The HIV Env variant N283 enhances macrophage tropism and is associated with brain infection and dementia

Abstract

HIV infects tissue macrophages and brain microglia, which express lower levels of CD4 and CCR5 than CD4+ T cells in peripheral blood. Mechanisms that enhance HIV tropism for macrophages in the CNS and other tissues are not well understood. Here, we identify an HIV envelope glycoprotein (Env) variant in the CD4-binding site of gp120, Asn 283 (N283), that is present at a high frequency in brain tissues from AIDS patients with HIV-associated dementia (HAD). N283 increases gp120 affinity for CD4 by decreasing the gp120-CD4 dissociation rate, enhancing the capacity of HIV Envs to use low levels of CD4 for virus entry and increasing viral replication in macrophages and microglia. Structural modeling suggests that the enhanced ability of Envs with N283 to use low levels of CD4 is due to a hydrogen bond formed with Gln 40 of CD4. N283 is significantly more frequent in brain-derived Envs from HAD patients (41%; n=330) compared with non-HAD patients (8%; n=151; P<0.001). These findings suggest that the macrophage-tropic HIV Env variant N283 is associated with brain infection and dementia in vivo, representing an example of a HIV variant associated with a specific AIDS-related complication.

Fig. 1.

N283 is associated with brain-derived Envs and low CD4 dependence. (A) 293T cells transfected with Env plasmids were analyzed by Western blotting with anti-gp120. (B) 293T cells expressing Env were analyzed in cell-cell fusion assays. (C) Cf2-Luc cells expressing different levels of CD4 and high CCR5 were infected with chimeric viruses expressing each Env, lysed 48 h postinfection, and analyzed for luciferase activity. Data in B and C were normalized to luciferase activity on cells expressing high CD4. Results shown are from duplicate samples and are representative of three Env clones from each viral isolate. Error bars represent standard deviations. (D) Amino acid sequences from the C2 region of gp120 were aligned by using Clustal X. Position 283 (numbered relative to the HXB2 reference sequence) is highlighted in gray. Env residues contacting CD4 in the HXB2 gp120 crystal structure (1G9M) (29) are indicated.

Fig. 2.

N283 enhances the capacity of Envs to use low levels of CD4. (A) 293T cells were transfected with Env plasmids and analyzed by Western blotting with anti-gp120 (Left) or incubated with HIV+ patient sera and analyzed for Env cell surface expression by flow cytometry (Right). (B) 293T cells expressing Env were analyzed in cell-cell fusion assays. (C) Cf2-Luc cells expressing different levels of CD4 and high CCR5 were infected with recombinant viruses expressing each Env, lysed 48 h postinfection, and analyzed for luciferase activity. Data in B and C were normalized to luciferase activity of the wild-type Env on cells expressing high CD4. Results shown are from duplicate samples. Error bars represent standard deviations.

Fig. 3.

N283 enhances macrophage and microglia tropism. PBMC, MDM, and microglia in primary brain cultures were infected as described in Materials and Methods. (A) Supernatants were collected every 3–7 days, and replication was monitored by p24 ELISA. Results shown are from triplicate samples. Error bars represent standard deviations. (B) Cytopathic effects in PBMC (day 11 postinfection), MDM (day 14 postinfection), and microglia (day 14 postinfection). Photographs were taken at a ×200 magnification.

Fig. 4.

Mutation Q40A in CD4 eliminates entry advantage of N283 in cells expressing low CD4. (A) T283N change increases potential for hydrogen bond to CD4 Q40. Swiss PDB Viewer was used to change Thr at position 283 (Left) to Asn (Right) in the JRFL gp120 crystal structure (2B4C) (31). Blue, gp120; yellow, CD4; red, position 283. Potential hydrogen bonds are indicated by dotted lines. (B) Cf2-Luc cells were transfected with plasmids expressing CCR5 and wild-type or Q40A mutant CD4, and CD4 cell surface expression was analyzed by flow cytometry. (C) Cf2-Luc cells expressing wild-type (Left) or Q40A mutant (Right) CD4 were infected with chimeric viruses expressing each Env, lysed 48 h postinfection, and analyzed for luciferase activity. Data were normalized to luciferase activity of the wild-type Env on cells expressing high wild-type CD4.