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. 2006 Feb;80(4):1939-48.
doi: 10.1128/JVI.80.4.1939-1948.2006.

Human immunodeficiency virus type 1 incorporated with fusion proteins consisting of integrase and the designed polydactyl zinc finger protein E2C can bias integration of viral DNA into a predetermined chromosomal region in human cells

Wenjie Tan  1 , Zheng Dong, Thomas A Wilkinson, Carlos F Barbas 3rd, Samson A Chow
Affiliations

Human immunodeficiency virus type 1 incorporated with fusion proteins consisting of integrase and the designed polydactyl zinc finger protein E2C can bias integration of viral DNA into a predetermined chromosomal region in human cells

Wenjie Tan et al. J Virol. 2006 Feb.

Abstract

In vitro studies using fusion proteins consisting of human immunodeficiency virus type 1 integrase (IN) and a synthetic polydactyl zinc finger protein E2C, a sequence-specific DNA-binding protein, showed that integration of retroviral DNA can be biased towards a contiguous 18-bp E2C-recognition site. To determine whether the fusion protein strategy can achieve site-specific integration in vivo, viruses were prepared by cotransfection and various IN-E2C fusion proteins were packaged in trans into virions. The resulting viruses incorporated with the IN-E2C fusion proteins were functional and capable of performing integration at a level ranging from 1 to 24% of that of viruses containing wild-type (WT) IN. Two of the more infectious viruses, which contained E2C fused to either the N (E2C/IN) or to the C (IN/E2C) terminus of IN, were tested for their ability to direct integration into a unique E2C-binding site present within the 5' untranslated region of erbB-2 gene on human chromosome 17. The copy number of proviral DNA was measured using a quantitative real-time nested-PCR assay, and the specificity of directed integration was determined by comparing the number of proviruses within the vicinity of the E2C-binding site to that in the whole genome. Viruses containing IN/E2C fusion proteins had sevenfold higher preference for integrating near the E2C-binding site than those viruses containing WT IN, whereas viruses containing E2C/IN had 10-fold higher preference. The results indicated that the IN-E2C fusion protein strategy is capable of directing integration of retroviral DNA into a predetermined chromosomal region in the human genome.

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Figures

FIG. 1.
FIG. 1.
Integration efficiency of viruses containing various IN-E2C fusion proteins. One million HeLa cells were infected for 4 h with equal amounts (ranging from 10 to 50 ng) of p24 equivalent of the indicated virus (panels a through g). Representative plates of the hygromycin resistance assay were shown. Dark spots on each plate are colonies that grew after selection with 200 μg/ml of hygromycin B for three weeks, beginning two days postinfection. The colonies are a result of provirus formation and stable expression of the hygromycin resistance gene. The number of resistant colonies per ng p24 for each virus was determined and expressed as a percentage of the colonies produced by the virus containing the WT IN in trans (panel b). Each determination at a particular p24 amount was performed in duplicate, and values on the upper-right corner of each panel are the mean ± standard error of the mean of three independent experiments. For the HXB-IN64 virus supplied with WT IN in trans (panel b), the number of resistant colonies per ng p24 was 11.6 ± 3.4.
FIG. 2.
FIG. 2.
A schematic diagram showing the real-time PCR assay for quantifying proviral DNA integrated in the entire genome (Alu-PCR) or near the E2C-binding site on chromosome 17 (e2c-PCR). HIV-1 DNA contains two LTRs, each located at the 5′ and 3′ ends of the viral genome. Integration of HIV-1 DNA can be upstream or downstream and in either orientations relative to a specified locus, Alu (open box) or e2c (shaded box). For simplicity, only one HIV-1 LTR and its subregions, U3, R, and U5, are shown, and the diagram depicts only the scenario in which the proviral DNA is integrated upstream of and in the same orientation as an Alu element or the e2c site. The quantitative assay consists of two rounds of PCR. Thick gray arrows represent the locations and orientations of the first-round PCR primers, whereas thin black arrows represent the locations and orientations of the second-round PCR primers. ZXF-P represents the location of the fluorescent probe. Primers LM652 and LM667 contain phage λ-specific sequences at their 5′ ends, and primer λT anneals specifically to phage λ sequences.
FIG. 3.
FIG. 3.
A standard curve generated by Alu-PCR for measuring the total integration events in the human genome. Alu-PCR standard DNA, isolated from HeLa cells infected with HXB-IN64-Hygro/Vpr-IN, was serially diluted to contain 18 to 1.8 × 104 copies of HIV-1 proviruses per reaction. The proviral copy number of the Alu-PCR standard was previously determined using the pCR-5L-e2c as the standard and the late RT primers set (MH531 and MH532). Fluorescence curves of the Alu-PCR standard and infected DNA samples were generated after two-step amplification (data not shown), and the PCR cycles at which the amplification signal entered the exponential range were determined (threshold cycle [Ct]). The reactions were performed in duplicate.
FIG. 4.
FIG. 4.
DNA constructs and standard curves generated by e2c-PCR for measuring proviral DNA integrated upstream or downstream of the E2C-binding site. (A) DNA constructs. An HIV-1 DNA fragment (open box) containing the 5′ LTR and part of the gag sequence or the 3′ LTR and part of the nef sequence was obtained by PCR. The 5′ LTR-gag fragment was cloned into pCR-e2c downstream of the 400-bp e2c-containing fragment (shaded box), resulting in pCR-5L-e2c. The 3′ LTR-nef fragment was cloned upstream of the 400-bp e2c-containing fragment to form pCR-3L-e2c. (B) A standard curve generated by e2c-PCR for quantifying proviral DNA copies downstream of the E2C-binding site. Quantitative dilution series of pCR-5L-e2c DNA ranging from 20 to 2.0 × 108 copies per reaction were prepared, and fluorescence curves were generated by two-step amplification (data not shown). Logarithmic regression of the threshold cycles for the various provirus copies per reaction was plotted for quantifying HIV-1 proviral DNA. The reactions were performed in duplicate. (C) A standard curve generated by e2c-PCR for quantifying proviral DNA copies upstream of the E2C-binding site. The reaction was performed as described in panel B except that a serial dilution of pCR-3L-e2c DNA ranging from 200 to 2.0 × 108 copies per reaction was prepared as the DNA standard.
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