Promiscuous RNA Binding Ensures Effective Encapsidation of APOBEC3 Proteins by HIV-1

<div><p>The apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) proteins are cell-encoded cytidine deaminases, some of which, such as APOBEC3G (A3G) and APOBEC3F (A3F), act as potent human immunodeficiency virus type-1 (HIV-1) restriction factors. These proteins require packaging into HIV-1 particles to exert their antiviral activities, but the molecular mechanism by which this occurs is incompletely understood. The nucleocapsid (NC) region of HIV-1 Gag is required for efficient incorporation of A3G and A3F, and the interaction between A3G and NC has previously been shown to be RNA-dependent. Here, we address this issue in detail by first determining which RNAs are able to bind to A3G and A3F in HV-1 infected cells, as well as in cell-free virions, using the unbiased individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) method. We show that A3G and A3F bind many different types of RNA, including HIV-1 RNA, cellular mRNAs and small non-coding RNAs such as the Y or 7SL RNAs. Interestingly, A3G/F incorporation is unaffected when the levels of packaged HIV-1 genomic RNA (gRNA) and 7SL RNA are reduced, implying that these RNAs are not essential for efficient A3G/F packaging. Confirming earlier work, HIV-1 particles formed with Gag lacking the NC domain (Gag ΔNC) fail to encapsidate A3G/F. Here, we exploit this system by demonstrating that the addition of an assortment of heterologous RNA-binding proteins and domains to Gag ΔNC efficiently restored A3G/F packaging, indicating that A3G and A3F have the ability to engage multiple RNAs to ensure viral encapsidation. We propose that the rather indiscriminate RNA binding characteristics of A3G and A3F promote functionality by enabling recruitment into a wide range of retroviral particles whose packaged RNA genomes comprise divergent sequences.</p></div

iCLIP reveals which RNAs are bound to A3G and A3F in living cells. (A)

<p>CEM-SS T cells stably expressing GFP, GFP-tagged A3G or A3F, or T7-tagged GFP, A3G or A3F were infected with <i>vif</i>-deficient HIV-1_IIIB. Cells were collected 48 h later, subjected to cross-linking with UV and lysed. A high concentration of RNase A was added to one sample (HR, lane 3) and a low concentration to the rest of the samples. Lysates were sonicated and the proteins of interest were immunoprecipitated with anti-GFP or anti-T7 antibodies bound to dynabeads. A linker was ligated to the nucleic acids, and these were radiolabeled with P³²-ϒ-ATP. The samples were resolved by SDS-PAGE, and the RNA was visualised by autoradiography. A representative gel is shown. <b>(B)</b> RNAs running at a higher molecular mass than the proteins of interest were extracted from the membrane and reverse transcribed using a bar coded primer annealing to the previously ligated linker. The cDNAs were multiplexed and run on a TBE-urea gel. Bands ranging from 70–85, 85–110 and >110 base pairs (that contain 20–35, 35–60 and >60 bp of insert) were excised and the nucleic acids were isolated. The cDNAs were circularised, digested with BamHI and amplified by PCR. The product was then run on a gel to assess the quality of the library. A representative library is shown. The fractions that did not contain primer dimers of each library were mixed and sequenced. <b>(C)</b> Reads obtained from sequencing were aligned to the human genome. Only reads that aligned once with the possibility of 1 mismatch were considered for further analysis. Reads aligning to each gene were divided by the total number of reads in the library and the relation for each of the replicates was determined (r>0.9) and for each of the differently tagged proteins (r>0.9). The reads were then sorted into categories: 3’-UTR, 5’-UTR, open reading frame (ORF), intergenic regions (inter), intron, non-coding RNAs (ncRNA) and telomers (telo) and the values compared with the GFP negative control. The graph shows the average fold of 4 independent replicates obtained for A3G and A3F compared with GFP for each category of sequence and their respective standard deviations. <b>(D)</b> Reads were aligned to the HIV-1 genome as described for panel C. Repeat masker was used to align reads to specific genes that are found in viral particles. Reads aligning to Y1, Y3, Y4 and Y5 RNAs were added and considered as total Y RNAs. Similarly, we considered the same for U RNAs and tRNAs. The average fold compared to GFP of the 4 independent libraries was then plotted with standard deviations. <b>(E)</b> Reads obtained in the libraries of HIV-1-packaged A3G and A3F were aligned as described in panel C. The percentage of aligned reads to the human and HIV-1 genomes for the iCLIP performed with cells or virions was then calculated for each sample. Here, we show the average of the 4 independent replicates with standard deviations.</p

