Methylation of Tat by PRMT6 regulates human immunodeficiency virus type 1 gene expression

Abstract

The human immunodeficiency virus (HIV) transactivator protein, Tat, stimulates transcription from the viral long terminal repeats via an arginine-rich transactivating domain. Since arginines are often known to be methylated, we investigated whether HIV type 1 (HIV-1) Tat was a substrate for known protein arginine methyltransferases (PRMTs). Here we identify Tat as a substrate for the arginine methyltransferase, PRMT6. Tat is specifically associated with and methylated by PRMT6 within cells. Overexpression of wild-type PRMT6, but not a methylase-inactive PRMT6 mutant, decreased Tat transactivation of an HIV-1 long terminal repeat luciferase reporter plasmid in a dose-dependent manner. Knocking down PRMT6 consistently increased HIV-1 production in HEK293T cells and also led to increased viral infectiousness as shown in multinuclear activation of a galactosidase indicator assays. Our study demonstrates that arginine methylation of Tat negatively regulates its transactivation activity and that PRMT6 acts as a restriction factor for HIV replication.

FIG. 1.

(A) Arginine methylation of Tat by PRMT6. Histidine-tagged recombinant Tat (HIS-Tat) was incubated with [methyl-³H]S-adenosyl-l-methionine in the absence (lanes 1 and 5) and presence of recombinant GST-CARM1 (lanes 2 and 6), GST-PRMT6 (lanes 3 and 7), and GST-PRMT7 (lanes 4 and 8). The proteins were separated by SDS-PAGE, and the gels were stained with Coomassie blue. The incorporation of ³H label on Tat was visualized by fluorography. The migration positions of the GST-PRMTs and HIS-Tat are indicated to the left of the gel. (B) In vivo methylation assay of Tat. HA epitope-tagged Tat (HA-Tat) was transfected with either empty pVAX vector or vector expressing PRMT6 or methyltransferase-inactive PRMT6 (VLD-KLA). The transfected cells were metabolically labeled with l-[methyl-³H]methionine. HA-Tat was immunopurified, bound HA-Tat was resolved by SDS-PAGE, and labeled Tat was detected by fluorography. HA-Tat was also transfected alone, and the cells were incubated with l-[³⁵S]methionine to ensure that protein synthesis was indeed inhibited (lane 4). (C) PRMT6 methylates the ARM. A schematic diagram of Tat with its cysteine-rich core (Cys-rich peptide Tat 25-39 [negative control]) and ARM (peptide 49-63). The sequences of the TAT peptides used for methylation are shown. The peptides were incubated with [methyl-³H]S-adenosyl-l-methionine in the presence of recombinant GST-PRMT6, and ³H incorporation was visualized by SDS-PAGE followed by fluorography.

FIG. 2.

Interaction of Tat with PRMT6. HeLa cells were transfected with expression vectors encoding HA-Tat and myc-PRMT6. The cells were lysed, and an aliquot was retained as total cell lysate (TCL). Cell extracts were incubated with either control immunoglobulin G (IgG) or with anti-HA or anti-Myc antibodies as indicated. The bound proteins were separated by SDS-PAGE and immunoblotted with anti-Myc (lanes 1 to 3) and anti-HA (lanes 4 to 6) antibodies. The migration of myc-PRMT6 and HA-Tat is shown. The bands at ∼60 kDa are the heavy chains of the antibodies. The bands in lane 4 above 55 kDa are often observed with our anti-HA (12CA5) antibodies and are nonspecific.

FIG. 3.

PRMT6 expression inhibits Tat-mediated transactivation of the HIV-LTR promoter. HEK293T cells were transfected with the HIV LTR luciferase reporter plasmid along with expression vectors for HA-Tat, PRMT6 (A), and methylase-inactive PRMT6:VLD-KLA (B) or PRMT6 siRNA (C) as indicated. In panels A and B, the amount (0.5 and 1.0 μg) of PRMT6 or PRMT6:VLD-KLA is indicated by the thickness of the triangle below the bar. PRL-TK was included to control for transfection efficiency. The transfected cells were lysed 48 h after DNA transfection and assayed for luciferase activity, which was normalized to the activity of renilla. The luciferase activity without Tat was normalized to 1, and Tat induction is shown as fold induction. The results shown represent the mean ± standard error of the mean from three separate experiments for each bar (n > 9).

FIG. 4.

Reduced PRMT6 expression enhances HIV-1 LTR expression. HeLa MAGI cells were transfected with mock siRNA or PRMT6 siRNA. After 48 h, the cells were lysed and the mRNA (A) and protein (B) extracts were analyzed by RT-PCR and immunoblotting with anti-PRMT6 or anti-Sam68 antibodies. The presence (+RT) or absence (−RT) of reverse transcriptase is indicated above the lanes. gapdh, glyceraldehyde-3-phosphate dehydrogenase. (C) HeLa MAGI cells containing a stably integrated copy of HIV-1 LTR-β-galactosidase and expressing the CD4 receptor were transfected with mock siRNA or PRMT6 siRNA. At 24 h posttransfection, the cells were infected with 2 or 10 ng of T-tropic HIV-1. The cells were incubated for 48 h, fixed, and stained with X-Gal. The bars represent the number of blue cells counted in three or four nonoverlapping fields.

FIG. 5.

HIVp24 production is increased with the knockdown of PRMT6 and decreased with PRMT6 overexpression. HEK293T cells were transfected with the BH10 proviral HIV-1 plasmid along with mock siRNA, PRMT6 siRNA, or myc-PRMT6 expression vector. Forty-eight hours after transfection, the knockdown of PRMT6 was examined by immunoblotting with anti-PRMT6 antibodies (A) or anti-Myc antibodies (B). The same membranes were also immunoblotted with anti-Sam68 antibodies to verify equivalent loading. (C) HIV-1 production was assessed by measuring the level of HIV p24 present in the supernatant of the transfected cells by an ELISA after transfection with PRMT6 siRNA or expression vectors. The results shown represent the mean ± standard error of the mean from three separate experiments (n > 6).