A dynamic view of hepatitis C virus replication complexes

Abstract

Hepatitis C virus (HCV) replicates its genome in a membrane-associated replication complex (RC). Specific membrane alterations, designated membranous webs, represent predominant sites of HCV RNA replication. The principles governing HCV RC and membranous web formation are poorly understood. Here, we used replicons harboring a green fluorescent protein (GFP) insertion in nonstructural protein 5A (NS5A) to study HCV RCs in live cells. Two distinct patterns of NS5A-GFP were observed. (i) Large structures, representing membranous webs, showed restricted motility, were stable over many hours, were partitioned among daughter cells during cell division, and displayed a static internal architecture without detectable exchange of NS5A-GFP. (ii) In contrast, small structures, presumably representing small RCs, showed fast, saltatory movements over long distances. Both populations were associated with endoplasmic reticulum (ER) tubules, but only small RCs showed ER-independent, microtubule (MT)-dependent transport. We suggest that this MT-dependent transport sustains two distinct RC populations, which are both required during the HCV life cycle.

FIG. 1.

Movement patterns of HCV RCs. Live Huh-7.5-I/5A-GFP-6 cells were imaged with an Axiovert 100M inverted microscope with a Pan-Apochromat 100× NA 1.4 objective. Fluorescence was recorded with an exposure time of 750 ms at a rate of 1 Hz over a period of 120 s. Images at the top of panels A and B represent the first images of the recordings included as Videos S1 and S2 in the supplemental material. (A) Time-lapse recording of a single Huh-7.5-I/5A-GFP-6 cell. Representative structures were selected and their movement tracked as specified in Materials and Methods. Tracks 1 to 11 depict small structures that show one or more saltatory movement events. Tracks 12 and 13 are examples of stationary small structures. Tracks 14 and 15 are examples of large structures that show only confined movement patterns. Velocity versus time graphs for three selected structures are shown at the bottom. The small structure of track A.3 (d_FWHM = 0.48 μm) shows three saltatory movement events starting at 0, 51, and 110 s. The small structure in track A.12 (d_FWHM = 0.39 μm) and the large structure in track A.14 (FWHM dimensions: 2.1 by 1.2 μm) display peak velocities (v_peak) < 0.3 μm/s. (B) Saltatory long distance movement of a small structure. Tracking of a small structure (d_FWHM = 0.62 μm) revealed five saltatory movement events which displaced the structure by a distance of 19.1 μm. The locations at various times during the 120-s recording are indicated by white arrows. The durations given at the white arrows indicate the period of time the structure remained stationary. The dashed box indicates the region for which images from various time points are shown in the middle of the panel. Five saltatory movement events can be identified in the velocity versus time graph shown at the bottom. The four images in the middle show the position of the structure immediately before the second movement event at time point t = 50 s, during a short stop before the third event (t = 55 s), immediately after the third event (t = 61 s), and after the fourth event (t = 93 s). The black arrows at the top of the graph reflect these time points. Note that the structure leaves the focal plane during the last frames. All images were processed as reflected by the intensity scale bars on the right or at the bottom of the images, respectively (see Materials and Methods for further details; γ = 0.75). Scale bars, 5 μm.

FIG. 2.

Small NS5A-GFP structures contain further HCV replicase components. Huh-7.5-I/5A-GFP-6 cells were fixed, processed for indirect immunofluorescence staining, and analyzed by confocal laser scanning microscopy as described in Materials and Methods. Viral proteins were labeled with the primary mouse MAb 9E10 anti-NS5A or 7019 anti-NS3, which were detected with an Alexa Fluor 546-labeled goat anti-mouse antibody (Alx546). (A) Colocalization between NS5A-GFP autofluorescence (green) and 9E10 anti-NS5A staining (red). Both NS5A specific signals colocalize well, which is also indicated by the high Pearson correlation coefficient (R_c = 0.90). (B) Colocalization between NS5A-GFP (green) and NS3 (red). NS5A and NS3 also colocalize well (R_c = 0.79). (C and D) Enlargement of the dashed box region in panel A (C) and enlargement of the dashed box region in panel C (D) focus on several small structures. Points a and b, as well as points c and d, define two line segments that each cross several structures. Intensity profiles along the line segments, shown on the right of the images, demonstrate that NS5-GFP and NS3 also colocalize in small structures. Scale bars indicate reference distances of 5 and 1 μm, respectively; the length of the line segments between points a and b, as well as points c and d, are depicted underneath the intensity profiles. Intensity adjustments are reflected by the intensity scale bars as detailed in Materials and Methods.

