Download Mutation of UL24 impedes the dissemination of acute herpes

Journal of General Virology (2015), 96, 2794 –2805
DOI 10.1099/vir.0.000189
Mutation of UL24 impedes the dissemination
of acute herpes simplex virus 1 infection from
the cornea to neurons of trigeminal ganglia
Pierre-Alexandre Rochette, Amélie Bourget,3 Carolina Sanabria-Solano,
Soumia Lahmidi, Gabriel Ouellet Lavallée and Angela Pearson
Correspondence
Angela Pearson
Université INRS, INRS-Institut Armand-Frappier, 531 boulevard des Prairies, Laval, Québec H7V
1B7, Canada
[email protected]
Received 16 December 2014
Accepted 17 May 2015
Herpes simplex virus 1 (human herpesvirus 1) initially infects epithelial cells of the mucosa and
then goes on to infect sensory neurons leading ultimately to a latent infection in trigeminal
ganglia (TG). UL24 is a core herpesvirus gene that has been identified as a determinant of
pathogenesis in several Alphaherpesvirinae, although the underlying mechanisms are unknown.
In a mouse model of ocular infection, a UL24-deficient virus exhibited a reduction in viral titres in
tear films of 1 log10, whilst titres in TG are often below the level of detection. Moreover, the
efficiency of reactivation from latency was also severely reduced. Herein, we investigated how
UL24 contributed to acute infection of TG. Our results comparing the impact of UL24 on viral
titres in eye tissue versus in tear films did not reveal a general defect in virus release from the
cornea. We also found that the impairment of replication seen in mouse primary embryonic
neurons with a UL24-deficient virus was not more severe than that observed in an epithelial cell
line. Rather, in situ histological analyses revealed that infection with a UL24-deficient virus led
to a significant reduction in the number of acutely infected neurons at 3 days post-infection
(p.i.). Moreover, there was a significant reduction in the number of neurons positive for viral DNA
at 2 days p.i. for the UL24-deficient virus as compared with that observed for WT or a rescue
virus. Our results supported a model whereby UL24 functions in the dissemination of acute
infection from the cornea to neurons in TG.
INTRODUCTION
Herpes simplex virus 1 (HSV-1; human herpesvirus 1) replicates in epithelial cells and then infects the ends of sensory neurons that form neuronal ganglions (Roizman et al.,
2007). HSV-1 persists in neurons as a life-long latent infection and may reactivate, often following stress, resulting in
recurrent infections (Roizman et al., 2007). In healthy individuals, symptoms typically vary from cold sores to viral keratitis and in rare cases infections can lead to a life-threatening
encephalitis (Whitley & Roizman, 2001). In people who are
immunocompromised, e.g. people living with AIDS, neonates or stem cell recipients, the symptoms are often severe
(Fatahzadeh & Schwartz, 2007). Many questions remain
regarding the spread of infection within the host, which
has hindered the development of treatments to prevent
recurrent infections.
UL24 is highly conserved across the family Herpesviridae
(Davison, 1993; Roizman et al., 2007). UL24 is involved in
nucleolar modifications during HSV-1 infection and impacts
3Present address: Montreal Chest Institute, McGill University Health
Centre, 3650 Saint-Urbain, Montreal, Québec H2X 2P4, Canada.
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the nuclear egress of capsids (Bertrand & Pearson, 2008;
Bertrand et al., 2010; Lymberopoulos et al., 2011). UL24
has also been shown to induce a G2/M cell cycle block
when expressed in the absence of infection (Nascimento
et al., 2009). Following infection of an epithelial-derived
cell line, a UL24-deficient HSV-1 strain produced five to
10 times fewer infectious virions and exhibited a reduced
plaque size as well as a temperature-sensitive syncytial phenotype (Jacobson et al., 1989a; Sanders et al., 1982; Tognon
et al., 1991).
In vivo, HSV-1 UL24 has been shown to be important for
pathogenesis (Jacobson et al., 1998). Mice infected with a
UL24-null virus exhibited a reduction in viral titres in trigeminal ganglia (TG) of up to 4 log10, a 2 log10 reduction
in viral DNA and a 1 log10 reduction in lat transcripts present
in latently infected TG, a drastic reduction in the severity of
disease, as well as a reduction in the frequency of reactivation
from latency in explant assays (Jacobson et al., 1998; LeivaTorres et al., 2010). Reduced pathogenesis has also been
observed for UL24 mutants of other alphaherpesviruses,
such as herpes simplex virus 2 (HSV-2; human herpesvirus 2)
(Blakeney et al., 2005), equid herpesvirus 1 (Kasem et al.,
2010) and varicella-zoster virus (human herpesvirus 3)
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Role of UL24 in acute infection of TG in vivo
(Ito et al., 2005). The exact mechanisms through which UL24
modulates pathogenesis are unknown.
In this study, we tested the hypothesis that the large
reduction in viral titres in mouse TG observed with a
UL24-deficient virus is the result of a defect in the dissemination of infection from the cornea to neurons of TG.
(a)
KOS
vUL24XRescue
UL24X
34 ºC
37 ºC
RESULTS
Construction and characterization of a UL24X
rescue virus
To confirm that reinsertion of the WT UL24 gene within
UL24X could correct the temperature-sensitive syncytial
plaque phenotype (Jacobson et al., 1989a; Tognon et al.,
1991), we infected Vero cells at low m.o.i. with KOS,
UL24X or vUL24X-Rescue (Fig. 1a) for 48 h. UL24X produced syncytial plaques at 37 and 39 uC, whilst vUL24XRescue produced non-syncytial plaques similar to those
of KOS at both temperatures.
