Regulation of human immunodeficiency virus type 1 infectivity by the ERK mitogen-activated protein kinase signaling pathway

Abstract

ERK1 and ERK2 mitogen-activated protein kinases (MAPK) play a critical role in regulation of cell proliferation and differentiation in response to mitogens and other extracellular stimuli. Mitogens and cytokines that activate MAPK in T cells have been shown to activate human immunodeficiency virus type 1 (HIV-1) replication. Little is known about the signal transduction pathways that activate HIV-1 replication in T cells upon activation by extracellular stimulation. Here, we report that activation of MAPK through the Ras/Raf/MEK signaling pathway enhances the infectivity of HIV-1 virions. Virus infectivity was enhanced by treatment of cells with MAPK stimulators, such as serum and phorbol myristate acetate, as well as by coexpression of constitutively activated Ras, Raf, or MEK (MAPK kinase) in the absence of extracellular stimulation. Treatment of cells with PD 098059, a specific inhibitor of MAPK activation, or with a MAPK antisense oligonucleotide reduced the infectivity of HIV-1 virions without significantly affecting virus production or the levels of virion-associated Gag and Env proteins. MAPK has been shown to regulate HIV-1 infectivity by phosphorylating Vif (X. Yang and D. Gabuzda, J. Biol. Chem. 273:29879-29887, 1998). However, MAPK activation enhanced virus infectivity in some cells lines that do not require Vif function. The HIV-1 Rev, Tat, p17(Gag), and Nef proteins were directly phosphorylated by MAPK in vitro, suggesting that other HIV-1 proteins are potential substrates for MAPK phosphorylation. These results suggest that activation of the ERK MAPK pathway plays a role in HIV-1 replication by enhancing the infectivity of HIV-1 virions through Vif-dependent as well as Vif-independent mechanisms. MAPK activation in producer cells may contribute to the activation of HIV-1 replication when T cells are activated by mitogens and other extracellular stimuli.

Figures

FIG. 1

Inhibition of HIV-1 infectivity by the MEK inhibitor PD 098059 (PD). H9 or SupT1 cells chronically infected with the HXB2 HIV-1 isolate were serum starved for 24 h, pretreated with the indicated concentrations of PD for 30 min, and then stimulated by addition of 20% FCS or 100 nM PMA. After incubation for 4 h (SupT1) or 6 h (H9) at 37°C, virus production was quantitated by measuring RT activity in the culture supernatants and virions were pelleted, resuspended in fresh medium, and used to infect fresh SupT1 cells cultured in media with 10% FCS. (A) Virus production in culture supernatants of chronically infected H9 and SupT1 cells. (B) Virus infectivity was determined by quantitation of RT activity in the supernatants of the acutely infected cells at the indicated time points. Values shown are expressed as 3H counts per minute per milliliter and are expressed as the means ± standard deviations of duplicates. As shown in panels C and D, PD inhibits MAPK activation by serum and PMA. (C) HIV-1-infected SupT1 cells were serum starved for 24 h and then stimulated for 4 h with 20% serum or 100 nM PMA in the presence or absence of PD at the indicated concentrations. For PMA-treated cells, PD was used at 50 μM. p44/42 MAPK activity and total MAPK expression levels were detected by immunoblotting equivalent amounts of protein in cell lysates with rabbit anti-phosphorylated MAPK (1:1,000 dilution; New England Biolabs) and rabbit anti-MAPK (anti-ERK1 and anti-ERK2, 1:1,500 dilution of each; Santa Cruz Biotechnology, Inc.), respectively, using the ECL system (Amersham) as described previously (55). (D) In vitro kinase assay for MAPK activity in cell lysates from SupT1 cells stimulated for 1.5 h with serum or PMA as described in panel C, using MBP as the MAPK substrate, was performed as described previously (55). DMSO, dimethyl sulfoxide.

