HIV-1 Vpu–mediated loss of tetherin is partially abrogated by MG132.

<p>(A) Co-expression of HIV-1 plasmids and wild-type (WT) N terminally tagged human tetherin and measurement of HIV-1 release in the presence or absence of HIV-1 Vpu-HA co-expression and proteasome inhibitor MG132 (0.8 µM for 12 hours) as shown. Errors are standard error of the mean of 2 experiments. (B) Tetherin was detected by western blot of Xpress tag in cleared RIPA extract supernatants and pellets as shown. Sizes of molecular weight markers are shown in kilodaltons. (C) Vpu-HA was detected in sonicated RIPA extract as shown (D) Blots in B have been stripped and re-probed for β actin as a loading control. (E) Measurement of HIV-1 p24 in the supernatant of transfected cells by western blot. Data are representative of 2 independent experiments.</p

Vpu expression leads to a loss of wild-type but not a quadruple mutant tetherin protein steady state levels.

<p>(A) Co-expression of HIV-1 plasmids and wild-type (WT) N terminally tagged human tetherin and measurement of HIV-1 release in the absence of HIV-1 Vpu (black bar), or presence (white bar). Mutation of positively selected residues I26V, V30G, I36L, T45I (ΔTHN) in the trans-membrane region of human tetherin results in reduced sensitivity to Vpu whilst maintaining antiviral activity. The effect of co-transfection of HIV-1 plasmids and untagged human tetherin is shown for comparison (Lane C). The titre of the unrestricted HIV-1 was 10⁷ infectious units/ml. Errors are standard error of the mean of 2 experiments. (B) Measurement of HIV-1 p24 in the supernatant of transfected 293T cells by western blot (C) Measurement of HIV-1 Gag levels in extracts from transfected 293T cells. Cell extract lysates (D) or pellets (F) were blotted for the Xpress tag to detect tetherin. Sizes of molecular weight markers are shown in kilodaltons. Blots in D (E) or F (G) were stripped and re-probed for β actin as a loading control. Data are representative of 3 independent experiments and similar results were seen with an N terminal HA tag.</p

Selection analyses reveal positively selected tetherin residues in the primate lineage.

<p>(A) Nucleotide alignment of nine primate tetherin sequences. HoSa (Homo Sapiens), Popy (Pongo Pygmaeus (Orangutan)) Patr (Pan Trolodytes (Chimpanzee)), Tan (Tantalus monkey), Ver (Vervet monkey), Mane (Macaque Nemestrina (pigtailed macaque)), Mafa (Macaque Fasicularis (cynomolgus monkey)), Mamu (Macaque Mulatta (Rhesus macaque)) Caja (Callithrix jacchus (white tufted ear marmoset). Codon positions under positive selection are indicated by shaded boxes. Secondary structure (SS) was predicted by PSIPRED <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000443#ppat.1000443-McGuffin1" target="_blank">[53]</a> and is symbolised as (+) cytoplasmic domain; (I) trans-membrane domain inner cap; (X) trans-membrane domain alpha helix; (O) trans-membrane domain outer cap; (−) extra-cellular domain. (B) Predicted structure of the trans-membrane helix performed using helical wheel projection (<a href="http://rzlab.ucr.edu/scripts/wheel/wheel.cgi" target="_blank">http://rzlab.ucr.edu/scripts/wheel/wheel.cgi</a>) suggests that residues 26, 30, and 36 are on the same side of the protein. Closed circles indicate positively selected amino acids, dashed circles indicate residues that are different between human and Tantalus monkey, but not positively selected. Human amino acids are shown in black and Tantalus monkey in grey italic.</p

Mutation of a single amino acid (T45I) leads to insensitivity to Vpu and persistence of tetherin protein.

<p>(A) Co-expression of HIV-1 plasmids and wild-type (WT) N terminally tagged human tetherin and measurement of HIV-1 release in the absence of HIV-1 Vpu (black bar), or presence (white bar). Mutation T45I in the trans-membrane region of human tetherin results in insensitivity to Vpu whilst maintaining antiviral activity. The titre of the unrestricted HIV-1 was 10⁷ infectious units/ml. Errors are standard error of the mean of 2 experiments. (B) Measurement of HIV-1 p24 in the supernatant of transfected 293T cells by western blot (C) Measurement of HIV-1 Gag levels in cleared RIPA extracts from transfected 293T cells. Tetherin was detected by western blot of N-terminal Xpress tag in the cleared RIPA extract supernatants (D) and pellets (F) as shown. Sizes of molecular weight markers are shown in kilodaltons. Blots in (E) and (G) have been stripped and re-probed for β actin as a loading control. Data are representative of 2 independent experiments.</p

The positively selected tetherin trans-membrane region residues impact on sensitivity to HIV-1 Vpu.

<p>(A) Co-expression of HIV-1 plasmids alone (C) with wild-type human tetherin (WT) and measurement of HIV-1 release in the absence of HIV-1 Vpu (white bar) or presence (black bar). Mutation of positively selected residues I26V, V30G, I36L, T45I (Quad) in the trans-membrane region of human tetherin results in reduced sensitivity to Vpu whilst maintaining similar antiviral activity. The effect of single mutations are also shown. Errors are standard error of the mean of 2 experiments. Equal amounts of tetherin plasmids were used (100 ng) (B) Measurement of HIV-1 p24 in the supernatant of transfected 293T cells by western blot (C) Measurement of HIV-1 Gag levels in extracts from transfected 293T cells. (D) Cell extract blots in C were stripped and re-probed for β actin as a loading control.</p

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

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

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

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

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