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Journal of Virology, June 2001, p. 5697-5702, Vol. 75, No. 12
Multi-Imaging Centre and Department of
Anatomy1 and Division of Virology,
Department of Pathology,2 University of
Cambridge, Cambridge, United Kingdom
Received 4 December 2000/Accepted 26 March 2001
Herpes simplex virus (HSV) nucleocapsids acquire an envelope by
budding through the inner nuclear membrane, but it is uncertain whether
this envelope is retained during virus maturation and egress or whether
mature progeny virions are derived by deenvelopment at the outer
nuclear membrane followed by reenvelopment in a cytoplasmic compartment. To resolve this issue, we used immunogold electron microscopy to examine the distribution of glycoprotein D
(gD) in cells infected with HSV-1 encoding a wild-type gD or a gD which is retrieved to the endoplasmic reticulum (ER). In cells infected with
wild-type HSV-1, extracellular virions and virions in the perinuclear
space bound approximately equal amounts of gD antibody. In cells
infected with HSV-1 encoding an ER-retrieved gD, the inner and outer
nuclear membranes were heavily gold labeled, as were perinuclear
enveloped virions. Extracellular virions exhibited very little gold
decoration (10- to 30-fold less than perinuclear virions). We conclude
that the envelope of perinuclear virions must be lost during maturation
and egress and that mature progeny virions must acquire an envelope
from a post-ER cytoplasmic compartment. We noted also that gD appears
to be excluded from the plasma membrane in cells infected with
wild-type virus.
Herpesvirus nucleocapsids assemble
in the nuclei of infected cells and acquire an envelope by budding
through the inner nuclear membrane, but the subsequent route of virus
maturation and egress has been a matter of controversy. Over 30 years
ago, Stackpole (19) proposed that enveloped virions in the
perinuclear space fused with the outer nuclear membrane, releasing into
the cytoplasm naked nucleocapsids which acquired a final envelope by
budding into a late cytoplasmic compartment. The observation that
infectious herpes simplex virions accumulated within cells in the
absence of a functional Golgi apparatus (11) implied that
virions in the perinuclear space were infectious and suggested that the
Golgi apparatus was required merely for egress of these virions. This "single envelopment" pathway, in which perinuclear enveloped
virions are transported to the cell surface via the secretory pathway and the envelope glycoproteins are processed in situ, has
the virtue of simplicity and became widely accepted as the route of egress of herpes simplex virus (HSV) (e.g., see reference
17). Studies of other alphaherpesviruses, notably
varicella-zoster virus and pseudorabies virus, have, however, supported
the view that the final envelope is acquired in a cytoplasmic
compartment, thus favoring the "two-step envelopment" route of
egress (6, 8, 12, 13, 22, 24). Indeed, several
observations are inconsistent with the view that HSV acquires its final
envelope from the nuclear membrane: the phospholipid composition of
secreted virions is different from that of the nuclear membrane
(21); naked nucleocapsids, not enveloped virions, are
observed in axons during virus egress (10, 15, 16); and a
major tegument component, VP22, is observed apparently exclusively in
the cytoplasm of live virus-infected cells (4). A detailed
analysis of the evidence for and against the alternative routes of
egress is provided by Enquist et al. (5).
In an attempt to resolve this controversy, we constructed HSVs in which
glycoprotein D (gD) or gH were targeted to the endoplasmic reticulum (ER) by addition of the ER retrieval signal KKXX to the
C-terminal cytoplasmic domain, and we reported that secreted progeny
virions were devoid of the targeted molecules (3, 23). The
simplest interpretation of these findings is that the virus acquires
its final envelope from a cytoplasmic compartment from which an
ER-retrieved molecule would be excluded. It is possible, however, that
the KKXX motif could result in reduced trafficking of the molecule to
the inner nuclear membrane or could exclude the molecule from the
budding process, and in either case the targeted molecule would be
excluded from progeny virions regardless of the route of egress. Formal
proof that progeny virions are enveloped in the cytoplasm requires us
to demonstrate that enveloped virions in the perinuclear space contain
the ER-targeted glycoprotein but that this molecule is
absent in progeny virus. Here, we report immunogold electron
microscopic studies which show that this is the case: cells infected
with an HSV-1 mutant encoding an ER-retrieved gD produce perinuclear
enveloped virions which contain gD, but the extracellular progeny
virions have lost this molecule.