FIG. 3.

Large HCV RCs are stable for many hours. Live Huh-7.5-I/5A-GFP-6 cells were analyzed with an inverted Zeiss LSM 510 confocal laser scanning microscope with a Plan-Apochromat 100× NA 1.4 objective for a time period of t = 14.7 h. An image stack comprising the full cell dimension in the z-direction was recorded every 6 min. Images were processed with a 3 × 3 median filter to reduce noise, for each time point a mean intensity z-projection of the image stack was generated, and pixel intensities were adjusted as specified in Materials and Methods (γ = 0.7). Projections of selected time points are shown as indicated in the captions. The recording can be found as Video S3 in the supplemental material. Two clusters of large structures can be identified in the perinuclear region. The size of the left cluster (angel bracket symbol) is about 7 by 10 μm, and about 15 individual large structures 1 to 2 μm in diameter can be counted in the projection image. The right cluster (arrow) has a size of 11 by 15 μm and contains about 20 large structures. While the left cluster disintegrates throughout the first 6 h of the time lapse, the right cluster can be followed throughout the 14.7-h recording. Of note, the settings used during acquisition and data processing do not allow the detection of the small structures shown in Fig. 1, 2, 6, 7, 8, and 9. Scale bar, 10 μm.

FIG. 4.

Large HCV RCs persist during cell division. Live Huh-7.5-I/5A-GFP-6 cells were analyzed with an inverted Zeiss LSM 510 confocal laser scanning microscope with a Plan-Neofluar 63× NA 1.25 objective for a time period of 10 h. An image stack comprising the full cell dimension in the z-direction was recorded every 15 min. Images were processed with a 3 × 3 median filter to reduce noise, and for each time point mean intensity z-projections were generated. Cell division occurred between 75 and 90 min of the recording. Projections of time points encompassing the cell division (white arrow) are shown, as indicated by the captions. Scale bar, 10 μm.

FIG. 5.

Static internal architecture of large HCV RCs. Live Huh-7.5-I/5A-GFP-6 cells were analyzed with an inverted Zeiss LSM 510 confocal laser scanning microscope with a Plan-Neofluar 63× NA 1.25 objective for a specified time period. Images of cells were recorded every 7 s. At the top, an overview image from the beginning of the experiment is shown. A rectangular region (dashed box) was bleached with a 488-nm laser pulse at time point t = −14 s for a duration of 14 s, as indicated in the time line. This area overlapped with one-half of a large structure representing a membranous web (d_FWHM = 1.7 μm; arrow). Enlargements of the continuous line box area for the time point before bleaching (t = −14 s), immediately after bleaching (t = 0 s), and 8 min after bleaching (t = 480 s) are shown in the middle panels. Directly after bleaching, no fluorescence could be detected in the right half of the large structure. The structure slowly migrated to the left during a time period of 8 min, as indicated by the distance to the dashed box, which was drawn at the original coordinates. However, no fluorescence recovery was detected in the bleached half. The nonbleached half, which was the only portion of the large structure that remained visible, sustained its fluorescence intensity and size, which indicates that NS5A-GFP was not redistributed between the bleached and nonbleached halves. Three potential outcomes of this experiment are illustrated at the bottom, as discussed in Results. Note that the outcome illustrated in diagram c was observed in this experiment. Scale bars indicate reference distances of 5 and 2 μm, respectively.

FIG. 6.

Saltatory movement of small HCV RCs is MT dependent. Huh-7.5-I/5A-GFP-6 cells were cultured in the presence of 20 μM nocodazole (A) or carrier alone (dimethyl sulfoxide, 0.1% final concentration) (B). Images were recorded with an acquisition rate of 3.5 Hz over a period of 4.5 min by spinning disk confocal microscopy, and pixel intensities were adjusted as specified in Materials and Methods (γ = 0.9). Long-distance saltatory movement events were tracked as specified in Materials and Methods. Tracks for representative structures, which display saltatory movement events, are shown as a white overlay on top of the images. Images shown in each panel are medium intensity t-projections of the first four images of the recordings to reduce noise. The entire recordings can be found as Videos S4 and S5 in the supplemental material. Cells treated with nocodazole did not display saltatory movement events. Scale bar, 5 μm.

