Several <i>N</i>-Glycans on the HIV Envelope Glycoprotein gp120 Preferentially Locate Near Disulphide Bridges and Are Required for Efficient Infectivity and Virus Transmission

<div><p>The HIV envelope glycoprotein gp120 contains nine disulphide bridges and is highly glycosylated, carrying on average 24 <i>N</i>-linked glycans. Using a probability calculation, we here demonstrate that there is a co-localization of disulphide bridges and <i>N</i>-linked glycans in HIV-1 gp120, with a predominance of <i>N</i>-linked glycans in close proximity to disulphide bridges, at the C-terminal side of the involved cysteines. Also, <i>N</i>-glycans are frequently found immediately adjacent to disulphide bridges in gp120 at the N-terminal side of the involved cysteines. In contrast, <i>N</i>-glycans at positions close to, but not immediately neighboring disulphide bridges seem to be disfavored at the N-terminal side of the involved cysteines. Such a pronounced co-localization of disulphide bridges and <i>N</i>-glycans was also found for the <i>N</i>-glycans on glycoprotein E1 of the hepatitis C virus (HCV) but not for other heavily glycosylated proteins such as E2 from HCV and the surface GP from Ebola virus. The potential functional role of the presence of <i>N</i>-glycans near disulphide bridges in HIV-1 gp120 was studied using site-directed mutagenesis, either by deleting conserved <i>N</i>-glycans or by inserting new <i>N</i>-glycosylation sites near disulphide bridges. The generated HIV-1_NL4.3 mutants were subjected to an array of assays, determining the envelope glycoprotein levels in mutant viral particles, their infectivity and the capture and transmission efficiencies of mutant virus particles by DC-SIGN. Three <i>N</i>-glycans located nearby disulphide bridges were found to be crucial for the preservation of several of these functions of gp120. In addition, introduction of new <i>N</i>-glycans upstream of several disulphide bridges, at locations where there was a significant absence of <i>N</i>-glycans in a broad variety of virus strains, was found to result in a complete loss of viral infectivity. It was shown that the <i>N</i>-glycan environment around well-defined disulphide bridges of gp120 is highly critical to allow efficient viral infection and transmission.</p></div

Capture efficiency of mutant gp120 virus strains, relative to capture efficiency of wild-type virus.

<p>Data are the means ± SEM of at least 2 independent experiments.</p><p>The difference between WT and mutant virus was considered to be significant when the <i>p</i> value calculated using the student’s t-test was <0.05 (* = p<0.05).</p><p>Capture efficiency of mutant gp120 virus strains, relative to capture efficiency of wild-type virus.</p

Schematic structure of HIV-1_NL4.3 gp120 with indication of the <i>N</i>-glycans and disulphide bridges that were deleted/inserted using site-directed mutagenesis.

<p>Disulphide bridges that were found in close proximity to one or two <i>N</i>-glycosylation sites, either directly neighboring or separated from each other by one other amino acid, are indicated with a red circle. Disulphide bridges that are not directly neighbored by <i>N</i>-glycans are indicated by a green circle. Mutations resulting in deleted disulphide bridges (Cys→Ala) or <i>N</i>-glycosylation sites (Asn→Gln) are indicated in black. Mutations resulting in the generation of novel <i>N</i>-glycosylation sites, due to the introduction of an Asn-X-Ser motif, are indicated in blue (only the glycosylated Asn is shown). The N-terminal signal sequence is colored in white, the conserved domaines (C1–C5) in light grey and the variable loops (V1–V5) in dark grey. Cysteines that are involved in a disulphide bridge are colored black. Figure based on Leonard <i>et al</i>. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130621#pone.0130621.ref003" target="_blank">3</a>]. Amino acid numbering according to HIV-1 strain HXB2.</p

Infectivity of mutant HIV-1_NL4.3 strains with <i>N</i>-glycosylation sites introduced in gp120 close to, and upstream of, the disulphide-involved C296 (A) and C385 (B).

<p>At day 0, CD4⁺ T lymphocyte C8166 cells were infected with similar viral loads of WT or mutant virus strains, based on equal amounts of the p24 capsid protein. For 7 consecutive days, samples were harvested, fixed and subjected to flow cytometry to analyse eGFP expression. The infectivity was quantified using a linear regression to part of the obtained eGFP-expression curves, from day 1 post infection to the peak of the curve. Asparagines involved in <i>N</i>-glycosylation sites (N-X-S/T) are coloured red. C296 and C385 are indicated in green. One introduced Asn (V290N) that was not part of an <i>N</i>-glycosylation site is indicated in bold <b>(A)</b>. Data are the mean ± SEM of 2–3 independent experiments. The difference between WT and mutant virus was considered to be significant when the <i>p</i> value calculated using the student’s t-test was <0.05 (* = p<0.05).</p

Western blot analysis of envelope glycoprotein incorporation in WT and mutant virus particles.

