The long-term objectives of this research program are to use comparative molecular studies of primate and nonprimate lentiviruses to discover principles of lentiviral biology and to apply them to lentivira/vector development. The laboratory studies HIV-1 and feline immunodeficiency virus (FIV). A central conviction is that judicious studies of selected molecular aspects of an AIDS-causing nonprimate lentivirus, FIV, will yield comparative virological insights into core features of lentiviral biology and evolution, and have particular value for features that have become subject to competing interpretations for HIV-I. Lentiviral nucleic acids undergo sequential processing within virions, beginning with selective encapsidation of a pair of full-length RNAs, which become dimerized. Subsequent reverse transcription of the mature, dimerized genome into linear double-stranded DNA generates a distinctive central plus strand discontinuity (flap structure). At this point the genome (pre-integration complex) is competent to enter the nucleus and integrate, even in interphase or post-mitotic cells. None of these sequential steps in lentiviral genome processing (RNA encapsidation, RNA dimerization, preintegration complex nuclear import) is understood well enough to permit effective therapeutic targeting. The mechanism of the signature ability to traverse an intact nuclear membrane, which is not shared by oncoretroviruses, is currently quite controversial; a role for the flap structure in the de novo-synthesized linear cDNA has been proposed, as has signal-mediated transport by a number of associated virion proteins. This project will determine how the linear nucleic acid molecules in FIV virions are sequentially processed, focusing on the three steps described above. The project will use a competitive genome encapsidation assay, an established FIV vector system that permits analysis of single-round infection, a native northern blot genome dimerization assay, ultrastructural (EM) studies, fluorescence-monitored PCR quantitation of viral cDNA intermediates, a rat brain transduction assay, new class I FIV integrase mutants, and mutagenesis of new FIV elements: encapsidation determinant boundaries that have been mapped to two discrete regions in the 5' end of the FIV genomic mRNA, and a central polypurine tract and central termination sequence that have been identified and shown to be generative of a central DNA flap in the FIV pre-integration complex. The specific aims are to definitively establish the encapsidation signal of this virus, determine which cis-acting genomic RNA sequences mediate dimerization, and establish the functional significance of the FIV central polypurine tract and central termination sequence. The project will therefore significantly augment existing molecular knowledge of this model lentivirus, focusing on aspects with strong potential to yield comparative insights into HIV-1 genome replication mechanisms and to further ground FIV vector development in rigorous basic virological understanding.

R01AI047536

1999-09-01

2009-07-31