Actinomycin D inhibits human immunodeficiency virus type 1 minus-strand transfer in in vitro and endogenous reverse transcriptase assays

Abstract

In this report we demonstrate that human immunodeficiency virus type 1 (HIV-1) minus-strand transfer, assayed in vitro and in endogenous reactions, is greatly inhibited by actinomycin D. Previously we showed that HIV-1 nucleocapsid (NC) protein (a nucleic acid chaperone catalyzing nucleic acid rearrangements which lead to more thermodynamically stable conformations) dramatically stimulates HIV-1 minus-strand transfer by preventing TAR-dependent self-priming from minus-strand strong-stop DNA [(-) SSDNA]. Despite this potent activity, the addition of NC to in vitro reactions with actinomycin D results in only a modest increase in the 50% inhibitory concentration (IC50) for the drug. PCR analysis of HIV-1 endogenous reactions indicates that minus-strand transfer is inhibited by the drug with an IC50 similar to that observed when NC is present in the in vitro system. Taken together, these results demonstrate that NC cannot overcome the inhibitory effect of actinomycin D on minus-strand transfer. Other experiments reveal that at actinomycin D concentrations which severely curtail minus-strand transfer, neither the synthesis of (-) SSDNA nor RNase H degradation of donor RNA is affected; however, the annealing of (-) SSDNA to acceptor RNA is significantly reduced. Thus, inhibition of the annealing reaction is responsible for actinomycin D-mediated inhibition of strand transfer. Since NC (but not reverse transcriptase) is required for efficient annealing, we conclude that actinomycin D inhibits minus-strand transfer by blocking the nucleic acid chaperone activity of NC. Our findings also suggest that actinomycin D, already approved for treatment of certain tumors, might be useful in combination therapy for AIDS.

FIG. 1

Selective inhibition of strand transfer by Act D. Reactions and analysis of DNA products were performed as described in Materials and Methods. (I) DNA-dependent DNA synthesis. (A) Gel analysis. The concentrations of Act D were as follows: lane 1, no drug; lane 2, 5 μM; lane 3, 10 μM; lane 4, 20 μM; lane 5, 40 μM; lane 6, 60 μM; lane 7, 80 μM; lane 8, 160 μM; and lane 9, 320 μM. The positions of the 36- and 50-nt products and the primer (P) are shown on the left. (B) Quantitative PhosphorImager analysis of gel data. The total amount of DNA synthesis (with the control value set at 100%) was plotted as a function of Act D concentration. (II) Minus-strand transfer. (A) Gel analysis. The concentrations of Act D used were as follows: lane 1, no drug; lane 2, 0.25 μM; lane 3, 0.5 μM; lane 4, 1 μM; lane 5, 2 μM; lane 6, 4 μM; lane 7, 8 μM; lane 8, 12 μM; lane 9, 16 μM; and lane 10, 20 μM. The positions of (−) SSDNA, the transfer product (T), SP DNAs (SP) (28), and primer are shown on the left. (B) Quantitative PhosphorImager analysis of gel data. To compare the amount of transfer product made in control and Act D-containing reaction mixtures, the percent transfer product represented in total DNA products was quantified for each reaction (with the control value set at 100%) and was plotted against the concentration of Act D.

FIG. 2

Effect of Act D on NC-catalyzed minus-strand transfer. Reactions were carried out in the presence of NC and Act D, as indicated, according to the procedures detailed in Materials and Methods. The data shown for reactions without NC were taken from Fig. 1, panel IIB. The percent transfer product represented in total DNA products was quantified with a PhosphorImager and was plotted against the concentration of Act D as described in the legend to Fig. 1, panel IIB. Symbols: circles, solid line, no NC; diamonds, dot-dash line, 0.4 μM NC; triangles, dotted line, 0.8 μM NC; and squares, dashed line, 1.6 μM NC.

