A New Approach to Produce HIV-1 Envelope Trimers: BOTH CLEAVAGE AND PROPER GLYCOSYLATION ARE ESSENTIAL TO GENERATE AUTHENTIC TRIMERS.
The trimeric envelope spike of HIV-1 mediates virus entry into human cells. The exposed part of the trimer, gp140, consists of two noncovalently associated subunits, gp120 and gp41 ectodomain. A recombinant vaccine that mimics the native trimer might elicit entry-blocking antibodies and prevent virus infection. However, preparation of authentic HIV-1 trimers has been challenging. Recently, an affinity column containing the broadly neutralizing antibody 2G12 has been used to capture recombinant gp140 and prepare trimers from clade A BG505 that naturally produces stable trimers. However, this antibody-based approach may not be as effective for the diverse HIV-1 strains with different epitope signatures. Here, we report a new and simple approach to produce HIV-1 envelope trimers. The C terminus of gp140 was attached to Strep-tag II with a long linker separating the tag from the massive trimer base and glycan shield. This allowed capture of nearly homogeneous gp140 directly from the culture medium. Cleaved, uncleaved, and fully or partially glycosylated trimers from different clade viruses were produced. Extensive biochemical characterizations showed that cleavage of gp140 was not essential for trimerization, but it triggered a conformational change that channels trimers into correct glycosylation pathways, generating compact three-blade propeller-shaped trimers. Uncleaved trimers entered aberrant pathways, resulting in hyperglycosylation, nonspecific cross-linking, and conformational heterogeneity. Even the cleaved trimers showed microheterogeneity in gp41 glycosylation. These studies established a broadly applicable HIV-1 trimer production system as well as generating new insights into their assembly and maturation that collectively bear on the HIV-1 vaccine design.
['AlSalmi|Wadad|W|', 'Mahalingam|Marthandan|M|', 'Ananthaswamy|Neeti|N|', 'Hamlin|Christopher|C|', 'Flores|Dalia|D|', 'Gao|Guofen|G|', 'Rao|Venigalla B|VB|']
D000595:Amino Acid Sequence D000906:Antibodies D000956:Antigens, Viral D004797:Enzyme-Linked Immunosorbent Assay D004926:Escherichia coli D015870:Gene Expression D006031:Glycosylation D015699:HIV Envelope Protein gp120 D015700:HIV Envelope Protein gp41 D015497:HIV-1 D008969:Molecular Sequence Data D009842:Oligopeptides D055503:Protein Multimerization D017433:Protein Structure, Secondary D017434:Protein Structure, Tertiary D059748:Proteolysis D011993:Recombinant Fusion Proteins D054299:env Gene Products, Human Immunodeficiency Virus
glycosylation vaccine AIDS envelope protein gp140 trimer human immunodeficiency virus (HIV) recombinant protein expression