Early Events during HSV-1 Entry
Due to the large size of HSV-1 virions and capsids, early virus cell interactions can be readily visualized by EM (Campadelli-Fiume et al., 1988
; Fuller et al., 1989
; Lycke et al., 1988
). We followed the incoming capsids at different stages of entry in Vero cells (Fig. ). The extracellular, surface-bound virus particles had the familiar features of a herpes virus: a membrane envelope with spikes, a capsid with an electron-dense DNA core, and a tegument layer between the capsid and the envelope (Fig. , a
). Images of fusion between the viral envelope and the plasma membrane were readily obtained (Fig. , c
). After fusion, the capsids appeared to separate from the bulk of the tegument since an electron-dense mass remained associated with the cytoplasmic surface of the plasma membrane (Fig. , e
; see also CampadelliFiume et al., 1988; Fuller et al., 1989
Figure 1 Entry and uncoating of HSV-1. Ultrathin Epon sections of Vero cells infected with HSV-1 at an MOI of 500 in the presence of cycloheximide (CH). After 2 h of virus binding at 4°C, the cells were either fixed immediately (a and b), or warmed up (more ...)
After 1 h, the first capsids had reached the nucleus and attached themselves to the nuclear pore complexes (Fig. , g
; see also Batterson et al., 1983
; Lyke et al., 1988). They seemed to bind to cytsosolic fibers emerging from the pores (see arrowheads
in Fig. h
). Since most of the capsids on the nuclear envelope lacked the electron-dense central mass, DNA release had apparently occurred. At later time points, some empty capsids were also seen free in the cytosol without connection to the nucleus. Occasionally, 1 h postinfection and later, intact and partially degraded virions were also seen in endosomes and lysosomes, indicating that a fraction of the inoculum was taken up by endocytosis.
The rate and efficiency of cell surface binding and subsequent internalization was assayed quantitatively using [3
H]thymidine-labeled HSV-1. To monitor binding, cells were incubated with labeled virus at an MOI of 10, 50, or 100 at 4°C, and the cell-associated radioactivity was measured at different times. As previously reported, a continuous increase in virus binding was observed up to 4 h with no signs of saturation (Fig. a
; McClain and Fuller, 1994
; Shieh et al., 1992
). At 2 h—the adsorption period used in all subsequent experiments—
~40% of the added virus was bound.
Figure 2 Efficiency and kinetics of HSV-1 binding and internalization. (a) Using [3H]thymidine-labeled virus at different multiplicities of infection (MOI), the amount of virus bound to cells at 4°C was quantified after various time points. There is no (more ...)
To follow internalization biochemically, a protease protection assay was used (Helenius et al., 1980
). After a 2-h binding period in the cold, the cells were warmed up to 37°C for different periods of time to initiate penetration. Thereafter, they were chilled to 4°C and treated with proteinase K to detach remaining cell surface viruses. The fraction of virus protected from the protease increased rapidly after warming (Fig. b
) with a half-time of internalization of 8 min. The overall protease resistance reached a level of 70% within 30 min. The efficiency and rate of internalization was identical over a multiplicity range from 3 × 10−4
–10 MOI. Treatment of the cells with nocodazole, a drug that depolymerizes MT (Fig. b
), or with cytochalasin D, which depolymerizes actin filaments (not shown), had no effect, either on the efficiency of binding and internalization or on the kinetics of internalization. Consistent with previous reports using acid inactivation and plaque assays to assess virus internalization (Huang and Wagner, 1964
), these results showed that HSV-1 penetration occurs rapidly and efficiently.
Transport of Capsids from the Plasma Membrane to the Nucleus
Indirect immunofluorescence microscopy was next used to analyze the transport of individual capsids from the cell periphery to the nucleus. The antibodies used were either against VP-5 (anti–NC-1) or VP19c (anti–NC-2), the two major capsid proteins, or against purified whole capsids (anti-HC and anti-LC). All the antibodies gave similar results and had in common that they stained capsids that were in the cytosol (Fig. , 1 h) but not capsids present in surface-bound intact viruses (Fig. , 0 h). Presumably, this useful selectivity was caused by antigen accessibility; the relevant capsid epitopes in the intact virus particles were probably obscured by envelope and/or tegument components. In contrast, anti-capsid antibodies labeled viral capsids on thawed cryosections (Sodeik, B., M.W. Ebersold, and A. Helenius, unpublished observations).
