An assisted evolution strategy to derive fit, rhTRIM5α-resistant HIV-1 strains.

<p>(A) Schematic representation of the experimental design used to select fit, rhTRIM5α resistant viruses. CA mutations shown to decrease TRIM5α-mediated restriction, without major fitness penalties were incorporated into a synthetic CA amplicon to generate a virus population containing random assortments of the mutations. (B) Serial passages of the virus population described in (A) in MT2-rhTRIM5α cells (filled symbols). Infection was monitored by measuring the proportion of cells expressing GFP over time, as described in the legend to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003667#ppat-1003667-g001" target="_blank">Figure 1</a>. In parallel, the last four of fifteen serial passages were also carried out in CEM-rhTRIM5α cells (open symbols).</p

Screen of random HIV-1 CA mutants for rhTRIM5α resistance.

<p>(A) NHG clones containing CA substitutions generated by random mutagenesis were used to challenge MT2 cells expressing an empty vector or rhTRIM5α using a single dose of each virus. Fold-restriction by rhTRIM5α (the ratio of the numbers of infected cells in each cell line) is plotted. Only data from viruses that gave measurable infectivity (>0.1% infected cells) in MT2-rhTRIM5α cells are plotted. The WT NHG is plotted as a red symbol and viruses restricted by less than 50-fold are represented by open symbols. (B) NHG mutants carrying CA mutations that decreased TRIM5α sensitivity were retested and infectious titers in MT2-vector (open symbols) and MT2-rhTRIM5α#8 (filled symbols, referencing the left Y-axis) cells, as well as the ratio of these titers (fold restriction red triangles, referencing the right Y-axis) are plotted. (C) Two views of the NL4-3 capsid hexamer <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003667#ppat.1003667-Pornillos1" target="_blank">[43]</a> are shown, as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003667#ppat-1003667-g002" target="_blank">Figure 2B</a>. The sites of amino acid substitutions found in (B) to increase infectious titer on MT2-rhTRIM5α cells (M10L, V83M, V86E, H87Q, I91T, R100S and A105T) regardless of effects on titer in vector control cells are indicated.</p

RhTRIM5α-resistant HIV-1 capsids are not recognized by rhTRIM5α.

<p>(A) The indicated CA sequences were tested for their ability to generate viral capsids that could saturate rhTRIM5α in a rhesus macaque cell line. FRhK-4 cells were infected with VSV-G pseudotyped virions containing HIV-1_NL4-3 GagPol proteins encoding either wildtype CA, SIV_mac239 CA, or the indicated mutant CA. An HIV-1 based vector that encoded Tat was packaged into these virions and ‘abrogating virus dose’ is given in TZM infectious units (I.U.). Cells were tested for rhTRIM5α abrogation by simultaneous infection with a fixed amount of a VSV-G pseudotyped virions carrying a WT HIV-1_NL4-3 GagPol and a GFP-reporter gene. (B) VSV-G pseudotyped virions carrying the indicated WT or mutant CA-encoding HIV-1_NL4-3 GagPol and a GFP-reporter gene were used to infect pgsA-745 cells or pgsA-745 cells stably expressing owl monkey TRIM-Cyp. Cells (1×10⁴) were infected with 2 µL of supernatant from transfected 293T cells.</p

RhTRIM5α-resistant mutants are immune to viral core disruption by rhTRIM5α.

<p>(A) Virions harvested from cells transfected with plasmids expressing HIV-1_NL4-3 GagPol (WT or CA mutants), VSV-G, and an HIV-1 vector carrying a GFP reporter were pelleted through 20% sucrose and CA protein was detected by western blotting. (B–F) PgsA (none) and pgsA-rhTRIM5α (rhT5α) cells were infected with VSV-G pseudotyped HIV-1_NL4-3 (WT or CA mutants), carrying a GFP reporter. Infected cells were harvested at T = 2 hr. after infection and post-nuclear supernatants were fractionated on 10–50% (w/v) sucrose gradients as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003667#s4" target="_blank">Materials and Methods</a>. Ten fractions were collected from the gradients. Q-PCR analysis of reverse transcription products and western blot analysis of CA and integrase (IN) in each fraction is represented for HIV-1_NL4-3 with WT (B), LNEIE (C), LMNEIE (D), INEIE (E) and NE (F) CA sequences.</p

HIV-1 adaptation in human cell lines expressing rhTRIM5α.

<p>(A–D) MT2 and MT4 cells expressing either rhTRIM5α (filled symbols) or an empty vector (open symbols) were infected with HIV-1_NL4-3/HIV-1_HXB2/GFP (NHG). Aliquots of cells from each culture were withdrawn and fixed for FACS analysis at the indicated times and the percentage of cells that were positive for GFP is plotted versus days after the infection of the first culture. Discontinuities indicate points at which cell-free supernatant was harvested and used to initiate the next passage in TRIM5α-expressing cell lines. NHG was passaged in four different cell lines expressing rhTRIM5α: MT2-rhTRIM5α#8 (A), MT2-rhTRIM5α#15 (B), MT4-rhTRIM5α#29 (C), and MT4-rhTRIM5α#32 (D). (E–H) Viral sensitivity to TRIM5α (Fold restriction) was calculated as the ratio of infectious titers present in culture supernatants at the end of each passage shown on the left, measured on MT2-vector and MT2-rhTRIM5α#8 cells.</p

Amino acid changes arising during HIV-1 passage in rhTRIM5α expressing cell lines.

