Virus egress occurs at specific sites on adherent surfaces of Vero cells.
To study the process of HSV-1 egress in Vero cells, electron microscopy was used to observe the pattern of progeny virus association with the cell surface. Glass-grown Vero cells were infected with KOS HSV-1 at an MOI of 10, and at 12 h postinfection cells were fixed on coverslips and processed for thin sectioning. At this time point, progeny virions that were not transferred to nearby cells were found to be associated with the parental cell surface with few virions released into the media. Micrographs showed that at 12 hpi, the majority of virions were observed at specific areas on the cell surface, rather than in a randomly dispersed release pattern ( to D). Virus-containing regions were located at cell-cell contact sites and at areas along the adherent cell surface. There were approximately 3-fold more virions at these locations than on the nonadherent upper cell surface, although this is likely an underestimation, since some virions along the upper cell surfaces are expected to be noninfectious parental virus particles that did not enter the cell. At both the substrate-adherent surface and at cell-cell contact site egress locations, additional membrane was present allowing a curvature in the membrane at the site and the creation of a pocket-like structure ( to C). This was not the case in uninfected cell samples; the adherent cell membrane of mock-infected Vero cells was tightly apposed to the coverslip surface ( and ). Although many virions were observed exterior to the plasma membrane in infected cells, none were observed near the interior side of the membrane ( to D). The few virions seen on other membrane surfaces were often in areas adjacent to cell-cell contacts (, bracket). Similar results were obtained whether the cells were in a confluent monolayer or if they were less densely plated; the movement of virions to the substrate-adherent surface was not induced by a lack of available cell-cell-adherent surfaces.
Fig 1 Location of progeny virions in HSV-1-infected Vero cells. (A to D) Thin-section electron micrographs of infected Vero cells fixed and processed on coverslips at 12 hpi. Note that the majority of virions are released in pockets along the adherent cell (more ...)
Confocal microscopy was utilized to observe the release of virions in larger numbers of infected Vero cells. Cells grown on glass coverslips were infected with a GFP-tagged VP26 mutant (K26GFP) and fixed at 12 hpi. Confocal Z-stack images showed that GFP-labeled virions were concentrated along the adherent surfaces of cells. A representative series is shown in . Most virions outside the nucleus were detected at the two planes closest to the coverslip (those marked by an asterisk). The majority of virions that were visible above the two planes were located at cell-cell contact points (, arrows in slices 4 to 6). The large GFP-containing areas located in the nucleus are capsid assembly compartments. In the following sections we further characterize the egress sites that form along the coverslip-adherent cell surface.
Viral egress sites are enriched in glycoproteins.
Due to a high cytoplasmic signal of viral proteins, egress sites along the adherent cell surface were best visualized using total internal reflection fluorescence (TIRF) microscopy. This method allowed the area of excitation to be restricted to a small plane above the coverslip. A standard TIRF field is 70 to 300 nm above the coverslip. The angle of the laser can be adjusted to obtain the desired excitation field within this range (50
). In the following studies, the laser was set at the maximum angle to allow the excitation of the largest area possible, which is 300 nm above the coverslip.
Using TIRF microscopy, we found that GFP-labeled virions clustered at specific sites along the adherent cell surface in a manner similar to that observed with EM (). In addition, infection induced the recruitment of glycoproteins to egress locations. Wheat germ agglutinin (WGA) binds to N
-acetylglucosamine and N
-acetylneuraminic acid (sialic acid) residues on glycoproteins, whether viral or cellular in origin (45
). When infected Vero cells were fixed, permeabilized, and stained with rhodamine-labeled WGA, it was observed that viral release sites were stained much more strongly than the surrounding cell membrane ( and ), indicating that the regions where cell-associated viral particles were observed had a much greater concentration of glycoproteins. Such focal concentrations were absent from uninfected cells, where the WGA stain was light and diffuse (). In fact, WGA staining outside the patches was much brighter in infected cells than in uninfected cells, suggesting a greater amount of surface glycoprotein expression overall. There were often several viral egress patches per infected cell, yet cells with a single large patch were also seen. Patches could be expansive and were generally peripheral, as shown in .
Fig 2 Glycoprotein enrichment at coverslip-adherent surface egress sites. (A) TIRF micrographs of a VP26-GFP HSV-1-infected Vero cell at 12 hpi fixed, permeabilized, and stained with rhodamine-conjugated WGA (Rh-WGA) to mark glycosylated proteins. As in EM (more ...)
