CRISPR/Cas9 to study virus-host
interactions
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Parolini F., Mutascio S., Serena M., Fochi S., Romanelli M. G., Zipeto D. Department of Neurosciences, Biomedicine and Movement Sciences, University of Verona, Italy
The new CRISPR/Cas9 technique enables the editing of specific DNA sequences of any given genome. It has found many applications in virology, allowing, among others, the viral DNA excision from latently infected cells and the generation of useful knock out cell lines.
Using the CRISPR/Cas9 system we originated β2microglobulin (β2m), human thioesterase 8 (ACOT8) and
histone deacetylase 6 (HDAC6) negative cells, to study their role in HIV-1 infection. We evaluated the editing efficiency by the T7 endonuclease I assay, western blot and flow cytometry analyses.
The β2m is crucial for the HLA molecules membrane
translocation. HLA-C interacts with HIV-1 Env on the cell membrane increasing viral infectivity (Zipeto & Beretta, Retrovirology 2012). We targeted the β2m gene in 293T,
HeLa-Lai (expressing HIV-1 Env), TZM-bl (HeLa cells susceptible to HIV-1 infection) and parental HeLa cells. We showed in 293T cells that β2m absence abrogates HLA-C
surface expression, even in the presence of HIV-1 proteins expression. We confirmed the observation in β2m negative
HeLa-Lai cells. Moreover, by comparing HIV-1 infectivity in 2m positive/negative TZM-bl cells, we observed that virions
produced in 2m negative 293T cells are significantly less
infectious than those produced in parental cells.
We reported that ACOT8 interacts with HIV-1 Nef preventing its degradation (Serena et al, Scientific Reports 2016). We observed in TZM-bl cells that ACOT8 absence did not affect HIV-1 infectivity. The ACOT8 role in HIV-1 production and infectivity is being tested using 293T ACOT8 negative cells. HDAC6 is an important regulator of membrane dynamics involved in HIV-1 infection. We inactivated the HDAC6 gene in 293T cells to evaluate its influence on HIV-1 infectivity and syncytia formation and analyses are in progress.
In conclusion, the CRISPR/Cas9 genome edited cell lines are powerful tools to study the molecular interaction required for HIV efficient infection.