We next tested the rescue virus in a one-step viral replication assay using m.o.i. 5 (Fig. 1b). Although levels for
vUL24X-Rescue were significantly lower than those
observed with KOS, we found that viral yields were
increased for vUL24X-Rescue as compared with UL24X,
resulting in significantly higher viral titres for the rescue
virus at both 12 and 24 h post-infection (p.i.).
Importantly, we demonstrated that vUL24X-Rescue behaved similarly to KOS in vivo. CD-1 mice (8 weeks old)
were infected with either 2|106 or 6|102 p.f.u. per eye
of KOS, UL24X or vUL24X-Rescue (Fig. 2). Viral titres
in tear films were determined for 1–3 days p.i. (Fig. 2a–c).
We found that the mean ocular viral titre for vUL24XRescue at an inoculum of 2|106 p.f.u. per eye was higher
than that for UL24X and similar to that for KOS.
At 3 days p.i., vUL24X-Rescue produced viral titres in TG
similar to those of KOS and values for both viruses were significantly higher than the viral titres produced by UL24X
http://vir.sgmjournals.org
39 ºC
(b)
108
Viral titre (p.f.u. ml–1)
UL24X contains stop codons in all three reading frames to
block translation at codon 44. This design ensures that the
mutant retains WT levels of the viral thymidine kinase
(TK), whose gene (UL23) is coded on the opposite strand
and partially overlaps UL24. UL24X was originally constructed by homologous recombination of the appropriate
transfer vector and the KOS-based strain tk LTRZ-1 (Davar
et al., 1994). The latter has a lacZ gene inserted within
UL23, which can be used to select for recombinants.
A rescue of tk LTRZ-1 has been described demonstrating
that this rescue virus has a WT phenotype (Jacobson
et al., 1998). Nevertheless, we wished to generate a direct
rescue of UL24X for use in our subsequent experiments.
A UL24X rescue virus (vUL24-Rescue) was generated by
homologous recombination of infectious DNA from UL24X
co-transfected with the transfer vector pAG5, which contains
UL22, UL23 and UL24 from KOS.
107
106
105
KOS
104
vUL24X-Rescue
UL24X
103
6
12
18
Time (h p.i.)
24
Fig. 1. Characterization of a UL24X rescue virus in cell culture.
(a) Comparison of plaque morphology for KOS, UL24X and
vUL24X-Rescue on Vero cells at 2 days p.i. grown at the indicated
temperatures. (b) One-step replication assay comparing KOS,
UL24X and vUL24X-Rescue. Data represent mean¡SD ; *statistically
significant differences between vUL24X-Rescue and UL24X.
(Fig. 2d). Moreover, there was no statistical difference in
titres obtained from mice infected with KOS or vUL24XRescue at 6|102 p.f.u. per eye.
Furthermore, the severity of disease was restored to WT
levels with vUL24X-Rescue at 2|106 and 6|102 p.f.u. per
eye (Table 1). We found no statistical difference in the
number of TG that reactivated ex vivo from vUL24XRescue-infected mice versus KOS-infected mice; thus the
defect in reactivation observed with UL24X was corrected
(data not shown). These data confirmed that in vivo defects
observed for UL24X were attributable to the UL24 mutation.
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(b)
7
10
107
106
106
(d)
7
10
107
106
106
cu
24
X
e
2×
6×
10
6
10
2
10
2
6×
S
vU
L2
UL
e
cu
6
2
10
10
24
UL
cu
es
X
e
2×
6×
10
6×
4X
-R
e
cu
vU
L2
4X
-R
es
KO
2
6
2×
S
2×
X
24
UL
es
L2
4X
-R
10
6
10
10
6×
e
cu
S
KO
vU
e
cu
es
4X
-R
L2
6×
10
10
2×
10
2×
S
KO
2
100
2
100
6
101
6
101
vU
4X
-R
es
102
S
102
103
KO
103
104
10
104
105
2×
105
6
Viral titre (p.f.u. ml–1)
(c)
4X
-R
es
10
6
2×
S
vU
L2
UL
KO
cu
24
X
e
2×
6×
6×
4X
-R
es
L2
vU
L2
4X
-R
es
KO
cu
e
S
2×
2×
S
KO
10
6
100
10
2
100
10
2
101
10
6
101
vU
Viral titre (p.f.u. ml–1)
102
L2
102
103
KO
103
104
vU
104
105
2×
105
10
6
Viral titre (p.f.u. ml–1)
(a)
10
6
Viral titre (p.f.u. ml–1)
P.-A. Rochette and others
Fig. 2. Rescue of UL24X mutation corrects in vivo defects observed in the mouse ocular infection model. (a–c) Viral titres in
tear films for (a) 1, (b) 2 and (c) 3 days p.i. (d) Viral titres in homogenized TG at 3 days p.i. *Statistically significant difference
between vUL24X-Rescue and UL24X. Dashed lines indicate the detection limit of the experiment.
Importance of UL24 for viral replication in primary
embryonic neurons is similar to that in epithelialderived cell lines
One possible explanation for the disparity between the
impact of UL24 mutations on viral titres in the corneal
epithelia and in TG might be a specific requirement of
UL24 for replication in neurons. To test this hypothesis, we
compared replication of KOS, vUL24X-Rescue and UL24X
in mouse primary embryonic neurons (Fig. 3a). Neurons
were infected at m.o.i. 0.1, and total virus was harvested
at 12, 24, 36, 48 and 72 h p.i. We found that the reduction
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in titres for UL24X in neurons was of the same magnitude
as the viral replication defect observed for UL24X in epithelial-derived cell lines (Jacobson et al., 1989a, 1998).
