Differential Vpu-Mediated CD4 and Tetherin Downregulation Functions among Major HIV-1 Group M Subtypes

Abstract

Downregulation of BST-2/tetherin and CD4 by HIV-1 viral protein U (Vpu) promotes viral egress and allows infected cells to evade host immunity. Little is known however about the natural variability in these Vpu functions among the genetically diverse viral subtypes that contribute to the HIV-1 pandemic. We collected Vpu isolates from 332 treatment-naive individuals living with chronic HIV-1 infection in Uganda, Rwanda, South Africa, and Canada. Together, these Vpu isolates represent four major HIV-1 group M subtypes (A [n = 63], B [n = 84], C [n = 94], and D [n = 59]) plus intersubtype recombinants and uncommon strains (n = 32). The ability of each Vpu clone to downregulate endogenous CD4 and tetherin was quantified using flow cytometry following transfection into an immortalized T-cell line and compared to that of a reference Vpu clone derived from HIV-1 subtype B NL4.3. Overall, the median CD4 downregulation function of natural Vpu isolates was similar to that of NL4.3 (1.01 [interquartile range {IQR}, 0.86 to 1.18]), while the median tetherin downregulation function was moderately lower than that of NL4.3 (0.90 [0.79 to 0.97]). Both Vpu functions varied significantly among HIV-1 subtypes (Kruskal-Wallis P < 0.0001). Specifically, subtype C clones exhibited the lowest CD4 and tetherin downregulation activities, while subtype D and B clones were most functional for both activities. We also identified Vpu polymorphisms associated with CD4 or tetherin downregulation function and validated six of these using site-directed mutagenesis. Our results highlight the marked extent to which Vpu function varies among global HIV-1 strains, raising the possibility that natural variation in this accessory protein may contribute to viral pathogenesis and/or spread.IMPORTANCE The HIV-1 accessory protein Vpu enhances viral spread by downregulating CD4 and BST-2/tetherin on the surface of infected cells. Natural variability in these Vpu functions may contribute to HIV-1 pathogenesis, but this has not been investigated among the diverse viral subtypes that contribute to the HIV-1 pandemic. In this study, we found that Vpu function differs significantly among HIV-1 subtypes A, B, C, and D. On average, subtype C clones displayed the lowest ability to downregulate both CD4 and tetherin, while subtype B and D clones were more functional. We also identified Vpu polymorphisms that associate with functional differences among HIV-1 isolates and subtypes. Our study suggests that genetic diversity in Vpu may play an important role in the differential pathogenesis and/or spread of HIV-1.

Keywords: CD4; Downregulation; HIV-1; Vpu; subtype; tetherin.

FIG 1

Vpu sequence diversity. (A) Maximum-likelihood phylogeny inferred from a nucleic acid sequence alignment of 300 HIV subtype A, B, C, and D vpu isolates analyzed in this study (32 vpu sequences encoding viral recombinants or other subtypes are not shown). Scale in estimated nucleotide substitutions per site. (B) Gap-stripped alignment of the Vpu consensus amino acid sequences for subtypes A, B, C, and D (defined as the most frequently observed residue at each position in our study sequences). Colors match the phylogeny in panel A. The inverted blue triangle denotes a common insertion that occurred exclusively in subtype C, usually seven amino acids in length (usually LA[K/R]VDYR). Major Vpu structural features are highlighted. β-TrCP, beta-transducin repeat-containing protein.

FIG 2

Analysis of CD4 and tetherin downregulation function. (A) Representative flow cytometry plots demonstrating downregulation of CD4 (top) or tetherin (bottom) following transfection of a negative control (pSelect empty vector), a positive control (NL4.3 Vpu), a representative functional clone, and a representative nonfunctional clone. Gray-shaded areas define GFP-negative (untransfected) and the GFP-high (Vpu-expressing) gates used for analysis. The median fluorescence intensity (MFI) of receptor expression is indicated at the top of each gate, and the downregulation function value (calculated as described in Materials and Methods) normalized to NL4.3 is indicated at the lower right. The absolute values of CD4 and tetherin reduction by NL4.3 Vpu were 69% ± 14% and 73% ± 13%. (B) Normalized CD4 and tetherin downregulation results for the 332 Vpu clones assessed in this study. Data are reported as the mean from at least three independent experiments. Horizontal lines and values report the median and interquartile ranges. (C) Association between CD4 and tetherin downregulation function for all 332 Vpu clones. Correlation was evaluated using Spearman’s rank sum test.

FIG 3

Differences in Vpu function among HIV group M subtypes. (A) CD4 downregulation activities of all Vpu clones, stratified by subtype. “Others” comprise non-A/B/C/D subtypes and unclassified/recombinant sequences. (B) Tetherin downregulation activities of all Vpu clones, stratified as in panel A. Horizontal lines denote median and interquartile ranges within. Significant variation was observed among groups for both data sets (Kruskal-Wallis P < 0.0001). Significant results based on pairwise Mann-Whitney U tests are indicated by asterisks: *, P < 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

FIG 4

Association between Vpu downregulation functions. Correlation between the CD4 and tetherin downregulation abilities of each clone, stratified by subtype, is shown. Correlation was evaluated using the Spearman rank sum test.

FIG 5

Vpu residues associated with variation in downregulation function. (A) Vpu residues associated with CD4 downregulation function. An aligned, gap-stripped Vpu consensus amino acid sequence, generated using all 332 vpu sequences in this study, is shown in the top row. Amino acids significantly associated with CD4 downregulation function (defined as at P < 0.05 and q < 0.1) are displayed in the bottom row in order of functional impact (from most negative to most positive). Dashes indicate gaps. Black dots denote residues uniquely associated with CD4, but not tetherin, downregulation at this statistical threshold. (B) Vpu residues associated with tetherin downregulation function, as described for panel A. Black dots denote residues uniquely associated with tetherin, but not CD4, downregulation at this statistical threshold.

FIG 6

Experimental verification of residues associated with Vpu function. (A) Amino acid alignment for NL4.3 Vpu and six site-directed mutants. (B) Normalized CD4 downregulation function of each NL4.3 Vpu mutant. Bars denote mean and standard deviation, calculated from a minimum of 7 replicate measurements per mutant. Results were evaluated using the one-sample t test (with NL4.3 = 1.0), and significant differences are indicated by asterisks: *, P < 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (C) Normalized tetherin downregulation functions of each Vpu mutant, as described for panel B.

FIG 7

Relationships between Vpu function and CD4⁺ T-cell count, stratified by subtype. (A) Spearman’s correlations between normalized Vpu-mediated CD4 downregulation and CD4⁺ T-cell count, colored by HIV subtype. A dotted trendline is drawn to visualize the statistically significant relationship observed for subtype D. (B) Spearman’s correlation between normalized Vpu-mediated tetherin downregulation and CD4⁺ T-cell count, colored by HIV subtype. A dotted trendline is drawn to visualize the statistically significant relationship observed for subtype C.