Previous Article | Next Article 
Journal of Virology, January 2000, p. 975-986, Vol. 74, No. 2
Departments of
Pediatrics,1 Biochemistry and Molecular
Genetics,2 Cell
Biology,3 and
Microbiology,4 The University of
Alabama at Birmingham, Birmingham, Alabama 35233
Received 3 August 1999/Accepted 11 October 1999
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.
As for other human herpesviruses,
the assembly of the infectious human cytomegalovirus (HCMV) particle is
a complex and poorly understood process. Extracellular virions produced
by HCMV-infected cells appear structurally similar to those of other
herpesviruses in that HCMV virions consist of three major structural
regions, the capsid, the tegument, and a lipid-containing envelope
(29). While it is generally accepted that capsid assembly
occurs in the nuclei of infected cells, the cellular compartments in
which tegument assembly takes place remain incompletely defined
(29). Several seemingly mutually exclusive pathways of
envelopment have been proposed for herpesviruses (6, 9, 11, 13,
16, 18, 20, 25, 27, 28, 33, 34, 39-42). The earliest model for
envelopment of herpes simplex virus (HSV) proposed that the viral
particle was enveloped during passage through the inner nuclear
membrane (18, 29, 32). Glycoproteins within the viral
envelope would then be modified as the particle traversed the Golgi
(9, 18). More recent models have suggested that envelopment
and deenvelopment occur at the nuclear envelope and that final
envelopment of the tegumented nucleocapsid occurs in the
trans-Golgi (TGN) following fusion with membranes containing processed envelope glycoproteins (6, 11, 13, 14, 16, 20, 25, 27,
39-43).
Addition of the tegument has been assumed to take place in the nucleus
in both models, although formal proof of such a site of tegument
acquisition is lacking. Previous studies of HCMV assembly utilizing
electron microscopy described coated particles budding into cytoplasmic
vacuoles; however, the site of tegument addition was not defined
(32, 36). A more recent study of the assembly of human
herpesvirus 6 suggested that tegumentation of the particle took place
by budding of nuclear capsids into cytoplasmic invaginations in the
nucleus (28). Some evidence consistent with the possibility that tegument proteins are incorporated into the viral particle outside
of the nucleus has been presented in a study of the UL11 tegument
protein of HSV. Electron micrographs of HSV-infected cells suggested
that this myristoylated protein was located in the nuclear
membrane, but the polarity of this association was not readily
apparent (2). Furthermore, the phenotype of a UL11 deletion
virus was characterized by significantly decreased infectious virion
production and accumulation of tegument-containing capsids on the inner
nuclear membrane (2). Recent studies of the morphogenesis of
a related alphaherpesvirus, varicella-zoster virus, have suggested that
the tegument of this virus is also partially assembled in the cytoplasm
of the infected cell, perhaps concurrently with final envelopment of
the maturing particle at the TGN (11, 42, 43). Studies of
the envelopment of HSV have suggested that if a cytoplasmic envelopment
follows envelopment/deenvelopment at the nuclear membrane, then this
step most likely occurs in the Golgi (9). A more definitive
analysis of the envelopment of pseudorabies virus suggested that two
pools of a major envelope glycoprotein, gE, were present within the
infected cell (35). One pool, derived by retrieval of
gE from the plasma membrane by the endocytic pathway, was not
necessary for assembly of infectious virions, suggesting that a second
intracellular pool of gE was incorporated into progeny virions in an as
yet undefined cytoplasmic compartment (35).
In HCMV-infected cells, the cellular distribution of several of the
better-characterized tegument proteins has suggested that tegument
assembly might occur in two different compartments, the nucleus and the
cytoplasm. Tegument proteins encoded by the UL25, UL99 (pp28), and UL32
(pp150) open reading frames (ORFs) have been shown to be present in the
cytoplasm of infected cells late in the viral replicative cycle
(3, 17, 21). Ultrastructural analysis by electron microscopy
has demonstrated nonenveloped particles apparently acquiring an
additional structural layer by budding into cytoplasmic structures
(12, 32, 36). This layer is assumed to be the envelope;
however, direct evidence for this is lacking, and it remains to be
determined whether these observations are relevant to the assembly of
infectious particles or represent a default pathway for noninfectious
particles destined for intracellular or extracellular degradation.
In this study, we examined the subcellular site of tegument
acquisition, using well-characterized HCMV-specific monoclonal and
polyclonal antibodies to describe both the expression and cellular
distribution of several tegument proteins. Our findings indicated that
tegument proteins were added to the subviral particle in both nuclear
and cytoplasmic compartments, suggesting that the final envelopment of
HCMV occurred in the cytoplasm of infected cells. Moreover, we have
identified a juxtanuclear structure which contained at least three
virion glycoproteins and three tegument proteins. This structure
colocalized with the microtubule organizing center (MTOC) and could be
disrupted with the microtubule-depolymerizing agent nocadazole but not
by brefeldin A. Although this structure failed to colocalize with
several markers of intracellular compartments, the viral
protein-containing structure was surrounded by elements of the Golgi,
suggesting a close proximity of soluble viral proteins and
Golgi-localized, transmembrane-containing viral glycoproteins. The
association of the viral proteins within this structure appeared to be
relatively stable since fractionation of infected cells by
discontinuous sucrose gradient centrifugation resulted in the enrichment of both tegument and envelope proteins in a single fraction.
Interestingly, this fraction contained at least one viral glycoprotein
but was devoid of markers for the endoplasmic reticulum (ER)-Golgi
intermediate compartment (ERGIC) or the Golgi, suggesting that viral
proteins might be sequestered in a membrane subdomain from which
cellular proteins were excluded. The presence of virion tegument and
envelope proteins within a single cytoplasmic compartment suggested
that tegumentation and envelopment likely occurred in the cytoplasm and
that the juxtanuclear compartment might represent a site of virion assembly.
Cells, viruses, plasmids, and antibodies.
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).
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
SDS-PAGE and immunoblotting. 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.).
