Investigation of HIV-1 Gag binding with RNAs and lipids using Atomic Force Microscopy

Atomic Force Microscopy was utilized to study the morphology of Gag, PsiRNA, and their binding complexes with lipids in a solution environment with 0.1A vertical and 1nm lateral resolution. TARpolyA RNA was used as a RNA control. The lipid used was phospha-tidylinositol-(4,5)-bisphosphate (PI(4,5)P2). The morphology of specific complexes Gag-PsiRNA, Gag-TARpolyA RNA, Gag-PI(4,5)P2 and PI(4,5)P2-PsiRNA-Gag were studied. They were imaged on either positively or negatively charged mica substrates depending on the net charges carried. Gag and its complexes consist of monomers, dimers and tetramers, which was confirmed by gel electrophoresis. The addition of specific PsiRNA to Gag is found to increase Gag multimerization. Non-specific TARpolyA RNA was found not to lead to an increase in Gag multimerization. The addition PI(4,5)P2 to Gag increases Gag multimerization, but to a lesser extent than PsiRNA. When both PsiRNA and PI(4,5)P2 are present Gag undergoes comformational changes and an even higher degree of multimerization.