title: Stoichiometric limitations in assembly of active recombinant retrovirus.
abstract: Although recombinant retroviruses are widely used in gene therapy and as gene transfer vehicles for basic biological studies, their titers are very low as compared to other recombinant viral systems, e.g., adenovirus. We investigated the rate-limiting steps in production of LacZ-encoding ecotropic (CRE BAG 2) and amphotropic (Psi-CRIP) retrovirus. We found that ecotropic retrovirus producer cells produced a large number of inactive viral particles because they were severely limited by the amount of mRNA that was packaged into viral capsids. Introduction of the gene for green fluorescence protein (GFP) increased retroviral titers 40-fold, without affecting the viral matrix protein, p30, or the activity of reverse transcriptase. Surprisingly, while transfer of GFP gene increased retrovirus production, beta-gal activity and X-gal titer decreased significantly. Quantitative real-time polymerase chain reaction (PCR) showed that although producer cells synthesized similar amounts of both mRNAs, retroviral supernatants contained significantly lower amount of LacZ mRNA, possibly due to competition between LacZ and GFP mRNAs for encapsidation into virions. In contrast to ecotropic producers, introduction of GFP gene copies into amphotropic producers resulted in a moderate twofold increase in retrovirus production. However, delivery of genes encoding for the viral proteins gp70 and p30 increased virus production by fivefold, suggesting that amphotropic producers may also be limited by synthesis of structural viral proteins. Our data show that in addition to the amount of viral genome or proteins, assembly of viral components into active viral particles may limit production of high titer retroviral preparations.
authors.affiliation: Bioengineering Laboratory, Department of Chemical and Biological Engineering, 908 Furnas Hall, University at Buffalo, The State University of New York, Amherst, New York 14260, USA.
authors.forename: Pedro
authors.initials: P
authors.lastname: Lei
authors.forename: Stelios T
authors.initials: ST
authors.lastname: Andreadis
mesh_terms: D002213:Capsid / Q000378:metabolism*; D015967:Gene Expression Regulation, Viral / Q000502:physiology*; D011995:Recombination, Genetic / Q000235:genetics*; D012190:Retroviridae / Q000502:physiology*; D014775:Virus Activation / Q000502:physiology*; D019065:Virus Assembly / Q000502:physiology*; D014776:Virus Cultivation / Q000379:methods*