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. 2001 Mar;75(6):2675-83.
doi: 10.1128/JVI.75.6.2675-2683.2001.

Role for human immunodeficiency virus type 1 Tat protein in suppression of viral reverse transcriptase activity during late stages of viral replication

M Kameoka  1 , L Rong, M Götte, C Liang, R S Russell, M A Wainberg
Affiliations

Role for human immunodeficiency virus type 1 Tat protein in suppression of viral reverse transcriptase activity during late stages of viral replication

M Kameoka et al. J Virol. 2001 Mar.

Abstract

We have examined the role of the human immunodeficiency virus type 1 (HIV-1) Tat protein in the regulation of reverse transcription. We show that a two-exon but not a one-exon form of Tat markedly suppressed cell-free reverse transcriptase (RT) activity. Conversely, viruses expressing two-exon Tat (pNL43 and pNL101) showed rapid replication kinetics and more efficient endogenous RT activity compared with viruses expressing one-exon Tat (pM1ex). The pM1ex virions, as well as pM1ex-infected cells, also contained higher levels of viral DNA than did either the pNL43 or pNL101 viruses, indicating that reverse transcription might have continued during later stages of viral replication in the absence of the second Tat exon. Moreover, degradation of viral genomic RNA was more apparent in the pM1ex virions. Accordingly, we propose that the two-exon Tat may help augment viral infectivity by suppressing the reverse transcription reaction during late stages of viral synthesis and by preventing the synthesis of potentially deleterious viral DNA products.

