Racentrifugation. CPSF6 binds HIV-1 CA [23] and CPSF6-358 inhibits HIV-1 in
Racentrifugation. CPSF6 binds HIV-1 CA [23] and CPSF6-358 inhibits HIV-1 in a CA-specific manner, with the block occurring at a step before theDe Iaco et al. Retrovirology 2013, 10:20 http://www.retrovirology.com/content/10/1/Page 9 ofACPSF6 ActinEmptyCPSFCPSF6 NLSCPSF6 NESBCPSF6 CPSF6 NLS CPSF6 NESCFigure 5 HIV-1 replication is inhibited when full-length CPSF6 is targeted to the cytoplasm. (A) Expression levels of CPSF6 in TZM-bl cells transduced with empty, CPSF6, CPSF6-NLS and CPSF6-NES vectors. Cell lysates were probed in western blots with anti-CPSF6 antibody (upper panel) and anti–actin antibody (lower panel). The upper panel shows the endogenous and exogenous full-length CPSF6 with an HA tag. (B) Localization of different forms of CPSF6 in TZM-bl cells stably expressing CPSF6, CPSF6 NLS or CPSF6 NES. The cells were stained with an anti-HA antibody (green) for the detection of the CPSF6 proteins. DAPI staining (blue) was used to mark the nuclear compartment. (C) TZM-bl stably expressing the different forms of CPSF6 were challenged with WT or CA mutant HIV-1NL4-3GFP reporter viruses. After 72 hours, GFP reporter expression was assessed by flow cytometry. Data represent one of at least three independent experiments. Error bars represent ?SEM (n = 3).virus reaches the nucleus (Additional file 1: Figure S1). CPSF6-358 might inhibit HIV-1 infectivity by altering the kinetics PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/25447644 of CA core uncoating, consequently delaying the nuclear import of the PIC. The stability of WT and A105T CA cores in the presence of CPSF6-358 was assessed using a kinetic assay for CA core stability in vivo (Figure 8A); variation in the amount of pelletable CA in this assay correlates with altered CA core stability [35]. 4, 10 and 16 hrs afterchallenge of TZM-bl cells with HIV-1 Env- virus pseudotyped with VSV G, and bearing either WT CA or the A105T CA mutant, cells were lysed and cytoplasmic capsid cores were pelleted through a 50 sucrose cushion. Virus without VSV G was used as a control for CA that had been taken up by cells non-specifically. 4 hrs after challenge with the WT, CA cores showed a Enzastaurin dose slight increase in stability when CPSF6-358 was expressed in the cell; A105T CA core stability was not altered. At 10 and 16 hrsDe Iaco et al. Retrovirology 2013, 10:20 http://www.retrovirology.com/content/10/1/Page 10 ofATNPO3 KD CPSF6 KD- -+ — ++ + TNPO3 CPSFActinBCPSFDAPICCytosol TNPO3 KD – + – Nuclei +Ctrl KDTubulinHCPSF6 TNPO3 KD TNPODFigure 6 TNPO3 KD inhibits HIV-1 replication by shifting CPSF6 to the cytoplasm. (A) Expression level of CPSF6 and TNPO3 in TZM-bl cells stably transduced with control or CPSF6 KD vectors and transfected with scrambled or TNPO3 specific small interfering RNA (siRNA). Cell lysate was probed in western blots with anti-TNPO3 antibody (upper panel), anti-CPSF6 antibody (middle panel) and anti–actin antibody (lower panel). (B) Immuno-fluorescence localization of endogenous CPSF6 (green) in control (Ctrl) KD or TNPO3 KD TZM-bl cells. DAPI staining (blue) was used to mark the nuclear compartment. (C) Cell fractionation to identify the cellular localization of endogenous CPSF6. Expression of tubulin in the cytoplasm and histone 3 (H3) in the nucleus was assessed to verify PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27488460 the fractionation. (D) Stable ctrl KD and CPSF6 KD cells transfected with scrambled or TNPO3 siRNA were challenged with WT or CA mutant HIV-1NL4-3GFP reporter viruses. After 72 hours GFP expression was checked by flow cytometry. The fold inhibition of infectivity due to.
