Cytoplasmic HIV-1 RNA is mainly transported by diffusion in the presence or absence of Gag protein

Abstract

Full-length HIV-1 RNA plays a central role in viral replication by serving as the mRNA for essential viral proteins and as the genome packaged into infectious virions. Proper RNA trafficking is required for the functions of RNA and its encoded proteins; however, the mechanism by which HIV-1 RNA is transported within the cytoplasm remains undefined. Full-length HIV-1 RNA transport is further complicated when group-specific antigen (Gag) protein is expressed, because a significant portion of HIV-1 RNA may be transported as Gag-RNA complexes, whose properties could differ greatly from Gag-free RNA. In this report, we visualized HIV-1 RNA and monitored its movement in the cytoplasm by using single-molecule tracking. We observed that most of the HIV-1 RNA molecules move in a nondirectional, random-walk manner, which does not require an intact cytoskeletal structure, and that the mean-squared distance traveled by the RNA increases linearly with time, indicative of diffusive movement. We also observed that a single HIV-1 RNA molecule can move at various speeds when traveling through the cytoplasm, indicating that its movement is strongly affected by the immediate environment. To examine the effect of Gag protein on HIV-1 RNA transport, we analyzed the cytoplasmic HIV-1 RNA movement in the presence of sufficient Gag for virion assembly and found that HIV-1 RNA is still transported by diffusion with mobility similar to the mobility of RNAs unable to express functional Gag. These studies define a major mechanism of HIV-1 gene expression and resolve the long-standing question of how the RNA genome is transported to the assembly site.

Keywords: HIV-1; RNA; cytoplasm; diffusion; transport.

Fig. 1.

Detection and tracking of HIV-1 RNA in living cells. (A) General structures of the modified HIV-1 genome and the construct that expresses the fusion RNA-binding protein Bgl-YFP. Stem–loop sequences (18 copies) recognized by BglG RNA-binding protein engineered into the HIV-1 genome are shown in red. A polyA sequence is illustrated by a circle. nef, negative regulatory factor; NLS, nuclear localization signal; Pro, RNA polymerase II promoter; rev, regulator of expression of viral proteins; tat, transactivator of transcription. (B) Maximum intensity projection of 14 consecutive frames (99–112) from a time-lapse movie (Movie S1) acquired at 23.8 Hz with a 40-ms integration time to follow HIV-1 RNA movement in the cytoplasm. (C) Trajectories generated by selected signals in the boxed region in B. (D) Snapshots of the boxed region in B showing the identification and tracking of three molecules marked by red, green, and blue circles in various panels. A corresponding time-lapse movie of this region is shown in Movie S2. The panel labeled “Max” is the maximum intensity projection of the 14 frames; numbers at the bottom left of each panel refer to frame numbers in Movie S1. A Laplacian of Gaussian filter was applied using ImageJ. Three additional black tracks shown in C are not marked in D.

Fig. 2.

Representative trajectories and quantitative analyses of HIV-1 RNA mobility. (A) Selected trajectories representing diverse diffusive movements (I–V) and one directional track (VI). Trajectories are depicted with changing colors from start (red) to end (yellow). The maximum intensity projection (Max) of each trajectory is also shown for comparison (Right). (Left) In VI, the time for directional movement (D), and random walk (RW) are shown. Time-lapse movies are shown in Movie S3. (B) Distribution of the one-step jump distance of 1-AAG RNA. Data were binned (40-nm bin size) and normalized to the bin that contained the most events, which was set to 100. x axis, one-step jump distance (displacement); y axis, frequency in arbitrary units (a.u.). The distribution was fitted with a three-component model using a constant diffusion coefficient (D1 = 0.01 μm²⋅s⁻¹) to represent the stagnant fraction (or mobility under the detection limit in our system) (Fig. S3). The solid red line represents the fitted curve, and the three dotted lines indicate distributions for each of the mobility fractions. The fitting errors for D2 and D3 are ±0.01; the percentages shown are the proportions of each fraction. (C) Comparisons of fitted curves of all 1-AAG trajectories (red line), with trajectories containing at least one step at a distance >250 nm (green line) or <40 nm (blue line). The three-component fittings of trajectories containing at least one step at a distance >250 nm or <40 nm are shown in Fig. S5 A and B, respectively.

