268 Dissecting the Blocks to HIV-1 Particle Production in Rodent Cells T. Hatziioannou*, J. Martin-Serrano, S. Cowan, Paul D. Bieniasz Aaron Diamond AIDS Res Ctr, Rockefeller Univ, New York, NY
Background: We and others have previously shown that most rodent cells lack a factor(s) critical for the production of HIV-1 virions. Rodent cells expressing HIV-1 receptors and a cyclin T1 that supports Tat function are able to support reverse transcription, integration, and early gene expression at levels comparable to those observed in human cells. However, significant defects are observed in unspliced viral genomic RNA production as well as a dramatic decrease in expression, processing and release of the HIV-1 Gag polyproteins. Furthermore, the few particles that are released from rodent cells are largely noninfectious. Here we present further studies on the basis of these defects.
Methods: Mouse 3T3 (engineered to support Tat function) and human HeLa and HOS cells were transfected with a proviral construct, or an intronless plasmid expressing a codon-optimized HIV-1 Gag-pol (Rev independent). Cell lysates and viral particles were analyzed by p24 ELISA and Western Blot. Co-transfection of an HIV-1 GFP vector and a VSV.G expression plasmid allowed the analysis of particle infectivity.
Results: We found that, compared to a proviral plasmid, transfection of a plasmid expressing a codon-optimized HIV-1 Gag-pol led to significantly higher levels of protein expression, processing, and particle release in mouse cells. Titration of the synthetic Gag-pol plasmid revealed that processing and particle production correlates with Gag-pol expression levels and is similar in both human and mouse cells. The HIV-1 matrix protein was modified by appending the Src membrane targeting signal or by deleting the matrix globular head. These mutants showed increased cell membrane localization, accompanied by increased Gag-pol processing and virion release particularly at low expression levels, in both human and mouse cells. However, regardless of the plasmid used, particles produced by mouse cells were still less infectious than the corresponding particles produced by human cells.
Conclusions: Mouse cells exhibit multiple blocks to HIV-1 replication. We show that blocks to virion production can be overcome by increasing the level of Gag-pol expression and by increasing the cell membrane targeting of Gag-pol. Nevertheless, the virions produced by mouse cells remain significantly less infectious than their counterparts produced by human cells, suggesting additional defects in virion morphogenesis.
