Time course of ATIp synthesis and ATI formation.
In previous studies performed with CPXV, the ATIp was detected starting at 6 h postinfection (hpi) and ATIs at 9 to 12 hpi (16
). We employed a recombinant VACV that expresses the full-length CPXV ATI protein with a C-terminal HA tag, in place of the truncated A25 homolog, and the A4 core protein fused to YFP (vATI+
.A4:YFP) under the control of their natural VACV promoters. The high sensitivity and high resolution provided by fluorescent confocal microscopy were used to visualize ATIp synthesis with anti-HA antibody. The A4 protein fused to YFP allowed us to simultaneously monitor viral core protein synthesis and MV formation (17
). VFs and nuclei were stained with DAPI. HeLa cells were synchronously infected with vATI+
.A4:YFP and fixed at 3, 5, 7, 9, and 11 hpi. The VFs appeared as juxtanuclear bodies that were initially compact but became more diffuse after 5 h (A). Numerous punctate bodies containing ATIp were distributed throughout the cytoplasm as early as 3 hpi, whereas A4:YFP was detected in or adjacent to VFs at the same time (A). The ATIs increased in size by 5 hpi and had a characteristic donut appearance due to peripheral staining, presumably because of an inability of the antibody to penetrate the dense structures (A). In contrast to A4:YFP, ATIp was not seen in VFs in any of the infected cells viewed (n
= 50). ATIs continued to increase in size throughout infection, although some small ATIs were still seen at 9 hpi (A, arrowhead and inset). MVs appeared as punctate YFP structures, and those associated with ATIs were mostly at their periphery at 7 hpi. At 11 h, MVs were dispersed throughout many of the ATIs, as determined by analyzing Z-sections, whereas other ATI had few MVs (A).
Fig 1 Changes in ATI size and number over time. HeLa cells were infected with VACV strain vATI+A26+ and fixed at 3, 5, 7, 9, and 11 hpi. (A) Representative stacked confocal images of ATIs in infected HeLa cells. Rabbit anti-HA antibodies, followed by Alexa (more ...) As ATIs increased in size, they decreased in number.
We had the impression that ATIs increased in size as their number decreased. To quantify this relationship, we measured the diameters of ATIs and counted them at consecutive times during the course of infection. HeLa cells were infected with vATI+A26+.A4:YFP and fixed at 5, 7, 9, and 11 hpi. ATIs were visualized by anti-HA antibody staining of ATIp in the fixed cells, which were imaged as Z-stacks. IMARIS Bitplane software was used to measure the diameter of each ATI in the Z-section at which the diameter was greatest. The lowest threshold of ATI diameters was set at 0.3 μm because of the limits of resolution; therefore, ATIs that measured less than 0.3 μm in diameter were not scored. The mean size of ATIs increased from 0.66 ± 0.02 to 1.36 ± 0.04 μm from 5 to 11 hpi (B). During the same time, the mean number of ATIs decreased (B). Plotting the distribution of individual ATI diameters provided further insight. At 5 hpi, ATI diameters distributed as a sharp peak, indicating relative uniformity of sizes (C). By 7 hpi, the major peak shifted, indicating that the population as a whole had larger diameters and that the distribution was more heterogeneous than at 5 hpi. At 9 and 11 hpi, large-diameter ATIs predominated (C). We considered that the increase in ATI size and reduction in number could have been at least partly due to coalescence, although some small ATIs persisted or continued to form, as shown in the inset at the 9-h time point in A.
Enlargement of ATIs is due to continued protein synthesis and ATI coalescence.
Next, we compared the effects of inhibiting protein synthesis and microtubular transport on the formation of ATIs. HeLa cells were infected with vATI+A26+.A4:YFP for 4 h, and then the medium was replaced with (i) drug-free medium; (ii) medium containing CHX, an inhibitor of protein synthesis; (iii) medium containing NOC, which depolymerizes microtubules; or (iv) medium containing both drugs. Infected cells were incubated for an additional 5 h and fixed. ATIs were stained with anti-HA antibody and imaged as Z-stacks using confocal microscopy. ATIs were enumerated and the diameters measured as described above.