In an initial series of experiments, we infected Vero cells with HSV-1
strain SC16 at a multiplicity of infection (MOI) of 10 and examined
thin sections of fixed embedded cells at various times after infection
during the productive phase (0 to 16 h). We found that after 8, 12, or 16 h, most cells contained many capsids in the nucleus and
many cytoplasmic and extracellular enveloped virions, but perinuclear
enveloped virions and virions budding at the inner nuclear membrane
were extremely rare Monolayers of BL1 cells were infected at an MOI of 10 with HSV-1 strain
SC16 or with SC16gDKKXX, a recombinant virus encoding an ER-targeted gD
(23). After a 1-h absorption period, residual inoculum was
removed using a low pH wash, and after 14 h, the monolayers were
harvested by scraping. The infected cells were pelleted, and small
droplets (0.5 µl) of packed cells were mounted onto resin films 3 mm
by 2 mm by 15 µm in dimension, which were quench-frozen by plunging
into liquid propane cooled in liquid nitrogen. Alternatively,
monolayers of BL1 cells were grown on Formvar-coated 100-mesh gold
transmission-electron microscope grids and infected at an MOI of 10 as
described above. After 14 h, the grids were blotted dry and
snap-frozen in liquid propane as described above. Subsequent
low-temperature embedding and antibody labeling was performed as
described in detail by Skepper (18). The frozen cells were
transferred into a Leica AFS freeze substitution unit in vials of
frozen dry methanol containing 0.5% uranyl acetate and were maintained
at In cells infected with wild-type virus, gD was widely distributed (Fig.
1). Extracellular virions and enveloped
virions in the perinuclear space and in cytoplasmic compartments were
invariably gold labeled, as were the nuclear membrane and cytoplasmic
membranes. Gold particles associated with enveloped virions in the
perinuclear and extracellular space were counted (Table
1), and the frequency of gold particles
per virion was similar in these two compartments (approximately two
particles per virion).
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.12.5697-5702.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Herpes Simplex Virus Nucleocapsids Mature to Progeny Virions by
an Envelopment
Deenvelopment Reenvelopment Pathway
ABSTRACT
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most sections contained no virions of this type.
It appears that in Vero cells, strain SC16 buds through the inner
nuclear membrane and traverses the perinuclear space very rapidly, and
it was apparent that it would be difficult or impossible to collect
adequate data on the composition of perinuclear virions using this
virus-cell system. Therefore, we examined infected BL1 cells
(7) since these cells are reported to accumulate
perinuclear virions (G. Kousoulas, personal communication), and we
found that these cells, when infected with strain SC16, contained a few
perinuclear enveloped virions in most cell profiles. These cells were
therefore used in subsequent experiments. This, clearly, is a
compromise. BL1 cells are derived from Vero cells and carry HSV-1
sequences corresponding to nucleotides 107951 to 113323, which contain
the UL51, UL52, and UL53 open reading frames. The presence of these
virus sequences may modify the virus replication cycle, but BL1 cells
have been used as complementing cell lines for UL52 mutants
(7), and we found that they produced the same virus
yields, with the same kinetics, as Vero cells following infection with
HSV-1 strain SC16 at a high MOI. We therefore consider it very unlikely
that these cells are grossly aberrant in the envelopment and processing of HSV virions. Furthermore, it is important to note that previous work
has established that gD-KKXX and gH-KKXX are excluded from extracellular virions in BHK or Vero cells (3, 23). The
main purpose of this study was to establish that the KKXX motif did not
exclude molecules from the envelope of particles in the perinuclear space.
90°C for 24 h, 70°C for 24 h, and 50°C for
24 h. The cells were infiltrated for 3 days with Lowicryl HM20,
which was then polymerized by UV irradiation for 48 h. Sections 50 nm thick were cut using a Leica Ultracut-S and mounted on
Formvar-coated nickel grids. The sections were incubated overnight in a
mixture of anti-gD monoclonal antibodies LP2, LP14, and AP7
(14), each at approximately 100 µg/ml in 10 mM
Tris-buffered saline (TBS) (pH 7.4) containing 0.1% Tween 20, 0.1%
Triton X-100, 10% fetal bovine serum, and 10% normal goat serum
(antibody diluent). The sections were then washed six times in TBS and
incubated for 1 h in goat anti-mouse immunoglobulin G conjugated
to 10-nm-diameter gold particles (British Biocell, Cardiff, United
Kingdom) diluted 1:100 in antibody diluent buffered to pH 8.5 and
without normal goat serum. They were then rinsed six times with TBS,
rinsed twice with deionized water, and stained with uranyl acetate and
lead citrate before examination using a Philips CM100 transmission electron microscope.
