Antiviral effects of human immunodeficiency virus type 1-specific small interfering RNAs against targets conserved in select neurotropic viral strains

Abstract

RNA interference, a natural biological phenomenon mediated by small interfering RNAs (siRNAs), has been demonstrated in recent studies to be an effective strategy against human immunodeficiency virus type 1 (HIV-1). In the present study, we used 21-bp chemically synthesized siRNA duplexes whose sequences were derived from the gp41 gene, nef, tat, and rev regions of viral RNA. These sequences are conserved in select neurotropic strains of HIV-1 (JR-FL, JR-CSF, and YU-2). The designed siRNAs exerted a potent antiviral effect on these HIV-1 strains. The antiviral effect was mediated at the RNA level (as observed by the down-regulation of the HIV-1-specific spliced transcript generating a 1.2-kbp reverse transcription [RT]-PCR product) as well as viral assembly on the cell membrane. Spliced transcripts (apart from the most abundant transcript generating a 1.2-kbp RT-PCR product) arising from an unspliced precursor likely contributed, albeit to a lesser extent, to the antiviral effect. The resultant progeny viruses had infectivities similar to that of input virus. We therefore conclude that these siRNAs interfere with the processing of the unspliced transcripts for the gp41 gene, tat, rev, and nef, eventually affecting viral assembly and leading to the overall inhibition of viral production. Apart from using the gp41 gene as a target, the conservation of each of these targets in the above-mentioned viral strains, as well as several primary isolates, would enable these siRNAs to be used as potent antiviral tools for investigations with cells derived from the central nervous system in order to evaluate their therapeutic potential and assess their utility in inhibiting HIV-1 neuropathogenesis and neuroinvasion.

FIG. 1.

HIV-1-specific siRNA sequences and their relative positions in select neurotropic strains. Twenty-one-base-pair siRNA duplexes were selected from within a region of the HIV-1 NL4-3 transcripts for the gp41 gene, tat, rev, and nef. (A) The positions of the siRNA cognate sequences in HIV-1 NL4-3 are shown by narrow rectangular bars (red bars indicate the positions of the three most potent siRNAs). These siRNA cognate sequences are conserved in NL4-3 and select neurotropic strains of HIV-1. (B) Relative positions of these sequences in NL4-3, YU-2, JR-FL, and JR-CSF. These sequences are also well conserved in a wide variety of primary isolates but lack significant homologies in the EST-human database.

FIG. 2.

Conservation of target sequences for the most potent siRNAs (5, 6, and 8) in select neurotropic strains of HIV-1 and NL4-3. Shown is an alignment of the select neurotropic strains in the region where the cognate sequences for siRNAs 5, 6, and 8 are present. Viral sequences are numbered with reference to the sense primer (HIV-5964-5983-F) for the NL4-3 genome and are from within a region of the transcripts for the gp41 gene, tat, rev, and nef. The primer nomenclature indicates the location of the sequence in the NL4-3 genome.

FIG. 3.

Antiviral efficacy of HIV-1-specific siRNAs for HIV-1 NL4-3 infection in HeLaCD4 cells. HeLaCD4 cells were transfected with siRNAs and then infected with HIV-1 NL4-3 (MOI, 0.1 pg of p24/cell). HIV-1 p24 antigen levels in culture supernatants were measured at 1 and 2 dpi. The means of three experiments and the standard deviations are indicated. siRNAs 5, 6, and 8 exerted the most potent inhibition of NL4-3 infection.

FIG. 4.

HIV-1-specific siRNAs affect viral assembly. Intracellular levels of HIV-1 p24 antigen are not down-regulated in a significant manner. When extracellular p24 antigen levels (progeny virion-associated p24) are analyzed as a percentage of intracellular p24 antigen levels, the efficiency of viral production can be assessed. Infection in the absence of HIV-1-specific siRNAs becomes increasingly inefficient. However, infection in cells transfected with HIV-1-specific siRNAs exhibits maximal inefficiency at 2 dpi and then increases in a statistically significant manner. The inefficiency of viral production seems to be correlated temporally with maximal interference by siRNAs. Bars: NL4-3, HeLaCD4 cells not transfected with siRNA but infected with HIV-1 NL4-3; Scrambled, HeLaCD4 cells transfected with a nonspecific siRNA and infected with HIV-1 NL4-3; 5, 6, and 8, HeLaCD4 cells transfected with HIV-1-specific siRNAs 5, 6, and 8, respectively, and infected with HIV-1 NL4-3; Multiplexed, HeLaCD4 cells transfected simultaneously with HIV-1-specific siRNAs 5, 6, and 8 and infected with HIV-1 NL4-3. The means of three experiments and the standard deviations are indicated.

