Poster abstracts

Poster number 67 submitted by Samantha Sarni

Interrogating the conformation of HIV-1 Gag and its stoichiometry of binding to genomic RNAs

Samantha Hinckley Sarni (OSBP), Erik Olson (OSBP), Karin Musier-Forsyth (Chemistry and Biochemistry, The Ohio State University), Vicki Wysocki (Chemistry and Biochemistry, The Ohio State University)

Abstract:
Gag, the main structural protein in HIV-1 virions, can bind nucleic acids and form virus-like particles.1-5 This promiscuous RNA-binding protein nonetheless selectively and efficiently packages the dimeric HIV-1 genome from amongst a vast excess of host RNAs. Gag recognizes and binds with high specificity to the Psi element within the 5’-untranslated region of genomic RNA.6 At least three motifs within Psi contribute to specific HIV-1 Gag interactions: two G-rich bulges within stem-loop 1 (SL1) and single-stranded G residues that flank SL3.7 It was determined via fluorescence anisotropy that as the concentration of ammonium acetate increased from 50 mM to 500 mM, the KD of GagΔp6 for non-Psi RNA (TARpolyA(A34U)) increased from 340 nM to 9 μM. In contrast, under identical conditions, the KD of GagΔp6 for PsiΔDIS (a monomeric form of the Psi element) increased from 8 to 39 nM. These data indicate that electrostatic interactions play a more dominant role in Gag:non-Psi interactions than in Gag-Psi interactions, consistent with previous studies using NaCl.7,8 Native mass spectrometry analysis of GagΔp6 alone on a QE+EMR Orbitrap platform revealed that multiple molecular weight species were present near the theoretical molecular weights of GagΔp6 monomer and dimer. This was further investigated by ion mobility MS on a G1 QTOF platform. The ion mobility data from the G1 indicated that the full-length GagΔp6 occupies two distinct drift time distributions, consistent with the multiple conformations Gag has previously been shown to adopt. The protein-RNA complexes were analyzed on the QE+EMR. The GagΔp6-PsiΔDIS complex presented a 3:1 GagΔp6:PsiΔDIS stoichiometry. In contrast, the Gag:TARpolyA(A34U) complex mixture showed an abundance of monomeric TARpolyA(A34U) and some 1:1 GagΔp6:TARpolyA(A34U) complex. These data are consistent with a model where Psi RNA supports a greater Gag binding stoichiometry than non-psi RNA, which could nucleate virion formation and ensure packaging of genomic RNA.

References:
1. Campbell et al., Journal of Virology 1999, 73 (3), 2270-2279.
2. Campbell et al., Proc Natl Acad Sci U S A 2001, 98 (19), 10875-9.
3. Campbell, et al. J Virol 1995, 69 (10), 6487-97.
4. Levin et al., J Virol 1974, 14 (1), 152-61.
5. Muriaux et al., Proc Natl Acad Sci U S A 2001, 98 (9), 5246-51.
6. Liu et al., J Mol Biol 2017, 429 (16), 2542-2555.
7. Rye-McCurdy et al., Viruses 2016, 8 (9).
8. Jones et al., Proc Natl Acad Sci U S A 2014, 111 (9), 3395-400.
9. Webb et al., RNA 2013, 19 (8), 1078-88.

Keywords: HIV-1 , Native Mass Spectrometry, Viral Assembly