Immunofluorescence microscopy. 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).Cell fractionation. 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.
| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
pp150 is expressed late during HCMV infection. 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).
|
pp150 accumulates in a juxtanuclear structure in HCMV-infected cells. 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.
|
|
The juxtanuclear structure containing pp150 is not a secretory or a degradative cellular compartment. 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.
|
The juxtanuclear structure containing pp150 surrounds the MTOC but is not an aggresome. 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).
|
-tubulin MAb, and
vimentin was stained with an antivimentin MAb as described in Materials
and Methods.
|
Tegument and envelope HCMV proteins colocalize in the juxtanuclear structure containing pp150. 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.
|
|
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
The assembly of HCMV, specifically the envelopment of the infectious particle, has been proposed to follow an assembly pathway similar to that for HSV, for which acquisition of the tegument proteins occurs within the nucleus and envelopment proceeds at the nuclear membrane (29). Yet little if any recent experimental data have been presented to support this model of assembly. As noted previously, recent studies of varicella-zoster virus and pseudorabies virus have suggested an alternate assembly pathway, in which envelopment is followed by deenvelopment at the nuclear membrane and final envelopment occurs in a cytoplasmic compartment (11, 14, 20, 39, 42). Similarly, studies of HCMV showed that at least two virion tegument proteins could be localized exclusively within the cytoplasm of infected cells, a finding contradictory with assembly at the nuclear membrane (3, 21). Furthermore, the cleavage of the major HCMV envelope glycoprotein, gB, into its mature forms has been shown to be accomplished by the proteolytic activity of host furin, an enzyme found within the late secretory/endocytic pathway, suggesting that envelopment was unlikely to occur at the nuclear membrane (23, 37, 38). Together, these findings strongly support a cytoplasmic phase in the assembly of the HCMV virion.
The results presented in this report suggested that the tegumentation and envelopment steps of virus assembly might occur within a cytoplasmic compartment. Specifically, we demonstrated that one of the most abundant and well-studied virion tegument protein, pp150, was detected only in the cytoplasm of infected cells throughout the course of infection. Rarely, pp150 could be detected within the nucleus of an infected cell, but this was evident only very late in infection, when excessive cytopathology was apparent in the cell. The findings in our study were not due to an antibody reactivity artifact since two different antibody preparations, a heterologous monospecific antiserum and a murine MAb, both detected pp150 only in the cytoplasm. In addition, after viewing countless number of infected cells with these reagents and only very infrequently noting a single cells with what could be considered as having a signal within the nucleus, we could not explain the discrepancy between our findings and those of an earlier report which suggested that pp150 was a nuclear protein (17). Furthermore, when the pp150 ORF was transiently expressed in Cos 7 cells, the protein remained entirely within the cytoplasm (data not shown). Taken together, the results indicated that at least two abundant virion tegument proteins, pp28 and pp150, remained exclusively cytoplasmic during the replicative cycle of HCMV. Since these proteins and the product of the UL25 ORF appeared to be restricted to the cytoplasm throughout the replicative cycle, our results suggested that tegumentation and envelopment (assuming that envelopment of the mature virion occurs after tegument acquisition) stages of virus assembly were likely completed within the cytoplasm.
Our results were consistent with these processes occurring within a single compartment, since pp150 and a number of other tegument and envelope viral proteins accumulated in a cytoplasmic juxtanuclear structure. We attempted to define the cellular compartment(s) that contributed to the formation of this juxtanuclear structure by using markers of known components of various cellular compartments. We observed that the ERGIC and the Golgi were altered in the infected cells late in infection, such that protein markers for these cellular compartments appeared to be localized on the periphery of the viral protein-containing juxtanuclear structure. Interestingly, pp150 within the virus-induced structure appeared to overlap with microtubules emanating from the MTOC, a finding suggesting that it was microtubule associated. In agreement, we found that the structural integrity of the virus-induced structure was dependent on microtubules, as demonstrated by its disruption following treatment with nocadazole. Thus, this intracellular collection of viral proteins appeared to displace normal cellular constituents from a compartment which would normally localize to an area adjacent to the MTOC. In uninfected cells, such a compartment would be consistent with either the Golgi, TGN, and/or the Golgi-ERGIC interface.
We considered the possibility that the juxtanuclear structure represented a cellular storage area or site for protein degradation. A recently described cellular response to overexpression of misfolded (and perhaps normal) proteins is the formation of a vimentin caged storage area, the aggresome (19). The location of the aggresome and its relative size was consistent with the virion protein-containing juxtanuclear structure which appeared late in infection, at a time when viral promoters would drive high expression of a number of viral proteins. However, two of the defining characteristics of aggresomes, the reorganization of cellular vimentin and the resistance of these structures to the microtubule-depolymerizing agent nocadazole, were not properties of the viral protein-containing juxtanuclear structure. It was unlikely that the juxtanuclear viral structure represented a degradative compartment since we failed to colocalize the lysosomal protein LAMP-1 to the pp150-containing structure and, perhaps more importantly, found no gross alteration in the cellular distribution of LAMP-1 in infected compared to uninfected cells. Thus, it appeared that the viral protein-containing juxtanuclear structure was not a known cellular site of protein storage or degradation.
The viral protein-containing structure appeared relatively stable since a number of tegument and envelope glycoproteins cosedimented in sucrose gradients following fractionation of infected cells. A trivial explanation for this finding was that this cofractionation represented an accumulation of cytoplasmic virions or subviral particles and that the results of our biochemical analysis represented a signal obtained from proteins incorporated into progeny virions which cosediment with a specific cellular membrane. However, the distribution of viral infectivity in all fractions of the gradient was not consistent with this interpretation. This was not unexpected because HCMV virions and/or noninfectious particles such as dense bodies would not be expected to sediment into specific fractions in the gradient and relative centrifugal field conditions used for separation of cellular membranes (22).
The finding of the cleaved form of gB within the fraction containing pp150 and pp65 was intriguing, as it suggested that this mature envelope glycoprotein was retrieved from a cellular compartment distal to the Golgi (10, 37). Although an explanation for this result was not obvious, the apparent proximity of the TGN and the pp150-containing juxtanuclear structure raised the possibility that regions of the TGN and the virion protein-containing structure can interface with one another. Finally, it should be noted that the juxtanuclear structure developed late in infection, at a time when the production of progeny virus was increasing rapidly. Interestingly, an abundant tegument protein and a true late protein encoded by HCMV, pp28 (UL99), was also transported to this juxtanuclear structure late in infection. In fact, pp28 was not readily detectable in infected cells until this structure developed, suggesting that accumulation of virion structural proteins within the juxtanuclear structure and the assembly of progeny virus might be temporally associated. Admittedly, several of these interpretations were based on correlative data, but they were consistent with the concept that assembly of HCMV included cytoplasmic tegumentation and envelopment.
