Deaminase-independent inhibition of HIV-1 reverse transcription by APOBEC3G

Abstract

APOBEC3G (A3G), a host protein that inhibits HIV-1 reverse transcription and replication in the absence of Vif, displays cytidine deaminase and single-stranded (ss) nucleic acid binding activities. HIV-1 nucleocapsid protein (NC) also binds nucleic acids and has a unique property, nucleic acid chaperone activity, which is crucial for efficient reverse transcription. Here we report the interplay between A3G, NC and reverse transcriptase (RT) and the effect of highly purified A3G on individual reactions that occur during reverse transcription. We find that A3G did not affect the kinetics of NC-mediated annealing reactions, nor did it inhibit RNase H cleavage. In sharp contrast, A3G significantly inhibited all RT-catalyzed DNA elongation reactions with or without NC. In the case of (-) strong-stop DNA synthesis, the inhibition was independent of A3G's catalytic activity. Fluorescence anisotropy and single molecule DNA stretching analyses indicated that NC has a higher nucleic acid binding affinity than A3G, but more importantly, displays faster association/disassociation kinetics. RT binds to ssDNA with a much lower affinity than either NC or A3G. These data support a novel mechanism for deaminase-independent inhibition of reverse transcription that is determined by critical differences in the nucleic acid binding properties of A3G, NC and RT.

Figure 1.

Schematic diagram of the events in reverse transcription. Step 1. Reverse transcription is initiated by a cellular tRNA primer (, in the case of HIV-1), following annealing of the 3′ 18 nt of the tRNA to the 18-nt PBS near the 5′ end of the genome. RT catalyzes synthesis of (−) SSDNA, which contains copies of the R sequence and the unique 5′ genomic sequence (U5). Step 2. As the primer is extended, the RNase H activity of RT degrades the genomic RNA sequences that have been reverse transcribed. Step 3. (−) SSDNA is transferred to the 3′ end of vRNA (minus-strand transfer). Step 4. Elongation of minus-strand DNA and RNase H degradation continue. Plus-strand synthesis is initiated by the 15-nt PPT immediately upstream of the unique 3′ genomic sequence (U3). Step 5. RT copies the u3, u5 and r regions in minus-strand DNA, as well as the 3′ 18 nt of the tRNA primer, thereby reconstituting the PBS. The product formed is termed (+) SSDNA. Step 6. RNase H removal of the tRNA and PPT primers from minus- and plus-strand DNAs, respectively. Step 7. Plus-strand transfer, facilitated by annealing of the complementary PBS sequences at the 3′ ends of (+) SSDNA and minus-strand DNA, is followed by circularization of the two DNA strands and displacement synthesis. Step 8. Minus- and plus-strand DNAs are elongated, resulting in a linear dsDNA with a long terminal repeat (LTR) at each end. vRNA is shown by an open rectangle and minus-and plus-strand DNAs are shown by black and gray rectangles, respectively. The tRNA primer is represented by a short open rectangle (3′ 18 nt of the tRNA) attached to a ‘clover-leaf’ (remaining tRNA bases). Minus- and plus-strand sequences are depicted in lower and upper case, respectively. The very short white rectangles represent fragments produced by RNase H cleavage of genomic RNA. Adapted from reference (43) with permission from Elsevier.

Figure 2.

Effect of A3G on -primed (−) SSDNA synthesis. (A) Time course of annealing to RNA UL244. Reactions were performed in the absence or presence of NC and A3G, as indicated by the headings at the top of the gel. The positions of the RNA UL244 template and the annealed RNA duplex are shown on the right. (B) The percentage of annealed product was calculated by dividing the amount of annealed RNA by the sum of annealed plus unannealed RNA, multiplied by 100. Symbols: no NC/no A3G (filled circles); +NC/no A3G (open squares); +NC/+hdA3G (open circles); and +NC/+A3G (open triangles). (C) A /RNA 244 complex was extended by HIV-1 RT in the absence (lane 1) or presence of hdA3G (lanes 2–4) or A3G (lanes 5–7). The positions of (−) SSDNA and initial pause products at bases +1, +3 and +5 are shown on the right. A3G concentrations: lane 1, 0 nM; lanes 2 and 5, 20 nM; lanes 3 and 6, 40 nM; lanes 4 and 7, 80 nM.

