Date of Award

5-2013

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Microbial Pathogenesis, Immunology, and Inflammation

Research Advisor

Lorraine M. Albritton, Ph.D.

Committee

Jonathan McCullers, M.D. Tiffany N. Seagroves, Ph.D. Stacey Schultz-Cherry, Ph.D. Michael A. Whitt, Ph.D.

Abstract

Attributes of both the viral glycoprotein and its cellular receptor play key roles in determining the outcome of infection. This body of work endeavors to illustrate how these two components influence the efficiency of virus infection in in vitro and in vivo systems with an emphasis on characterizing the transduction capacity of a novel entry-targeting glycoprotein. In previous work, the Moloney murine leukemia virus (MLV) envelope glycoprotein (Env) was modified to generate the Sst-RBS glycoprotein. This glycoprotein was created by replacing the wild type (WT) receptor binding site (RBS), located on the surface subunit (SU) of the Env, with the somatostatin peptide hormone sequence SST-14. The modifications resulted in abrogating transduction via the natural MLV receptor and redirecting transduction to a family of five somatostatin receptors (SSTR). I demonstrate that structural characteristics of the Sst-RBS glycoprotein and the intracellular fate of the SSTR receptor influence the infection efficiency of pseudotyped MLV and human immunodeficiency virus type 1 (HIV-1) based lentiviral (LV) particles. Infection and western blot assays indicate that Sst-RBS retains the structural requirements for mediating levels of transduction that are comparable to WT and approach within 5-fold that of transduction mediated by vesicular stomatitis virus (VSV) G protein when each envelope protein is pseudotyped on MLV particles. To address the contribution of receptor characteristics on infection efficiency, HEK 293 cell lines stably expressing comparable cell surface levels of SSTR-2, SSTR-3 and SSTR-5; which have natural differences in intracellular trafficking; were generated. Infection assays revealed that distinctive SSTR subtype-specific destinations correlated with observable differences in the level of Sst-RBS MLV and LV transduction. Taken together the results of virus binding, internalization kinetics, pH-neutralizing agents, protease inhibitor and penetration assays support that SSTR-5 allows a greater level of transduction because viruses internalized by this subtype are exposed to more permissive intracellular compartments. Specifically, SSTR-5-associated virions are directed to compartments that are more favorable to cytosolic penetration of viral cores than the compartment(s) to which virions bound to subtypes 2 and 3 are directed; possibly due to a more beneficial complement of host cell proteases. These data suggested that receptor characteristics such as the intracellular fate of internalized virus-receptor complexes exert a strong influence on the efficiency of infection. Surprisingly, even though the difference in the in vitro transduction capacity of Sst-RBS and VSV G pseudotyped LV particles was greater than that of the MLV pseudotypes, the difference did not translate to a reduction in in vivo transduction capacity. A pilot study examining the feasibility of in vivo transduction demonstrated proof of principle and identified regions of the murine brain with endogenous surface expression of SSTRs that were as efficiently transduced by Sst-RBS LV as by VSV G LV.

DOI

10.21007/etd.cghs.2013.0166

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