Based on the obtained results, we proposed that the juxtanuclear accumulation of tegument and envelope proteins might represent a cytoplasmic site of virion assembly. It appears inherently reasonable that the host cell would sequester tegument and envelope viral proteins destined for cytoplasmic virion assembly into a compartment adjacent to a cellular site where ERGIC or Golgi membranes and microtubular transport converged. In support of our hypothesis, we found that three tegument proteins (UL99, UL83, and UL32) whose primary sequence did not contain motifs which have been described as signals for entry into the cellular secretory pathway and that viral envelope glycoproteins which do transit the secretory pathway could be colocalized to this juxtanuclear compartment. The proximity of this structure to ERGIC or Golgi elements suggested that tegument proteins could interface with envelope glycoproteins within such structures.
It remains unclear how infectious particles would leave this juxtanuclear compartment unless membrane-bound vacuoles containing mature virions could enter the exocytic pathway. Although we and other have observed such cytoplasmic collections of virions, often in the site of a morphologically altered Golgi, we cannot argue with any certainty that this represented a major route of virion production (data not shown). However, the finding of relatively high titers of infectious virus in the membrane fraction containing pp150, pp65, and gB was consistent with assembly of virions within this juxtanuclear compartment followed by export from the cell as membrane-bound virus-containing vacuoles. Taken together, our results strongly supported a cytoplasmic model of HCMV tegumentation and envelopment. Future studies will be aimed at further characterizing the nature of the viral protein-containing compartment in which virion assembly is likely to occur.
| |
ACKNOWLEDGMENTS |
|---|
V. Sanchez and K. D. Greis contributed equally to this study.
This work was supported by grant R01 AI35602 to W.J.B. from NIAID, National Institutes of Health.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Pediatrics, The University of Alabama at Birmingham, 1600 7th Ave. South, Suite 752, Birmingham, AL 35233. Phone: (205) 939-9590. Fax: (205) 975-6549. E-mail: wbritt{at}peds.uab.edu.
Present address: Department of Biology, University of California,
San Diego, La Jolla, Calif.
Present address: Procter and Gamble Pharmaceuticals, Mason, Ohio.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
| 1. | Andreoni, M., M. Faircloth, L. Vugler, and W. J. Britt. 1989. A rapid microneutralization assay for the measurement of neutralizing antibody reactive with human cytomegalovirus. J. Virol. Methods 23:157-168[CrossRef][Medline]. |
| 2. |
Baines, J. D., and B. Roizman.
1992.
The UL11 gene of herpes simplex virus 1 encodes a function that facilitates nucleocapsid envelopment and egress from cells.
J. Virol.
66:5168-5174 |
| 3. |
Battista, M. C.,
G. Bergamini,
M. C. Boccuni,
F. Campanini,
A. Ripalti, and M. P. Landini.
1999.
Expression and characterization of a novel structural protein of human cytomegalovirus, pUL25.
J. Virol.
73:3800-3809 |
| 4. | Billstrom, M. A., and W. J. Britt. 1995. Postoligomerization folding of human cytomegalovirus glycoprotein B: identification of folding intermediates and importance of disulfide bonding. J. Virol. 69:7015-7022[Abstract]. |
| 5. | Britt, W. J., and D. Auger. 1985. Identification of a 65,000 dalton virion envelope of human cytomegalovirus. Virus Res. 4:31-36[CrossRef][Medline]. |
| 6. | Browne, H., S. Bell, T. Minson, and D. W. Wilson. 1996. An endoplasmic reticulum-retained herpes simplex virus glycoprotein H is absent from secreted virions: evidence for reenvelopment during egress. J. Virol. 70:4311-4316[Abstract]. |
| 7. |
Chee, M.,
S. Rudolf,
B. Plachter,
B. Barrell, and G. Jahn.
1989.
Identification of the major capsid protein gene of human cytomegalovirus.
J. Virol.
63:1345-1353 |
| 8. |
Chen, C., and H. Okayama.
1987.
High-efficiency transformation of mammalian cells by plasmid DNA.
Mol. Cell. Biol.
7:2745-2752 |
| 9. | Di Lazarro, C., G. Campadelli-Fiume, and M. R. Torrisi. 1995. Intermediate forms of glycoconjugates are present in the envelope of herpes simplex virions during their transport along the exocytic pathway. Virology 214:619-623[CrossRef][Medline]. |
| 10. |
Fish, K. N.,
C. Soderberg-Naucler, and J. A. Nelson.
1998.
Steady-state plasma membrane expression of human cytomegalovirus gB is determined by the phosphorylation state of Ser900.
J. Virol.
72:6657-6664 |
| 11. |
Gershon, A. A.,
D. L. Sherman,
Z. Zhu,
C. A. Gabel,
R. T. Ambron, and M. Gershon.
1994.
Intracellular transport of newly synthesized varicella-zoster virus: final envelopment in the trans-Golgi network.
J. Virol.
68:6372-6390 |
| 12. | Gibson, W. 1996. Structure and assembly of the virion. Intervirology 39:389-400[Medline]. |
| 13. |
Gong, M., and E. Kieff.
1990.
Intracellular trafficking of two major Epstein-Barr virus glycoproteins gp350/220 and gp110.
J. Virol.
64:1507-1516 |
| 14. | Granzow, H., F. Weiland, A. Jones, B. G. Klupp, A. Karger, and T. C. Mettenleitner. 1997. Ultrastructural analysis of the replication cycle of pseudorabies virus in cell culture: a reassessment. J. Virol. 71:2072-2082[Abstract]. |
| 15. |
Greis, K. D.,
W. Gibson, and G. W. Hart.
1994.
Site-specific glycosylation of the human cytomegalovirus tegument basic phosphoprotein (UL32) at serine 921 and serine 952.
J. Virol.
68:8339-8349 |
| 16. | Grose, C. 1990. Glycoproteins encoded by varicella-zoster virus: biosynthesis, phosphorylation, and intracellular trafficking. Annu. Rev. Microbiol. 44:59-80[CrossRef][Medline]. |
| 17. |
Hensel, G.,
H. Meyer,
S. Gartner,
G. Brand, and H. F. Kern.
1995.
Nuclear localization of the human cytomegalovirus tegument protein pp150 (ppUL32).
