HIV-1 VprB and C RNA cleavage by potent 10-23 DNAzymes that also cause reversal of G2 cell cycle arrest mediated by Vpr genes

Accessory Vpr protein of HIV-1 is known to influence several key cellular functions that also impacts on the HIV-1 replication cycle. Besides other activities, it alone causes cell cycle arrest at the G2 phase and thus potentially contribute to the overall pathology. We designed several 10-23 catalytic motifs containing DNAzymes (Dzs) against the full-length Vpr gene from subtype B and checked its activity against VprC gene from one of the Indian HIV-1 isolates. Among several Dzs that showed sequence-specific cleavage activities, Dz-94 was very potent and equally efficient in its ability to cleave full-length VprB and C RNA to completion under standard conditions of cleavage. Although Dz-90 target sequence was fully conserved between VprB and C genes, it was more effective on latter genes, suggesting that spatial structures of RNA at other regions of Vpr can also influence the cleavage activity for this Dz. HIV-1 VprB and C encoding genes under the powerful CMV promoter, when cotransfected into mammalian cells with Dz-94, a potent intracellular inhibition, was observed, which also resulted in reversing the G2 cell cycle arrest mediated by VprB and C proteins. Thus, Dz-94 could potentially be developed to prevent Vpr-mediated cytopathic effects caused by HIV-1 subtype B and C isolates.     

RNA heptamers that direct RNA cleavage by mammalian tRNA 3' processing endoribonuclease.

Mammalian tRNA 3' processing endoribonuclease (3' tRNase) can recognize and cleave any target RNA that forms a precursor tRNA-like complex with another RNA. Various sets of RNA molecules were tested to identify the smallest RNA that can direct target RNA cleavage by 3' tRNase. A 3' half tRNAArgwas cleaved efficiently by 3' tRNase in the presence of small 5' half tRNAArgvariants, the D stem-loop region of which was partially deleted. Remarkably, 3' tRNase also cleaved the 3' half tRNAArgin the presence of a 7 nt 5' tRNAArg composed only of the acceptor stem region with a catalytic efficiency comparable with that of cleavage directed by an intact 5' half tRNAArg. The catalytic efficiency of cleavage directed by the heptamer decreased as the stability of the T stem-loop structures of 3' half tRNAArg variants decreased. No heptamer-directed cleavage of a 3' half tRNAArg without T stem base pairs was detected. A heptamer also directed cleavage of an HIV-1 RNA containing a stable hairpin structure. These findings suggest that in the presence of an RNA heptamer, 3' tRNase can discriminate and eliminate target RNAs that possess a stable hairpin adjacent to the heptamer binding sequence from a large complex RNA pool.     

The effect of structure in a long target RNA on ribozyme cleavage efficiency.

Inhibition of gene expression by catalytic RNA (ribozymes) requires that ribozymes efficiently cleave specific sites within large target RNAs. However, the cleavage of long target RNAs by ribozymes is much less efficient than cleavage of short oligonucleotide substrates because of higher order structure in the long target RNA. To further study the effects of long target RNA structure on ribozyme cleavage efficiency, we determined the accessibility of seven hammerhead ribozyme cleavage sites in a target RNA that contained human immunodeficiency virus type 1 (HIV-1) vif - vpr . The base pairing-availability of individual nucleotides at each cleavage site was then assessed by chemical modification mapping. The ability of hammerhead ribozymes to cleave the long target RNA was most strongly correlated with the availability of nucleotides near the cleavage site for base pairing with the ribozyme. Moreover, the accessibility of the seven hammerhead ribozyme cleavage sites in the long target RNA varied by up to 400-fold but was directly determined by the availability of cleavage sites for base pairing with the ribozyme. It is therefore unlikely that steric interference affected hammerhead ribozyme cleavage. Chemical modification mapping of cleavage site structure may therefore provide a means to identify efficient hammerhead ribozyme cleavage sites in long target RNAs.     

Specific cleavage of target RNAs from HIV-1 with 5' half tRNA by mammalian tRNA 3' processing endoribonuclease.

