Figure 2
From: Heterogeneous susceptibility of circulating SIV isolate capsids to HIV-interacting factors
Chimeric constructs that express lentiviral capsids and stable expression of primate TRIM5 in CHO cells reproduce TRIM5α restriction specificity. (A) Wild-type SIVmac GagPol expression vector and chimeric GagPol constructions were used to obtain single-round infectious virions, containing an SIVmac vector with eGFP and the vesicular stomatitis virus G fusion protein. Supernatants were harvested 48 h later and pelleted by ultracentrifugation through a 20% sucrose cushion. Viral preparations that gave 20% GFP positive cells on CHO cells were immunoblotted. Gag proteins were detected using sera from HIV-2 infected patients. (B) Stable expression of TRIM5α proteins in CHO cells. Cell extracts were obtained from CHO cells and CHO cells transduced with vectors encoding HA-tagged TRIM5α from human (hu-TRIM5α), chimpanzee (cpz-TRIM5α), African green monkey (agm-TRIM5α), or rhesus monkey (rh-TRIM5α). Immunoblotting was performed with an anti-HA monoclonal antibody. Actin was probed as loading control. (C) Cell extracts were obtained from CHO cells transduced with vectors encoding HA-tagged TRIM5CypA proteins from owl monkey (owl-TRIMCypA), Macaca mulatta (mamu-TRIMCypA) or Macaca fascicularis (mamu-TRIMCypA). TRIM-RanBP2Cyp corresponds to a synthetic protein constructed after fusing TRIM5 Linker 2 region of owl-TRIMcypA with the human RanBP2-Cyp sequence. (D) Infection efficiency of serial dilutions for each chimera in CHO hamster cells. (E) Restriction specificity in TRIM5α-expressing CHO cell lines. CHO cells stably expressing hu-TRIM5α (solid bars) or agm-TRIM5α (open bars) were challenged with virions harboring wild-type SIVmac or HIV-1 CA, or chimeric SIVmac-HIV-1 (MACA or CA) Gag proteins. GFP-positive cells were enumerated by flow cytometry. Ratios of percentage of infected cells in TRIM5α-expressing CHO cells over CHO control cells were determined. Results are expressed as means from at least three independent experiments. Error bars represent the standard error of the mean. Unpaired two-tailed Student’s t-test was used to assess significance and P-values < 0.001) are indicated with asterisks.