Download Glycoprotein gH of pseudorabies virus is essential for penetration

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Glycoprotein gH of pseudorabies virus is essential for
penetration and propagation in cell culture and in the nervous
system of mice
Nathalie Babic, I B a r b a r a G. Klupp, 2 Birgit Makoschey, I Axel Karger, 2 A n n e Flamand I
and T h o m a s C. M e t t e n l e i t e r 2
Laboratoire de G6n4tique des Virus, CNRS,91198 Gif-sur-Yvette CEdex, France
2 Institute of Molecular and Cellular Virology, Friedrich-Loeffler-lnstitutes, Federal Research Centre for Virus Diseases of Animals,
D-17498 Insel Riems, Germany
Glycoprotein H (gH) of pseudorabies virus (PrV)
is a structural component of the virion and forms
a complex with another glycoprotein, gL. For
a detailed analysis of the function of PrV gH,
we isolated a gH-deficient mutant on transcomplementing gH-expressing cells after insertion
of a p-galactosidase expression cassette into a
partially deleted gH gene. The absence of gH did not
affect primary or secondary attachment of PrV but
the mutant was not infectious. The defect in infectivity could partially be overcome by experimentally induced membrane fusion using PEG, which
suggests that gH was necessary for fusion between
virion and cellular membranes. After intranasal
inoculation into mice, the LDso of complemented
Introduction
Herpesviruses express a large number of glycoproteins that
are incorporated into the virion envelope; of these, four (gB,
gH, gL and gM) have been found in every member of the
herpesvirus family analysed so far (Mettenleiter, 1994; Spear,
1993). The highest degree of conservation is found within the
gB homologues, followed by glycoproteins homologous to
gM and gH of herpes simplex virus type 1 (HSV-1). It is likely
that homologues play similar roles in the life cycles of their
respective viruses.
Herpesvirus glycoproteins have been shown to execute
important steps during the initiation of infection of target celIs.
In pseudorabies virus (PrV), gC binds cell-surface heparan
sulphate, leading to primary attachment of flee virions to cells
Author for correspondence: Anne Flamand.
Fax + 33 1 69 82 43 08, e-mail [email protected]
0 0 0 1 - 3 9 5 3 © 1996 SGM
gH PrV was more than four orders of magnitude
higher than that of wild-type PrV. Infection of the
respiratory epithelium was much less efficient with
complemented gH PrV as compared with rescued
PrV, reflecting the lack of direct cell-to-cell spread.
Complemented gH PrV was able to penetrate into a
few trigeminal and sympathetic first order neurons
accessible from the nasal cavity, whereas transneuronal transfer in the second order neurons was
not observed. In summary, gH is essential for entry
and cell-to-cell spread in cell culture, and for
propagation in the nervous system of mice. This
substantiates the hypothesis that transneuronal
spread in vivo and direct cell-to-cell spread in cell
culture are governed by similar mechanisms.
(Mettenleiter et al., 1990; Sawitzky eta]., 1990). For a
secondary, more stable association of virion and target cell, gD
has been shown to be required and it has been demonstrated
that primary, heparin-sensitive, attachment converts into
secondary heparin-resistant binding (Karger & Mettenleiter,
1993). As has also been observed for respective HSV-I
glycoproteins (Cai et al., 1988; Ligas & Johnson, 1988), PrV gB
and gD are both necessary for infectious entry of the attached
virions (Peeters et aI., 1992a; Rauh & Mettenleiter, 1991)
presumably by triggering fusion between the virion envelope
and cellular cytoplasmic membrane. Glycoprotein B is also
essential for direct cell-to-cell spread of HSV-1 and PrV. In
contrast, direct transmission of infectivity from cell to cell can
occur in the absence of gD in PrV (Heffner eta]., 1993 ; Peeters
et al., 1993), but is dependent on the presence of gD in HSV1 (Ligas & Johnson, 1988) pointing to fundamental differences
in the regulation of direct cell-to-cell spread in these two
alphaherpesviruses.
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Fig. 1. Construction of gH PrV. Below a schematic diagram of the PrY genome indicating location of unique long (UL) and
unique short (Us) portions as well as internal (IR) and terminal repeats (TR), a BamHI restriction fragment map is shown.
Enlarged is the area containing BamHI fragments 11, 16 and 15 which encompass the gH gene including relevant restriction
enzyme cleavage sites. Also depicted is the extent of the Notl deletion introduced into the gH gene and the insertion of the
gG-,g-gal expression cassette. (Sail) and (BamHI) denote restriction sites which were destroyed during insertional mutagenesis.
Arrow shows transcriptional direction of the gH and fl-gal genes.
We previously showed in an in vivo model after infection of
mice either intranasally or into the hypoglossal (XII) nerve that
PrV gB is required for transneuronal transfer from first to
second order neurons, whereas gD is not necessary for
this process (Babic eta]., 1993). We have also found that
glycoprotein gE is necessary for transneuronal transfer in the
four pathways supplying the nasal cavity: i.e. the olfactory,
trigeminal, sympathetic and parasympathetic pathways (Babic
et al., 1996). These results confirm and extend observations
from other laboratories that PrV or HSV-1 gE is implicated in
transneuronal transfer rather than in penetration and multiplication in first order neurons (Card et aL, 1992; Ba[an et aI.,
1994; Kritas et al., 1994, Mulder et a]., 1994; Dingwell et al.,
1995; Standish et al., 1994).
Recently, PrV gH has been described as a 95 kDa structural
component of virions, and it has been shown to contain Nlinked carbohydrates (Klupp et a]., 1992). In PrV and HSV-I,
gH appears to form a complex with gL and virions devoid of
gH also lack gL (Klupp et al., 1994; Forrester et aL, 1992).
