A New Approach to Produce HIV-1 Envelope Trimers: BOTH CLEAVAGE AND PROPER GLYCOSYLATION ARE ESSENTIAL TO GENERATE AUTHENTIC TRIMERS.
Bottom Line: However, this antibody-based approach may not be as effective for the diverse HIV-1 strains with different epitope signatures.Uncleaved trimers entered aberrant pathways, resulting in hyperglycosylation, nonspecific cross-linking, and conformational heterogeneity.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.
Affiliation: From the Department of Biology, The Catholic University of America, Washington, D. C. 20064.Show MeSH
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Mentions: Codon-optimized gp140 DNAs from JRFL-FD, SF162-FD, and CONPEP-FD were provided by Dr. Peter Kwong (Vaccine Research Center, National Institutes of Health). These DNAs contained the sequence corresponding to gp120 and gp41 ectodomain up to aa 683. In addition, they have the human CD5 secretion signal at the 5′-end, furin cleavage-resistant mutation SEKS at the junction of gp120 and gp41, and FD followed by the hexahistidine tag at the C terminus (49). Using the JRFL-FD as the starting template, a series of additional mutations were introduced. These include, for instance, SOSIP mutations (50, 51), stabilizing mutations (52), enhanced furin cleavage site RRRRRR (53), and various truncations shown in Fig. 2 and described under “Results.” The JRFL gp120 clone was also constructed from the same template by PCR amplification of the appropriate sequence corresponding to gp120.
Affiliation: From the Department of Biology, The Catholic University of America, Washington, D. C. 20064.