Skip to main content
. 2009 Sep 8;119(10):3035–3047. doi: 10.1172/JCI39354

Figure 2. HIV-1 restriction activity correlates with CypA fusion to the TRIM5α specificity determinant and the ability to form cytoplasmic bodies.

Figure 2

(A) Expression of hT5Cyp fusion proteins. FLAG-hT5Cyp fusion proteins synthesized in 293T cells were immunoprecipitated with anti-FLAG antibodies and immunoblotted with anti-CypA antibodies. (B) HIV-1 CA binding activity of hT5Cyp fusion proteins. FLAG-hT5Cyp and GST-CA fusion proteins were co-expressed in 293T cells, pulled out on glutathione-sepharose beads in the presence or absence of 20 μM CsA, and immunoblotted with anti-CypA and anti–p24-CA antibodies. (C) Model of the PRYSPRY domain of hTRIM5α based on crystal structures of PRYSPRY, GUSTAVUS, and TRIM21. Two ribbon representations showing the position of hCypA fusions (spheres) to the hTRIM5α PRYSPRY domain. The grey transparent ribbon indicates regions of the model that would be replaced by CypA in hT5-S331-Cyp. (D) Restrictive T5Cyp fusion proteins formed discrete puncta in the cytoplasm. Indirect immunofluorescence images of CRFK cells stably expressing the indicated T5Cyp fusions. Fixed samples were stained with anti-TRIM5 antibody (green) and anti-tubulin antibody (red), followed by counterstain with DAPI (blue) to visualize the nuclear DNA. For each color, an individual stack of twenty 0.35-μm optical sections was acquired and subjected to maximum intensity projection along the optical axis. Images represent 3-color overlays. Scale bar: 5 μm. All panels are color coded for restriction phenotype (red, restrictive; orange, variably restrictive; green, permissive).