Indeed, the kinetics of CypA-DsRed loss from IN-sfGFP particles was considerably faster (half-time ~10 min) in the presence than in the absence of CsA (compare Fig 3A and 3B). proteins are shown. Scale bar 50 m. (D) Pseudoviruses co-labeled with IN-sfGFP and Oxaliplatin (Eloxatin) CypA-mRFP or CypA-DsRed were adhered to coverslips and subjected to mild permeabilization with saponin (100 g/ml). Images were acquired immediately before and 5 min after application of saponin. Scale bar 5 m. (E) Analysis of the CypA-mRFP and CypA-DsRed loss from saponin-permeabilized viruses in panel D. Error bars represent standard error from 3 independent experiments.(TIFF) ppat.1005709.s001.tiff (7.1M) GUID:?8A1A97D7-5554-4675-98AC-035790FD3336 S2 Fig: Oligomerization and Oxaliplatin (Eloxatin) virus incorporation of fluorescently tagged CypA constructs. (A) Western blot analysis Rabbit Polyclonal to Collagen IX alpha2 of oligomerization of mRFP, DsRed and CypA fusions with either of fluorescent proteins transiently expressed in 293T cells. Cytosolic extracts were obtained by digitonin treatment as described in Material and Methods. Samples containing 0.25 g of total protein were boiled for 5 min at 95C or left at room temperature prior to loading on a 12% PAGE and immunoblot developed using either rabbit anti-mCherry antibody (1:500 dilution, Abcam) or rabbit anti-Cyclophilin A antibody (1:500 dilution, Millipore). (B) Western blot analysis of pseudoviruses produced by transfection of 293T cells with pR8Env plasmid and either CypA, CypA-DsRed or CypA-mCherry vector. Control CypA-DsRed-labeled samples were produced in the presence of 500 nM HIV-1 protease inhibitor Saquinavir (SQV). Virus samples were purified through 20% sucrose cushion and quantified for p24 content. Equal p24 content containing viral suspension was loaded on a 12% PAGE and immunoblot developed using antibodies against HIV-1 CA, CypA. Lower panels showing CypA expression and loading control tubulin in producer cell lysates. (C) Densitometric quantification of CypA-DsRed and CypA-mCherry incorporation into virions (panel B, top). The intensity of the respective CypA bands was normalized to the total intensity of Pr55 and p24 bands using Image Lab software (Bio-Rad).(TIFF) ppat.1005709.s002.tiff (1.4M) GUID:?1062A190-F774-4196-9321-41006A947BA9 S3 Fig: The effect of CypA-DsRed on infection of parental and CypA-/- Jurkat cells. Shown are raw infectivity results for NL4-3/VSV-G pseudoviruses in parental Jurkat cells (A) and in CypA-/- Jurkat cells (B) pertaining to the main Fig 1D. Ten thousand cells were inoculated with 400, 80, or 40 pg of p24 of VSV-G pseudotyped pNLR-E-Luc virus that contained or lacked CypA-DsRed. NL-Cyp1 and NL-Cyp2 denote two different virus preparations containing CypA-DsRed. Luciferase signal was measured at 48 h post infection. Average RLU and SD from duplicate samples of a representative experiment of 4 independent experiments are shown.(TIFF) ppat.1005709.s003.tiff (1.7M) GUID:?E1999438-DE43-4D0A-9CE7-4D84D882E43E S4 Fig: CypA-DsRed expressed in target cells does not restrict HIV-1 Oxaliplatin (Eloxatin) infection. 293T cells were transfected with plasmids expressing DsRed, CypA-mRFP and CypA-DsRed, as well as TRIMCyp-eCFP (positive control). Twenty four hours post transfection, the cells were re-plated into 96-well plate, and 16 hours later infected with different dilutions of VSV-G pseudotyped pNL4.3 R-E- Luc virus (based on the RT activity) in the absence (A) or in the presence (B) of 5 M CsA. Two days after infection, the luciferase signal (RLU) was measured. A representative triplicate experiment from 3 independent experiments is shown. Error bar represents SD. Note the less potent restriction of infection by the TRIMCyp-eCFP fusion protein as compared to unlabeled TRIMCyp reported in the literature (Perez-Caballero et al., and in living cells. The rate of loss is modulated by the core stability and is accelerated upon the initiation of reverse transcription. We show that the majority of single cores lose CypA-DsRed shortly after viral fusion, while a small fraction remains intact for several hours. Single particle tracking at late times post-infection reveals a gradual loss of CypA-DsRed which is dependent on reverse transcription. Uncoating occurs both in the cytoplasm and at the nuclear membrane. Our novel imaging assay thus enables time-resolved visualization of single HIV-1 uncoating in living cells, and reveals the previously unappreciated spatio-temporal features of this incompletely understood process. Author Summary HIV-1 genome and key enzymes required for establishing productive infection are encased in a cone-shaped shell made of the capsid protein (CA). After being released into the cytosol of target cells, the cone-shaped core complex undergoes a series of carefully orchestrated steps, including uncoating (loss of CA). HIV-1 uncoating remains poorly understood, due in part to the lack of direct assays enabling studies of this process in living cells. Here, we introduce a novel strategy for labeling the HIV-1 capsid without genetically modifying the CA protein. We designed a novel fluorescent cyclophilin A construct that binds the capsid with an extremely high avidity and (1) efficiently incorporates into virions without compromising infectivity; (2) remains bound to cores after.