L2, dominated by subtype B Envs. The subtype C Envs from India were distributed throughout the Heatmap and are labeled on the right in green text.
FIG. 5
FIG. 5
Heatmap 2. Hierarchical clustering analysis of neutralization results generated with Indian HIV-1-positive plasma samples. Plasma samples from 18 chronic HIV-1 infected individuals in Pune, India were assayed against a multi-subtype panel of Env-pseudotyped viruses, including viruses pseudotyped with each of the 10 Indian Env clones. Description of the Heatmap is as described in the legend to Figure 4. Indian Envs are shown in green. The highly neutralization-sensitive tier 1 Envs (SF162.LS and MN) cluster at the bottom of the Heatmap.
FIG. 6
FIG. 6
Signature amino acid positions associated with neutralization clusters. Shown are all signature positions having q <0.20 as aligned with M group ancestor gp160 sequence from the Los Alamos database. Above each signature is its numerical position (HXB2 numbering). Colors indicate the group of the association in Heatmap 1: U, yellow; U1, dark orange; U2, light orange; U3, red; L, light blue; L1, medium blue; L2, dark blue. Upper case letters are used denote that an amino acid is associated with the group. Lower case letters are used to denote that the amino acid tends to be mutated away in the group. If a subcluster and cluster were both associated with the same amino acid pattern, we assumed they were related statistics and show only the most significant one. The complete list of sites and statistical details can be found in Supplemental Materials (Table S1).
FIG. 7
FIG. 7
Neutralization cluster-associated amino acid signatures mapped on gp120 and gp41. Color coding is the same as is in Figure 6. Top left: Signature positions mapped on a CD4-bound gp120 core structure with modeled loops; the three PNLG signature positions are shown separately for clarity. Top right: Signature positions shown on a cartoon model of gp41. Bottom left: Signature positions 281 and 121 in relationship to the receptor and 17B binding domains on gp120 crystal structure (53). Bottom right: Signature position 281 in relationship to the b12 binding domain on gp120 crystal structure [REF 107]. In the bottom figures, CD4 and antibody contact regions in gp120 are marked in light green and magneta, respectively. Contacts are defined based on crystal structures with a definition of heavy atom distance less than 7Å.
FIG. 8
FIG. 8
Length of gp120 and its variable regions in association with neutralization phenotype clusters U and L. The top p-value given is based on a direct comparison that does not correct for the phylogeny, and uses a non-parametric rank sum test to compare distributions of sequence lengths in the more neutralization-sensitive U cluster (grey bars) and the more neutralization-resistant L cluster (open bars). V3 is not shown because it does not often vary in length. The lower p-value in each panel is calculated using a GLM, a statistic that indicated that the loop length associations were highly predictive of the neutralization susceptibility profile and cluster, independent of the subtype.
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References

    1. Agnihotri KD, Tripathy SP, Jere AJ, Kale SM, Paranjape RS. Molecular analysis of gp41 sequences of HIV-1 type 1 subtype C from India. J Acquir Immune Defic Syndr. 2006;41:345–351. - PubMed
    1. Agnihorti K, Tripathy S, Jere A, Jadhav S, Kurle S, Paranjape R. gp120 sequences from HIV type 1 subtype C early seroconverters in India. AIDS Res Hum Retroviruses. 2004;20:889–894. - PubMed
    1. Barbato G, Bianchi E, Ingallinella P, Hurn WH, Miller MD, Ciliberto G, Cortese R, Bazzo R, Shiver JW, Pessi A. Structural analysis of the epitope of the anti-HIV antibody 2F5 sheds light into its mechanism of neutralization and HIV fusion. J Mol Biol. 2003;330:1101–1115. - PubMed
    1. Baskar PV, Ray SC, Rao R, Quinn TC, Hildreth JE, Bollinger RC. Presence in India of HIV type 1 similar to North American strains. AIDS Res Hum Retrovir. 1994;10:1039–1041. - PubMed
    1. Berlioz-Torrent C, Shacklett BL, Erdtmann L, Delamarre L, Bouchaert I, Sonigo P, Dokhelar MC, Benarous R. Interactions of the cytoplasmic domains of human and simian retroviral transmembrane proteins with components of the clathrin adaptor complexs modulate intracellular and cell surface expression of envelope glycoproteins. J Virol. 1999;73:1350–1361. - PMC - PubMed

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