To effectively apply hammerhead ribozymes as therapeutic agents it is necessary to co-localize them with the desired target. Human immunodeficiency virus type1 (HIV- 1) infectivity is dependent on env gene expression. HIV-1 Rev protein binds to a higher ordered RNA structure within the env transcript termed the Rev Binding Element (RBE). In anti-HIV gene therapy employing ribozymes to increase the co-localization of anti- HIV ribozymes with target HIV mRNAs, it has been proposed that when the native HIV- 1 RBE is appended to a ribozyme as a decoy molecule, simultaneous binding of Rev monomers to the RBE sequences in both HIV-1 genome and in the ribozyme-RBE fusion molecule and their subsequent multimerization may serve to increase the co-localization of ribozyme with HIV-1 mRNA. In this respect, Yamada et al. (1996) have combined the native HIV-1 RBE sequence with a hairpin ribozyme targeted to the U5 region of HIV-1. Their data have demonstrated a substantial enhancement of antiviral activity in vivo when both RBE and ribozyme were present in comparison to either one alone. But their studies never demonstrated co-localization in vitro. In this study we have tested the concept of Rev mediated co-localization in vitro. First of all, we have detected the most accessible sites for hammerhead ribozymes targeting a region of the HIV-1 env gene encoding gpl20 and gp41 proteins using antisense & RNAseH mapping in cell extracts prepared from the HIV-1 infected CEM cells. We have next designed anti-ercv hammerhead ribozymes against the best sites and fused them with the native HIV-1 RBE sequences. Using Rev binding and gel shift retardation assays we have tested whether or not RNP complexes which include Rev, the RBE and the HIV-1 mRNA are formed as a result of Rev multimerization. Our results here demonstrate simultaneous binding of Rev monomers to the RBE sequences in both HIV-1 genome and in the fusion molecule and their subsequent multimerization can co-localize ribozyme and target RNAs.
","abstract_html":"<p>To effectively apply hammerhead ribozymes as therapeutic agents it is necessary to co-localize them with the desired target. Human immunodeficiency virus type1 (HIV- 1) infectivity is dependent on <em>env</em> gene expression. HIV-1 Rev protein binds to a higher ordered RNA structure within the <em>env</em> transcript termed the Rev Binding Element (RBE). In anti-HIV gene therapy employing ribozymes to increase the co-localization of anti- HIV ribozymes with target HIV mRNAs, it has been proposed that when the native HIV- 1 RBE is appended to a ribozyme as a decoy molecule, simultaneous binding of Rev monomers to the RBE sequences in both HIV-1 genome and in the ribozyme-RBE fusion molecule and their subsequent multimerization may serve to increase the co-localization of ribozyme with HIV-1 mRNA. In this respect, Yamada et al. (1996) have combined the native HIV-1 RBE sequence with a hairpin ribozyme targeted to the U5 region of HIV-1. Their data have demonstrated a substantial enhancement of antiviral activity in vivo when both RBE and ribozyme were present in comparison to either one alone. But their studies never demonstrated co-localization in vitro. In this study we have tested the concept of Rev mediated co-localization in vitro. First of all, we have detected the most accessible sites for hammerhead ribozymes targeting a region of the HIV-1 env gene encoding gpl20 and gp41 proteins using antisense & RNAseH mapping in cell extracts prepared from the HIV-1 infected CEM cells. We have next designed anti-ercv hammerhead ribozymes against the best sites and fused them with the native HIV-1 RBE sequences. Using Rev binding and gel shift retardation assays we have tested whether or not RNP complexes which include Rev, the RBE and the HIV-1 mRNA are formed as a result of Rev multimerization. Our results here demonstrate simultaneous binding of Rev monomers to the RBE sequences in both HIV-1 genome and in the fusion molecule and their subsequent multimerization can co-localize ribozyme and target RNAs.</p>","abstract_has_math":false,"creators":["Yildiz, Yuksel"],"institution":null,"degree_name":"Doctor of Philosophy (PhD)","degree_level":"Dissertation","degree_discipline":"Physiology","degree_department":null,"school":null,"contributors":["John J. Rossi","Marino De Leon","George T. Javor","Donna D. Strong","Anthony J. Zuccarelli"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2002,"date_issued":"2002-03-01T08:00:00Z","date_published":"2002-03-01T08:00:00Z","updated_at":"2026-07-24T02:53:37Z","subjects":["Immune System Diseases","Infectious Disease","Physiology","HIV Infections -- genetics; Gene Therapy -- methods; Acquired Immunodeficiency Syndrome -- immunotherapy"],"languages":["English"],"rights":["This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights."],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://scholarsrepository.llu.edu/etd/972","outbound_label":"Repository record","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["John J. Rossi","Marino De Leon","George T. Javor","Donna D. 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The author retains all other copyrights."]