The construction of cyclophilin-binding loop substitutions in the HIV-1 capsid.

<p><b>A)</b> (top) Crystal structures of hexameric (pdb:3GV2) and monomeric (pdb:1AK4) HIV-1 capsid. The region shown in red is the cyclophilin-binding loop. (middle) Location of the cyclophilin-binding loop within gag-pol. (bottom) An alignment of various cyclophilin-binding loops. Asterisks (*) indicate conserved residues. The cyclophilin-binding loop of HIV-1 group M was replaced with the corresponding loop (in gray box) from each indicated virus. <b>B)</b> VSV-G pseudotyped HIV-1 (containing cyclophilin-binding loop substitutions as indicated and a GFP reporter) was produced and used to infect CRFK cells. Percent infection is shown over a viral dose curve. Regression slopes were calculated for each curve and no significant differences were found for any pairwise comparison. <b>C)</b> Western blot of whole cell extracts and virions from virus-producing 293T cells. Viral packaging plasmids were transfected into 293T cells and samples were analyzed 48 hours post transfection by immunoblotting with anti-p24 and anti-β-actin. <b>D)</b> Single-cycle infection assays were performed in the indicated TRIM-RanCyp stable cell lines (bottom) with viruses that have the indicated cyclophilin-binding loop (top of graph) and a GFP reporter in the HIV-1 backbone. The percentage cells infected in each sample was normalized to the empty vector control. Infections were performed in triplicate and error bars represent twice the standard error of the mean.</p>