J. Gen. Virol.
76:1591-1601 |
| 18. | Johnson, D. C., and P. G. Spear. 1983. O-linked oligosaccharides are acquired by herpes simplex virus glycoproteins in the Golgi apparatus. Cell 32:987-997[CrossRef][Medline]. |
| 19. |
Johnston, J. A.,
C. L. Ward, and R. R. Kopito.
1998.
Aggresomes: a cellular response to misfolded proteins.
J. Cell Biol.
143:1883-1898 |
| 20. |
Jones, F., and C. Grose.
1988.
Role of cytoplasmic vacuoles in varicella-zoster virus glycoprotein trafficking and virion envelopment.
J. Virol.
62:2701-2711 |
| 21. | Landini, M. P., B. Severi, G. Furlini, and D. G. L. Badiali. 1987. Human cytomegalovirus structural components: intracellular and intraviral localization of p28 and p65-69 by immunoelectron microscopy. Virus Res. 8:15-23[CrossRef][Medline]. |
| 22. | Li, L., J. A. Nelson, and W. J. Britt. 1997. Glycoprotein H related complexes of human cytomegalovirus: identification of a third protein in the gCIII complex. J. Virol. 71:3090-3097[Abstract]. |
| 23. | Molloy, S. S., E. D. Anderson, F. Jean, and G. Thomas. 1999. Bi-cycling the furin pathway: from TGN localization to pathogen activation and embryogenesis. Trends Cell Biol. 9:28-35[CrossRef][Medline]. |
| 24. |
Nelson, D. S.,
C. Alvarez,
Y. S. Gao,
R. Garcia-Mata,
E. Fialkowski, and E. Sztul.
1998.
The membrane transport factor TAP/p115 cycles between the Golgi and earlier secretory compartments and contains distinct domains required for its localization and function.
J. Cell Biol.
143:319-331 |
| 25. | Nii, S., M. Yoshida, F. Uno, T. Kurata, Y. Ikuta, and K. Yamanishi. 1990. Replication of human herpesvirus 6 (HHV-6): morphological aspects. Adv. Exp. Med. Biol. 278:19-28[Medline]. |
| 26. | Plachter, B., W. Britt, R. Vornhagen, T. Stamminger, and G. Jahn. 1993. Analysis of proteins encoded by IEI regions 1 and 2 of human cytomegalovirus using monoclonal antibodies generated against recombinant antigens. Virology 193:642-652[CrossRef][Medline]. |
| 27. |
Rixon, F. J.,
C. Addison, and J. McLauchlan.
1992.
Assembly of enveloped tegument structures (L particles) can occur independently of virion maturation in herpes simplex type 1-infected cells.
J. Gen. Virol.
73:277-284 |
| 28. |
Roffman, E.,
J. P. Albert,
J. P. Goff, and N. Frenkel.
1990.
Putative site for the acquisition of human herpesvirus 6 virion tegument.
J. Virol.
64:6308-6313 |
| 29. | Roizman, B. 1996. Herpesviridae, p. 2221-2230. In B. N. Fields, D. M. Knipe, P. M. Howley, R. M. Chanock, J. L. Melnick, T. P. Monath, B. Roizman, and S. E. Straus (ed.), Virology, 3rd ed. Lippincott-Raven, Philadelphia, Pa. |
| 30. |
Sanchez, V.,
P. C. Angeletti,
J. A. Engler, and W. J. Britt.
1998.
Localization of human cytomegalovirus structural proteins to the nuclear matrix of infected human fibroblasts.
J. Virol.
72:3321-3329 |
| 31. |
Simpson, J. A.,
J. C. Chow,
J. Baker,
N. Avdalovi,
S. Yuan,
D. Au,
M. S. Co,
M. Vasquez,
W. Britt, and K. L. Coelingh.
1993.
Neutralizing monoclonal antibodies that distinguish three antigenic sites on the human cytomegalovirus glycoprotein H have conformationally-distinct binding sites.
J. Virol.
67:489-496 |
| 32. |
Smith, J. D., and E. DeHarven.
1973.
Herpes simplex virus and human cytomegalovirus replication in WI-38 cells. I. Sequence of viral replication.
J. Virol.
12:919-930 |
| 33. | Spear, P. G. 1985. Glycoproteins specified by herpes simplex viruses, p. 315-356. In B. Roizman (ed.), The herpesviruses, vol. 3. Plenum Press, New York, N.Y. |
| 34. |
Stackpole, C. W.
1969.
Herpes-type virus of the frog renal adenocarcinoma. I. Virus development in tumor transplants maintained at low temperature.
J. Virol.
4:75-93 |
| 35. |
Tirabassi, R. S., and L. W. Enquist.
1999.
Mutation of the YXXL endocytosis motif in the cytoplasmic tail of pseudorabies virus gE.
J. Virol.
73:2717-2728 |
| 36. | Tooze, J., M. Hollinshead, B. Reis, K. Radsak, and H. Kern. 1993. Progeny vaccinia and human cytomegalovirus particles utilize early endosomal cisternae for their envelopes. Eur. J. Cell Biol. 601:163-178. |
| 37. | Vey, M., W. Schafer, B. Reis, R. Ohuchi, W. Britt, W. Garten, H. D. Klenk, and K. Radsak. 1995. Proteolytic processing of human cytomegalovirus glycoprotein B (gpUL55) is mediated by the human endoprotease furin. Virology 206:746-749[CrossRef][Medline]. |
| 38. | Wan, L., S. S. Molloy, L. Thomas, G. Liu, Y. Xiang, S. L. Rybak, and G. Thomas. 1998. PACS-1 defines a novel gene family of cytosolic sorting proteins required for trans-Golgi network localization. Cell 94:205-216[CrossRef][Medline]. |
| 39. |
Whealy, M. E.,
J. P. Card,
R. P. Meade,
A. K. Robbins, and L. W. Enquist.
1991.
Effect of brefeldin A on alphaherpesvirus membrane protein glycosylation and virus egress.
J. Virol.
65:1066-1081 |
| 40. |
Whealy, M. E.,
A. K. Robbins, and L. W. Enquist.
1990.
The export pathway of the pseudorabies virus gB homolog gII involves oligomer formation in the endoplasmic reticulum and protease processing in the Golgi apparatus.
J. Virol.
64:1946-1955 |
| 41. |
Whealy, M. E.,
A. K. Robbins,
F. Tufaro, and L. W. Enquist.
1992.