The ATI diameters seen at 9 hpi in cells incubated with CHX were smaller than those in untreated cells at the same time point but larger than ATI diameters in cells fixed at 4 hpi (time zero) (A). The impression that new protein synthesis contributed to ATI size was corroborated by measurements showing that the peak of ATI diameters in cells incubated with CHX did not shift as far toward a value representing larger sizes as the peak of ATI in untreated cells (B). In addition to some increase in ATI sizes, the average ATI number per cell decreased in the presence of CHX (C).
Fig 2 Contribution of new protein synthesis and microtubular transport to ATI size and number. (A) Stacked confocal images. CHX (10 μg/ml) or NOC (30 μm) or both or untreated medium (No drug) was added to vATI+A26+.A4:YFP-infected HeLa cells (more ...)
We considered that the increase in size and decrease in number of ATIs during infection even in the presence of CHX might have been due to coalescence and might require microtubules. NOC treatment depolymerized microtubules, as shown in A, and resulted in a relatively narrow peak of ATIs representing structures of greater uniformity and smaller diameter than occurred without drug (B). Furthermore, the number of ATIs in NOC-treated cells was greater than in untreated cells (C), consistent with an inhibition of coalescence.
The ATI diameters after incubation with both NOC and CHX were more drastically reduced and resembled the peak of diameters in cells fixed at 4 hpi (time zero) (A and B). With CHX and NOC treatment, the mean number of ATIs per cell was slightly reduced compared to the number seen at time zero, likely reflecting the effects of the combination of inhibition of new ATIp synthesis and prevention of coalescence (C). The data suggested that both new protein synthesis and intact microtubules contributed to ATI enlargement.
Visualization of ATI coalescence.
The finding that ATIs enlarged while the mean number per cell decreased with time suggested that inclusion bodies were merging and coalescing. Moreover, the inhibition by NOC implied a role for microtubules. To visualize ATI dynamics over time, we transfected plasmids expressing mCherry fused to the N terminus of ATIp (pCherry:ATIHA) into cells infected with VACV expressing YFP fused to the A4 core protein. We tracked individual mCherry-fluorescing ATIs and YFP-fluorescing MVs by collecting Z-stack data with a confocal microscope at 15- to 30-min intervals between 12 and 16 hpi. A shows an image of a 3D reconstruction of Z-stacks at 12 hpi. The lower ATI shown in the box is filled with MVs, whereas the upper ATI has MVs mostly at the periphery. B contains individual sectional images taken at 30-min intervals, showing merger of the two ATIs. An intermediate bilobed structure was evident at 12.5 hpi. The YFP fluorescence gradually redistributed within the merged ATIs over the 4-h interval. The box in the right panel of A shows an image of a 3D reconstruction of the merged ATI at 16 h.
Fig 3 Visualization of coalescing ATIs. (A) Images of confocal Z-sections reconstructed in 3D, showing a portion of a vA25−A26+.A4:YFP-infected cell at 12 h posttransfection (left) and 16 h posttransfection (right) with plasmids expressing mCherry fused (more ...)
We conducted similar experiments to determine if ATIs coalesce in CPXV-infected cells. Cells infected with CPXV-BR or vATI+A26+ were transfected with pCherry:ATIHA to visualize ATIs and imaged from 8 to 18 hpi. A total of 20 ATIs from each infection in two separate experiments were selected randomly and monitored for coalescence. As shown in Fig. S1 in the supplemental material, 16 and 14 ATIs in CPXV and recombinant VACV infections, respectively, were observed to coalesce with at least one other ATI. Anti-ATI antibodies and mCherry colabeled ATIs in cells infected with CPXV and transfected with pCherry:ATIHA in fixed-cell experiments (data not shown). Since CPXV-BR lacks a functional A26 protein, MVs were not associated with ATIs. Nevertheless, coalescence of ATI occurred, indicating that MVs were not required for this event.
Live imaging of cells infected with a vATI+A26+.A4:YFP was performed with a confocoal microscope at 8 hpi. At that time, MVs began to associate with the peripheries of ATIs, which could be recognized as spherical structures studded with YFP-fluorescing MVs. In contrast, VFs containing A4:YFP appeared as amorphous structures and were easily distinguishable from ATIs. This distinction was confirmed by analysis of cells infected with a similar virus expressing the truncated ATIp, which did not contain the spherical structures (data not shown), and in experiments using fluorescently labeled ATIp, as shown subsequently (A, right panel). As shown in C and Movie S1 in the supplemental material, two mobile ATIs studded with MVs on their exteriors contacted and merged into a larger, spherical ATI.