Moreover, we obtained similar results using neurons
derived from the human neuroblastoma cell line LA-N-5.
Following differentiation by retinoic acid (RA), these cells
develop into cholinergenic neurons. By day 8 post-RA
treatment, w95 % of cells stained positive for the neuronal
maturation marker Neurofilament M (Fig. 3b). Neurons
were either mock-infected or infected with the WT virus
KOS or UL24X at a m.o.i. 10. At 9 h p.i., cells were immunostained for the viral ssDNA-binding protein ICP8, a
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Journal of General Virology 96
Role of UL24 in acute infection of TG in vivo
Table 1. Restoration of WT clinical signs with vUL24X-Rescue
Disease score 0–4. Each row represents the clinical scores for one animal.
Virus (p.f.u. per eye)
Time (days p.i.)
1
2
3
4
5
6
7
8
9
10
KOS 26106
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
+
+
0
+
++
0
++
++
+
++
+
+
++
+++
+++
+
++
++
++
++
++
++
++
+++
+++
+
++
+++
+++
+++
+++
+++
+++
++++
+++
++
++
+++
+++
+++
+++
+++
+++
++++
++++
++
+++
+++
+++
+++
+++
+++
+++
++++
++++
++
+++
+++
+++
+++
+++
+++
+++
++++
++++
++
vUL24X-Rescue 26106
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
+
0
+
0
0
0
++
+
+
+
++
++
++
++
++
++
++
++
++
+
++
++
+++
+++
++
++
++
++
++
++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
++
+++
+++
+++
+++
+++
+++
++++
+++
+++
++
+++
+++
+++
+++
+++
+++
++++
+++
+++
KOS 66102
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
+
+
0
0
0
0
0
0
0
0
+
+
0
0
0
0
0
0
0
+
+
++
0
0
0
0
0
0
0
++
++
+++
0
0
0
0
0
0
0
++
++
+++
0
0
0
0
0
0
0
++
++
+++
0
0
0
0
0
0
0
++
++
+++
0
0
0
0
0
0
0
vUL24X-Rescue 66102
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
+
0
0
+
0
0
0
0
0
0
+
0
0
+
0
0
0
0
0
0
+
0
0
++
0
0
0
0
0
0
0
+
+
+++
0
0
0
0
0
0
0
+
+
+++
0
0
0
0
0
++
0
0
+
++
0
0
0
0
0
++
0
0
+
++
0
0
0
0
0
UL24X 26106
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
+
0
0
0
+
0
0
0
+
0
0
0
0
0
0
0
0
0
0
0
0
0
marker for viral replication compartments (VRCs). We
observed similar large VRCs in both KOS- and UL24Xinfected LA-N-5 neurons (Fig. 3c). Next, differentiated LAN-5 cells were infected with KOS or UL24X at m.o.i. 10,
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and titres determined at 6, 12, 18 and 24 h p.i. (Fig. 3d).
In the absence of UL24, we observed a 10-fold reduction in
viral titres as compared with KOS. Here too, reduction was
of the same magnitude as the viral replication defect
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P.-A. Rochette and others
Viral titre (p.f.u. ml–1)
(a) 107
(b) Day 1
Day 5
Day 8
106
105
KOS
104
vUL24X-Rescue
UL24X
104
102
12
24
36
48
72
Time (h p.i.)
ICP8
Draq5
Merge
(d)
Viral titre (p.f.u. ml–1)
(c)
Mock
KOS
108
107
106
105
KOS
UL24X
104
103
6
12
18
Time (h p.i.)
24
UL24X
Fig. 3. Replication of a UL24-deficient virus in neurons. (a) Multi-step replication assay in murine embryonic neurons. Data
represent mean¡SD ; *statistically significant differences between vUL24X-Rescue and UL24X. (b) Differentiation of LA-N-5
neurons. Cells were immunostained for the neuronal maturation marker Neurofilament M (green). Nuclei were stained with
Draq5 (blue). (c) Formation of VRCs. Infected neurons were immunostained for ICP8 (green) and nuclei stained with Draq5
(blue). Merged images are shown in the right-hand panels. (d) One-step replication assay in LA-N-5 neurons comparing
UL24X with the WT virus KOS.
Reduction of the ocular viral load in the absence
of UL24 is not sufficient to explain the drastic
reduction of viral titres in TG
with 2|106 p.f.u. per eye of KOS, vUL24X-Rescue or
UL24X. Eyes were harvested post-mortem 2 days p.i. and
homogenized. We found that, similar to the reduction previously observed in tear films, viral titres were reduced 10fold in eye homogenates for mice infected with UL24X as
compared with KOS and vUL24X-Rescue (data now
shown). Thus, we did not observe a defect in virus release
into tear films with UL24X and therefore concluded that
the magnitude of the reduction of viral titres in tear films
reflected the reduction of the viral load within cornea.
Successful dissemination of HSV-1 infection from the eye to
TG depends in part on the amount of virus present in the
cornea (Chen et al., 2004; Leib et al., 1989; Sawtell, 1998).
