Cellular RNase Research Articles

Relationship between Retroviral Replication and RNA Interference Machineries

Small interfering RNAs (siRNAs) associated with gene silencing are cellular defense mechanisms against invading viruses. The viruses fight back by suppressors or escape mechanisms. The retroviruses developed a unique escape mechanism by disguising as DNA proviruses. An evolutionary relationship between the siRNA machinery and the replication machinery of retroviruses is likely. The RNA cleavage enzymes PIWI and RNase H proteins are structurally related. This relationship can be extended from structure to function, since the retroviral reverse transcriptase (RT)/RNase H can also cause silencing of viral RNA by siRNA. Thus, both enzymes can cleave RNA-DNA hybrids and double-stranded RNA (dsRNA) with various efficiencies shown previously and here, demonstrating that their specificities are not absolute. Other similarities may exist, for example between PAZ and the RT and between RNA-binding proteins and the viral nucleocapsid protein. Dicer has some similarities with the viral integrase, since both specifically generate dinucleotide 3'-overhanging ends. We described previously the destruction of the human immunodeficiency virus (HIV) RNA by a DNA oligonucleotide ODN (oligodeoxynucleotide). Variants of the ODN indicated high length and sequence specificities, which is reminiscent of siRNA and designated here as "siDNA." Cleavage of the viral RNA in the presence of the ODN is caused by the retroviral RT/RNase H and cellular RNase H activities. Several siRNA-mediated antiviral defense mechanisms resemble the interferon system.

ICP0 Prevents RNase L-Independent rRNA Cleavage in Herpes Simplex Virus Type 1-Infected Cells

The classical interferon (IFN)-dependent antiviral response to viral infection involves the regulation of IFN-stimulated genes (ISGs), one being the gene encoding cellular endoribonuclease RNase L, which arrests protein synthesis and induces apoptosis by nonspecifically cleaving rRNA. Recently, the herpes simplex virus type 1 (HSV-1) protein ICP0 has been shown to block the induction of ISGs by subverting the IFN pathway upstream of the 2'-5'-oligoadenylate synthetase (OAS)/RNase L pathway. We report that ICP0 also prevents rRNA degradation at late stages of HSV-1 infection, independent of its E3 ubiquitin ligase activity, and that the resultant rRNA degradation is independent of the classical RNase L antiviral pathway. Moreover, the degradation is independent of the viral RNase vhs and is independent of IFN response factor 3. These studies indicate the existence of another, previously unidentified, RNase that is part of the host antiviral response to viral infection.

Silencing of HIV RNA by a Hairpin-loop DNA

We describe an RNA silencing, which inhibits HIV replication by a hairpin-loop DNA. A partially double-stranded 54mer DNA oligonucleotide (ODN) was targeted to the polypurine tract, PPT of HIV. It inhibits virus replication. We demonstrate that it prevents steps before DNA provirus formation. The effect of the ODN on HIV replication in cell culture is highly sequence-specific and sensitive to changes in length and single mismatches on either strand of the DNA. An ODN against HIV-IIIB was ineffective against HIV-Ba-L and vice versa, whereby their PPT's differ by two of 24 nucleotides. Thus, the structure and sequence of both strands of the ODN are important in cellular assays. In vitro the ODN leads to an RNA-DNA hybrid formation at the PPT, a structure which is cleaved by the RT/ RNase H in permeabilized virus particles. The hybrid at the PPT is preferentially recognized by the RT/RNase H for initiation of the second-strand DNA synthesis. This recognition is its normal biological function and shown here with the ODN. A cell extract containing cellular RNase H activities or RISC proteins, is unable to induce such a cleavage. In summary, the ODN mimicks a real step in viral replication, whereby the viral RNA is cleaved prematurely before DNA transcription is completed. The mechanism is reminiscent of RNA silencing by siRNA and supported by its relationship with RNaseH. The ODN may be a basis for drug design, because of low tendency for escape mutations. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005

Functional Characterization of XendoU, the Endoribonuclease Involved in Small Nucleolar RNA Biosynthesis