Fig 4 Contribution of coalescence to MV distribution within ATI. (A) Representative images of filled and unfilled ATIs. HeLa cells were infected with vA25−A26+.A4:YFP and transfected with plasmids expressing mCherry fused to ATIp. Images shown represent (more ...) Correlation of ATI coalescence with increased numbers of MVs within ATIs.
The matrix of one of the two ATIs coalescing in B was filled with MVs whereas the other had MVs mainly on the periphery. Immediately after coalescence, the MVs remained in the portion of the matrix that the filled ATI contributed to the merger. Gradually, however, the MVs became more evenly distributed within the merged ATI (B). The latter image and others like it suggested that coalescence drives MVs into the interior of the ATI matrix. To further assess the effects of coalescence on MV embedment, we used live-cell microscopy to follow individual ATIs over time. We infected cells with vA25−A26+.A4:YFP and transfected them with plasmids expressing mCherry fused to ATIp. A illustrates a filled ATI with MVs in the interior (left panel) and an unfilled ATI with MVs only at the surface (right panel). Note that a Z-stack analysis is necessary to distinguish the two types of ATIs. A time series of images was made from 10 to 18 h after transfection. We randomly picked filled and unfilled ATIs present at 18 h and then traced them back in time. Eight ATIs from each group that were not filled with MVs at 10 h were analyzed over the entire 8-h time period. Only two of the ATIs that remained unfilled at 18 h had coalesced with at least one other ATI during the 8-h time course, whereas seven of the eight filled ATIs merged with at least one other ATI (). The filled and unfilled ATIs were defined as regions of interest using the IMARIS Surfaces feature that allows measurements for the mCherry and YFP channels within those ATIs to be extracted over time (see Movie S2 in the supplemental material). The time series for the 16 ATIs scored for volume (calculated as the sum of the mCherry pixels within the ATI) and MV content (calculated as the YFP fluorescent intensity sum) are shown in B and C. The ATIs observed visually to have been filled also had the greatest increase in YFP fluorescence over time (C). Most filled inclusion bodies underwent several coalescence events, as is reflected in the overall greater increase in volume of the filled inclusion bodies relative to the unfilled ATIs (B). An exception, filled ATI 7 (F_7) was not observed to coalesce (), and we did not observe an increase in volume with ATI F_7 relative to the unfilled ATIs (B). F_6 and F_2 were graphed separately, because the increases in volume and YFP fluorescent intensities were out of range relative to the data determined for the other ATIs (D). With both F_2 and F_6, steep increases of YFP fluorescence correlated with coalescence with ATIs containing MV, as indicated by the concomitant increase in ATI volume. The cumulative mean YFP intensity at 18 h posttransfection of coalesced ATIs was higher than that of noncoalesced ATIs (E).
Incidence of coalescence of unfilled and filled ATIsa
Microtubules are important for MV embedment within ATIs.
Thus far, our focus had been on changes in the size of ATI, and we showed that the changes resulted from enlargement and coalescence due to protein synthesis and microtubules, respectively. In addition, coalescence contributed to the internalization of MVs from the periphery of the ATIs. Further experiments were carried out to more directly assess the contributions of microtubules and protein synthesis to embedment of MVs. HeLa cells were infected with vA25−A26+.A4:YFP and transfected with plasmids expressing mCherry fused to ATIp. At 8 hpi, cells were treated with NOC with or without CHX or with CHX alone. After an additional 2 h, we selected ATIs containing MVs only at the peripheries and collected Z-stacks at 20-min intervals for 8 h. We then compared the effects of the drugs on MV embedment within the matrix of 10 ATIs at 18 h posttransfection. The mean fluorescent intensity of YFP in ATIs determined as described in the preceding section was lower in cells treated with NOC or CHX alone or together compared to that seen in ATIs in untreated cells (A), demonstrating that the microtubular network and new protein synthesis are important for the embedment of MVs within ATIs.