We first verified that reduction in viral titres detected in
tear films of mice infected with UL24X reflected a defect
in viral replication within the cornea and not a defect in
virus release from corneal epithelial cells. Mice were infected
We next tested if, under our conditions, the 10-fold
reduction of the ocular viral load as observed for UL24X
was sufficient to cause the up to 4 log10 reduction in viral
titres in TG, possibly by a threshold effect. Thus, we
attempted to infect mice with an amount of WT virus
that would produce titres in tear films similar to those
seen for UL24X under our standard infection conditions.
observed for UL24X in epithelial-derived cell lines
(Jacobson et al., 1989a, 1998). These data suggested that
there was no inherent difference with respect to the
importance of UL24 for viral replication in neurons as
compared with epithelial-derived cells.
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Journal of General Virology 96
Role of UL24 in acute infection of TG in vivo
We infected mice either with 2|106 p.f.u. per eye of
UL24X or with quantities of KOS virus decreasing from
2|106 to 2|102 p.f.u. per eye (Fig. 4). As described previously, new infectious virus is first detected in the eye and
tear films at 1 day p.i., whilst viral replication in neurons is
first detected at 2 days p.i. (Summers et al., 2001). At 1 day
p.i. (Fig. 4a), a time when we would expect the impact on
TG titres to be the greatest, there was no significant difference between the viral titres in tear films for mice infected
with KOS at 2|106, 2|105 and 2|104 p.f.u. per eye; for
each condition, titres were *10-fold higher than for mice
infected with UL24X at 2|106 p.f.u. per eye. At the other
end of the spectrum, infection with 1|103 p.f.u. per eye or
less led to viral titres in tear films on 1 day p.i. that were
significantly below the levels produced upon infection
(a)
with 2|106 p.f.u. per eye of UL24X, with most viral
titres falling below our detection threshold. Interestingly,
although we were unable to detect infectious virus in tear
films at 2 and 3 days p.i. in many of the mice that were
infected with j1|103 p.f.u. per eye, in those where
virus was detected, titres rose and surpassed levels seen
with UL24X by day 3 p.i. (Fig. 4b, c). Furthermore, we
found that a dose of 2|103 p.f.u. per eye of KOS was
the only dose tested that resulted in tear films titres
that were down to levels on day 1 p.i. similar to those
seen for UL24X inoculated at 2|106 p.f.u. per eye and
where infectious virus was detected in all mice tested.
However, despite this similarity to UL24X, titres in TG
at 3 days p.i. rose to levels seen for higher inocula of
KOS, far surpassing titres obtained for UL24X (Fig. 4d).
(b)
106
106
Viral titre (p.f.u. ml–1)
107
Viral titre (p.f.u. ml–1)
107
105
105
104
104
103
103
(c)
8
10
6
1×
10
2
KOS
2×
10
2
2×
10
3
6×
10
3
1×
10
4
UL24X
2×
10
5
2×
2×
10
10
8
10
6
10
1×
2
KOS
2×
10
2
10
2×
3
6×
10
10
1×
2×
10
10
2×
2×
10
2×
3
100
4
100
5
101
6
101
6
102
2×
102
UL24X
(d)
106
106
Viral titre (p.f.u. ml–1)
107
Viral titre (p.f.u. ml–1)
107
105
105
104
104
103
103
UL24X
KOS
8
1×
10
6
2×
10
2
2×
10
2
6×
10
3
1×
10
3
2×
10
4
10
2×
10
2×
10
2×
8
10
6
10
2×
1×
2
10
2
10
6×
2×
3
10
10
KOS
1×
2×
10
10
2×
2×
10
2×
3
100
4
100
5
101
6
101
5
102
6
102
UL24X
Fig. 4. Reduction of the ocular viral load as observed in the absence of UL24 is not sufficient to explain the up to 4 log10
reduction of viral titres in TG. The inoculum (expressed in p.f.u. per eye of the indicated virus) used for each group is indicated
at the bottom of each graph. (a–c) Virus in tear films was titrated at (a) 1, (b) 2 and (c) 3 days p.i. (d) Virus in homogenized TG
was titrated at 3 days p.i. Dashed lines represent the detection limit of the experiment. *Statistically significant difference in viral
titres obtained for the indicated group as compared with the group of mice infected with 26106 p.f.u. per eye of UL24X.
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P.-A. Rochette and others
Thus, we concluded that the 1 log10 reduction in tear film
titres with UL24X was not sufficient to explain the drastic
reduction in viral titres in TG. Although the similarity in
titres seen with the inoculum of 2|103 p.f.u. of KOS and
2|106 p.f.u. of UL24X was lost by 3 days p.i. (Fig. 4c),
we would not expect virus levels in tear films to have
an impact on TG titres of the same day. The reverse strategy of using an inoculum of UL24X (1|108 p.f.u. per
eye) that was higher than that used for KOS did not produce higher viral titres in the eye or in TG.
Although lowering the infectious dose of KOS to 1|103 p.f.u.
per eye reduced the incidence of viral reactivation to levels
similar to those observed for UL24X (Table 2), unlike
UL24X, this reduction could be explained by a reduction in
the likelihood of establishing a productive infection in the
eye as demonstrated by the large number of mice infected
with j1|103 p.f.u. per eye in which levels of virus in tear
films were below the detection limit of the experiment.
Similarly, we also noted a difference between UL24X and
KOS with regard to the link between acute replication in the
eye and the development of clinical signs. Lowering the
infectious dose of KOS reduced the incidence of acute infection, but severe clinical signs were still evident in many
animals; in contrast, for UL24X, acute replication in the eye
was detected in all animals, but most animals exhibited no
clinical signs and none exhibited severe clinical signs (Table 1).
