Determination of essential amino acids involved in the CD4-independent tropism of the X4 human immunodeficiency virus type 1 m7NDK isolate: role of potential N glycosylations in the C2 and V3 regions of gp120

Abstract

Seven mutations in the C2, V3, and C3 regions of gp120 are implicated in the tropism of the first CD4-independent human immunodeficiency virus type 1 isolate, m7NDK. Site-directed mutagenesis revealed that three amino acids are essential to maintain this tropism, one in the C2 region and two in the V3 loop. Two mutations implied N glycosylation modifications.

FIG. 1

Comparison of amino acids of the C2, V3, and C3 regions of the wtNDK and m7NDK HIV-1 isolates. Amino acid differences are in boldface, and dashes indicate identical amino acids. Positions of the reverted mutations and the different substitutions and their positions are indicated and numbered according to the HIV-1 wtNDK Env sequence.

FIG. 2

Fusion phenotype analysis of mutated gp120s. Mutated env expressors were transiently transfected in HEK293 cells. Fusion analysis was performed after a coculture of the transfected cells with HeLa_LTRLacZ cells expressing (P42) or not expressing (Z24) the CD4 protein. Analysis was performed using a quantitative CPRG test (10). The CD4 independence index was stated as the ratio of A₅₇₀ of Z24 cells/A₅₇₀ of P42 cells relative to the 100% value corresponding to the m7NDK gp120 CD4-independent fusion efficiency. Experiments were performed in quadruplicate in 96-well plates. The data are representative of three experiments. (A) Each of the seven mutations was reverted one by one in the C2, V3, and C3 regions. These regions were reintroduced in the expression vector. (B) Substitutions of the three essential mutations. Groups 1 to 3 represent revertants G307R, N192D, and I298T and their related mutants, respectively.

FIG. 3

Alteration of migration patterns of mutated gp120 after modification of potential N glycosylation sites. Expression plasmids were transfected in HEK293 cells. Cell extracts were prepared for a Western blot experiment and were analyzed using a monoclonal anti-gp120 antibody (1). C2, V3, and C3 regions from m7NDK virus were inserted in the wtNDK isolate env gene (one N glycosylation site) (2), N192D gp120 mutant (no N glycosylation site) (3), and I298T gp120 mutant (two N glycosylation sites). M, molecular mass marker (in kilodaltons) (BenchMark Prestained Protein Ladder; Life Technologies). Arrow, gp120.