Development and characterization of a novel single-cycle recombinant-virus assay to determine human immunodeficiency virus type 1 coreceptor tropism

Abstract

Most human immunodeficiency virus type 1 (HIV-1) strains require either the CXCR4 or CCR5 chemokine receptor to efficiently enter cells. Blocking viral binding to these coreceptors is an attractive therapeutic target. Currently, several coreceptor antagonists are being evaluated in clinical trials that require characterization of coreceptor tropism for enrollment. In this report, we describe the development of an automated and accurate procedure for determining HIV-1 coreceptor tropism (Trofile) and its validation for routine laboratory testing. HIV-1 pseudoviruses are generated using full-length env genes derived from patient virus populations. Coreceptor tropism is determined by measuring the abilities of these pseudovirus populations to efficiently infect CD4+/U87 cells expressing either the CXCR4 or CCR5 coreceptor. Viruses exclusively and efficiently infecting CXCR4+/CD4+/U87 cells are designated X4-tropic. Conversely, viruses exclusively and efficiently infecting CCR5+/CD4+/U87 cells are designated R5-tropic. Viruses capable of infecting both CXCR4+/CD4+/U87 and CCR5+/CD4+/U87 cells are designated dual/mixed-tropic. Assay accuracy and reproducibility were established by evaluating the tropisms of well-characterized viruses and the variability among replicate results from samples tested repeatedly. The viral subtype, hepatitis B virus or hepatitis C virus coinfection, and the plasma viral load did not affect assay performance. Minority subpopulations with alternate tropisms were reliably detected when present at 5 to 10%. The plasma viral load above which samples can be amplified efficiently in the Trofile assay is 1,000 copies per ml of plasma. Trofile has been automated for high-throughput use; it can be used to identify patients most likely to benefit from treatment regimens that include a coreceptor inhibitor and to monitor patients on treatment for the emergence of resistant virus populations that switch coreceptor tropism.

FIG. 1.

Structures of vectors used in the Trofile assay. (A) Patient eETVs were constructed by cloning the amplified env genes from patient plasma samples into pCXAS-PXMX (see the text for details). The amplified fragment comprised the entire open reading frame of HIV-1 gp160. (B) A replication-defective genomic vector, RTV1.F-lucP.CNDOΔU3, was constructed with a luciferase cassette inserted into a deleted region of the env gene of the NL4-3 strain of HIV-1.

FIG. 2.

Schematic diagram of the assay. Replication-defective virus particles were produced by cotransfecting HEK 293 cells with eETV and RTV1.F-lucP.CNDOΔU3. Following transfection, the virus particles were harvested and used to infect target cells (U87 cells expressing CD4 and either the CCR5 or CXCR4 coreceptor). The abilities of virus particles to complete a single round of replication in the presence or absence of CRAs was assessed by measuring the luciferase activities in target cells.

FIG. 3.

Trofile coreceptor tropism determinations of well-characterized reference strains. CXCR4⁺ and CCR5⁺ cells were infected with (A) the X4-tropic strain HXB2, (B) the R5-tropic strain JRCSF, and (C) the dual-tropic strain 92TH594. Infectivity was analyzed in the presence and absence of either CXCR4 or CCR5 antagonists. The percent inhibition by these compounds is given above the appropriate columns. (A) bRLU from inoculation of CXCR4⁺ and CCR5⁺ cell lines with strain HXB2. (B) bRLU from inoculation of CXCR4⁺ and CCR5⁺ cell lines with strain JRCSF. (C) bRLU from inoculation of CXCR4⁺ and CCR5⁺ cell lines with strain 92TH594.

FIG. 4.

Concordance of duplicate tropism determinations. Two replicates of 46 plasma samples were evaluated in the Trofile assay. The infectivities (luciferase activities) of paired replicates on CCR5⁺/CD4⁺/U87 cells (crosses) and CXCR4⁺/CD4⁺/U87 cells (circles) were graphed pairwise to demonstrate the concordance between measurements.

FIG. 5.

Sensitivity to detect minor variants within mixed populations of clones derived from four patients (244, 417, 1354, and 2188). The results are displayed as median RLU from replicates (see the text for details); the error bars are 1 standard deviation from the median (see the text for details). (A) Mixture 244. Minority species of X4- and R5-tropic viruses were both detected in mixed viral stocks at the lowest frequency tested (5% of the population). (B) Mixture 417. Minority species of R5-tropic viruses were detected in mixed viral stocks at the lowest frequencies tested (5% of the population). The minority X4-tropic variant was detectable only when its frequency was greater than 10% of the total population. (C) Mixture 1354. The minority species of X4- and R5-tropic viruses were both detected in mixed viral stocks at the lowest frequency tested (5% of the population). (D) Mixture 2188. The minority species of X4- and R5-tropic viruses were both detected in mixed viral stocks at the lowest frequency tested (5% of the population).