Figure 10.

Endogenous SUN2 is not required for wild type HIV-1 infection in human HAP-1 cells. (A) Construction of human HAP-1 cells that do not express SUN1, SUN2, or both (SUN1/SUN2). HAP-1 cells were transiently transfected with CRISPR-Cas9 system using a specific RNA guide that targets the exon 2 of SUN1 and/or exon 4 of SUN2. Sequencing of SUN1, SUN2, and SUN1/2 KO clones revealed frame shifts that resulted in the creation of early stop codons. (B) HAP-1 KO cells were tested for expression of endogenous SUN1 and SUN2 by Western blotting using anti-SUN1 and SUN2 antibodies. To test for the bona fide recognition of SUN1 and SUN2 by anti-SUN1/2 antibodies, we performed similar Western blots using lysates from human HEK293T cells that were transiently transfected with SUN1-FLAG or SUN2-FLAG proteins. As loading control, extracts were also Western blotted using anti-GAPDH antibodies. (C) HIV-1 infection of HAP-1 SUN1 KO (clone 10), SUN2 KO (clone 3), and SUN1/SUN2 KO (clone 9) cell lines. HAP-1 control cells and HAP-1 KO cell lines were challenged with increasing amounts of HIV-1-GFP. Forty-eight hours post-infection, the percentage of GFP-positive cells was measured using a flow cytometer. Experiments were repeated at three times and a representative experiment is shown. Fold differences in restriction are shown as the ratio of the area under the curve of the SUN variant to the empty vector pLPCX. Fold values greater than 2 are shown.