Zinc finger endonuclease targeting PSIP1 inhibits HIV-1 integration

Abstract

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.

FIG 1

Experimental design and specificity of ZFN_LEDGF targeting the PSIP1 gene. (a) Schematic representation of LEDGF/p75 protein highlighting the position of the ZFN_LEDGF targeted region. The cutting site of ZFN_LEDGF is located near the sequence coding for the integrase binding domain (IBD) of the LEDGF/p75. NLS, nuclear localization signal; AT, AT-hook-like domains. (b) Experimental design/process used to generate and evaluate LEDGF/p75 knockout cells. (c) Flow cytometry plots of wild-type cells, mock-treated cells, and cells transfected with ZFN_LEDGF plasmids and challenged with VSV-pseudotyped NL4-3 GFP-expressing virus. WT, wild type; MOCKtr, mock-transfected cells; FSC, forward scatter; FITC, fluorescein isothiocyanate. (d) Gene editing by ZFN_LEDGF induces heteroduplex formation determined by the surveyor mutation assay (CEL-I). After genomic DNA extraction, heteroduplex formation due to the generation of insertions or deletions was assessed by the surveyor mutation assay. DNA fragments were resolved in a 10% Tris-borate-EDTA (TBE)-PAGE gel. The lower migrating products (arrows) are a direct measure of ZFN-mediated gene disruption. +/−, clone 1 (heterozygous); −/−, clone 2 (homozygous).

FIG 2

Generation and phenotypic characterization of LEDGF/p75 knockout cells. (a) Sequence analysis of the insertions and deletions identified in the three cell lines selected after ZFN_LEDGF treatment. At least 16 different sequences from each selected cell line were sequenced and aligned. The consensus sequences of the modifications identified in each of the two alleles of the selected cell lines are depicted as allele A and allele B of cell lines 1 to 3. WT, wild type. (b and c) Protein alignments of in silico-predicted sequences based on the sequencing data obtained from the two alleles of the ZFN_LEDGF cell lines that introduce a premature stop codon (b) or of cell line 1 allele B that harbors an in-frame deletion of 3 exons (c). The IBD is highlighted by a red box. (d) Gene expression of PSIP1 mRNA corresponding to the 5′ and 3′ regions. Shown is an agarose gel with which the presence or absence of 5′ and 3′ fragments of PSIP1 mRNA was identified. In the case of cell line 1, the full-length and a truncated form of the 3′ mRNA fragment were identified. Ctrl, control. (e) Quantification of gene expression (mRNA) corresponding to the 3′ region of PSIP1 in the selected cell lines. Expression of LEDGF mRNA was completely inhibited in LEDGF^−/− KO cell lines tested compared to untreated or mock-transfected (MOCKtr) cells. LEDGF^+/− (cell line 1) showed a 50% decrease of the LEDGF mRNA compared to control cells. The mean ± SD values of three independent determinations are shown. (f) Assessment of protein levels of LEDGF/p75 determined by Western blot assay in the selected LEDGF/p75^+/− and LEDGF/p75^−/− KO cell lines compared to the control cells (upper panel, antibody recognizing the C terminus [C-ter] of LEDGF/p75 protein; lower panel, antibody recognizing the N terminus [N-ter] of the LEDGF/p75 and LEDGF/p52 proteins. Molecular weight (in thousands) markers are depicted. The red arrows indicate putative truncated proteins.

FIG 3

Infectivity of HIV in ZFN_LEDGF-treated cells and susceptibility to antiretroviral compounds. (a and b) Relative (Rel.) infection of ZFN_LEDGF-treated cells compared to wild-type (WT) and mock-transfected (MOCKtr) controls for the NL4-3 (a) and BaL (b) viral strains. The mean ± SD values of three independent experiments are shown. ***, P < 0.001; **, P < 0.01; *, P < 0.05. (c) Percentage of HIV X4 replication using strain NL4-3 in LEDGF^+/− and LEDGF^−/− KO mutants relative to the results for the wild-type and mock-treated controls treated or not with the reverse transcriptase inhibitor AZT, the CXCR4 antagonist AMD3100, and the IN strand transfer inhibitor RAL. The mean ± SD values of three independent experiments are shown. (d) Percentage of HIV R5 replication using strain BaL in LEDGF^+/− and LEDGF^−/− KO mutants relative to the results for the wild-type and mock-treated controls treated or not with the reverse transcriptase inhibitor AZT, the CXCR4 antagonist AMD3100, and the IN strand transfer inhibitor RAL. The mean ± SD values of three independent experiments are shown. WT, wild-type.

FIG 4

Integration of HIV NL4-3 is impaired in LEDGF^−/− KO cells. (a) Viral DNA in LEDGF^−/− KO cells (white bars) and mock-treated cells (black bars) with or without the reverse transcriptase inhibitor AZT (4 μM) and the IN inhibitor raltegravir (RAL) (2 μM). The values are expressed relative to those of the mock-transfected cells. The mean ± SD values of three independent experiments are shown. (b) Integrated viral DNA in LEDGF^−/− KO cells (white bars) and mock-treated cells (black bars) with or without the RT inhibitor AZT (4 μM) and increasing concentrations of the IN inhibitor raltegravir. The values are expressed relative to those of the mock-transfected cells. The mean ± SD values of three independent experiments are shown. Prov, total proviral DNA; UN, uninfected; INF, infected.