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://scholarsrepository.llu.edu/etd/972"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["To effectively apply hammerhead ribozymes as therapeutic agents it is necessary to co-localize them with the desired target. Human immunodeficiency virus type1 (HIV- 1) infectivity is dependent on env gene expression. HIV-1 Rev protein binds to a higher ordered RNA structure within the env transcript termed the Rev Binding Element (RBE). In anti-HIV gene therapy employing ribozymes to increase the co-localization of anti- HIV ribozymes with target HIV mRNAs, it has been proposed that when the native HIV- 1 RBE is appended to a ribozyme as a decoy molecule, simultaneous binding of Rev monomers to the RBE sequences in both HIV-1 genome and in the ribozyme-RBE fusion molecule and their subsequent multimerization may serve to increase the co-localization of ribozyme with HIV-1 mRNA. In this respect, Yamada et al. (1996) have combined the native HIV-1 RBE sequence with a hairpin ribozyme targeted to the U5 region of HIV-1. Their data have demonstrated a substantial enhancement of antiviral activity in vivo when both RBE and ribozyme were present in comparison to either one alone. But their studies never demonstrated co-localization in vitro. In this study we have tested the concept of Rev mediated co-localization in vitro. First of all, we have detected the most accessible sites for hammerhead ribozymes targeting a region of the HIV-1 env gene encoding gpl20 and gp41 proteins using antisense & RNAseH mapping in cell extracts prepared from the HIV-1 infected CEM cells. We have next designed anti-ercv hammerhead ribozymes against the best sites and fused them with the native HIV-1 RBE sequences. Using Rev binding and gel shift retardation assays we have tested whether or not RNP complexes which include Rev, the RBE and the HIV-1 mRNA are formed as a result of Rev multimerization. Our results here demonstrate simultaneous binding of Rev monomers to the RBE sequences in both HIV-1 genome and in the fusion molecule and their subsequent multimerization can co-localize ribozyme and target RNAs.
"]},{"key":"dc:title","label":"Title","values":["Enhancement of anti-HIV-1 Ribozyme Activities by Rev Binding and Multimerization"]}]}],"canonical_facts":{"dc:contributor":["John J. Rossi","Marino De Leon","George T. Javor","Donna D. Strong","Anthony J. Zuccarelli"],"dc:creator":["Yildiz, Yuksel"],"dc:description.abstract":["To effectively apply hammerhead ribozymes as therapeutic agents it is necessary to co-localize them with the desired target. Human immunodeficiency virus type1 (HIV- 1) infectivity is dependent on env gene expression. HIV-1 Rev protein binds to a higher ordered RNA structure within the env transcript termed the Rev Binding Element (RBE). In anti-HIV gene therapy employing ribozymes to increase the co-localization of anti- HIV ribozymes with target HIV mRNAs, it has been proposed that when the native HIV- 1 RBE is appended to a ribozyme as a decoy molecule, simultaneous binding of Rev monomers to the RBE sequences in both HIV-1 genome and in the ribozyme-RBE fusion molecule and their subsequent multimerization may serve to increase the co-localization of ribozyme with HIV-1 mRNA. In this respect, Yamada et al. (1996) have combined the native HIV-1 RBE sequence with a hairpin ribozyme targeted to the U5 region of HIV-1. Their data have demonstrated a substantial enhancement of antiviral activity in vivo when both RBE and ribozyme were present in comparison to either one alone. But their studies never demonstrated co-localization in vitro. In this study we have tested the concept of Rev mediated co-localization in vitro. First of all, we have detected the most accessible sites for hammerhead ribozymes targeting a region of the HIV-1 env gene encoding gpl20 and gp41 proteins using antisense & RNAseH mapping in cell extracts prepared from the HIV-1 infected CEM cells. We have next designed anti-ercv hammerhead ribozymes against the best sites and fused them with the native HIV-1 RBE sequences. Using Rev binding and gel shift retardation assays we have tested whether or not RNP complexes which include Rev, the RBE and the HIV-1 mRNA are formed as a result of Rev multimerization. Our results here demonstrate simultaneous binding of Rev monomers to the RBE sequences in both HIV-1 genome and in the fusion molecule and their subsequent multimerization can co-localize ribozyme and target RNAs.
"],"dc:identifier":["https://scholarsrepository.llu.edu/etd/972"],"dc:language":["English"],"dc:rights":["This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights."],"dc:subject":["Immune System Diseases","Infectious Disease","Physiology","HIV Infections -- genetics; Gene Therapy -- methods; Acquired Immunodeficiency Syndrome -- immunotherapy"],"dc:title":["Enhancement of anti-HIV-1 Ribozyme Activities by Rev Binding and Multimerization"],"thesis:degree_discipline":["Physiology"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Doctor of Philosophy (PhD)"]},"updated_at":"2026-07-24T02:53:37Z"}