XendoU is the endoribonuclease involved in the biosynthesis of a specific subclass of Xenopus laevis intron-encoded small nucleolar RNAs. XendoU has no homology to any known cellular RNase, although it has sequence similarity with proteins tentatively annotated as serine proteases. It has been recently shown that XendoU represents the cellular counterpart of a nidovirus replicative endoribonuclease (NendoU), which plays a critical role in viral replication and transcription. In this paper, we combined prediction and experimental data to define the amino acid residues directly involved in XendoU catalysis. Specifically, we find that XendoU residues Glu-161, Glu-167, His-162, His-178, and Lys-224 are essential for RNA cleavage, which occurs in the presence of manganese ions. Furthermore, we identified the RNA sequence required for XendoU binding and showed that the formation of XendoU-RNA complex is Mn2+-independent.

Antisense RNA regulation by stable complex formation in the Enterococcus faecalis plasmid pAD1 par addiction system.

The par stability determinant, encoded by the Enterococcus faecalis plasmid pAD1, is the only antisense RNA regulated postsegregational killing system identified in gram-positive bacteria. Because of the unique organization of the par locus, the par antisense RNA, RNA II, binds to its target, RNA I, at relatively small, interspersed regions of complementarity. The results of this study suggest that, rather than targeting the antisense bound message for rapid degradation, as occurs in most other antisense RNA regulated systems, RNA I and RNA II form a relatively stable, presumably translationally inactive complex. The stability of the RNA I-RNA II complex would allow RNA I to persist in an untranslated state unless or until the encoding plasmid was lost. After plasmid loss, RNA II would be removed from the complex, allowing translational activation of RNA I. The mechanism of RNA I activation in vivo is unknown, but in vitro dissociation experiments suggest that active removal of RNA II, for example by a cellular RNase, may be required.

An RNA external guide sequence ribozyme targeting human interleukin-4 receptor α mRNA

RNA oligonucleotides termed External Guide Sequence (EGS) and RNAi have been described that target specific gene expression by site-specific cleavage of mRNA. EGS serve as an RNA catalyst or ribozyme by directing bound mRNA to the ubiquitous cellular enzyme RNAse P. We describe an EGS targeting human interleukin (IL)-4 receptor α mRNA, an important cytokine receptor in the pathogenesis of asthma and allergic disease expressed in pulmonary tissues. This EGS was designed to explore pulmonary delivery of catalytic RNA oligonucleotides as a novel therapy in asthma and other atopic diseases. Inhaled DNA oligonucleotides termed Respirable Antisense OligoNucleotide Sequences (RASONS) are selectively internalized in lung tissues in a complex with endogenous lipid surfactants present in normal lung and can alter pulmonary gene expression. Potential applications of inhaled RNA oligonucleotides in therapy of pulmonary and related systemic diseases are discussed.

Selective inhibition of HIV-1 reverse transcriptase (HIV-1 RT) RNase H by small RNA hairpins and dumbbells.

We present here the design of a novel class of RNA inhibitors of the RNase H domain of HIV-1 RT, a ribonuclease activity that is essential for viral replication in vivo. Specifically, we show that small RNA hairpins and dumbbells can selectively inhibit the RNase H activity of HIV-1 RT without affecting other cellular RNases H (e.g., E. coli and human RNase H). These results suggest that the inhibitors do not interact with the nucleic acid binding site of RT RNase H, as this region should be well conserved among the various enzymes. The most potent inhibitors displayed IC50 values in the 3-8 microM range. Remarkably, the DNA polymerase activity, an intrinsic property of HIV RT, was not inhibited by the hairpin and dumbbell aptamers, a property not previously observed for any nucleic acid aptamer directed against RT RNase H. The results described here suggest a noncompetitive binding mechanism, as outlined in the differential inhibitory characteristics of each of the nucleic acid aptamers against the bacterial, human, and viral RNase H homologues.