FIG. 7.

HCV RCs are associated with MTs and the ER. Huh-7.5-I/5A-GFP-6 cells were fixed and processed for indirect immunofluorescence staining as described in Materials and Methods. Primary mouse MAbs against α-tubulin or calnexin were used to detect MTs and the ER, respectively. Stacks of images were acquired by confocal laser scanning microscopy and deconvoluted. (A) Association of HCV RCs with MTs. An overview image is shown at the top. MTs (red) form a dense network in Huh-7.5 cells. NS5A-GFP (green) is found in large and small structures. The area marked by the dashed box is enlarged below and shows that the structures are located in close proximity to MTs. Green intensity settings were adjusted in the enlargement to match the ER signal in this area. (B) Association of HCV RCs with the ER. An enlarged region from a deconvoluted image stack is shown (see Fig. 8C for an overview image). Small NS5A-GFP structures (green) are closely associated with the ER (red) and are found either on top or directly adjacent to ER tubules; however, colocalization on a per pixel basis is hardly detectable. NS5A-GFP structures often bridge a gap in the ER marker signal (arrow), which suggests that they may exclude the ER marker protein. Scale bars indicate reference distances of 20 and 1 μm, respectively.

FIG. 8.

HCV RCs are not associated with ERES. Huh-7.5-I/5A-GFP-6 cells were fixed, processed for indirect immunofluorescence staining, and analyzed by confocal laser scanning microscopy as described in Materials and Methods. Antibodies against (A) Sec13 and Sec31 (B) were used as markers for ERES, and antibodies specific for calnexin (C) and golgin-97 (D) were used as markers for ER and the Golgi apparatus, respectively. Primary antibodies were detected with Alexa Fluor 546-labeled secondary antibodies (Alx546). Interestingly, no colocalization was found between NS5A-GFP and ERES. The dot plots show that image pixels were only positive for either NS5A-GFP or the ER exit marker (R_c = 0.13 or R_c = 0.17). Slightly better colocalization was found with the ER marker calnexin (R_c = 0.43). No colocalization was found with golgin-97 (R_c = 0.28). Scale bars, 5 μm.

FIG. 9.

Association of HCV RCs with the ER in live cells. Huh-7.5-I/5A-GFP-6 cells were examined 36 h after transfection with the red fluorescent ER marker IgLdR1kdel. (A) Confocal laser scanning microscopy was used to acquire green NS5A-GFP and red ER fluorescence simultaneously with a rate of 0.1 Hz. An overview image is shown on the left. The area in the dashed box is shown on the right for four different time points, as indicated by the captions. The entire recording is shown in Video S6 in the supplemental material. Arrows indicate two NS5A-GFP structures that were monitored over time. Both large and small structures follow the highly dynamic ER movement. Of note, fast saltatory movements could not be recorded due to the slow frame rate. (B to D) Spinning disk confocal microscopy in combination with a beam splitter was used to simultaneously detect NS5A-GFP and the red ER marker at high acquisition rates of 2 Hz. Images of the time points indicated are shown. For panels B and C, intensity profiles along the indicated dashed lines are shown on the right for all time points. (B) Fast saltatory movement of small structures was found to occur independent of ER movement. The highlighted structure moves along ER tubules (v_peak = 1.8 μm/s), but no ER movement can be detected in parallel. The entire recording is shown in Video S7 in the supplemental material. (C) Comigration of ER (v_peak = 0.8 μm/s) was observed only in some instances. (D) Small NS5A-GFP structures were also found to follow reorganization or growth of ER tubules either directly at the tip (angel bracket symbol) or at some distance from the tip (arrow). All images were processes as specified in Materials and Methods. For noise reduction, a 3 × 3 median filter was applied to images in panel A. In the images in panels B to D, a Kalman filter was applied. This better preserved spatial separation perpendicular to the direction of movement but resulted in artificial trail formation along the axis of migration. Therefore, the intensity track profiles shown are based on images that were filtered by a spatial Gaussian blur filter only.