Selective translational repression of HIV-1 RNA by Sam68DeltaC occurs by altering PABP1 binding to unspliced viral RNA

HIV-1 structural proteins are translated from incompletely spliced 9 kb and 4 kb mRNAs, which are transported to the cytoplasm by Crm1. It has been assumed that once in the cytoplasm, translation of incompletely spliced HIV-1 mRNAs occurs in the same manner as host mRNAs. Previous analyses have demonstrated that Sam68 and a mutant thereof, Sam68ΔC, have dramatic effects on HIV gene expression, strongly enhancing and inhibiting viral structural protein synthesis, respectively. While investigating the inhibition of incompletely spliced HIV-1 mRNAs by Sam68ΔC, we determined that the effect was independent of the perinuclear bundling of the viral RNA. Inhibition was dependent upon the nuclear export pathway used, as translation of viral RNA exported via the Tap/CTE export pathway was not blocked by Sam68ΔC. We demonstrate that inhibition of HIV expression by Sam68ΔC is correlated with a loss of PABP1 binding with no attendant change in polyadenosine tail length of the affected RNAs. The capacity of Sam68ΔC to selectively inhibit translation of HIV-1 RNAs exported by Crm1 suggests that it is able to recognize unique characteristics of these viral RNPs, a property that could lead to new therapeutic approaches to controlling HIV-1 replication

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

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

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

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

Compensatory Link between Fusion and Endocytosis of Human Immunodeficiency Virus Type 1 in Human CD4 T Lymphocytes

Virions of the type 1 human immunodeficiency virus (HIV-1) can enter target cells by fusion or endocytosis, with sharply different functional consequences. Fusion promotes productive infection of the target cell, while endocytosis generally leads to virion inactivation in acidified endosomes or degradation in lysosomes. Virion fusion and endocytosis occur equally in T cells, but these pathways have been regarded as independent because endocytosis of HIV virions requires neither CD4 nor CCR5/CXCR4 engagement in HeLa-CD4 cells. Using flow cytometric techniques to assess the binding and entry of green fluorescent protein (GFP)-Vpr-labeled HIV virions into primary peripheral blood mononuclear cells, we have found that HIV fusion and endocytosis are restricted to the CD4-expressing subset of cells and that both pathways commonly require the initial binding of HIV virions to surface CD4 receptors. Blockade of CXCR4-tropic HIV virion fusion with AMD3100, a CXCR4-specific entry inhibitor, increased virion entry via the endocytic pathway. Similarly, inhibition of endosome acidification with bafilomycin A1, concanamycin A, or NH(4)Cl enhanced entry via the fusion pathway. Although fusion remained dependent on CD4 and chemokine receptor binding, the endosome inhibitors did not alter surface expression of CD4 and CXCR4. These results suggest that fusion in the presence of the endosome inhibitors likely occurs within nonacidified endosomes. However, the ability of these inhibitors to impair vesicle trafficking from early to late endosomes in some cells could also increase the recycling of these virion-containing endosomes to the cell surface, where fusion occurs. In summary, our results reveal an unexpected, CD4-mediated reciprocal relationship between the pathways governing HIV virion fusion and endocytosis

Newly Synthesized APOBEC3G Is Incorporated into HIV Virions, Inhibited by HIV RNA, and Subsequently Activated by RNase H

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

Virion-Incorporated HA-A3G Associates with Viral Genomic RNA

<div><p>(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.</p><p>(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.</p><p>(C) Incorporation of HA-A3G into virions enhances the recruitment of NC into the IVAC.</p><p>(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.</p></div

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

<div><p>(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).</p><p>(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.</p><p>(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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0030015#ppat-0030015-g001" target="_blank">Figure 1</a>A. The immunoblots (IB) were also probed for A3G to determine the amount packaged into virion cores.</p></div

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

<div><p>(A) HA-A3G was immunoprecipitated from IVAC fraction 7 (F7) of virion lysates (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0030015#ppat-0030015-g003" target="_blank">Figure 3</a>A) or from a lower fraction, F17, generated by treatment of the virion lysates with RNase A (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0030015#ppat-0030015-g003" target="_blank">Figure 3</a>B). 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.</p><p>(B) Lysates of virions containing or lacking A3G were assessed in the deaminase assay, with or without RNase A treatment.</p><p>(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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0030015#ppat-0030015-g001" target="_blank">Figure 1</a>A.</p><p>(A–C) All deaminase reactions were carried out in 50 mM Tris (pH 7.4) with (+) or without (−) RNase A, as indicated.</p><p>(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).</p></div