Affinofile profiling: how efficiency of CD4/CCR5 usage impacts the biological and pathogenic phenotype of HIV

Abstract

HIV-1 envelope (Env) uses CD4 and a coreceptor (CCR5 and/or CXCR4) for viral entry. The efficiency of receptor/coreceptor mediated entry has important implications for HIV pathogenesis and transmission. The advent of CCR5 inhibitors in clinical use also underscores the need for quantitative and predictive tools that can guide therapeutic management. Historically, measuring the efficiency of CD4/CCR5 mediated HIV entry has relied on surrogate and relatively slow throughput assays that cannot adequately capture the full spectrum of Env phenotypes. In this review, we discuss the details of the Affinofile receptor affinity profiling system that has provided a quantitative and higher throughput method to characterize viral entry efficiency as a function of CD4 and CCR5 expression levels. We will then review how the Affinofile system has been used to reveal the distinct pathophysiological properties associated with Env entry phenotypes and discuss potential shortcomings of the current system.

Figure 1

CD4 expression is controlled by the Tet-On system in which constitutive expression of the tet transactivator from pcDNA/TR (1) represses expression from the Tet-O-CMV promoter (3) in the absence of tetracycline. In the presence of tetracycline, the tet transactivator is released from the Tet-O-CMV promoter on pcDNA5/TO-CD4 (3), allowing CMV driven expression of CD4. CCR5 expression is controlled by the synthetic ecdysone-inducible system in which the pon A transactivator, VgRXR (2), comprises of the modified heterodimeric subunits of the insect nuclear hormone receptors VgEcR and RXR. VgEcR and RXR are independently driven by constitutive promoters on the same plasmid (pcVgRXR) (2), but are shown simply as “ponTransAct” for clarity. In the presence of ponasterone A, the two subunits dimerize (VgRXR) and bind to the pon A inducible promoter on pIND-R5 (4) consisting of 5× Ecdysone/Glucocorticoid Response Element (5× E/GREs), driving expression of CCR5. Thus, the addition of varying amounts of tetracycline and/or ponasterone A induces CD4 and/or CCR5 expression, respectively. The induced surface levels of CD4 and CCR5 is quantified by qFACS as described in the text. The inset tables represent the publications that specifically state the maximal number of CD4 (red) and/or CCR5 (green) Antibody Binding Sites (ABS)/cell induced. The natural logarithms of CD4 and CCR5 cell surface concentrations (ABS/cell) are indicated here for convenience. We will refer to these numbers when discussing the analytic methods used to quantify the efficiency of CD4/CCR5 usage.

Figure 2

(A) A prototypic R5 Env pseudotyped virus was used to infect Affinofile cells induced with varying concentration of tetracycline and ponasterone A. The infectivity profile generated in (A) can be mathematically fitted to the surface function F(x, y) shown in (B), where x and y are the mathematically rescaled values for the relevant CD4 and CCR5 concentrations (see text for details). Essentially, the surface plot in (B) represents the infectivity response of Env as a function of CD4 and CCR5 expression level, which is described by the continuous polynomial function F(x, y). As indicated in the text, three parameters can be found to represent the salient features of the surface function to capture the essential phenotypic behavior of Env in response to varying levels of CD4 and CCR5. The three metrics (1) mean infectivity M, (2) sensitivity vector angle θ, and (3) sensitivity vector amplitude Δ, are graphically represented in (B) and (C). The mathematical formulation of these metrics, their biophysical correlates, and how these metrics can be used to quantify the entry phenotype of Env are detailed in the text. (D) A reproduction of Fig. 5 from Agrawal-Gamse et al. (2009) illustrating how the nature of the infectivity plot is reflected in the angular metric. See text for details.