Accumulation of Virion Tegument and Envelope Proteins in a Stable Cytoplasmic Compartment during Human Cytomegalovirus Replication: Characterization of a Potential Site of Virus Assembly

Primary human foreskin fibroblasts (HF cells) were prepared, propagated, and infected as previously described (30). HCMV strain AD169 was used for all experiments. Infectious stock were prepared from supernatants of infected HF cells which exhibited 100% cytopathic effect and were titered as described elsewhere (1); fractions collected from cell fractionation gradients were titered by the same method. The ORFs encoding pp150 (UL32) and pp28 (UL99) were cloned into the expression vector pcDNA 3 (Invitrogen, San Diego, Calif.). Orientation and sequence were verified by nucleotide sequencing. Human 293T cells or monkey Cos 7 cells were used for transient expression of pp150 (UL32) following calcium phosphate-mediated transfection (8).

HCMV-encoded proteins were detected with monoclonal antibodies (MAbs) as previously described (30). MAbs used in this study included those specific for IE-1 (UL123, MAb P63-27), MCP (UL86, MAb 28-4), ppUL69 (UL69, MAb 69), pp65 (UL83, MAbs 28-19 and 65-8), pp150 (UL32, MAb 36-14), pp28 (UL99, MAb 41-18), gB (UL55, MAb 58-15), gH (UL75, MAb 14-4b), and gp65 (MAb 14-16) (4, 5, 7, 26, 30, 31). The generation of the monospecific rabbit anti-serum reactive with pp150 has been previously described (15). MAb 36-14 was generated from spleen cells obtained from mice immunized with prokaryotic expressed fragments of pp150.

The antibodies reactive with cellular markers included a MAb specific for ERGIC53 (generously provided by Peter Hauri, University of Basel, Basel, Switzerland), a rabbit antiserum specific for mannosidase II (provided by Marilyn Farquhar, University of California, San Diego), a rabbit antiserum against TGN46 (from George Banting, University of Manchester, Manchester, United Kingdom), and a rabbit antiserum reactive with the lysosomal membrane protein LAMP-1 (from Minoru Fukuda, La Jolla Cancer Research Center, La Jolla, Calif.). The rabbit antiserum against the Golgi protein GM130 and the murine MAb reactive with the ER-resident protein RAP have been described previously (24). A murine MAb specific for vimentin, clone V9, was purchased from Sigma Chemical Co., St. Louis, Mo. The murine MAb reactive with tubulin was obtained from Tom Howard, University of Alabama, Birmingham. Actin was stained with fluorochrome-conjugated phalloidin (Molecular Probes, Eugene, Oreg.). Fluorochrome-conjugated secondary antibodies, including murine immunoglobulin G (IgG) subclass antibodies, were purchased from Southern Biotechnology Associates, Birmingham, Ala.

Electrophoresis under reducing conditions and immunoblotting were carried out as described elsewhere (30). Virus-infected cell proteins were obtained from HCMV-infected HF cells grown in 35-mm-diameter tissue culture dishes. Following washing in phosphate-buffered isotonic saline (PBS; pH 7.4), the cells were lysed in sample buffer containing 5% 2-mercaptoethanol and 2% sodium dodecyl sulfate (SDS) and heated to 100°C. The solubilized proteins were then subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to either nitrocellulose membranes or polyvinylidene fluoride filters. Murine MAbs or in some cases a 1:500 dilution of the IgG fraction of the rabbit anti-pp150 serum were used to detect specific proteins. Antibody binding was detected by ¹²⁵I-protein A followed by autoradiography or alternatively by reaction with horseradish peroxidase-conjugated goat anti-mouse or rabbit IgG followed by enhanced chemiluminescence (ECL) fluorography (ECL kit; Amersham, Arlington Heights, Ill.).

HF cells were grown in 24-well tissue culture plates containing a 13-mm-diameter coverslip. After the cells had reached at least 90% confluency, the cells were infected with HCMV strain AD169 for 1 to 2 h and then washed once and incubated for the indicated time. The coverslips were harvested by first washing the cells with PBS and then fixing them for 30 min at room temperature in 2% paraformaldehyde (PFA) freshly prepared in PBS. In some cases the cells were incubated for 5 min prior to PFA fixation in a 100 mM HEPES-buffered solution containing 5 mM EGTA and 5% polyethylene glycol (pH 6.9) to stabilize microtubules. The coverslips were then washed in PBS and permeabilized with 0.05% NP-40 in PBS at 4°C for 10 min. The coverslips were then blocked with PBS containing 20% normal goat serum for 45 min at 37°C. After a wash in PBS, primary antibody was added and the coverslips were incubated at 37°C for 60 min. The coverslips were then washed in PBS and then incubated for 45 min in fluorochrome-conjugated secondary antibody diluted in PBS containing 10% normal goat serum. Following repeated washings in PBS the coverslip was fixed in 2% PFA as described above and then washed once in PBS immediately prior to viewing.

