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. 2013 Mar;87(5):2401-11.
doi: 10.1128/JVI.02964-12. Epub 2012 Dec 26.

Transmitted/founder and chronic HIV-1 envelope proteins are distinguished by differential utilization of CCR5

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Transmitted/founder and chronic HIV-1 envelope proteins are distinguished by differential utilization of CCR5

Zahra F Parker et al. J Virol. 2013 Mar.

Abstract

Infection by HIV-1 most often results from the successful transmission and propagation of a single virus variant, termed the transmitted/founder (T/F) virus. Here, we compared the attachment and entry properties of envelope (Env) glycoproteins from T/F and chronic control (CC) viruses. Using a panel of 40 T/F and 47 CC Envs, all derived by single genome amplification, we found that 52% of clade C and B CC Envs exhibited partial resistance to the CCR5 antagonist maraviroc (MVC) on cells expressing high levels of CCR5, while only 15% of T/F Envs exhibited this same property. Moreover, subtle differences in the magnitude with which MVC inhibited infection on cells expressing low levels of CCR5, including primary CD4(+) T cells, were highly predictive of MVC resistance when CCR5 expression levels were high. These results are consistent with previous observations showing a greater sensitivity of T/F Envs to MVC inhibition on cells expressing very high levels of CCR5 and indicate that CC Envs are often capable of recognizing MVC-bound CCR5, albeit inefficiently on cells expressing physiologic levels of CCR5. When CCR5 expression levels are high, this phenotype becomes readily detectable. The utilization of drug-bound CCR5 conformations by many CC Envs was seen with other CCR5 antagonists, with replication-competent viruses, and did not obviously correlate with other phenotypic traits. The striking ability of clade C and B CC Envs to use MVC-bound CCR5 relative to T/F Envs argues that the more promiscuous use of CCR5 by these Env proteins is selected against at the level of virus transmission and is selected for during chronic infection.

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Figures

Fig 1
Fig 1
Partial resistance to saturating amounts of maraviroc on Affinofile cells expressing high levels of CCR5. (A) Quantitative assessment of cell surface expression of CCR5 was determined by staining cells with PE-labeled CCR5 MAb 2D7 followed by quantitative flow cytometry, making it possible to calculate the number of 2D7 binding sites per cell. (B) MVC saturation was confirmed by measuring residual infection between the two highest concentrations used. No differences in virus inhibition were observed between 2 μM and 6 μM MVC (P = 0.70). (C) MVC-sensitive pseudoviruses displayed an MPI of >95% on both low-CCR5 (gray) and high-CCR5 (black) 293-Affinofile cells. Two phenotypes were observed. Fifty-seven pseudoviruses and the JRFL control virus displayed an MPI of >95% regardless of CCR5 expression levels (top and bottom panels). However, 30 pseudoviruses were efficiently inhibited by MVC on cells expressing low CCR5 but exhibited MPI values of ≤95% on high-CCR5 Affinofile cells (middle panel). All experiments were done at least in triplicate in each of at least three independent experiments. Error bars represent standard deviations.
Fig 2
Fig 2
Partial resistance is enriched in chronic HIV-1 Envs compared to T/F Envs, irrespective of clade and CCR5 antagonist. (A) Eighty-seven pseudoviruses were tested on high-CCR5 Affinofile cells. There was a higher frequency of partial resistance to MVC in CC Envs than to T/F Envs (P < 0.0001). (B) Data were segregated by clade (52 clade B and 35 clade C) and independently confirmed an enrichment of the partially resistant phenotype in CC Envs (clade B, P = 0.02; clade C, P = 0.02). (C) Representative resistant (704010330.G5h) and sensitive (700010040.C9.4520) pseudoviruses were tested for sensitivity to other CCR5 antagonists on high-CCR5 Affinofile cells. Cells were pretreated with various concentrations of MVC, APL, CMPD-167, TAK779, and VVC prior to infection. MPI values of MVC-resistant (gray) and MVC-sensitive (black) pseudovirus are shown. Data were analyzed by Fisher's exact test. All infections were done at least in triplicate in each of at least three independent experiments. Error bars represent standard deviations.
Fig 3
Fig 3
Residual infection on cells expressing different levels of CCR5. All pseudoviruses were tested on low-CCR5 Affinofile cells, NP2/CD4/CCR5 cells, and high-CCR5 Affinofile cells, and the residual infection in the presence of saturating MVC was plotted for all cell types evaluated. There was a significant difference between T/F (black) and chronic (gray) residual infection in both NP2/CD4/CCR5 and high-CCR5 Affinofile cells (NP2/CD4/CCR5, P = 0.01; high-CCR5 Affinofiles, P = 0.0003), but not on low-CCR5 Affinofile cells (P = 0.94). Data were analyzed by Mann-Whitney test. All experiments were done at least in triplicate in each of at least two independent experiments.
Fig 4
Fig 4
Residual infection on cells expressing low levels of CCR5 is predictive of MVC resistance on cells expressing high levels of CCR5. (A) The residual infection on low-CCR5 Affinofile cells was plotted against that on high-CCR5 Affinofiles in the presence of saturating MVC. Residual infection on low-CCR5 Affinofiles was predictive of partial MVC resistance on high-CCR5 Affinofile cells (Spearman's correlation coefficient = 0.22; P = 0.04). (B) The residual infection on high-CCR5 Affinofile cells was also plotted against that on NP2/CD4/CCR5 in the presence of saturating MVC. Similarly, residual infection on NP2/CD4/CCR5 was predictive of residual infection on high-CCR5 Affinofile cells (Spearman's correlation coefficient = 0.65; P < 0.0001).
Fig 5
Fig 5
CC Envs exhibit greater residual infection compared to T/F Envs on primary human CD4+ T cells. Levels of residual infection for three T/F (black) and three CC (gray) Envs on CD4+ T cells were assessed in the presence of saturating MVC. Higher levels of residual infection were mediated by CC Envs in the presence of MVC than to T/F Envs (CC median, 1.1%, versus T/F median, 0.51%; P = 0.10). Residual infection for the three T/F and CC Envs were compared between CD4+ T cells, high-CCR5 Affinofile cells, and NP2/CD4/CCR5 cells. In all three cell types, CC Envs displayed higher residual infection than T/F Envs. All primary cell infections were done with at least two donors in triplicate in at least two independent experiments.
Fig 6
Fig 6
Infectivity is not associated with the partial resistance phenotype. Pseudoviral infectivity in the absence of MVC was plotted against residual infection in the presence of saturating MVC to determine whether partial resistance correlated with infectivity. There was no correlation (Spearman's correlation coefficient = 0.02; P = 0.84).

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