Functional domains of Tat required for efficient human immunodeficiency virus type 1 reverse transcription

Abstract

Tat expression is required for efficient human immunodeficiency virus type 1 (HIV-1) reverse transcription. In the present study, we generated a series of 293 cell lines that contained a provirus with a tat gene deletion (Deltatat). Cell lines that contained Deltatat and stably transfected vectors containing either wild-type tat or a number of tat mutants were obtained so that the abilities of these tat genes to stimulate HIV-1 gene expression and reverse transcription could be compared. tat genes with mutations in the amino terminus did not stimulate either viral gene expression or HIV-1 reverse transcription. In contrast, tat mutants in the activation, core, and basic domains of Tat did not stimulate HIV-1 gene expression but markedly stimulated HIV-1 reverse transcription. No differences in the levels of virion genomic RNA or tRNA3Lys were seen in the HIV-1 Deltatat viruses complemented with either mutant or wild-type tat. Finally, overexpression of the Tat-associated kinases CDK7 and CDK9, which are involved in Tat activation of HIV-1 transcription, was not able to complement the reverse transcription defects associated with the lack of a functional tat gene. These results indicate that the mechanism by which tat modulates HIV-1 reverse transcription is distinct from its ability to activate HIV-1 gene expression.

FIG. 1

Schematic of the first exon of the HIV-1 Tat protein. The amino acid changes are shown boxed below the native amino acid sequence. Multiple mutations are indicated by solid lines between boxed amino acids. The mutations in the tat gene product which were constructed are [E2G, D5G, E9G], P3L, P[6, 10]L, P[10, 14]L, C27S, K41A, and K/R[50-57]G.

FIG. 2

Activation of HIV-1 gene expression by Tat. HeLa cells were cotransfected with the reporter constructs HIV-1 LTR-CAT and pCH110 (β-Gal) together with plasmids (pDex) that expressed either the β-globin gene (bar 1), the wild type tat gene (bar 2), or the mutated tat genes corresponding to [E2G, D5G, E9G] (bar 3), P3L (bar 4), P[6, 10]L (bar 5), P[10, 14]L (bar 6), C27S (bar 7), K41A (bar 8), and K/R[50-57]G (bar 9). The cells were harvested at 48 h posttransfection, and equal amounts of protein were normalized to β-Gal activity and assayed for CAT protein by ELISA. The transfections were performed three times with the standard deviations indicated.

FIG. 3

RT-PCR analysis of wild-type and mutant tat genes. Total RNA was obtained from uninfected 293 cells (lanes 1), 293 cells stably transfected with HIV-1 wild-type (lanes 2) or HIV-1 Δtat (lanes 3), and 293 cells containing both HIV-1 Δtat and wild type tat (lanes 4) or the mutated tat genes corresponding to [E2G, D5G, E9G], P3L, P[6, 10]L, P[10, 14]L, C27S, K41A, and K/R[50-57]G (lanes 5 to 11, respectively). Primers specific for plasmid-derived tat mRNA or cellular β-actin mRNA were annealed to RNA obtained from each of the 293 cell lines, and a reverse transcription reaction was performed in the presence (A and B) or absence (C and D) of M-MLV RT. PCR was performed on each cDNA reaction mixture to detect either the tat (A and C) or β-actin (B and D) gene. PCR products were resolved on a 1.5% agarose gel. Molecular mass markers are shown for each gel (lanes M). PCRs with a plasmid containing the tat gene (panel A, lanes 12 and 13) (equivalent to 0.1 and 0.5 pg) or serially diluted β-globin cDNA (panel B, lanes 12 and 13) are shown.