The fixed coverslips were incubated for 5 min in Slowfade (Molecular Probes) and then inverted and sealed on a glass microscope slide with fingernail polish. The sealed coverslips were viewed under a Leitz Dialux epifluorescence microscope at a magnification of ×50, and the images were captured with a digital camera (Photometrics, Tucson, Ariz.). The images were processed with Image Pro software (Media Cybernetics, Silver Spring, Md.). Deconvolution was accomplished with Hazebuster (Vaytek, Fairfield, Iowa).

Three 150-cm² flasks of HF cells were infected at a multiplicity of infection (MOI) of 3 and harvested on day 6 postinfection by scraping the cells from the flask. The cell pellet was washed twice with cold PBS plus 5 mM EDTA and then resuspended in 1 ml of Tris-buffered isotonic saline (pH 7.4) containing 5 mM EDTA and 0.25 M sucrose. The cell suspension was repeatedly passed through a 27-gauge needle until there were no intact cells in the suspension as determined by light microscopy. This material was then centrifuged at 1,000 × g for 10 min, and the supernatant was transferred to a new tube. This solution, termed the postnuclear supernatant, was then applied to a preformed sucrose step gradient consisting of the following amounts of sucrose in 10 mM Tris-buffered isotonic saline, pH 7.4: (i) 0.5 ml, 2 M; (ii) 1.5 ml, 1.6 M; (iii) 2.5 ml, 1.4 M; (iv) 3.5 ml, 1.2 M; and (v) 1.5 ml, 0.8 M. The postnuclear supernatant was layered on the top of the gradient and centrifuged for 3 h at 100,000 × g in an SW41 rotor at 4°C. The gradient was fractionated from the bottom, and individual fractions were assayed for virus infectivity as described above and for viral protein by immunoblotting.

The kinetics of pp150 expression were initially studied by immunoblotting of HF cell lysates harvested on days 1 to 6 postinfection. HCMV-specific MAbs reactive with the major immediate-early protein, IE-1 (UL123, pp72), and the tegument protein, pp65 (UL83), were used to detect proteins of the immediate and early-late kinetic classes, respectively. As previously shown, the IE-1 protein was expressed by day 1 postinfection and could be detected at approximately similar levels throughout the duration of the infection (Fig. 1). In contrast, pp65 was first detected on day 3 and appeared to increase in amount during subsequent days (Fig. 1). Although incoming virion pp65 has been reported to be detectable immediately after infection, at the low MOI used in this experiment, we failed to detect pp65 until day 3 postinfection. Similarly, pp150 was first detected on day 3 and appeared to increase in amount during subsequent days (Fig. 1). These results suggested that pp150 was expressed with kinetics consistent with either an early-late or a late protein. The lack of detectable pp150 in infected HF cells treated with phosphonoformic acid further supported the classification of pp150 as an early-late protein (data not shown).

