Yuksel Yildiz

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.

LLU Discipline

Physiology

Department

Physiology

School

Graduate School

First Advisor

John J. Rossi

Second Advisor

Marino De Leon

Third Advisor

George T. Javor

Fourth Advisor

Donna D. Strong

Fifth Advisor

Anthony J. Zuccarelli

Degree Name

Doctor of Philosophy (PhD)

Degree Level

Ph.D.

Year Degree Awarded

2002

Date (Title Page)

3-2002

Language

English

Library of Congress/MESH Subject Headings

HIV Infections -- genetics; Gene Therapy -- methods; Acquired Immunodeficiency Syndrome -- immunotherapy

Type

Dissertation

Page Count

xii; 119

Digital Format

PDF

Digital Publisher

Loma Linda University Libraries

Usage 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.

Collection

Loma Linda University Electronic Theses and Dissertations

Collection Website

http://scholarsrepository.llu.edu/etd/

Repository

Loma Linda University. Del E. Webb Memorial Library. University Archives

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