VLPs with reduced 7SL RNA content package A3G and A3F. (A)

<p>The <i>vif</i>-deficient NL4-3 provirus was co-transfected with a plasmid expressing SRP19 or a control plasmid into 293T cells stably expressing HA-tagged A3G or A3F. Viruses were harvested 48 h later and concentrated through a sucrose cushion. Gag and A3G/F were detected by immunoblot with representative data from one of at least 3 independent experiments shown. <b>(B)</b> 293T cells stably expressing HA-tagged A3G or A3F were co-transfected with Gag expression constructs and with a plasmid expressing SRP19 or an empty vector. VLPs were harvested 48 h later and concentrated through a sucrose cushion. Gag proteins and A3G or A3F were visualised by immunoblot. Proteins in VLPs were quantified as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004609#ppat.1004609.g002" target="_blank">Fig. 2B</a>. The graph shows the average of 3 independent experiments and the respective standard deviation. <b>(C)</b> RNAs were extracted from VLPs and 7SL RNA was quantified by qPCR. The average and standard deviation of 3 experiments are shown, where the value obtained for Wt Gag was set to 1 and the others compared to it.</p

A3G and A3F are packaged into VLPs doubly depleted for genomic RNA and 7SL RNA.

<p>VLPs were produced in 293T by co-transfection with expression vectors for HA-tagged A3G or A3F and a plasmid expressing Gag-Pol, Gag-Pol ΔΨ or Gag ΔNC. SRP19 (or a negative control) was over-expressed where indicated. Gag and A3G/F proteins in concentrated VLPs were visualised by immunoblot. Proteins in VLPs were quantified as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004609#ppat.1004609.g002" target="_blank">Fig. 2A</a>. The average of 3 replicates with standard deviation is shown here with a representative immunoblot.</p

Incorporation of A3G and A3F into Gag-SRP19 and Gag-Ro60 containing VLPs. (A)

<p>293T cells were co-transfected at a 1:5 ratio with HA-tagged A3G or A3F expression vectors and Wt Gag or the different Gag ΔNC constructs indicated. Gag and A3G/F proteins from concentrated VLPs were then visualised by immunobloblot. <b>(B)</b> RNA was extracted from VLPs and 7SL RNA was quantified by qPCR. The average of 3 replicates with standard deviation is shown. <b>(C)</b> Expression vectors for HA-tagged A3G or A3F were co-transfected with Gag constructs at a ratio of 1:5 into 293T cells. Where indicated, Wt Gag or Gag ΔNC were co-transfected with Gag ΔNC fused to Ro60 at a ratio of 1:5 but maintaining the overall levels of Gag expression plasmid between samples. VLPs were harvested and concentrated. Gag and A3G/F were visualised by immunoblot. A representative result from 3 independent experiments is shown. <b>(D)</b> VLPs were lysed and the RNAs extracted. Y3 RNA was detected by qPCR. The average of 3 replicates with standard deviation is shown.</p

A3G and A3F concentrations are similarly distributed between cells and VLPs by RNA. (A)

<p>293T cells were co-transfected at a 1:5 ratio with expression vectors for T7-tagged A3G or A3F, and Wt Gag. Cell lysates (CL) were kept for analysis. Supernatant from cells transfected with an empty plasmid (negative control) and VLPs were harvested and isolated by ultracentrifugation through a continuous sucrose gradient (20–60%). Fractions were harvested and p24^Gag-containing fractions were identified by immunoblot. T7-tagged A3G was purified and quantified as specified in the material and methods. Cell lysates, fractions containing VLPs (or the corresponding fraction from the negative control) and purified A3G were analysed by immunoblot. Gag and HSP90 were visualised using anti-p24^Gag and anti-HSP90 antibodies, respectively. A3G and A3F were visualised using an anti-T7 antibody. APOBEC3 proteins were quantified by densiometry. This figure shows a representative example. <b>(B)</b> Standard curve of purified T7-A3G visualised and quantified by densiometry of the immunoblot of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004609#ppat.1004609.g007" target="_blank">Fig. 7A</a>. <b>(C)</b> RNA was extracted from cell lysates (CL) and VLP samples and quantified. Total A3G/F protein in CL and VLPs was quantified using a standard curve. The RNA in the negative control was below the detection threshold of the method. <b>(D)</b> The total amount of A3G or A3F was divided by the total concentration of extracted RNA. The graph shows the average of 4 independent experiments. Error bars indicate standard deviation. There was no statistically significant difference between the ratios in lysates and VLPs (p>0.5).</p

A3G and A3F are incorporated in VLPs when Gag is fused to different RNA-binding domains.