Interfering with hepatitis C virus RNA replication

The emergence of RNA interference (RNAi) as a powerful tool for silencing gene expression has spurred considerable interest in its experimental and therapeutic potential. RNAi is a cellular process of gene silencing in which small duplexes of RNA specifically target a homologous sequence for cleavage by cellular ribonucleases. The introduction of 21–23 nucleotide RNA duplexes, termed small interfering RNAs (siRNAs), into mammalian cells can specifically degrade homologous mRNAs. RNAi efficiently silences the expression of both cellular and viral RNAs. A number of groups have demonstrated that siRNAs interfere with hepatitis C virus (HCV) gene expression and replication. Additionally, cellular genes are efficiently silenced in the presence of replicating HCV. These studies lay the foundation for using RNAi as an experimental tool for studying HCV replication and defining host genes that are significant for viral replication. The potential for RNAi as an antiviral therapy remains less clear, as it will face many of the challenges that have hindered nucleic acid therapies in the past.

SiRNA-mediated inhibition of HBV replication and expression.

RNA interference (RNAi) is a newly discovered phenomenon provoked by dsRNA. The dsRNA is initially cleaved by Dicer into 21-23 nt small interfering RNA (siRNA) and can then specifically target homologous mRNA for degradation by cellular ribonucleases. RNAi has been successfully utilized to down-regulate the endogenous gene expression or suppress the replication of various pathogens in mammalian cells. In this study, we investigated whether vector-based siRNA promoted by U6 (pSilencer1.0-U6) could efficiently inhibit HBV replication in cell culture. pSilencer vectors with inserts targeting on different regions of HBV genome were constructed. These plasmids were co-transfected with pHBV3.8 into Huh-7 cells via lipofection and viral antigens were measured by ELISA. Viral RNA was analyzed by Northern blot. The mRNA of MxA and 2'-5'OAS was reverse transcribed and quantified by real-time PCR. Vector-based siRNA could potently reduce hepatitis B virus antigen expression in transient replicative cell culture. Furthermore, Northern blot analysis showed that viral RNA was effectively degraded, thus eliminating the messengers for protein expression as well as template for reverse transcription. Real-time PCR analysis of cellular MxA and 2'-5'OAS gene expression revealed that vector-based siRNA did not provoke the interferon pathway which reassured the specificity of the vector-based RNA interference technique. Our results indicate that RNA interference may be a potential tool to control HBV infection.

HIV-1 encoded candidate micro-RNAs and their cellular targets

MicroRNAs (miRNAs) are small RNAs of 21–25 nucleotides that specifically regulate cellular gene expression at the post-transcriptional level. miRNAs are derived from the maturation by cellular RNases III of imperfect stem loop structures of ~ 70 nucleotides. Evidence for hundreds of miRNAs and their corresponding targets has been reported in the literature for plants, insects, invertebrate animals, and mammals. While not all of these miRNA/target pairs have been functionally verified, some clearly serve roles in regulating normal development and physiology. Recently, it has been queried whether the genome of human viruses like their cellular counterpart also encode miRNA. To date, there has been only one report pertaining to this question. The Epstein-Barr virus (EBV) has been shown to encode five miRNAs. Here, we extend the analysis of miRNA-encoding potential to the human immunodeficiency virus (HIV). Using computer-directed analyses, we found that HIV putatively encodes five candidate pre-miRNAs. We then matched deduced mature miRNA sequences from these 5 pre-miRNAs against a database of 3' untranslated sequences (UTR) from the human genome. These searches revealed a large number of cellular transcripts that could potentially be targeted by these viral miRNA (vmiRNA) sequences. We propose that HIV has evolved to use vmiRNAs as a means to regulate cellular milieu for its benefit.

Potent inhibition of respiratory syncytial virus by combination treatment with 2–5A antisense and ribavirin