Viral glycoproteins were found to be a component of WGA-stained patches. Infected Vero cells were fixed at 12 hpi and stained with α-gB, α-gD, α-gH, or α-gE monoclonal antibody. Staining for gB and gD are shown in and , while gH and gE staining is not shown. All viral glycoproteins were found in greater amounts in the regions where cell-associated virus was concentrated compared to the surrounding membrane. Viral glycoprotein staining in infected cells entirely overlapped with WGA staining (). Viral proteins appear to compose a large portion of the glycoprotein patches, but cellular proteins also may be recruited. Since both viral glycoprotein staining and WGA staining similarly define the size and shape of egress locations, patches were labeled using rhodamine-WGA in the following figures. Patches were observed in all cells at 12 hpi that were labeled with viral glycoprotein antibodies, suggesting that the majority of infected cells form these structures (data not shown). Many glycoprotein-stained patches resembled a donut in shape (, arrowheads). This phenomenon was due to the pocket-like structure of these sites. The holes in the donuts were the result of the membrane rising above the 300-nm excitation limit in the center of the patches.
Concentrations of virions and glycoproteins along the adherent surface were not specific to Vero cells but seemed to be associated with a nonpolarized state. Similar adherent surface glycoprotein patches were observed in infected HeLa cells (a nonpolarized cell type), but glycoproteins appeared to accumulate only at cell-cell junctions in polarized HEC-1A cells (data not shown).
Glycoprotein patches form independently from trafficking virions.
Vero cells infected at an MOI of 10 showed neither glycoprotein staining nor VP26-GFP virus signal at 4 hpi (, top). At 6 h postinfection, small glycoprotein patches were detectable, although virions were rarely seen associating with these areas (, middle). By 8 hpi, glycoprotein patches were more widespread and low levels of virus were consistently detected associating with the patches (, bottom). This observation indicates that release sites were determined early in infection. Glycoproteins arrived first, modifying the composition of the egress site membrane. Virions then associated with these membranes at later time points.
Fig 3 TIRF micrographs showing formation of glycoprotein patch sites uncoupled from virus egress. (A) Vero cells infected with VP26-GFP HSV-1 were fixed at 4, 6, and 8 hpi. Patch glycoproteins were labeled with rhodamine-WGA. All images were taken at the same (more ...)
To further explore the differential trafficking between virions and glycoproteins, Vero cells were infected with a ΔUL25 mutant (KUL25NS). The gene product of UL25 is needed for the packaging of DNA into the capsid; when it is absent, DNA is not packaged and capsids do not exit the nucleus. However, protein expression continues normally (31
). If glycoprotein movement to the cell surface was induced by virus envelopment and trafficking, then we would expect to see a drop in the size of glycoprotein patches in cells infected with the ΔUL25 virus compared to the WT. If the glycoprotein concentrations observed were actually glycoproteins on virion envelopes, then we would expect to see no glycoproteins on the surface with the ΔUL25 mutant.
To test this idea, Vero cells were infected with either WT KOS-HSV-1 or the ΔUL25 mutant for 12 h. Cells were then fixed, permeabilized, and stained with WGA. Capsids were labeled with α-VP5 monoclonal antibody and Alexa 488-labeled secondary antibody. In the KOS-infected cells, virus and glycoproteins were found to colocalize at release site patches as expected (, top). In ΔUL25 mutant-infected cells, glycoproteins formed normal patches even though capsids were not present (, bottom). In addition, the patches were approximately equal in size to those formed in the infection with wild-type UL25 gene product (data not shown). These results provide evidence that patch glycoproteins are in the cell membrane of infected Vero cells, and glycoproteins traffic to and accumulate at viral egress sites independently of virions.
Release sites are depleted of cytoskeletal elements.
It has been previously shown that the cellular cytoskeleton is altered during HSV-1 infection. The microtubule organizing center (MTOC) is disrupted and microtubules become sparse and disorganized (23
). Although peripheral anti-alpha-tubulin-stained microtubules could be seen in our samples, results showed that these microtubules did not colocalize with virions at peripheral egress sites (). Actin stress fibers are also largely depolymerized during infection (16
). When infected Vero cells were stained for actin with Texas Red-labeled phalloidin, few stress fibers were visible but the actin cortex (the unbundled, highly branched actin lining the cell membrane) could still be seen in most cases (). Contrary to our expectations, the actin cortex was depleted at the viral egress sites. It is unlikely that the actin holes observed were due to limitations of the TIRF laser; depleted areas were still apparent when the entire membrane was within range of the laser. In addition, the majority of areas where actin was cleared contained a glycoprotein patch whose edges closely followed the line of actin depletion even if few virions were visible (data not shown). A similar result was observed with focal adhesions (). Although patches often formed shapes that mimicked large focal adhesions, focal adhesions were not present at the viral egress sites; they were depleted in areas where the patches formed. Areas of microtubule, actin, and focal adhesion depletion were rarely detected in mock-infected cells ( to F). They appeared only after HSV infection.
