Effects of CCR5 and CD4 cell surface concentrations on infections by macrophagetropic isolates of human immunodeficiency virus type 1 - PubMed (original) (raw)
Effects of CCR5 and CD4 cell surface concentrations on infections by macrophagetropic isolates of human immunodeficiency virus type 1
E J Platt et al. J Virol. 1998 Apr.
Abstract
It has been proposed that changes in cell surface concentrations of coreceptors may control infections by human immunodeficiency virus type 1 (HIV-1), but the mechanisms of coreceptor function and the concentration dependencies of their activities are unknown. To study these issues and to generate stable clones of adherent cells able to efficiently titer diverse isolates of HIV-1, we generated two panels of HeLa-CD4/CCR5 cells in which individual clones express either large or small quantities of CD4 and distinct amounts of CCR5. The panels were made by transducing parental HeLa-CD4 cells with the retroviral vector SFF-CCR5. Derivative clones expressed a wide range of CCR5 quantities which were between 7.0 x 10(2) and 1.3 x 10(5) molecules/cell as measured by binding antibodies specific for CCR5 and the chemokine [125I]MIP1beta. CCR5 was mobile in the membranes, as indicated by antibody-induced patching. In cells with a large amount of CD4, an unexpectedly low trace of CCR5 (between 7 x 10(2) and 2.0 x 10(3) molecules/cell) was sufficient for maximal susceptibility to all tested HIV-1, including primary patient macrophagetropic and T-cell-tropic isolates. Indeed, the titers as indicated by immunoperoxidase staining of infected foci were as high as the tissue culture infectious doses measured in human peripheral blood mononuclear cells. In contrast, cells with a small amount of CD4 required a much larger quantity of CCR5 for maximal infection by macrophagetropic HIV-1 (ca. 1.0 x 10(4) to 2.0 x 10(4) molecules/cell). Cells that expressed low and high amounts of CD4 were infected with equal efficiencies when CCR5 concentrations were above threshold levels for maximal infection. Our results suggest that the concentrations of CD4 and CCR5 required for efficient infections by macrophagetropic HIV-1 are interdependent and that the requirements for each are increased when the other component is present in a limiting amount. We conclude that CD4 and CCR5 directly or indirectly interact in a concentration-dependent manner within a pathway that is essential for infection by macrophagetropic HIV-1. In addition, our results suggest that multivalent virus-receptor bonds and diffusion in the membrane contribute to HIV-1 infections.
Figures
FIG. 1
Detection of CCR5 cell surface expression on viable cells by immunofluorescence microscopy. The left and right hand panels depict fluorescence and phase-contrast imaging, respectively. Representative CCR5-expressing cell clones stained with rabbit anti-CCR5 show strong fluorescence, while the parent cell line, HI-J, shows only weak reactivity. Similarly, the high-CCR5-expressing clone JC.53 shows only weak staining with preimmune serum. The two representative clones shown (JC.53 and JC.48) depict the uniformity of CCR5 expression level within a clonal population, as well as differences in CCR5 expression levels among cell clones.
FIG. 2
Scatchard and competitive binding analysis of [125I]MIP1β onto HeLa-CD4/CCR5 clone JC.53. At the time of assay, the cells had grown to approximately 7 × 104/well. Specific [125I]MIP1β binding in counts/minute was calculated by subtracting the nonspecific binding (approximately 300 cpm) of [125I]MIP1β measured in the presence of 1 μM unlabeled MIP1β. Background binding of [125I]MIP1β, measured on HI-J cells lacking CCR5, gave binding values of approximately 300 cpm at all concentrations of unlabeled MIP1β. A Scatchard analysis of the binding data (inset) was performed to determine the number of CCR5 molecules/cell. The x intercept yields the number of accessible MIP1β binding sites/cell and equals approximately 1.45 × 105 CCR5 molecules/cell. The competition curve yields an 50% inhibitory concentration value of 15 nM, in close agreement with a KD of 17 nM which was obtained when the same binding data were used to generate a Scatchard plot with the x axis presented in molar concentration. Data points represent the means of triplicate assays.
FIG. 3
Foci of infection of HeLa-CD4/CCR5 cells by M-tropic HIV-1 clones or isolates. HIV-1 p24 antigen in infected cells was detected by indirect immunoperoxidase staining. p24-positive foci consisted of single or grouped multinucleated giant cells, with occasional adjacent mononuclear cells. (A) M-tropic HIV-1 clone 81A, containing the V1, V2, and V3 regions of the env gene of clone Ba-L in the background of clone NL4-3 (68). One small syncytium with four nuclei and three large syncytia containing greater than 20 nuclei are shown. (B) Field showing 10 typical foci of cells infected by M-tropic clone JR-CSF containing 1 to 10 nuclei each. (C) Numerous small foci of cells infected by an HIV-1 isolate obtained from a patient on day 7 of illness due to primary HIV-1 infection.
FIG. 4
Infectivities of three HIV-1 strains in JC clones and PBMCs. M-tropic HIV-1 strain JR-CSF and Ba-L and T-tropic HIV-1 NL4-3 were used to infect nine HeLa-CD4/CCR5 JC clones with differing levels of CCR5 expression, as well as the CCR5-negative CD4-positive parental clone HI-J and PBMCs. JC clones with CCR5 expression ranging from 2 × 103 to 1.3 × 105 molecules/cell gave similar titers for the two M-tropic strains. In contrast, HI-J was negative and clone JC.20, with background CCR5 expression (<7 × 102 molecules/cell), had markedly reduced titers. Although M-tropic HIV-1 did not infect HI-J cells, titers are indicated as <5 to account for the virus dilutions used in the assays. Titers in PBMCs expressed as TCID50/0.05 ml were numerically similar to titers (foci/0.05 ml) seen on the highly sensitive JC clones.
FIG. 5
M-tropic HIV-1 infectivity on JC (high CD4) and RC (low CD4) clonal panels. Infectivity assays were performed as described in Materials and Methods on the 11 JC clones (A) and the 14 RC clones (B) whose CCR5 expression was above background (Table 1). Approximately 500 focus-forming units of diluted HIV-1 Ba-L was added per well (1 cm2), in 0.1 ml. Titers were calculated by multiplying foci/well by the dilution factor at which the virus was used. Points represent the mean of duplicate assays.
FIG. 6
Infectivities of M-tropic and T-cell-tropic HIV-1 isolates on HeLa-CD4/CCR5 cells. Infectivity assays were performed as described in Materials and Methods. M-tropic (SF162, JR-FL, Ba-L, and ADA), primary T-cell-tropic (ELI 1 and K4), and laboratory-adapted T-cell-tropic (NL4-3) titers were determined on clones RC.49 and JC.24, expressing low CD4/high CCR5 and high CD4/high CCR5 cell surface amounts, respectively, and on parental HI-R and HI-J clones, expressing low and high amounts of CD4, respectively. Titers are expressed as percent titer obtained on JC.24 cells. Approximately 500 focus-forming units of diluted HIV-1 isolates were added per well, in 0.1 ml. Assays were performed in duplicate except for SF162 and JR-FL infections of JC.24, which are presented as single assays. Error bars represent the range of values obtained.
References
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