Inhibition Of HIV-1 Gene Expression Research Articles

SERINC5 Mediates a Postintegration Block to HIV-1 Gene Expression in Macrophages.

HIV-1 antagonizes SERINC5 by redundant mechanisms, primarily through Nef and additionally via envelope glycoprotein. Paradoxically, HIV-1 preserves Nef function to ensure the exclusion of SERINC5 from virion incorporation regardless of the availability of envelope that can confer resistance, suggesting additional roles of the virion-incorporated host factor. Here, we report an unusual mode of SERINC5 action in inhibiting viral gene expression. This inhibition is observed only in the myeloid lineage cells but not in the cells of epithelial or lymphoid origin. We found that SERINC5-bearing viruses induce the expression of RPL35 and DRAP1 in macrophages, and these host proteins intercept HIV-1 Tat from binding to and recruiting a mammalian capping enzyme (MCE1) to the HIV-1 transcriptional complex. As a result, uncapped viral transcripts are synthesized, leading to the inhibition of viral protein synthesis and subsequent progeny virion biogenesis. Cell-type-specific inhibition of HIV-1 gene expression thus exemplifies a novel antiviral function of virion-incorporated SERINC5. IMPORTANCE In addition to Nef, HIV-1 envelope glycoprotein has been shown to modulate SERINC5-mediated inhibition. Counterintuitively, Nef from the same isolates preserves the ability to prevent SERINC5 incorporation into virions, implying additional functions of the host protein. We identify that virion-associated SERINC5 can manifest an antiviral mechanism independent of the envelope glycoprotein to regulate HIV-1 gene expression in macrophages. This mechanism is exhibited by affecting the viral RNA capping and is plausibly adopted by the host to overcome the envelope glycoprotein-mediated resistance to SERINC5 restriction.

An activator of G protein-coupled receptor and MEK1/2-ERK1/2 signaling inhibits HIV-1 replication by altering viral RNA processing.

The ability of HIV-1 to evolve resistance to combined antiretroviral therapies (cARTs) has stimulated research into alternative means of controlling this infection. We assayed >60 modulators of RNA alternative splicing (AS) to identify new inhibitors of HIV-1 RNA processing-a segment of the viral lifecycle not targeted by current drugs-and discovered compound N-[4-chloro-3-(trifluoromethyl)phenyl]-7-nitro-2,1,3-benzoxadiazol-4-amine (5342191) as a potent inhibitor of both wild-type (Ba-L, NL4-3, LAI, IIIB, and N54) and drug-resistant strains of HIV-1 (IC50: ~700 nM) with no significant effect on cell viability at doses tested. 5342191 blocks expression of four essential HIV-1 structural and regulatory proteins (Gag, Env, Tat, and Rev) without affecting total protein synthesis of the cell. This response is associated with altered unspliced (US) and singly-spliced (SS) HIV-1 RNA accumulation (~60% reduction) and transport to the cytoplasm (loss of Rev) whereas parallel analysis of cellular RNAs revealed less than a 0.7% of host alternative splicing (AS) events (0.25-0.67% by ≥ 10-20%), gene expression (0.01-0.46% by ≥ 2-5 fold), and protein abundance (0.02-0.34% by ≥ 1.5-2 fold) being affected. Decreased expression of Tat, but not Gag/Env, upon 5342191 treatment was reversed by a proteasome inhibitor, suggesting that this compound alters the synthesis/degradation of this key viral factor. Consistent with an affect on HIV-1 RNA processing, 5342191 treatment of cells altered the abundance and phosphorylation of serine/arginine-rich splicing factor (SRSF) 1, 3, and 4. Despite the activation of several intracellular signaling pathways by 5342191 (Ras, MEK1/2-ERK1/2, and JNK1/2/3), inhibition of HIV-1 gene expression by this compound could be reversed by pre-treatment with either a G-protein α-subunit inhibitor or two different MEK1/2 inhibitors. These observations demonstrate enhanced sensitivity of HIV-1 gene expression to small changes in host RNA processing and highlights the potential of modulating host intracellular signaling as an alternative approach for controlling HIV-1 infection.