A cellular function is required for pseudorabies virus envelope glycoprotein processing and virus egress.
J. Virol.
66:3803-3810 |
| 42. | Zhu, Z., M. D. Gershon, Y. Hao, R. T. Ambron, C. A. Gabel, and A. A. Gershon. 1995. Envelopment of varicella-zoster virus: targeting of viral glycoproteins to the trans-Golgi network. J. Virol. 69:7951-7959[Abstract]. |
| 43. |
Zhu, Z.,
Y. Hao,
M. D. Gershon,
R. T. Ambron, and A. A. Gershon.
1996.
Targeting of glycoprotein I (gE) of varicella-zoster virus to the trans-Golgi network by an AYRV sequence and an acidic amino acid-rich patch in the cytosolic domain of the molecule.
J. Virol.
70:6563-6575 |
Journal of Virology, January 2000, p. 975-986, Vol. 74, No. 2
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Buchkovich, N. J., Maguire, T. G., Paton, A. W., Paton, J. C., Alwine, J. C.
(2009). The Endoplasmic Reticulum Chaperone BiP/GRP78 Is Important in the Structure and Function of the Human Cytomegalovirus Assembly Compartment. J. Virol.
83: 11421-11428
[Abstract] [Full Text] -
Tandon, R., AuCoin, D. P., Mocarski, E. S.
(2009). Human Cytomegalovirus Exploits ESCRT Machinery in the Process of Virion Maturation. J. Virol.
83: 10797-10807
[Abstract] [Full Text] -
Chevillotte, M., Schubert, A., Mertens, T., von Einem, J.
(2009). Fluorescence-Based Assay for Phenotypic Characterization of Human Cytomegalovirus Polymerase Mutations Regarding Drug Susceptibility and Viral Replicative Fitness. Antimicrob. Agents Chemother.
53: 3752-3761
[Abstract] [Full Text] -
Hanson, L. K., Slater, J. S., Cavanaugh, V. J., Newcomb, W. W., Bolin, L. L., Nelson, C. N., Fetters, L. D., Tang, Q., Brown, J. C., Maul, G. G., Campbell, A. E.
(2009). Murine Cytomegalovirus Capsid Assembly Is Dependent on US22 Family Gene M140 in Infected Macrophages. J. Virol.
83: 7449-7456
[Abstract] [Full Text] -
Walker, J. D., Maier, C. L., Pober, J. S.
(2009). Cytomegalovirus-Infected Human Endothelial Cells Can Stimulate Allogeneic CD4+ Memory T Cells by Releasing Antigenic Exosomes. J. Immunol.
182: 1548-1559
[Abstract] [Full Text] -
Schroer, J., Shenk, T.
(2008). Inhibition of cyclooxygenase activity blocks cell-to-cell spread of human cytomegalovirus. Proc. Natl. Acad. Sci. USA
105: 19468-19473
[Abstract] [Full Text] -
Tandon, R., Mocarski, E. S.
(2008). Control of Cytoplasmic Maturation Events by Cytomegalovirus Tegument Protein pp150. J. Virol.
82: 9433-9444
[Abstract] [Full Text] -
Seo, J.-Y., Britt, W. J.
(2008). Multimerization of Tegument Protein pp28 within the Assembly Compartment Is Required for Cytoplasmic Envelopment of Human Cytomegalovirus. J. Virol.
82: 6272-6287
[Abstract] [Full Text] -
Kalejta, R. F.
(2008). Tegument Proteins of Human Cytomegalovirus. Microbiol. Mol. Biol. Rev.
72: 249-265
[Abstract] [Full Text] -
Sugimoto, K., Uema, M., Sagara, H., Tanaka, M., Sata, T., Hashimoto, Y., Kawaguchi, Y.
(2008). Simultaneous Tracking of Capsid, Tegument, and Envelope Protein Localization in Living Cells Infected with Triply Fluorescent Herpes Simplex Virus 1. J. Virol.
82: 5198-5211
[Abstract] [Full Text] -
Raftery, M. J., Hitzler, M., Winau, F., Giese, T., Plachter, B., Kaufmann, S. H. E., Schonrich, G.
(2008). Inhibition of CD1 Antigen Presentation by Human Cytomegalovirus. J. Virol.
82: 4308-4319
[Abstract] [Full Text] -
Borst, E. M., Wagner, K., Binz, A., Sodeik, B., Messerle, M.
(2008). The Essential Human Cytomegalovirus Gene UL52 Is Required for Cleavage-Packaging of the Viral Genome. J. Virol.
82: 2065-2078
[Abstract] [Full Text] -
Ryckman, B. J., Rainish, B. L., Chase, M. C., Borton, J. A., Nelson, J. A., Jarvis, M. A., Johnson, D. C.
(2008). Characterization of the Human Cytomegalovirus gH/gL/UL128-131 Complex That Mediates Entry into Epithelial and Endothelial Cells. J. Virol.
82: 60-70
[Abstract] [Full Text] -
Sadaoka, T., Yoshii, H., Imazawa, T., Yamanishi, K., Mori, Y.
(2007). Deletion in Open Reading Frame 49 of Varicella-Zoster Virus Reduces Virus Growth in Human Malignant Melanoma Cells but Not in Human Embryonic Fibroblasts. J. Virol.
81: 12654-12665
[Abstract] [Full Text] -
Das, S., Vasanji, A., Pellett, P. E.
(2007). Three-Dimensional Structure of the Human Cytomegalovirus Cytoplasmic Virion Assembly Complex Includes a Reoriented Secretory Apparatus. J. Virol.
81: 11861-11869
[Abstract] [Full Text] -
Sanchez, V., Mahr, J. A., Orazio, N. I., Spector, D. H.
(2007). Nuclear Export of the Human Cytomegalovirus Tegument Protein pp65 Requires Cyclin-Dependent Kinase Activity and the Crm1 Exporter. J. Virol.
81: 11730-11736
[Abstract] [Full Text] -
Krzyzaniak, M., Mach, M., Britt, W. J.
(2007). The Cytoplasmic Tail of Glycoprotein M (gpUL100) Expresses Trafficking Signals Required for Human Cytomegalovirus Assembly and Replication. J. Virol.
81: 10316-10328
[Abstract] [Full Text] -
Sourvinos, G., Tavalai, N., Berndt, A., Spandidos, D. A., Stamminger, T.