<p>Amino acids symbols are <b>bold</b> where the WT codon was not detected in the sequence chromatogram. In other cases, the mutant was present as a mixture with the WT codon.</p

Selection of fit, rhTRIM5α-resistant viral clones from a synthetic CA library containing assortments of mutations conferring partial rhTRIM5α resistance.

<p>(A) Two views of the HIV-1 capsid hexamer <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003667#ppat.1003667-Pornillos1" target="_blank">[43]</a> are shown, as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003667#ppat-1003667-g002" target="_blank">Figure 2B</a>. The sites of amino acid substitutions in the species (LNEIE) that became dominant in MT2-rhTRIM5α cells, or codominant in CEM-rhTRIM5α cells (LNEIE and INEIE) following the assisted evolution experiment described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003667#ppat-1003667-g005" target="_blank">Figure 5</a> are shown. (B) For comparison, the sites of amino acid substitutions in the best performing mutant (NE) derived in the standard selection experiment described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003667#ppat-1003667-g001" target="_blank">Figure 1</a> as well as an LNEIE derivative containing an additional substitution (LMNEIE) are also shown. (C) NHG, an NHG derivative encoding an SIV_mac239 capsid, or NHG derivatives carrying the indicated combination of amino acid substitutions were used to infect MT2-rhTRIM5α#8 cells (filled symbols) or MT2 cells transduced with empty vector (open symbols).</p

HIV-1 CA mutations selected during passage in rhTRIM5α-expressing cells decrease sensitivity to rhTRIM5α.

<p>(A) Positions of amino acid residues at which mutations arose during replication in cells expressing rhTRIM5α are indicated on the HIV-1_NL4-3 capsid hexameric structure <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003667#ppat.1003667-Pornillos1" target="_blank">[43]</a>. V86E and I91N/T are in the cyclophilin-binding loop and G116E is in helix six. The capsid hexamer shown on the left represents the exterior of the viral core, viewed from the cytoplasm of an infected cell. The hexamer on the right is oriented with the cytoplasmic face toward the top of the picture and the residues that face toward the interior of the core on the bottom. Images were generated using MacPyMOL. (B) NHG (WT) or NHG derivatives carrying the indicated mutations were titrated on MT2-vector (open symbols) or MT2-rhTRIM5α#8 (filled symbols) cells. Single-cycle infectivity was determined by FACS analysis. Mutant CA sequences that were found in the passaged viral population are marked with a green asterisk while mutants that were not found in the passaged viral population but were constructed using site-directed mutagenesis are marked with a red asterisk.</p

A single gp120 residue can affect HIV-1 tropism in macaques

<div><p>Species-dependent variation in proteins that aid or limit virus replication determines the ability of lentiviruses to jump between host species. Identifying and overcoming these differences facilitates the development of animal models for HIV-1, including models based on chimeric SIVs that express HIV-1 envelope (Env) glycoproteins, (SHIVs) and simian-tropic HIV-1 (stHIV) strains. Here, we demonstrate that the inherently poor ability of most HIV-1 Env proteins to use macaque CD4 as a receptor is improved during adaptation by virus passage in macaques. We identify a single amino acid, A281, in HIV-1 Env that consistently changes during adaptation in macaques and affects the ability of HIV-1 Env to use macaque CD4. Importantly, mutations at A281 do not markedly affect HIV-1 Env neutralization properties. Our findings should facilitate the design of HIV-1 Env proteins for use in non-human primate models and thus expedite the development of clinically relevant reagents for testing interventions against HIV-1.</p></div

Amino acid changes in the CD4 binding site of gp120.

<p><b>(A)</b> Amino acid sequence alignments of the CD4 binding site of HIV-1 gp120 proteins prior to (top) and following passage in macaques of 1054. Clones obtained from animal P1A at weeks 2, 9 and 35 post-inoculation are represented by sequences P1(Aw2), P1(Aw9) and P1A(w35) respectively. Individual clones from animal P1A obtained from week 17 post-inoculation and subsequent weeks and passages are shown. Residues are numbered as in [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006572#ppat.1006572.ref037" target="_blank">37</a>] and residues shown to interact directly with CD4 are in black. (B) Amino acid sequence alignments as in (A) of AD8, SF162, 89.6, SF33 and C109 HIV-1 Env aa sequences prior to (top) and following <i>in vivo</i> adaptation (bottom). <b>(C)</b> Structure of gp120 bound to soluble human CD4 (modified from [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006572#ppat.1006572.ref037" target="_blank">37</a>] using PyMol). Surface representation of HIV-1 gp120 is shown in grey, with aa A281 with in red. Stick representation of huCD4 in light purple with residue N39 shown as green spheres. Calculated distance between gp120-A281 and huCD4-N39 is shown. The magenta asterisk marks Phe43 in CD4 (that is conserved between human and macaque proteins) involved in the interaction with gp120.</p