View larger version (104K):
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FIG. 1.
Immunogold labeling of cells infected with HSV-1
expressing wild-type gD. (A) Decoration of inner and outer nuclear
membranes and particles in the perinuclear space; (B and C) decoration
of extracellular particles. Bars, 100 nm.
TABLE 1.
Frequency of gold labeling in perinuclear and
extracellular compartments
In contrast, cells infected with SC16gDKKXX showed a much more limited
distribution of gD (Fig.
2). Inner and outer
nuclear membranes were heavily decorated, and we occasionally observed decorated cisternae which probably correspond to ER membranes. Most
cytoplasmic membranes, however, exhibited little or no decoration, consistent with the ER retrieval of gD expressed by this virus. Crucially, for the purpose of this study, enveloped particles in the
perinuclear space were almost invariably associated with gold
particles, whereas extracellular virions were not. Perinuclear virions
were decorated with, on average, 3.2 gold particles per virion, whereas
extracellular virions were associated with 0.26 gold particles/virion
(Table 1). The experiment using SC16gDKKXX-infected BL1 cells was
repeated, and similar results were obtained. Gold particles associated
with perinuclear and extracellular virions were counted (Table 1), and
on this occasion, perinuclear virions were labeled more heavily, by a
factor of 30, than extracellular virions. We noted that the low level
of labeling of extracellular SC16gDKKXX virions appeared asymmetric;
the large majority of virions were unlabeled, but rare virions
decorated with more than one gold particle were present (Fig. 2F). In
contrast, wild-type extracellular virions were more randomly
labeled (Fig. 1C). The source of extracellular gD-positive SC16gDKKXX
virions is unclear, but previous work has shown that they contain
endoglycosidase H-sensitive gD (23). It seems
likely, therefore, that these rare virions are derived directly from
the perinuclear space, either as a result of cell lysis or via a
secretory pathway which bypasses the Golgi.
|
These data confirm previous findings, namely that an ER-targeted gD is excluded from progeny virions (23). Crucially, however, they show that this molecule is present on the inner nuclear membrane, is acquired during the initial budding event, and is present on perinuclear enveloped virions. It follows that the envelope present on perinuclear virions must be lost during the subsequent maturation and egress of these virus particles. In the face of these data, we consider that the single envelopment route of egress is untenable and that the final envelope is acquired in a cytoplasmic compartment.
Many questions remain to be answered. Since progeny virions assemble in the cytoplasm, it follows that the enveloped virions in the perinuclear space are of unknown composition. It has been noted that the tegument of alphaherpesvirus virions in the perinuclear space appears less electron dense than that of progeny virions (8, 19), and it appears that perinuclear virions lack the tegument protein VP22 (4). A study of the effects of monensin on HSV-infected cells implies that perinuclear virions are infectious (11), but similar studies of the effects of brefeldin A on pseudorabies virus infection suggest that these virions are noninfectious (22).
The loss of the envelope from perinuclear virions and the release of
nucleocapsids into the cytoplasm must involve a fusion event, but this
cannot be mediated by those envelope proteins (gD, gB, and gH:L) known
to be required for plasma membrane fusion (20) because
virus mutants lacking these proteins are processed and secreted
normally from infected cells. The UL20 gene product is a possible
candidate for this fusion function because HSV-1 mutants lacking this
gene accumulate in the perinuclear space (2). The site of
final envelopment of alphaherpesviruses is uncertain. Studies of
varicella-zoster virus favor the trans-Golgi network
(6, 24), whereas a recent study of HSV-1 implicates a late
endosomal compartment (9), but regardless of the site, the
mechanism whereby some 10 or more envelope membrane proteins accumulate
in the relevant compartment is entirely unknown. When expressed alone,
different HSV-1 envelope proteins exhibit different trafficking
properties. Thus, gE is localized to the trans-Golgi network
(1), whereas gD appears to traffic to the cell surface via
the default pathway (e.g., see reference 23). We noticed, however, that in the context of wild-type virus infection, gD is
present at very low levels on the cell surface (Fig. 1B). To confirm
this impression, we quantified gold decoration on nuclear membranes,
cytoplasmic membranes, and plasma membranes in the same
wild-type-infected cells. Table 2 shows
that, per unit membrane length, plasma membranes contain about 50-fold
less gD than cytoplasmic membranes. We conclude that in the context of
virus infection, gD is excluded from the plasma membrane by a mechanism
which is at present entirely obscure.
