CRISPR/Cas9 as a tool for studying the interactions between viral and cellular proteins

The CRISPR/Cas9 system is a new promising technique that allows editing of DNA sequences in the cell genome. We used CRISPR/Cas9 to produce β2microglobulin (β2m) and human thioesterase 8 (ACOT8) knock-out cell lines to study their role in HIV-1 infection. It has been reported that HLA-C interacts with HIV-1 Env, increasing virus infectivity (D. Zipeto, A. Beretta. 2012), while ACOT8 interacts with HIV-1 Nef, increasing Nef stability (M. Serena et al. 2016). The loss of β2m expression in 293T, HeLa-Lai (expressing HIV-1 Env) and parental HeLa cells was assessed by western blot and flow cytometry; β2m negative cells were finally sorted. Cytofluorimetric analysis indicated that, in the absence of β2m, HLA-C expression on the cell membrane was abrogated, both in HeLa and in HeLa-Lai cells. The presence of HIV-1 Env did not restore HLA-C on the cell surface, suggesting that HLA-C needs b2m to be expressed on the cell membrane, where the Env/HLA-C interaction occurs. In addition, we observed that HIV-1 viruses produced in 293T b2m negative cells were three fold less infectious than those produced in the parental cells, suggesting that the presence of HLA-C on the cell surface is required for the association with Env, thus resulting in an increased virus infectivity. The loss of ACOT8 expression in 293T cells and in TZM-bl, a cell line highly sensitive to infection with HIV-1, was assessed by western blot. Cells were clonally expanded and ACOT8 knock out was further confirmed in immunoblot. The role of Nef/ACOT8 association will be explored comparing the infectivity of HIV-1 viruses produced in the presence or in the absence of ACOT8 (293T), as well as comp