Student Theses and Dissertations

Date of Award

2009

Document Type

Thesis

RU Laboratory

Simon Laboratory

Keywords

gene therapy, virus-host interactions, adeno-associated virus type 2, heparan sulfate proteoglycan

Abstract

The study of virus-host interactions has not only produced insights relevant to both preventative drug design and the emerging field of gene therapy, but it has also served as a valuable tool to the field of cell biology. The biological roles of many pathogen recognized cell-surface receptors have yet to be determined, yet are being exploited by pathogens to facilitate their entry and infection. Specifically a small family of sugar modified membrane protein known as heparan sulfate proteoglycans, have been shown to be a critical determinant in the efficiency of infection of many important pathogens. Adeno-associated virus type 2 (AAV) is a small (25nm) single stranded DNA, non-enveloped parvovirus that is currently being used as a vector for gene therapy. Valued for its lack of pathogenicity and ability to stably transduce a variety of terminal differentiated and quiescent cell types, the use of AAV during clinical trails has faced many complications. Consequently, the search for knowledge about this vector is more critical now then ever. Although extensive work on the safety of the in vivo application of this vector has been established, the cell-biological basis of AAV’s life cycle remains to be fully elucidated. Binding and entry of AAV requires heparan sulfate proteoglycan (HSPG) as well as two secondary receptors, fibroblast growth factor receptor and αv/5 β5/1 integrins. In this study, we follow the cellular modifications induced upon AAV binding and subsequent internalization into host cells. Earlier reports implicated receptor-mediated endocytosis as the sole pathway for AAV entry. We show that AAV’s primary receptor, HSPG, mediates an alternate entry pathway that is clathrin-independent and caveolar-independent. We show that inhibitors of macropinocytosis, which did not inhibit the clathrin-dependent entry pathways, blocked the entry of AAV and HSPG as well as blocked AAV transduction. In cells that are deficient in cell surface HSPG, transduction by AAV was reduced but unaffected by drugs that block macropinocytosis. Membrane ruffles induced upon AAV binding were enriched in activated PKC alpha, actin and dynamin. Treatment with dynosore, expression of dominant negative dynamin 2, or an HSPG construct lacking the PKC recruitment domain greatly diminished the size of macropinosomes and AAV internalization. Finally, silencing of clathrin heavy chain by siRNA did not affect AAV infection. Our data shows that HSPGs play a critical role in chaperoning AAV into a macropinocytic pathway, which leads to productive infection.

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

Permanent URL

http://hdl.handle.net/10209/407

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