Enhancement of anti-HIV-1 Ribozyme Activities by Rev Binding and Multimerization

<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>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Physiology

Year

2002

Author dc:creator

• Yildiz, Yuksel

Contributors dc:contributor

• John J. Rossi
• Marino De Leon
• George T. Javor
• Donna D. Strong
• Anthony J. Zuccarelli

• Immune System Diseases
• Infectious Disease
• Physiology
• HIV Infections -- genetics; Gene Therapy -- methods; Acquired Immunodeficiency Syndrome -- immunotherapy

Statement 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.

Language dc:language

English

Repository record dc:identifier

https://scholarsrepository.llu.edu/etd/972

OAI identifier oai:identifier

oai:scholarsrepository.llu.edu:etd-2072