Figure 3.

Effect of A3G on (−) SSDNA synthesis primed by D18. (A) Time course of (−) SSDNA synthesis in reactions containing ³²P-labeled D18 and RNA 244 in the presence of increasing concentrations of A3G. Positions of (−) SSDNA and D18 are shown on the right. (B) Graph of percent (−) SSDNA formed plotted versus incubation time. The percentage of (−) SSDNA product was calculated by dividing the amount of (−) SSDNA by the total amount of DNA, multiplied by 100. Symbols: 0 nM (filled circles); 20 nM (open squares); 40 nM (open circles); and 80 nM A3G (open triangles). (C) Mapping of pause sites on the RNA 244 template in A3G-containing reactions was based on the data shown in Figure S1. The arrows point to the pause sites.

Figure 4.

A3G inhibition of (−) SSDNA synthesis in the absence of deaminase activity. Symbols: no A3G (filled circles); WT A3G (80 nM) (open squares) and A3G C291S (80 nM) (open triangles).

Figure 5.

Effect of A3G on minus-strand transfer reactions. (A) Effect of A3G on the time course of RNase H cleavage in the absence or presence of NC. ³²P-labeled TAR RNA (0.1 pmol) and TAR DNA (0.2 pmol) were heat annealed and the hybrid was incubated at 37°C in reaction buffer (see above) with 0.4 pmol HIV-1 RT with or without NC (7 nt/NC, 0.1 µM), with or without A3G (80 nM). Samples were loaded on a 15% denaturing gel. Positions of the major cleavage products are indicated on the right. (B) Time course of annealing of ³²P-labeled DNA 128 to RNA 148 incubated in the absence or presence of A3G (80 nM) with or without NC (3.5 nt/NC, 0.4 µM). Symbols: no NC/+A3G (filled circles); +NC/no A3G (open squares); and +NC/+A3G (open circles). (C) Schematic diagram illustrating the minus-strand transfer assay system. The R homology is 94 nt; U5 and U3 are 34 and 54 nt, respectively. (D) Graph of percent transfer product plotted versus incubation time. To quantify the percentage of strand transfer, the amount of transfer product was divided by the total amount of DNA, multiplied by 100. Symbols: no NC/no A3G (filled circles); no NC/+A3G (open squares); +NC/no A3G (open circles); and +NC/+A3G (open triangles).

Figure 6.

Effect of A3G on PPT initiation and plus-strand transfer. (A) Time course of PPT-primed plus-strand DNA synthesis. The 15-nt PPT RNA was heat-annealed to a 35-nt minus-strand DNA template and was then extended by HIV-1 RT. The 20-nt DNA product was internally labeled with [α-³²P]dCTP in the absence (filled circles) and presence (open squares) of A3G (80 nM). The amount of 20-nt DNA was plotted as Relative Extension (%) versus Time (min), where 100% represents the end point value for the ‘no A3G’ reaction. (B) Time course of plus-strand transfer. The percentage of 80-nt plus-strand DNA product was calculated as described in the legend to Figure 5D. Symbols: no NC/no A3G (filled circles); +NC/no A3G (open circles); and +NC/+A3G (open triangles).

Figure 7.

Examples of lambda DNA stretching (continuous) and relaxation (dashed) curves. (A) A3G. 0 nM (black); 150 nM (red). (B) NC. 0 nM (black); 10 nM (red). All stretching experiments were conducted at 20°C in 10 mM HEPES, pH 7.5 and 50 mM Na⁺.