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ISOLATION AND CHARACTERIZATION OF RETARGETED FELINE LEUKEMIA VIRUS ENVELOPE PROTEINS AND ISOLATION OF HOST FACTORS INVOLVE IN ENVELOPE SPECIFIC ENTRY

by
Leonardo Valdivieso-Torres
BS, University of Puerto Rico - Aquadilla, May 2008

Thesis Advisor: Monica Roth, PhD

Friday, November 20, 2015
12:00 PM


Abstract

Retroviral vectors are exceptional vehicles for gene delivery. However, the engineered viral vectors must be highly specific for the appropriate tissue or recognize disease specific markers. The specificity of retroviral vectors is conferred by the envelope (Env) protein present on the surface of virus particles. We have succeeded in altering the virus tissue specificity by engineering a library of Env proteins in which we randomize 11 amino acids of the receptor-binding domain of feline leukemia virus (FeLV) Env and screening for functional Envs. The overall goal of our research is to identify novel retroviral Env: host cell-receptor pairs to facilitate targeted gene delivery.
Studies herein, aims at understanding the biochemistry interaction between host-cell receptor and Env proteins, improve the backbone in which we build our library of Envs and to isolate proteins involved in Env-mediated specific entry. The CP isolate is the best Env yet obtained from our library of Envs, in terms of high transduction efficiencies and capacity of infecting a broad types of human cells. The CP receptor was identified as PAR1 also known as GPR172A. The same receptor is used by the natural occurring porcine endogenous retrovirus (PERV-A). PERV-A receptor utilization extends to HuPAR2 receptor. Thus, studies were carried to investigate if CP receptor utilization can extend also to HuPAR2 and using direct site mutagenesis we mapped the receptor-extracellular loop involve in CP entry. To improve the backbone of our library of Envs, a series of constructs were tested for their ability to increase CP/ neomycin titers. The selected construct was then used in a new Env library and three new isolates were obtained on 143B osteosarcoma cells. Similar to CP, two of the new Env isolates, AII and BV2 were constrained by the presence of a cysteine pair within the randomized region. Thus, studies were conducted using mutagenesis and mass spectrometry to understand the role of these cysteines in viral titer and envelope stability. In addition, major efforts have been conducted to isolate the host-cell receptor used by one of our Env protein L1, which exhibit a narrowed tropism infecting mainly HEK293T and osteosarcoma cells.


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