Fig 4 Location of cytoskeletal elements in relation to viral egress sites on the coverslip-adherent surface of HSV-1-infected Vero cells as shown by TIRF microscopy. (A to C) VP26-GFP HSV-infected Vero cells stained with α-tubulin antibody to label (more ...) Both actin and microtubules contribute to glycoprotein patch formation and virion trafficking to release sites.
Although viral egress sites were depleted of cytoskeletal elements, it was likely that microtubules, actin, or both were involved in virus trafficking. The expected size and density of virus-containing vesicles along with the nonrandom release of virions suggested that a structural element was assisting their movement and delivery to their destination. The possible role of microtubules and actin was investigated by depolymerizing their filaments at increasing intervals after infection. VP26-GFP HSV-1-infected cells were treated at 4 hpi (before patches were visible and infectious virus detectable), 8 hpi (patches were expanding, a few virions could be seen), or 11.5 hpi (patches were large and contained many virus particles). Treatment included either 10 μg/ml nocodazole to depolymerize microtubules, 1.7 μg/ml cytochalasin B to depolymerize actin, or 3.3 μl/ml DMSO as a control. Each drug concentration used was the minimal amount needed to completely depolymerize the cytoskeleton. Treated cells were fixed at 12 hpi and stained with rhodamine-WGA to define the patch outlines. Resulting TIRF images ( to C) were then analyzed as described in Materials and Methods.
Fig 5 Role of microtubules and actin in virus/glycoprotein trafficking and patch formation. Cells were infected with VP26-GFP HSV-1 and fixed at 12 hpi. (A) TIRF micrograph of DMSO-treated sample stained with rhodamine-WGA to mark glycoprotein patches. (B) (more ...)
It was found that the depolymerization of the microtubules during infection caused a significant reduction in the percentage of cell membrane covered by glycoprotein patches. Greater effects were observed at the earlier time point addition ( and ). In addition, depolymerization at early time points caused a reduction in the total number of virions on the visible cell membrane at the adherent surface ( and ). No significant effect was observed when nocodazole was added later in infection after patches had formed and many virions were present. The determination of virus titers showed that there was no statistically significant effect on titer throughout several experimental repetitions (data not shown), although small reductions in infectious virus are difficult to measure. The results are interpreted to indicate that the lack of microtubules impedes the trafficking of glycoproteins to the cell surface. The decrease in virus at the cell membrane is also likely due to inhibited transport to the surface, although the depolymerization of microtubules could also be hindering the envelopment of virus.
Surprisingly, it was observed that disrupting the actin cytoskeleton decreased the number and size of egress patches at all time points tested ( and ). This effect was more pronounced when the cytoskeleton was depolymerized early in infection, but it was also seen when actin was depolymerized after patches had already formed. As actin was disrupted, the patches that had formed were dispersed into very small glycoprotein aggregations with the occasional associated virion. The result that previously formed patches disassemble when filamentous actin is removed is interpreted to indicate that actin filaments are maintaining the structure of egress sites.
Treatment with cytochalasin B early in infection decreased the amount of virus on the cell surface (). This suggested that actin is involved in trafficking of virus to egress locations as well as maintaining patch integrity. There was no statistically significant difference in titers of cell-associated or supernatant virus for all treatments, so dispersing the patches after formation likely did not induce the release of the surface virions into the supernatant (data not shown). The observed decrease in detectable virus on the membrane when cells were treated from 11.5 to 12 hpi was probably due to the diffusion of virions onto the top membrane, where they could not be detected using TIRF microscopy. Actin could be assisting the trafficking of virions from the cytoplasm to the cell surface, or it could be assisting newly secreted virus to move to patch locations through actin surfing. We conclude that actin contributes to both virus trafficking to egress sites and to the maintenance of viral and possibly cellular components at these sites.
The structure of egress sites can reform after disruption.
During the process of secretion in secretory cell types, actin is locally depolymerized in the cortex, allowing the passage of a vesicle. Existing studies have found that the depolymerized areas are often not much larger than the secretory vesicle, and the cytoskeleton generally reforms after exocytosis has occurred (13
). One explanation that we considered for the existence of actin holes at viral egress sites was that actin depolymerized to allow the exocytosis of the virus, and the holes remained due to a global block in actin repolymerization. We also considered the possibility that the actin cortex as a whole is lost during infection (likely through a block in actin polymerization) and viral release is directed to areas where this has occurred.
