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

RESEARCH ARTICLE Several N-Glycans on the HIV Envelope Glycoprotein gp120 Preferentially Locate Near Disulphide Bridges and Are Required for Efficient Infectivity and Virus Transmission Leen Mathys, Jan Balzarini* Rega Institute for Medical Research, KU Leuven, Leuven, Belgium * a11111 Abstract OPEN ACCESS Citation: Mathys L, Balzarini J (2015) Several NGlycans on the HIV Envelope Glycoprotein gp120 Preferentially Locate Near Disulphide Bridges and Are Required for Efficient Infectivity and Virus Transmission. PLoS ONE 10(6): e0130621. doi:10.1371/journal.pone.0130621 Editor: William A Paxton, Institute of Infection and Global Health, UNITED KINGDOM Received: March 6, 2015 Accepted: May 21, 2015 Published: June 29, 2015 Copyright: © 2015 Mathys, Balzarini. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by KU Leuven (PF 10/018, GOA 10/14) and the "Fonds voor Wetenschappelijk Onderzoek" (FWO) (G.0528.12N). LM is supported by a fellowship from L'OréalUNESCO, in collaboration with the Belgian FWO. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 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. PLOS ONE | DOI:10.1371/journal.pone.0130621 June 29, 2015 1 / 24 Co-Localization of N-Glycans and C-C Bridges in HIV-1 gp120 Competing Interests: The authors have declared that no competing interests exist. Introduction The envelope of the human immunodeficiency virus (HIV) carries two virus-encoded glycoproteins: the surface gp120 and the non-covalently associated transmembrane gp41. The former is highly glycosylated, carrying 18 to up to 32 N-linked glycans (24 on average) which occur clustered on the protein and constitute about 50% of the molecular weight of gp120 [1– 3]. Although still subject of discussion, it is nowadays assumed that at least 56–73% of these Nlinked glycans on gp120 are high-mannose-type glycans [4], which is unusual as compared to cellular glycoproteins containing mostly, if not exclusively, complex-type glycans [5]. In addition to N-glycans, gp120 is also characterized by 18 cysteines which form 9 disulphide bridges. It has been shown that 5 disulphide bridges are crucial for the correct folding of gp120 and six or seven of the disulphide bridges in gp120 are found to be indispensable for appropriate envelope function [6]. During glycoprotein biosynthesis, N-glycosylation and disulphide bridge formation in the endoplasmic reticulum (ER) are considered to have a reciprocal influence on each other. Nglycans may sterically hinder the formation of disulphide bridges [7] and disulphide bridges in turn may prevent the use of a nearby N-glycosylation site [8]. Normally, the addition of Nlinked glycans to the asparagine of an N-glycosylation motif (Asn-X-Thr/Ser, in which X is not proline) by the ER oligosacharyl transferase in the nascent protein backbone initiates the recognition of glycoproteins by the ER resident lectins calreticulin and calnexin. These lectins act as chaperones and enhance folding of glycoproteins by the recruitment and activation of the disulphide isomerase ERp57, which is responsible for the formation of disulphide bridges in glycoproteins (reviewed in [9]). Together, calnexin, calreticulin and ERp57 are responsible for the correct folding of the HIV precursor envelope glycoprotein gp160 in the ER, after which it is cleaved to gp120 and gp41 in the Golgi apparatus (reviewed in [10]). By inspecting the structure of HIV-1NL4.3 gp120, we noticed that more than half of the disulphide bridges are immediately surrounded by N-linked glycans (Fig 1). These observations may suggest that the presence of N-glycans immediately near, or close to the disulphide bridges in gp120 is not a coincidence and that there might exist a specific functional interference between the location of disulphide bridges and these N-linked glycosylation sites in HIV gp120. Given the important role of disulphide bridges and N-glycans during glycoprotein biosynthesis, we wondered whether neighboring disulphide bridges and N-glycans could have a central, indispensable role in gp120 biosynthesis and functionality. To address this issue, the distribution and appearance of neighboring disulphide bridges and N-glycans in gp120 was first investigated using an alignment of a wide variety of different HIV-1 subtypes. Next, a broad variety of mutant HIV-1NL4.3 strains lacking disulphide bridges and/or the neighboring N-glycans in gp120 was generated. In addition, new N-glycosylation sites were introduced in HIV-1NL4.3 gp120 at positions that were found to be disfavored for glycosylation in wild-type (WT) gp120. We examined the influence of these (...truncated)