Journal
of General Virology (1999), 80, 51–56. Printed in Great Britain
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SHORT COMMUNICATION
US3 protein kinase of herpes simplex virus type 2 plays a role
in protecting corneal epithelial cells from apoptosis in infected
mice
Shinya Asano,1, 2 Takashi Honda,3 Fumi Goshima,1 Daisuke Watanabe,1 Yozo Miyake,2
Yasuo Sugiura3 and Yukihiro Nishiyama1
1
Laboratory of Virology, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, 65 Tsurumai-cho,
Showa-ku, Nagoya 466, Japan
2, 3
Departments of Ophthalmology2 and Anatomy3, Nagoya University School of Medicine, Nagoya 466, Japan
To clarify the biological role of US3 protein kinase
of herpes simplex virus type 2 (HSV-2) in vivo, the
expression of the viral antigen, the appearance of
apoptotic bodies and DNA fragmentation were
examined immunohistologically after corneal infection of mice with three different kinds of HSV-2
strain 186 : the wild-type virus, a US3-deficient
mutant (L1BR1) and its revertant (L1BN11). In both
wild-type 186- and L1BN11-infected mice, viral
antigen was diffusely found in the corneal epithelium ; no apoptotic changes were detected in the
epithelial cells. Whereas, in L1BR1-infected mice,
HSV-immunoreactivity was localized around the
virus-inoculated sites, and a large number of apoptotic bodies were observed in the corneal epithelium with dual-positive reactions for both HSVimmunostaining and TUNEL staining. These results
suggest that the US3 protein kinase plays an
important role in protecting HSV-2-infected cells
from apoptotic death in vivo.
Apoptosis, also known as programmed cell death, is a
morphologically distinct form of cell death and has been
shown to be a common host-cell response to a wide variety of
virus infections (Young et al., 1997). Such a response is clearly
of benefit to the host, since premature death of infected cells
can potentially limit the production of progeny virus. However, many viruses encode genes which protect cells from
apoptosis sufficiently to allow virus replication (Rao et al.,
1992 ; Ray et al., 1992 ; Zhu et al., 1995). With respect to herpes
simplex virus type 1 (HSV-1), two viral genes, ICP4 (Leopardi
& Roizman, 1996) and γ 34.5 (Chou & Roizman, 1992), have
"
Author for correspondence : Yukihiro Nishiyama.
Fax j81 52 744 2452. e-mail ynishiya!tsuru.med.nagoya-u.ac.jp
0001-5760 # 1999 SGM
been shown to be involved in anti-apoptosis functions. In
addition, a recent study has reported that the US3 gene of
HSV-1 plays an important role in blocking apoptosis in cell
culture (Leopardi & Roizman, 1997). The present study was
therefore designed to confirm the role of US3 protein kinase
(PK) in vivo, and for this purpose we examined the expression
of HSV-immunoreactivity and histopathological changes in
the cornea of mice infected with three different kinds of
HSV-2 strain 186 : the parental wild-type virus, a US3-deficient
mutant (L1BR1) and its revertant (L1B−11).
Experiments were conducted in 56 specific-pathogen-free
male ICR mice (postnatal 3–4 weeks). As reported previously
(Kurachi et al., 1993 ; Yamamoto et al., 1996), the spread of the
US3-deficient mutant L1BR1 in mice is highly restricted, and
thus we have chosen the corneal route of infection because the
primarily infected site can easily be identified. Under avertin
(2,2,2-tribromoethanol) anaesthesia (200 mg\kg, intraperitoneal injection), the corneal surface of both eyes was
scratched and perforated with a fine 27-gauge hypodermic
needle and 10 µl virus suspension (1n7i10% p.f.u. per mouse)
was dropped on the right cornea. The same volume of PBS
without virus was dropped on the left cornea. After virus
inoculation, mice were kept under conventional laboratory
conditions. At indicated times post-infection, the mice were
reanaesthetized and perfused with saline, followed by 4 %
paraformaldehyde in 0n1 M PBS at pH 7n4. Whole bilateral eye
bulb tissue was removed and serial 20 µm thick sections cut on
a cryostat. For detection of HSV-2 antigen, immunohistochemical staining was performed as described previously
(Kurachi et al., 1993) using anti-HSV-2 rabbit serum (Biomeda)
and biotinylated anti-rabbit IgG goat serum (Vector Laboratories). For detection of DNA fragmentation, the terminal
deoxynucleotidal transferase-mediated dUTP nick-end labelling (TUNEL) assay was performed by using fluorescein-12
dUTP-labelled DNA (Apoptosis Detection System kit,
Promega). Paraffin-embedded sections (3 µm thick) were also
made and stained with haematoxylin and eosin (H&E) for
histopathological examination.
