Exclusive and persistent use of the entry coreceptor CXCR4 by human immunodeficiency virus type 1 from a subject homozygous for CCR5 delta32

Abstract

Individuals who are homozygous for the 32-bp deletion in the gene coding for the chemokine receptor and major human immunodeficiency virus type 1 (HIV-1) coreceptor CCR5 (CCR5 -/-) lack functional cell surface CCR5 molecules and are relatively resistant to HIV-1 infection. HIV-1 infection in CCR5 -/- individuals, although rare, has been increasingly documented. We now report that the viral quasispecies from one such individual throughout disease is homogenous, T cell line tropic, and phenotypically syncytium inducing (SI); exclusively uses CXCR4; and replicates well in CCR5 -/- primary T cells. The recently discovered coreceptors BOB and Bonzo are not used. Although early and persistent SI variants have been described in longitudinal studies, this is the first demonstration of exclusive and persistent CXCR4 usage. With the caveat that the earliest viruses available from this subject were from approximately 4 years following primary infection, these data suggest that HIV-1 infection can be mediated and persistently maintained by viruses which exclusively utilize CXCR4. The lack of evolution toward the available minor coreceptors in this subject underscores the dominant biological roles of the major coreceptors CCR5 and CXCR4. This and two similar subjects (R. Biti, R. Ffrench, J. Young, B. Bennetts, G. Stewart, and T. Liang, Nat. Med. 3:252-253, 1997; I. Theodoreu, L. Meyer, M. Magierowska, C. Katlama, and C. Rouzioux, Lancet 349:1219-1220, 1997) showed relatively rapid CD4+ T-cell declines despite average or low initial viral RNA load. Since viruses which use CXCR4 exclusively cannot infect macrophages, these data have implications for the relative infection of the T-cell compartment versus the macrophage compartment in vivo and for the development of CCR5-based therapeutics.

FIG. 1

Construction of isogenic recombinant proviruses. (a) Exogenous V3 region env sequences were inserted into a portion of the HIV-1_HXB2 env gene contained within the SalI-BamHI fragment and then inserted into the HIV-1_HXB2 proviral background. (b) Exogenous sequences V3 through V5 contained by BglII sites were inserted into the HIV-1_JR-FL background bounded by SacI and XhoI sites and then inserted into the HIV-1_NL4-3 proviral background.

FIG. 2

Analysis by V3-HTA. V3 loop RT-PCR products from the June 1986 serum sample from patient UNC116 (lane 86) and control YU-2 and NL4-3 recombinant proviral clones were annealed with the JR-FL V3 probe that was ³⁵S labeled on one strand. The heteroduplexes were resolved on a native polyacrylamide gel and visualized by autoradiography. Only the bottom half of the autoradiograph is shown. The positions of the single-stranded probe (upper arrow) and double-stranded probe homoduplexes (lower arrow) are indicated. Heteroduplexes in each lane resolved between the single-stranded probe and probe homoduplexes.

FIG. 3

HIV-1 entry coreceptor typing of viral stocks in GHOST cells. HOS cells nonpermissive for HIV-1 infections were transduced with human CD4 and the GFP under the control of the HIV-2 LTR to generate parental GHOST cells. These cells were then individually transduced with the HIV-1 entry coreceptor cDNAs indicated at the top. HIV-1 entry and subsequent tat gene expression transactivates the HIV-2 LTR and upregulates the expression of GFP. Following infection of parental and coreceptor-transduced cells with equivalent amounts of specific viral stocks (indicated at the left), the cells were fixed and analyzed by flow cytometry. For each fluorescence-activated cell sorter profile, linear forward side scatter is plotted against log fluorescence intensity for GFP in the FL1 window. Basal-level production of GFP is red; transactivated GFP expression is green.

FIG. 4

Replication kinetics of viral stocks in CCR5 +/+ (a) and CCR5 −/− (b) PBMC and CCR5 +/+ macrophages (c). One hundred and 500 TCID₅₀ of PBMC-derived viral stocks were used to infect PHA- and IL-2-stimulated PBMC and monocyte-derived macrophages, respectively. Virus replication was monitored by supernatant p24 antigen production.