(2007). Recruitment of Human Cytomegalovirus Immediate-Early 2 Protein onto Parental Viral Genomes in Association with ND10 in Live-Infected Cells. J. Virol.
81: 10123-10136
[Abstract] [Full Text] -
Seo, J.-Y., Britt, W. J.
(2007). Cytoplasmic Envelopment of Human Cytomegalovirus Requires the Postlocalization Function of Tegument Protein pp28 within the Assembly Compartment. J. Virol.
81: 6536-6547
[Abstract] [Full Text] -
Mach, M., Osinski, K., Kropff, B., Schloetzer-Schrehardt, U., Krzyzaniak, M., Britt, W.
(2007). The Carboxy-Terminal Domain of Glycoprotein N of Human Cytomegalovirus Is Required for Virion Morphogenesis. J. Virol.
81: 5212-5224
[Abstract] [Full Text] -
Adler, B., Scrivano, L., Ruzcics, Z., Rupp, B., Sinzger, C., Koszinowski, U.
(2006). Role of human cytomegalovirus UL131A in cell type-specific virus entry and release. J. Gen. Virol.
87: 2451-2460
[Abstract] [Full Text] -
Feng, X., Schroer, J., Yu, D., Shenk, T.
(2006). Human Cytomegalovirus pUS24 Is a Virion Protein That Functions Very Early in the Replication Cycle.. J. Virol.
80: 8371-8378
[Abstract] [Full Text] -
AuCoin, D. P., Smith, G. B., Meiering, C. D., Mocarski, E. S.
(2006). Betaherpesvirus-Conserved Cytomegalovirus Tegument Protein ppUL32 (pp150) Controls Cytoplasmic Events during Virion Maturation.. J. Virol.
80: 8199-8210
[Abstract] [Full Text] -
Seo, J.-Y., Britt, W. J.
(2006). Sequence Requirements for Localization of Human Cytomegalovirus Tegument Protein pp28 to the Virus Assembly Compartment and for Assembly of Infectious Virus.. J. Virol.
80: 5611-5626
[Abstract] [Full Text] -
Shimamura, M., Mach, M., Britt, W. J.
(2006). Human Cytomegalovirus Infection Elicits a Glycoprotein M (gM)/gN-Specific Virus-Neutralizing Antibody Response. J. Virol.
80: 4591-4600
[Abstract] [Full Text] -
Munger, J., Yu, D., Shenk, T.
(2006). UL26-Deficient Human Cytomegalovirus Produces Virions with Hypophosphorylated pp28 Tegument Protein That Is Unstable within Newly Infected Cells.. J. Virol.
80: 3541-3548
[Abstract] [Full Text] -
Gaspar, M., Shenk, T.
(2006). Human cytomegalovirus inhibits a DNA damage response by mislocalizing checkpoint proteins. Proc. Natl. Acad. Sci. USA
103: 2821-2826
[Abstract] [Full Text] -
Das, S., Skomorovska-Prokvolit, Y., Wang, F.-Z., Pellett, P. E.
(2006). Infection-Dependent Nuclear Localization of US17, a Member of the US12 Family of Human Cytomegalovirus-Encoded Seven-Transmembrane Proteins. J. Virol.
80: 1191-1203
[Abstract] [Full Text] -
Prichard, M. N., Britt, W. J., Daily, S. L., Hartline, C. B., Kern, E. R.
(2005). Human Cytomegalovirus UL97 Kinase Is Required for the Normal Intranuclear Distribution of pp65 and Virion Morphogenesis. J. Virol.
79: 15494-15502
[Abstract] [Full Text] -
Hegde, N. R., Dunn, C., Lewinsohn, D. M., Jarvis, M. A., Nelson, J. A., Johnson, D. C.
(2005). Endogenous human cytomegalovirus gB is presented efficiently by MHC class II molecules to CD4+ CTL. JEM
202: 1109-1119
[Abstract] [Full Text] -
Spaderna, S., Kropff, B., Kodel, Y., Shen, S., Coley, S., Lu, S., Britt, W., Mach, M.
(2005). Deletion of gpUL132, a Structural Component of Human Cytomegalovirus, Results in Impaired Virus Replication in Fibroblasts. J. Virol.
79: 11837-11847
[Abstract] [Full Text] -
Cassady, K. A.
(2005). Human Cytomegalovirus TRS1 and IRS1 Gene Products Block the Double-Stranded-RNA-Activated Host Protein Shutoff Response Induced by Herpes Simplex Virus Type 1 Infection. J. Virol.
79: 8707-8715
[Abstract] [Full Text] -
Turcotte, S., Letellier, J., Lippe, R.
(2005). Herpes Simplex Virus Type 1 Capsids Transit by the trans-Golgi Network, Where Viral Glycoproteins Accumulate Independently of Capsid Egress. J. Virol.
79: 8847-8860
[Abstract] [Full Text] -
Netterwald, J., Yang, S., Wang, W., Ghanny, S., Cody, M., Soteropoulos, P., Tian, B., Dunn, W., Liu, F., Zhu, H.
(2005). Two Gamma Interferon-Activated Site-Like Elements in the Human Cytomegalovirus Major Immediate-Early Promoter/Enhancer Are Important for Viral Replication. J. Virol.
79: 5035-5046
[Abstract] [Full Text] -
Karabekian, Z., Hanson, L. K., Slater, J. S., Krishna, N. K., Bolin, L. L., Kerry, J. A., Campbell, A. E.
(2005). Complex Formation among Murine Cytomegalovirus US22 Proteins Encoded by Genes M139, M140, and M141. J. Virol.
79: 3525-3535
[Abstract] [Full Text] -
Sampaio, K. L., Cavignac, Y., Stierhof, Y.-D., Sinzger, C.
(2005). Human Cytomegalovirus Labeled with Green Fluorescent Protein for Live Analysis of Intracellular Particle Movements. J. Virol.
79: 2754-2767
[Abstract] [Full Text] -
Schierling, K., Buser, C., Mertens, T., Winkler, M.
(2005). Human Cytomegalovirus Tegument Protein ppUL35 Is Important for Viral Replication and Particle Formation. J. Virol.
79: 3084-3096
[Abstract] [Full Text] -
Mach, M., Kropff, B., Kryzaniak, M., Britt, W.