Fig. 3.

Effects of disrupting the cytoskeleton on 1-AAG RNA mobility. Distribution of one-step jump distances of 1-AAG tracks when cells were treated with cytochalasin-D (A) or nocodazole (B). Distribution and subpopulations of one-step jump distances were determined as described in Fig. 2B. The solid red line represents the fitted curve, and the three dotted lines indicate distributions for each of the mobility fractions. (C) Overlay of fitted curves of 1-AAG tracks without drug treatment (red), treated with cytochalasin-D (Cyto-D, blue), or treated with nocodazole (Noco, green).

Fig. 4.

Detection and tracking of HIV-1 RNA encoding Gag in living cells. (A) General structure of the modified HIV-1 genomes used for simultaneous detection of viral RNA and Gag. (B) Representative images of Gag and RNA signals. The first frame of a 20-s movie (Movie S4) of different channels is shown: Gag-mCherry (Left), RNA (Middle; YFP), and Gag and RNA (Right) signals. (Insets) Maximum intensity projection of 50 consecutive frames (frames 1–50) of the boxed region showing accumulated movement of puncta signals over 2 s. White arrows indicate colocalized Gag/RNA puncta at the cell periphery that showed little mobility. (Scale bars: images, 4 μm; insets, 2 μm.) (C) Distribution of one-step jump distances of RNA in the cytoplasm that did not colocalize with Gag puncta. (D) Overlay of fitted curves of 1-AAG tracks and 1-Gag-BSL tracks, which contained RNA tracks from 1-Gag-BSL and 1-GagmCherry-BSL. (E) General structures of the HIV-1 genomes encoding mutant Gag proteins in which the nucleocapsid domains were replaced with an LZ motif. (F) Distribution of one-step jump distances of 1-GagLZ-BSL RNA. (G) Overlay of fitted curves of 1-AAG tracks and 1-GagLZ-BSL tracks, which contained RNA tracks from 1-GagLZ-BSL and 1-GagLZmCherry-BSL. The distribution of each set of one-step jump distance analysis was fitted with a three-component model as described in Fig. 2B. The solid red line represents the fitted curve, and the three dotted lines indicate distributions for each of the mobility fractions.

Fig. 5.

Internalized assembled particles contribute to the observed directionally transported Gag–RNA complexes. (A) Visualization of directional cotrafficking of Gag/RNA subsequent to HIV-1 particle assembly at the plasma membrane. Representative images of Gag (Left), RNA (Middle), and both signals (Right) are shown. These panels are the first frame of a 19-s movie (Movie S6) acquired 17–18 h posttransfection and focus close to the bottom of the cell. (Insets) Maximum intensity projections of 50 consecutive frames (frames 1–50) of the boxed region showing accumulated movement of signals over 2 s. White arrows indicate five directional cotrafficking of Gag/RNA puncta. (Scale bars: images, 4 μm; insets, 2 μm.) (B) Representative images of Gag-mCherry and Rab7-YFP (Top) and the effects of nocodazole treatment (Bottom; 60 min, 10 μg/mL). Images show the first frame of 20-s movies (Movies S7 and S8, respectively) acquired 20–21 h posttransfection. (Insets) Maximum intensity projections showing the accumulated movement over 20 s of the boxed region. (Scale bars: images, 4 μm; insets, 2 μm.) (C) Representative images of Gag and RNA in cells cotransfected with WT dynamin (Top) or a dominant-negative K44A dynamin mutant (Bottom). Images in the panels are the first frames of the 20-s movies (Movie S9, Top and Movie S10, Bottom) acquired 17–18 h posttransfection and focus close to the bottom of the cells. (Insets) Maximum intensity projections of the boxed region showing Gag and/or RNA signal movement over 20 s. (Scale bars: images, 4 μm; insets, 2 μm.) (D) Factors influencing the proportions of RNA tracks that undergo directional movement. The percentage of directional RNA tracks analyzed from cells expressing 1-AAG-BSL or 1-GagmCherry-BSL/1-Gag-BSL (labeled as 1-Gag-BSL) is shown. The percentage was calculated by dividing the number of tracks containing at least one 18-step segment at a persistence index of ≥0.7 by the total number of tracks. The results marked as the 1-Gag-BSL late time point are from data presented in A.