Abstract
Atomic Force Microscopy was utilized to study the morphology of Gag, ΨRNA, and their binding complexes with lipids in a solution environment with 0.1Å 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-ΨRNA, Gag-TARpolyA RNA, Gag-PI(4,5)P2 and PI(4,5)P2-ΨRNA-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 ΨRNA 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 ΨRNA. When both ΨRNA and PI(4,5)P2 are present Gag undergoes comformational changes and an even higher degree of multimerization.
Highlights
Human immunodeficiency virus (HIV) is a retrovirus with a diploid genome of singlestranded RNA [1,2,3,4]
In the Atomic Force Microscopy (AFM) experiments here, we started with size and morphology measurements of the RNAs (CRNA and TARpolyA RNA) to benchmark and validate the measurement and analysis software developed
The AFM technique was utilized to study the morphology of GagΔP6 (0.5μM), CRNA (0.5μM), and their binding complexes with the lipid PI(4,5)P2 in HEPES buffer with 0.1 Å vertical and 1nm lateral resolution
Summary
Human immunodeficiency virus (HIV) is a retrovirus with a diploid genome of singlestranded RNA [1,2,3,4]. The NC domain binds Gag to the RNA genome through nonspecific interaction as well as specific binding to the stem-loop 3 (SL3) in the packaging signal C (CRNA) [20]. Within the NC domain, there are two CCHC type zinc fingers, which are crucial for specific CRNA binding and genomic viral RNA packaging [22, 23, 24]. At the 5’ untranslated region (UTR) of HIV-1 genomic RNA, there are many critical regions which are thought necessary for genome dimerization and binding with Gag: the transactivation response stem-loop (TAR), the polyadenylation stem-loop (polyA), the prime binding site (PBS) and the packaging signal domain C. Within CRNA, SL1 contains a palindromic sequence DIS that is responsible for HIV genomic RNA dimerization and Gag binding [30,31,32]. The influence of the addition of both CRNA and PI(4,5)P2 was examined to understand their collective effect on Gag
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