Figure 3 Subcellular distribution of incoming HSV-1 capsids. Conventional immunofluorescence microscopy of Vero cells infected with HSV-1 at an MOI of 50 in the presence of CH. The cells were fixed in 3% PFA, permeabilized with 0.1% TX-100, and labeled with (more ...)
The capsids in the cytoplasm appeared as small, intensely labeled spots (Fig. , arrows, 1 h). Using immunoelectron microscopy on thawed ultrathin cryosections, we confirmed that these spots represented single capsids (not shown). All the structures labeled in the cytosol or at the nuclear membrane by antibodies directed against the capsid or against VP5 represented single viral capsids (not shown). The number of fluorescently labeled spots increased continuously during the first hour of warming. In addition, their distribution within the cell changed with increasing time: from the cell periphery toward the nucleus where they accumulated at the nuclear rim (Fig. , 2 h). After 4 h, virtually all the spots were localized at the nuclear membrane (Fig. , 4 h) where several hundred capsids could be seen. Thus, incoming cytosolic capsids were transported efficiently from the periphery to the host nucleus.
Capsids Bind to Microtubules
Double immunofluorescence microscopy using anti-capsid and anti-tubulin antibodies showed that most of the capsids that were not bound to the nucleus were associated with MT (Fig. , a and b). Moreover, in many cells, capsids were seen to concentrate around the MT organizing center (MTOC) (Fig. b). The close association of capsids with MT was confirmed by EM (Fig. , c–e). Numerous cytoplasmic capsids were seen in close proximity to cytoplasmic filaments with the expected 24-nm width and MT-like morphology. Typically, the distance between the capsid surface and the tubules was ~50 nm, suggesting the presence of additional tethering components. The viral DNA inside the MT-associated capsids could be seen as a darkly stained material.
Figure 4 Incoming HSV-1 capsids colocalize with MTs. (a) Conventional immunofluorescence microscopy. Vero cells infected at an MOI of 50 in the presence of CH were fixed in methanol at 2 h PI, and double labeled with anti-VP5 (NC-1; FITC anti–rabbit) (more ...)
The tubules were unambiguously identified as MT by antitubulin labeling of specimen that had been preextracted with TX-100 before fixation. After such extraction, thicker sections could be viewed, allowing the analysis of MT over longer distances. The MT-associated capsids generally contained the viral DNA (Fig. f, arrows). At later time points, most of the capsids were empty and typically located at the nuclear pores and not at the nuclear envelope membrane (Fig. g). Most of these capsids did not contain DNA (Fig. , g and h). At 4 h postinfection, there were occasionally empty capsids localized on MT in close proximity to the nucleus (not shown). Whenever extracellular viruses were observed in these TX-100 extracted samples, they were surrounded by very-electron dense material, which probably represented the viral glycoproteins and tegument proteins that were apparently not removed during extraction (Fig. h, curved arrow).
Quantitation of HSV-1 Entry and Capsid Transport
To quantify the kinetics of the cytosolic capsid transport, we determined the subcellular localization of the incoming virus by EM at various time points postinfection at an MOI of 500 (Fig. ). To compare this experiment with the immunofluorescence microscopy, ultrathin sections were cut parallel to the substrate through the basal region of the cells (see Fig. a). For each time point, 25 electron micrographs were taken in a systematic random fashion, and the number and localization of cytosolic capsids was determined. Cytosolic capsids (Fig. b) were localized at the plasma membrane (Fig. c), in the cytosol without obvious connection to any organelle (Fig. d), at MT (Fig. e), or at the nucleus (Fig. f).
After warming, the total number of cytosolic capsids increased rapidly, reaching a peak after 60 min (Fig. b). At later times the number of cytosolic capsids declined, suggesting that the capsids were ultimately disassembled. We also scored whether the cytosolic capsids contained the electron-dense viral DNA core or whether they appeared empty (see Fig. ). Up to 60 min after internalization, no empty capsids were detected, whereas after 2 h 20% and after 4 h 64% had lost their electron-dense DNA core (Fig. b). Virions in endosomes peaked at 1 h and sharply declined thereafter. They may have been degraded or recycled back to the cell surface, or capsids could have been released by fusion into the cytosol.
At 15 min postinfection, almost 70% of all cytosolic capsids were localized within 100 nm of the plasma membrane (see Figs. , c–f, and 5 c). All of the capsids close to the plasma membrane contained the viral DNA core. At 1 h postinfection, most of the cytosolic capsids (almost 80%) had an intermediate location; they were neither close to the plasma membrane or to the nucleus (Fig. , d and e). A significant fraction (10% of all cytosolic capsids) colocalized with MT (see Figs. , c–e, and 5 e). Since the capsids (125 nm) are considerably larger than the section thickness (60–70 nm), the figure for colocalization with the MT was most likely an underestimation. Of the cytosolic capsids, only one capsid (0.9% of all capsids at 4 h postinfection) was seen to colocalize with intermediate filaments (not shown).
