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

Mathys L, Balzarini J - PLoS ONE (2015)

Bottom Line: Such a pronounced co-localization of disulphide bridges and N-glycans was also found for the N-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 generated HIV-1NL4.3 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.In addition, introduction of new N-glycans upstream of several disulphide bridges, at locations where there was a significant absence of N-glycans in a broad variety of virus strains, was found to result in a complete loss of viral infectivity.

View Article: PubMed Central - PubMed

Affiliation: Rega Institute for Medical Research, KU Leuven, Leuven, Belgium.

ABSTRACT
The HIV envelope glycoprotein gp120 contains nine disulphide bridges and is highly glycosylated, carrying on average 24 N-linked glycans. Using a probability calculation, we here demonstrate that there is a co-localization of disulphide bridges and N-linked glycans in HIV-1 gp120, with a predominance of N-linked glycans in close proximity to disulphide bridges, at the C-terminal side of the involved cysteines. Also, N-glycans are frequently found immediately adjacent to disulphide bridges in gp120 at the N-terminal side of the involved cysteines. In contrast, N-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 N-glycans was also found for the N-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 N-glycans near disulphide bridges in HIV-1 gp120 was studied using site-directed mutagenesis, either by deleting conserved N-glycans or by inserting new N-glycosylation sites near disulphide bridges. The generated HIV-1NL4.3 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 N-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 N-glycans upstream of several disulphide bridges, at locations where there was a significant absence of N-glycans in a broad variety of virus strains, was found to result in a complete loss of viral infectivity. It was shown that the N-glycan environment around well-defined disulphide bridges of gp120 is highly critical to allow efficient viral infection and transmission.

No MeSH data available.


Related in: MedlinePlus

HIV-1 gp120 amino acid alignment with indication of N-glycosylation sites that were mutated in this study.An alignment of consensus sequences (2004) of the amino acid sequence of gp120 was obtained through the HIV sequence database [11] and was analyzed with the N-glycosite software [11] to locate N-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 N-glycosylation sites that were found in HIV-1NL4.3 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.
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pone.0130621.g003: HIV-1 gp120 amino acid alignment with indication of N-glycosylation sites that were mutated in this study.An alignment of consensus sequences (2004) of the amino acid sequence of gp120 was obtained through the HIV sequence database [11] and was analyzed with the N-glycosite software [11] to locate N-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 N-glycosylation sites that were found in HIV-1NL4.3 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.

Mentions: Fig 3 represents the alignment of HIV-1 gp120, obtained from the HIV sequence database [11], with indication of the N-glycans that were deleted using site-directed mutagenesis (vertical black lines with amino acid numbering). It was shown that 6 out of the 7 deleted N-glycans were at least 75% conserved [4 were fully (100%) conserved (i.e. N156, N197, N241 and N386), one was 88% conserved (i.e. N332) and the other N-glycosylation site was 75% conserved (i.e. N295)]. In contrast, the glycan on N230 which is present in HIV-1NL4.3 and included in our study, was only 17% conserved among all strains in the HIV sequence database [11].


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

Mathys L, Balzarini J - PLoS ONE (2015)

HIV-1 gp120 amino acid alignment with indication of N-glycosylation sites that were mutated in this study.An alignment of consensus sequences (2004) of the amino acid sequence of gp120 was obtained through the HIV sequence database [11] and was analyzed with the N-glycosite software [11] to locate N-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 N-glycosylation sites that were found in HIV-1NL4.3 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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4488071&req=5

pone.0130621.g003: HIV-1 gp120 amino acid alignment with indication of N-glycosylation sites that were mutated in this study.An alignment of consensus sequences (2004) of the amino acid sequence of gp120 was obtained through the HIV sequence database [11] and was analyzed with the N-glycosite software [11] to locate N-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 N-glycosylation sites that were found in HIV-1NL4.3 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.
Mentions: Fig 3 represents the alignment of HIV-1 gp120, obtained from the HIV sequence database [11], with indication of the N-glycans that were deleted using site-directed mutagenesis (vertical black lines with amino acid numbering). It was shown that 6 out of the 7 deleted N-glycans were at least 75% conserved [4 were fully (100%) conserved (i.e. N156, N197, N241 and N386), one was 88% conserved (i.e. N332) and the other N-glycosylation site was 75% conserved (i.e. N295)]. In contrast, the glycan on N230 which is present in HIV-1NL4.3 and included in our study, was only 17% conserved among all strains in the HIV sequence database [11].

Bottom Line: Such a pronounced co-localization of disulphide bridges and N-glycans was also found for the N-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 generated HIV-1NL4.3 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.In addition, introduction of new N-glycans upstream of several disulphide bridges, at locations where there was a significant absence of N-glycans in a broad variety of virus strains, was found to result in a complete loss of viral infectivity.

View Article: PubMed Central - PubMed

Affiliation: Rega Institute for Medical Research, KU Leuven, Leuven, Belgium.

ABSTRACT
The HIV envelope glycoprotein gp120 contains nine disulphide bridges and is highly glycosylated, carrying on average 24 N-linked glycans. Using a probability calculation, we here demonstrate that there is a co-localization of disulphide bridges and N-linked glycans in HIV-1 gp120, with a predominance of N-linked glycans in close proximity to disulphide bridges, at the C-terminal side of the involved cysteines. Also, N-glycans are frequently found immediately adjacent to disulphide bridges in gp120 at the N-terminal side of the involved cysteines. In contrast, N-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 N-glycans was also found for the N-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 N-glycans near disulphide bridges in HIV-1 gp120 was studied using site-directed mutagenesis, either by deleting conserved N-glycans or by inserting new N-glycosylation sites near disulphide bridges. The generated HIV-1NL4.3 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 N-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 N-glycans upstream of several disulphide bridges, at locations where there was a significant absence of N-glycans in a broad variety of virus strains, was found to result in a complete loss of viral infectivity. It was shown that the N-glycan environment around well-defined disulphide bridges of gp120 is highly critical to allow efficient viral infection and transmission.

No MeSH data available.


Related in: MedlinePlus