The Frequency of Cytidine Editing of Viral DNA Is Differentially Influenced by Vpx and Nucleosides during HIV-1 or SIVMAC Infection of Dendritic Cells.

Two cellular factors are currently known to modulate lentiviral infection specifically in myeloid cells: SAMHD1 and APOBEC3A (A3A). SAMHD1 is a deoxynucleoside triphosphohydrolase that interferes with viral infection mostly by limiting the intracellular concentrations of dNTPs, while A3A is a cytidine deaminase that has been described to edit incoming vDNA. The restrictive phenotype of myeloid cells can be alleviated through the direct degradation of SAMHD1 by the HIV-2/SIVSM Vpx protein or else, at least in the case of HIV-1, by the exogenous supplementation of nucleosides that artificially overcome the catabolic activity of SAMHD1 on dNTPs. Here, we have used Vpx and dNs to explore the relationship existing between vDNA cytidine deamination and SAMHD1 during HIV-1 or SIVMAC infection of primary dendritic cells. Our results reveal an interesting inverse correlation between conditions that promote efficient infection of DCs and the extent of vDNA editing that may reflect the different susceptibility of vDNA to cytoplasmic effectors during the infection of myeloid cells

The susceptibility of primate lentiviruses to nucleosides and Vpx during infection of dendritic cells is regulated by CA

International audienceThe block toward human immunodeficiency virus type 1 (HIV-1) infection of dendritic cells (DCs) can be relieved by Vpx (viral protein X), which degrades sterile alpha motif-hydroxylase domain 1 (SAMHD1) or by exogenously added deoxynucleosides (dNs), lending support to the hypothesis that SAMHD1 acts by limiting deoxynucleoside triphosphates (dNTPs). This notion has, however, been questioned. We show that while dNs and Vpx increase the infectivity of HIV-1, only the latter restores the infectivity of a simian immunodeficiency virus of macaques variant, SIVMACΔVpx virus. This distinct behavior seems to map to CA, suggesting that species-specific CA interactors modulate infection of DCs

Identification of a New Ribonucleoside Inhibitor of Ebola Virus Replication

The current outbreak of Ebola virus (EBOV) in West Africa has claimed the lives of more than 15,000 people and highlights an urgent need for therapeutics capable of preventing virus replication. In this study we screened known nucleoside analogues for their ability to interfere with EBOV replication. Among them, the cytidine analogue β-d-N4-hydroxycytidine (NHC) demonstrated potent inhibitory activities against EBOV replication and spread at non-cytotoxic concentrations. Thus, NHC constitutes an interesting candidate for the development of a suitable drug treatment against EBOV

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

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

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

Protection against henipavirus infection by use of recombinant adeno-associated virus-vector vaccines.

International audienceNipah virus (NiV) and Hendra virus (HeV) are closely related, recently emerged paramyxoviruses that are capable of causing considerable morbidity and mortality in several mammalian species, including humans. Henipavirus-specific vaccines are still commercially unavailable, and development of novel antiviral strategies to prevent lethal infections due to henipaviruses is highly desirable. Here we describe the development of adeno-associated virus (AAV) vaccines expressing the NiV G protein. Characterization of these vaccines in mice demonstrated that a single intramuscular AAV injection was sufficient to induce a potent and long-lasting antibody response. Translational studies in hamsters further demonstrated that all vaccinated animals were protected against lethal challenge with NiV. In addition, this vaccine induced a cross-protective immune response that was able to protect 50% of the animals against a challenge by HeV. This study presents a new efficient vaccination strategy against henipaviruses and opens novel perspectives on the use of AAV vectors as vaccines against emergent diseases