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ACKNOWLEDGMENTS |
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We thank Susanne Bell and Janet Powell for excellent technical support. We thank S. Weller for permission to use BL1 cells and K. Kousoulas for providing them.
This work was supported by the Wellcome Trust, United Kingdom.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Pathology, University of Cambridge, Tennis Court Rd., Cambridge CB2 1QP, United Kingdom. Phone: 44 1223-336920. Fax: 44 1223-336926. E-mail: acm{at}mole.bio.cam.ac.uk.
Present address: Marie Curie Research Institute, The Chart, Oxted,
Surrey RH9 0TL, United Kingdom.
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Journal of Virology, June 2001, p. 5697-5702, Vol. 75, No. 12
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.12.5697-5702.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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80: 4264-4275
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Farnsworth, A., Johnson, D. C.
(2006). Herpes Simplex Virus gE/gI Must Accumulate in the trans-Golgi Network at Early Times and Then Redistribute to Cell Junctions To Promote Cell-Cell Spread.. J. Virol.
80: 3167-3179
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Saksena, M. M., Wakisaka, H., Tijono, B., Boadle, R. A., Rixon, F., Takahashi, H., Cunningham, A. L.
(2006). Herpes simplex virus type 1 accumulation, envelopment, and exit in growth cones and varicosities in mid-distal regions of axons.. J. Virol.
80: 3592-3606
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von Einem, J., Schumacher, D., O'Callaghan, D. J., Osterrieder, N.
(2006). The {alpha}-TIF (VP16) Homologue (ETIF) of Equine Herpesvirus 1 Is Essential for Secondary Envelopment and Virus Egress. J. Virol.
80: 2609-2620
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Naldinho-Souto, R., Browne, H., Minson, T.
(2006). Herpes Simplex Virus Tegument Protein VP16 Is a Component of Primary Enveloped Virions. J. Virol.
80: 2582-2584
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Mettenleiter, T. C., Minson, T., Wild, P.
(2006). Egress of Alphaherpesviruses. J. Virol.
80: 1610-1612
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Leuzinger, H., Ziegler, U., Schraner, E. M., Fraefel, C., Glauser, D. L., Heid, I., Ackermann, M., Mueller, M., Wild, P.
(2005). Herpes Simplex Virus 1 Envelopment Follows Two Diverse Pathways. J. Virol.
79: 13047-13059
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Nozawa, N., Kawaguchi, Y., Tanaka, M., Kato, A., Kato, A., Kimura, H., Nishiyama, Y.
(2005). Herpes Simplex Virus Type 1 UL51 Protein Is Involved in Maturation and Egress of Virus Particles. J. Virol.
79: 6947-6956
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Kamen, D. E., Gross, S. T., Girvin, M. E., Wilson, D. W.
(2005). Structural Basis for the Physiological Temperature Dependence of the Association of VP16 with the Cytoplasmic Tail of Herpes Simplex Virus Glycoprotein H. J. Virol.
79: 6134-6141
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Liang, L., Baines, J. D.
(2005). Identification of an Essential Domain in the Herpes Simplex Virus 1 UL34 Protein That Is Necessary and Sufficient To Interact with UL31 Protein. J. Virol.
79: 3797-3806
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Crump, C. M., Bruun, B., Bell, S., Pomeranz, L. E., Minson, T., Browne, H. M.
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85: 3517-3527
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Foster, T. P., Melancon, J. M., Olivier, T. L., Kousoulas, K. G.
(2004). Herpes Simplex Virus Type 1 Glycoprotein K and the UL20 Protein Are Interdependent for Intracellular Trafficking and trans-Golgi Network Localization. J. Virol.
78: 13262-13277
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Wisner, T. W., Johnson, D. C.
(2004). Redistribution of Cellular and Herpes Simplex Virus Proteins from the Trans-Golgi Network to Cell Junctions without Enveloped Capsids. J. Virol.