FIG. 5.

Infectivity of progeny virions generated from HeLaCD4 cells treated with HIV-1-specific siRNAs. Supernatants containing progeny virions generated from HeLaCD4 cells transfected with HIV-1-specific siRNAs at 2 dpi were tested to determine the infectivity of the viral particles in a MAGI assay. HeLaCD4-LTR-β-Gal indicator cells were stained for β-galactosidase enzyme activity with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal). The data represent the infectivity of progeny virions generated from HeLaCD4 cells transfected with no siRNA (N/2dpi), scrambled (negative control) siRNA (S/2dpi), siRNA 5 (5/2dpi), siRNA 6 (6/2dpi), siRNA 8 (8/2dpi), and siRNAs 5, 6, and 8 (multiplexed) (M/2dpi). I.U., infectious units. The means of three experiments and the standard deviations are indicated.

FIG. 6.

HIV-1-specific siRNAs down-regulate the major spliced transcript. (A) Southern blot hybridization of RT-PCR products with three internal probes (used simultaneously) corresponding to the siRNA 5, 6, and 8 target sequences. (B) RT-PCR products generated by intron-spanning primers within the unspliced transcripts for the gp41 gene, tat, rev, and nef in the HIV-1 NL4-3 genome. The major spliced transcript generated a 1.2-kbp RT-PCR product observed on agarose gels or by hybridization. (C) RT-PCR products generated by β-globin primers. Lanes for panels A to C: lane 1, uninfected; lane 2, HeLaCD4 cells infected with NL4-3; lane 3, HeLaCD4 cells transfected with scrambled (negative control) siRNA and infected with NL4-3; lanes 4, 5, and 6, HeLaCD4 cells transfected with siRNAs 5, 6, and 8, respectively, and infected with NL4-3; lane 7, HeLaCD4 cells transfected with siRNAs 5, 6, and 8 simultaneously (multiplexed) and infected with NL4-3. (D) Schematic representation of the HIV-1 NL4-3 genome. Red bars indicate the positions of the oligonucleotide probes used for Southern blotting in panel A. Light-blue bars indicate the positions of intron-spanning primers. The unspliced transcript would generate a 4.5-kbp RT-PCR product. The major spliced transcript would generate a 1.2-kbp RT-PCR product.

FIG. 7.

HIV-1-specific siRNAs inhibit the replication of HIV-1 NL4-3, YU-2, and JR-CSF. To assess the potency of HIV-1-specific siRNAs in inhibiting viral replication, siRNAs (multiplexed) were cotransfected into 293T cells along with various quantities (62.5 to 1,000 ng) of plasmid DNAs for NL4-3, YU-2, and JR-CSF. HIV-1 p24 antigen levels in culture supernatants were measured at 48 h posttransfection. Parallel experiments were also performed with HeLaCD4 cells. Control data are for 293T cells transfected with plasmid DNA forHIV-1 alone (one set). Scrambled siRNA data are for 293T cells cotransfected with scrambled (negative control) siRNA and plasmid DNA for HIV-1 (one set). HIV-1-specific siRNA data are for 293T cells cotransfected with HIV-1-specific siRNAs (multiplexed) and plasmid DNA for HIV-1 (in triplicate; the means and standard deviations are indicated).

FIG. 8.

Antiviral efficacy of HIV-1-specific siRNAs for HIV-1 YU-2 infection in human MDMs. Primary human macrophages were transfected with siRNAs and then infected with HIV-1 YU-2 (MOI, 0.1 pg of p24/cell). HIV-1 p24 antigen levels in culture supernatants were measured at 2 and 9 dpi. The data show the levels of viral production in macrophages infected with HIV-1 YU-2 and not transfected with siRNA (YU-2) and in macrophages infected with HIV-1 YU-2 and transfected with HIV-1-specific siRNAs 5, 6, and 8 (YU-2 + HIV-1-specific siRNA). The means of three experiments and the standard deviations are indicated.