FIG. 4

Analysis of HIV-1 gene expression from 293 cells. Culture supernatants were obtained from either 293 cells (bars 1), 293 cells stably infected with HIV-1 wild-type virus (bars 2), 293 cells infected with an HIV-1 Δtat virus (bars 3), or the Δtat cell line stably transfected with pBK-RSV containing the wild-type tat gene (bars 4) or the mutated tat genes corresponding to [E2G, D5G, E9G] (bars 5), P3L (bars 6), P[6, 10]L (bars 7), P[10, 14]L (bars 8), C27S (bars 9), K41A (bars 10), and K/R[50-57]G (bars 11). The amounts of p24 Ag and reverse transcriptase activity in each virus stock were determined as described in Materials and Methods. The data from four to six independent virus stocks were averaged and the standard deviation for each assay indicated.

FIG. 5

Reverse transcription of HIV-1 lacking tat. Activated PBMCs were infected for 2 h with culture supernatant from transfected 293 cells containing 90 mU of RT activity for either wild-type HIV-1 (lanes 2), Δtat virus (lanes 3), or virus produced from 293 HIV-1 Δtat cells stably transfected with an RSV expression vector containing the wild-type tat gene (lanes 4) or the mutated tat genes corresponding to [E2G, D5G, E9G], P3L, P[6, 10]L, P[10, 14]L, C27S, K41A, and K/R[50-57]G (panels A to C and E, lanes 5 to 11, respectively), and mock supernatant (panels A to C and E, lanes 1). PBMCs were infected with aliquots of the same viruses that were heat inactivated at 60°C (D). At 2 h postinfection, residual virus was removed and Hirt lysates were prepared from half of the infected cells, while the remaining PBMCs were cultured for 24 h before Hirt lysates were prepared. The recovered nucleic acids were assayed for HIV-1 negative-strand strong-stop DNA in 2-h (A) and 24-h (B) lysates and for full-length DNA in 24-h lysates (C) by quantitative PCR. PCR analysis of the Cyt-OxyII content in Hirt lysates was used to standardize the DNA recovery (E). All PCRs were performed within the linear range of the assay as determined by assays of HIV-1 DNA copy number (10, 10², 10³, and 10⁴) or cell number (4 × 10², 2 × 10³, 1 × 10⁴, and 5 × 10⁴). This analysis is representative of PCRs performed for four separate infections with independently prepared virus stocks.

FIG. 6

NERT assay for HIV-1 wild-type and tat mutant viruses. Virus stocks for wild-type virus (lanes 1), Δtat virus trans-complemented with wild-type tat (lanes 2), Δtat virus (lanes 3), or Δtat virus produced in the presence of tat mutants [E2G, D5G, E9G], P3L, P[6, 10]L, P[10, 14]L, C27S, K41A, and K/R[50-57]G (lanes 4 to 10, respectively) were analyzed for endogenous reverse transcription. Culture supernatant (200 μl) containing approximately 0.75 mU of RT activity was treated with 100 U of DNase I. Half of each reaction mixture was added to 150 μl of stop solution, incubated at 37°C for 10 min, and then boiled for 10 min (B). The remaining half of each reaction mixture was supplemented with 50 μM dNTPs and incubated at 37°C for 90 minutes before the reaction was terminated as described above. (A) PCR to detect HIV-1 negative-strand strong-stop DNA was performed on NERT reaction mixtures as described in Materials and Methods. All PCRs were performed within the linear range of the assay as determined by assays of HIV-1 DNA copy number (10, 10², 10³, and 10⁴).

FIG. 7

Cyclin-dependent kinases do not complement reverse transcription defects associated with Δtat viruses. (A) Viral supernatants from 293 cells producing Δtat virus (lanes 1 to 5) or wild-type HIV-1 (lanes 6 to 10) following transfection of wild-type tat (lanes 1 and 6), an empty RSV expression vector (lanes 2 and 7), a wild-type cdk7 expression vector (lanes 3 and 8), a wild-type cdk9 expression vector (lanes 4 and 9), a wild-type cdc5 expression vector (lanes 5 and 10), mock supernatant (lane 11), or heat-inactivated wild-type HIV-1 (lane 12) were used to infect 5 × 10⁶ activated PBMCs. At 2 h postinfection, residual virus was removed by washing, and Hirt lysates were prepared at 24 h postinfection. The recovered nucleic acids were assayed for HIV-1 negative-strand strong-stop DNA. (B) Quantitative PCR analysis of Cyt-OxyII content in Hirt lysates was used to standardize the DNA recovery. All PCRs were performed within the linear range as determined by assays of HIV-1 DNA copy number (0, 10, 50, 250, and 1,000). This analysis is representative of PCRs performed for three separate HIV-1 infections with independently prepared virus stocks.

