Experimental setup and susceptibility of DCs to HIV-1 and SIV_MAC infection in the presence of Vpx and dNs.

<p>A) DCs were differentiated by incubation of primary blood monocytes in GM-CSF/IL4 for 4 days prior to infection with VSVg-pseudotyped and exo-RT normalized HIV-1 and SIV_MACΔVpx viruses coding GFP at an MOI comprised between 0.5 and 1, according to the presented scheme. Cells were then either harvested 3 days later for flow cytometry analysis (B), or lysed twenty-four hours after infection for WB or DNA extraction and amplification (C and D, respectively). The results of the effects of Vpx and dNs on HIV-1 and SIV_MAC infection of DCs have been published in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140561#pone.0140561.ref032" target="_blank">32</a>] and representative FACS panels are shown here only for clarity’s sake from a total of more than 10 independent experiments conducted with cells of different donors. C) DCs were challenged with HIV-1 under the conditions specified in the figure prior to cell lysis twenty-four hours later and WB analysis. Note that the anti-A3A antibody used here recognizes two isoforms of the protein, the shorter one formed by translation at an internal ATG site. Similar results were obtained following infection with SIV_MACΔVpx (not shown). D) Identical amounts of cell lysates were amplified with primers specific for <i>gfp</i>, using denaturation temperatures ranging from 94°C to 82°C in a direct 3D-PCR. DNA amplified in the conditions of fraction 4 (denaturation temperature of 89°C) was cloned and individual clones sequenced. This fraction represents the lowest denaturation temperature at which DNA amplification is reproducibly observed. Representative agarose gel panels from 4 independent experiments are shown here.</p

Analysis of the possible modulation between editing activity of A3s and overall vDNA levels.

<p>A) DCs were challenged with the indicated viruses and twenty-four hours post infection the amount of vDNA was quantified by qPCR using oligonucleotides specific for <i>gfp</i> carried on both HIV-1 and SIV_MAC genomes. B) The average frequencies of editing measured in the experiments of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140561#pone.0140561.g002" target="_blank">Fig 2</a> in the case of HIV-1 alone were then used to calculate an expected frequency of deamination following normalization for vDNA levels. This value represents the deamination that could be expected if this activity depended only on the amount of vDNA present during infection. Both expected and measured values are presented here.</p

Cytidines present in a TC dinucleotide context account for the majority of editing signatures retrieved in vDNA produced following infection of DCs.

<p>A) The data presented in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140561#pone.0140561.g001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140561#pone.0140561.g002" target="_blank">2</a> was analyzed to determine the dinucleotide context of mutated cytidines found in the different conditions. For each virus, the size of the pie is proportional to the absolute number of mutations. B) Spatial distribution of all the mutations obtained within the reference sequence. Cytidines present in a TC context are marked with a dot. The height of the black bars represents the frequency of mutations at a specific site.</p

IFITM3 is a <i>bona fide</i> virion-associated protein.

<p>Virion particles produced as described above were then analyzed by immuno-gold electron microscopy. Briefly, unfixed viral preparations purified by ultracentrifugation and produced in the presence or absence of IFITM3 were incubated with anti-Flag antibodies, followed by incubation with a gold-conjugated secondary antibody (arrows). Representative pictures are shown here. The graph displays the number of gold particles counted on a per virion basis.</p

IFITMs associate to viral particles of most, albeit not all, viruses tested and do not induce detectable changes in mature glycoproteins incorporation.

<p>A) Virion particles produced in presence or absence of IFITMs and their corresponding cellular lysates were analyzed by WB. Virions were harvested from the cell supernatant and purified by ultracentrifugation through sucrose, with the exception of AAV for which cells were first lysed by freeze/thaw and then virions were purified by ultracentrifugation on a four-step iodixanol gradient. Load.; loading controls using either anti-actin, -tubulin or -EF1α antibodies. Virus-specific antibodies were used according to their availability. In the case of MPMV for which antibodies were not available, CA was identified by comparison with control supernatants and quantified after <i>Coomassie</i> staining and densitometry. The panels display representative results of 3 to 5 independent experiments. B) When available, the same viral preparations displayed in A were probed with antibodies specific to the indicated mature glycoproteins. The panels present representative results. The intracellular processing pathways of the viral glycoproteins analyzed is shown schematically at the right.</p

Experimental overview of the present study.

<p>Snapshot presentation of the viruses used in this study and schematic color-coded representation of the intracellular locations of virion particle assembly (A). Experimental setup used to determine effects of IFITMs on the formation of infectious viral particles (B). A comprehensive table of the viral systems used is provided in Supplementary <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006610#ppat.1006610.s003" target="_blank">S1 Fig</a>.</p