Cytosolic capsids arrived at the nucleus at 2 h postinfection, and at 4 h, the majority of cytosolic capsids (>60%) was localized to the nucleus (Fig. f). At the ealier time points (up to 1 h), <4% of the cytosolic capsids were located at the nucleus. Of the nuclear capsids, 68% at 2 h and 87% at 4 h were empty, suggesting that DNA release occurred after arrival at the nucleus. The vast majority of empty capsids were localized to the nucleus (Fig. g). All capsids at the nucleus were localized to the nuclear pore complexes (see Fig. , g and h).
In summary, the electron microscopic experiment confirmed the immunofluorescence microscopy data. Cytosolic capsids were efficiently transported from the plasma membrane via the cytosol to the nuclear pore complexes. During transit, a significant fraction attached to MT. Empty capsids appeared soon after their arrival at the nucleus, and some of these were released into the cytosol. Ultimately, the capsids seemed to disassemble and disappear because the overall number of cytosolic capsids decreased late in infection. Interestingly, the appearance of cytosolic capsids under the plasma membrane peaked before the transport of virions into endosomes, strongly suggesting that the early cytosolic capsids were indeed derived from fusion of the virus at the plasma membrane rather than by fusion from endosomes.
Reduced Capsid Transport to the Nucleus without Microtubules
To determine whether efficient capsid transport via the cytosol depended on an intact cytoskeleton, cells were infected at an MOI of 50 for 2 h in the presence of drugs that affect MT or actin filaments. Immunofluorescence microscopy showed that neither taxol, a drug that prevents the disassembly of MT (Wilson and Jordan, 1994
), nor cytochalasin D, a drug that causes depolymerization of actin filaments (Cooper, 1987
), affected capsid transport to the nucleus (Fig. a
, upper panels
). In contrast, capsid accumulation at the nuclear membrane was significantly reduced by colchicine, vinblastine, and nocodazole (Fig. a
, lower panels
). These drugs interact with tubulin by different molecular mechanisms (Wilson and Jordan, 1994
). Labeling with anti-tubulin confirmed that colchicine and nocodazole depolymerized MT, whereas vinblastine caused tubulin paracrystal formation (not shown). In the presence of these drugs, the majority of capsids remained scattered throughout the cytosol. To test whether the effect of nocodazole was reversible, the cells were analyzed 2 h after drug removal. At this time, the MT had repolymerized, and the previously dispersed capsids were concentrated on the nuclear rim (Fig. b
Figure 6 Reduced transport of capsids to the nucleus in the absence of MTs. Immunofluorescence microscopy of Vero cells infected with HSV-1 for 2 h at an MOI of 50 in the presence of CH and drugs affecting the cytoskeleton. (a) Cytoskeletal drugs. Under control (more ...)
Since we were unable to quantify the localization of the fluorescent spots representing cytosolic capsids reliably, we infected Vero cells at an MOI of 150 in the presence or absence of nocodazole and analyzed the embedded cell pellets by quantitative EM. For each experimental condition, 50 electron negatives were taken in a systematic, random fashion and the localization of cytosolic capsids was determined (Fig. , a and b). After 2 h of infection, 31% of the cytosolic capsids had reached the nucleus, and only 25% were still in close proximity to the plasma membrane. In contrast in the absence of MT, none had reached the nucleus, and 58% were still in the region close to the plasma membrane. After 4 h postinfection, 75% of all cytosolic capsids had reached the nucleus, 14% were at the plasma membrane, and the remaining 11% were present in the cytosol. In the presence of nocodazole, only 47% of the cytosolic capsids had reached the nucleus, 18% were still at the plasma membrane, and 36% were in the cytosol distant from both the plasma membrane and the nucleus.
As nocodazole, vinblastine, and colchicine all affect the network of MT (Wilson and Jordan, 1994
), we concluded that MT are directly or indirectly involved in the rapid transport of capsids from the cell periphery to the nucleus in Vero cells. In cells devoid of functional MT, the arrival of capsids at the nucleus was significantly delayed.