FIG. 1

Inhibition of HIV-1 infectivity by the MEK inhibitor PD 098059 (PD). H9 or SupT1 cells chronically infected with the HXB2 HIV-1 isolate were serum starved for 24 h, pretreated with the indicated concentrations of PD for 30 min, and then stimulated by addition of 20% FCS or 100 nM PMA. After incubation for 4 h (SupT1) or 6 h (H9) at 37°C, virus production was quantitated by measuring RT activity in the culture supernatants and virions were pelleted, resuspended in fresh medium, and used to infect fresh SupT1 cells cultured in media with 10% FCS. (A) Virus production in culture supernatants of chronically infected H9 and SupT1 cells. (B) Virus infectivity was determined by quantitation of RT activity in the supernatants of the acutely infected cells at the indicated time points. Values shown are expressed as 3H counts per minute per milliliter and are expressed as the means ± standard deviations of duplicates. As shown in panels C and D, PD inhibits MAPK activation by serum and PMA. (C) HIV-1-infected SupT1 cells were serum starved for 24 h and then stimulated for 4 h with 20% serum or 100 nM PMA in the presence or absence of PD at the indicated concentrations. For PMA-treated cells, PD was used at 50 μM. p44/42 MAPK activity and total MAPK expression levels were detected by immunoblotting equivalent amounts of protein in cell lysates with rabbit anti-phosphorylated MAPK (1:1,000 dilution; New England Biolabs) and rabbit anti-MAPK (anti-ERK1 and anti-ERK2, 1:1,500 dilution of each; Santa Cruz Biotechnology, Inc.), respectively, using the ECL system (Amersham) as described previously (55). (D) In vitro kinase assay for MAPK activity in cell lysates from SupT1 cells stimulated for 1.5 h with serum or PMA as described in panel C, using MBP as the MAPK substrate, was performed as described previously (55). DMSO, dimethyl sulfoxide.

FIG. 2

Activation or inhibition of MAPK does not affect the levels of virion-associated Gag and Env proteins. Equivalent amounts of HIV-1 virions (15,000 cpm RT units) were pelleted from the supernatants of chronically infected SupT1 cells after serum starvation followed by stimulation with 20% FCS or 100 nM PMA in the presence or absence of PD 098059 as in the experiment for which results are presented in Fig. 1, lysed, and analyzed for the levels of virion-associated gp120Env, p24Gag, and p17Gag proteins by immunoblotting with rabbit anti-gp120Env (1:1,000 dilution; a gift of Richard Wyatt and Joseph Sodroski), rabbit anti-p24Gag (1:2,000 dilution; Intracell), or mouse monoclonal anti-p17Gag (1:2,000 dilution; Advanced Biotechnologies Inc.) as described in the legend for Fig. 1. Lanes 1 through 6 contain proteins from cells treated the same as indicated for the lanes (from left to right) in Fig. 1C.

FIG. 3

Coexpression of constitutively activated Ras, Raf, or MEK enhances the infectivity of HIV-1 virions. (A) p44/42 MAPK activity (upper panel) and total MAPK expression levels (lower panel) in 293T cells transfected with constitutively active kinase mutants (MEK-R4F, Ras-12V, or Raf-BXB) or control vector pSG5 (Stratagene) DNA (control) and pNL4-3 containing HIV-1 proviral DNA by the calcium phosphate method. After transfection, cells were incubated with Dulbecco’s minimal essential medium containing 0.5% serum. At 48 h after transfection, cell lysates were prepared and equivalent amounts of proteins were subjected to electrophoresis and immunoblotting with anti-phosphorylated MAPK (α-phospho-MAPK) or anti-MAPK (anti-ERK1 and anti-ERK2) (α-MAPK). (B) MAGI assays were performed as described previously (28, 40) to quantitate the infectivity of HIV-1 virions produced in 293T cells under the same experimental conditions as used in the experiment for which activities are presented in panel A. Two days after transfection, culture supernatants containing virus were collected, passed through a 0.45-μm-pore-size filter, and assayed for RT activity. Equivalent amounts of virus stock (10,000 cpm RT units) were used to infect HeLa CD4-LTR/β-galactosidase indicator cells. After overnight incubation at 37°C, the medium was replaced with fresh medium. At 48 h after infection, cells were stained for β-galactosidase expression (28, 40). The total number of cells with blue nuclei in 16 random fields were counted under a microscope by using a 10× objective. A cluster of cells containing multiple blue nuclei was counted as one syncytium. Results are expressed as the means ± standard deviations of duplicates.

