Antisense-mediated inhibition of human immunodeficiency virus (HIV) replication by use of an HIV type 1-based vector results in severely attenuated mutants incapable of developing resistance

Abstract

We have constructed a human immunodeficiency virus type 1 (HIV-1)-based lentiviral vector expressing a 937-base antisense sequence against the HIV-1 envelope gene. Transduction of CD4(+) T lymphocytes with this vector results in expression of the therapeutic antisense sequence and subsequent inhibition of productive HIV-1 replication. In this report, we examined the effect of antisense-mediated suppression on the potential development of virus escape mutants using a permissive T-cell line cultured under conditions that over serial passages specifically allowed for generation and amplification of mutants selected for by antisense pressure. In the resulting virus clones, we found a significant increase in the number of deletions at the envelope target region (91% compared to 27.5% in wild-type HIV). Deletions were most often greater than 1 kb in length. These data demonstrate for the first time that during antisense-mediated suppression of HIV, mutants develop as a direct result of selective pressure on the HIV genomic RNA. Interestingly, in clones where deletions were not observed, there was a high rate of A-G transitions in mutants at the antisense target region but not outside this region, which is consistent with those mutations that are predicted as a result of antisense-mediated modification of double-stranded RNA by the enzyme double-stranded RNA-specific adenosine deaminase. These clones were not found to be escape mutants, as their replicative ability was severely attenuated, and they did not replicate in the presence of vector.

FIG. 1.

Schematic representation of HIV-1-based vector expressing antisense to the HIV-1 envelope region. (A) The antisense-expressing vector contains a 5′ and 3′ LTR, splice donor site (SD), splice acceptor site (SA), packaging signal (PSI), a cPPT (CPT) and central termination sequence (CTS), RRE, EGFP as a marker gene, a 937-base antisense sequence to the HIV envelope gene, and a 3′ PPT. (B) PCR primers were designed to amplify both the antisense target region and part of the envelope (Table 1; 1,936 bases), or the entire envelope region (Table 2; 2,544 bases). (C) Flow cytometry of untransduced and VRX494-transduced primary CD4⁺ T lymphocytes at 20 TU per cell. Upper panels show a density blot of untransduced (left) and transduced (right) lymphocytes, and relative percentages are shown. The bottom panel shows the mean fluorescent intensity (MFI) from the upper panels for untransduced (dotted line) and transduced (solid line) cultures. (D) Flow cytometric analysis of primary CD4 T lymphocytes transduced with VRX494 at 5, 10, and 20 TU per cell. Shown at the top is the transduction level, average vector copy number, and MFI in cells at each level of transduction. The lower panel shows the shift in MFI in graph format, in comparison to untransduced cells (dark black line). (E) Challenge of transduced (closed circles) and untransduced (open squares) primary CD4⁺ T-lymphocyte cultures with various HIV strains at an MOI of 0.001. Top and bottom panels show challenge with X4 and R5 strains of HIV, respectively. Virus strains are shown above each panel, and virus replication was measured by ELISA for p24 production. Bars represent standard deviations (n = 3). (F) Challenge with HIV at an MOI of 0.3 in untransduced SupT1 cells (open circles), SupT1 cells transduced at 20 TU per cell with an empty vector without antisense to the HIV env gene (closed circles), or SupT1 cells transduced at 20 TU per cell with the same vector containing a 937-base antisense sequence to the HIV env gene (squares). Transduction rates and MFI in Sup T1 cells for antisense and control vectors were 97% and 586 and 96% and 887, respectively. n = 3.

FIG. 2.

Selection of HIV by antisense in SupT1 cells. (A) Isolation of breakthrough wt HIV from antisense selective pressure can only be measured after challenge of SupT1 cells transduced at suboptimal doses of vector. SupT1 cells were transduced with vector at an MOI of 5, 10, or 20 TU/ml and then challenged with HIV_NL4-3 at an MOI of 0.1. Virus production was measured by a p24 ELISA. (B) Schematic representation of selection procedure. SupT1 cells were mock transduced or transduced with vector at an MOI of 5 and then challenged with HIV_NL4-3 at an MOI of 0.1. Culture supernatants were collected twice weekly and were tested for p24 protein levels in order to determine the point of peak replication. Culture supernatants from each group were collected during peak replication, normalized for p24, and subsequently used to infect naïve and transduced SupT1 cells for a total of three rounds of selection reflecting a selection period of 56 days. (C) After three rounds of selection, naïve SupT1 cells were infected with p24-normalized supernatants collected from the final passage of the third selection during peak replication. Supernatants were collected at the indicated times postinfection and assayed for p24 production by ELISA.

FIG. 3.

Deletion analysis of selected viruses. Viruses obtained from serial selection in SupT1 cells as described in the legend for Fig. 2 were subcloned and sequenced. Representative clones that contain large deletions are shown. Deletions are represented by the dotted lines, and the antisense target region is indicated at the top of the figure in grey. Please note that the figure is not drawn to scale. Most deletions observed were larger than 1 kb in length, and all deletions overlapped with the target region of the envelope.

FIG. 4.

Sequence of the antisense target region of three clones with a high mutation rate. Three clones, BT-P1, BT-P2, and BT-P3 (Table 2) that had significantly higher mutation rates than wt HIV were sequenced. The nucleotide changes are shown and are predominantly A-G transitions. The antisense target region is shown in bold black, and the underlined region represents the V3 loop. Mutations were concentrated within the antisense target region.

FIG. 5.

Replicative ability of selected mutants. (A) Virus variants selected for by antisense pressure are not fit. Viruses were produced by transfecting 293T cells with the mutant virus plasmids. Supernatants were collected at 48 h posttransfection for determination of virus titer by TCID₅₀ in SupT1 cells. Actual values were as follows: 3.88 × 10⁶ for wt HIV, 6.56 × 10⁵ for BT-P2, and 0 for both BT-P3 and BT-P1 (ND is shown on the graph to indicate not detected). Mutation rates in the antisense region are shown in parentheses. BT-P2 and BT-P3 were full-length envelopes that were inserted back into HIV_NL4-3. (B) Selected viruses cannot replicate in SupT1 cells transduced with vector expressing antisense. SupT1 cells were transduced with vector at an MOI of 11 and then challenged with either wt HIV or BT-P2 at an MOI of 0.01 (WT HIV Td and PT-P2 Td, respectively). Virus replication was measured by p24 ELISA. wt HIV mock-infected cultures (WT HIV Mock) were terminated after day 27 due to virus-induced cell death. Cells transduced with vector showed no evidence of HIV replication. Standard error is shown for WT HIV Td, but not for WT HIV Mock, since only one culture was used as a replication control. BT-P1 and BT-P3 were not tested because they do not replicate.