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Journal of Virology, February 2000, p. 2005-2010, Vol. 74, No. 4
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Varicella-Zoster Virus Proteins in Skin Lesions: Implications for
a Novel Role of ORF29p in Chickenpox
Paula W.
Annunziato,1,*
Octavian
Lungu,2
Christos
Panagiotidis,2,
Jing H.
Zhang,1
David N.
Silvers,3
Anne A.
Gershon,1 and
Saul J.
Silverstein2
Departments of
Pediatrics,1
Microbiology,2 and Dermatology
and Pathology,3 College of Physicians and
Surgeons, Columbia University, New York, New York
Received 10 August 1999/Accepted 8 November 1999
 |
ABSTRACT |
Skin biopsy samples from varicella-zoster virus (VZV)-infected
patients examined by immunohistochemistry demonstrated VZV replication
in nonepithelial cell types. ORF29p, a nonstructural nuclear protein,
was found in nerves of two of six patients with chickenpox. In tissue
culture, ORF29p was secreted by VZV-infected fibroblasts. Extracellular
ORF29p can be taken up through endocytosis by human neurons, implying a
novel role for this protein in pathogenesis.
| |
TEXT |
Varicella-zoster virus (VZV)
infects dorsal root ganglia (DRG), enters latency, and may later
reactivate to cause zoster. Studies have detected VZV in specific sites
at different stages of infection. VZV DNA is present in the oropharynx
(27) and in peripheral blood mononuclear cells (PBMCs) of
patients with chickenpox (3, 16, 20). Virus DNA, the
glycoproteins gE and gB, and the immediate-early protein 63 (IE63p) are
found in skin biopsy samples obtained from patients with chickenpox or zoster (1, 23-25). VZV is found in keratinocytes,
antigen-presenting cells, and endothelial cells during acute zoster
(23, 25) and in keratinocytes and inflammatory cells during
chickenpox (1). VZV is present in neurons and satellite
cells of DRG years following primary infection (6-8, 12,
17, 22) and has been observed by electron microscopy in
sensory nerves during zoster (10). Other details of VZV
pathogenesis remain speculative, including how the virus spreads from
respiratory epithelial cells to PBMCs, keratinocytes, and DRG. Because
PBMCs, sensory nerves, and epithelial cells are in close proximity
in the dermis and epidermis, the skin is likely the site where
virus enters the nervous system.
By analogy with herpes simplex virus (HSV), it is thought that VZV
transcription is temporally regulated. Immediate-early (IE) genes are
expressed first, followed by early (E) genes and lastly late (L) genes
(5). Some VZV proteins encoded by IE and L genes are
incorporated in the virion, including transactivators such as IE63p and
structural proteins such as gC (14, 15). ORF29p (for open
reading frame 29 protein), the major VZV DNA binding protein, is
encoded by a putative E gene and is not detected in purified virions
(13). During latency, VZV exhibits limited gene expression
(6-9, 22), with the accumulation of specific IE and E
gene-encoded proteins in neurons (18, 19). During reactivation, all kinetic classes of VZV genes are expressed in neurons
(18). Whether VZV is in the lytic or latent state is reflected by the localization of expressed VZV gene products. VZV IE
and E proteins that are present in both the nucleus and cytoplasm
during productive infection are detected only in the cytoplasm
of neurons during latency (18).
Early observations suggested that there were inclusion bodies in
endothelial cells present in varicella lesions (29);
however, there was no known association between histology and
viral etiology at that time. To determine if, during primary infection,
as in zoster, VZV infects endothelial cells and nerves in the dermis and to characterize the inflammatory cells in the epidermis and dermis
infected by VZV, we performed comparative immunohistochemical analyses of skin biopsy samples obtained from patients with chickenpox and zoster.
Comparative immunohistochemical analysis of chickenpox and zoster
lesions.
Six cases of chickenpox and eight cases of zoster were
analyzed by immunohistochemistry using purified polyclonal antibodies generated against VZV proteins (18). Each specimen was
analyzed for the presence of gC, a late gene product and component of
the virus envelope (15); IE63p, a regulatory protein and
component of the virus tegument (14); and ORF29p
(13). In these specimens, antibodies to IE63p, ORF29p, and
gC detected proteins in the expected intracellular compartments: ORF29p
was found in cell nuclei (Fig. 1), gC was
found in cell membranes and the cytoplasm, and IE63p was found in both
the cell nucleus and cytoplasm (data not shown). VZV proteins were
detected in epithelial cells, endothelial cells, nerves, and
CD43+/CD68+ inflammatory cells in the epidermis
and dermis in both chickenpox and zoster cases (Fig. 1 and
2). Among the six chickenpox specimens, all were positive for ORF29p, four were positive for IE63p, and five
were positive for gC. Among the eight zoster specimens, all were
positive for ORF29p, five were positive for IE63p, and five were
positive for gC (Table 1). These results are consistent with our
experience concerning the affinities of these antibodies for their
target proteins. Five cases of Grover's disease, a noninfectious dermatosis, and three cases of HSV infection were included as controls
and were negative (Table 1), confirming that each antibody signal was
specific. Detection of ORF29p, a DNA binding protein not present in the
virion (13), in the nucleus of infected cells demonstrates
that VZV replication occurs in endothelial cells, epithelial cells, and
cells of the monocyte/macrophage lineage expressing surface CD43 and
CD68. Although VZV genomes were detected in circulating lymphocytes of
patients with chickenpox and zoster (16, 20) we detected VZV
proteins only in CD43+/CD68+ cells and not in
cells expressing CD3 or CD20 in these specimens.