Iron overload down-regulates the expression of the HIV-1 Rev cofactor eIF5A in infected T lymphocytes

BackgroundChanges in iron metabolism frequently accompany HIV-1 infection. However, while many clinical and in vitro studies report iron overload exacerbates the development of infection, many others have found no correlation. Therefore, the multi-faceted role of iron in HIV-1 infection remains enigmatic.MethodsRT-qPCR targeting the LTR region, gag, Tat and Rev were performed to measure the levels of viral RNAs in response to iron overload. Spike-in SILAC proteomics comparing i) iron-treated, ii) HIV-1-infected and iii) HIV-1-infected/iron treated T lymphocytes was performed to define modifications in the host cell proteome. Data from quantitative proteomics were integrated with the HIV-1 Human Interaction Database for assessing any viral cofactors modulated by iron overload in infected T lymphocytes.ResultsHere, we demonstrate that the iron overload down-regulates HIV-1 gene expression by decreasing the levels of viral RNAs. In addition, we found that iron overload modulates the expression of many viral cofactors. Among them, the downregulation of the REV cofactor eIF5A may correlate with the iron-induced inhibition of HIV-1 gene expression. Therefore, we demonstrated that eiF5A downregulation by shRNA resulted in a significant decrease of Nef levels, thus hampering HIV-1 replication.ConclusionsOur study indicates that HIV-1 cofactors influenced by iron metabolism represent potential targets for antiretroviral therapy and suggests eIF5A as a selective target for drug development.

Cdk9 phosphorylates Pirh2 protein and prevents degradation of p53 protein

Several reports have pointed to the negative involvement of p53 in transcriptional regulation of the human immunodeficiency virus type 1 long-terminal repeat (HIV-1 LTR). We recently demonstrated that through their physical interaction, cdk9 phosphorylates p53 on Ser-392, leading to p53 stability and accumulation. As a result, p53 stalled transcriptional elongation of the HIV-1 LTR and significantly reduced HIV-1 replication in primary microglia and astrocytes. Therefore, we sought to identify the mechanisms used by cdk9 to allow this p53 function. Using western blot analysis, we found that cdk9 promotes inhibition and phosphorylation of Mdm2 on Ser-395, thus preventing degradation of p53, a protein that is directly involved in promoting p53 ubiquitination. On the other hand, we showed that cdk9 phosphorylates Pirh2 on Ser-211 and Thr-217 residues through their physical interaction. Phosphorylation of Pirh2 renders it inactive and may contribute to p53-inhibition of transcriptional elongation of the HIV-1 LTR. Hence, we suggest that phosphorylation of Pirh2 may be a novel target for the inhibition of HIV-1 gene expression.

HIV RNA dimerisation interference by antisense oligonucleotides targeted to the 5′ UTR structural elements

The HIV-1 genome consists of two identical RNA molecules non-covalently linked by their 5′ unstranslatable regions (5′ UTR). The high level of sequence and structural conservation of this region correlates with its important functional involvement in the viral cycle, making it an attractive target for antiviral treatments based on antisense technology. Ten unmodified DNA antisense oligonucleotides (ODNs) targeted against different conserved structural elements within the 5′ UTR were assayed for their capacity to interfere with HIV-1 RNA dimerisation, inhibit gene expression, and prevent virus production in cell cultures. The results show that, in addition to the well-characterised dimerisation initiation site (DIS), targeting of the AUG-containing structural element may reflect its direct role in HIV-1 genomic RNA dimerisation in vitro. Similarly, blocking the 3′ end sequences of the stem-loop domain containing the primer biding site interferes with RNA dimerisation. Targeting the apical portion of the TAR element, however, appears to promote dimerisation. ODNs targeted against the conserved polyadenylation signal [Poly(A)], the primer binding site (PBS), the major splicing donor (SD) or the major packaging signal (Psi), and AUG-containing structural elements led to a highly efficient inhibition of HIV-1 gene expression and virus production in cell culture. Together, these results support the idea that ODNs possess great potential as molecular tools for the functional characterisation of viral RNA structural domains. Moreover, the targeting of these domains leads to the potent inhibition of viral replication, underscoring the potential of conserved structural RNA elements as antiviral targets.

Transcriptional regulation of HIV-1 gene expression by p53

Several reports have pointed to the negative involvement of p53 in transcriptional regulation of the human immunodeficiency virus type 1 long terminal repeat (HIV-1 LTR). However, the mechanisms of this negative effect remain unclear. In here, we showed that over expression of p53 wild type prevented the phosphorylation of serine 2 in the carboxyl terminal domain (CTD) of RNA polymerase II. As a result of this inhibition, p53 stalled transcriptional elongation on the HIV-1 LTR leading to a significant reduction of HIV-1 replication in primary microglia and astrocytes. However, despite the delay/pause caused by p53, viral transcription and replication decreased and then salvaged. These studies suggest that the negative effect of p53 is alleviated by a third factor. In this regard, our Preliminary Data point to the involvement of the Pirh2 protein in p53 inhibition. Therefore, we suggest that p53 may be a novel therapeutic target for the inhibition of HIV-1 gene expression and replication and the treatment of AIDS.

