Date of Award

12-2008

Document Type

Thesis

Degree Name

Master of Science (MS)

Program

Biomedical Sciences

Research Advisor

Lorraine M. Albritton Ph.D.

Committee

Ramareddy V. Guntaka, Ph.D Jonathan A. McCullers, MD Michael A. Whitt, Ph.D

Keywords

Cathepsin, Deaminoacylation, MLV, Retrovirus, Splicing, tRNA

Abstract

The study of retroviruses and their lifecycle has contributed immensely to our knowledge of the world of biology and medicine. The central dogma of the basic flow of genetic information was shattered when the discovery that retroviruses copy their RNA genome into DNA was made. The same enzyme that performs this step, reverse transcriptase (RT), also revolutionized molecular biology when it was used as a tool to generate full length cDNA clones of expressed genes. The impact of retroviruses on the medical field has been extremely exciting as the ideas of using retroviral vectors to deliver genes providing long term expression is becoming a reality in the field of gene therapy.

The following study delves further into many aspects of the retroviral lifecycle including entry, reverse transcription, and alternative splicing. Recent evidence has shown that cathepsin B cleaves the Moloney Murine Leukemia Virus (MoMLV) surface unit (SU) and may be important for the membrane fusion step during viral entry. Here I present evidence that other host cell cathepsins including L, S, and D are able cleave MoMLV SU at specific sites. Many of the cathepsins tested appear to cleave in the very same region as cathepsin B, suggesting they may perform a similar function.

The second phase of this study focuses on the reverse transcription step of the retroviral lifecycle. Similar to other polymerases, retroviral RT requires a primer to initiate transcription. Retroviruses use host cell tRNAs which must first be deaminoacylated before the RT enzyme can begin reverse transcription. This work examines the crystal structure of human immunodeficiency virus type one (HIV-1) RT for a possible hydrolase site that is capable of deaminoacylating its primer tRNA. The hydrolase activity of HIV-1 RT was investigated when a charged tRNA was annealed to a genomic template. Furthermore recombinant HIV-1 virions were investigated for the presence of two cellular proteins histidine triad nucleotide-binding protein and human peptidyl-tRNA hydrolase 2 that may also play a role in deaminoacylation of the primer tRNA.

This study culminates by examining the cis-elements that control alternative splicing in MoMLV. The splice donor (SD) and splice acceptor (SA) sites within MoMLV poorly match the consensus sequence for optimal splicing. Mutagenesis was performed using a MoMLV envelope expression plasmid to alter the nucleotide sequence of the SD and SA to match the consensus sequence. This resulted in an increase in envelope expression and viral infectivity of recombinant MoMLV.

DOI

10.21007/etd.cghs.2008.0305

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