Comparative analyses of intracellularly expressed antisense RNAs as inhibitors of human immunodeficiency virus type 1 replication

Abstract

The antiviral activities of intracellularly expressed antisense RNAs complementary to the human immunodeficiency virus type 1 (HIV-1) pol, vif, and env genes and the 3' long terminal repeat (LTR) sequence were evaluated in this comparative study. Retroviral vectors expressing the antisense RNAs as part of the Moloney murine leukemia virus LTR promoter-directed retroviral transcript were constructed. The CD4+ T-cell line CEM-SS was transduced with retroviral constructs, and Northern blot analyses showed high steady-state antisense RNA expression levels. The most efficient inhibition of HIV-1 replication was observed with the env antisense RNA, followed by the pol complementary sequence, leading to 2- to 3-log10 reductions in p24 antigen production even at high inoculation doses (4 x 10(4) 50% tissue culture infective doses) of the HIV-1 strain HXB3. The strong antiviral effect correlated with a reduction of HIV-1 steady-state RNA levels, and with intracellular Tat protein production, suggesting that antisense transcripts act at an early step of HIV-1 replication. A lower steady-state antisense RNA level was detected in transduced primary CD4+ lymphocytes than in CEM-SS cells. Nevertheless, replication of the HIV-1 JR-CSF isolate was reduced with both the pol and env antisense RNA. Intracellularly expressed antisense sequences demonstrated more pronounced antiviral efficacy than the transdominant RevM10 protein, making these antisense RNAs a promising gene therapy strategy for HIV-1.

FIG. 1

Schematic representation of the HIV-1 genome. The nucleotide positions, sizes, and positions of the restriction fragments used for antisense-vector construction are indicated.

FIG. 2

(A) Structure of the retroviral vectors containing the antisense sequences. The neomycin phosphotransferase gene and the Lyt2 gene were used as selectable marker genes. The antisense sequence together with the marker gene was expressed from the MoMLV LTR promoter. The arrow indicates the antisense orientation of the inserted HIV-1 sequences. (B) Northern blot analyses of antisense RNA expression in transduced CEM-SS cells. The recombinant transcripts carrying the antisense sequences were detected with a neo-specific probe. The lower panel shows the same blot hybridized with a GAPDH-specific probe as a internal standard. Lane 1, pLN vector; lane 2, pLN-gag/AS; lane 3, pLN-pol1/AS; lane 4, pLN-pol2/AS; lane 5, pLN-vif/AS; lane 6, pLN-env/AS; lane 7, pLN-3′LTR/AS; lane 8, pLN-pol12/AS.

FIG. 3

Inhibition of HIV-1 replication in transduced CEM-SS cells. (A) CEM-SS cell populations (10⁶ cells/ml) were inoculated with 4 × 10² TCID₅₀ of HIV-1 strain HXB3 per ml. B. Infection of transduced CEM-SS cell populations with a high HIV-1 inoculation dose, 4 × 10⁴ TCID₅₀/ml. The culture supernatants were tested for p24 antigen production by ELISA. Experiments were done in duplicate. (C) CEM-SS cell populations (10⁶ cells/ml) were inoculated with 4 × 10³ TCID₅₀ of HIV-1 strain HXB3 per ml and HIV-1 replication was monitored for 50 days.

FIG. 4

Evaluation of anti-HIV-1 efficacies of vectors containing different-length complementary pol sequences. (A) Anti-HIV-1 efficacies of pol1 deletion constructs. CEM-SS cells expressing the 1,400-nt pol1 antisense construct (pLN-pol1/AS), the 790-nt pol antisense construct (pLN-790pol/AS), and the sense pol1 construct (pLN-pol1/S) were inoculated with 4 × 10³ TCID₅₀ of HIV-1 strain HXB3 per ml. (B) CEM-SS cells expressing the 1,400-nt pol1 (pLN-pol1/AS) and the 2,600-nt pol12 (pLN-pol12/AS) antisense sequences were inoculated with 4 × 10³ TCID₅₀ of HIV-1 strain HXB3 per ml. The corresponding sense constructs (pLN-pol1/S and pLN-pol12/S) were used as controls.

FIG. 5

Antisense RNA expression and inhibition of HIV-1 replication in transduced PBLs. (A) Total cellular RNA was isolated from activated, CD4-enriched PBLs transduced with the pL-Lyt2-pol1/AS, pL-Lyt2/pol1/S, pL-Lyt2-env/AS, and pL-Lyt2/env/S vectors and selected for Lyt2 expression. The antisense transcripts were analyzed by Northern blotting with a radiolabeled Lyt2-specific probe. A GAPDH-specific probe was used to monitor the amount of RNA loaded. Lane 1, pL-Lyt2-pol1/AS; lane 2, pL-Lyt2-pol1/S; lane 3, pL-Lyt2-env/AS; lane 4, pL-Lyt2-env/S; lane 5, pL-Lyt2-pol1/AS. (B) Transduced CD4⁺, Lyt2-selected PBLs were activated (36) and infected with HIV-1 JR-CSF (600 TCID₅₀/ml). Cultures were inoculated in quadruplicate, and p24 antigen production was measured.

FIG. 6

Comparison of trans-dominant RevM10 and intracellularly expressed vif, pol1, and env antisense RNAs in high-inoculation-dose HIV-1 infection experiments. CEM-SS cells (10⁶/ml) were inoculated with 10⁵ TCID₅₀ of HIV-1 strain HXB3 per ml, and viral replication was monitored by measuring p24^Gag antigen production in the culture supernatant.

FIG. 7

Detection of HIV-1, antisense, and RevM10 transcripts in CEM-SS cells inoculated with 10⁵ TCID₅₀ of HIV-1 strain HXB3 per ml. Total cellular RNA was isolated from 10⁶ CEM-SS cells at days 4 (A), 6 (B), and 8 (C) postinfection and analyzed by Northern blotting. The HIV-specific transcripts were detected with a radiolabeled TAR-specific oligonucleotide probe, and expression of the ΔRevM10 and RevM10 transcripts was detected with a Rev-specific probe. After the filter was washed, a neo-specific probe which detected both RevM10 and ΔRevM10 and the antisense transcripts was used. A GAPDH-specific probe was used to monitor the amount of RNA loaded. Lane 1, RevM10; lane 2, ΔRevM10; lane 3, pLN (vector control); lane 4, pLN-vif/AS; lane 5, pLN-pol1/AS; lane 6, pLN-env/AS.

FIG. 8

Analyses of intracellular p24^Gag and Tat expression in HIV-1-infected CEM-SS cells. (A) Intracellular p24^Gag expression was measured at day 8 postinoculation. The mean fluorescence intensities reflect the relative intracellular p24^Gag expression levels. (B) Detection of Tat protein in transduced and HIV-1-infected CEM-SS cells. Aliquots of CEM-SS cells at day 8 postinfection were fixed in methanol, stained with a Tat-specific antibody, and analyzed with a FACScan.