Cyclin T1 stabilizes expression levels of HIV‐1 Tat in cells

Transcription from HIV-1 proviral DNA is a rate-determining step for HIV-1 replication. Interaction between the cyclin T1 (CycT1) subunit of positive transcription elongation factor b (P-TEFb) and the Tat transactivator protein of HIV-1 is crucial for viral transcription. CycT1 also interacts directly with the transactivation-responsive element (TAR) located on the 5'end of viral mRNA, as well as with Tat through the Tat-TAR recognition motif (TRM). These molecular interactions represent a critical step for stimulation of HIV transcription. Thus, Tat and CycT1 are considered to be feasible targets for the development of novel anti-HIV therapies. In this study, we demonstrate that CycT1 is positively involved in the Tat protein stability. Selective degradation of CycT1 by small interfering RNA (siRNA) culminated in proteasome-mediated degradation of Tat and eventual inhibition of HIV-1 gene expression. We noted that the siRNA-mediated knockdown of CycT1 could inhibit HIV-1 transcription without affecting cell viability and Tat mRNA levels. These findings clearly indicate that CycT1 is a feasible therapeutic target, and inactivation or depletion of CycT1 should effectively inhibit HIV replication by destabilizing Tat and suppressing Tat-mediated HIV transcription.

Inhibition of HIV-1 gene expression by Ciclopirox and Deferiprone, drugs that prevent hypusination of eukaryotic initiation factor 5A

BackgroundEukaryotic translation initiation factor eIF5A has been implicated in HIV-1 replication. This protein contains the apparently unique amino acid hypusine that is formed by the post-translational modification of a lysine residue catalyzed by deoxyhypusine synthase and deoxyhypusine hydroxylase (DOHH). DOHH activity is inhibited by two clinically used drugs, the topical fungicide ciclopirox and the systemic medicinal iron chelator deferiprone. Deferiprone has been reported to inhibit HIV-1 replication in tissue culture.ResultsCiclopirox and deferiprone blocked HIV-1 replication in PBMCs. To examine the underlying mechanisms, we investigated the action of the drugs on eIF5A modification and HIV-1 gene expression in model systems. At early times after drug exposure, both drugs inhibited substrate binding to DOHH and prevented the formation of mature eIF5A. Viral gene expression from HIV-1 molecular clones was suppressed at the RNA level independently of all viral genes. The inhibition was specific for the viral promoter and occurred at the level of HIV-1 transcription initiation. Partial knockdown of eIF5A-1 by siRNA led to inhibition of HIV-1 gene expression that was non-additive with drug action. These data support the importance of eIF5A and hypusine formation in HIV-1 gene expression.ConclusionAt clinically relevant concentrations, two widely used drugs blocked HIV-1 replication ex vivo. They specifically inhibited expression from the HIV-1 promoter at the level of transcription initiation. Both drugs interfered with the hydroxylation step in the hypusine modification of eIF5A. These results have profound implications for the potential therapeutic use of these drugs as antiretrovirals and for the development of optimized analogs.

Novel HIV-1 therapeutics through targeting altered host cell pathways

The emergence of drug-resistant HIV-1 strains presents a challenge for the design of new drugs. Anti-HIV compounds currently in use are the subject of advanced clinical trials using either HIV-1 reverse transcriptase, viral protease or integrase inhibitors. Recent studies show an increase in the number of HIV-1 variants resistant to anti-retroviral agents in newly infected individuals. Targeting host cell factors involved in the regulation of HIV-1 replication might be one way to combat HIV-1 resistance to the currently available anti-viral agents. A specific inhibition of HIV-1 gene expression could be expected from the development of compounds targeting host cell factors that participate in the activation of the HIV-1 LTR promoter. Here we discuss how targeting the host can be accomplished either by using small molecules to alter the function of the host's proteins such as p53 or cdk9, or by utilizing new advances in siRNA therapies to knock down essential host factors such as CCR5 and CXCR4. Finally, we will discuss how the viral protein interactomes should be used to better design therapeutics against HIV-1.

Inhibition of HIV-1 gene expression by Sam68ΔC: multiple targets but a common mechanism?