(2005). Complex Formation by Glycoproteins M and N of Human Cytomegalovirus: Structural and Functional Aspects. J. Virol.
79: 2160-2170
[Abstract] [Full Text] -
Silva, M. C., Schroer, J., Shenk, T.
(2005). Human cytomegalovirus cell-to-cell spread in the absence of an essential assembly protein. Proc. Natl. Acad. Sci. USA
102: 2081-2086
[Abstract] [Full Text] -
Maresova, L., Pasieka, T. J., Homan, E., Gerday, E., Grose, C.
(2005). Incorporation of Three Endocytosed Varicella-Zoster Virus Glycoproteins, gE, gH, and gB, into the Virion Envelope. J. Virol.
79: 997-1007
[Abstract] [Full Text] -
Hertel, L., Mocarski, E. S.
(2004). Global Analysis of Host Cell Gene Expression Late during Cytomegalovirus Infection Reveals Extensive Dysregulation of Cell Cycle Gene Expression and Induction of Pseudomitosis Independent of US28 Function. J. Virol.
78: 11988-12011
[Abstract] [Full Text] -
Terhune, S. S., Schroer, J., Shenk, T.
(2004). RNAs Are Packaged into Human Cytomegalovirus Virions in Proportion to Their Intracellular Concentration. J. Virol.
78: 10390-10398
[Abstract] [Full Text] -
Wang, Z., La Rosa, C., Mekhoubad, S., Lacey, S. F., Villacres, M. C., Markel, S., Longmate, J., Ellenhorn, J. D. I., Siliciano, R. F., Buck, C., Britt, W. J., Diamond, D. J.
(2004). Attenuated poxviruses generate clinically relevant frequencies of CMV-specific T cells. Blood
104: 847-856
[Abstract] [Full Text] -
Netterwald, J. R., Jones, T. R., Britt, W. J., Yang, S.-J., McCrone, I. P., Zhu, H.
(2004). Postattachment Events Associated with Viral Entry Are Necessary for Induction of Interferon-Stimulated Genes by Human Cytomegalovirus. J. Virol.
78: 6688-6691
[Abstract] [Full Text] -
Maglova, L. M., Crowe, W. E., Russell, J. M.
(2004). Perinuclear localization of Na-K-Cl-cotransporter protein after human cytomegalovirus infection. Am. J. Physiol. Cell Physiol.
286: C1324-C1334
[Abstract] [Full Text] -
Jones, T. R., Lee, S.-W.
(2004). An Acidic Cluster of Human Cytomegalovirus UL99 Tegument Protein Is Required for Trafficking and Function. J. Virol.
78: 1488-1502
[Abstract] [Full Text] -
Jarvis, M. A., Jones, T. R., Drummond, D. D., Smith, P. P., Britt, W. J., Nelson, J. A., Baldick, C. J.
(2004). Phosphorylation of Human Cytomegalovirus Glycoprotein B (gB) at the Acidic Cluster Casein Kinase 2 Site (Ser900) Is Required for Localization of gB to the trans-Golgi Network and Efficient Virus Replication. J. Virol.
78: 285-293
[Abstract] [Full Text] -
Britt, W. J., Jarvis, M., Seo, J.-Y., Drummond, D., Nelson, J.
(2004). Rapid Genetic Engineering of Human Cytomegalovirus by Using a Lambda Phage Linear Recombination System: Demonstration that pp28 (UL99) Is Essential for Production of Infectious Virus. J. Virol.
78: 539-543
[Abstract] [Full Text] -
Patrone, M., Percivalle, E., Secchi, M., Fiorina, L., Pedrali-Noy, G., Zoppe, M., Baldanti, F., Hahn, G., Koszinowski, U. H., Milanesi, G., Gallina, A.
(2003). The human cytomegalovirus UL45 gene product is a late, virion-associated protein and influences virus growth at low multiplicities of infection. J. Gen. Virol.
84: 3359-3370
[Abstract] [Full Text] -
Crump, C. M., Hung, C.-H., Thomas, L., Wan, L., Thomas, G.
(2003). Role of PACS-1 in Trafficking of Human Cytomegalovirus Glycoprotein B and Virus Production. J. Virol.
77: 11105-11113
[Abstract] [Full Text] -
Silva, M. C., Yu, Q.-C., Enquist, L., Shenk, T.
(2003). Human Cytomegalovirus UL99-Encoded pp28 Is Required for the Cytoplasmic Envelopment of Tegument-Associated Capsids. J. Virol.
77: 10594-10605
[Abstract] [Full Text] -
Terauchi, M., Koi, H., Hayano, C., Toyama-Sorimachi, N., Karasuyama, H., Yamanashi, Y., Aso, T., Shirakata, M.
(2003). Placental Extravillous Cytotrophoblasts Persistently Express Class I Major Histocompatibility Complex Molecules after Human Cytomegalovirus Infection. J. Virol.
77: 8187-8195
[Abstract] [Full Text] -
Baillie, J., Sahlender, D. A., Sinclair, J. H.
(2003). Human Cytomegalovirus Infection Inhibits Tumor Necrosis Factor Alpha (TNF-{alpha}) Signaling by Targeting the 55-Kilodalton TNF-{alpha} Receptor. J. Virol.
77: 7007-7016
[Abstract] [Full Text] -
Kopp, M., Granzow, H., Fuchs, W., Klupp, B. G., Mundt, E., Karger, A., Mettenleiter, T. C.
(2003). The Pseudorabies Virus UL11 Protein Is a Virion Component Involved in Secondary Envelopment in the Cytoplasm. J. Virol.
77: 5339-5351
[Abstract] [Full Text] -
Brignati, M. J., Loomis, J. S., Wills, J. W., Courtney, R. J.
(2003). Membrane Association of VP22, a Herpes Simplex Virus Type 1 Tegument Protein. J. Virol.
77: 4888-4898
[Abstract] [Full Text] -
Homman-Loudiyi, M., Hultenby, K., Britt, W., Soderberg-Naucler, C.
(2003). Envelopment of Human Cytomegalovirus Occurs by Budding into Golgi-Derived Vacuole Compartments Positive for gB, Rab 3, Trans-Golgi Network 46, and Mannosidase II. J. Virol.
77: 3191-3203
[Abstract] [Full Text] -
Lai, L., Britt, W. J.
