The interaction of APOBEC3G with human immunodeficiency virus type 1 nucleocapsid inhibits tRNA3Lys annealing to viral RNA

Abstract

Human immunodeficiency virus type 1 (HIV-1) containing human APOBEC3G (hA3G) has a reduced ability to produce viral DNA in newly infected cells. At least part of this hA3G-facilitated inhibition is due to a cytidine deamination-independent reduction in the ability to initiate reverse transcription. HIV-1 nucleocapsid (NCp7) is required both for the incorporation of hA3G into virions and for the annealing between viral RNA and tRNA(3)(Lys), the primer tRNA for reverse transcription. Herein we present evidence that the interaction of hA3G with nucleocapsid is required for the inhibition of reverse transcription initiation. A tRNA(3)(Lys) priming complex was produced in vitro by the NCp7-facilitated annealing of tRNA(3)(Lys) to synthetic viral RNA in the absence or presence of hA3G. The effect of hA3G on the annealing of tRNA(3)(Lys) to viral RNA and the ability of tRNA(3)(Lys) to initiate reverse transcription was measured. Our results show the following. (i) Electrophoretic band shift and primer binding site assays show that hA3G reduces the annealing of tRNA(3)(Lys) 44 and 60%, respectively, but does not disrupt the annealed complex once formed. (ii) hA3G inhibits tRNA(3)(Lys) priming 70 to 80%. (iii) Inhibition of tRNA(3)(Lys) priming by hA3G requires an interaction between hA3G and NCp7 during annealing. Thus, annealing of tRNA(3)(Lys) is insensitive to hA3G inhibition when facilitated by a zinc finger mutant of NCp7 unable to interact with hA3G. NCp7-independent annealing of DNA to viral RNA also is insensitive to hA3G inhibition. These results indicate that hA3G does not sterically block tRNA(3)(Lys) annealing by binding to viral RNA. Annealing and priming are not affected by another RNA binding protein, QKI-6.

FIG. 1.

Inhibition of NCp7-facilitated tRNA₃^Lys priming of reverse transcription by hA3G. Synthetic viral genomic RNA and purified human placental tRNA₃^Lys were incubated with NCp7 as described in Materials and Methods. The resulting tRNA₃^Lys/viral RNA (vRNA) complex (annealed complex), either with the NCp7 used for annealing or deproteinized, was then used as a source of primer tRNA₃^Lys/template viral genomic RNA in an in vitro reverse transcription system containing HIV-1 RT and deoxynucleotides. (A) The upper portion shows 1D PAGE patterns of tRNA₃^Lys extended by 6 bases in the presence of dCTP, dTTP, 5 μCi of [α-³²P]dGTP, and ddATP, as described in Materials and Methods. Lanes a to c, tRNA₃^Lys priming carried out in the presence of the NCp7 used for the annealing; d and e, tRNA₃^Lys priming carried out using deproteinized tRNA₃^Lys/viral RNA annealed complex. The lower portion shows Western blots of end reactions probed with anti-NCp7. (B) The upper portion shows 1D PAGE patterns of tRNA₃^Lys extended by 6 bases. The effect of hA3G upon tRNA₃^Lys priming is shown. During formation of the annealed complex, either hA3G or BSA was added. Lanes a and b, tRNA₃^Lys priming carried out using an annealed complex not deproteinized; c and d, tRNA₃^Lys priming carried out using a deproteinized annealed complex. The middle and lower portions show Western blots of end reactions probed with either anti-hA3G or anti-NCp7. (C) 1D PAGE patterns of tRNA₃^Lys extended by 6 bases. The specificity of hA3G inhibition of tRNA₃^Lys priming is shown. Annealing reaction mixtures contained either BSA (lanes a and b), the RNA binding protein QKI-6 (lane c), 16S rRNA (lanes d and f), or hA3G (lanes e and f). tRNA₃^Lys priming was carried out using deproteinized annealed complexes.

FIG. 2.

Inhibition of NCp7-facilitated tRNA₃^Lys annealing to viral RNA by hA3G. (A and B) Radioactive tRNA₃^Lys (A) or a radioactive 18-nt DNA sequence (B) was annealed to synthetic viral RNA (vRNA), as facilitated by NCp7. The annealing reaction mixtures also contained either BSA, QKI-6, 16S rRNA, or hA3G. Free tRNA₃^Lys or the DNA oligomer was separated from annealed complexes electrophoretically. The ratios of free tRNA₃^Lys or DNA to viral RNA are listed below each lane. (C) Illustration showing the strategy used to measure the percentage of the PBSs occupied with tRNA₃^Lys after tRNA₃^Lys is annealed to synthetic viral RNA using NCp7. A 5′-³²P-end-labeled DNA primer annealed downstream of the PBS in viral RNA was extended with reverse transcription. The full-length extension product is 226 nt, while a truncated product, the extension of which is blocked by the presence of tRNA₃^Lys on the PBS, is 46 nt. (D) Separation of the full-length and truncated extension products by 1D PAGE. The NCp7-facilitated annealing of tRNA₃^Lys to viral RNA occurred in the presence of either BSA, QKI-6, 16S rRNA, or hA3G. The percentages of PBSs occupied by tRNA₃^Lys are listed at the bottom of each lane, and they were determined by multiplying the ratio of truncated product to full-length product by 100.

