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. 2009 May 10;387(2):341-52.
doi: 10.1016/j.virol.2009.02.037. Epub 2009 Mar 12.

Characterization of a myristoylated, monomeric HIV Gag protein

Jun Dou  1 , Jaang-Jiun Wang, Xuemin Chen, Hua Li, Lingmei Ding, Paul Spearman
Affiliations

Characterization of a myristoylated, monomeric HIV Gag protein

Jun Dou et al. Virology. .

Abstract

The process of HIV assembly requires extensive homomultimerization of the Gag polyprotein on cellular membranes to generate the nascent particle bud. Here we generated a full-length, monomeric Gag polyprotein bearing mutations that eliminated multimerization in living cells as indicated by fluorescence resonance energy transfer (FRET). Monomeric Gag resembled non-myristoylated Gag in its weak membrane binding characteristics and lack of association with detergent-resistant membranes (DRMs or lipid rafts). Monomeric Gag failed to assemble virus-like particles, but was inefficiently rescued into particles by wildtype Gag through the influence of the matrix domain. The subcellular distribution of monomeric Gag was remarkably different than either non-myristoylated Gag or wildtype Gag. Monomeric Gag was found on intracellular membranes and at the plasma membrane, where it highlighted plasma membrane extensions and ruffles. This study indicates that monomeric Gag can traffic to assembly sites in the cell, where it interacts weakly with membranes.

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Figures

Fig. 1
Fig. 1. Emission scans representing FRET assay for the indicated pairs of Gag-CFP/YFP constructs
SrcCANC (open circles) serves as a positive FRET control in both panels, Myr(−) SrcCANC (triangles) as a negative control that illustrates a CFP curve. Total YFP emission values are not shown in this figure but were measured separately, and were of approximately equal intensity for each comparison. (A) SrcCANC(M39A/W184A/M185A)NC15A pair fails to generate FRET (“X”). (B) Matrix and p6 regions do not restore FRET to monomeric Gag (curve marked by “X”). Data were obtained on a PTI scanning cuvette fluorometer following excitation at 433nm.
Fig. 2
Fig. 2. Myristoylation of monomeric Gag constructs
(A) 3H-myristic acid labeling of wildtype and monomeric Gag. Gag proteins were labeled for 8 hours, then harvested and immunoprecipitated with HIV patient sera and analyzed by SDS-PAGE and autoradiography. (B) A fraction of the lysates were analyzed by Western blotting using CA183 monoclonal antibody to demonstrate that all Gag proteins were produced in this experiment.
Fig. 3
Fig. 3. Comparison of particle production by wildtype and monomeric Gag protein
(A) Wildtype (open bars) and monomeric Gag (closed bars) were expressed in 293T cells. Cells and supernatants were harvested at 24 hours post-transfection, and the p24 antigen present in cells (C) or supernatants (S) was measured by antigen capture ELISA. Results are presented as total p24 (left) and as percentage of particle release (right). (B) Pelleted supernatants were analyzed after equilibrium density centrifugation on linear 20–60% sucrose gradients. Note that monomer Gag was not detectable on the same scale as wildtype Gag (left panel, closed vs. open circles). Using a different scale, a peak of p24 antigen was detected at a low particle density (1.138 g/ml, bottom gradient).
Fig. 4
Fig. 4. Transmission electron microscopic analysis of particle production
(A) Wildtype Gag particles were easily detected in all fields; shown is one representative image at low power (A, bar = 2.4 mm) and higher magnification from the boxed region is shown below (B, bar = 305 nm). (C) No virus-like particles were seen in cells expressing monomeric Gag. Shown is a representative cell, with prominent cell ruffles and filamentous extensions of the plasma membrane. Bar = 3.2 mm in C, 750 nm in D.
Fig. 5
Fig. 5. Membrane flotation and detergent-resistant membrane formation
(A) Membrane flotation in the absence of detergent. Cell lysates were layered onto the bottom of a 50%–40%–10% iodixanol gradient, and equilibrium flotation centrifugation was carried out to separate membranes (40%–10% interface, fraction 4) from cytosol (fraction 10–12), using YFP fluorescence as a marker of total Gag protein in the membrane or cytosol. (B) Flotation following treatment of cell lysates with 1% TX-100 on ice for 30 minutes. Fraction 4 represents Gag-YFP on detergent-resistant membranes or putative rafts. Top of gradients is to the left.
Fig. 6
Fig. 6. FRET rescue experiments
The ability of wildtype Gag-CFP to interact with (rescue) a series of test constructs expressed as YFP fusions was assessed by scanning cuvette fluorometry as described in Materials and Methods. Wildtype Gag-YFP (triangles) serves as positive control. (A) Myr(−) full-length monomeric Gag-YFP and the indicated constructs were assessed for interaction with Gag-CFP by FRET. (B) Myr(+) full-length monomeric Gag-YFP and other indicated constructs were assessed for interaction with wildtype Gag-CFP by FRET.
Fig. 7
Fig. 7. Particle rescue experiments
A FLAG-tagged wildtype Gag protein was co-expressed with HA-tagged Gag test constructs. Cell lysates and particles (pelleted through sucrose) were evaluated by Western blotting with anti-FLAG or anti-HA antibody. Top panel shows the wildtype Gag in cell lysates (left) and particles (right). Bottom panel shows protein production in cell lysates (left); the right panel here represents rescue of test constructs into particles by wildtype Gag. Blot shown is representative of three independent experiments.
Fig. 8
Fig. 8. Role of MA in particle rescue
FLAG-tagged wildtype Gag was co-expressed with HA-tagged Gag test constructs as in Fig. 7. Inefficient but real rescue of the myristoylated, monomeric Gag is demonstrated in particle blot, while DMA monomeric Gag (faster migrating band, lanes 4 and 7) was not rescued (lane 14). Blot shown is representative of three independent experiments.
Fig. 9
Fig. 9. Subcellular distribution of monomeric Gag differs markedly from wildtype
Cells expressing Gag constructs were fixed and stained with anti-Gag or with antibodies against epitope tags and examined by confocal fluorescence microscopy. (A) Monomeric Gag-HA stained with anti-HA antisera. (B) Non-myristoylated, untagged Gag stained with anti-MA antisera. (C) Wildtype Gag stained with anti-p24 monoclonal antibody. (D) Monomer Gag is shown in red in this panel, and endoplasmic reticulum staining in green. (E) pECFP-MEM is shown in blue, marking the plasma membrane, and monomer Gag in red. Areas of light blue/white signal indicate colocalization. (F) Wildtype Gag stained with anti-MA antibody is shown in red, and pECFP-MEM in blue. (G–I) Coexpression of monomeric Gag-HA and wildtype Gag-FLAG. Monomeric Gag-HA is shown in green (G and I). Wildtype Gag-FLAG is shown in red (H and I). (K–L) Coexpression of monomeric Gag-HA and wildtype Gag-FLAG, same technique as in (G–I). Size bars indicate 16 mm in each panel.
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