A Novel HIV-1 Genomic RNA Packaging Element and its Role in Interplay between RNAPackaging and Gag-Pol Ribosomal Frameshifting

Retroviruses belong to a diverse family of RNA viruses that utilize the eukaryotic translational machinery for viral protein synthesis during infection of its target cells. In the cytoplasm, the unspliced version of retroviral RNA takes one of two pathways: 1) to serve as mRNA template for Gag protein translation, or 2) to act as genomic RNA that is packaged into virions. Packaging of two genomic RNAs in the retroviral particle involves interaction between a cis-acting packaging signal and GagNC. Various studies have mapped the canonical packaging signal or ¿ of most retroviruses such as HIV-1 to the 5’untraslated region (UTR) containing four hairpin structures. However, most of these studies only focused on small fragments of the 5’UTR and therefore the evidences were not sufficient to consider the ¿ as the sole packaging element. A central goal of this dissertation was to identify additional packaging determinant(s) in the coding region of HIV-1 genome.The ability to orchestrate genomic RNA packaging and gene expression is central to production of infectious and morphologically correct retrovirus containing sufficient amount of genomic (g) RNA required for further recombination and integration. Aside from housing the ¿, 5’UTR forms a complex RNA structure that interacts with various8host and viral proteins to mediate multiple steps of viral life cycle. Although the ¿ packaging signal is often described as a set of static, discrete RNA stem loops, there is considerable evidence that HIV-1 genome and its 5’UTR is capable of adopting multiple conformations that could regulate dimerization, translation and genome packaging.The ribosomal frameshifting step of translation is critical for efficient synthesis of Gag and Gag-pol proteins through shifting of ribosome in -1 nt direction at the ribosomal frameshift signal (RFS). After identification of a Genomic RNA Packaging Element (GRPE) that overlapped with RFS using a dual RNA complementation system, another important goal of this study was to determine the role of GRPE in coordination of translation at the level of frameshifting and RNA packaging which is an enduring question in the field of retroviral biology. The studies presented here reveal a new understanding of retroviral genomic RNA packaging by showing that RNA that was used for translation of Gag-pol by ribosomal frameshifting can escape the nonsense-mediated mRNA decay (NMD) pathway and be selected as genomic RNA for encapsidation into the assembled viruses. These findings provide broad knowledge for optimizing the inhibitors of packaging step and also basic understanding of retroviral replication and host translational control system. It is also important for lentiviral gene therapy system since current lentiviruses lack the GRPE element and all lentiviruses utilize a similar -1 ribosomal slippage mechanism to express the Gag-Pol precursor proteins. The following brief overview of the retroviral replication cycle and the adaptation of retroviruses as vectors for gene transfer will serve as background for these studies and emphasize two post-transcriptional steps of the lifecycle: RNA packaging, and ribosomal frameshifting.

Name thesis:degree_name

Doctor of Philosophy

Level thesis:degree_level

doctoral

Discipline thesis:degree_discipline

Molecular Virology

Grantor dc:publisher

Case Western Reserve University School of Graduate Studies

Year dc:date

2013

Author dc:creator

• Chamanian, Mastooreh

Contributors dc:contributor

• Arts, Eric

• Molecular Biology
• Virology

Statement dc:rights

• unrestricted
• This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws.

Language dc:language

English

Repository record dc:identifier

http://rave.ohiolink.edu/etdc/view?acc_num=case1363902886

OAI identifier oai:identifier

oai:etd.ohiolink.edu:case1363902886