A lentiviral cDNA library employing lambda recombination used to clone an inhibitor of human immunodeficiency virus type 1-induced cell death

Abstract

Expression cloning technology of cDNAs is a suitable tool for identifying novel functional properties of genes. Here, we generated a lentiviral cDNA library-expressing system for human T cells based on a site-specific recombination system of phage lambda for transferring cDNA libraries with a minimum loss of its complexity. The library-transduced CD4(+) T cells were challenged with wild-type human immunodeficiency virus type 1 (HIV-1), and the cells that acquired resistance to HIV-1-induced cytopathic effect (CPE) were selected. From these cells, CD14 was isolated and proved to inhibit the entry of HIV-1 and the HIV-1-induced CPE. This cloning system allows rapid identification of genes encoding novel properties in human T cells and probably other mammalian cells.

FIG. 1.

Histogram analysis of lengths of individual cDNA fragments in each library. The lengths of cDNA fragments were determined as described in Materials and Methods and are plotted in 250-bp increments on the x axes. Percentages of individual clones are indicated on the y axes.

FIG. 2.

Scheme for strategy used to select genes that arm cells with resistance to HIV-induced CPE. MT-4 cells were infected with the viral cDNA library and then challenged with HIV-1_NL4-3, which is highly cytopathic to MT-4 cells. If the introduced gene has anti-CPE, the cell will survive in the presence of HIV-1.

FIG. 3.

Characterization of a cDNA clone that confers T-cell resistance to HIV-1-induced CPE. Cells transduced with the CD14-carrying vector isolated from this viral cDNA library were stained with anti-CD4 antibody (A), anti-CD14 antibody (B), or isotype-matched control antibody (C) and analyzed by flow cytometry. The results shown are data from one flow cytometry experiment, which is representative of three independent experiments. PE, phycoerythrin.

FIG.4.

Resistance to HIV-1-induced cell death in CD14-transduced T cells. (A) Flow cytometric analysis of MT-4 cells infected with a CD14- and GFP-expressing lentivirus vector was performed. These cells (GFP⁺ H-2K^k−) were mixed with cells infected with a lentivirus vector expressing H-2K^k alone (GFP⁻ H-2K^k+) and uninfected cells (GFP⁻ H-2K^k−) and then challenged with HIV-1_NL4-3. Flow cytometric analysis of the mixed culture is shown before HIV-1 challenge (B), 8 days after HIV-1 challenge (C), and 8 days after mock infection (D). Trypan blue staining (E) and fluorescent microscopic examination (F) were performed 3 days after HIV-1 challenge. A merged image of panels E and F is shown in panel G. Magnification, ×200. Flow cytometric analysis of the HIV-1-challenged culture 10 days after infection (H) or of an uninfected culture (I) was performed by staining with anti-HIV human serum. The results shown are data from one experiment, which is representative of three independent experiments.

FIG.4.

Resistance to HIV-1-induced cell death in CD14-transduced T cells. (A) Flow cytometric analysis of MT-4 cells infected with a CD14- and GFP-expressing lentivirus vector was performed. These cells (GFP⁺ H-2K^k−) were mixed with cells infected with a lentivirus vector expressing H-2K^k alone (GFP⁻ H-2K^k+) and uninfected cells (GFP⁻ H-2K^k−) and then challenged with HIV-1_NL4-3. Flow cytometric analysis of the mixed culture is shown before HIV-1 challenge (B), 8 days after HIV-1 challenge (C), and 8 days after mock infection (D). Trypan blue staining (E) and fluorescent microscopic examination (F) were performed 3 days after HIV-1 challenge. A merged image of panels E and F is shown in panel G. Magnification, ×200. Flow cytometric analysis of the HIV-1-challenged culture 10 days after infection (H) or of an uninfected culture (I) was performed by staining with anti-HIV human serum. The results shown are data from one experiment, which is representative of three independent experiments.

FIG. 5.

Inhibition of HIV-1 replication in CD14-transduced cells. HIV-1 replication was evaluated by production of p24^gag antigen in the culture supernatant of CD14- or empty vector-transduced MT-4 (A) or CD4⁺ CCR5⁺ HeLa cells (B) with a lentivirus vector expressing CD14 and H-2K^k or H-2K^k alone, respectively. To determine the level of HIV-1 entry efficiency, CD14- or empty vector-transfected CD4⁺ CCR5⁺ HeLa cells were challenged with DNase-treated HIV-1_NL4-3. Target cell DNA was isolated at the indicated time and used to detect early reverse transcripts (C), late reverse transcripts (D), 2-LTR circle (E), and the integrated form (F). Data are the means ± SD from duplicate experiments. The levels of the p24^gag antigen or HIV-1 DNA in the CD14-transduced cultures were significantly lower than those of the empty vector-transduced cultures (P < 0.05, Mann-Whitney U test). Lines with open circles, CD14 vector; lines with filled circles, empty vector.