Two recent publications have explored the mechanisms by which a mutant of the host protein Sam68 blocks HIV-1 structural protein synthesis and expands its activity to encompass Nef. Although the two studies propose different mechanisms for the responses observed, it is possible that a common activity is responsible. Understanding how this Sam68 mutant discriminates among the multiple viral mRNAs promises to reveal unique properties of HIV-1 RNA metabolism.

Potent inhibition of HIV-1 gene expression and TAT-mediated apoptosis in human T cells by novel mono- and multitarget anti-TAT/Rev/Env ribozymes and a general purpose RNA-cleaving DNA-enzyme

One of the hallmarks of progression of HIV-1/AIDS is the rapid depletion of CD4 + T cells that is known to occur at the late stages of the disease when usually X4 tropic HIV-1 predominates. Besides direct killing of T lymphocytes, HIV-1 infection leads to extensive apoptosis of naïve/uninfected bystander T cells, which is predominantly mediated by HIV-1 TAT protein. Therefore, reduction of HIV-1 TAT protein is likely to reduce substantially the pathogenesis associated with HIV-1 infection. We designed two non-GUX hammerhead ribozymes (Rzs) and a Di-Rz by placing them in direct tandem. These were targeted against the most conserved second exon of HIV-1 TAT/Rev/Env region. Although very impressive in vitro cleavage of the target RNA by the two hammerhead Rzs was obtained under standard conditions of cleavage, only one of them was active under simulated physiological conditions. Sequence-specific cleavage by the Di-Rz was most efficient under standard conditions. Cleavage reactions carried out under simulated physiological conditions by the Di-Rz, however, indicated that both mono-Rzs were functional. We also assembled a 10–23 catalytic motif containing general purpose RNA-cleaving DNA-enzyme (Dz) against the same region, which cleaved the target RNA very efficiently. Both Rzs and Dz showed not only potent inhibition of HIV-1 gene expression but also showed remarkable protection against HIV-1 TAT protein-mediated apoptosis in Jurkat T cells. Possible implications of these findings are discussed.

Novel Pol II Fusion Promoter Directs Human Immunodeficiency Virus Type 1-Inducible Coexpression of a Short Hairpin RNA and Protein

We demonstrate a novel approach for coexpression of a short hairpin RNA (shRNA) with an open reading frame which exploits transcriptional read-through of a minimal polyadenylation signal from a Pol II promoter. We first observed efficient inducible expression of enhanced green fluorescent protein along with an anti-rev shRNA. We took advantage of this observation to test coexpression of the transdominant negative mutant (humanized) of human immunodeficiency type 1 (HIV-1) Rev (huRevM10) along with an anti-rev shRNA via an HIV-1-inducible fusion promoter. The coexpression of the shRNA and transdominant protein resulted in potent, long-term inhibition of HIV-1 gene expression and suppression of shRNA-resistant mutants. This dual expression system has broad-based potential for other shRNA applications, such as cases where simultaneous knockdown of mutant and wild-type transcripts must be accompanied by replacement of the wild-type protein.

Inhibition of HIV-1 gene expression by retroviral vector-mediated small-guide RNAs that direct specific RNA cleavage by tRNase ZL.

The tRNA 3′-processing endoribonuclease (tRNase Z or 3′ tRNase; EC 3.1.26.11) is an essential enzyme that removes the 3′ trailer from pre-tRNA. The long form (tRNase ZL) can cleave a target RNA in vitro at the site directed by an appropriate small-guide RNA (sgRNA). Here, we investigated whether this sgRNA/tRNase ZL strategy could be applied to gene therapy for AIDS. We tested the ability of four sgRNA-expression plasmids to inhibit HIV-1 gene expression in COS cells, using a transient-expression assay. The three sgRNAs guide inhibition of HIV-1 gene expression in cultured COS cells. Analysis of the HIV-1 mRNA levels suggested that sgRNA directed the tRNase ZL to mediate the degradation of target RNA. The observation that sgRNA was localized primarily in nuclei suggests that tRNase ZL cleaves the HIV-1 mRNA when complexed with sgRNA in this location. We also examined the ability of two retroviral vectors expressing sgRNA to suppress HIV-1 expression in HIV-1-infected Jurkat T cells. sgRNA-SL4 suppressed HIV-1 expression almost completely in infected cells for up to 18 days. These results suggest that the sgRNA/tRNase ZL approach is effective in downregulating HIV-1 gene expression.

