Macrophage inflammatory protein 1alpha inhibits postentry steps of human immunodeficiency virus type 1 infection via suppression of intracellular cyclic AMP

Abstract

Primary isolates of human immunodeficiency virus type 1 (HIV-1) predominantly use chemokine receptor CCR5 to enter target cells. The natural ligands of CCR5, the beta-chemokines macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, and RANTES, interfere with HIV-1 binding to CCR5 receptors and decrease the amount of virions entering cells. Although the inhibition of HIV-1 entry by beta-chemokines is well documented, their effects on postentry steps of the viral life cycle and on host cell components that control the outcome of infection after viral entry are not well defined. Here, we show that all three beta-chemokines, and MIP-1alpha in particular, inhibit postentry steps of the HIV-1 life cycle in primary lymphocytes, presumably via suppression of intracellular levels of cyclic AMP (cAMP). Productive HIV-1 infection of primary lymphocytes requires cellular activation. Cell activation increases intracellular cAMP, which is required for efficient synthesis of proviral DNA during early steps of viral infection. Binding of MIP-1alpha to cognate receptors decreases activation-induced intracellular cAMP levels through the activation of inhibitory G proteins. Furthermore, inhibition of one of the downstream targets of cAMP, cAMP-dependent PKA, significantly inhibits synthesis of HIV-1-specific DNA without affecting virus entry. These data reveal that beta-chemokine-mediated inhibition of virus replication in primary lymphocytes combines inhibitory effects at the entry and postentry levels and imply the involvement of beta-chemokine-induced signaling in postentry inhibition of HIV-1 infection.

FIG. 1.

β-Chemokines inhibit postentry steps of HIV-1 infection in primary lymphocytes. (A) PHA-activated lymphocytes were infected with HIV-1 ADA and treated with 200 ng/ml of MIP-1α, MIP-1β, or RANTES before, during, and after (Pre+post), before and during (Pre), or only after (Post) infection. Cell lysates were prepared 24 h later and analyzed by PCR using primers specific for the HIV-1 pol gene. Amplification of the α-tubulin gene was used to control for the amount of DNA. Data are expressed in counts per minute (cpm) and represent the average of four experimental samples (obtained from two independent experiments) ± standard deviation. Dilutions of 8E5/LAI cells containing one HIV-1 genome per cell were used as PCR standards (bottom panel). (B) Quiescent lymphocytes were infected with primary isolate HIV-1 92US660. Three days later, cells were activated with anti-CD3 and treated with β-chemokines according to the schematic (upper panel). HIV-1-specific pol transcripts were analyzed by PCR. Data show results of one representative experiment out of three, each performed in duplicate. Standard deviations are indicated as vertical bars (lower panel).

FIG. 2.

Donor-dependent variation in the magnitude of β-chemokine-mediated postentry inhibition of HIV-1. Lymphocytes isolated from five different blood donors were infected with HIV-1 92US660. After infection, cells were treated with 200 ng/ml of MIP-1α, MIP-1β, or RANTES. Cell lysates prepared 24 h later were analyzed by PCR using primers specific for the HIV-1 pol gene. Data are expressed in counts per minute (cpm) and represent an average of two experimental samples.

FIG. 3.

MIP-1α-mediated postentry inhibition of HIV-1 is dose dependent. Lymphocytes were infected with HIV-1 92US660 for 2 h. After infection, cells were treated with the indicated concentrations of MIP-1α. Cell lysates prepared 24 h later were analyzed by PCR using primers specific for the HIV-1 pol gene. Data are expressed in counts per minute (cpm) and show results of one representative experiment out of three, each performed in duplicate.

FIG. 4.

PTX abolishes MIP-1α-mediated inhibition of HIV-1-specific DNA synthesis. Lymphocytes were treated with 500 ng/ml of PTX for 18 h before infection with HIV-1 92US660 and treatment with MIP-1α. Cell lysates were prepared 24 h later and analyzed by PCR. Data show means ± standard deviations of HIV-1-specific pol gene transcript levels from one representative experiment out of two.

