Inhibition of tRNA₃(Lys)-primed reverse transcription by human APOBEC3G during human immunodeficiency virus type 1 replication - PubMed (original) (raw)
Inhibition of tRNA₃(Lys)-primed reverse transcription by human APOBEC3G during human immunodeficiency virus type 1 replication
Fei Guo et al. J Virol. 2006 Dec.
Abstract
Cells are categorized as being permissive or nonpermissive according to their ability to produce infectious human immunodeficiency virus type 1 (HIV-1) lacking the viral protein Vif. Nonpermissive cells express the human cytidine deaminase APOBEC3G (hA3G), and Vif has been shown to bind to APOBEC3G and facilitate its degradation. Vif-negative HIV-1 virions produced in nonpermissive cells incorporate hA3G and have a severely reduced ability to produce viral DNA in newly infected cells. While it has been proposed that the reduction in DNA production is due to hA3G-facilitated deamination of cytidine, followed by DNA degradation, we provide evidence here that a decrease in the synthesis of the DNA by reverse transcriptase may account for a significant part of this reduction. During the infection of cells with Vif-negative HIV-1 produced from 293T cells transiently expressing hA3G, much of the inhibition of early (> or =50% reduction) and late (> or =95% reduction) viral DNA production, and of viral infectivity (> or =95% reduction), can occur independently of DNA deamination. The inhibition of the production of early minus-sense strong stop DNA is also correlated with a similar inability of tRNA(3)(Lys) to prime reverse transcription. A similar reduction in tRNA(3)(Lys) priming and viral infectivity is also seen in the naturally nonpermissive cell H9, albeit at significantly lower levels of hA3G expression.
Genomic RNA packaging, packaging, and priming in Vif-positive and Vif-negative HIV-1 produced from the nonpermissive cell lines H9 and MT2 and the permissive cell line MT4. A. The /genomic RNA annealing complex. The first six deoxyribonucleotides incorporated during reverse transcription are underlined. B. (Left) Relative incorporation of viral genomic RNA and into BH10Env- and BH10Env-Vif- produced in MT2, H9, or MT4 cells, as determined by dot blot hybridization with a probe specific for viral RNA or . (Right) Relative priming from equal amounts of viral RNA isolated from BH10Env- and BH10Env-Vif- produced in MT2, H9, or MT4 cells, using either the annealed present in the total viral RNA or an exogenous 30-mer DNA annealed in vitro as primer. The values were determined from the data in panel C. C. 1D-PAGE of radioactive reverse transcription products. (Left) The in vitro reverse transcription reaction, described in Materials and Methods, uses either purified heat annealed in vitro to synthetic viral genomic RNA (lane 1) or total viral RNA, extracted from BH10Env- and BH10Env-Vif- produced in either MT2, H9, or MT4 cells, as the source of primer /viral RNA template. (Right) A 30-mer DNA was annealed at room temperature to the same samples of total viral RNA, and initiation of reverse transcription from this DNA was measured using the same reaction conditions as for -primed reverse transcription.
FIG. 2.
Genomic RNA packaging, packaging, and priming in Vif-positive and Vif-negative HIV-1 produced from 293T cells in the presence or absence of hA3G. (A and B) Incorporation of protein. 293T cells were cotransfected with 2 μg of plasmid containing either BH10 or BH10Vif- DNA, and 1 μg pcDNA3.1, either empty or containing DNA coding for hA3G. Western blots of cell (A) or viral (B) lysates were normalized to β-actin and p24, respectively. (A) Blots were probed with anti-Vif, anti-HA, anti-CA, and anti-β-actin. (B) Blots were probed with anti-HA and anti-CA. (C) Data for incorporation of probes specific for and viral RNA. (D) priming and 1D-PAGE of radioactive reverse transcription products. (Left) The in vitro reverse transcription reaction, described in Materials and Methods, using either purified heat annealed in vitro to synthetic viral genomic RNA (lane 1) or total viral RNA (containing equal amounts of viral genomic RNA), extracted from the four types of virions as the source of primer /viral RNA template. Quantitation of RT products by phosphorimaging is shown on the right. Bars: a, BH10 and pcDNA3.1; b, BH10Vif- and pcDNA3.1; c, BH10 and hA3G; d, BH10Vif- and hA3G.
FIG. 3.
Real-time PCR quantitation of newly synthesized HIV-1 DNA. DNA was extracted at different times postinfection from SupT1 cells infected with the four viral types: BH10, plus or minus hA3G, and BH10Vif-, plus or minus hA3G. Early (R-U5) and late (U5-gag) minus-strand cDNA production was monitored by real-time PCR, as described in Materials and Methods. (A) Arrows indicate the PCR primers used to detect early (U5a-R) and late (gag-U5b) minus-strand DNA. (B and C) Graphs on the right show the postinfection time course of production of viral early (B) and late (C) DNA in SupT1 cells infected with one of the four viral types. Data on the left were normalized to peak DNA production for BH10 in the absence of hA3G. Bars: a, BH10 and pcDNA3.1; b, BH10Vif- and pcDNA3.1; c, BH10 and hA3G; d, BH10Vif- and hA3G.