Inhibition of HIV-1 gene expression by novel DNA enzymes targeted to cleave HIV-1 TAR RNA: potential effectiveness against all HIV-1 isolates

Nucleic acid-based antiviral approaches have been tried against multiple HIV-1 genes with the purpose of down-regulating its replication. A unique stem–loop structure called TAR is present at the 5′-end of all HIV-1 transcripts; Tat and other cellular proteins bind to TAR and thus govern transcription. Therefore, HIV-1 TAR is an attractive target against which various antiviral approaches could be tried. We screened several DNA enzymes (Dz's) containing the 10-23 catalytic motif and a single Dz containing the 8-17 catalytic motif against the HIV-1 TAR RNA. Dz's directed against the predicted single-stranded bulge regions showed sequence-specific cleavage activities. One of the two Dz's, namely Dz-475, showed moderate cleavage activity in complete absence of Mg2+. Addition of unrelated sequences at the 5′-end of the HIV-1 TAR RNA rendered it susceptible to four additional Dz-mediated cleavages. Both Dz's (470 and 475) showed significant intracellular reduction of HIV-1 gene expression. Dz-475-treated cells showed significant protection against T-tropic and macrophage-tropic HIV-1 challenge. Dz-475-transfected T-lymphocytes, human PBMCs, or chronically infected cell lines showed marked viral resistance. Unique features of this antiviral strategy with respect to HIV-1 gene inhibition are discussed.

Direct participation of Sam68, the 68-kilodalton Src-associated protein in mitosis, in the CRM1-mediated Rev nuclear export pathway.

Human immunodeficiency virus type 1 (HIV-1) replication requires efficient nuclear export of incompletely spliced and unspliced HIV-1 mRNA transcripts, which is achieved by Rev expression at an early stage of the viral life cycle. We have recently shown that expression of Sam68, the 68-kDa Src-associated protein in mitosis, is able to alleviate Rev function block in astrocytes by promoting Rev nuclear export. In the present study, we utilized an antisense RNA expression strategy to down-modulate constitutive Sam68 expression and examined its effect on Rev function, HIV-1 gene expression, and viral replication. These results showed that down-modulation of constitutive Sam68 expression markedly inhibited HIV-1 production in 293T cells and viral replication in T lymphocytes such as Jurkat and CEM cells, as well as human peripheral blood mononuclear cells (PBMCs). Rev-dependent in trans complementation and reporter gene assays further demonstrated that inhibition of HIV-1 gene expression by Sam68 down-modulation was due to impeded Rev activity. Moreover, digital fluorescence microscopic imaging revealed that down-modulation of Sam68 expression caused exclusive nuclear retention and colocalization of both Rev and CRM1. Taken together, these data suggest that adequate Sam68 expression is required for Rev function and, thereby, for HIV-1 gene expression and viral replication, and they support the notion that Sam68 is directly involved in the CRM1-mediated Rev nuclear export pathway.

Specific inhibition of HIV-1 gene expression by double-stranded RNA.

RNA interference (RNAi) is phenomenon in which introduced double-stranded RNAs (ds-RNAs) silence gene expression through specific degradation of their cognate mRNAs. RNAi has been observed in a wide variety of organisms and is considered to be a feature of nearly all eukaryotes. The mediators of sequence specific messenger RNA degradation are 21-23 nucleotide small interfering RNAs generated by ribonucleaseIII cleavage from longer dsRNAs. To investigate the potential of dsRNA to interfere with the function of HIV-1 genes, we have designed six longer dsRNAs containing the HIV-1 gag and env genes. Double-stranded RNAs were tested for inhibitory effects using monkey COS cells. In these anti-HIV-1 activity tests, the dsRNAs targeted to the HIV-1 env sequence showed more than 90% anti-HIV-1 efficiency. Our results show that dsRNA interference can be used as a powerful method for the inhibition of HIV-1 gene expression.

Inhibition of HIV-1 gene expression by novel macrophage-tropic DNA enzymes targeted to cleave HIV-1 TAT/Rev RNA

