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Species-specific vulnerability of RanBP2 shaped the evolution of SIV as it transmitted in African apes

Fig 3

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

A) (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) 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. C) 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. D) 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.

Fig 3