<p>Virus was concentrated, lysed and subjected to western blotting. Gp120, gp41 and p24 were detected in virus lysates of (A) mutants lacking a disulphide bridge and (B) <i>N</i>-glycosylation site mutants. (C-D) Quantification of protein levels was performed using the BioRad Image Lab Software, based on panels A and B. Gp160, gp120 and gp41 levels are presented after normalization to the p24 levels. Graphs represent the mean ± SEM based on 2–4 independent experiments. The difference between WT and mutant virus was considered to be significant when the <i>p</i> value calculated using the student’s t-test was <0.05 (* = p<0.05, for both gp120+gp160 and gp41).</p

HIV-1 gp120 amino acid alignment with indication of <i>N</i>-glycosylation sites that were mutated in this study.

<p>An alignment of consensus sequences (2004) of the amino acid sequence of gp120 was obtained through the HIV sequence database [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130621#pone.0130621.ref011" target="_blank">11</a>] and was analyzed with the N-glycosite software [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130621#pone.0130621.ref011" target="_blank">11</a>] to locate <i>N</i>-glycosylation sites. For our analysis, we did not include the consensus of consensus sequences nor the ancestral sequences, but focused on the consensus sequences for HIV-1 subtypes A1, A2, B, C, D, F1, F2, G, H, CRF01-AE, CRF02-AG, CRF03-AB, CRF04-cpx, CRF06-cpx, CRF08-BC, CRF10-CD, CRF11-cpx, CRF12-BF, and CRF14-BG. The height of the bars indicates the level of conservation among these HIV-1 subtypes. The 7 black bars represent <i>N</i>-glycosylation sites that were found in HIV-1_NL4.3 gp120 to be located near a disulphide bridge (directly neighbouring one of the disulphide cysteines or separated by only one amino acid) and were mutated by site-directed mutagenesis.</p

Probability of finding an <i>N</i>-linked glycosylation site at a position 1–5 amino acids from a disulphide bridge in gp120.

<p>The positions and levels of conservation of <i>N</i>-linked glycosylation sites in HIV-1 gp120 were determined using the N-glycosite software available on the HIV sequence database website [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130621#pone.0130621.ref011" target="_blank">11</a>], based on an alignment of consensus sequences of 19 HIV-1 subtypes (2004) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130621#pone.0130621.ref011" target="_blank">11</a>]. The probability of finding <i>N</i>-glycosylation sites closeby cysteines involved in disulphide bridges was calculated. The graph shows the relative probabilities of a glycosylated asparagine at 1, 2, 3, 4 or 5 amino acid positions away from the cysteines involved in disulphide bridges. Negative amino acid positions correspond to positions at the N-terminal site of the cysteine, positive amino acid positions correspond to positions at the C-terminal site of the cysteine. The cysteine itself is shown as a black bar. The striped line indicates the probablities in case of random distribution of <i>N</i>-glycosylation sites. (A) <i>N</i>-glycosylation sites with at least 50% conservation. (B) <i>N</i>-glycosylation sites with less than 50% conservation.</p

Infectivity of WT and mutant gp120 HIV-1 strains.

<p>At day 0, CD4⁺ T lymphocyte C8166 cells were infected with similar viral loads of WT or mutant virus strains, based on equal amounts of the p24 capsid protein. For 7 consecutive days, samples were harvested, fixed and subjected to flow cytometry to analyse eGFP expression. (A) Infectivity of mutant viruses lacking disulphide bridges in gp120, due to the mutation of one of the involved cysteines into an alanine. (B) Infectivity of mutant viruses lacking one <i>N</i>-glycosylation site in gp120 due to the mutation of the asparagine of the glycosylation motif into a glutamine. (C) The infectivity as shown in panel B was quantified using a linear regression to part of the curves, from day 1 post infection to the peak of the infectivity curve. Data are the means ± SEM of at least 2 independent experiments. The difference between WT and mutant virus was considered to be significant when the <i>p</i> value calculated using the student’s t-test was <0.05 (* = p<0.05).</p

Transmission efficiency of wild-type and mutant virus strains.

<p>Virus-exposed Raji/DC-SIGN cells were cocultivated with C8166 CD4⁺ T cells for 72 h. Production of virus particles by the C8166 cells (following transmission of HIV from the virus-captured Raji/DC-SIGN cells) was quantified using a p24 ELISA and was used as a measurement for transmission efficiency. Data were normalized to capture efficiency and presented as relative to transmission of WT virus. Data are the means ± SEM of at least 2 independent experiments. The difference between WT and mutant virus was considered to be significant when the <i>p</i> value calculated using the student’s t-test was <0.05 (* = p<0.05).</p

Correlation between the infectivity and transmission efficiency of the gp120 <i>N</i>-glycan deleted mutant virus strains, relative to WT virus.

<p>The black dashed line represents a curve where infectivity and transmission would have been equally influenced by the mutations. White quadrant: both transmission and infectivity are decreased. Full grey quadrant: both transmission and infectivity are increased. Quadrant with horizontal striping: transmission efficiency is increased while infectivity is decreased. Quadrant with vertical striping: infectivity is increased while transmission efficiency is decreased.</p