Human immunodeficiency virus type 1 tat protein activates transcription factor NF-kappaB through the cellular interferon-inducible, double-stranded RNA-dependent protein kinase, PKR

Abstract

The transactivator protein of human immunodeficiency virus type 1 (HIV-1) (Tat) is a powerful activator of nuclear factor-kappaB (NF-kappaB), acting through degradation of the inhibitor IkappaB-alpha (F. Demarchi, F. d'Adda di Fagagna, A. Falaschi, and M. Giacca, J. Virol. 70:4427-4437, 1996). Here, we show that this activity of Tat requires the function of the cellular interferon-inducible protein kinase PKR. Tat-mediated NF-kappaB activation and transcriptional induction of the HIV-1 long terminal repeat were impaired in murine cells in which the PKR gene was knocked out. Both functions were restored by cotransfection of Tat with the cDNA for PKR. Expression of a dominant-negative mutant of PKR specifically reduced the levels of Tat transactivation in different human cell types. Activation of NF-kappaB by Tat required integrity of the basic domain of Tat; previous studies have indicated that this domain is necessary for specific Tat-PKR interaction.

FIG. 1

Tat proteins and mutants. The Tat protein of HIV-1 and its functional domains are schematically shown. Tat 101 is the full-length, two-exon Tat of most clinical isolates; Tat 86, lacking 15 amino acids at the C terminus, derives from clone HXB2 and is fully active for LTR transcription activation. The mutant proteins include Tat 86 Δ(1-21), which has a truncation in the first 21 amino acids; Tat 86 C(22-27)A, in which cysteines 22, 25, and 27 were mutated to alanines; and Tat 86 R(49-57)A, in which arginines at positions 49, 52, 53, 55, 56, and 57 were mutated to alanines. Asterisks indicate the positions of the mutated amino acids.

FIG. 2

The basic domain of Tat is required for NF-κB activation. The figure shows the results of gel retardation analysis of NF-κB complexes present in nuclear extracts of HL3T1 cells either treated with wild-type and mutant Tat proteins (A) or transfected with plasmids expressing the respective cDNAs (B). The arrows indicate the specific NF-κB- or USF-containing complexes, or the free probe, as specified. The asterisks mark unspecific bands. The specificity and identity of the NF-κB-containing complex have been previously verified (16). (A) Seven micrograms of nuclear extracts from control cells (lane 7) or from cells in which 9 μg of GST protein/15-cm plate (lane 6), or the same amount of the Tat 86 protein (lane 1) and mutant derivatives (lanes 3 to 6), had been delivered by lipofection was used in a gel retardation assay with a γ-³²P-labeled double-stranded oligonucleotide specific for NF-κB (16). (B) HL3T1 cells were transfected with 1 μg of empty vector/15-cm plate (lanes 1 and 2) or with 1 μg of expression vectors for Tat 86, Tat 86 Δ(1-21) (lane 4), and Tat 86 R(49-57)A (lane 5). Sixteen hours after transfection, serum concentration was lowered to 0.5% for an additional 24 h. Nuclear extracts were prepared and used in a gel retardation assay as described above. Two hours before harvesting, 10% serum was added to control cells (lane 2).

FIG. 3

Gel retardation analysis of NF-κB complexes induced by Tat in NIH 3T3 and PKR knockout mouse cells. (A) NIH 3T3 cells plated in 15-cm dishes were transiently transfected with 5 μg of the different cDNA constructs as indicated in the figure. The control samples (lanes 1 to 3) were transfected with the empty vector pCDNAIII. After 16 h, cells were serum starved (0.5% serum) for 24 h to lower the endogenous NF-κB levels (37). Gel retardation assays were performed with 7.5 μg of nuclear extract by using an NF-κB-specific oligonucleotide probe. Two hours before cells harvesting, 10% serum was added to control cells in lane 2. Lane 1, endogenous levels of NF-κB in cycling (Cycl.) cells grown in 10% serum. (B) Results of transfection of primary mouse PKR^0/0 fibroblasts with plasmids expressing Tat 101 (lane 1), PKR (lane 2), or both (lane 3). Cells were transfected and serum starved as described above. Lane 4, same as lane 2 in panel A. (C) Results of supershift assay to ascertain the identity of the retarded band detected with the κB probe in gel retardation assays with nuclear extract from mouse PKR^0/0 fibroblasts. The assay was performed with antibodies against the p50 and p65 subunits of NF-κB as indicated in the figure and nuclear extracts from PKR^0/0 fibroblasts cotransfected with PKR and Tat. In all panels, the arrow indicates the specific NF-κB-containing complexes and the asterisks mark the unspecific band.

FIG. 4

PKR is functionally required for Tat transactivation of the LTR promoter in mouse fibroblasts. NIH 3T3 mouse cells and primary mouse fibroblasts (panels A and B, respectively) from PKR knockouts were transiently transfected with a reporter plasmid containing the LTR linked to the CAT gene. Five micrograms of Tat 101 and/or 5 μg of PKR expression constructs was cotransfected, as indicated. The empty vector pCDNAIII was added in order to keep constant the amount of transfected DNA. Forty-eight hours after transfection, cell extracts were prepared and monitored for CAT activity after normalization for transfection efficiency. The bars show the percentage of chloramphenicol acetylation obtained with each extract; the data are the averages of three independent experiments, and the standard deviations are indicated. Standardization for transfection efficiency was also obtained by cotransfection of 0.5 μg of a luciferase expression vector per 60-mm plate.

FIG. 5

Dominant-negative PKR impairs LTR activation induced by Tat. HL3T1, BF24, and Jurkat cells (panels A, B, and C, respectively) were transfected with 2.5 μg of pCDNAIII empty vector (first lanes of each panel) or with 500 ng of Tat 101 expression vector and 2 μg of the dominant-negative mutant cDNAs for Ras (plasmid Ras N17), Rac (Rac N17), RhoA (Rho T19N), CDC42 (Cdc42 T17N), and PKR (PKR-M) as indicated. Jurkat cells were also cotransfected with 1 μg of an LTR-CAT-containing plasmid. Twenty hours after transfection, cell extracts were prepared and used for CAT assays after normalization for transfection efficiency. The results are the mean values from three independent experiments, with error bars indicating standard deviations.

FIG. 6

An intact LTR enhancer region is required for downregulation of Tat transactivation by dominant-negative PKR. HeLa cells were transfected with the wild-type LTR-CAT reporter (1 μg; left panel) or plasmid pNFA-CAT (1 μg; right panel). The latter plasmid, described by Leonard et al. (30), was a kind gift of G. Scala and contains a deletion of the NF-κB sites of the LTR. These plasmids were cotransfected with 0.5 μg of pCDNAIII-Tat101 and 1 μg of pPKR-M, as indicated. The empty vector pCDNAIII was added in order to keep constant the amount of transfected DNA in each plate. Experiments were performed in duplicate.