These results indicated that the *1 log10 defect in viral
replication of UL24X observed in the eye could not account
for the large reduction in viral titres in TG and in clinical
signs, and suggested a specific role for UL24 in the acute
infection of TG.
UL24 is important to establish a productive
infection in a high number of neurons
at 3 days p.i.
The low viral titres in TG observed with UL24X could
theoretically result from there being the same number of
infected neurons as seen with KOS, but a lower viral
yield, or due to fewer acutely infected neurons with the
same or a lower viral yield than for KOS. We next tested
Table 2. Frequency of ex vivo viral reactivation from latently
infected TG comparing different inocula of KOS and UL24X
Virus (p.f.u. per eye)
6
KOS 2610
KOS 26104
KOS 16103
KOS 66102
KOS 26102
UL24X 26106
UL24X 16108
Mock
Reactivated/total TG (%)
16/16 (100)*
10/10 (100)*
2/6 (33)
2/6 (33)
2/28 (7)
5/18 (27)
4/10 (40)
0/8 (0)
*Statistically different from UL24X with Fisher’s exact test.
2800
the hypothesis that the defect was at the level of establishment of lytic infection in neurons. TG were harvested from
mice infected with KOS, UL24X or vUL24X-Rescue at
3 days p.i., sectioned, stained using an antibody against
HSV-1, and visualized by immunohistofluorescence using
a stereoscope. We found that for UL24X, the size of the
infected area in TG, when one could be detected, was typically smaller than that observed for KOS or vUL24XRescue (Fig. 5a). Furthermore, for both KOS and
vUL24X-Rescue, we often observed multiple large foci of
infection per TG, which was in contrast to our observations
for UL24X where we typically detected either none or a
single centre of infection. Moreover, all TG from KOSand vUL24X-Rescue-infected mice stained positive for
viral lytic antigens, whilst for UL24X, lytic replication
was detected in only half of TG from infected mice.
For quantification purposes, sections were then analysed
by confocal microscopy. Foci of infection consisted primarily of large pseudomonopolar neurons as identified by their
characteristic morphology (Krastev et al., 2008; Mochizuki
et al., 1995). For each virus, infected neurons were quantified from individual TG harvested from at least three
infected mice. We found that the mean number of infected
neurons per TG section for KOS and vUL24X-Rescue was
*7 and *6, respectively, with individual values ranging
from 1 to 16 (Fig. 5b). In contrast, in the absence of
UL24, there was a mean 30-fold reduction in the number
of infected neurons detected per section as compared with
that observed for both KOS and vUL24X-Rescue.
Absence of UL24 leads to a reduced number
of TG neurons staining positive for HSV-1 DNA
at 2 days p.i.
At 3 days p.i., the total number of neurons expressing late
viral proteins was the cumulative result of newly infected
cells from the periphery, as well as possible interneuronal
dissemination of the infection within TG. Moreover, detection of infected neurons by immunohistofluorescence using
our anti-HSV-1 antibody depended on the expression of late
viral genes. Therefore, using this technique, we could miss
neurons where the genome gained access to the nucleus
and began to be replicated, but there was little expression
of late proteins. In order to have a better idea of the
number of neurons that were infected prior to extensive
spread of the virus, we used whole-mount in situ hybridization (WmISH) to quantify the number of neurons with
HSV DNA at 2 days p.i. (Fig. 5c, d). At this time point,
we could expect that on average the virus could have replicated once in epithelial cells and then gone on to infect neurons of TG. Thus, infected neurons would mostly represent
cells infected from the periphery. We found that in the
absence of UL24, the size of the infected area in TG, when
one could be detected, was typically smaller than that
observed for KOS or vUL24X-Rescue (Fig. 5c). For each
virus, infected neurons were quantified from individual
TG harvested from at least three infected mice (Fig. 5d).
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Journal of General Virology 96
Role of UL24 in acute infection of TG in vivo
(a)
Autofluo
HSV-1
Merge
(c)
KOS
vUL24X-Rescue
UL24XG
KOS
vUL24X-Rescue
UL24XG
(b)
(d)
6
15
No. of infected neurons
10
5
4
2
X
vU
vU
L2
UL
24
e
cu
es
4X
-R
X
24
es
4X
-R
L2
UL
cu
e
S
KO
S
0
0
KO
No. of infected neurons
20
Fig. 5. UL24 is important for dissemination of the acute infection to a high number of neurons. (a) Sections of TG from mice
infected with KOS, UL24X or vUL24X-Rescue at 3 days p.i. were analysed by immunohistofluorescence for HSV-1. Lefthand panels show the tissue autofluorescence (green), centre panels show HSV-1-infected cells (red) and right-hand panels
show merged images. (b) The number of positive neurons in (a) was quantified. Each point represents the mean number of
HSV-1-positive neurons per section for a given TG. Results presented were pooled from two independent experiments.
*Statistically significant difference as compared with the values obtained for UL24X. (c) TG from mice infected with KOS,
UL24X or vUL24X-Rescue were harvested at 2 days p.i., hybridized against HSV-1 DNA, immunochemically stained, sectioned and analysed by phase-contrast microscopy. Boxes in the upper panels indicate areas with neurons harbouring HSV-1
DNA and are enlarged in the corresponding lower panels. Arrowheads indicate other infected neurons outside the boxed
area. Bar, 0.5 mm. (d) The number of positive neurons in (c) was quantified as described in (b).