78: 11519-11535
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Fuchs, W., Klupp, B. G., Granzow, H., Mettenleiter, T. C.
(2004). Essential Function of the Pseudorabies Virus UL36 Gene Product Is Independent of Its Interaction with the UL37 Protein. J. Virol.
78: 11879-11889
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Terhune, S. S., Schroer, J., Shenk, T.
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78: 10390-10398
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Simpson-Holley, M., Baines, J., Roller, R., Knipe, D. M.
(2004). Herpes Simplex Virus 1 UL31 and UL34 Gene Products Promote the Late Maturation of Viral Replication Compartments to the Nuclear Periphery. J. Virol.
78: 5591-5600
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Jones, T. R., Lee, S.-W.
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78: 1488-1502
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Beitia Ortiz de Zarate, I., Kaelin, K., Rozenberg, F.
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78: 1540-1551
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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
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Ryckman, B. J., Roller, R. J.
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78: 399-412
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Grunewald, K., Desai, P., Winkler, D. C., Heymann, J. B., Belnap, D. M., Baumeister, W., Steven, A. C.
(2003). Three-Dimensional Structure of Herpes Simplex Virus from Cryo-Electron Tomography. Science
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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
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Farnsworth, A., Goldsmith, K., Johnson, D. C.
(2003). Herpes Simplex Virus Glycoproteins gD and gE/gI Serve Essential but Redundant Functions during Acquisition of the Virion Envelope in the Cytoplasm. J. Virol.
77: 8481-8494
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Aleman, N., Quiroga, M. I., Lopez-Pena, M., Vazquez, S., Guerrero, F. H., Nieto, J. M.
(2003). L-Particle Production during Primary Replication of Pseudorabies Virus in the Nasal Mucosa of Swine. J. Virol.
77: 5657-5667
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Browne, H., Bruun, B., Whiteley, A., Minson, T.
(2003). Analysis of the role of the membrane-spanning and cytoplasmic tail domains of herpes simplex virus type 1 glycoprotein D in membrane fusion. J. Gen. Virol.
84: 1085-1089
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Brignati, M. J., Loomis, J. S., Wills, J. W., Courtney, R. J.
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77: 4888-4898
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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
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Hutchinson, I., Whiteley, A., Browne, H., Elliott, G.
(2002). Sequential Localization of Two Herpes Simplex Virus Tegument Proteins to Punctate Nuclear Dots Adjacent to ICP0 Domains. J. Virol.
76: 10365-10373
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Miranda-Saksena, M., Boadle, R. A., Armati, P., Cunningham, A. L.
(2002). In Rat Dorsal Root Ganglion Neurons, Herpes Simplex Virus Type 1 Tegument Forms in the Cytoplasm of the Cell Body. J. Virol.
76: 9934-9951
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Reynolds, A. E., Wills, E. G., Roller, R. J., Ryckman, B. J., Baines, J. D.
(2002). Ultrastructural Localization of the Herpes Simplex Virus Type 1 UL31, UL34, and US3 Proteins Suggests Specific Roles in Primary Envelopment and Egress of Nucleocapsids. J. Virol.
76: 8939-8952
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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
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Klupp, B. G., Fuchs, W., Granzow, H., Nixdorf, R., Mettenleiter, T. C.
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76: 3065-3071
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Mettenleiter, T. C.
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Heineman, T. C., Hall, S. L.
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76: 591-599
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Johnson, D. C., Huber, M. T.
(2002). Directed Egress of Animal Viruses Promotes Cell-to-Cell Spread. J. Virol.
76: 1-8
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Fuchs, W., Klupp, B. G., Granzow, H., Osterrieder, N., Mettenleiter, T. C.
(2002). The Interacting UL31 and UL34 Gene Products of Pseudorabies Virus Are Involved in Egress from the Host-Cell Nucleus and Represent Components of Primary Enveloped but Not Mature Virions. J. Virol.
76: 364-378
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Foster, T. P., Rybachuk, G. V., Kousoulas, K. G.
(2001). Glycoprotein K Specified by Herpes Simplex Virus Type 1 Is Expressed on Virions as a Golgi Complex-Dependent Glycosylated Species and Functions in Virion Entry. J. Virol.
75: 12431-12438
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Desai, P., Sexton, G. L., McCaffery, J. M., Person, S.
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75: 10259-10271
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