Mammalian tRNA 3' processing endoribonuclease (3' tRNase) can be converted to an RNA cutter that recognizes four bases, with about a 65-nt 3'-truncated tRNA(Arg) or tRNA(Ala). The 3'-truncated tRNA recognizes the target RNA via four base pairings between the 5'terminal sequence and a sequence 1-nt upstream of the cleavage site, resulting in a pre-tRNA-like complex (Nashimoto M, 1995, Nucleic Acids Res 23:3642-3647). Here I developed a general method for more specific RNA cleavage using 3' tRNase. In the presence of a 36-nt 5' half tRNA(Arg) truncated after the anticodon, 3' tRNase cleaved the remaining 56-nt 3' half tRNA(Arg) with a 19-nt 3' trailer after the discriminator. This enzyme also cleaved its derivatives with a 5' extra sequence or nucleotide changes or deletions in the T stem-loop and extra loop regions, although the cleavage efficiency decreases as the degree of structural change increases. This suggests that any target RNA can be cleaved site-specifically by 3'tRNase in the presence of a 5' half tRNA modified to form a pre-tRNA-like complex with the target. Using this method, two partial HIV-1 RNA targets were cleaved site-specifically in vitro. These results also indicate that the sequence and structure of the T stem-loop domain are important, but not essential, for the recognition of pre-tRNAs by 3' tRNase.     

Requirements for cleavage by a modified RNase P of a small model substrate.

M1 RNA, the catalytic RNA subunit of RNase P from Escherichia coli, has been covalently linked at its 3' terminus to oligonucleotides (guide sequences) that guide the enzyme to target RNAs through hybridization with the target sequences. These constructs (M1GS RNAs) have been used to determine some minimal features of model substrates. As few as 3 bp on the 3' side of the site of cleavage in a substrate complex and 1 nt on the 5' side are required for cleavage to occur. The cytosines in the 3' terminal CCA sequence of the model substrates are important for cleavage efficiency but not cleavage site selection. A purine (base-paired or not) at the 3' side of the cleavage site is important both for cleavage site selection and efficiency. M1GS RNAs provide both a simple system for characterization of the reaction governed by M1 RNA and a tool for gene therapy.     

The inhibitory efficacy of RNA POL III-expressed long hairpin RNAs targeted to untranslated regions of the HIV-1 5' long terminal repeat

Human immunodeficiency virus type 1 (HIV-1) is a lentivirus that causes persistent infection resulting in the demise of immune regulatory cells, and ensuing diseases associated with acquired immune deficiency syndrome (AIDS). Although current therapeutic modalities have had a significant impact on mortality rates, novel therapies are constantly needed to prevent the emergence of resistant viral variants that escape the effects of antivirals. RNA Interference (RNAi) is a promising therapeutic modality for the inhibition of HIV-1 RNAs. Traditionally, RNAi effector sequences include expressed short hairpin RNAs (shRNAs) or short interfering RNAs (siRNAs). Recently, expressed long hairpin RNAs (lhRNAs) have been used with the aim of generating multiple independent siRNAs, which simultaneously target different susceptible sites on HIV-1. Here, modified lhRNAs expressed from U6 RNA Pol III promoters were targeted to sites within the first transcribed sequences of the HIV-1 5' long terminal repeat (LTR) region. Both Tat-dependent and independent suppressive efficacy was demonstrated against subtype B and C reporter sequences; however, lhRNAs complementary to the TAR stem-loop were refractory to silencing. None of the lhRNAs induced an unwanted interferon response as measured by interferon beta levels. Silencing by the lhRNAs was not equal across the extent of its cognate sequence, with the greatest efficacy observed for sequences located at the base of the stem. Nevertheless, direct antireplicative activity was seen when targeting lhRNAs to a subtype B HIV clone pNL4-3 Luc and a subtype C wild-type HIV-1 strain, FV5. These data highlight distinct target loci within the 5' LTR of HIV-1 that are susceptible to lhRNA targeting, and may prove to have an important advantage over other RNAi target sites within HIV-1. Although lhRNAs themselves require further manipulation to improve their overall efficacy in generating multiple functioning siRNAs, they may prove useful in any combinatorial-based approach to treating HIV-1 infection.     

