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. 2011 May;85(9):4111-21.
doi: 10.1128/JVI.00006-11. Epub 2011 Feb 16.

On the role of the SP1 domain in HIV-1 particle assembly: a molecular switch?

Siddhartha A K Datta  1 , Lakew G Temeselew, Rachael M Crist, Ferri Soheilian, Anne Kamata, Jane Mirro, Demetria Harvin, Kunio Nagashima, Raul E Cachau, Alan Rein
Affiliations

On the role of the SP1 domain in HIV-1 particle assembly: a molecular switch?

Siddhartha A K Datta et al. J Virol. 2011 May.

Abstract

Expression of a retroviral protein, Gag, in mammalian cells is sufficient for assembly of immature virus-like particles (VLPs). VLP assembly is mediated largely by interactions between the capsid (CA) domains of Gag molecules but is facilitated by binding of the nucleocapsid (NC) domain to nucleic acid. We have investigated the role of SP1, a spacer between CA and NC in HIV-1 Gag, in VLP assembly. Mutational analysis showed that even subtle changes in the first 4 residues of SP1 destroy the ability of Gag to assemble correctly, frequently leading to formation of tubes or other misassembled structures rather than proper VLPs. We also studied the conformation of the CA-SP1 junction region in solution, using both molecular dynamics simulations and circular dichroism. Consonant with nuclear magnetic resonance (NMR) studies from other laboratories, we found that SP1 is nearly unstructured in aqueous solution but undergoes a concerted change to an α-helical conformation when the polarity of the environment is reduced by addition of dimethyl sulfoxide (DMSO), trifluoroethanol, or ethanol. Remarkably, such a coil-to-helix transition is also recapitulated in an aqueous medium at high peptide concentrations. The exquisite sensitivity of SP1 to mutational changes and its ability to undergo a concentration-dependent structural transition raise the possibility that SP1 could act as a molecular switch to prime HIV-1 Gag for VLP assembly. We suggest that changes in the local environment of SP1 when Gag oligomerizes on nucleic acid might trigger this switch.

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Figures

Fig. 1.
Fig. 1.
(A) Schematic representation of HIV-1 Gag, showing location and sequence of SP1. (B) Thin-section EM images of 293T cells following transfection with the indicated Gag expression plasmids. In panel H, the arrow indicates a proper VLP. Scale bar, 100 nm.
Fig. 2.
Fig. 2.
Thin-section EM images of 293T cells following transfection with Gag expression plasmids as indicated. Scale bar, 100 nm.
Fig. 3.
Fig. 3.
Assembly properties of G2A ΔA364 Gag. (A) Thin-section EM image of cells expressing G2A Gag. In contrast, EM images of cells expressing G2A ΔA364 Gag could not be distinguished from those of mock-transfected cells (data not shown). (B) Inefficient coassembly of G2A ΔA364 Gag with Gag-Z. Cells were transfected with the indicated Gag expression plasmids, and VLPs and cell lysates were then analyzed by immunoblotting with anti-p24 antiserum. (C) Thin-section EM image of cells expressing both Gag-Z and G2A ΔA364 Gag. (D) Thin-section EM image of cells expressing both Gag-Z and G2A Gag. Scale bar, 100 nm.
Fig. 4.
Fig. 4.
In vitro assembly properties of Δ16–99 ΔA364 Gag and Δ16–99 ΔV370 Gag. (A) Δ16–99 ΔA364 Gag (ΔA364) does not assemble efficiently upon addition of nucleic acid. Increasing amounts of tRNA were added to Δ16–99 ΔA364 Gag or Δ16–99 ΔV370 Gag (ΔV370), and the reactions were fractionated into supernatant and pellet. Protein in the pellets was quantitated by absorbance measurements as described in Materials and Methods. (B) Negative-stain EM image of pellet from Δ16–99 ΔA364 Gag plus tRNA. (C) Negative-stain EM image of pellet from Δ16–99 ΔV370 Gag plus tRNA. Scale bar, 100 nm.
Fig. 5.
Fig. 5.
(A) Result of MD simulation of the helical content of residues P356 to Q386 of Gag in a series of water-DMSO mixtures. Points on the graph represent the mean helical contents; 50% of the events are contained within the error bars. (B) Helical content of individual residues between A364 and M378 in water (open symbols) or 80% DMSO (closed symbols). (C) Helical wheel depiction of residues P356 to A378. Polar residues are black, and nonpolar residues are gray.
Fig. 6.
Fig. 6.
Solvent-dependent change in secondary structure of the P356-T373 peptide, determined by CD spectroscopy as described in Materials and Methods. (A) CD spectra of peptide at 0.052 mM as a function of TFE concentration. (B) Helix and coil contents from panel A, estimated using Pepfit. (C) CD spectra of peptide at 0.052 mM as a function of EtOH concentration. (D) Helix and coil contents from panel C. (E) CD spectra of peptide at 0.52 mM as a function of EtOH concentration. Insets show magnified spectra from 0 to 25% EtOH and 50 to 80% EtOH. (F) Ellipticities at 198 nm versus ellipticities at 222 nm for peptide at 0.052 mM (from panel C) and 0.52 mM (from panel E) over a range of EtOH concentrations. Values from 10% and 50% EtOH are indicated. deg, degree.
Fig. 7.
Fig. 7.
Concentration-dependent changes in secondary structure of the P356-T373 peptide. Representative CD spectra of the peptide diluted to 0.052 mM, 2.6 mM, and 5.2 mM from a 10.4 mM stock solution.
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