Journal: Retrovirology

Article Title: Functional analysis of the secondary HIV-1 capsid binding site in the host protein cyclophilin A

doi: 10.1186/s12977-019-0471-4

Figure Lengend Snippet: Functional analysis of CypA mutants in Hela-P4 cells. The PPIA gene was disrupted in Hela-P4 cells with CRISPR technology. The cloned cell line was transduced with a lentiviral vector encoding the indicated CypA mutant cDNAs. a Analysis of the extent of HIV-1 infection of the indicated cell lines by the A92E CA mutant in the presence and absence of CsA. A higher ratio indicates less enhancement of infection by CsA. Shown are the results of two independent determinations. b Quantitative analysis of CypA expression by immunoblotting relative to wild type HeLa-P4 cells. All samples were separated on the same gel and CypA and GAPDH were stained on the same blot membrane. CypA signals were normalized by the corresponding GAPDH signals

Article Snippet: For CypA reconstitution, pEF-IRES-bsd, a lentiviral vector containing the eEF1 promoter upstream of an IRES-Bsd cassette, was constructed from the EF.CMV.RFP lentiviral vector (Addgene plasmid # 17619; a gift from Linzhao Cheng) by substituting the IRES-Bsd cassette region, amplified by PCR from pMXs-IRES-Bsd (Cell Biolabs, cat. RTV-016), for the CMV-RFP region.

Techniques: Functional Assay, CRISPR, Clone Assay, Transduction, Plasmid Preparation, Mutagenesis, Infection, Expressing, Western Blot, Staining, Membrane