Short hairpin-looped oligodeoxynucleotides reduce hepatitis C virus replication

ABSTRACT: BACKGROUND: Persistent infection with hepatitis C virus (HCV) is a leading cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Standard therapy consists of a combination of interferon-alpha and ribavirin, but many patients respond poorly, especially those infected with HCV genotypes 1 and 4. Furthermore, standard therapy is associated with severe side-effects. Thus, alternative therapeutic approaches against HCV are needed. FINDINGS: Here, we studied the effect of a new class of antiviral agents against HCV, short, partially double-stranded oligodeoxynucleotides (ODNs), on viral replication. We targeted the 5' nontranslated region (5' NTR) of the HCV genome that has previously been shown as effective target for small interfering RNAs (siRNAs) in vitro. One of the investigated ODNs, ODN 320, significantly and efficiently reduced replication of HCV replicons in a sequence-, time- and dose-dependent manner. ODN 320 targets a genomic region highly conserved among different HCV genotypes and might thus be able to inhibit a broad range of genotypes and subtypes. CONCLUSIONS: ODNs provide an additional approach for inhibition of HCV, might be superior to siRNAs in terms of stability and cellular delivery, and suitable against HCV resistant to standard therapy. This study underlines the potential of partially double-stranded ODNs as antiviral agents.     

Generation of catalytic human Ago4 identifies structural elements important for RNA cleavage

Argonaute proteins bind small RNAs and mediate cleavage of complementary target RNAs. The human Argonaute protein Ago4 is catalytically inactive, although it is highly similar to catalytic Ago2. Here, we have generated Ago2-Ago4 chimeras and analyzed their cleavage activity in vitro. We identify several specific features that inactivate Ago4: the catalytic center, short sequence elements in the N-terminal domain, and an Ago4-specific insertion in the catalytic domain. In addition, we show that Ago2-mediated cleavage of the noncanonical miR-451 precursor can be carried out by any catalytic human Ago protein. Finally, phylogenetic analyses establish evolutionary distances between the Ago proteins. Interestingly, these distances do not fully correlate with the structural changes inactivating them, suggesting functional adaptations of individual human Ago proteins.     

Mechanism of RNA cleavage catalyzed by sequence specific polyamide nucleic acid-neamine conjugate

In earlier studies, we found that a conjugate of neamine-polyamide nucleic acid targeting transactivation response element of HIV-1 RNA genome (HIV-1 TAR) displayed anti-HIV-1 activity and sequence-specific cleavage of the target RNA in vitro. Here we show that both the position of conjugation of polyamide nucleic acid (PNA) on neamine and the length of the spacer are critical parameters for conferring cleavage activity to the conjugate. The conjugation of PNA via a spacer incorporating 11 atoms to the 5-position of ring I of the neamine core conferred sequence-specific RNA cleavage activity on the conjugate, while conjugation to the 4'-position of ring II abolished this activity. Similarly, 5-neamine PNA complementary to TAR sequence of HIV-1 genome (PNA(TAR)) conjugates having either a 23-atom spacer or a bulky dansyl group between PNA and the neamine core also resulted in complete loss of cleavage activity. Based on these observations, we propose a mechanism for the observed RNA cleavage catalyzed by the conjugate involving unprotonated and protonated amino groups at the 3-position of ring I and the 6'-position of ring II of the neamine core, respectively.     

Dimerization of HIV-1 genomic RNA of subtypes A and B: RNA loop structure and magnesium binding.

Retroviruses encapsidate their genome as a dimer of homologous RNA molecules noncovalently linked close to their 5' ends. The dimerization initiation site (DIS) of human immunodeficiency virus type 1 (HIV-1) RNA is a hairpin structure that contains in the loop a 6-nt self-complementary sequence flanked by two 5' and one 3' purines. The self-complementary sequence, as well as the flanking purines, are crucial for dimerization of HIV-1 RNA, which is mediated by formation of a "kissing-loop" complex between the DIS of each monomer. Here, we used chemical modification interference, lead-induced cleavage, and three-dimensional modeling to compare dimerization of subtype A and B HIV-1 RNAs. The DIS loop sequences of these RNAs are AGGUGCACA and AAGCGCGCA, respectively. In both RNAs, ethylation of most but not all phosphate groups in the loop and methylation of the N7 position of the G residues in the self-complementary sequence inhibited dimerization. These results demonstrate that small perturbations of the loop structure are detrimental to dimerization. Conversely, methylation of the N1 position of the first and last As in the loop were neutral or enhanced dimerization, a result consistent with these residues forming a noncanonical sheared base pair. Phosphorothioate interference, lead-induced cleavage, and Brownian-dynamics simulation revealed an unexpected difference in the dimerization mechanism of these RNAs. Unlike subtype B, subtype A requires binding of a divalent cation in the loop to promote RNA dimerization. This difference should be taken into consideration in the design of antidimerization molecules aimed at inhibiting HIV-1 replication.     

