Effective suppression of human immunodeficiency virus type 1 through a combination of short- or long-hairpin RNAs targeting essential sequences for retroviral integration

Abstract

Small interfering RNA (siRNA) could provide a new therapeutic approach to treating human immunodeficiency virus type 1 (HIV-1) infection. For long-term suppression of HIV-1, emergence of siRNA escape variants must be controlled. Here, we constructed lentiviral vectors encoding short-hairpin RNAs (shRNA) corresponding to conserved target sequences within the integrase (int) and the attachment site (att) genes, both of which are essential for HIV-1 integration. Compared to shRNA targeting of the HIV-1 transcription factor tat (shTat), shRNA against int (shIN) or the U3 region of att (shU3) showed a more potent inhibitory effect on HIV-1 replication in human CD4+ T cells. Infection with a high dose of HIV-1 resulted in the emergence of escape mutants during long-term culture. Of note, limited genetic variation was observed in the viruses resistant to shIN. A combination of shINs against wild-type and escape mutant sequences had a negative effect on their antiviral activities, indicating a potentially detrimental effect when administering multiple shRNA targeting the same region to combat HIV-1 variants. The combination of shIN and shU3 att exhibited the strongest anti-HIV-1 activity, as seen by complete abrogation of viral DNA synthesis and viral integration. In addition, a modified long-hairpin RNA spanning the 50 nucleotides in the shIN target region effectively suppressed wild-type and shIN-resistant mutant HIV-1. These results suggest that targeting of incoming viral RNA before proviral DNA formation occurs through the use of nonoverlapping multiple siRNAs is a potent approach to achieving sustained, efficient suppression of highly mutable viruses, such as HIV-1.

FIG. 1.

shRNA target sequences of HIV-1 int and the U3 and U5 att sites. (A) Schematic representation of HIV-1 integrase (top squares) and shIN target region (bold bar). The conserved HHCC residues in the zinc-binding motif and enzyme active-site residues (DDE) located in the N terminus of the core domain of HIV-1 IN are indicated. The sequence of the shIN target region corresponding to the HIV-1 NL43 clone (1) and used in the present study is shown on the first line. Sequences of this region in 345 different isolates were aligned according to the HIV-1 sequence database published by Los Alamos National Laboratory (http://hiv-web.lanl.gov). Nucleotide differences in comparison with NL43 and numbers of clones that carried them are indicated. (B) Target sequences of shU3 and shU5 in the HIV-1 NL43 clone are shown. Location of each target region in a whole HIV-1 genome is indicated by the bold bar. Conserved sequences in the U3 att and U5 att regions among all HIV-1 strains are underlined.

FIG. 2.

HIV-1 replication in shRNA-transduced MT-4 or primary CD4⁺ T cells. (A) MT-4 cells were transduced by the indicated shRNA using a lentivirus vector system (shLuc, shTat, shIN, shU3, and shU5). Seven days later, transduced MT-4 cells were infected by HIV-1_NL-EGFP at an input dose of 100 pg of p24 (HIV-1 core antigen) per 10⁶ cells. Culture supernatants were collected periodically after infection as indicated. HIV-1 replication was monitored by measuring percent EGFP-positive cells by fluorescence-activated cell sorting (left) or level of HIV-1 p24 by enzyme-linked immunosorbent assay (right). The cross symbol indicates cell death associated with HIV-1 replication. Representative results of three independent experiments are shown. (B) Inhibition of HIV-1 replication by each shRNA in primary CD4⁺ T cells. CD4⁺ T cells (1 × 10⁶) were transduced with the indicated lentiviral vectors by low centrifugation at 2,000 rpm for 1 h in the presence of 10 μg/ml polybrene. Transduced cells were infected with HIV-1_NL43 for 3 h, and p24 antigen levels in culture supernatants 4 days postinfection were measured. Values represent the means and standard deviations for three independent experiments. (C) MT-4 cells transduced with the indicated shRNAs (shLuc, shTat, shIN, shU3, or shU5) were infected by HIV-1_NL-EGFP at a high input dose (1,000 pg of p24 per 10⁶ cells), and HIV-1 replication was monitored as described for panel A. (C) MT-4 cells transduced with each shRNA (shLuc, shTat, shIN, shU3, or shU5) were infected by HIV-1_NL-EGFP with a high input dose (1,000 pg of p24 per 10⁶ cells), and HIV-1 replication was monitored as described for panel A.

FIG. 3.

shRNA-specific resistance of HIV-1 escape mutants in shRNA-transduced MT-4 cells. Culture supernatants of shTat-transduced MT-4 cells infected with a low dose of HIV-1 (100 pg of p24) (A) or shIN (B)- or shU3 (C)-transduced MT-4 cells infected with a high dose of HIV-1 (1,000 pg of p24 per 10⁶ cells) were harvested at 12 days after challenge infection. Culture supernatants containing shRNA-resistant virus (100 pg of p24) were inoculated to newly prepared MT-4 cells transduced by shLuc, shTat, shIN, or shU3, and replication of HIV-1 in these cells was monitored by measuring percent EGFP-positive cells (left). Representative results of three independent experiments are shown. Culture supernatants of shTat-transduced MT-4 cells infected with shTat-resistant virus (A), shIN-transduced MT-4 cells infected with shIN-resistant virus (B), and shU3-transduced MT-4 cells infected with shU3-resistant virus (C) were harvested at 6 days postinfection. Each culture supernatant containing shRNA-resistant viruses (100 pg of p24) was infected with newly prepared MT-4 cells transduced by shTat, shIN, or shU3. Total DNA was extracted from these MT-4 cells at 4 days postinfection. A fragment of viral DNA spanning each shRNA target region was amplified by PCR followed by TA cloning. Then, several clones from each were subjected to DNA sequence analysis. Nucleotide changes in the target sequence for shTat (nucleotides 5888 to 5906 of the tat gene), shIN (nucleotides 4285 to 4304 of the int gene), and shU3 (nucleotides 9073 to 9091 of the U3 att region) are shown on the right, along with the expected amino acid changes. Relative numbers of each clone are indicated in parentheses.

