Inhibition of human immunodeficiency virus type 1 replication with artificial transcription factors targeting the highly conserved primer-binding site

Abstract

The human immunodeficiency virus type 1 (HIV-1) primer-binding site (PBS) is a highly conserved region in the HIV genome and represents an attractive target for the development of new anti-HIV therapies. In this study, we designed four artificial zinc finger transcription factors to bind at or adjacent to the PBS and repress transcription from the HIV-1 long terminal repeat (LTR). These proteins bound to the LTR in vivo, as demonstrated by the chromatin immunoprecipitation assay. In transient reporter assays, three of the four proteins repressed transcription of a reporter driven by the HIV-1 LTR. Only one of these proteins, however, designated KRAB-PBS2, was able to prevent virus production when transduced into primary lymphocytes. We observed >90% inhibition of viral replication over the course of several weeks compared to untransduced cells, and no significant cytotoxicity was observed. Long-term exposure of HIV-1 to KRAB-PBS2 induced mutations in the HIV-1 PBS that reduced the effectiveness of the repressor, but these mutations also resulted in decreased rates of viral replication. These results show that KRAB-PBS2 has the potential to be used in antiviral therapy for AIDS patients and might complement other gene-based strategies.

FIG. 1.

Binding sites of PBS zinc finger proteins on the HIV-1 LTR. The sequence shown is from an HXB2 reference strain.

FIG. 2.

PBS zinc finger-binding constants and inhibition of HIV-1 LTR in a transient transfection assay. (A) Gel shifts of the PBS zinc fingers. A ³²P-labeled DNA hairpin oligonucleotide containing the PBS sequence was incubated with decreasing amounts of protein. The density of free and bound DNA was quantitated using ImageQuant software, and the K_D for each protein was calculated. (B) Transient reporter assays comparing repression of the PBS proteins fused to the KRAB repression domains. The graph on the left shows results from transfection of KRAB-zinc finger proteins with an HIV-1 LTR-driven luciferase reporter and a plasmid expressing the Tat protein. The graph on the right shows control transfection of KRAB-zinc finger proteins with an SV40 promoter-driven luciferase reporter. (C) Protein expression levels of PBS zinc finger proteins. Cells were transiently transfected with zinc finger proteins as in panel B, and cell extracts were prepared. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Blots were probed with an antibody recognizing the HA tag on the zinc finger proteins or a β-actin antibody. Relative protein expression was calculated by normalizing zinc finger expression to β-actin expression in each sample.

FIG. 3.

Repression and in vivo binding of PBS zinc finger proteins on a chromosomally integrated HIV-1 LTR reporter. (A) KRAB-PBS zinc finger proteins and Tat were transiently expressed in the TZM-bl cell line, a HeLa cell line containing chromosomally integrated HIV constructs that drive expression of luciferase and lacZ genes. (B) Chromatin immunoprecipitation of zinc finger protein-bound HIV-1 LTR. Zinc finger proteins were expressed by retroviral transduction in TZM-bl cells. Cells were cross-linked with formaldehyde, and nuclear extract was prepared. The extract was incubated with RNA polymerase II (pol II) or an antibody recognizing an Sp1 consensus zinc finger (Sp1C) and precipitated with Staph A cells. Immunoprecipitated DNA was purified and analyzed by PCR for the presence of the HIV-1 LTR using primers specific for the LTR. (C) Northern blot of . HeLa cells were transfected with the indicated KRAB-ZF proteins, and total RNA was extracted. RNA was separated by denaturing polyacrylamide gels and transferred to a membrane, which was probed with radiolabeled oligonucleotides for and tRNA^Phe. The numbers below each lane represent the relative expression of after normalization to tRNA^Phe.

FIG. 4.

Transient inhibition of virus production by PBS zinc finger proteins. Plasmids expressing the indicated KRAB-PBS zinc finger and the genome of the NL4-3 strain of HIV-1 were cotransfected into 293T cells. Virus production was monitored by a p24 ELISA.

FIG. 5.

Inhibition of HIV virus production by PBS zinc fingers in primary cells. (A) Flow cytometry analysis of zinc finger expression in PBMCs. PBMCs were transduced with lentiviral vectors that express KRAB-PBS zinc finger proteins. Cells were fixed and stained with an antibody that recognizes the C-terminal HA tag of the ZFs and analyzed by flow cytometry for zinc finger expression. The right panel shows flow cytometry analyses of KRAB-PBS2-transduced cells stained for CD4 and HA antibodies. (B) PBMCs were transduced with the indicated KRAB-PBS lentiviral vector and then challenged with infection by the HIV-1 strain NL4-3 at an MOI of 0.1. p24 levels were monitored at 3, 7, 12, and 18 days after infection.

FIG. 6.

Transduction of PBMCs with ZF proteins did not affect cell viability. PBMCs transduced with the indicated ZF transcription factor or nontransduced cells were challenged with HIV-1. At the indicated time points, cell viability was determined by the WST-1 assay (50).

FIG. 7.

Reduced effectiveness of KRAB-HLTR3 on the F-subtype LTR. (A) Sequence comparison of the HLTR3 and PBS2 ZF-binding sites in different HIV-1 M-group subtypes. Dashes indicate sequence identity, and dots indicate deletions (sequence information is taken from reference 14). (B) PBMCs transduced with either KRAB-HLTR3 (left) or KRAB-PBS2 (right) were challenged with B-subtype viruses containing either wild-type or F-subtype LTR. Virus replication was monitored by measuring p24 production.

FIG. 8.

Mutation of the primer-binding site in response to zinc finger repression. (A) PBMCs were transduced with KRAB-PBS2 and infected with HIV-1. Virus was collected from the supernatant and used to reinfect a new population of transduced cells. After several rounds, output virus was cloned, and the primer-binding site regions from 20 clones were sequenced. The sites of mutation are indicated by underlining. (B) Infection of KRAB-PBS-transduced PBMCs by wild-type and Mut-3 HIV. (C) Gel shift of PBS2 with wild-type and Mut-3 hairpin oligonucleotides.