FIG. 8

Analysis of genomic RNA packaging. (A) Supernatants containing wild-type virus (lanes 10 to 12), Δtat virus (lanes 1 to 3), or Δtat virus complemented with wild-type tat (lanes 7 to 9) or [E2G, D5G, E9G] (lanes 4 to 6) or mock complemented (lanes 13 to 16) were pelleted through 20% sucrose and suspended in PBS-BSA buffer. An IC RNA was added to purified virus that contained 100 ng of p24 Ag, and both RNAs were copurified. cDNA reactions were performed in either the presence or absence of M-MLV with a first-strand primer that annealed to sequences located downstream from the Gag initiating methionine shown in panel C. The cDNA was serially diluted in fivefold increments and assayed by PCR for HIV-1 DNA with primers indicated in panel C. PCRs were performed on HIV-1 DNA present at 0, 10¹, 10², 10³, and 10⁴ copies (lanes 16 to 20). (B) The RNA recovery and cDNA synthesis were similar for each cDNA reaction corresponding to Δtat (lanes 1 and 2), Δtat plus [E2G, D5G, E9G] (lanes 3 and 4), Δtat plus wild-type tat (lanes 5 and 6), wild-type virus (lanes 7 and 8), and mock virus (lanes 9 and 10). IC RNA was reverse transcribed in either the presence (lanes 1, 3, 5, 7, and 9) or absence (lanes 2, 4, 6, 8, and 10) of M-MLV and detected by PCR with the primers shown in panel C (dotted lines). IC plasmid DNA standards present at 20, 100, 300, and 1,000 copies are shown (lanes 11 to 14). (C) Model showing HIV-1 RNA and IC RNA. An internal deletion from +80 to +151 in IC RNA allows detection of IC cDNA from HIV-1 cDNA by PCR with the indicated primers. Solid arrow, first-strand cDNA primer; dotted arrows, PCR primers; dotted line, pGem4Z RNA; solid line, HIV-1 RNA.

FIG. 9

Analysis of tRNA₃^Lys packaging in wild-type and tat mutant viruses. RNA was extracted from pelleted virus that contained 100 ng of p24 Ag, and cDNA was synthesized in the presence (+) or absence (−) of AMV RT primed with an antisense oligonucleotide that hybridized to either the 3′-terminal 18 nucleotides of the tRNA₃^Lys molecule (A) or HIV-1 sequences extending from +242 to +219 (B). (A) tRNA₃^Lys cDNA was detected by PCR with primers that hybridize to internal tRNA₃^Lys sequences. Total HeLa cell RNA (lanes 1 and 2) or wild-type HIV-1 (lanes 3 and 4), Δtat virus (lanes 5 and 6), and Δtat virus produced following transfection with a wild-type tat expression vector (lanes 7 and 8) contain similar amounts of tRNA₃^Lys. An in vitro-transcribed tRNA₃^Lys molecule was added as a positive control for the reactions (lanes 10 and 11). A PCR-negative control is shown in lane 9. (B) As a control for virus load, HIV-1 cDNA was detected by PCR with a nested antisense primer corresponding to HIV-1 sequences +236 to +214 and a sense primer corresponding to +96 to +118 for HeLa cells (lanes 1 and 2), wild-type HIV-1 (lanes 3 and 4), Δtat virus (lanes 5 and 6), or Δtat virus produced following transfection with a wild-type tat expression vector (lanes 7 and 8). A negative PCR control is shown in lane 9.