Newly synthesized APOBEC3G is incorporated into HIV virions, inhibited by HIV RNA, and subsequently activated by RNase H - PubMed (original) (raw)

Newly synthesized APOBEC3G is incorporated into HIV virions, inhibited by HIV RNA, and subsequently activated by RNase H

Vanessa B Soros et al. PLoS Pathog. 2007 Feb.

Abstract

APOBEC3G (A3G) is a potent antiretroviral deoxycytidine deaminase that, when incorporated into HIV virions, hypermutates nascent viral DNA formed during reverse transcription. HIV Vif counters the effect of A3G by depleting intracellular stores of the enzyme, thereby blocking its virion incorporation. Through pulse-chase analyses, we demonstrate that virion A3G is mainly recruited from the cellular pool of newly synthesized enzyme compared to older "mature" A3G already residing in high-molecular-mass RNA-protein complexes. Virion-incorporated A3G forms a large complex with viral genomic RNA that is clearly distinct from cellular HMM A3G complexes, as revealed by both gel filtration and biochemical fractionation. Unexpectedly, the enzymatic activity of virion-incorporated A3G is lost upon its stable association with HIV RNA. The activity of the latent A3G enzyme is ultimately restored during reverse transcription by the action of HIV RNase H. Degradation of the viral genomic RNA by RNase H not only generates the minus-strand DNA substrate targeted by A3G for hypermutation but also removes the inhibitory RNA bound to A3G, thereby enabling its function as a deoxycytidine deaminase. These findings highlight an unexpected interplay between host and virus where initiation of antiviral enzymatic activity is dependent on the action of an essential viral enzyme.

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Conflict of interest statement

Competing interests. The authors have declared that no competing interests exist.

Figures

Figure 1. A3G Is Incorporated into Virion Cores but A3G Overexpression in Cells Results in Additional A3G Packaging Outside of the Core

(A) HA-A3G–containing ΔVif virions were generated from 293T cells transfected with a fixed amount of proviral plasmid (60 μg) and increasing doses of HA-A3G (0 to 20 μg). Empty HA vector (0 to 20 μg) was used as balance DNA in the transfections. Sample number 1 = 0 (μg of HA-A3G):60 (μg of pNL4-3ΔVif), 2 = 1:60, 3 = 2:60, 4 = 5:60, 5 = 10:60, 6 = 20:60. ΔVif virions were also derived from the H9 T cell line and primary CD4 T cells, which endogenously express A3G. The virion lysates were subjected to immunoblotting with antibodies specific for p24-CA and A3G. The immunoblot is representative of several independent analyses used to generate the graph in (B). (B) Graphical representation of quantification from immunoblots in (A) and unpublished data. Data are averaged from three independent transfections of 293T cells, five independent spinoculations of activated primary CD4 T cells, and three independent spinoculations of H9 T cells. The error bars represent standard deviation. The relative ratio of packaged A3G to p24-CA is plotted, with virions derived from CD4 T cells assigned a value of 1. (C) Virions containing increasing amounts of HA-A3G relative to p24-CA were solubilized by brief Triton X-100 treatment to generate virion cores containing p24-CA, IN, RT, and NC and supernatants containing gp41 and p24-CA. The triangles represent the increasing dose of A3G and correspond exactly to the numbered samples presented in Figure 1A. The immunoblots (IB) were also probed for A3G to determine the amount packaged into virion cores.

Figure 2. Newly Synthesized HA-A3G Is Recruited into HIV-1 Virions

(A) Cells transfected with HA-A3G were pulse radiolabeled for 10 min, chased with cold medium, and harvested at 0, 0.5, and 1 h. Chase lysate was subjected to Sepharose CL-6B gel filtration (10 fractions collected per lysate), HA-A3G was immunoprecipitated from each fraction, and the relative amount of radiolabeled HA-A3G in each fraction was determined by autoradiography. Fraction 4 corresponds to HMM complexes, and fractions 6 to 8 contain proteins similar in size to LMM A3G. See Figure S2A for Sepharose CL-6B fractionation performance. (B) Cells transfected with HA-A3G and pNL4–3ΔVif were assessed as in (A). (C) Cells transfected with HA-A3G were assessed as in (A), except that the pulse was extended to 30 min. Plotted is the t = 0-h pulse sample. (D) Cells transfected with HA-A3G and pNL4–3ΔVif were assessed as in (C). (A–D) Data shown are representative of three independent experiments for each panel. (E) Virus-producing cells were pulse radiolabeled and chased with cold medium, and both cells and virus-containing supernatants were collected at 0.5, 1, 2, and 4 h. As all of the supernatant was collected at the indicated time points, the virions harvested represent only those that budded during the intervening time period. Plotted is the percent density of the immunoprecipitated protein for a given time point relative to the total radioactive density of all time points. Upper panels, immunoprecipitates of HA-A3G from the producer cells. Middle panels, immunoprecipitates of HA-A3G from virions. Lower panels, immunoprecipitates of p24-CA from virions. Data are from three experiments performed independently.

Figure 3. Virion-Incorporated HA-A3G Resides in a Large RNase A–Sensitive Complex and Biochemically Fractionates with Viral RNP Proteins

(A) Virions collected from cells expressing HIV-1ΔVif contain HA-A3G that predominantly fractionates in a large complex (fractions 6 to 8) as assessed by gel filtration. (B) The IVAC is sensitive to RNase A treatment which shifts HA-A3G into lower fractions (fractions 15 to 19). (C) Virion cores obtained in Figure 1 were subjected to further biochemical fractionation to generate viral RNPs. Shown are the viral RNPs from virions either lacking or containing A3G, as indicated, and containing viral RT, IN, and NC but not p24-CA, as detected by immunoblotting (IB). The triangles represent the increasing dose of A3G relative to provirus and correspond exactly to the sample numbers in Figure 1A.