To confirm the immunoblotting results and to examine the cellular distribution of pp150 within HF cells at various times after infection, a murine MAb reactive with pp150 was generated. This antibody was specific for pp150; it detected viral pp150 in virions and infected cells as well as recombinant pp150 produced in transfected Cos 7 cells as determined by immunoblot analysis (Fig.2). The MAb did not cross-react with cellular proteins in Cos 7 and HF cells or cells expressing pp28 (Fig.2). The anti-pp150 MAb and a MAb specific for pp65 were used to examine the localization of the corresponding antigens during productive infection of HF cells. As noted previously by other investigators, pp65 was rapidly translocated to the nucleus and could be readily detected in the nucleus as early as 120 min postinfection when cells were infected at a high MOI (data not shown). Within 24 h postinfection, pp65 was detectable in the nucleus of almost every cell in the culture (Fig. 3). In contrast, pp150 was present in small punctate structures scattered throughout the cytoplasm. Although some punctate structures could be seen overlying the nucleus, these were not localized to the nucleus when multiple focal planes of the same image were collected (data not shown). Within 48 h postinfection, pp65 expression continued to be restricted to the nucleus (Fig.3). At that time, pp150 was still detected in clearly separate punctate structures but with an increase in the intensity of the signal in an area adjacent to the nucleus. At 72 and 96 h postinfection, pp65 was present in both the nucleus and the cytoplasm of infected cells. At these times postinfection, pp150 localization was still limited to the cytoplasm, but it appeared that the punctate structures coalesced into a large juxtanuclear structure. In the early periods postinfection, we could not distinguish between incoming virion pp150 and newly synthesized pp150; however, the cytoplasmic distribution and the increased intensity of the signal between 48 and 72 h postinfection suggested that newly synthesized pp150 was accumulating in the juxtanuclear structure (Fig. 3). In the interval between 120 and 144 h postinfection, pp65 was present in both the cytoplasm and the nucleus, with partial overlap with pp150 at 144 h postinfection. In this final time interval, pp150 remained exclusively cytoplasmic, the intensity of the signal associated with the juxtanuclear structure increased, and the structures appeared more compact (Fig. 3). Additional analysis by immunoelectron microscopy was consistent with these findings in that anti-pp150 antibodies labeled particles only in the cytoplasm and not in the nucleus (data not shown). These results suggested that the abundant tegument protein pp150 was expressed as a cytoplasmic protein throughout the productive infection of HF cells with HCMV. Significantly, the intracellular distribution of pp150 varied with the time postinfection, ranging from a dispersed punctate distribution early in infection, to a concentration of the protein in a large juxtanuclear structure late in infection.

Late in infection, during the time interval of maximal production of infectious progeny virions, pp150 was localized predominantly to a large juxtanuclear compartment. We attempted to characterize this structure by using a panel of antibodies reactive with specific subcellular marker proteins. Although the juxtanuclear structure did not have the morphologic appearance of the ER, we used an antibody reactive with a resident lumenal ER protein, RAP, to demonstrate that the juxtanuclear structure did not represent a structurally aberrant ER (Fig.4). The pp150-containing structure did not colocalize with ERGIC53, an integral membrane protein of the ERGIC positioned between the ER and the Golgi (Fig. 4). Similarly, the pp150-containing compartment was not labeled with antibodies reactive with two Golgi marker proteins, GM130 and mannosidase II (Fig. 4). Antibodies to the TGN marker TGN 46 revealed overlap in signal with the pp150-containing structure, although there was not complete colocalization (Fig. 4). Interestingly, the distribution of the ERGIC, Golgi, and TGN markers appeared displaced by the juxtanuclear pp150-containing structure and resulted in the accumulation of the markers on the periphery of the virus induced structure (Fig. 4). The intracellular distribution of the ERGIC53 appeared most similar to that seen in uninfected cells, yet even this protein appeared to be more concentrated in the periphery of the pp150-containing juxtanuclear structure instead of its more diffuse distribution in uninfected cells (Fig. 4). The changes in the morphology of the Golgi and the TGN in infected cells were very striking, with change of the normal lacy, tubular appearance in uninfected cells to more compact fragments surrounding the juxtanuclear structure in infected cells. The appearance of the Golgi and the TGN suggested that they were displaced radially by the pp150-containing structure. Together, these results indicated that the morphology of the intracellular compartments of the secretory system was altered during the late phases of HCMV infection.

An obvious possibility that could account for the accumulation of pp150 in the perinuclear location was that this compartment represented a degradative pathway for viral proteins overexpressed during productive viral infection. We examined this by attempting to colocalize the LAMP-1 protein, an integral membrane protein of lysosomes, with the pp150-containing structure. No colocalization was observed (Fig. 4). Furthermore, comparison of the distributions of LAMP-1-containing structures in infected and uninfected cells indicated no significant alteration of distribution (Fig. 4).

The displacement of Golgi elements that usually surround the MTOC to around the pp150-containing structure suggested that the viral proteins might be concentrated in the region of the MTOC. In agreement, the pp150-containing structure did not colocalize with the cytoskeletal proteins, actin, or vimentin but appeared to be in close proximity to a structure whose morphologic appearance was suggestive of that of the MTOC (Fig.5). A signal overlap between the base of microtubules arising from the periphery of this structure and the pp150-containing juxtanuclear structure was also noted (Fig. 5).

