Genome editing using
zinc finger nucleases (ZFNs) has been successfully applied to disrupt CCR5 or CXCR4 host factors and inhibit viral entry and
infection. Gene therapy using ZFNs to modify the PSIP1 gene, which encodes the
lens epithelium-derived growth factor (
LEDGF)
protein, might restrain an early step of the viral replication cycle at the integration level. ZFNs targeting the PSIP1 gene (ZFNLEDGF) were designed to specifically recognize the sequence after the
integrase binding domain (IBD) of the
LEDGF/p75
protein. ZFNLEDGF successfully recognized the target region of the PSIP1 gene in TZM-bl cells by heteroduplex formation and DNA sequence analysis. Gene editing induced a frameshift of the coding region and resulted in the abolishment of
LEDGF expression at the
mRNA and
protein levels. Functional assays revealed that
infection with the HIV-1 R5 BaL or X4 NL4-3 viral strains was impaired in
LEDGF/p75 knockout cells regardless of entry tropism due to a blockade in HIV-1 proviral integration into the host genome. However, residual
infection was detected in the
LEDGF knockout cells. Indeed,
LEDGF knockout restriction was overcome at a high multiplicity of
infection, suggesting alternative mechanisms for HIV-1 genome integration rather than through
LEDGF/p75. However, the observed residual integration was sensitive to the
integrase inhibitor raltegravir. These results demonstrate that the described ZFNLEDGF effectively targets the PSIP1 gene, which is involved in the early steps of the viral replication cycle; thus, ZFNLEDGF may become a potential
antiviral agent for restricting HIV-1 integration. Moreover,
LEDGF knockout cells represent a potent tool for elucidating the role of HIV integration cofactors in virus replication.