FIG. 3.

Effect of the order of addition of reactants upon tRNA₃^Lys priming. (A) The radioactive tRNA₃^Lys extended 6 bases by reverse transcription and resolved by 1D PAGE. (B) The quantitation of the gels shown in panel A, using the major middle band. Lanes a, c, e, g, and i represent reverse transcription reactions using annealed complexes (tRNA₃^Lys annealed to viral RNA [vRNA]) exposed to BSA during their formation, while lanes b, d, f, h, and j represent reverse transcription reactions using annealed complexes exposed to hA3G during their formation. In all cases, after reverse transcription the reaction products were deproteinized, alcohol precipitated, and resolved by 1D PAGE. Lanes: a and b, annealed complexes formed in the presence of BSA (a) or hA3G (b), deproteinized, and used in the reverse transcription reaction; c and d, annealed complex formed, deproteinized, and used in the reverse transcription reaction in the presence of either BSA (c) or hA3G (d); e and f, annealed complex formed, deproteinized, and exposed to either BSA (e) or hA3G (f) for 90 min (the annealed complex was then deproteinized and used in the reverse transcription reaction); g and h, tRNA₃^Lys heat annealed to viral RNA and then exposed to either BSA (g) or hA3G (h) for 90 min and used in reverse transcription reactions; i and j, tRNA₃^Lys heat annealed to viral RNA and then exposed to either BSA (i) or hA3G (j) for 90 min, deproteinized, and used in the reverse transcription reaction.

FIG. 4.

Inhibition of tRNA₃^Lys priming is dependent upon the amount of hA3G used and can be rescued with increasing amounts of NCp7. The effect of increasing concentrations of hA3G (A) or NCp7 (B) upon the ability of the annealed complex to initiate reverse transcription is shown. (A) The upper portion shows 1D PAGE patterns of tRNA₃^Lys extended by 6 bases. The nucleocapsid concentration used for annealing was held constant. Lanes a to d, increasing concentrations of BSA; lanes e to h, increasing concentrations of hA3G. The middle and lower portions show Western blots of end reactions probed with either anti-hA3G or anti-NCp7. (B) The upper portion shows 1D PAGE patterns of tRNA₃^Lys extended by 6 bases. Lanes a to f represent increasing amounts of NCp7 used for annealing. Lanes a to d, annealing reaction mixture exposed to BSA; lanes e to h, annealing reaction mixture exposed to hA3G. The middle and lower portions show Western blots of end reactions probed with either anti-hA3G or anti-NCp7. vRNA, viral RNA.

FIG. 5.

Interaction between NCp7 and hA3G. (A) Sequences of wild-type and mutant nucleocapsids are shown. Sequences: a, wild type; b and c, the N-terminal five basic amino acids replaced with either alanine (b) or glycine (c); d, the three cysteines in each zinc finger replaced with alanines; e, F16 and W37 in the first and second zinc fingers, respectively, replaced with alanines; f, four basic amino acids in the linker region between the two zinc fingers replaced with alanine. (B) For the experiment shown in the upper portion, wild-type or mutant NCp7 was incubated with hA3G, and its ability to be coimmunoprecipitated with anti-hA3G was detected by Western blots (WB) probed with anti-NCp7. The middle and lower portions show Western blots of input reactions probed with either anti-NCp7 or anti-hA3G. (C) Role of RNA in the interaction between wild-type NCp7 and hA3G. The upper portion shows coimmunoprecipitation experiments that were performed similarly to those shown in panel B, except that for lanes b and c, RNase A was added to hA3G and wild-type NCp7 before (b) or after (c) their mixing. The middle and lower portions show Western blots of input reactions probed with either anti-NCp7 or anti-hA3G. IP, immunoprecipitation.

FIG. 6.

hA3G-facilitated inhibition of tRNA₃^Lys priming requires an interaction between hA3G and NCp7. (A) 1D PAGE of tRNA₃^Lys extended by 6 bases in an in vitro reverse transcription system, described in the legend to Fig. 1. Lanes a to f and lanes a′ to f′ represent the wild-type and mutant NCp7 species (shown in Fig. 5A) used to anneal tRNA₃^Lys to viral RNA in the absence (a to f) or presence (a′ to f′) of hA3G. (B and C) The major band in each lane was quantitated by phosphorimaging. (B) tRNA₃^Lys priming in the absence of hA3G relative (rel.) to tRNA₃^Lys priming from the annealed complex produced with wild-type NCp7. (C) Comparison of tRNA₃^Lys priming produced from each type of annealed complex in the presence of hA3G to that in its absence. vRNA, viral RNA.