FIG. 4.
Effects of increasing amounts of hA3G upon -primed reverse transcription and viral infectivity in wild-type and Vif-negative HIV-1 produced from 293T cells. (A and B) Western blots of cell (A) or viral (B) lysates. In panel A, blots were probed with anti-HA and anti-β-actin. In panel B, blots were probed with anti-HA and anti-CA. (C, left) 1D-PAGE of radioactive reverse transcription products ( extended 6 bases, as described in Materials and Methods), using total viral RNA as the source of primer /viral RNA template. (Right) Quantitation of RT products by phosphorimaging. (D) Western blots of lysates of BH10Env-, with or without Vif, produced in H9 cells and of lysates of BH10, with or without Vif, produced in 293T cells also transiently transfected with 1 μg pAPOBEC3G. Blots were probed with anti-hA3G and anti-CAp24. (E) Infectivity in HeLa-CD4+ cells of HIV-1 produced in 293T cells containing increasing amounts of hA3G, as measured in a MAGI assay.
FIG. 5.
hA3G incorporation, and genomic RNA packaging, and priming in Vif-positive and Vif-negative HIV-1 produced from H9 cells, 293T cells transiently expressing hA3G, and a 293 cell line stably expressing APOBEC3G. Cells were transfected with DNA coding for either BH10Env- or BH10Vif-Env- as described in Materials and Methods. The cells included a 293 cell line stably expressing hA3G and 293T cells transiently expressing hA3G. H9 cells were also infected with BH10Env- or BH10Vif-Env- pseudotyped with VSV-G. (A) Western blots of lysates of BH10Env-, with or without Vif, produced from the different cell types. Blots were probed with anti-hA3G and anti-CAp24. (B) 1D-PAGE of radioactive reverse transcription products ( extended 6 bases, as described in Materials and Methods), using viral RNA extracted from the different types of virions as the source of primer /viral RNA template. (C, left) Relative incorporation of viral genomic RNA and into BH10Env- and BH10Env-Vif- produced in the three cell types, as determined by dot blot hybridization with a probe specific for viral RNA or . (Right) Relative priming from equal amounts of viral RNA isolated from BH10Env- and BH10Env-Vif- produced in the three cell types, as determined from the data in panel B.
FIG. 6.
Viral early and late DNA production and priming in SupT1 cells infected with BH10Vif- containing either wild-type or mutant hA3G. SupT1 cells were infected with BH10Vif- containing either no hA3G (a), wild-type hA3G (b), or mutant hA3G (c to f). (A) Graphic representation of the wild-type and mutant APOBEC3G variants tested. a, no hA3G; b, wild-type hA3G; c, hA3G105-384; d, hA3G157-384; e, hA3G1-156; f, hA3G104-246. The filled rectangles represent the two catalytic sites (zinc coordination units) in hA3G, and the numbers represent the amino acid positions. (B and C) Early and late viral DNA production. DNA was extracted at different times postinfection from SupT1 cells infected with the different viruses. Early (R-U5) and late (U5-gag) minus-strand cDNA production was monitored by real-time PCR, as described in Materials and Methods, using the same PCR primers as shown in Fig. 3A. Production of viral early DNA (B) and late DNA (C) in SupT1 cells infected with the different viruses was normalized to DNA production for BH10Vif- in the absence of hA3G (a). (D) priming in BH10Vif- containing wild-type or mutant hA3G. Total viral RNA was extracted from BH10Vif- containing either no hA3G (a), wild-type hA3G (b), or mutant hA3G (c to f). Synthesis of the six-base-extended in an in vitro reverse transcription reaction, using total viral RNA as the source of primer /viral RNA template, is described in Materials and Methods. extension was normalized to that obtained for BH10Vif- containing no hA3G.
References
Alce, T. M., and W. Popik. 2004. APOBEC3G is incorporated into virus-like particles by a direct interaction with HIV-1 Gag nucleocapsid protein. J. Biol. Chem. 279:34083-34086. - PubMed
Arts, E. J., X. Li, Z. Gu, L. Kleiman, M. A. Parniak, and M. A. Wainberg. 1994. Comparison of deoxyoligonucleotide and tRNA(Lys-3) as primers in an endogenous human immunodeficiency virus-1 in vitro reverse transcription/template-switching reaction. J. Biol. Chem. 269:14672-14680. - PubMed
Bishop, K. N., R. K. Holmes, A. M. Sheehy, and M. H. Malim. 2004. APOBEC-mediated editing of viral RNA. Science 305:645. - PubMed
Bogerd, H. P., H. L. Wiegand, B. P. Doehle, K. K. Lueders, and B. R. Cullen. 2006. APOBEC3A and APOBEC3B are potent inhibitors of LTR-retrotransposon function in human cells. Nucleic Acids Res. 34:89-95. - PMC - PubMed
Borman, A. M., C. Quillent, P. Charneau, C. Dauguet, and F. Clavel. 1995. Human immunodeficiency virus type 1 Vif- mutant particles from restrictive cells: role of Vif in correct particle assembly and infectivity. J. Virol. 69:2058-2067. - PMC - PubMed
