Imaging the interaction of HIV-1 genomes and Gag during assembly of individual viral particles

Abstract

The incorporation of viral genomes into particles has never previously been imaged in live infected cells. Thus, for many viruses it is unknown how the recruitment and packaging of genomes into virions is temporally and spatially related to particle assembly. Here, we devised approaches to simultaneously image HIV-1 genomes, as well as the major HIV-1 structural protein, Gag, to reveal their dynamics and functional interactions during the assembly of individual viral particles. In the absence of Gag, HIV-1 RNA was highly dynamic, moving in and out of the proximity of the plasma membrane. Conversely, in the presence of Gag, RNA molecules docked at the membrane where their lateral movement slowed and then ceased as Gag assembled around them and they became irreversibly anchored. Viral genomes were not retained at the membrane when their packaging signals were mutated, nor when expressed with a Gag mutant that was not myristoylated. In the presence of a Gag mutant that retained membrane- and RNA-binding activities but could not assemble into particles, the viral RNA docked at the membrane but continued to drift laterally and then often dissociated from the membrane. These results, which provide visualization of the recruitment and packaging of genomes into individual virus particles, demonstrate that a small number of Gag molecules recruit viral genomes to the plasma membrane where they nucleate the assembly of complete virions.

Fig. 1.

HIV-1 genomes carrying MS2 binding sites are packaged and infectious. (A) Schematic of the MS2-tagged HIV-1 genome, V1B-MS2, used in these studies. The positions of critical cis-acting sequences, including packaging sequence (Ψ), the central polypurine tract (cPPT), and the Rev response element (RRE) are indicated, as are the positions of the salient splice donors (d) and acceptors (a). (B) Infectivity of virions generated by 293T cells coexpressing HIV-1 Gag-Pol, VSV-G envelope, and V1B-MS2 or V1B-ΔΨ-MS2 constructs. β-galactosidase activity following infection of TZM indicator cells was measured and plotted as the mean± SD relative light units (RLU). (C) The mean percentage ± SD of mCherry-VLPs that contained GFP-labeled genomes for V1B, V1B-MS2, or V1B-ΔΨ-MS2 (cumulative n >1000 VLPs, three independent experiments).

Fig. 2.

Visualization of individual HIV-1 genomes in live HeLa cells. (A) MS2-NLS-GFP localizes primarily in the nucleus in the absence of HIV-1 genomes as imaged in epifluorescence (EPI) illumination. (Scale bar, 10 μm.) (B) Cells stably expressing MS2-NLS-GFP were transfected with V1B-MS2, fixed 24 h later. A deconvolved optical section from the center of the vertical dimension of the cell is shown. (Scale bar, 10 μm.) The inset shows an expanded segment of the image. The right panel shows the same image with nuclei revealed by staining with Hoechst 33258. (C) Cells stably expressing MS2-NLS-GFP were observed under TIR-FM 6 h after transfection with V1B-MS2. An RNA punctum was tracked and the numbers indicate the elapsed time (in seconds). Images are 5 × 5 μm. Far right image shows the track (green) of the RNA punctum.

Fig. 3.

Retention of viral RNA at the plasma membrane and VLP assembly on RNA. (A and B) Cells stably expressing MS2-NLS-GFP were transfected with Gag/Gag-mCherry and V1B-MS2 and observed live under TIR-FM beginning at 6 h post-transfection. (A) Images of an appearing GFP-labeled RNA punctum on which a VLP subsequently assembles. Images are 2.5 × 2.5 μm. Elapsed time is in minutes:seconds. (B) Plots of fluorescence intensity in arbitrary units (a.u) for the GFP-labeled RNA and Gag-mCherry signals for the VLP shown in (A). (C) Time for which GFP-labeled HIV-1 genomes remain in the TIR field. Each point represents an individual GFP-labeled RNA punctum, and the total time of residence in the TIR field is plotted. However, for “V1B-MS2 + WT Gag” only RNAs on which a VLP assembled are indicated, and the time taken from the appearance of the RNA to completion of the corresponding VLP assembly is plotted. For “V1B-MS2 + Gag-ΔCTD” only RNAs that became anchored at the plasma membrane, but both appeared and disappeared during the period of observation are plotted.

Fig. 4.

Lateral movement of HIV-1 genomes in the absence and presence of Gag. (A) Images showing the track taken by a GFP-labeled RNA punctum before (top) and after (bottom) the Gag punctum became detectable coincident with the RNA. Images are 5 × 5 μm. (B) Plots of fluorescence intensities of Gag and RNA in arbitrary units (a.u), are plotted for the VLP shown in (A) Also plotted is the lateral velocity of the RNA, averaged using a sliding window of 21 frames (3.5 min). (C) Lateral velocity of RNAs in the TIR field under various conditions. Each point represents the velocity of an individual GFP-labeled RNA punctum. Velocities were averaged over the entire time the RNA molecule was observed in the field in the absence of Gag (No Gag) or in the presence of Gag-ΔCTD. For RNAs anchored at the membrane in the presence of WT Gag, velocities were averaged over (i) the time that Gag was undetectable (‘pre’-assembly), (ii) the time during which Gag fluorescence increased (assembly), and (iii) the time during which Gag fluorescent reached a plateau (postassembly). The same thirteen GFP-labeled RNA puncta, from three different cells were evaluated for each phase of assembly.