Although we could not colocalize the juxtanuclear structure with cellular markers of the Golgi, the localization of pp150 in the proximity of the MTOC appeared similar to that of the Golgi. To further explore the possibility that the juxtanuclear structure colocalized with the Golgi, we incubated infected cells with brefeldin A and compared the distribution of the pp150 to that of the Golgi marker, GM130. Brefeldin A treatment resulted in the vesiculation of the Golgi as revealed by staining of the cells with anti-GM130 but failed to cause a similar change in the appearance of the pp150-containing juxtanuclear structure compared to control, untreated infected cells (Fig. 6A).

In recent studies of COS cells transfected with expression vectors encoding mutant proteins, a novel subcellular compartment has been identified as the site of accumulation of misfolded and overexpressed proteins (19). This compartment, termed an aggresome, appeared to represent a cellular site for storage of abundantly expressed proteins. Characteristics of the aggresome included its stability and a capacity to remain as a morphologically defined structure following treatment with the microtubule-destabilizing drug nocadazole (19). In addition, the aggresome has been shown to be encased in a vimentin cage. We analyzed the pp150-containing juxtanuclear structure formed in the late stages of HCMV infection in HF cells for these properties of aggresomes. Treatment of infected cells with nocadazole dispersed the juxtanuclear structure containing pp150 (Fig. 6B). When we examined the effect on multiple fields from nocadazole-treated cells, we could not find an intact juxtanuclear pp150-containing structure (data not shown). Furthermore, the pp150-containing structures were not encased in vimentin cages, and the vimentin pattern in infected cells was not significantly different from that in uninfected cells (Fig. 6B). Together, these data support the conclusion that the juxtanuclear structures that contained viral proteins were not collections of lysosomes or aggresomes. Moreover, these results indicated that integrity of the microtubule network is essential for maintenance of the pp150-containing structure, a finding consistent with the association of this structure with the MTOC.

The finding of an abundant virion protein within a discrete cytoplasmic compartment raised the possibility that other virion proteins could also be present within this structure. We used several different antibodies reactive with both structural and nonstructural HCMV-encoded proteins along with antibodies against pp150 to compare the intracellular distribution of these proteins. The nonstructural proteins IE-1 and the polymerase accessory protein UL44 were nuclear in their distribution and did not colocalize with pp150 in the perinuclear structure (data not shown). Similarly, we failed to detect a signal from antibodies reactive with the nuclear tegument protein UL69 or the major capsid protein (UL86) in the perinuclear structure (Fig. 7). In contrast, we have already shown that pp65 colocalized with pp150 (Fig.3) and could readily colocalize another tegument pp28 (UL99) within the pp150-containing juxtanuclear structures (Fig. 7). Likewise, we could also colocalize signals from three virion envelope glycoproteins, gB, gH, and gp65, within the perinuclear pp150-containing structure (Fig.7). Together, these data indicated that virion proteins of the tegument and the envelope of HCMV accumulated in juxtanuclear structures late in infection and raised the possibility that this structure has a role in the cytoplasmic assembly of subviral particles or infectious virions.

To determine whether the juxtanuclear structure containing viral proteins represented a distinct and stable subcompartment, we sedimented a postnuclear supernatant of HCMV infected cells harvested late in infection over a discontinuous sucrose gradient and then analyzed individual fractions for the presence of three viral proteins, the tegument proteins pp150 and pp65 and the envelope glycoprotein gB. We could detect all three proteins, including the proteolytically cleaved form of gB, in few fractions near the bottom of the gradient (Fig. 8). Analysis of the remaining fractions failed to show the presence of either pp150 or gB; however, lesser amounts of pp65 were detected throughout the entire gradient, a finding consistent with the widespread cellular distribution of this protein late in infection as observed in our imaging studies. Consistent with our immunofluorescence imaging findings, we could not detect the Golgi marker protein GM130 within the fraction (fraction 6) that contained the three viral proteins, although GM130 could be detected in other fractions (fractions 12 to 14) recovered from the gradient (data not shown).

Although the high sucrose concentrations and relative centrifugal field used in this cell fractionation were not sufficient to sediment HCMV virions, we explored the possibility that this profile of virus-encoded proteins reflected the concentration of assembled virions instead of cosedimentation of several virion proteins within an intracellular compartment. To test this possibility, we determined the titer of infectious virus in each fraction. Although a large amount of infectious virus was found associated with the fraction containing pp150, pp65, and gB, this fraction did not represent the peak fraction of viral infectivity (Fig. 8). Infectious virus of similar titers could be found throughout most of the gradient. Thus, it appeared that following fractionation of infected HF cells, we could enrich for a cellular compartment which contained virion structural proteins.