FIG. 4

Inhibition of HIV-1 infectivity by a MAPK antisense oligonucleotide (AS) or scrambled control oligonucleotide (SC). (A) Depletion of p44/42 MAPK expression by AS. HeLa cells were treated with AS or SC at the indicated concentrations. HeLa cells were grown to 80% confluence in Dulbecco’s minimal essential medium (DMEM) containing 10% FCS in 35-mm-diameter six-well plates, and washed twice with serum-free DMEM. Appropriate concentrations of oligonucleotides in 125 μl of serum-free DMEM were preincubated at room temperature for 15 min with 125 μl of serum-free DMEM containing 40 μg of DOTMA (Lipofectin) (Gibco-BRL) per ml. This solution was then mixed with an additional 250 μl of serum-free DMEM. Cells were incubated with the mixture for 6 h at 37°C in the presence of 5% CO2. Subsequently, the medium containing DOTMA was removed and the incubation was continued for 48 h in fresh medium containing 10% FCS. After 48 h incubation, MAPK expression was determined by immunoblotting of equivalent amounts of total protein with anti-MAPK (anti-ERK1 [p44] and anti-ERK2 [p42]). (B) HeLa cells were transfected with 3 μg of pHXB2 containing HIV-1 proviral DNA following depletion of MAPK by treatment with MAPK antisense oligonucleotide as described for panel A. Virus production was determined by quantitation of RT activity in the supernatants of the transfected HeLa cell cultures at 48 h after transfection. Virus infectivity was determined by infection of CEM cells and quantitation of RT activity in the supernatants of the newly infected cells on day 7 after infection. (C) Treatment with AS does not inhibit expression of an HIV-1 LTR CAT reporter plasmid transactivated by coexpression of HIV-1 Tat. HeLa cells were treated with the oligonucleotides at 0.2 μM and cotransfected with 0.5 μg of pUIIIRCAT and 0 or 0.5 μg of the HIV-1 Tat expressor plasmid pSVLtat by the same method as described for panels A and B. Shown is CAT activity in the transfected cell lysate at 48 h posttransfection, as determined by the conversion of chloramphenicol to its acetylated forms.

FIG. 5

Phosphorylation of HIV-1 proteins by MAPK. In vitro kinase assays were performed using recombinant p42 MAPK (A) or MAPK immunoprecipitated from COS-1 cells stimulated with PMA (B). MAPK immunocomplexes were isolated from cell lysates by using anti-ERK1 and anti-ERK2. MAPK was incubated with 2 μg of each indicated recombinant HIV-1 protein for in vitro kinase assays performed in the presence of [γ-32P]ATP. Recombinant Rev and p7Gag were from the NIH AIDS Research and Reference Reagent Program, Tat and p24Gag were from Immunodiagnostics, Inc. (kindly provided by J. Raina), and p17Gag and Nef were from Intracell. Vif, Rev, Tat, p24Gag, and p7Gag were derived from the HXB2 HIV-1 isolate, p7Gag was derived from the MN HIV-1 isolate, and Nef was derived from the LAI HIV-1 isolate. The results of phosphorylated amino acid analysis performed as described previously (54) are shown on the right. The positions of phosphorylated Ser, Thr, and Tyr (pSer, pThr, and pTyr) markers are indicated. Phosphorylated amino acids detected in the recombinant HIV-1 proteins comigrated with pSer and pThr but not with pTyr.