Journal of virology 2017 02 15() pii 10.1128/JVI.02428-16
HIV-1 envelope glycoprotein (Env) glycosylation is important because individual glycans are components of multiple broadly neutralizing antibody epitopes, while shielding other sites that might otherwise be immunogenic. The glycosylation on Env is influenced by a variety of factors, including the genotype of the protein, the cell line used for its expression, and the details of the construct design. Here, we used a mass spectrometry-based approach to map the complete glycosylation profile at every site in multiple HIV-1 Env trimers, accomplishing two goals: 1) We determined which glycosylation sites contain conserved glycan profiles across many trimeric Envs. 2) We identified the variables that impact Env’s glycosylation profile at sites with divergent glycosylation. Over half of the gp120 glycosylation sites on eleven different trimeric Env proteins have a conserved glycan profile, indicating that a native consensus glycosylation profile does indeed exist among trimers. We showed that some soluble gp120s and gp140s exhibit highly divergent glycosylation profiles compared to trimeric Env. We also assessed the impact of several variables on Env glycosylation: truncating the full-length Env; producing Env in CHO cells, instead of more virologically relevant T lymphocytes; and purifying Env with different chromatographic platforms: Ni-NTA, 2G12, or PGT151 affinity. This study provides the first consensus glycosylation profile of Env trimers, which should serve as a useful benchmark for HIV-1 vaccine developers. This study also defines the sites where glycosylation may be impacted when Env trimers are truncated or produced in CHO cells.IMPORTANCE: A protective HIV-1 vaccine will likely include a recombinant version of the viral envelope glycoprotein (Env). Env is highly glycosylated, yet vaccine developers have lacked guidance on how to assess whether their immunogens have optimal glycosylation. Important questions are still unanswered: 1) What is the "target" glycosylation profile, when the goal is to generate a natively glycosylated protein? 2) What variables exert the greatest influence on Env glycosylation? We identified numerous sites on Env where the glycosylation profile does not deviate in eleven different Env trimers, and we investigated the impact on the divergent glycosylation profiles of changing the genotype of the Env sequence, the construct design, the purification method, and the producer cell type. The data herein give vaccine developers a "glycosylation target" for their immunogens, and they show how protein production variables can impact Env glycosylation.