FIG. 3

Effect of Act D on endogenous HIV-1 reverse transcription. Reaction mixtures were incubated for 6 h with increasing concentrations of Act D (A) or in the absence or presence of Act D at 5 μM for increasing times (B), as described in Materials and Methods. Samples were analyzed on a 6% sequencing gel to visualize (−) and (+) SSDNA products still attached to the tRNA or PPT RNA primers, respectively. (A) Dose response. Lane 1, no drug; lanes 2 to 7, Act D at 1, 5, 10, 20, 40, and 80 μM, respectively. (B) Time course. Lanes 1 to 5, no Act D; lanes 6 to 10, 5 μM Act D. The incubation times were 0.5 (lanes 1 and 6), 1 (lanes 2 and 7), 2 (lanes 3 and 8), 4 (lanes 4 and 9), and 6 (lanes 5 and 10) h. The sizes of the products were verified by running a sequencing ladder generated with MP18 DNA, which is included in the Sequenase kit.

FIG. 4

Detection of total and transferred minus-strand DNA synthesized during HIV-1 endogenous reverse transcription. The DNA products made in the reactions shown in Fig. 3A (Act D dose response) were stored for approximately 6 months, which allowed the original radioactivity to decay. The DNAs were then amplified by PCR with two sets of PCR primers (one primer of each set was labeled at its 5′ end with ³²P), as described in Materials and Methods. Shown are gel analyses of the 123- and 159-bp PCR products (IA and IIA, respectively) and dilution analyses of known amounts of the 890-bp template (expressed as copy number) to indicate the sensitivity of the assay (IB and IIB). (IIC) PhosphorImager analysis. The amount of the 159-bp PCR product generated from the reaction without drug was set at 100%. The percent transfer relative to the control was plotted against the concentration of Act D. For panels IA and IIA, the designation of lanes 1 to 7 is the same as that given in the legend for Fig. 3A.

FIG. 5

Effect of Act D on RNase H activity in the absence of NC. In vitro strand transfer reaction mixtures were incubated with donor RNA labeled at its 5′ end with ³²P and increasing concentrations of Act D, as described in Materials and Methods. (A) Gel analysis. (B) Quantitative PhosphorImager analysis. The amount of the 8-nt RNA cleavage product was plotted against the concentration of Act D. Lanes: 2, no drug; lanes 3 to 8, Act D at 1, 5, 10, 20, 40, and 80 μM, respectively. Lane 1 is a control showing the position of the uncleaved labeled donor RNA from a reaction without RT and Act D.

FIG. 6

Effect of Act D on the kinetics of annealing of (−) SSDNA and acceptor RNA in the absence or presence of NC protein. In the absence of NC (A and B), the annealing reaction was carried out with Act D (as indicated), as described in Materials and Methods. Aliquots were removed for gel analysis at 0.5, 1, 2, 3, 4, 5, and 6 h. In panel C, reaction mixtures were preincubated with Act D (as indicated) for 10 min at 37°C prior to the addition of NC. Aliquots were removed for gel analysis at 0.25, 0.5, 1, 2, 3, 5, and 10 min. (A) Quantitative PhosphorImager analysis of annealing in the absence of NC. The concentration of the DNA-RNA hybrid was plotted against the time of incubation. (B) Kinetic plot of the PhosphorImager data. The data from panel A were plotted as a semilogarithmic plot of the concentration of hybrid formed with time (35). A_∞ and A_t are the concentrations of hybrid formed at infinite time and at the indicated time, respectively. A_∞ is a theoretical value; A_t was determined by PhosphorImager analysis of the gel data. (C) Quantitative PhosphorImager analysis of annealing in the presence of 0.4 μM NC. The concentration of the DNA-RNA hybrid was plotted against the time of incubation. Since the reactions performed in the presence of Act D and NC showed little or no progression to final hybridization after the first 15 to 30 s, the data did not lend themselves to a kinetic analysis. Symbols for panels A and B: diamonds, no drug; squares, 0.5 μM Act D; triangles, 1 μM Act D; circles, 8 μM Act D. Symbols for panel C: diamonds, no drug; squares, 1 μM Act D; triangles, 2 μM Act D; circles, 16 μM Act D.