Figure 4. Intravirion A3G Enzymatic Activity Is Negatively Regulated by Binding to Genomic HIV RNA

(A) HA-A3G was immunoprecipitated from IVAC fraction 7 (F7) of virion lysates (Figure 3A) or from a lower fraction, F17, generated by treatment of the virion lysates with RNase A (Figure 3B). Immunoprecipitates (IPs) were tested for enzymatic activity in an in vitro deoxycytidine deaminase assay with or without RNase A addition and contained equivalent amounts of HA-A3G as shown in the corresponding immunoblot. The generation of a shorter cleavage product from the input ssDNA substrate reveals A3G deoxycytidine deaminase activity. Data shown are representative of multiple experiments. (B) Lysates of virions containing or lacking A3G were assessed in the deaminase assay, with or without RNase A treatment. (C) Lysates of virions containing increasing amounts of HA-A3G (as shown in the corresponding immunoblot) were assessed in the deaminase assay, with or without RNase A treatment. The asterisk marks bleed-through of marker loaded to the left of the samples. The triangles represent the increasing dose of A3G relative to provirus and correspond to the sample numbers presented in Figure 1A. (A–C) All deaminase reactions were carried out in 50 mM Tris (pH 7.4) with (+) or without (−) RNase A, as indicated. (D) IPs of HMM or LMM HA-A3G from producer cell lysates were similarly assessed in the deaminase assay, with (+) or without (−) added RNase A. The IPs contained equivalent amounts of HA-A3G as shown in the corresponding immunoblot (IB).

Figure 5. Virion-Incorporated HA-A3G Associates with Viral Genomic RNA

(A) Viral genomic RNA, detected by RT-PCR, was detected in virions and virus-producing cells but not in lysates of uninfected cells. Genomic RNA was also detected in the IVAC derived from virions (fraction 7) and coimmunoprecipitated with HA-A3G from both virions and producer cell lysates. RT was performed using RNA derived from either whole lysates (L) or anti-HA immunoprecipitates (IP). Control reactions were performed in the absence of RT (–RT). Control PCRs were performed using proviral plasmid DNA, in the absence or presence of Taq, as indicated. (B) Viral genomic RNA, detected by RT-PCR, was assessed from size-fractionated virion lysates that lacked (HA) or contained HA-A3G. Amplicons generated probed across the TAR/Gag region or Pol/Vpu regions, as indicated. (C) Incorporation of HA-A3G into virions enhances the recruitment of NC into the IVAC. (D) HA-A3G from virus-producing cells is HMM and is converted to LMM form after RNase A treatment. “IB” indicates immunoblotting with the indicated antibody.

Figure 6. Enzymatically Inactive Virion-Incorporated HA-A3G Is Activated by Viral RNase H

(A) Recombinant RTs containing either a WT or mutant (E478Q) RNase H catalytic domain were assessed for RNase H activity in vitro in the absence or presence of the RNase H inhibitor Compound I (final concentration of 1, 10, or 100 μM). The RNA of an RNA–DNA hybrid remains intact unless RNase H digests the RNA into a smaller cleavage product that is distinguishable from the more complete cleavage product generated by RNase A. WT RNase H cannot digest ssDNA or DNA of an RNA–DNA hybrid, or RNA–RNA hybrids (data not shown). RNase H assays were performed in RNase H buffer (50 mM Tris [pH 8.0], 60 mM KCl) with (+) or without (−) 5 mM MgCl2 or RNase A, as indicated. (B) Viruses bearing the RNase H E478Q mutation are compromised for in vitro RNase H activity. RNase H assays were performed in RNase H buffer with (+) or without (−) 5 mM MgCl2 or RNase A, as indicated. (C) Virion lysates were subjected to endogenous reverse transcription (enRT) conditions with or without Compound I (final concentration of 0.1, 1, 10, or 100 μM), and A3G activity in these samples assessed in the in vitro deoxycytidine deaminase assay. Deaminase assays were performed in RNase H buffer either supplemented (enRT:+) or not (enRT:−) with 4 mM MgCl2 and 1 mM dNTPs. (D) Compound I does not inhibit the intrinsic deoxycytidine deaminase activity of A3G. HA-A3G from RNase A–treated virion lysates was assessed for in vitro deaminase activity in the presence of increasing doses of Compound I (0.1, 1, 10, and 100 μM). Deaminase assay was performed in RNase H buffer supplemented with RNase A only. (E) Virions containing WT RNase H or the E478Q mutation in the RNase H catalytic domain were subjected to the enRT reaction followed by assessment of A3G enzymatic activity. Deaminase assays were performed in RNase H buffer either supplemented (enRT:+) or not (enRT:−) with 4 mM MgCl2 and 1 mM dNTPs. (F) WT and RNase H–compromised ΔVif virions containing WT or mutant RNase H displayed equivalent A3G activity when RNase A was added to the virion lysate. Deaminase assay was performed in RNase H buffer with (+) or without (−) RNase A, as indicated. All data are representative of multiple experiments.

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Newly synthesized APOBEC3G is incorporated into HIV virions, inhibited by HIV RNA, and subsequently activated by RNase H - PubMed (original) (raw)