Diverse pathways of escape from all well-characterized VRC01-class broadly neutralizing HIV-1 antibodies
Fig 1
Construction of a HIV-1 proviral library with a soft-randomized Env.
(A) A representation of the HIV-1 env gene. Regions containing CD4 binding sites (colored boxes)—Loop D, CD4 binding loop, bridging sheet and V5—are expanded with NIH45-46 epitopes indicated by filled red ovals. Residues of Loop D and V5 regions of the HIV-1 isolate ADA, soft randomized in this study, are shown below. (B) Sequences of primers used to soft-randomize the Loop D and V5 regions of ADA Env are shown. Amino-acid numbers are based on HXB2 numbering. Soft randomizing primers were synthesized by hand mixing 88% of the original nucleotide and 4% each of the other three nucleotides (88:4:4:4) for the first two codon positions (nucleotides in lower case) of Loop D residues, and 91:3:3:3 ratio for the first two codon positions of V5 residues. For wobble positions of Loop D residues, an equimolar mix (50:50) of G and T (indicated as K) was used for 4- and 6-codon amino acids. For V5 primer, because it is anti-sense, C and A (indicated as M) were used. Nucleotides subject to soft randomization are colored in red. Both the 5’ and 3′ of the primers outside the randomized regions were extended (shown in green) to have a melting temperature matching the pairing primers (shown in blue).