To test these ideas, Vero cells with observable patches () were treated with 1.7 μg/ml cytochalasin B for 30 min. Actin was depolymerized as expected, as shown in . The drug was then rinsed out and infection continued for another 45 min. Samples were stained with either WGA or phalloidin. The results showed that the actin cortex was able to reform after depolymerization; there was no apparent block in actin filament polymerization or elongation (). In addition, the viral egress sites were able to reform after disruption; patches consisting of dense glycoproteins and GFP-labeled virus were visible after the 45-min recovery period (). It is unclear whether the dispersed virus/glycoproteins reformed into patches or the visible patches were simply new virus released since the restoration of the actin cortex. In either case, we conclude that actin was able to polymerize normally and infected cells were able to create new egress sites late in infection. The clearance of actin at these sites appears to be locally rather than globally induced.
Fig 6 TIRF micrographs showing egress site reformation after actin depolymerization-induced disruption. VP26-GFP-infected Vero cells at 11.5 hpi (A) were treated with 1.7 μg/ml cytochalasin B for 30 min (B). (C) Toxin was then rinsed out and the infection (more ...) Glycoprotein E is necessary for glycoprotein patch formation but not trafficking of virus to release sites.
Directed egress in polarized cells such as keratinocytes and neurons is dependent upon viral glycoprotein E (gE). gE null mutants produce normal levels of progeny virus, but the trafficking of virions to egress sites is disrupted (8
). We therefore wanted to test the role of this protein in HSV-1-infected nonpolarized cells. To test the function of gE in the trafficking of viral components to the coverslip-adherent cell surface, we ultilized ΔgE, gEΔCT, and gEΔCT rescue mutants. Using a bacterial artificial chromosome system, the start codon was replaced with a stop codon in the gE gene sequence. To create the gE cytoplasmic tail deletion mutant, two stop codons were added at residue 446 directly after the transmembrane region (14
). The expression of gE was rescued by removing these stop codons.
The mutants were verified by harvesting the supernatant and cell lysates of infected Vero cells and staining for the appropriate protein (). The results showed that staining for gE was seen in the WT and rescue infection but was absent from the cell lysates and virions of the gE deletion mutants. The gE cytoplasmic tail deletion protein was incorporated into the virion at a much lower level and was less stable than its full-length counterpart. In cell lysate samples, the gE band was shifted compared to that of virions due to the presence of large amounts of immature gE ().
Fig 7 Western blot analysis of gE expression in deletion and rescue mutants. Cell lysates and supernatants of wild-type HSV- and gE mutant-infected Vero cells were collected at 18 hpi. Samples were run on an SDS-polyacrylamide gel, transferred, and blotted (more ...)
To test the role of gE in the creation of release sites and the egress of virions at these sites, Vero cells grown on glass coverslips were infected with ΔgE, gEΔCT, and gEΔCT rescue mutants. A low MOI of 0.3 was used to ensure infections arose from a single virion. To compare virus production, titer samples were collected at 10 hpi, a time at which each infected cell was producing virus but second-generation infected cells were not yet doing so. In this way, the effect of the gE deletions on the formation of infectious virus could be determined separately from effects on cell-to-cell spread. At 12 hpi, infected Vero cells were fixed with 4% PFA and treated with α-VP5 antibody (labeling major capsid protein) and Alexa 488-conjugated secondary antibody. Samples were then stained with rhodamine-WGA to label glycoprotein patches and viewed using TIRF microscopy. Twenty cells per infection sample were measured as described in Materials and Methods for both patch coverage and density of virions on the cell surface.
The results showed that the lack of gE or the gE cytoplasmic tail caused a reduction in the percentage of cell surface covered in glycoprotein patches compared to that of the rescue virus ( and ). However, while there was an inhibition in glycoprotein patch formation, the number of virions on the cell surface of the ΔgE mutants increased slightly ( and ) for unknown reasons. There was no statistical difference in titers, indicating that the production of progeny infectious virus was not increased (). The results are interpreted to indicate that gE is important for the expansion of glycoprotein patches at egress locations, but unlike in polarized epithelial cells, gE does not direct virions to these sites. The gE protein appears to play different roles in the spread of infection in polarized and nonpolarized epithelial cells.
Fig 8 Effect of gE and gE cytoplasmic tail deletions on patch formation and progeny virion trafficking. (A) TIRF images of Vero cells infected with the ΔgE mutant, the gEΔCT mutant, or the gEΔCT rescue virus at an MOI of 0.3 for 12 h. (more ...)