We have identified an RNA catalytic domain within the sequence of the 359 base long negative-strand satellite RNA of tobacco ringspot virus. The catalytic domain contains two minimal sequences of satellite RNA, a 50-base catalytic RNA sequence, and a 14-base substrate RNA sequence. The catalytic complex of catalytic RNA/substrate RNA represents a structure not previously found in any RNA catalytic reaction described to date. The reaction is truly catalytic since the catalytic RNA has multiple substrate cleavage events and is not consumed during the course of the reaction. A linear relationship is seen between reaction rate and catalytic RNA concentration. The reaction has a Km of 0.03 microM, a kcat of 2.1/min, a temperature optimum of near 37 degrees C, and an energy of activation of 19 kcal/mol.

We have identified the catalytic domain within the sequence of the negative strand of the satellite RNA of tobacco ringspot virus. Minimum energy RNA folding calculations predict a two dimensional model with four major helical regions which are supported by mutagenesis experiments. This model for the catalytic complex consists of a 50 base catalytic RNA and a 14 base substrate RNA folded together in a type of hairpin two dimensional structure. Part of the recognition region between the catalyst and substrate is two helices of 6 bases and 4 bases respectively. Catalytic activity remains when the bases in these two helices are changed but base pairing is maintained. Thus an appropriately engineered 'hairpin' catalyst is capable of cleaving heterologous RNA.

Extensive in vitro mutagenesis studies have been performed on the hairpin ribozyme and substrate in an effort to refine the overall secondary structure of the molecule and provide further insight into what elements are essential for activity. A secondary structure consisting of four helices and five loop regions remains the basic model as originally proposed. Two helices, helix 1 and 2, form between the substrate and ribozyme while helices 3 and 4 are within the ribozyme itself. Our results suggest that helices 3 and 4 are smaller than previously proposed, consisting of four base pairs and three base pairs respectively. Helix 4 can be extended without loss of activity and loop 3 at the closed end of the hairpin model can be varied in sequence with retention of activity. There is an unpaired nucleotide between helices 2 and 3 consisting of a single A base, suggesting the opportunity for flexibility within the tertiary structure at this point. Comparisons are made between the new data and previously published mutagenesis and phylogenetic data. Substrate targeting rules require base pairing between helices 1 and 2 with cleavage (*) occurring in a preferred 5'(g/c/u)n*guc3' sequence of the substrate.

CorrectionsMICROBIOLOGY. For the article ''Identification of eIF2B␥ and eIF2␥ as cofactors of hepatitis C virus internal ribosome entry site-mediated translation using a functional genomics approach''

HBV, a partially double-stranded DNA virus, replicates modifications were performed on these two Rzs, with the through a pregenomic RNA (pgRNA) intermediate, which goal of increasing catalytic efficiency both in vitro and in provides a therapeutic opportunity for a novel antiviral gene cells. To determine the Rz activities in liver cells, the therapy based on ribozyme RNA cleavage. Three hairpin cDNAs for each of the anti-HBV Rzs (and their catalytically ribozymes (Rzs) were designed which have the potential disabled negative controls) were cloned into retroviral vecto disrupt HBV replication by targeting the pgRNA as well tors. Unmodified ribozymes co-expressed with HBV in as specific mRNAs encoding the HBV surface antigen human liver Huh7 cells reduced the level of viral particle (HBsAg), the polymerase and the X protein. The ability of production by up to 66% based on the endogenous polyeach ribozyme to cleave approximately 0.3 kb HBV merase assay, while the structurally modified ribozymes subgenomic RNA fragments was tested in vitro. Two of the inhibited HBV production up to 83%. These encouraging three Rzs tested (BR1 and BR3) were capable of cleaving results indicate the feasibility of ribozyme-mediated gene their respective RNA substrates, while their catalytically therapy for the treatment of HBV infections. disabled mutated counterpart Rzs were not. Structural Keywords: plasmid-independent transcription; endogenous polymerase assay; ribozyme kinetics; RNA cleavage; antiviral problems associated with the direct injection of antisense Introduction DNA. Stable expression of the anti-HBV ribozymes in Hepatitis B is a major worldwide health problem.liver cells could theoretically lead to lifelong intracellular Although a vaccine is currently available for hepatitis B immunity against HBV. virus (HBV), it is estimated that no less than two billion HBV undergoes replication through reverse transcrippeople are infected globally and 300 million are chrontion of a pregenomic RNA intermediate 5,6 and is thus ically infected carriers. 1,2 Approximately 10% of infected somewhat analogous to HIV and other retroviruses. This adults will develop a chronic, life-long infection often offers an excellent rationale for the use of ribozymes as with devastating consequences. 1,2 About 2 million deaths antiviral agents for HBV based on the successful preclinioccur each year as a direct result of chronic HBV infection cal studies of ribozymes against HIV infections (for due to either cirrhosis or primary liver cancer. Presently review see Refs 7 and 8). An anti-HBV ribozyme can the only partially effective treatment for hepatitis B is potentially be multifunctional, targeting both the pregeninterferon-␣, which is effective in only less than half of omic RNA as well as the viral mRNAs (see Figure 1a). all patients.In addition, by targeting the ribozymes to small highly One recently investigated approach to the treatment of conserved regions of the viral genome, the possibility of HBV infection has been the utilization of antise...

CCR-5 is a major cellular coreceptor for R5 strains of HIV-1. Individuals carrying a homozygous 32-base-pair deletion in this gene are apparently healthy and are relatively resistant to HIV-1 infection. Since CCR5 appears to be dispensable for the host, but important for initial HIV-1 infection, CCR5 mRNA is an excellent therapeutic target for inhibiting HIV-1 replication via ribozyme knockout. We report here that hairpin ribozymes are able to reduce cellular CCR5 mRNA and cell surface CCR5 when stably introduced into PM1 cells by transduction with recombinant adenoassociated viral vector. The ribozymes effectively protect the cells from infection by R5 HIV-1 strains or non-syncytium-inducing clinical isolates commensurate with a reduction in CCR5 mRNA. These results suggest a novel gene therapy approach to preventing or slowing the disease progression of HIV-1 infection.

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The gene for phosphatidylinositol-4-phosphate adaptor-2 (FAPP2) encodes a cytoplasmic lipid transferase with a plekstrin homology domain that has been implicated in vesicle maturation and transport from trans-Golgi to the plasma membrane. The introduction of ribozymes targeting the FAPP2 gene in colon carcinoma cells induced their apoptosis in the presence of Fas agonistic antibody. Furthermore, by quantitative PCR we showed that a siRNA specific to FAPP2, but not a randomized siRNA control, reduced FAPP2 gene expression in tumor cells. Transfection of FAPP2 siRNA into human tumor cells then incubated with FasL resulted in reduction of viable cell numbers. Also, FAPP2 siRNA transfected glioma and breast tumor cells showed significant increases in apoptosis upon incubation with soluble FasL, but the apoptosis did not necessarily correlate with increased Fas expression. These data demonstrate a previously unknown role for FAPP2 in conferring resistance to apoptosis and indicate that FAPP2 may be a target for cancer therapy.