(2003). The Interaction between the Major Capsid Protein and the Smallest Capsid Protein of Human Cytomegalovirus Is Dependent on Two Linear Sequences in the Smallest Capsid Protein. J. Virol.
77: 2730-2735
[Abstract] [Full Text] -
Hegde, N. R., Tomazin, R. A., Wisner, T. W., Dunn, C., Boname, J. M., Lewinsohn, D. M., Johnson, D. C.
(2002). Inhibition of HLA-DR Assembly, Transport, and Loading by Human Cytomegalovirus Glycoprotein US3: a Novel Mechanism for Evading Major Histocompatibility Complex Class II Antigen Presentation. J. Virol.
76: 10929-10941
[Abstract] [Full Text] -
Adair, R., Douglas, E. R., Maclean, J. B., Graham, S. Y., Aitken, J. D., Jamieson, F. E., Dargan, D. J.
(2002). The products of human cytomegalovirus genes UL23, UL24, UL43 and US22 are tegument components. J. Gen. Virol.
83: 1315-1324
[Abstract] [Full Text] -
Dal Monte, P., Pignatelli, S., Zini, N., Maraldi, N. M., Perret, E., Prevost, M. C., Landini, M. P.
(2002). Analysis of intracellular and intraviral localization of the human cytomegalovirus UL53 protein. J. Gen. Virol.
83: 1005-1012
[Abstract] [Full Text] -
Jarvis, M. A., Fish, K. N., Soderberg-Naucler, C., Streblow, D. N., Meyers, H. L., Thomas, G., Nelson, J. A.
(2002). Retrieval of Human Cytomegalovirus Glycoprotein B from Cell Surface Is Not Required for Virus Envelopment in Astrocytoma Cells. J. Virol.
76: 5147-5155
[Abstract] [Full Text] -
Ogawa-Goto, K., Irie, S., Omori, A., Miura, Y., Katano, H., Hasegawa, H., Kurata, T., Sata, T., Arao, Y.
(2002). An Endoplasmic Reticulum Protein, p180, Is Highly Expressed in Human Cytomegalovirus-Permissive Cells and Interacts with the Tegument Protein Encoded by UL48. J. Virol.
76: 2350-2362
[Abstract] [Full Text] -
Liu, Y., Biegalke, B. J.
(2002). The Human Cytomegalovirus UL35 Gene Encodes Two Proteins with Different Functions. J. Virol.
76: 2460-2468
[Abstract] [Full Text] -
Theiler, R. N., Compton, T.
(2002). Distinct Glycoprotein O Complexes Arise in a Post-Golgi Compartment of Cytomegalovirus-Infected Cells. J. Virol.
76: 2890-2898
[Abstract] [Full Text] -
Sanchez, V., Clark, C. L., Yen, J. Y., Dwarakanath, R., Spector, D. H.
(2002). Viable Human Cytomegalovirus Recombinant Virus with an Internal Deletion of the IE2 86 Gene Affects Late Stages of Viral Replication. J. Virol.
76: 2973-2989
[Abstract] [Full Text] -
Mettenleiter, T. C.
(2002). Herpesvirus Assembly and Egress. J. Virol.
76: 1537-1547
[Full Text] -
Spaderna, S., Blessing, H., Bogner, E., Britt, W., Mach, M.
(2002). Identification of Glycoprotein gpTRL10 as a Structural Component of Human Cytomegalovirus. J. Virol.
76: 1450-1460
[Abstract] [Full Text] -
Chin, K.-C., Cresswell, P.
(2001). Inaugural Article: Viperin (cig5), an IFN-inducible antiviral protein directly induced by human cytomegalovirus. Proc. Natl. Acad. Sci. USA
98: 15125-15130
[Abstract] [Full Text] -
Loomis, J. S., Bowzard, J. B., Courtney, R. J., Wills, J. W.
(2001). Intracellular Trafficking of the UL11 Tegument Protein of Herpes Simplex Virus Type 1. J. Virol.
75: 12209-12219
[Abstract] [Full Text] -
Ren, X., Harms, J. S., Splitter, G. A.
(2001). Tyrosine Phosphorylation of Bovine Herpesvirus 1 Tegument Protein VP22 Correlates with the Incorporation of VP22 into Virions. J. Virol.
75: 9010-9017
[Abstract] [Full Text] -
Kinchington, P. R., Fite, K., Seman, A., Turse, S. E.
(2001). Virion Association of IE62, the Varicella-Zoster Virus (VZV) Major Transcriptional Regulatory Protein, Requires Expression of the VZV Open Reading Frame 66 Protein Kinase. J. Virol.
75: 9106-9113
[Abstract] [Full Text] -
Baxter, M. K., Gibson, W.
(2001). Cytomegalovirus Basic Phosphoprotein (pUL32) Binds to Capsids In Vitro through Its Amino One-Third. J. Virol.
75: 6865-6873
[Abstract] [Full Text] -
Fraile-Ramos, A., Kledal, T. N., Pelchen-Matthews, A., Bowers, K., Schwartz, T. W., Marsh, M.
(2001). The Human Cytomegalovirus US28 Protein Is Located in Endocytic Vesicles and Undergoes Constitutive Endocytosis and Recycling. Mol. Biol. Cell
12: 1737-1749
[Abstract] [Full Text] -
Oliveira, S. A., Shenk, T. E.
(2001). Murine cytomegalovirus M78 protein, a G protein-coupled receptor homologue, is a constituent of the virion and facilitates accumulation of immediate-early viral mRNA. Proc. Natl. Acad. Sci. USA
98: 3237-3242
[Abstract] [Full Text] -
Borst, E.-M., Mathys, S., Wagner, M., Muranyi, W., Messerle, M.
(2001). Genetic Evidence of an Essential Role for Cytomegalovirus Small Capsid Protein in Viral Growth. J. Virol.
75: 1450-1458
[Abstract] [Full Text] -
Mach, M., Kropff, B., Dal Monte, P., Britt, W.
(2000). Complex Formation by Human Cytomegalovirus Glycoproteins M (gpUL100) and N (gpUL73). J. Virol.
74: 11881-11892
[Abstract] [Full Text] -
Sanchez, V., Sztul, E., Britt, W. J.
(2000). Human Cytomegalovirus pp28 (UL99) Localizes to a Cytoplasmic Compartment Which Overlaps the Endoplasmic Reticulum-Golgi-Intermediate Compartment. J. Virol.
74: 3842-3851
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»