For all the viruses tested, the number of cells for which HSV1 DNA was detected at 2 days p.i. was much lower than the
number of cells expressing viral proteins at 3 days p.i.
We found that the mean number of HSV-DNA-positive
neurons per TG section for KOS and vUL24X-Rescue was
*1.5 and *3, respectively, with individual values ranging
from 0.19 to 5. In stark contrast, in the absence of UL24,
there was a mean 50- and 100-fold reduction in the
http://vir.sgmjournals.org
number of infected neurons detected per section as compared with that observed for both KOS and vUL24XRescue, respectively, with a mean value of 0.03 and individual values ranging from 0.018 to 0.18. Owing to the limited
sensitivity of the in situ approach, we also analysed
the relative amount of viral DNA in TG by quantitative
real-time (qRT)-PCR. TG were harvested at 1.5 days p.i.
before the expected production of new infectious viral
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2801
P.-A. Rochette and others
particles (Summers et al., 2001), total DNA isolated and viral
DNA quantified by qRT-PCR (Fig. 6). As expected (Sawtell,
1997), there was a large drop in the relative amount of viral
DNA in TG for KOS and vUL24X-Rescue when the inoculum was reduced from 2|106 to 2|103 p.f.u. per eye.
Nevertheless, even at the lower inoculum – one that led to
titres in tear films similar to those obtained with
2|106 p.f.u. per eye of UL24X – there was still more viral
DNA detected in TG from mice infected with the WT or
rescue virus than when mice were infected with
2|106 p.f.u. per eye of UL24X (five and 3.5 times more,
respectively). Thus, in the absence of UL24, reduced
spread of acute infection from the cornea to neurons of
TG appeared to be a major factor leading to the drastic
reduction in viral titres seen in TG at 3 days p.i.
DISCUSSION
Reduced viral titres in the eye cannot explain
the drastic reduction of viral titres in TG seen
with UL24X
It is known that a decrease in the viral load within the cornea
can impact the amount of virus reaching TG (Chen et al.,
8
Relative quantification of viral DNA
7
6
5
4
3
2
1
6
10
3
24
es
c
L2
4
XR
UL
ue
2×
2×
10
3
2×
10
KO
S
vU
vU
L2
4
XR
es
c
KO
S
ue
2×
2×
10
10
6
6
0
Fig. 6. Reduced amounts of viral DNA in TG for UL24-deficient
virus even upon normalization of viral titres in the eye. Relative
amounts of viral DNA from TG harvested at 1.5 days p.i. were
determined by qRT-PCR. The results for the KOS 26103 p.f.u.
samples were defined as 1. Data represent mean¡SEM for two
mice per condition.
2802
2004; Leib et al., 1989; Sawtell, 1998); however, we identified
inocula for KOS where titres at 1 day p.i. were below those
for UL24X, but nevertheless resulted in TG titres that far
surpassed those for UL24X. Thus, our results do not support
a model whereby a reduction of viral load in tear films is the
cause of the drastic reduction in viral titres observed in TG
for the UL24 mutant. Rather, our results point to UL24
playing a direct role in neurovirulence.
A UL24 mutant replicates similarly in epitheliaderived cells, in murine embryonic neurons
and in LA-N-5-derived neurons
HSV-1 infection of neurons depends on a select subset of
genes required to compensate for the lack of robust nucleotide metabolism in these non-dividing cells, e.g. the viral
ribonucleotide reductase gene (Goldstein & Weller, 1988;
Jacobson et al., 1989b; Perkins et al., 2002). Infection of
primary mouse embryonic neurons can reveal neuronal
replication defects of HSV-1 strains harbouring critical
mutations in such neurovirulence genes (Perkins et al.,
2002). We found that infection of such primary neurons
in the absence of UL24 led to a decrease in viral titres as
compared with KOS, but this decrease of *1 log10 was
no greater than what is observed in an epithelial cell line.
Similar results were obtained after infection of neurons
derived from a human neuroblastoma cell line (LA-N-5).
Thus, whilst UL24 does seem to play a role in the efficiency
of viral replication in a variety of cell types, our results do
not support a model whereby UL24 is critical for productive infection in neurons.
It has recently been shown that infection of neurons at the
level of the cell stroma favours an acute infection; in contrast, infecting neurons through their axonal termini
favours a latent infection (Hafezi et al., 2012). This effect
is possibly due to loss of specific transcription factors
from the tegument of the incoming virion as it makes its
way along the axon to the nucleus, thus shifting the balance
between viral lytic gene expression and latency (Hafezi
et al., 2012; Roizman & Sears, 1987). In our experiments,
we did not restrict infection of neurons to a specific substructure of the cell. Thus, we cannot rule out the possibility of a UL24-specific function related to infection of
neurons via their axonal termini. Although such a function
could theoretically account for the discrepancy between the
in vivo observations and our results obtained for UL24X
infections in neuronal cultures, UL24 of HSV-1 has never
been detected in extracellular virions (Loret et al., 2008;
Pearson & Coen, 2002) and thus is unlikely to play a
direct role in retrograde transport to the neuronal body following entry. Nevertheless, we cannot exclude the possibility that absence of UL24 during cellular infection has
an impact on tegument composition, which could ultimately impact subsequent transport within neuronal axons.
