(282f) Analysis of Retroviral Decay: Protein and RNA Stability

Gene therapy, which requires efficient gene transfer into target cells, has great potential for the treatment of many inherited and acquired disorders. Retroviral vectors are powerful tools for the introduction and long-term expression of transgenes into mammalian cells, but their application is often limited by a rapid loss of bioactivity. It has been well-established that retroviruses spontaneously lose activity at 37°C, with a half-life of 3-8 hours, depending on the retrovirus type. We sought to determine which components of the retrovirus are responsible for this loss in bioactivity. To this end, we focused on the viral proteins and RNA, two major components that we hypothesized may undergo degradation and negatively influence viral infectivity. CD spectroscopy and 2D-SDS-PAGE analysis of viral proteins did not show any change in secondary structure or evidence of proteolysis. Reverse transcription PCR targeting RNA encoding portions of the viral genome clearly demonstrated time-dependent degradation of RNA, which was correlated with the loss in viral bioactivity. The mechanism underlying the degradation of viral RNA was investigated by site-directed mutagenesis of the viral genome. Reverse transcriptase and protease mutants exhibited enhanced RNA stability in comparison to wild type recombinant virus, suggesting that the reverse transcriptase enzyme may be responsible for RNA degradation. On the basis of these results, we propose a mechanism for retroviral decay that involves viral RNA degradation via the RNA-RNA (RNase H*) nuclease activity of the viral reverse transcriptase.