228 Lentivirus Packaging and Gene Transfer Vectors S. K. Arya*, J. E. Cho, A. Zamani Natl Cancer Inst, NIH, Bethesda, MD
Background: Packaging of viral RNA into particles is a complex process involving recognition of the packaging signal in the candidate viral RNA by the nucleocapsid protein. A non-reciprocal relationship between HIV-1 and HIV-2 cross-packaging was reported. Furthermore, HIV-2 packaging machinery reportedly failed to encapsidate HIV-2 RNA transcribed from a subgenomic vector. It was proposed that HIV-2 RNA encapsidation occur co-translationally in cis, such that the vector RNA cannot gain access to the packaging machinery. Why this mechanism is used in HIV-2 and not in HIV-1 was unexplained. A simpler hypothesis would be that self- and cross-packaging is governed by the relative affinities between the packaging signals and nucleocapsid proteins.
Methods: HIV-1 and HIV-2 vectors were produced by transient transfection into 293T cells and GFP vectors were titrated on the same cells. Self-packaging consisted of co-transfecting the vector with the packaging-signal-deleted, env-truncated homologous provirus and cross-packaging by co-transfecting with the similarly-modified provirus of the other HIV. Vectors containing the amino acid decarboxylase (AADC) gene for Parkinson's disease model were similarly constructed.
Results: The titer of HIV-1 vector packaged with HIV-1 core was 2.7 ±1.0 x 10e3 TU/ng and that packaged with HIV-2 cores was 0.12 ±0.04 x 10e3 TU/ng. The titer of HIV-2 vector packaged with HIV-2 cores was 1.2 ±0.6 x10e3 TU/ng and that packaged with HIV-1 cores was 0.16 ±0.06 x 10e3 TU/ng. For HIV-1 and HIV-2 vectors carrying the AADC gene, 2 vectors were roughly equivalent in imparting the ability to human fetal brain cells to convert L-dopa into dopamine. In a pilot in vivo experiment, injection of these vectors into the brains of Parkinson's rat caused effective gene transfer.
Conclusions: Examined at the level of functional vector assembly, the difference in cross-packaging of HIV-1 by HIV-2 and HIV-2 by HIV-1 was only 2-3 folds. Thus, in the subgenomic vector context, the concept of co-translational encapsidation may not be important. Our data are more consistent with preferential interaction of the nucleocapsid protein with the homologous than with the heterologous packaging signal. Our ongoing studies on chimera and on physical association between 3 partners may shed additional light on this phenomenon. HIV-1 and HIV-2 vectors carrying "real" genes caused effective gene transfer in the relevant target cells.