<p>Gag ΔNC was fused to RNA-binding domains (RBD) of hnRNP C1, hnRNP K, SRSF2 and Staufen-1. These constructs were co-transfected into 293T cells with vectors expressing either Wt Gag or Gag ΔNC at a ratio of 5:1 and also with HA-tagged A3G or A3F. VLPs were harvested and concentrated, and proteins were visualised by immunoblot. A representative blot of 3 independent experiments is shown.</p

Site-specific editing frequencies in infected cells from single-cycle APOBEC3 titration transfection experiments.

<p>The single-cycle substitution rate for HIV-1 in the absence of human APOBEC3 was 8.6×10⁻⁴ mutations per nucleotide, whereas the mean single-cycle substitution rate for HIV-1 in the presence of human APOBEC3 ranged from about 1×10⁻³ to 2×10⁻² per nucleotide substitution (<b>A</b>). The frequency of substitutions increased significantly in the region of the Gag gene of HIV-1 we sequenced in accord with increasing concentrations of the APOBEC3 proteins. The maximum single-cycle substitution rate for HIV-1 was 2×10⁻³ substitutions per site in the presence of APOBEC3D, 1.4×10⁻² substitutions per site in the presence of APOBEC3F, 2.7×10⁻² substitutions per site in the presence of APOBEC3G and 1.1×10⁻⁴ substitutions per site in the presence of APOBEC3H. The concentration of APOBEC3 at which we observed half of the estimated maximum substitution rate was 0.02 for APOBEC3D, 2.09 for APOBEC3F, 0.23 for APOBEC3G, and 0.22 µg for APOBEC3H. The single-cycle substitution rate for each mutation type (transition = Ts or transversion = Tv) of HIV-1 in the titration transfection experiments differed by 1.2 order of magnitude (<b>B</b>). For each of the human APOBEC3 proteins, we show the positions in the Gag gene of HIV-1 where the frequency of G-to-A mutation increased with increasing amounts of human APOBEC3 protein (Spearman rank correlation coefficient, <i>P</i>-value<0.05) (<b>C</b>). The G-to-A mutations are shown in a number of contiguous nucleotide sequence editing contexts. We used a sliding window to deduce the base frequency of G-to-A mutations (in each contiguous nucleotide context of the edited sites for each APOBEC3 protein (APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H) using the total G-to-A mutation frequency at increasing concentration. Positions with a non-significant increase in G-to-A mutations were excluded from the calculations.</p

Diversifying selection at individual nucleotide sites.

<p>A summary of the number of codon sites identified by the SLAC method implemented in by HyPhy <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004281#ppat.1004281-Pond1" target="_blank">[50]</a> that show positive or negative selection at the APOBEC3 and non-APOBEC3 motifs in the regions of the Gag and Vif genes of HIV-1 we sequenced (<i>P</i>-value<0.02). The Vif gene of HIV-1 was over-represented with G-to-A mutations in an APOBEC3 editing context at positively selected sites (<b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004281#ppat.1004281.s008" target="_blank">Table S5</a></b>).</p

Single-cycle titration transfection conditions and mutation rates per conditions.

a<p>3 µg of Vif deficient HIV-1 pIIIB/Δvif construct and 0.15 µg each of Vesicular Stomatitis Virus-G (VSV-G) envelope construct were co-transfected with wild type APOBEC3 expression vector and non-editing mutant APOBEC3 or empty vector in various conditions.</p>b<p>Wild-type APOBEC3 construct in pcDNA3.1 expression vector.</p>c<p>Non-editing mutant APOBEC3 or absent APOBEC3 construct.</p>d<p>Empty pcDNA3.1 vector.</p