Accumulation of virion tegument and envelope proteins in a stable cytoplasmic compartment during human cytomegalovirus replication: characterization of a potential site of virus assembly

Abstract

The assembly of human cytomegalovirus (HCMV) is thought to be similar to that which has been proposed for alphaherpesviruses and involve envelopment of tegumented subviral particles at the nuclear membrane followed by export from the cell by a poorly defined pathway. However, several studies have shown that at least two tegument virion proteins remain in the cytoplasm during the HCMV replicative cycle, thereby suggesting that HCMV cannot acquire its final envelope at the nuclear envelope. We investigated the assembly of HCMV by determining the intracellular trafficking of the abundant tegument protein pp150 (UL32) in productively infected human fibroblasts. Our results indicated that pp150 remained within the cytoplasm throughout the replicative cycle of HCMV and accumulated in a stable, juxtanuclear structure late in infection. Image analysis using a variety of cell protein-specific antibodies indicated that the pp150-containing structure was not a component of the endoplasmic reticulum, (ER), ER-Golgi intermediate compartment, cis or medial Golgi, or lysosomes. Partial colocalization of the structure was noted with the trans-Golgi network, and it appeared to lie in close proximity to the microtubule organizing center. Two additional tegument proteins (pp28 and pp65) and three envelope glycoproteins (gB, gH, and gp65) localized in this same structure late infection. This compartment appeared to be relatively stable since pp150, pp65, and the processed form of gB could be coisolated following cell fractionation. Our findings indicated that pp150 was expressed exclusively within the cytoplasm throughout the infectious cycle of HCMV and that the accumulation of the pp150 in this cytoplasmic structure was accompanied by at least five other virion proteins. These results suggested the possibility that this virus-induced structure represented a cytoplasmic site of virus assembly.

FIG. 1

Time course of pp150 (UL32) expression in virus-infected HF cells. HF cells were infected with HCMV strain AD169 at an MOI of approximately 0.5 and harvested on the indicated day postinfection (p.i.). Following solubilization, the cell lysates were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were then developed with antibodies reactive with the pp72 (UL123) major immediate-early protein, and antibody binding was detected by ECL as described in Materials and Methods. The membrane was then stripped of bound antibody, and a 1:500 dilution of rabbit antibodies reactive with the carboxyl terminus of pp150 was used to detect pp150. Similarly, the membrane was stripped and reacted a third time with antibodies reactive with pp65 (UL83).

FIG. 2

Characterization of a murine MAb reactive with pp150 (UL32). Cell lysates from uninfected HF cells (HF), HCMV-infected HF cells (AD169 HF), Cos 7 cells transfected with an expression plasmid encoding pp28 (Cos-pp28), or Cos 7 cells transfected with an expression plasmid encoding pp150 (Cos-pp150) were solubilized, subjected to SDS-PAGE, and transferred to a nitrocellulose membrane. Gradient-purified HCMV strain AD169 virions (VIRUS) were solubilized in a similar fashion and loaded in the same gel. Following transfer, the membrane was incubated with MAb 36-14, and antibody binding was detected as described in Materials and Methods.

FIG. 3

Time course of pp150 (UL32) expression in virus-infected HF cells. HF cells grown on glass coverslips were infected at an MOI of between 3 and 5 and harvested at 24-h intervals. Following fixation, the expression of pp65 (UL83) and pp150 (UL32) was determined by using murine MAbs followed by IgG subclass-specific fluorochrome-conjugated second antibodies. The signals from the red and green channels were merged to determine colocalization.

FIG. 4

Accumulation of pp150 (UL32) in a juxtanuclear compartment. HF cells grown on glass coverslips were infected with HCMV at an MOI of 3 to 5 and fixed on day 6 postinfection. Uninfected HF cells were processed in a similar fashion. The various cellular compartments were stained with the following antibodies: ER, anti-RAP; ERGIC, anti-ERGIC53; Golgi, anti-GM130; Golgi, anti-mannosidase (MAN) II; TGN, anti-TGN46; and lysosome, anti-LAMP-1. Infected cells were also reacted with the anti-pp150 MAb, 36-14. The cellular markers were detected with a fluorescein isothiocyanate-conjugated secondary antibody, and pp150 was detected with a Texas red-conjugated secondary antibody. Colocalization is indicated by a yellow signal in the merge column. (A) Uninfected cells; (B) infected cells.

FIG. 5

The pp150 (UL32)-containing juxtanuclear compartment is associated with the MTOC. HF cells were infected with HCMV as described for Fig. 4. Immediately prior to fixation, the cells were incubated in a microtubule-stabilizing solution and then fixed as described for Fig. 4. In all cases pp150 (UL32) was detected with MAb 36-14 followed by a Texas red-conjugated secondary antibody. Actin was stained with phalloidin, tubulin was stained with an anti-α-tubulin MAb, and vimentin was stained with an antivimentin MAb as described in Materials and Methods.

FIG. 6

The pp150-containing juxtanuclear structure is resistant to treatment with brefeldin A but is dispersed by treatment with nocadazole. (A) Infected cells were incubated in the presence of 2 μg of brefeldin A (BFA) per ml for 2 h or left untreated and then processed as described for Fig. 4. (B) Infected cells were incubated in 2 μM nocadazole for 1.5 h prior to fixation or left untreated and processed as described for Fig. 5. Vimentin and pp150 were detected as described for Fig. 5.

FIG. 7

Distribution of virion proteins in infected HF cells. HF cells were infected and processed as described for Fig. 4. Following fixation, the glass coverslips were incubated in MAbs specific for pp150 and the antibodies against the indicated virus-encoded proteins. pp150 reactivity was detected with a Texas red-conjugated secondary antibody, and the second viral protein was detected with fluorescein isothiocyanate conjugated secondary antibody. Colocalization was indicated by a yellow signal in the merge channel.

FIG. 8

Cell fractionation of HCMV-infected HF cells. (A) HF cells were infected with HCMV at an MOI of 3 to 5 and harvested on day 6 postinfection as described in Materials and Methods. The postnuclear supernatant was applied to a discontinuous sucrose gradient; following centrifugation at 100,000 × g for 3 h, individual 0.3-ml fractions were collected from the bottom of the gradient. An aliquot of each fraction was either analyzed by immunoblotting or titered for virus infectivity. Murine MAbs specific for pp150 (UL32) and pp65 (UL83) were used to probe the membranes, and antibody was detected as described in Materials and Methods. The filter probed with anti-gB was made by cutting the original filter into two pieces such that proteins migrating faster than 100 kDa were present in the membrane probed with anti-gB and those above 100 kDa probed were present in the membrane probed with anti-TAP antibody. The positions of migration of pp150, pp65, and gB are indicated at the right. (B) Infectivity of the gradient fractions.