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Effects of stoichiometry of retroviral components on virus production

Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK¹
Oxford BioMedica (UK) Ltd, The Medawar Centre, The Oxford Science Park, Oxford OX4 4GA, UK²

Author for correspondence: Alan Kingsman (at Oxford BioMedica). Fax +44 1865 783001. e-mail A.Kingsman{at}OxfordBiomedica.co.uk

A study was conducted to investigate the effects of increasing the amount of each retroviral component on vector production. It was found that, while the components of both amphotropic and ecotropic vectors were expressed independently of each other in a transient transfection system, increasing the amount of the gag/gag–pol component resulted in a decrease in virus titres for the amphotropic particles but not ecotropic particles. Analyses of the virus stocks produced indicated that the negative effect on titres was closely linked to the availability of envelope proteins for virion incorporation. The negative effect was not observed for ecotropic particle production in 293T cells, where the ecotropic receptor was absent, but was manifested when production was conducted in 293/12 cells expressing the ecotropic receptor. This suggested that the premature interaction between envelope and receptor in producer cells could limit the amount of envelope available for virion incorporation. In designing optimal vector production systems it is essential, therefore, to balance the concentration of the vector components and to ensure that there is never an excess of Gag/Gag–Pol.