FIG. 5.

Exogenously supplied cAMP overrides the inhibitory effect of MIP-1α. Lymphocytes infected with HIV-1 92US660 were treated with the indicated concentrations of caged decyl cAMP and cultivated for 24 h in the presence or absence of MIP-1α. Synthesis of HIV-1-specific pol transcripts was analyzed by PCR. Each bar represents the mean ± standard deviation of HIV-1-specific pol gene transcript levels from one representative experiment out of two.

FIG. 6.

Activation-induced levels of intracellular cAMP correlate with susceptibility of HIV-1 to MIP-1α-mediated postentry inhibition. (A) Donor-dependent variability of intracellular cAMP levels after lymphocyte activation. Intracellular cAMP levels were measured in cell lysates prepared from lymphocytes isolated from five different blood donors 3 days after activation with PHA. Each bar represents the mean ± standard deviation of cAMP levels measured in samples isolated from the same donor. (B) Regression analysis of the correlation between activation-induced intracellular cAMP levels and MIP-1α-mediated postentry inhibition of HIV-1-specific DNA synthesis in lymphocytes isolated from 10 blood donors. Lymphocytes were activated with PHA for 3 days. Afterwards, half of the cells were lysed and analyzed for intracellular cAMP. The other half of the cells were infected with HIV-1 92US660 and treated with MIP-1α after infection. Cell lysates were prepared 24 h later and analyzed by PCR using primers specific for the HIV-1 pol gene. Percent inhibition was calculated for MIP-1α-treated samples relative to untreated controls and plotted against cAMP concentrations determined in cells isolated from the same donor. Each point represents the average of at least two experimental values obtained with cells isolated from the same donor. (C) PHA-activated lymphocytes isolated from seven blood donors were incubated with HIV-1 92US660 for 1 h at 4°C before treatment with MIP-1α. Intracellular cAMP was measured in cell lysates prepared 2 h after MIP-1α treatment. Data are a regression analysis of the dependence of MIP-1α-mediated decrease of intracellular cAMP on initial cAMP levels after HIV-1 infection. Each point represents the average of at least two experimental values obtained with cells isolated from the same donor.

FIG. 7.

Inhibition of cAMP-dependent PKA suppresses synthesis of HIV-1-specific DNA without affecting virus entry. (A) Activated lymphocytes were pretreated with the PKA inhibitors PKA 14-22 (0.1 μM and 1 μM) and H89 (1 μM and 10 μM; DMSO soluble) 1 h before infection with HIV-1 92US660. Infected and DMSO-treated samples served as a control for H89 treatment. Synthesis of HIV-1-specific pol transcripts was analyzed 24 h later. Amplification of the α-tubulin gene was used to control for the amount of DNA. (B) PKA inhibitors do not affect HIV-1 entry into primary lymphocytes. Activated lymphocytes pretreated with PKA inhibitors and infected with HIV-1 92US660 were analyzed for HIV-1-specific strong-stop DNA (LTR RU5) 2 h after infection. MIP-1α-treated samples served as a control. Amplification of the α-tubulin gene was used to control for the amount of DNA. Data show results of one representative experiment out of three, each performed in duplicate.

FIG. 8.

Mechanistic model for the antiviral activity of MIP-1α. Cell activation increases activity of G_s proteins, activators of adenylyl cyclase (AC), resulting in increased intracellular cAMP, and activation of cAMP-dependent PKA, which enhances synthesis of HIV-1-specific DNA. The presence of MIP-1α during infection results in a substantial decrease in the amount of the virus entering the cells, as they compete with the virus for CCR5 binding and induce CCR5 internalization (1). Still, a small amount of virus enters cells in the presence of β-chemokine. These viruses, however, might not proceed efficiently through the early steps of viral DNA synthesis, considering the involvement of intracellular cAMP, which is suppressed by MIP-1α-mediated activation of G_i proteins, in the enhancement of synthesis of proviral DNA (2).