Increased Adenovirus Type 5 Mediated Transgene Expression Due to RhoB Down-Regulation

Dragomira Majhen,Andreja Ambriović-Ristov,Maja Osmak,Chloé Leduc,Dunja Vukić,Nikolina Stojanović,Chantal Pichon,Carmen San Martin

doi:10.1371/journal.pone.0086698

Abstract

Adenovirus type 5 (Ad5) is a non-enveloped DNA virus frequently used as a gene transfer vector. Efficient Ad5 cell entry depends on the availability of its primary receptor, coxsackie and adenovirus receptor, which is responsible for attachment, and integrins, secondary receptors responsible for adenovirus internalization via clathrin-mediated endocytosis. However, efficacious adenovirus-mediated transgene expression also depends on successful trafficking of Ad5 particles to the nucleus of the target cell. It has been shown that changes occurring in tumor cells during development of resistance to anticancer drugs can be beneficial for adenovirus mediated transgene expression. In this study, using an in vitro model consisting of a parental cell line, human laryngeal carcinoma HEp2 cells, and a cisplatin-resistant clone CK2, we investigated the cause of increased Ad5-mediated transgene expression in CK2 as compared to HEp2 cells. We show that the primary cause of increased Ad5-mediated transgene expression in CK2 cells is not modulation of receptors on the cell surface or change in Ad5wt attachment and/or internalization, but is rather the consequence of decreased RhoB expression. We propose that RhoB plays an important role in Ad5 post-internalization events and more particularly in Ad5 intracellular trafficking. To the best of our knowledge, this is the first study showing changed Ad5 trafficking pattern between cells expressing different amount of RhoB, indicating the role of RhoB in Ad5 intracellular trafficking.

Highlights

Adenovirus-based vectors are leading vectors used in gene therapy clinical trials today
In order to find out whether development of cisplatin resistance in the CK2 cell line was accompanied by changes in the repertoire of the cell surface receptors involved in adenovirus type 5 (Ad5) infection, we analyzed their expression in HEp2 and CK2 cells by flow cytometry
No significant difference in av integrin subunit expression was found between HEp2 and CK2 cells, there was a slight increase in expression of coxsackie-adenovirus receptor (CAR) and b1 integrin subunit and an approximately 2-fold increase in expression of a3b1 integrin heterodimer and a5 integrin subunit

Summary

Introduction

Adenovirus-based vectors are leading vectors used in gene therapy clinical trials today. Ad5 infection begins with high-affinity binding of the fiber protein to the coxsackie-adenovirus receptor (CAR) on the cell surface [2]. Ad5 internalization is mostly mediated by dynamin- and clathrin-dependent receptor-mediated endocytosis [7], there is evidence that some capsid-modified Ad5based vectors can enter the cell by using lipid raft- and caveolaemediated endocytosis [8]. Ad5 continues part of its intracellular journey in the endosome. Lowering pH in the endosome allows dismantling of the Ad5 capsid and release of the membrane-lytic internal protein VI, which triggers penetration of the endosome. It has been shown that integrin avb plays an important role in the release of Ad5 from the endosome [9,10,11]. The process of adenovirus endocytosis is regulated by lipid kinases and actinmodulating small GTPases, and has been shown to require assembly of the actin cytoskeleton, an event initiated by activation of PI3K and, subsequently, Rac and Cdc, members of the Rho GTPase family [13]

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Conclusion