Respiratory syncytial virus (RSV) is a major cause of lower respiratory diseases in infants, young children, and the elderly. Ribavirin, the only currently approved drug for the treatment of RSV infections in the U.S., requires high doses to be effective. Therefore, it has only a limited clinical efficacy in the treatment of RSV infections. It has been shown that a cellular ribonuclease, RNase L, can be recruited by 2′–5′ linked tetra-adenylates (2–5A) attached to an antisense sequence complementary to the RSV genome to specifically cleave RSV genomic RNA. Here we confirm the antiviral activity of the lead 2–5A antisense compound, RBI034, by using several different viral assays. We demonstrate that RBI034 is more efficient than antisense lacking 2–5A or small interfering dsRNA (siRNA) in inhibiting RSV replication. Although the best antiviral activity of RBI034 was observed with co-treatment of RSV infection, it remained effective even when administrated 24 h after the initiation of infection. Interestingly, the activity of RBI034 can be further enhanced by a combination treatment with ribavirin. At suboptimal concentrations, neither ribavirin nor RBI034 was effective in suppressing RSV replication. However, a combination of these two drugs at the same suboptimal concentrations showed a potent inhibitory activity. The potent reduction of RSV replication by combination treatment was also confirmed in primary human airway epithelial cells. Therefore, a combination therapy of the 2–5A antisense compound RBI034 and ribavirin might be a more effective therapeutic approach for treating RSV infections than ribavirin alone.

Gene Silencing Mediated by Small Interfering RNAs in Mammalian Cells

RNA interference (RNAi) as a protecting mechanism against invasion by foreign genes was first described in C. elegans and has subsequently been demonstrated in diverse eukaryotes such as insects, plants, fungi and vertebrates. RNAi is the mechanism of sequence-specific, post-transcriptional gene silencing initiated by double-stranded RNAs (dsRNA) homologous to the gene being suppressed. dsRNAs are processed by Dicer, a cellular ribonuclease III, to generate duplexes of about 21 nt with 3'-overhangs (small interfering RNA, siRNA) which mediate sequence-specific mRNA degradation. In mammalian cells siRNA molecules are capable of specifically silencing gene expression without induction of the unspecific interferon response pathway. Thus, siRNAs have become a new and powerful alternative to other genetic tools such as antisense oligonucleotides and ribozymes to analyze loss-of-function phenotypes. Application of siRNA duplexes to interfere with the expression of a specific gene requires knowledge of target accessibility, highly effective delivery of siRNAs into target cells and for some applications long-term siRNA expression. Effective strategies to deliver siRNAs to target cells in cell culture include transduction by physical or chemical transfection. An alternative strategy uses the endogenous expression of siRNAs by various Pol III promoter expression cassettes that allow transcription of functional siRNAs or their precursors. This review summarizes some genetic and biochemical aspects of RNAi, the delivery and application of siRNAs to target cells, the kinetics of RNAi and the utility of siRNAs as analytical and potential therapeutic tools.

Clearance of replicating hepatitis C virus replicon RNAs in cell culture by small interfering RNAs.

RNA interference is a cellular process of gene silencing in which small duplexes of RNA specifically target a homologous sequence for cleavage by cellular ribonucleases. The introduction of approximately 22-nt small interfering RNAs (siRNAs) into mammalian cells can specifically silence cellular mRNAs without induction of the nonspecific IFN responses that are activated by longer RNA duplexes. We investigate in this article whether siRNAs can also silence the expression of the cytoplasmically replicating hepatitis C virus (HCV) RNAs by using a replicon system that supports robust HCV replication, but not the production of infectious virions. We report the efficient silencing of both cellular lamin AC and HCV RNAs in Huh-7 hepatoma cell lines supporting HCV replication. Silencing of HCV RNAs was dose dependent and specific, inasmuch as two HCV variants that differ by 3 nt within the target sequence were only silenced by the exact homologous sequence for each. siRNAs designed to target HCV RNA triggered an exponential decrease in HCV RNA, resulting in an 80-fold decrease in HCV RNA after 4 days. The introduction of siRNAs into cells with established HCV replication cured >98% of these cells of detectable HCV antigen and replication-competent HCV RNAs. These data support the principle of siRNA-based HCV antiviral therapy.

Critical Features of a Conserved RNA Stem-loop Important for Feedback Regulation of RNase E Synthesis

RNase E is an important regulatory enzyme that governs the principal pathway for mRNA degradation in Escherichia coli. This endonuclease controls its own synthesis via a feedback mechanism in which the longevity of rne (RNase E) mRNA is modulated by a cis-acting sensory element that responds to changes in cellular RNase E activity. Previous research has shown that this element is an RNA stem-loop (hp2) within the 5'-untranslated region of the rne transcript. Here we report studies involving mutational analysis and phylogenetic comparison that have identified the features of rne hp2 important for its function. These comprise an internal loop flanked on one side by a 2-bp stem and a hairpin loop and on the other side by a longer stem whose sequence is inconsequential. A search of bacterial genome sequences suggests that regulation by an hp2-like element may be a unique evolutionary adaptation of the rne transcript that is not shared by other mRNAs.

