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.
Highlights
The envelope of the human immunodeficiency virus (HIV) carries two virus-encoded glycoproteins: the surface gp120 and the non-covalently associated transmembrane gp41
Several highly conserved N-linked glycans of gp120 are preferentially localized near disulphide bridges
We demonstrated that conserved N-linked glycans appear preferentially near, or in close proximity to, disulphide bridges in HIV-1 gp120, either at asparagines directly neighboring the involved cysteines or at asparagines located at the C-terminal side of the cysteine
Summary
The envelope of the human immunodeficiency virus (HIV) carries two virus-encoded glycoproteins: the surface gp120 and the non-covalently associated transmembrane gp. 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]). 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 gp in the Golgi apparatus (reviewed in [10])
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