Impact of E1 and Cre on Adenovirus Vector Amplification: Developing MDCK CAV-2-E1 and E1-Cre Transcomplementing Cell Lines

Paulo Fernandes,Virgínia M Santiago,Ana S Coroadinha,Paula M Alves,Eric J Kremer,Ana F Rodrigues,Hélio Tomás

doi:10.1371/journal.pone.0060342

Abstract

Adenovirus vectors have been extensively studied through the manipulation of viral genome. However, little attention is being paid to their producer cell-lines; cells are selected according to virus yields, neglecting the expression profile of transcomplementing gene products underlying cell performance. This work evaluates the impact of E1 (E1A and E1B) and Cre recombinase levels in the production of E1-deleted and helper-dependent canine adenovirus type 2 (CAV-2) vectors using MDCK cells. E1A and E1B gene expression and Cre activity were evaluated in different cell clones and compared with the corresponding cell productivity and susceptibility to oxidative stress injury. CAV-2 production was proportional to E1A expression (the highest levels of E1A corresponding to productivities of 3000–5000 I.P./cell), while E1B prolonged host cell viability after infection, conferring protection against apoptosis. Cre recombinase counteracted E1B anti-apoptotic properties, however viral production was maintained under high levels of Cre. Yet, Cre recombinase side effects can be reduced using cell lines with lower Cre-activities, without compromising the excision efficiency of helper vector packaging signal. These results highlight the influence of transcomplementing gene products on CAV-2 producer cell line performance, and the ability to express high levels of E1A and E1B as an important feature for cell line establishment and high adenovirus titers.

Highlights

Adenovirus vectors (AdV) are efficient gene transfer vectors due to the ability to efficiently infect a wide variety of quiescent and proliferating cell types leading to high-level gene expression [1]
We studied the impact of E1 expression in the development of MDCK-E1 and MDCK-E1-Cre cell lines for E1deleted and HD canine adenovirus type 2 (CAV-2) vectors
E1 Gene Expression and DE1 CAV-2 Amplification MDCK cells were stably transfected with the plasmid encoding

Summary

Introduction

Adenovirus vectors (AdV) are efficient gene transfer vectors due to the ability to efficiently infect a wide variety of quiescent and proliferating cell types leading to high-level gene expression [1]. Adenovirus E1A gene expression products stimulate infected cells to enter S phase of the cell cycle and provide an intracellular environment for viral replication. E1B gene encodes two major proteins generally designated according to adenovirus terminology by E1B 19 kDa and E1B 55 kDa, whose main functions are inhibition of apoptosis, protection of viral and cellular DNA from degradation during infection and further intracellular environment modification, to make the cell more hospitable to viral protein production and viral DNA replication [7,8,9,10]

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Conclusion