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FIG. 1.
Immunohistochemical detection of ORF29p in skin biopsy
samples. Chickenpox (A), zoster (B), and Grover's disease (C) skin
lesions were analyzed for ORF29p as previously described
(18), with the following exceptions. All washes were
performed in Tris-buffered saline, and the signal was developed for 10 min in AP substrate (Vector Laboratories, Inc., Burlingame, Calif.),
according to the manufacturer's recommendations, in the presence of
levamisole to inhibit endogenous alkaline phosphatase activity. Arrows
indicate positive epithelial cells. Magnification, ×100.
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FIG. 2.
Immunohistochemical detection of ORF29p and CD43 in skin
biopsy samples. Skin biopsy samples from a patient with chickenpox (A
and B) or a patient with zoster (C and D) were probed for the presence
of ORF29p (A and C) or ORF29p and CD43 (B and D) as described in the
legend to Fig. 1. Red arrows indicate endothelial cells containing
ORF29p. Black arrows indicate cells expressing CD43 that contain
ORF29p. Magnification, ×600.
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|
No differences in immunohistochemical staining were appreciated between
specimens of chickenpox and zoster except for the
presence of ORF29p in
peripheral nerves in the dermis in two of
the chickenpox specimens.
ORF29p was detected in the Schwann cells
and axons of nerves in these
two cases (Fig.
3A), which included
a biopsy sample that was obtained 2 days following the onset of
the
rash. Cytoplasmic localization of ORF29p is not consistent
with the
presence of replicating VZV or of formed virions. In
contrast, ORF29p
was not detected in axons or Schwann cells in
the seven zoster biopsy
samples with peripheral nerves apparent
in the analyzed sections (Fig.
3C). The absence of ORF29p from
axons during zoster was not surprising,
as this protein localizes
to the nucleus of productively infected
cells, including neurons
containing reactivating virus (
18),
and is not detected in virions
(
13). As expected, gC was
found in both axons and Schwann cells
of nerves in three chickenpox
cases and four zoster cases (Fig.
3B and D). Detection of gC indicates
the presence of virions or
replicating VZV at these sites. The
significance of infection
of Schwann cells in chickenpox and zoster is
unclear. Schwann
cells cultivated in tissue culture are permissive for
VZV infection
(
4), but the role of these cells in
pathogenesis is not known.
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FIG. 3.
Immunohistochemical detection of ORF29p and gC in skin
biopsy samples. Sections of nerves in the dermis underlying chickenpox
(A and B) or zoster (C and D) lesions underwent immunohistochemistry
for ORF29p (A and C) or gC (B and D) as described in the legend to Fig.
1. Magnification, ×400.
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|
Secretion of ORF29p by infected cells in tissue culture.
Clinical and laboratory evidence suggests that DRG are infected via the
peripheral nerves during the exanthem of chickenpox (11, 21,
26). Although hematogenous and axonal spread of virus are not
mutually exclusive, given the small numbers of VZV-infected circulating
PBMCs (16, 20) it seems unlikely that a substantial number
of neurons are infected without amplification in the epidermis and
dermis. Nonetheless, the exact mechanism by which VZV reaches DRG
remains unsettled.
Entry of virus particles into peripheral axons during chickenpox cannot
account for the presence of ORF29p at this site because
this protein is
not a component of the virion (
13). Assuming
that the virus
spreads from the skin to the peripheral nerves
during the exanthem, the
appearance of ORF29p in the nerve within
2 days of rash onset is
surprising because of the distance between
the peripheral axon and the
sensory neuron in the DRG. By analogy
with HSV, it is thought that VZV
entering the axon in the epidermis
travels by retrograde axonal
transport (
2) at a rate of 200
to 400 mm/day
(
28). Additional time would be required for VZV
proteins to
be produced in the neuron in the DRG and then to travel
to the dermis
and epidermis by anterograde axonal transport. If
the DRG were infected
during viremia prior to the onset of the
rash, virus replication in the
neuron and anterograde axonal transport
of VZV proteins could occur.