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S. Asano and others
Fig. 1. Histopathological changes in the corneal epithelium of mice infected with wild-type HSV-2 (strain 186) (a–c) and a
US3-deficient mutant (L1BR1) (d and e) (H&E stain). In mice infected with the wild-type virus, a large number of lymphoid
cells infiltrated into the substantia propria (a). The epithelial cells nearby the virus-inoculated site slightly swelled and contained
FC
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HSV-2 US3 protein kinase and apoptosis
Fig. 2. HSV-2-immunostaining (a, c and e) and TUNEL-staining (b, d and f ) of the corneal epithelium of mice infected with
wild-type HSV-2 (a and b), L1BR1 (c and d ) and L1B−11 (e and f ). Each neighbouring section was stained for detection of
HSV-2 antigen and DNA fragmentation. Diffuse immunolabelling was seen in the epithelium infected with the wild-type virus
and L1B−11 (a and e), but the immunolabelling in L1BR1-infected mice was localized in the area around the corneal erosion
(asterisk) caused by scratching (c). TUNEL-positive epithelial cells were detected only in mice infected with L1BR1 (d ). Bars,
5 µm.
large vacuoles (b ; arrows) and inclusion bodies (c ; arrows) 3 days after infection. In L1BR1-infected mice, small round
basophilic granules (apoptotic bodies) were observed in the corneal epithelium (d ; arrow heads) 2 days after infection. On day
5, the corneal epithelium showed an atypical bizarre contour and hyalinous degeneration of the epithelial cells was found at the
centre of the ulcer (e ; asterisk). Instead of disappearance of nuclei, a large number of small round basophilic granules
(apoptotic bodies) were found in the epithelium (e ; arrows). Bars : 10 µm (a), 1 µm (b–e).
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S. Asano and others
Table 1. HSV immunoreactivity in the orbital tissues of mice
HSV-immunoreactivity was graded from k to jjj, depending on the extent of the spread. , Not done.
Time after virus inoculation (days)
Virus
strain
Intraorbital
tissues
1
Strain 186
Epithelium
Substantia propria
Endothelium
2
Cornea
Iris
Choroid
Retina
No. of animals
4
3
1
L1BR1
Cornea
2
Epithelium
Substantia propria
Endothelium
Iris
Choroid
Retina
No. of animals
4
3
1
L1B−11
2
Cornea
Iris
Choroid
Retina
No. of animals
4
3
Epithelium
Substantia propria
Endothelium
2
3
jj
j
j
j
j
k
5
j
k
k
k
k
k
5
jj
j
j
j
k
k
3
jj
jj
j
j
j
k
3
j
k
k
j
k
k
4






0
The results of histopathological examination and immunostaining are shown in Figs 1 and 2. In mice infected with the
wild-type strain 186 virus, the corneal epithelial cells disappeared from the scratched site of virus inoculation and
resulted in obvious ulceration by day 2. The epithelial cells
nearby this site slightly swelled and contained large vacuoles
in the cytoplasm. A large number of lymphoid cells infiltrated
into the substantia propria under the vacuolated epithelial cells,
and slight to moderate HSV-immunolabelling was found in the
corneal epithelium and the substantia propria. On day 3, the
lymphoid cells infiltrating into the substantia propria increased
in number and swelling of the cornea was clearly observed (Fig.
1 a). In the corneal epithelium, a number of vacuoles (Fig. 1 b)
and inclusion bodies (Fig. 1 c) were detected, and HSVimmunoreactivity was found widely spread over the corneal
epithelium (Fig. 2 a). Viral antigen was found not only in both
the cornea and the iris but also in a part of the choroid.
Pigmented epithelium, sphincter pupillae muscle and stroma of
the iris were also immunolabelled. Although infiltrated lymphoid cells appeared to be TUNEL-positive, almost all HSVimmunoreactive epithelial cells were TUNEL-negative (Fig.
2 b). On day 4, slight proliferation of epithelial cells was seen at
the edge of the corneal ulcer. Morphological changes of
apoptosis were found only in the connective tissues and
infiltrated cells in the substantia propria. Marked HSVFE
4
jj
jj
j
j
j
j
4
j
j
j
j
k
k
5
jj
jj
j
j
j
j
3
5
5
6
5
6
6
Death
8
7
Death
7
8
0
j
j
j
j
j
k
4
5
6
5
0
jj
k
k
j
j
k
2
6
Death
8
7
0
Death
7
8
0
immunolabelling was found in the corneal epithelium, infiltrated lymphoid cells and the stromal cells in the substantia
propria. The iris and the choroid also showed strong HSVimmunoreactivity. Moderate labelling was found in the corneal
endothelium. In the inner granular layer of the retina, only a
small number of cells were HSV-immunopositive. However,
HSV-immunoreactive epithelial cells remained TUNEL-negative.
