Research in our laboratory has focused on factors which govern HIV-1 infectivity and, in particular, viral determinants which facilitate transport of viral nucleic acids from the point of virus entry to the host cell nucleus. The HIV-1 gag MA protein, a major structural virion protein, is a component of the nucleoprotein reverse transcription complex and facilities nuclear transport of viral nucleic acids. In addition, gag mA myristoylation is required for transport of gag polyproteins to the membrane for incorporation into maturing virions. Thus, gag MA exhibits both membrane and nuclear targeting functions. We have determined that phosphorylation of gag MA regulates these opposing targeting functions by facilitating dissociation of reverse transcription complexes from the target cell membrane thereby allowing the complex to undergo nuclear import. Kinase inhibitors which prevent phosphorylation of gag MA inhibit translocation of the reverse transcription complex to the host cell nucleus thereby preventing efficient infection of the target cell. The object of the virology and reagent development component is to support efforts to develop specific inhibitors of kinases which phosphorylate gag MA and to clone these kinases respectively. In addition, this component will conduct mechanistic analysis of kinase inhibitors and transdominant kinase mutants.
Specific aims of the virology and reagent support components include:
Aim 1 : Identify suitable gag MA phosphorylation substrates and kinase sources which will be used in efforts to purify and clone the involved kinases. These reagents will additionally be used for derivation of in vitro assays for high throughput screening of compounds which inhibit kinase activity in vitro.
Aim 2 : Examine antiviral properties of lead kinase inhibitors and their analogs in vitro and confirm their mechanism of inhibition.
Aim 3 : Examine antiviral properties and specificities of transdominant mutants of the cellular kinases that phosphorylate gag MA.
| Purohit, P; Dupont, S; Stevenson, M et al. (2001) Sequence-specific interaction between HIV-1 matrix protein and viral genomic RNA revealed by in vitro genetic selection. RNA 7:576-84 |