Many regions of the HIV-1 genome have been targeted in earlier studies by RNA-cleaving DNA enzymes possessing the 10–23 catalytic motif, and efficient inhibition of HIV-1 gene expression was reported. All these studies employed charged synthetic lipids to introduce the catalytic DNA into the mammalian cells, which severely limits its practical application and usefulness in vivo. Taking advantage of the ability of G residues to interact directly with the scavenger receptors on the macrophages, we synthesized a DNA enzyme 5970 that contained 10 G residues at the 3′ end. With the aim of improving the intracellular stability of the DNA enzyme 5970, we added two short stretches of stem-loop structures that were 12 bases long on either side of the DNA enzyme 5970. DNA enzyme 5970 without the poly-G tracts cleaved the synthetic RNA of HIV-1 TAT/Rev, two important regulatory proteins of HIV, very efficiently in a sequence-specific manner. Addition of 10 G residues at the 3′ end of the DNA enzyme affected the cleavage efficiency only marginally whereas the same DNA enzyme with stem-loop structures on either end was significantly less efficient. The DNA enzyme with the poly-G tract at its 3′ end was taken up specifically by a human macrophage-specific cell line directly in the absence of Lipofectin and was also able to inhibit HIV-1 gene expression in a transient-expression system as well as when challenged with the virus. The potential applications of these novel macrophage-tropic DNA enzymes are discussed.

Inhibition of HIV-1 gene expression by novel macrophage-tropic DNA enzymes targeted to cleave HIV-1 TAT/Rev RNA.

Many regions of the HIV-1 genome have been targeted in earlier studies by RNA-cleaving DNA enzymes possessing the 10-23 catalytic motif, and efficient inhibition of HIV-1 gene expression was reported. All these studies employed charged synthetic lipids to introduce the catalytic DNA into the mammalian cells, which severely limits its practical application and usefulness in vivo. Taking advantage of the ability of G residues to interact directly with the scavenger receptors on the macrophages, we synthesized a DNA enzyme 5970 that contained 10 G residues at the 3' end. With the aim of improving the intracellular stability of the DNA enzyme 5970, we added two short stretches of stem-loop structures that were 12 bases long on either side of the DNA enzyme 5970. DNA enzyme 5970 without the poly-G tracts cleaved the synthetic RNA of HIV-1 TAT/Rev, two important regulatory proteins of HIV, very efficiently in a sequence-specific manner. Addition of 10 G residues at the 3' end of the DNA enzyme affected the cleavage efficiency only marginally whereas the same DNA enzyme with stem-loop structures on either end was significantly less efficient. The DNA enzyme with the poly-G tract at its 3' end was taken up specifically by a human macrophage-specific cell line directly in the absence of Lipofectin and was also able to inhibit HIV-1 gene expression in a transient-expression system as well as when challenged with the virus. The potential applications of these novel macrophage-tropic DNA enzymes are discussed.

Relative Levels of Inhibition of p24 Gene Expression by Different 20-mer Antisense Oligonucleotide Sequences Targeting Nucleotides +1129 to +1268 of the HIV-1gagGenome: An Analysis of Mechanism

GPI2A is a 20-mer antisense oligonucleotide sequence that is complementary to a region of the HIV-1gaggene. An analysis of viral core antigen p24 protein synthesis inhibition was performed with cells expressing HIV-1 proteins, following treatment with GPI2A or eight other unique antisense constructs designed to bind to regions of thegaggene, at positions that 5′ or 3′ flank the GPI2A target site. GPI2A was found to be the most effective construct, indicating that the GPI2A target region is a particularly sensitive site for antisense activity. An analysis of energy-related parameters important in complementary duplex formation was performed for each antisense construct. Also, the potential of each antisense sequence to exhibit self-complementarity or to self-dimerize was assessed. The results from these analyses provided an explanation for the high specificity and the superior inhibitory characteristics of GPIA when compared to the eight other antisense oligonucleotides. GPI2A exhibited the second most favorable energy-related characteristics for hybridization reactions, and most importantly, unlike the other eight antisense sequences, it did not show the potential to self-complement or to dimerize. The results of this study and a previous investigation of sequence specificity requirements for GPI2A inhibition of HIV-1 gene expression provide strong evidence for an antisense mode of action for this oligonucleotide construct, a useful tool for analysis of viral gene expression and perhaps a potential therapeutic agent.

Phosphorothioate Oligonucleotides Block Reverse Transcription by the RNase-H Activity Associated with the HIV-1 Polymerase

We demonstrate the degradation of RNA bound to an antisense oligonucleotide by a reverse transcriptase enzyme-associated RNase H activity. We found that phosphorothioate oligonucleotides inhibit the RNase H activity by binding to AMV RT, rather than to the template RNA, whereas the RNase H activity of HIV-1 RT is not affected by the antisense phosphorothioate oligonucleotide. Selective inhibition of HIV-1 gene expression involves the degradation of the template RNA bound to the antisense phosphorothioate oligonucleotide by the RNase H activity associated with the HIV-1 polymerase.