FIG. 4.

shRNA-specific resistance of HIV-1 molecular clones carrying point mutations within each shRNA target site. Point mutations within the shTat target site (Tat-T5901C) or shIN target site (IN-G4288A or IN-A4293T) were introduced into the parental HIV-1 clone (pNL-EGFP) through mutagenesis. Each recombinant mutant clone was transfected into 293T cells, and the culture supernatant was harvested and inoculated to MT-4 cells expressing the corresponding shRNA. Replication of each mutant clone was monitored by measuring percent EGFP-positive cells at the indicated days. Representative results of three independent experiments are shown.

FIG. 5.

Reversion of escape mutants in the presence of modified shRNAs targeting mutant sequences. (A) MT-4 cells were transduced with shIN or its modified shIN, which targeted escape mutant sequences (shIN-G4288A or shIN-A4293T). As a negative control, shLuc was introduced into MT-4 cells as well. The transduced cells were infected by HIV-1_NL-G4288A or HIV-1_NL-A4293T at a dose of 1,000 pg of p24 antigen per 10⁶ cells. Ten or 12 days after challenge infection, total DNA was isolated, viral DNA spanning the shIN target region was amplified by PCR and subjected to TA cloning, and sequences were analyzed. WT, wild type. (B) Effect of sequential transduction of shRNAs targeting int from wild-type and escape mutant viruses. MT-4 cells were transduced with shIN, shIN-G4288A, and shIN-T4293A sequentially (shIN/G4288A/T4293A). In parallel, MT-4 cells were transduced with shIN or shLuc alone. Transduced MT-4 cells were infected with HIV-1_NL-EGFP at a dose of 100 pg of p24 per 10⁶ cells. Virus replication was monitored by measuring percent EGFP-positive cells at the indicated days. Representative results of three independent experiments are shown.

FIG. 6.

Efficient antiviral activity with a combination of shRNAs targeting different sites in the HIV-1 genome. (A) MT-4 cells were transduced with combinations of shIN and shU3 (shIN/shU3), shTat and shU3 (shTat/shU3), or shTat and shIN (shTat/shIN). The dual-transduced cells were infected with DNase I-treated HIV-1_NL-EGFP at a dose of 1,000 pg of p24 per 10⁶ cells. Virus replication was monitored by measuring percent EGFP-positive cells at the indicated days postinfection. The cross symbol indicates cell death associated with HIV-1 replication. (B) In parallel, total DNA was isolated from MT-4 cells 6 h, 24 h, or 8 days postinfection. Level of viral DNA was determined by quantitative PCR as described previously (27). For PCR, virus incubated at 65°C for 30 min prior to inoculation was used as the heat-inactivated control (HI), and for the HIV-1 DNA standard, a linearized HIV-1 molecular clone (pNL43lucΔenv) was amplified. Human β-globin DNA was used as the internal control (17). The gel image was taken by using Image Saver System AE-6905C (ATTO, Tokyo, Japan), and the intensities of the PCR products were quantified by using Adobe Photoshop 7.0 software. The values shown are the intensity of each band relative to that in the control shLuc-transduced MT-4 cells, taken as 1.0.

FIG. 7.

Emergence of escape mutants from combinations of shRNAs targeting different sites following a high dose of HIV-1 infection. (A) MT-4 cells were transduced with combinations of two shRNAs as described for Fig. 6. The dual-transduced cells were infected with HIV-1_NL-EGFP at 10,000 pg of p24 per 10⁶ cells. Virus replication was monitored by measuring percent EGFP-positive cells at the indicated days postinfection. (B) Viruses were harvested from the culture supernatants of dual-transduced MT-4 cells 12 days postinfection for shTat/shIN, 16 days for shTat/shU3, and 22 days for shIN/shU3. Viruses resistant to each combination of shRNAs were inoculated into MT-4 cells freshly transduced by each combination of the two shRNAs. Virus replication was monitored by measuring percent EGFP-positive cells at the indicated days. The cross symbol indicates cell death associated with HIV-1 replication. Total DNA was harvested from the infected cells when virus replication became evident. Viral DNA spanning each shRNA target region was amplified by PCR and subjected to TA-cloning followed by sequence analysis, as described for Fig. 3. WT, wild type.

FIG. 8.

Inhibitory effect of lhRNA on wild-type or shIN-resistant viral clones. (A) The target sites of lhRNAs against the HIV-1 int gene (lhIN). The target sites of two lhINs (lhIN50#1 and lhIN50#2) are indicated by bold bars. lhIN#1 was designed to target the 50 nucleotides upstream of the shIN target sequence. lhIN50#2 targets 50 nucleotides that include the shIN target sequence. (B) Lentiviral vectors expressing each lhRNA under the control of the human H1 promoter or U6 promoter were constructed. As a negative control, lhRNA targeting 50 nucleotides of HCV genome (U6-lhHCV50) was used (38). Transduction of MT-4 cells with each lhRNA was performed as described for Fig. 2. Transduced MT-4 cells were infected by the parental clone (HIV-1_NL-EGFP) or the shIN-resistant clone (HIV-1_G4288A or HIV-1_A4293T) at a dose of 100 pg of p24 per 10⁶ cells. Virus replication was monitored by measuring levels of p24 antigen in culture supernatants 4 days postinfection. Values are the means plus standard deviations for three independent experiments.