Fig 5 Effects of NOC and CHX on occlusion. (A) HeLa cells were infected with vA25−A26+.A4:YFP and transfected with plasmids expressing mCherry fused to ATIp. Bars indicate the mean intensities of YFP at 10 h posttransfection within ATIs in cells treated (more ...)
We also compared the effects of NOC and cytochalasin D, an inhibitor of actin polymerization, on the association of MVs with ATIs following virion assembly. HeLa cells were infected with vATI+
.A4:YFP in the presence of rifampin, which reversibly prevents virus assembly (11
) without blocking ATI formation (14
). After 10 h, the rifampin was washed out and the cells were incubated for an additional 10 h in the presence of either NOC or cytochalasin D. The cells were fixed and stained with anti-HA antibodies to detect ATIp; occlusion of MVs was monitored using A4p:YFP fluorescence (B). The effects of the drugs on microtubules and actin were visualized using both mouse anti-α- and anti-β-tubulin antibody staining and 594-phalloidin staining, respectively (data not shown). ATIs in randomly selected cells from three separate experiments were scored as positive (+) if MVs were present at the periphery or within the matrix or negative (−) for occlusion if MVs were unassociated. Cytochalasin D caused cells to shrink and have a misshapen appearance but did not have much of an effect on occlusion (B). NOC, however, decreased the number of occluded ATIs by more than 60% (B).
MVs use microtubules for transport to ATIs.
Although microtubules could enhance MV embedment by increasing coalescence of ATIs, they might also have a more direct role in MV movement to ATIs. To facilitate the analysis of MV movement, we used the drug ST-246 to suppress the formation of WVs (33
), which are known to move on microtubules. HeLa cells were infected with vATI+
.A4:YFP in the presence of ST-246 and were imaged live at 8 hpi, a time at which ATIs encrusted with YFP-fluorescing MVs could be recognized by their characteristically spherical pattern. These ATIs were easily distinguishable from clusters of virions, as verified by imaging cells infected with VACV expressing a truncated ATIp and A4:YFP (data not shown). MVs were tracked by imaging them in the X, Y, and Z planes at 1 frame/1.573 s. The individual Z-stacks were typically about 4 μm thick. Some MVs were observed to associate with an ATI and then disassociate and move away from inclusion bodies. MVs were tracked with the IMARIS Spots function, and those that visibly adhered to inclusion bodies corresponding to at least 10 time points following contact were used for calculations. MVs moved to inclusion bodies at an average speed of 0.501 ± 0.637 μm/s (), which is consistent with transport on microtubules (31
). Particle velocities alternated between high and low, but particles did not pause as often as the MVs previously observed moving between VFs and sites of wrapping (31
Frame-by-frame measurements of movement of four individual virionsa
A26 is not required for MV movement but contributes to particle velocity.
A26 associates with MVs and is required for their occlusion, suggesting that A26 might be a movement protein (22
). We therefore compared MV particle dynamics in the presence and absence of A26 to see if there was a difference in movement. VACVs that expressed the truncated form of ATIp (A25) instead of full-length ATIp were used to prevent ATI formation, since this would lead to occlusion of A26+
but not A26−
particles. HeLa cells were infected with a VACV expressing an HA-tagged A25 and a V5-tagged A26 (vA25+
.A4:YFP) or an A26 deletion VACV expressing an HA-tagged A25 (vA25+
.A4:YFP). Following adsorption, the medium was replaced with medium containing ST-246 to prevent formation and movement of WVs. At 7 to 8 hpi, MVs were imaged live in regions of cells that were distal from factories and from virion aggregates in order to allow tracking of individual particles. Tracking was carried out using IMARIS Spots software, and the movement of MVs in the presence and absence of A26 was analyzed. MV tracks are shown in A and the track lengths and speeds in B. The deletion of A26 did not affect overall track lengths; however, mean particle speeds were significantly lower.
Fig 6 MV movement dynamics in the absence of A26. (A) Images of tracks in two representative cells are shown with color coding indicating speed. HeLa cells were infected with vA25+A26+.A4:YFP or vA25+A26−.A4:YFP in media containing 10 μm ST-246 (more ...)