On the other hand, histopathological changes in L1BR1infected mice were summarized as follows. On day 2, the nuclei
of the corneal epithelial cells near the edge of the ulcer swelled
and showed an atypical contour, and small vacuoles were
found in these nuclei. In some epithelial cells, small round
basophilic granules, recognized as apoptotic bodies (Kerr et al.,
1972 ; Cohen, 1993), were observed (Fig. 1 d). HSVimmunoreactivity was found only in the epithelial cells
adjacent to the ulcer (Fig. 2 c) and these immunoreactive
epithelial cells were TUNEL-positive (Fig. 2 d). Although a few
of the inflammatory cells were HSV-antigen-positive, no
immunolabelling was detected in the stromal cells of the
substantia propria. On day 3, basal cells of the squamous
epithelium near the ulcer piled up and showed a hyperplastic
appearance. Only a small number of inflammatory cells, i.e.
neutrophilic leukocytes and lymphocytes, infiltrated into the
oedematous stroma of the substantia propria, and HSV-
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HSV-2 US3 protein kinase and apoptosis
immunolabelling was still restricted in the corneal epithelium.
On day 5, hyalinous degeneration of the epithelial cells was
found in the central part of the ulcer and a large number of
apoptotic bodies were detected in the degenerated epithelium
around the ulcer (Fig. 1 e). Although many lymphoid cells or
neutrophilic leukocytes infiltrated the substantia propria,
inflammation was localized in the region underlying the
degenerated epithelium.
In mice infected with L1B−11, histopathological changes
and the expression of HSV-immunoreactivity were similar to
those observed in mice infected with wild-type virus (Fig. 2 e).
No TUNEL-positive cells were found in the corneal epithelium
of the infected mice (Fig. 2 f ). The expression of HSVimmunoreactivity in mice infected with each virus strain is
summarized in Table 1.
Apoptosis is induced in response to a wide variety of
stimuli (Thatte & Dahanukar, 1997). In virus-infected hosts,
apoptosis can be a direct or indirect result of virus infection ;
uninfected cells can also cause apoptosis in response to
cytokines induced by virus infection (McQuaid et al., 1997). In
the present study, we have observed that a number of
lymphoid cells infiltrating the infected cornea were TUNELpositive but negative for HSV-immunoreactivity. These
TUNEL-positive lymphoid cells could be observed in any mice
infected with the wild-type virus, the US3-deficient mutant or
its revertant. It seems that apoptosis of these cells is an indirect
result of HSV-2 infection : cytokines or Fas-mediated pathways
might contribute to the induction of apoptosis of these
lymphoid cells (Kaplan & Sieg, 1998). On the other hand,
TUNEL-positive epithelial cells of the cornea could be
observed only in mice infected with the US3-deficient mutant,
and almost all of these epithelial cells were also positive for
HSV-immunostaining. These results suggest that the US3 gene
of HSV-2 plays a role in blocking apoptosis of infected cells in
vivo. However, it appears that such an effect of the US3 PK on
apoptosis is dependent on cell type.
Our previous studies have shown that an HSV-2 US3deficient mutant, L1BR1, displays a highly attenuated phenotype of virulence in mice when given by peripheral routes of
infection (Nishiyama et al., 1992). Since L1BR1, unlike the wildtype virus, cannot grow in peritoneal macrophages (Kurachi et
al., 1993 ; Nishiyama et al., 1992), we have proposed that the
attenuation of the mutant may be due to the inability to
overcome the mononuclear phagocytic system of the host. The
present study provides an additional reason to explain its
attenuated phenotype : L1BR1 cannot spread well because of
the induction of early cell death. In this regard, it should be
noted that the mutant can grow as efficiently as the wild-type
virus in Vero cells, in which both cannot induce apoptosis until
the very late stage of infection (unpublished observation).
Since the US3 gene product is a serine\threonine kinase
(Daikoku et al., 1993), the phosphorylation of a protein(s) by
the US3 PK is possibly involved in suppressing apoptosis of
infected cells. Although several viral proteins have been shown
to be substrates for the US3 PK (Daikoku et al., 1994 ; Purves
et al., 1991), there is no evidence that they are involved in
blocking apoptosis. In summary, our present results strongly
suggest a role for the US3 PK in blocking apoptosis in vivo,
although the possible involvement of an additional mutation
cannot completely be ruled out.
We thank E. Iwata and T. Tsuruguchi for their technical assistance.
This work was supported by a grant from the Japan Society for the
Promotion of Science (JSPS-RFTF97L00703) and also by a grant-in-aid
for Scientific Research from the Ministry of Education, Science, and
Culture of Japan.
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