Structure of the transferrin receptor TfR1.

<p>The dimeric, butterfly-shaped protein TfR1 comprises three domains (colored yellow, red, and green in one protomer; the other protomer is shown in cyan). The receptor-binding domain of the Machupo virus GP1 protein (purple) is shown bound to the TfR1 apical domain. The purple and blue ovals illustrate the sites of Machupo GP1 and MMTV envelope surface (SU) protein binding, respectively, as well as the sites of positive selection. Reprinted with modification from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001574#pbio.1001574-Abraham1" target="_blank">[12]</a>, with permission.</p

Beneficial viral mutants (red) arise in the “effective” virus subpopulation (<i>N</i>_eff, pink circle) and spread gradually to the entire “census” population (blue circle).

<p>For a number of reasons (see the text), the effective population may be much smaller than the census population.</p

Lower pre-ART intra-participant HIV-1 <i>pol</i> diversity may not be associated with virologic failure in adults

<div><p>Background</p><p>Identifying pre-ART factors associated with the emergence of HIV-1 drug resistance is critical for optimizing strategies to prevent virologic failure. A previous study reported that lower pre-ART HIV-1 <i>pol</i> diversity was associated with higher risk of virologic failure in HIV-1-infected children. To investigate this association in adults, we measured HIV-1 diversity with deep sequencing in pre-ART samples from adults with well-characterized virologic outcomes in a study (A5142) of initial ART conducted by the AIDS Clinical Trials Group (ACTG).</p><p>Methods</p><p>We identified 22 cases in ACTG A5142 who experienced virologic failure with drug resistance mutations in RT and 44 matched controls who did not experience virologic failure. cDNA was synthesized from plasma HIV-1 RNA. Each cDNA molecule was tagged with a unique primer ID and RT codons 41–103 were amplified and deep sequenced. Sequences with the same tag were aligned and a consensus was generated to reduce PCR and sequencing errors. Diversity was calculated by measuring average pairwise distance (APD) of the consensus sequences. An exact conditional logistic regression model with percent APD as the risk factor estimated the odds ratio for VF and the corresponding 95% confidence interval.</p><p>Results</p><p>Consensus single-genome sequences and diversity estimates of <i>pol</i> were obtained for pre-ART samples from 21 cases and 42 controls. The median (IQR) pre-ART percent APD was 0.71 (0.31–1.13) in cases and 0.58 (0.32–0.94) in controls. A possible trend was found for higher diversity being associated with greater risk of virologic failure in adults (OR = 2.2 per one percent APD increase, 95% CI = [0.8, 7.2]; p = 0.15).</p><p>Conclusions</p><p>This study in adults suggests there is a positive association between higher pre-ART <i>pol</i> diversity and the risk of virologic failure in adults rather than an inverse relationship reported in children.</p></div

Effect of VPA and intensified ART on resting cell infection and low-level viremia.

<p>*All results represent pooled assays at entry/week −4, and week 12/16. Baseline ART assays for patients 6, 9, and 12 represent pooled assays from entry/week −4 and 2 prior time points. Weeks 32, 48, 96 are assays from only those time points.</p><p>**Simultaneous Amplicor assays at all SCA time points were <50 copies, except for patient 3 at day of study entry when Amplicor = 58 and SCA>1000.</p><p>†Declined VPA dose escalation.</p><p>††Intermittent non-adherence to study medication.</p><p>§Early study discontinuation.</p

Comparison of the infectivity of the viruses with the synthesis of the viral DNA.

<p>Viruses were prepared by co-transfection of 293T-hygro cells (which contain pLXSH) with viral clones, pBABE-Luc, and 0, 3, or 10 μg of APOBEC3 DNA. 293-mCAT1 cells were then infected with the resulting culture fluids. Relative specific infectivities (black lines) were determined as in Fig. 2. Parallel cultures were lysed and assayed for <i>hph</i> DNA (red lines) and strong stop DNA (green lines) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038190#s4" target="_blank">Materials and Methods</a>. Values in each assay were divided by the value for the virus produced with no APOBEC3 plasmid.</p

Effects of the different APOBEC3s on the infectivity of MoMLV and chimera viruses.

<p>Virus particles were produced by transient transfection of viral clones together with pBABE-Luc and 0, 3, or 10 μg of either mA3 or hA3G expression plasmids. Specific infectivities were calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038190#s4" target="_blank">Materials and Methods</a>, dividing the luciferase activity values by the RT activity values; specific infectivity of samples produced without APOBEC3 is set to 100%. Black line, MoMLV with mA3; green, chimera with mA3; red, MoMLV with hA3G; blue, chimera with hA3G. Results are plotted <i>vs.</i> the quantity of APOBEC3 plasmid used in the transfections to generate the viruses.</p

Specific infectivity of the viruses.

<p>293T cells were transiently transfected with an infectious MoMLV or chimera proviral genome together with the reporter plasmid pBABE-Luc. 293T-mCAT1 cells were then infected with culture supernatants from the transfectants, and lysates of these cells were assayed for luciferase activity. Virions were assayed for RT activity following precipitation from the culture supernatants with polyethylene glycol. The graph shows the luciferase activity divided by the RT activity of the viruses, with the value for MoMLV set to 100%; thus the data represent the relative specific infectivities of the samples.</p

Immunoblotting of virus particles. A) Western blot on produced virus.

<p>Chimera (lanes 1 to 5) or MoMLV (lanes 6 to 10) were prepared by transient transfection of 293T cells, using 0, 3, or 10 μg APOBEC3 DNA as well as viral DNA; control cells were transfected with DNA of pGCcos3Neo (a derivative of pSV2Neo) (lane 11). The culture supernatants were fractionated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038190#s4" target="_blank">Materials and Methods</a> and equal volumes of culture fluid were loaded into the lanes and analyzed by immunoblotting against P30^CA. M, molecular weight markers. <b>B) Encapsidation of different APOBEC3s in MoMLV and chimera viruses.</b> Virus particles shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038190#pone-0038190-g003" target="_blank">figure 3A</a> were analyzed by immunoblotting against the HA tag. Equal volumes of culture fluid were loaded into the lanes.</p

Effect of APOBEC3s on ΔVif HIV-1 DNA synthesis.

<p>293-mCAT1 cells were infected with ΔVif HIV-1. Infectivity and RT activity were assayed as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038190#pone.0038190-Derse1" target="_blank">[25]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038190#pone.0038190-Gorelick1" target="_blank">[29]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038190#pone.0038190-Gorelick2" target="_blank">[30]</a>. Twenty-four hours after infection, the cells were lysed and assayed by real-time PCR for <i>Luciferase</i> DNA (black and green lines) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038190#s4" target="_blank">Materials and Methods </a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038190#pone.0038190-Gherghe1" target="_blank">[32]</a>. Specific infectivity is represented with red and blue lines.</p

Neighbor-joining tree of 25 consensus sequences from each pre-ART participant sample using a requirement of 5 sequences containing the same primer ID to generate the consensus.

<p>Bootstrap values are shown for each branch. Cases are shown in blue and controls in black. The width of each triangle is indicative of the APD of HIV sequences in each participant.</p