However, this would not explain the
presence of ORF29p in peripheral
axons, because ORF29p localizes
to the nucleus rather than to the
cytoplasm during productive
infection (
18). Moreover, ORF29p
was not found in peripheral
axons during
zoster.
We therefore postulated that ORF29p may be secreted by
VZV-infected cells in the dermis or epidermis and enter peripheral
axons by endocytosis. In order to test whether ORF29p was
secreted
by VZV-infected cells, tissue culture media from uninfected
human
embryonic lung fibroblasts (HELF) or HELF infected with VZV or
HSV-1 was clarified by centrifugation and filtration to remove
detached
cells. Immunoprecipitation with antibodies to ORF29p;
ORF21p, a
putative VZV accessory DNA binding protein; or ICP8,
the HSV-1
homologue of ORF29p, was performed and the precipitated
proteins were
analyzed by sodium dodecyl sulfate-polyacrylamide
gel
electrophoresis and Western blotting. ORF29p was detected
in culture
supernatants of infected cells but not in culture supernatants
of
uninfected cells (Fig.
4). ORF21p was not
detected in supernatants
of VZV-infected cells, and ICP8 was not
detected in supernatants
of HSV-1-infected cells. Thus, ORF29p is
secreted by infected
fibroblasts in tissue culture.
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FIG. 4.
Western blot analyses of VZV and HSV-1 proteins. ORF29p,
ORF21p, and ICP8 were detected in mock-infected cell extracts (M Cell)
and supernatants (M Sup) or cell extracts (I Cell) and supernatants (I
Sup) infected with the viruses denoted on the left. The proteins were
immunoprecipitated and detected using the antibodies denoted on the
right. Arrowheads denote the proteins of interest.
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|
Endocytosis of secreted ORF29p by neurons in tissue
culture.
Filtered tissue culture medium from VZV-infected
HELF and LysoTracker Red DND-99 (Molecular Probes, Eugene,
Oreg.), a label for acidic endocytic vesicles, were applied to
cultivated human neurons (hNTs) (Stratagene, La Jolla, Calif.) to
determine if secreted ORF29p entered neurons by endocytosis. After
incubating the hNTs with the filtered culture medium and LysoTracker
for 2 h, the cells were examined by immunohistochemistry for the
presence of ORF29p. ORF29p was detected in cytoplasmic vesicles (Fig.
5A and D) that
colocalized with LysoTracker (Fig. 5C). ORF29p was not found in
untreated hNTs (Fig. 5E). Therefore, extracellular ORF29p can enter
hNTs by endocytosis, supporting our hypothesis that the presence of
this protein in peripheral axons may result from its assimilation from
surrounding cells that are infected with VZV.
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FIG. 5.
Immunohistochemical detection of ORF29p in hNTs. hNTs
treated with VZV-infected cell supernatants and LysoTracker were
analyzed by immunohistochemistry for the presence of ORF29p. Yellow
arrows indicate ORF29p (A), LysoTracker (B), and colocalization of
ORF29p and LysoTracker in the merged image (C). White arrows indicate
an endocytic vesicle that does not contain ORF29p (B and C). ORF 29p is
restricted to cyptoplasmic vesicles in the treated hNTs (D). Untreated
hNTs do not contain ORF29p (E).
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Our results illustrate key steps of VZV pathogenesis. During
chickenpox, VZV infects epithelial cells, endothelial cells,
cells of
the monocyte/macrophage lineage, and nerves of the skin.
After
infecting the neuron, the virus enters latency. In some
individuals,
the virus reactivates in one or more neurons, travels
via the axon to
the skin, and infects the epithelial cells. In
addition, endothelial
cells are infected in zoster, which could
potentially spread virus to
other areas. That VZV does not typically
spread outside of the
dermatome during zoster implies that host
immunity effectively halts
cell-to-cell spread. This study suggests
that entry of VZV into the
nervous system during primary infection
may not rely solely on axonal
transport of mature virions from
the skin during chickenpox, because
ORF29p was present in axons
early in the course of the
rash.
| |
ACKNOWLEDGMENTS |
This work was supported by NIH grants AI-01409 (to P.W.A.) and
AI-124021 (to A.A.G. and S.J.S.) and by a Pilot Award from the Columbia
Presbyterian Medical Center Office of Clinical Trials (to P.W.A.).
We thank Michael Gershon, Giorgio Catoretti, and Sharon Steinberg for
assistance with this work.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Pediatrics, 650 West 168th St., New York, NY 10032. Phone: (212)
305-2876. Fax: (212) 342-5218. E-mail: paa5{at}columbia.edu.
Present address: Department of Pharmaceutical Sciences, Aristotle
University, Thessaloniki 5006, Greece.
| |
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Journal of Virology, February 2000, p. 2005-2010, Vol. 74, No. 4
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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