Control of human immunodeficiency virus type-1 protease activity in insect cells expressing Gag-Pol rescues assembly of immature but not mature virus-like particles.
Expression of human immunodeficiency virus type 1 (HIV-1) Gag protein in insect cells using baculovirus vectors leads to the abundant production of virus-like particles (VLPs) that represent the immature form of the virus. When Gag-Pol is included, however, VLP production is abolished, a result attributed to premature protease activation degrading the intracellular pool of Gag precursor before particle assembly can occur. As large-scale synthesis of mature noninfectious VLPs would be useful, we have sought to control HIV protease activity in insect cells to give a balance of Gag and Gag-Pol that is compatible with mature particle formation. We show here that intermediate levels of protease activity in insect cells can be attained through site-directed mutagenesis of the protease and through antiprotease drug treatment. However, despite Gag cleavage patterns that mimicked those seen in mammalian cells, VLP synthesis exhibited an essentially all-or-none response in which VLP synthesis occurred but was immature or failed completely. Our data are consistent with a requirement for specific cellular factors in addition to the correct ratio of Gag and Gag-Pol for assembly of mature retrovirus particles in heterologous cell types.
Adamson
Catherine S
CS
School of Animal and Microbial Sciences, The University of Reading, Reading RG6 6AJ, UK. c.s.adamson@rdg.ac.uk
Nermut
Milan
M
Jones
Ian M
IM
D000818:Animals; D016367:Baculoviridae / Q000378:metabolism; D001665:Binding Sites; D015685:Fusion Proteins, gag-pol / Q000378:metabolism*; D016333:HIV Protease / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology / Q000472:pathogenicity / Q000502:physiology*; D007313:Insecta; D016297:Mutagenesis, Site-Directed; D011480:Protease Inhibitors / Q000494:pharmacology; D011498:Protein Precursors / Q000378:metabolism*; D011995:Recombination, Genetic; D019258:Saquinavir / Q000494:pharmacology; D019065:Virus Assembly
D015685:Fusion Proteins, gag-pol; D011480:Protease Inhibitors; D011498:Protein Precursors; D016333:HIV Protease; D019258:Saquinavir