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Biotechnology and Applied Biochemistry (2007) 48, (35–43) (Printed in Great Britain)

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Molecular construction of bionanoparticles: chimaeric SIV p17–HIV I p6 nanoparticles with minimal viral protein content

Maria J. L. Costa*†¹, Luísa Pedro‡¹, António P. Alves De Matos§, Maria R. Aires-Barros†, José A. Belo‡¶, João Goncalves*² and Guilherme N. M. Ferreira‡^2,3

*URIA (Unidade dos Retrovírus e Infecções Associadas), Faculdade de Farmácia Universidade de Lisboa, Lisbon, Portugal, †IBB (Institute for Biotechnology and Bioengineering), Centro de Engenharia Biológica e Química, Instituto Superior Técnico Lisboa, Lisbon, Portugal, ‡IBB (Institute for Biotechnology and Bioengineering), Centro de Biomedicina Molecular e Estrutural, Universidade do Algarve-FERN (Faculdade de Engenharia de Recursos Naturais), Campus de Gambelas, 8005-139 Faro, Portugal, §Departamento de Anatomia Patológica, Hospital Curry Cabral, Lisbon, Portugal, Departamento de Biomateriais, Escola de Medicina Dentária, Universidade de Lisboa, Lisbon, Portugal, and ¶Instituto Gulbenkian de Ciência, Oeiras, Portugal

Key words: bionanoparticle, delivery vector, gene therapy, human and simian immunodeficiency virus (HIV and SIV), molecular therapy, virus-like particle.

Abbreviations used: CHO cell, Chinese-hamster ovary cell; DM, double mutant; DMEM, Dulbecco's modified Eagle's medium; EF1, human elongation factor 1 promoter; HA, haemagglutinin; HEK-293 cell, human embryonic kidney-293 cell; HEK-293T cells, HEK-293 cells expressing the large T-antigen of SV40 (simian virus 40); HRP, horseradish peroxidase; SIV, simian immunodeficiency virus; VLP, virus-like particle; Vpr, viral protein R; VSV-G, vesicular-stomatitis-virus glycoprotein; Zeo, zeocin selection marker.

¹These authors contributed equally to this work.

²These authors contributed equally to this work as senior authors.

³To whom correspondence should be addressed (email gferrei@ualg.pt).

VLPs (virus-like particles) are promising delivery vectors for molecular therapy, since they combine the major advantages of viral vectors with significantly fewer viral vector disadvantages. The present paper describes the molecular construction of chimaeric VLPs based on minimal SIV (simian immunodeficiency virus) and HIV1 components. A chimaeric protein was constructed by fusion of SIV matrix protein (p17) and HIV1 p6 protein, and we demonstrated that the chimaeric proteins assemble as 80 nm nanoparticles containing ~7700 chimaeric protein units. Chimaeric VLPs are released from HEK-293T cells (human embryonic kidney cells expressing the large T-antigen of simian virus 40) and are fully encapsulated with lipid membrane. Chimaeric VLPs are produced at 3.7-fold higher levels when compared with SIV p17 VLPs owing to duplication of a PTAP (Pro-Thr-Ala-Pro) domain previously shown as essential for virus particle release. The chimaeric VLPs constructed in the present paper were efficiently pseudotyped with vesicular-stomatitis-virus glycoprotein, as shown by immunoprecipitation assays.

Received 23 October 2006/28 February 2007; accepted 29 March 2007

Published as Immediate Publication 29 March 2007, doi:10.1042/BA20060208

© 2007 Portland Press Ltd