Consequences of RNase E scarcity in Escherichia coli.

The endoribonuclease RNase E plays an important role in RNA processing and degradation in Escherichia coli. The construction of an E. coli strain in which the cellular concentration of RNase E can be precisely controlled has made it possible to examine and quantify the effect of RNase E scarcity on RNA decay, gene regulation and cell growth. These studies show that RNase E participates in a step in the degradation of its RNA substrates that is partially or fully rate-determining. Our data also indicate that E. coli growth requires a cellular RNase E concentration at least 10-20% of normal and that the feedback mechanism that limits overproduction of RNase E is also able to increase its synthesis when its concentration drops below normal. The magnitude of the in-crease in RNA longevity under conditions of RNase E scarcity may be limited by an alternative pathway for RNA degradation. Additional experiments show that RNase E is a stable protein in E. coli. No other E. coli gene product, when either mutated or cloned on a multicopy plasmid, seems to be capable of compensating for an inadequate supply of this essential protein.

Antigen Presenting Cells Transfected with LMP2a RNA Induce CD4 + LMP2a-specific Cytotoxic T Lymphocytes which Kill via a Fas-independent Mechanism

Recent reports have demonstrated that EBV can be used as a target of specific CTL-based treatments in severe chronic EBV, immunoblastic B cell lymphoma and Hodgkin's disease (HD). Based upon the promising results form these in vivo studies, it has been suggested that an antigen-specific CTL-based immunotherapy may be of benefit in treating EBV-associated tumors such as HD and nasopharyngeal carcinoma (NPC) which express the potentially immunogenic antigens, LMP1 and LMP2a. Recent work form our group has demonstrated that LMP2a-specific CTLs may be generated in vitro using autologous antigen presenting cells which have been transfected with polyadenylated LMP2a RNA in the presence of a cationic lipid. In this study, we demonstrate that the presence of the lipid enhances dendritic cell (DC) transfection efficiency and appears to protect the intracellular LMP2a RNA from degradation by cellular RNAses. Significantly, these improvements resulted in the transfected DCs having a superior ability to stimulate autologous T cell proliferation. These LMP2a+DCs were used to stimulate LMP2a-specific effector cells which were predominantly a mixture of cytotoxic and helper CD4 + T cells. The molecular mechanisms whereby these CD4 + T cells lyzed their LMP2a-expressing targets was investigated and we show that, although expressing Fas ligand on their surface, LMP2a-specific CD4 + effector cells kill their targets using the Ca 2+ -dependent perforin/granzyme pathway which is the same mechanism used by CD8 + CTLs.

The 2–5A/RNase L/RNase L Inhibitor (RNI) Pathway Regulates Mitochondrial mRNAs Stability in Interferon α-treated H9 Cells

Interferon alpha (IFNalpha) belongs to a cytokine family that exhibits antiviral properties, immuno-modulating effects, and antiproliferative activity on normal and neoplasic cells in vitro and in vivo. IFNalpha exerts antitumor action by inducing direct cytotoxicity against tumor cells. This toxicity is at least partly due to induction of apoptosis. Although the molecular basis of the inhibition of cell growth by IFNalpha is only partially understood, there is a direct correlation between the sensitivity of cells to the antiproliferative action of IFNalpha and the down-regulation of their mitochondrial mRNAs. Here, we studied the role of the 2-5A/RNase L system and its inhibitor RLI in this regulation of the mitochondrial mRNAs by IFNalpha. We found that a fraction of cellular RNase L and RLI is localized in the mitochondria. Thus, we down-regulated RNase L activity in human H9 cells by stably transfecting (i) RNase L antisense cDNA or (ii) RLI sense cDNA constructions. In contrast to control cells, no post-transcriptional down-regulation of mitochondrial mRNAs and no cell growth inhibition were observed after IFNalpha treatment in these transfectants. These results demonstrate that IFNalpha exerts its antiproliferative effect on H9 cells at least in part via the degradation of mitochondrial mRNAs by RNase L.

