Two N-Linked Glycosylation Sites in the V2 and C2 Regions of Human Immunodeficiency Virus Type 1 CRF01_AE Envelope Glycoprotein gp120 Regulate Viral Neutralization Susceptibility to the Human Monoclonal Antibody Specific for the CD4 Binding Domain▿

A recombinant human monoclonal antibody, IgG1 b12 (b12), recognizes a conformational epitope on human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) gp120 that overlaps the CD4 binding domain. Although b12 is able to broadly neutralize HIV-1 subtype B, C, and D viruses, many HIV-1 CRF01_AE viruses are resistant to b12-mediated neutralization. In this report, we examined the molecular mechanisms underlying the low neutralization susceptibility of CRF01_AE viruses to b12, using recently established CRF01_AE Env recombinant viruses. Our results showed that two potential N-linked glycosylation (PNLG) sites in the V2 and C2 regions of Env gp120 played an important role in regulating the susceptibility of CRF01_AE Env to b12. The locations of these PNLG sites correspond to amino acid positions 186 and 197 in HXB2 Env gp120; thus, they are designated N186 and N197 in this study. Removal of N186 significantly conferred the b12 susceptibility of 2 resistant CRF01_AE Env clones, 65CC4 and 107CC2, while the introduction of N186 reduced the b12 susceptibility of a susceptible CRF01_AE Env clone, 65CC1. In addition, removal of both N186 and N197 conferred the b12 susceptibility of 3 resistant CRF01_AE Env clones, 45PB1, 62PL1, and 101PL1, whereas the removal of either N186 or N197 was not sufficient to confer the b12 susceptibility of these CRF01_AE Env clones. Finally, removal of N197 conferred the b12 susceptibility of 2 resistant CRF01_AE Env clones lacking N186, 55PL1 and 102CC2. Taken together, we propose that two PNLG sites, N186 and N197, in Env gp120 are important determinants of the b12 resistance of CRF01_AE viruses

Anti-HIV-1 neutralizing activity of plasma derived from 33 rapid progressors against AE-Env-recombinant viruses.

<p>Neutralizing activity of plasma samples against 8 AE-Env-recombinant viruses was evaluated and reciprocal plasma dilution at which viral replication was suppressed by 50% (50% inhibitory dilution, ID50) was calculated, as described in Methods. Data are presented as the means of at least three independent experiments. Plasma IDs and AE-Env-recombinant viruses tested are denoted on the left side and above the panel, respectively. In addition, mean ID50 values and the percentages of virus/plasma combinations (% neutralization) in which viral neutralization was observed among the data sets in horizontal and vertical directions are shown on the right side and bottom of the panel, respectively. ID50 values >500, values 100–500, and values 20–100 are highlighted in red, orange and yellow, respectively. In addition, no neutralization (ID50 values <20) of a recombinant virus is denoted by a gray background.</p

Anti-HIV-1 neutralizing activity of 6 selected plasma samples against B-Env- and C-Env-recombinant viruses.

<p>Neutralizing activity of 6 plasma samples against 5 B-Env- and 6 C-Env-recombinant viruses was evaluated as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053920#pone-0053920-g001" target="_blank">Figure 1</a>. Data are presented as the means of at least three independent experiments. Plasma IDs and Env-recombinant viruses tested are denoted on the left side and above the panel, respectively. ID50 values 100–500 and 20–100 are highlighted in orange and yellow, respectively. No neutralization (ID50 values <20) of a recombinant virus is denoted by a gray background.</p

Design and evaluation of antiretroviral peptides corresponding to the C-terminal heptad repeat region (C-HR) of human immunodeficiency virus type 1 envelope glycoprotein gp41

AbstractTwo α-helical heptad repeats, N-HR and C-HR, located in the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp41, play an important role in membrane fusion by forming a 6-helix bundle. C34, a peptide mimicking C-HR, inhibits the formation of the 6-helix bundle; thus, it has potential as a novel antiretroviral compound. In order to improve the inhibitory effect of C34 on HIV-1 replication, we designed new C34-derived peptides based on computational analysis of the stable conformation of the 6-helix bundle. Newly designed peptides showed a stronger inhibitory effect on the replication of recombinant viruses containing CRF01_AE, subtype B or subtype C Env than C34 or a fusion inhibitor, T-20. In addition, these peptides inhibited the replication of a T-20-resistant virus. We propose that these peptides could be applied to develop novel antiretroviral compounds to inhibit the replication of various subtypes of HIV-1 as well as of T-20-resistant variants

Anti-HIV-1 neutralizing activity of plasma derived from 34 slow progressors against AE-Env-recombinant viruses.

<p>Neutralizing activity of plasma samples against 8 AE-Env-recombinant viruses was evaluated as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053920#pone-0053920-g001" target="_blank">Figure 1</a>. Data are presented as the means of at least three independent experiments. Plasma IDs and AE-Env-recombinant viruses tested are denoted on the left side and above the panel, respectively. In addition, mean ID50 values and the percentages of virus/plasma combinations (% neutralization) in which viral neutralization was observed among the data sets in horizontal and vertical directions are shown on the right side and bottom of the panel, respectively. ID50 values >500, values 100–500 and values 20–100 are highlighted in red, orange and yellow, respectively. No neutralization (ID50 values <20) of a recombinant virus is denoted by a gray background. Plasma samples that neutralized all recombinant viruses tested are highlighted in green.</p

Comparison of the neutralization breadth between plasma derived from rapid and slow progressors.

<p>The proportion of AE-Env-recombinant viruses in which replication was inhibited by a plasma sample was calculated and plotted. The levels of plasma-mediated neutralization against 60% and 80% of recombinant viruses tested are highlighted by horizontal blue and red grid lines, respectively. Plasma IDs are denoted below the panels.</p