Poster abstracts
Poster number 22 submitted by Samantha Hinckley
Characterization of HIV-1 Gag and Gag-RNA interactions by native mass spectrometry
Samantha H. Hinckley (Chemistry and Biochemistry, The Ohio State University), Erik D. Olson (Chemistry and Biochemistry, The Ohio State University), Karin Musier-Forsyth (Chemistry and Biochemistry, The Ohio State University), Vicki H. Wysocki (Chemistry and Biochemistry, The Ohio State University)
Abstract:
Most HIV-1 therapies are focused on targeting viral enzymes. Due to the common drug resistance which occurs when HIV-1 therapies based on targeting viral enzymes are employed new strategies for treatment must be developed. The HIV-1 Gag polyprotein and its mature protein products are critical for the lifecycle of this retrovirus. It is established that the presence of both the membrane and nucleic acids lead to linear Gag conformations associated with infectious viral particle formation a defined mechanism has not been established. To better understand the lifecycle of the HIV-1 retrovirus the oligomeric states of Gag and the binding stoichiometry of Gag in the presence of the trans activating response (TAR) element and the packaging signal sequence (PSI) have been determined with native mass spectrometry. A systematic approach has been used to dissect the interactions and of Gag with TAR-polyA and PSI.
In vitro transcribed TAR-polyA (A34U) RNA (81 µM) was obtained from the Musier Forsyth lab and folded in ammonium acetate (50 mM). The solution was heated up to 80oC for 2 minutes, subsequently incubated at 60oC for 2 minutes and then magnesium acetate (10mM) was added before chilling on ice for 30 minutes. The folded TAR-polyA was then incubated with GagΔp6, GagΔsp1Δp6, nucleocapsid, or matrix (7 µM each) in 500 mM ammonium acetate for 10 minutes at 25oC. The same protocol was followed for forming the Gag-Psi complex. The complex was then analyzed on an in house modified Thermo Q-Exactive plus EMR with an installed quadrupole and SID device.
Upon analysis, the complex was seen with a mass of 86 kDa, indicating a one to one stoichiometry in these conditions. The Psi-Gag mixture was observed to yield a 200 kDa complex indicating that either higher oligomeric states of Gag or a three to one stoichiometry in these conditions. Apo-gag polyprotein was observed to have multiple oligomeric states ranging from monomer to hexamer.
This is the first high resolution mass spectrum of the intact Gag: TAR-polyA and Gag: Psi RNA-protein complex and observation of the various oligomeric states of Apo-gag.
References:
Kilmarx, P. H. Global epidemiology of HIV. Curr Opin HIV AIDS 4, 240-246, doi:10.1097/COH.0b013e32832c06db (2009).
Webb, J.A., Jones, C.P., Parent, L.J., Rouzina, I. & Musier-Forsyth, K. Distinct binding interactions of HIV-1 Gag to Psi and non-Psi RNAs: Implications for viral genomic RNA packaging. RNA (2013).
Keywords: HIV-1 Packaging, Native Mass Spectrometry, RNA Protein Complexes