Viral Infection in the Absence of Microtubules
Whether nocodazole would have an effect on productive infection was determined by monitoring the onset of viral protein synthesis in the presence or absence of the drug. Cell extracts prepared after different periods of infection were subjected to immunoblotting using antibodies against an early HSV-1 protein, ICP4, and a late structural protein, VP5. The results in Fig. c
show that the synthesis of both viral proteins was delayed and reduced in the absence of MT. Calnexin, an integral membrane protein of the ER (Hammond and Helenius, 1994
), was used as a control and remained constant. Labeling with [35
S]methionine demonstrated that nocodazole had no overall effect on protein synthesis in uninfected or HSV-1–infected Vero cells (not shown).
When immunofluorescence microscopy using ICP4 was performed, we found that, under control conditions, viral protein synthesis commenced in many cells already at 2 h postinfection. In contrast, in the presence of nocodazole, only very few cells showed ICP4 labeling. At 4 h postinfection, all control cells were strongly labeled for ICP4, whereas in the absence of MT, significantly less cells show strong labeling for ICP4 (data not shown). However, at later time points, all cells synthezised ICP4, in the presence and absence of MT.
Thus, depolymerization of MT delayed the onset of viral protein synthesis in cultured fibroblasts, but did not prevent viral infection per se.
Dynein Colocalizes with Incoming Capsids
Our results strongly suggested that incoming herpes capsids move along MT toward the nucleus. Therefore, we ask whether they would use host MT-dependent motors for their transport. Since the antibody against tubulin often obscured the morphology of putative tethering factors located between the viral capsids and the MT (see Fig. ), we repeated these experiments without immunolabeling. Electron-dense material was usually attached to the vertices of the capsids that were associated with MT (Fig. ). The morphology of the appendages was variable, but, in some cases (Fig. d
), they had the dimensions and the shape of cytoplasmic dynein, a minus end–directed MT-dependent motor. Dynein is a Y-shaped protein complex with a length of ~50 nm. It is connected to its cargo via the stalk and to MT through two 14-nm globular domains (Gilbert and Sloboda, 1989
; Vale, 1990
; Vallee et al., 1988
Figure 8 Incoming HSV-1 capsids display appendices at their vertices. At 1 h PI, Vero cells infected at an MOI of 500 in the presence of CH were extracted with 0.5% TX-100 in MT buffer for 2 min at 37°C and fixed in 1% GA in MT buffer. They were labeled (more ...)
To test whether cytoplasmic dynein was, indeed, associated with the capsids, we used an affinity-purified antibody directed against the heavy chain of cytoplasmic dynein (Vaisberg et al., 1993
). Immunoelectron microscopy with the anti-dynein antibody was performed on ultrathin cryosections of in situ fixed cells 1 or 2 h after initiation of virus entry. In addition to a low level of labeling of the cytoplasm, dynein was found to be localized on the surface of endosomes (Fig. h
), mitochondria (Fig. a
), and the Golgi apparatus (Fig. a
). Very few gold particles were seen over the nucleus and the mitochondrial matrix (Fig. , a
, and d
). The mitochondria, which do not contain dynein, served as a background control. Compared with the mitochondrial matrix, there was a slightly higher labeling in the nuclei, and the cytoplasm was labeled 2.3-fold higher than background (Table ).
Figure 9 Anti-dynein antibodies label incoming viral HSV-1 capsids. Thawed cryosections of Vero cells infected for 1 (c) or 2 h (a, b, and d–h) in the presence of CH at an MOI of 500 were labeled with rabbit antibodies followed by protein A–5 (more ...)
Quantitation of Dynein Immunolabeling
In cells containing incoming HSV-1 capsids, dynein was, in addition, found associated with many of the capsids (Fig. , a–f
). Capsids directly underneath the plasma membrane (Fig. g
) and within the cytoplasm (Fig. b
), as well as capsids in close proximity to the nucleus (Fig. , a
, and d
), were labeled with anti-dynein. On average, 13% of the cytoplasmic capsids were labeled (Table ). Capsids of intact virions, either extracellular (Fig. f
) or within endosomes (Fig. , g
), were not labeled. Only 1.5% of all capsids present in virions had colloidal gold particles in close proximity (Table ). Thus, the labeling density was eightfold higher on cytosolic capsids compared with capsids within virions, ruling out the possibility that the antidynein antibody showed cross-reactivity to capsid proteins. Given that the labeling efficiency using this technique does not exceed 10%, e.g., only 10% of the primary antibody present on the sections is detected by protein A–gold (Griffiths, 1993a
), these results showed that incoming cytosolic viral capsids associated with cytoplasmic dynein.