Tat protein of human immunodeficiency virus type 1 subtype C strains is a defective chemokine

Abstract

Human immunodeficiency virus type 1 (HIV-1)-associated dementia (HAD) is correlated with increased monocyte migration to the brain, and the incidence of HAD among otherwise asymptomatic subjects appears to be lower in India than in the United States and Europe (1 to 2% versus 15 to 30%). Because of the genetic differences between HIV-1 strains circulating in these regions, we sought to identify viral determinants associated with this difference. We targeted Tat protein for these studies in view of its association with monocyte chemotactic function. Analyses of Tat sequences representing nine subtypes revealed that at least six amino acid residues are differentially conserved in subtype C Tat (C-Tat). Of these, cysteine (at position 31) was highly (>99%) conserved in non-subtype C viruses and more than 90% of subtype C viruses encoded a serine. We hypothesized a compromised chemotactic function of C-Tat due to the disruption of CC motif and tested it with the wild type C-Tat (CS) and its two isogenic variants (CC and SC) derived by site-directed mutagenesis. We found that the CS natural variant was defective for monocyte chemotactic activity without a loss in the transactivation property. While the CC mutant is functionally competent for both the functions, in contrast, the SC mutant was defective in both. Therefore, the loss of the C-Tat chemotactic property may underlie the reduced incidence of HAD; although not presenting conclusive evidence, this study provides the first evidence for a potential epidemiologic phenomenon associated with biological differences in the subtype C viruses.

FIG. 1.

Genetic features of HIV-1 Tat sequences. (A) Phylogenetic relationships among group M HIV-1 Tat exon 1 sequences, illustrating the monophyletic subtype-specific clustering of sequences. Subtypes corresponding to each of the clusters in the neighbor-joining phylogram are shown. (B) Dicysteine motif within the cysteine-rich domain (Tat1 amino acids 25 to 37) of HIV-1, HIV-2, and SIV. Sequences representing different subtypes of HIV-1, HIV-2, and SIV are shown; dots indicate identity with the sequence at the top, and the dicysteine motif is highlighted. Asterisks (*) indicate sequence data obtained from consensus sequences derived in this study; the other sequence data were obtained from the database. (C) Subtype C sequences from different countries, illustrating the level of conservation of the C31S substitution regardless of geographic origin. Similar findings for the uniform substitution of Q35L and the high level of heterogeneity at position 29 (as identified in panel D) are also evident. The number of sequences obtained from each country and used in these analyses and the number that carried C or G at position 31 are shown. (D) Signature amino acids present in 70% or more subtype C sequences. Corresponding amino acids for non-subtype C sequences as well as their frequencies are illustrated. Position 29 in C-Tat is shown because of the high level of diversity that appears to be unique to subtype C viruses. A total of 14 different amino acids are seen at this position. (E) Phylogenetic analysis consistent with independent evolution of subtype C sequences with C31. The ML phylogram was generated using 146 subtype C sequences and non-subtype C representative sequences. The sole subtype H sequence that encoded S31 (AF190128) was used.

FIG. 2.

Functional evaluation of Tat transactivation using isogenic variants of C-Tat. (A) Schematic diagram of the Tat mammalian expression vectors encoding the isogenic C-Tat proteins. Differences within the dicysteine motif of these vectors are highlighted. Cys, cysteine; CMV, cytomegalovirus; IRES, internal ribosome entry site; Puro, puromycin; C-term, carboxy terminus. (B) Transactivation of LTR-driven GFP expression by different Tat vectors in 293 cells. (C) Transactivation of LTR-driven SEAP expression by different Tat vectors in 293 cells. SEAP in the culture medium was quantified on day 1 (open bars) and day 3 (filled bars). (D) Rescue of the Tat-defective virus by isogenic C-Tat proteins. HLM-1 cells were transfected with different C-Tat variant expression vectors. Culture supernatants were collected on days 1, 3, 5, and 7 following transfection, and p24 levels in the culture supernatants were determined using a commercial kit. Results of experiments using samples from day 3 (when antigen levels peaked) are presented; similar results were observed for samples from other days. Abs, absorbance; −VE, parental vector.

FIG. 3.

Monocyte migration induced by isogenic Tat proteins. f-MLP peptide was used as a positive control at 10⁻⁷ and 10⁻⁸ M concentrations. Tat proteins were used at concentrations of 100 and 20 ng/ml (12 and 2.4 nM, respectively) as indicated. No grad, wells with 100 ng of CC-Tat protein/ml in both the compartments. Differences in the numbers of monocytes that migrated with Tat-CC and Tat-CS were statistically significant (odds ratio = 4.95; confidence interval = 0.95 [3.4 to 7.2]; P < 0.0001 at 100 ng/ml).