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Figures

FIG. 1
FIG. 1
(A) Tat 86 and Tat 101, but not Tat 72, suppress DNA synthesis primed with tRNAformula image. The annealed tRNAformula image (1 pmol)-template (2 pmol) complex and RT (3 pmol) were mixed with various amounts of Tat 72 (lanes 3 to 6), Tat 86 (lanes 7 to 10), or Tat 101 (lanes 11 to 14). Reverse transcription was then initiated and allowed to proceed at 37°C for 30 min as described in Materials and Methods. Lane 1 represents the control reaction without the addition of MgCl2. The amounts of Tat used in these reactions were 0 pmol (lanes 1 and 2), 3 pmol (lanes 3, 7, and 11), 10 pmol (lanes 4, 8, and 12), 30 pmol (lanes 5, 9, and 13), and 100 pmol (lanes 6, 10, and 14) per lane. (B) The polypeptide within the second exon of Tat cannot suppress the RT reaction. RT reactions were carried out as described in panel A. The amounts of the synthetic peptide, corresponding to aa 61 to 86 of Tat (Tat[61–86]), used in these experiments were 50 pmol (lane 1), 100 pmol (lane 2), and 200 pmol (lane 3) per reaction. (C) Tat suppresses cell-free RT reactions in a TAR-independent manner. RT reactions were carried out as described in panel A, except that TAR(−) (lanes 6 to 10) and TAR(+) (lanes 1 to 5) templates were used. The amounts of Tat 86 in these reactions were 0 pmol (lanes 1 and 6), 3 pmol (lanes 2 and 7), 10 pmol (lanes 3 and 8), 30 pmol (lanes 4 and 9), and 100 pmol (lanes 5 and 10) per lane. Reaction products were analyzed on 8% polyacrylamide–7 M urea gels and visualized by autoradiography. The runoff bands in panels A and B and the runoff (TAR+) and run-off (TAR−) bands in panel C indicate full-length synthesis of DNA, i.e., 239, 258, and 208 nt, respectively. The tRNAformula image band indicates unprocessed 32P-labeled tRNAformula image (76 nt).
FIG. 2
FIG. 2
Tat 86, but not Tat 72, suppresses DNA synthesis primed with a DNA primer in an in vitro RT reaction. The annealed DNA primer (2.5 pmol)-template (5.0 pmol) complex and RT (2.5 pmol) were mixed with various amounts of Tat 72 (lanes 2 to 5) or Tat 86 (lanes 6 to 9). The amounts of Tat preparations used were 0 pmol (lane 1), 7.5 pmol (lanes 2 and 6), 25 pmol (lanes 3 and 7), 75 pmol (lanes 4 and 8), and 250 pmol (lanes 5 and 9) per reaction. Note that the ratios of Tat to primer were 3:1 (lanes 2 and 6), 10:1 (lanes 3 and 7), 30:1 (lanes 4 and 8), and 100:1 (lanes 5 and 9). RT reactions were carried out as described in the legend to Fig. 1A, except that the reactions were allowed to proceed at 37°C for 10 min. The runoff and DNA primer bands indicate full-length products and unprocessed 32P-labeled DNA primer, respectively.
FIG. 3
FIG. 3
Tat forms a supershifted complex with primer-template and RT. The annealed, 32P-labeled tRNA (1 pmol)-template (2 pmol) complex was incubated with Tat 72 (lanes 2 to 4 and lanes 9 to 11) or Tat 86 (lanes 5 to 7 and lanes 12 to 14) in the presence (lanes 8 to 14) or absence (lanes 1 to 7) of RT (3 pmol). The amounts of Tat in the reaction mixtures were 0 pmol (lanes 1 and 8), 3 pmol (lanes 2, 5, 9, and 12), 10 pmol (lanes 3, 6, 10, and 13), and 30 pmol (lanes 4, 7, 11, and 14). After incubation at room temperature for 20 min, the samples were separated on 5% polyacrylamide gels at 4°C, and then the gels were dried and the bands were visualized on X-ray film.
FIG. 4
FIG. 4
Tat 86, but not Tat 72, suppresses the endogenous RT reaction. HIV-1 virions (HTLV-IIIB strain) were pelleted through a 20% sucrose cushion at 40,000 rpm for 1 h at 4°C. The permeabilized virions containing 250 ng of p24 were then mixed with Tat 72 (100 and 300 pmol, lanes 2 and 3, respectively) or Tat 86 (100 and 300 pmol, lanes 4 and 5, respectively), and endogenous RT reactions were performed as described in Materials and Methods. Lane 1 represents a reaction performed without Tat.
FIG. 5
FIG. 5
Comparison of replication capacity and endogenous RT activity among pM1ex, pNL43 and pNL101 viruses. (A) Jurkat cells (6 × 105 cells) were infected with equivalent amounts (10 ng of p24 content) of viruses. The production of progeny virus was monitored by measuring de novo RT activity in the culture supernatant. (B) HeLa–LTR–β-Gal cells were infected with equivalent amounts (3 ng of p24 content) of viruses. After 48 h, cells were fixed and stained as described in Materials and Methods. The number of blue-stained cells was scored, and the results are expressed as the average ± the standard deviation. Three independent infections were performed for each viral preparation. (C) pM1ex, pNL43, and pNL101 virions were isolated from culture supernatants of transfected Cos-7 cells. Endogenous RT reactions were carried out using pelleted viruses containing 250 ng of p24, as described in Materials and Methods.
FIG. 6
FIG. 6
PCR analysis of HIV-1 DNA. (A) PCR analysis of HIV-1 DNA in cytosolic fractions of infected cells. Cytosolic DNA was isolated from infected Jurkat cells at days 9, 12, and 15 postinfection, as described in Materials and Methods, and aliquots corresponding to 104 cells were subjected to quantitative PCR using the R-U5 and R-5NC primer pairs. To monitor the efficiency of cytosolic DNA isolation, the mitochondrial CytOxy II gene was amplified. (B) PCR analysis of virion-associated HIV-1 DNA. Virion-associated nucleic acids were extracted, and aliquots corresponding to 10 and 100 pg of p24 were subjected to quantitative PCR using the R-U5 and R-5NC primer pairs. To analyze the amount of viral genomic RNA in the extracted nucleic acids, samples corresponding to 100 ng of p24 were reverse transcribed with Moloney murine leukemia virus RT, and an aliquot of synthesized viral cDNA, corresponding to 10 pg of p24, was subjected to PCR, using the R-5NC primer pair (shown as RT-PCR in the figure). (C) Aliquots of pM1ex virion-associated nucleic acids, corresponding to 10, 40, and 200 pg of p24, were subjected to quantitative PCR, using the indicated primer pairs. Primer pair R-U5 was designed to detect early RT products synthesized either before or immediately after the first template switch. With primer pairs U3-U5, Env1-Env2, Tat1-Tat2, and p7-RT1, a specific PCR product was expected only if negative-strand DNA of increased length, relative to wild-type, had been synthesized after the first template switch. Primer pair R-5NC was predicted to amplify only late RT products synthesized after the second template switch. Serial 10-fold dilutions of pNL43 plasmid were used as positive controls. PCR products were separated on 6% polyacrylamide gels (0.5× TBE) at 4°C, and then the gels were dried and the bands were visualized on X-ray film.
FIG. 7
FIG. 7
Nondenaturing Northern blot analysis of HIV-1 genomic RNA. Viral RNA samples corresponding to 500 ng of p24 were separated on nondenaturing 0.9% agarose gels and were transferred onto a nylon membrane. Hybridization with the denaturing HIV-1 probe was carried out as described in Materials and Methods. As a control experiment, RNA samples were heated at 50°C for 10 min before electrophoresis (heated pNL101).
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