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Journal of General Virology 96
Role of UL24 in acute infection of TG in vivo
UL24 affects the spread of infection from the
cornea to neurons of TG
Whilst the five- to 10-fold reduction in the efficiency of
viral replication of UL24X in neurons, as observed in cell
culture, undoubtedly contributes in part to the drop in
viral titres in TG, it cannot by itself account for the severity
of the defect in neurovirulence seen with UL24X. Rather,
the huge drop in TG viral titres appears to be due to a significant reduction both in the number of neurons and in
the number of TG where acute infection takes hold.
To identify infected neurons where viral replication may not
have progressed to the late phase of gene expression, we also
analysed TG at 2 days p.i. using WmISH targeting the HSV1 genome. Of note, we could not distinguish between
incoming and newly synthesized DNA in these experiments,
so we cannot formally rule out the possibility that the UL24
mutant entered and released its genome into neurons as efficiently as the WT virus, but then failed to replicate its
genome. Nevertheless, this strategy enabled us to identify
infected neurons prior to a major contribution of any intragangliolar spread of the infection. We observed a significant
decrease in the number of neurons harbouring HSV-1 DNA
in the absence of UL24. In all our experiments aimed at
detecting HSV-1 proteins or DNA in situ, we never detected
infected neurons in more than half of TG isolated from
UL24X-infected mice. Importantly, we also found that
levels of viral DNA in TG at 1.5 days p.i. were lower for
UL24X even when inoculation doses of KOS or vUL24Xrescue were reduced 1000-fold.
Our results point to UL24 being a novel player in the dissemination of HSV-1 infection from the cornea to TG
neurons. Further studies will be required to uncover the
precise mechanism underlying the role of UL24 in the neuropathogenesis of HSV-1.
METHODS
Viruses and cells. Virus was propagated and titrated on Vero cells as
described previously (Lymberopoulos & Pearson, 2007). Primary
embryonic neurons were harvested from E16-18 CD-1 mice (Jacomy
et al., 2006). LA-N-5 cells were maintained and differentiated with RA
(Hill & Robertson, 1998). The HSV-1 strains KOS and UL24XG
(Jacobson et al., 1998) were originally provided by D. M. Coen
(Harvard Medical School). vUL24X-Rescue was generated by homologous recombination essentially as described previously by cotransfecting
UL24X-infectious DNA with the linearized transfer vector pAG5 containing the Bam HI fragment of KOS from nt 45 001 to 48 575 (Griffiths
& Coen, 2003) into Vero cells using Lipofectamine (Life technologies).
Screening for recombinant virus was done by PCR designed to detect
the 24 bp insertion within the UL24 ORF of UL24X (59-TACGAAGCCATACGCGC-39 and 59-GTCAACAGCGTGCCGC-39). The UL24
ORF of vUL24X-Rescue was sequenced to ensure the absence of
undesired mutations.
Viral yield in LA-N-5 neurons was assessed in a one-step replication
assay. LA-N-5 cells (4|104 cells per well) were seeded in duplicate in
six-well Cell+ plates and differentiated into neurons. Neurons were
infected at m.o.i. 10 in 2 ml of preheated complete RPMI 1640
medium containing 1 % FBS. Viral replication assays in Vero cells
were described previously (Lymberopoulos & Pearson, 2007). Statistical analysis was performed using Student’s t-test, a50.05.
Immunofluorescence on cells in culture. LA-N-5 cells were dif-
ferentiated on #1 coverslips pre-treated with 0.1 % cold water fish skin
gelatin (Sigma-Aldrich). Neurons were infected at m.o.i. 10 for 9 h in
200 ml preheated RPMI 1640 medium containing 1 % FBS. Cells were
fixed with PBS containing 4 % (w/v) paraformaldehyde, permeabilized
with chilled methanol for 5 min at 220 uC, washed and immunostained.
Coverslips were mounted using ProlongGold antifade reagent (Life
Technologies). The following antibodies were used: mouse monoclonal
anti-ICP8 (Abcam), rabbit polyclonal anti-Neurofilament M (Millipore)
and Alexa Fluor 488-conjugated goat polyclonal antibodies directed
against rabbit or mouse IgG. Nuclei were stained using Draq5 (Biostatus). Confocal microscopy was carried out at the INRS-Institut
Armand-Frappier imaging facility using a Bio-Rad Radiance 2000 confocal system with an argon/krypton laser at 488 and 568 nm (diode
638 nm) mounted on a Nikon E800 microscope using a |60 objective,
with an aperture of 1.4, and |1.6 software magnification in Lasersharp
software (Bio-Rad). Images were prepared using Adobe Photoshop.
Murine model of ocular infection. Animal experiments were conducted at the INRS-Centre for Experimental Biology in accordance
with institutional good animal care practices. Ocular infection of CD-1
mice deeply anaesthetized with a mixture of ketamine and xylazine,
analyses of acute replication in the eye and TG, and ex vivo reactivation
following latency were conducted essentially as described previously
(Coen et al., 1989; Leib et al., 1989; Leiva-Torres et al., 2010). Backtiters of inocula were verified following infection of the mice. Viral
titres were determined for at least three mice per condition. Data
points below the level of detection of the experiment were treated as
the limit values for calculation of means. Statistical analysis was performed using Student’s t-test, a50.05. For the reactivation assays, the
statistical analysis was performed using Fisher’s exact test.
Disease scoring. Clinical disease was assessed as described pre-
viously (Leiva-Torres et al., 2010). Five animals per group were followed daily for the indicated periods of time.