DNA aptamers selected against the HIV-1 RNase H display in vitro antiviral activity.

The DNA polymerase of the human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) is a target widely used to inhibit HIV-1 replication. In contrast, very few inhibitors of the RNase H activity associated with RT have been described, despite the crucial role played by this activity in viral proliferation. DNA ligands with a high affinity for the RNase H domain of HIV-1 RT were isolated by systematic evolution of ligands by an exponential enrichment strategy (SELEX), using recombinant RTs with or without the RNase H domain. The selected oligonucleotides (ODNs) were able to inhibit in vitro the HIV-1 RNase H activity, while no effect was observed on cellular RNase H. We focused our interest on two G-rich inhibitory oligonucleotides. Model studies of the secondary structure of these ODNs strongly suggested that they were able to form G-quartets. In addition to the inhibition of HIV-1 RNase H observed in a cell free system, these ODNs were able to strongly diminish the infectivity of HIV-1 in human infected cells. Oligonucleotides described here may serve as leading compounds for the development of specific inhibitors of this key retroviral enzyme activity.

Drosophila RNase H1 is essential for development but not for proliferation.

Ribonucleases H (RNases H) recognize and specifically degrade RNA that is bound to complementary DNA and are thought to be involved in DNA replication and transcriptional regulation. Though it was previously shown that bacterial RNases H participate in DNA synthesis, none of the known mutations in RNase H genes in either prokaryotes or lower eukaryotes is lethal. Here, we report the characterization of the first loss-of-function mutation in an RNase H1 gene in a metazoan organism, Drosophila melanogaster. Genetic studies of this mutant showed that this gene is essential for metamorphosis in Drosophila. However, disruption of the RNase H1 gene does not affect proliferation, but probably alters the regulation of gene expression. The lethal phenotype of this mutant also demonstrates that RNase H1 activity in Drosophila cannot be provided by other cellular RNase H activities. Analysis of the developmental and spatial expression profiles of a reporter gene placed under the control of the RNase H1 promoter revealed increased expression in several larval tissues. In salivary glands this increase was shown to be inducible by treatment with ecdysone.

Caspase-dependent apoptosis by 2',5'-oligoadenylate activation of RNase L is enhanced by IFN-beta.

The 2',5'-oligoadenylate (2-5A) system is an interferon (IFN)-regulated RNA decay pathway that provides innate immunity against viral infections. The biologic action of the 2-5A system is mediated by RNase L, an endoribonuclease that becomes enzymatically active after binding to 2-5A. RNase L is also implicated in mediating apoptosis in response to both viral and nonviral inducers. To study the cellular effects of RNase L activation directly, 2-5A was transfected into the human ovarian cancer cell line, Hey1B. Activation of RNase L by 2-5A resulted in specific 18S rRNA cleavage and induction of apoptosis, as measured by TUNEL and annexin V binding assays. In contrast, the dimeric form of 2-5A, ppA2'p5'A, neither activated RNase L nor caused apoptosis. Treatment with IFN-beta prior to 2-5A transfection enhanced cellular RNase L levels (< or = 2.2-fold) and increased the proportion of cells undergoing apoptosis (by < or =40%). However, rRNA cleavages after 2-5A transfections were not enhanced by IFN-beta pretreatments, indicating that basal levels of RNase L were sufficient for this activity. Apoptosis in response to RNase L activation was accompanied by cytochrome c release from mitochondria. Induction of apoptosis by either 2-5A alone or by the combination of 2-5A and IFN-beta was effectively blocked with either the pancaspase inhibitor, Z-VAD-fmk, or with the caspase 3 inhibitor, DEVD-fmk. Therefore, activation of RNase L by 2-5A leads to cytochrome c release into the cytoplasm and then to caspase activation and apoptosis. These results suggest potential uses for 2-5A in augmenting the anticancer activities of IFN.