Upstream and downstream cis-acting elements for cleavage at the L4 polyadenylation site of adenovirus-2.

A study of the cis-acting elements involved in the 3' end formation of the RNAs from the major late L4 family of adenovirus-2 was undertaken. Series of 5' or 3' end deletion mutants and mutants harboring either internal deletions or substitutions were prepared and assayed for in vitro cleavage. This first allowed the demonstration of a sequence, located at -6 to -29, relative to AAUAAA, whose deletion or substitution reduces cleavage efficiency at the L4 polyadenylation site two to three fold. This upstream efficiency element 5' AUCUUUGUUGUC/AUCUCUGUGCUG 3' is constituted of a partially repeated 12 nucleotide long, UCG rich sequence. The activities of the 2 sequence elements in cleavage are additive. We also searched for regulatory sequences downstream of the L4 polyadenylation site. We found that the deletion or substitution of a 30 nucleotide long UCG rich sequence, between nucleotides +7 and +35 relative to the cleavage site and harboring a UCCUGU repeat reduces cleavage efficiency at least ten fold. A GUUUUU sequence, starting at +35 had no influence. Thus, the usage of the L4 polyadenylation site requires down-stream sequences different from the canonical GU or U boxes and is regulated by upstream sequence elements.     

Lead-catalysed specific cleavage of ribosomal RNAs.

Ribosomes have long been known to require divalent metal ions for their functional integrity. Pb2+-induced cleavage of the sugar-phosphate backbone has now been used to probe for metal binding sites in rRNA. Only three prominent Pb2+cleavages have been detected, with cleavage sites 5' of G240 in 16S rRNA and two sites 5' of A505 and C2347 in 23S rRNA. All cleavages occur in non-paired regions of the secondary structure models of the rRNAs and can be competed for by high concentrations of Mg2+, Mn2+, Ca2+ and Zn2+ ions, suggesting that lead is bound to general metal binding sites. Although Pb2+ cleavage is very efficient, ribosomes with fragmented RNAs are still functional in binding tRNA and in peptidyl transferase activity, indicating that the scissions do not significantly alter ribosomal structure. One of the lead cleavage sites (C2347 in 23S RNA) occurs in the vicinity of a region which is implicated in tRNA binding and peptidyl transferase activity. These results are discussed in the light of a recent model which proposes that peptide bond formation might be a metal-catalysed process.     

Sequence-specific RNase H cleavage of gag mRNA from HIV-1 infected cells by an antisense oligonucleotide in vitro.

We have used a ribonuclease protection assay to investigate RNase H cleavage of HIV-1 mRNA mediated by phosphorothioate antisense oligonucleotides complementary to the gag region of the HIV-1 genome in vitro. Cell lysate experiments in H9 and U937 cells chronically infected with HIV-1 IIIB showed RNase H cleavage of unspliced gag message but no cleavage of spliced message which did not contain the target gag region. RNase H cleavage products were detected at oligonucleotide concentrations as low as 0.01 microM and the RNase H activity was seen to be concentration dependent. Similar experiments with 1-, 3- and 5-mismatch oligonucleotides demonstrated sequence specificity at low concentrations, with cleavage of gag mRNA correlating with the predicted activities of the parent and mismatch oligonucleotides based on their hybridization melting temperatures. Experiments in living cells suggested that RNase H-specific antisense activity was largely determined by the amount of oligonucleotide taken up by the different cell lines studied. RNase H cleavage products were detected in antisense oligonucleotide treated MT-4 cells acutely infected with HIV-1 IIIB, but not in infected H9 cells treated with oligonucleotide under the same conditions. The data presented demonstrate potent and specific RNase H cleavage of HIV-1 mRNA mediated by an antisense oligonucleotide targeted to HIV-1 gag mRNA, and are in agreement with previous reports that the major obstacle to demonstrating antisense activity in living cells remains the lack of penetration of these agents into the desired cellular compartment.