HIV-1 Gag protein with or without p6 specifically dimerizes on the viral RNA packaging signal

Abstract

The HIV-1 Gag protein is responsible for genomic RNA (gRNA) packaging and immature viral particle assembly. Although the presence of gRNA in virions is required for viral infectivity, in its absence, Gag can assemble around cellular RNAs and form particles resembling gRNA-containing particles. When gRNA is expressed, it is selectively packaged despite the presence of excess host RNA, but how it is selectively packaged is not understood. Specific recognition of a gRNA packaging signal (Psi) has been proposed to stimulate the efficient nucleation of viral assembly. However, the heterogeneity of Gag-RNA interactions renders capturing this transient nucleation complex using traditional structural biology approaches challenging. Here, we used native MS to investigate RNA binding of wild-type (WT) Gag and Gag lacking the p6 domain (GagΔp6). Both proteins bind to Psi RNA primarily as dimers, but to a control RNA primarily as monomers. The dimeric complexes on Psi RNA require an intact dimer interface within Gag. GagΔp6 binds to Psi RNA with high specificity in vitro and also selectively packages gRNA in particles produced in mammalian cells. These studies provide direct support for the idea that Gag binding to Psi specifically promotes nucleation of Gag-Gag interactions at the early stages of immature viral particle assembly in a p6-independent manner.

Keywords: Gag; Gag p6 domain; HIV; MS; RNA; RNA binding protein; RNA-protein interaction; genomic RNA packaging; human immunodeficiency virus; mass spectrometry; native mass spectrometry; virus assembly.

Figure 1.

A, domain structure of WT and mutant Gag proteins investigated in this work. B, sequence and predicted secondary structures of TARpolyA (left) and Psi (right) RNAs. To ensure a monomeric state, the SL1 loop of Psi was mutated to a GAGA tetraloop as indicated. The gray nucleotides at the 5′ end of Psi (GG) are not encoded by HIV-1 but were added to improve the yield of in vitro transcription.

Figure 2.

Zero-charge mass spectra of WT Gag (A1), GagΔp6 (B1), and WM-GagΔp6 (C1) at 16 μm; WT Gag (A2), GagΔp6 (B2), and WM-GagΔp6 (C2) at 3 μm; 500 nm TARpolyA in the presence of 3 μm WT Gag (A3), GagΔp6 (B3), and WM-GagΔp6 (C3); 500 nm Psi in the presence of 3 μm WT Gag (A4), GagΔp6 (B4), and WM-GagΔp6 (C4). Notation is as follows: G = Gag, G₂ = two Gag, G₃ = three Gag, TpA = TARpolyA. Molecular masses for all analytes are listed in Table S1. The number of replicates (n) per experiment are indicated on each spectrum with additional repeats shown in Figs. S1–S6.

Figure 3.

Fluorescence anisotropy results for WT Gag and GagΔp6 binding to Psi and TARPolyA RNAs as a function of NaCl concentration (n = 3, error bars reflect the S.D. of three independent measurements). See Figs. S7 and S8 for data acquired in ammonium acetate.

Figure 4.

A and B, viral pellets (A) and cell lysates (B) from a representative transfection experiment were analyzed for Gag by Western blot analysis as described in “Experimental Procedures.” The cell lysates (B) were also probed for β-actin. A dilution series of recombinant GagΔp6 protein was loaded adjacent to the viral pellets (A) so that a standard curve could be constructed (Fig. S12). In the “mock” samples, no Gag plasmid was transfected, but the vector (Psi⁺ or Psi⁻, as indicated) was. The vertical lines in panels A and B indicate where lanes unrelated to this study were spliced out of the gel photo.

Figure 5.

RNA packaging by mutant Gag proteins. The figure shows the results of three separate transfections. In each case, viral pellets were assayed for copies of the vector RNA and for Gag content as described in “Experimental Procedures.” A, the ratio of these two quantities in the WT control was set to 100. B, for each Gag, the ratio of these two quantities for Psi⁺ was set to 100.

Figure 6.

Model showing WT Gag and GagΔp6 binding to Psi and TARpolyA based on the results of this work. In the presence of TARpolyA (orange), WT Gag (panel A) and GagΔp6 (panel C) bind primarily as monomers, whereas the presence of Psi (teal) promotes dimerization of both proteins (panels B and D). Gag domains are colored as follows: MA (green), CA (red), NC (blue), and p6 (purple).