Multiple components of the nuclear pore complex interact with the amino-terminus of MX2 to facilitate HIV-1 restriction
Fig 5
Endogenous MX2 requires NUP214 and TNPO1 for a full anti-viral function.
(A) U87-MG cells transduced with a CRISPR-Cas9 control guide RNA were transfected twice with a control siRNA (CTRL) or siRNAs targeting MX2, NUP214 and/or TNPO1, and treated or not with 1000 U/ml of IFNα, prior to infection with an HIV-1/GFP lentiviral vector. On the left, the percentage of infected cells calculated by flow cytometry and the fold inhibition of infection due to IFNα treatment are shown (n = 4; mean ± SD; *p-value < 0.05; (ns) non-significant; paired t-test). On the right is an immunoblot from a representative experiment, showing depletion of the indicated proteins. (B) Same experiment as in A, but using U87-MG cells where the MX2 alleles were disrupted using CRISPR-Cas9 genome editing (n = 4; mean ± SD; *p-value < 0.05; (ns) non-significant; paired t-test). (C) Primary CD4+ T cells were isolated from 4 independent donors, transduced with shRNAs targeting MX2, NUP214, TNPO1 or a control shRNA (CTRL) and treated or not with 3000 U/ml of IFNα. 24 h later, cells were challenged with NL4.3/Nef-IRES-Renilla and luciferase activity determined 48 h later. The mean of three technical replicates are shown for each donor on the left, and the fold inhibition of infection (no IFN/IFN) on the right. (D) Efficiency of MX2, NUP214 and TNPO1 depletion in primary CD4+ T cells following shRNA transduction was quantitated by qPCR and normalized to GAPDH. Data shown represent 4 donors used in (C).