Histological sectioning. TG were harvested 3 days p.i. and processed for histological sectioning. Each TG was entirely processed into
8 mm sections on a Microm HM525 cryostat using replaceable blades
(Edge-Rite, Thermo Scientific). All sections were serially placed on
slides and overlaid with PBS.
Immunohistofluorescence. Immediately following tissue section-
ing, samples were permeabilized, dried briefly and incubated overnight at 4 uC in a humid chamber with a rabbit polyclonal anti-HSV-1
(Abcam) antibody and Hoechst (Life technologies) for nuclear
staining. The next day, slides were washed three times with PBS
containing 5 % newborn calf serum (NCS), incubated for 1.5 h at
room temperature with a goat polyclonal anti-rabbit IgG conjugated to
Alexa Fluor 568 and washed, and coverslips were mounted using
ProlongGold. Antibodies were diluted in PBS containing 5 % NCS.
Analysis of stained histological sections by fluorescence
microscopy. Low-magnification imaging of whole TG sections
Viral replication assays. Viral yield in primary mouse embryonic
was performed using a stereoscope fluorescence microscopy system (Nikon SMZ800; X-Cite 120 series) and a Q-Imaging camera
(01-RET-2000R-F-M-12-C) at |6.3 optical magnification.
neurons was assessed in a multi-step replication assay at m.o.i. 0.1.
Primary neurons (2|105 cells cm22) were seeded in duplicate in sixwell Cell+ plates (Sarstedt) that were pretreated with poly-D -lysine.
Quantification of the number of infected neurons per section was
performed using a high-magnification epifluorescence microscope
(Nikon Eclipse TE2000-U) at |40 optical magnification. Neurons
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2803
P.-A. Rochette and others
were identified qualitatively by the large size of the nucleus, their large
cell body size and their inherent autofluorescence that emits in the
green spectrum. Neurons were identified as infected by positive antiHSV-1 immunohistofluorescence staining. For each ganglion analysed, the number of infected TG neurons was determined for each
section produced. Results are presented as the total number of
infected neurons per number of sections obtained for a given TG.
Statistical analysis was performed using Student’s t-test, a50.05.
Chen, S. H., Pearson, A., Coen, D. M. & Chen, S. H. (2004). Failure of
Probe preparation for WmISH. Samples of 3 mg of EcoR1b plasmid
Cohrs, R. J., Randall, J., Smith, J., Gilden, D. H., Dabrowski, C., van
Der Keyl, H. & Tal-Singer, R. (2000). Analysis of individual human
(Goldin et al., 1981) containing a 20 kb fragment of HSV-1 DNA were
used in conjunction with a DIG-High-Prime DNA labelling kit
(Roche) using a 1 : 2 ratio of DIG-labelled dUTP to dTTP.
WmISH analysis of TG sections. Detection of cells that stained
positive for HSV-1 DNA in histological sections was performed by
WmISH as described previously (Escalante & Loomis, 1995). Quantification of the number of infected neurons per section was performed
using a high-magnification phase-contrast microscope (Nikon Eclipse
TE2000-U). For each ganglion analysed, the number of infected TG
neurons was determined for each section produced. Results are presented as the mean number of infected neurons per section of TG.
Statistical analysis was performed using Student’s t-test, a50.05.
qRT-PCR. TG harvested from each mouse were pooled and placed on
dry ice. DNA was extracted using a DNeasy Blood and Tissue kit
(Qiagen) (Cohrs et al., 2000). Reactions were performed in 10 ml
using iTaq Universal SYBR Green Master Mix (Bio-Rad), 100 ng of
ganglionic DNA, 500 nM each primer. For TK: 59-CCCAACGGCGACCTGTATAAC; 59-CCGGAGGTAAGTTGCAGCAG. MT-1 and
MT-2 primers for mouse adipsin were described previously (Kramer
& Coen, 1995). qRT-PCR was performed using the Rotor-Gene
RG3000 (Corbett Research). The relative amount of viral DNA normalized against adipsin was determined using the 22DDCt method
(Livak & Schmittgen, 2001). All samples were run in triplicate.
thymidine kinase-negative herpes simplex virus to reactivate from
latency following efficient establishment. J Virol 78, 520–523.
Coen, D. M., Kosz-Vnenchak, M., Jacobson, J. G., Leib, D. A., Bogard,
C. L., Schaffer, P. A., Tyler, K. L. & Knipe, D. M. (1989). Thymidine
kinase-negative herpes simplex virus mutants establish latency in
mouse trigeminal ganglia but do not reactivate. Proc Natl Acad Sci
U S A 86, 4736–4740.
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Bebrin, W., Coen, D. M., Kosz-Vnenchak, M., Knipe, D. M. & other
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ACKNOWLEDGEMENTS
We thank D. Favreau and P. Talbot (INRS-Institut Armand-Frappier)
for assistance with the setup of LA-N-5 neuronal cultures. DNA
sequencing was carried out by the McGill University and Génome
Québec Innovation Centre. P.-A. R. is the recipient of scholarships
from the Fondation Armand-Frappier (FAF) and the Fonds pour la
Recherche en Santé du Québec. A. B. was the recipient of scholarships
from the FAF and the Canadian Institutes for Health Research
(CIHR). G. O. L and C. S.-S are recipients of scholarships from the
FAF. This work was supported by an operating grant from CIHR
(MOP 82924), by Institut Pasteur International Network–Paribas
funds, and infrastructure and equipment grants from the Canada
Foundation for Innovation (9991) and the National Science and
Engineering Research Council (300745) to A. P.
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