Analysis of a gH- null mutant of HSV-1, isolated on a gHexpressing transcomplementing cell line, showed that gH is an
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essential viral glycoprotein which is involved in fusion between
virion and cellular membranes during viral entry and in cell-tocell spread of the virus (Forrester et al, 1992). Recently, a gHPrV mutant was found to exhibit a defect in productive
replication in cell culture, in particular in virus cell-to-cell
spread (Peeters et al., 1992 b). In addition, virions lacking gH
were found to be non-infectious but the nature of the defect
was not conclusively elucidated. To further analyse the role of
gH in cell culture and in animal models, we isolated a gHdeficient mutant from the PrV Kaplan strain (Kaplan & Vatter,
1959) on transcomplementing cells expressing gH. The mutant
attached normally onto MDBK ceils but penetration was
impaired, probably because virions devoid of gH could not
fuse at the cell membrane. The absence of gH also prevented
penetration and propagation of the virus in the nervous system
of adult mice after intranasal inoculation.
Methods
• Viruses and ceils. For construction of a gH-expressing cell line,
fragments BamHI-11and BamHI-16(Fig. 1) were inserted in the correct
relative orientation into BamHl-cleavedplasmid TN-47, a pBR322
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Wild-type
gH-
Fig. 2. Attachment of gH- PrV onto MDBK cells. Radiolabelled purified
wild-type or gH virions were bound to cells for 2 h at O °C. Thereafter,
three washes with PBS determined total binding (open bars), three
washes with PBS supplemented by 50 gg/ml heparin identified heparinresistant binding (striped bars). Presence of 50 gg/ml heparin during the
attachment process demonstrated heparin-independent binding (hatched
bars). Indicated is the percentage of input radioactive material which
remained stably bound to the cells after the respective treatment (Karger
& Mettenleiter, 1993). Data were derived from two independent
experiments. Standard deviations are indicated.
derivative containing the multiple cloning site of phage MI3mp18 (T. C.
Mettenleiter, unpublished data), giving rise to plasmid pbk1116. After
cleavage with XhoI and EcoRI, a 2"6 kb fragment encompassing 336 bp of
upstream sequences including the promoter, the gH ORF, and ~he
putative polyadenylation site of the PrV gH transcript, was excised. After
blunt-ending of non-compatible ends with Klenow polymerase, this
fragment was inserted into EcoRI-cleaved plasmid pSV2-neo-MTgB
containing the bacterial neomycin-resistance gene under control of the
SV40 early promoter (Southern & Berg, 1982) and the PrV gB gene under
control of the mouse metallothionein promoter (Rauh et al., 1991). After
transfection into Vero cells by calcium phosphate coprecipitation
(Graham & van der Eb, 1973), transfectants were selected on the basis of
their resistance to 500 ~g/ml geneticin. Colonies were picked and
analysed for their ability to complement growth of a gB- PrV mutant.
Positive clones were selected and used in attempts to purify gH- PrV. A
number of cell lines proved to allow the purification of the gH- mutant.
One of them, Veto SW78, was selected at random for further experiments.
To isolate a gH-deficient PrV mutant, pbk1116 was cleaved with
EcoRI thereby releasing a 3"4 kb fragment which was then subjected to
incomplete cleavage with HphI to obtain a 2'3 kb HphI-EcoRI fragment
containing the gH gene which was in turn cloned into TN-47. After
cleavage with NotI, which led to deletion of a 481 bp fragment from
within the gH gene, a SalI-BamHI gG-fl-gal expression cassette
(Mettenleiter & Rauh, 1990) was inserted by blunt-end ligation after fillin of 5" overhangs with Klenow polymerase. Transcriptional orientation
of the gG-fl-galactosidase (fl-gaI) cassette paralleled that of the gH gene
(Fig. 1). After cotransfection with wild-type PrV Kaplan DNA, virus
progeny was screened for a blue plaque phenotype under a chromogenic
agarose overlay containing 300 lag/ml Bluo-Gal (BRL). Blue plaques were
picked and purified three times on Vero SW78 cells. Correct integration
of the gG-fl-gal cassette into the gH gene was verified by Southern Blot
hybridization (data not shown) and absence of gH was demonstrated by
Western Blot (Klupp eta]., 1994).
To obtain a gH-rescued virus, genomic DNA of gH- PrV was
cotransfected with a plasmid carrying the 2"6 kb XhoI-EcoRI fragment
used for construction of the gH-expressing cell line (see above).
Transfection progeny were screened for the appearance of a white plaque
phenotype since restoration of gH expression should eliminate the gG-flgal cassette. White plaques were picked, purified, and analysed by
Southern Blot hybridization for restoration of the gH gene and by
Western Blot for restoration of gH expression (data not shown).
Glycoprotein H-rescued virus was propagated on normal Vero cells,
whereas the gH- PrV mutant was grown on complementing Veto SW78
cells. Cell culture supernatants were harvested at 72 h p.i. Infected cells
were sonicated for 30 s and pelleted, and supernatants were concentrated
by ultracentrifugation (Coulon et aL, 1989). To obtain virions devoid of
gH, normal Vero cells were infected at a m.o.i, of 5 with phenotypically
complemented gH- PrV and harvested after exhibiting pronounced CPE.
Supematants were collected and analysed.
•
Attachment and penetration of gH PrV onto MDBK cells.
Attachment assays on bovine kidney (MDBK) cells using radiolabelled
virions as well as the differentiation of heparin-sensitive, heparin-resistant
and heparin-independent binding have been described (Karger &
Mettenleiter, 1993). Polyethylene glycol (PEG)-induced fusion of
attached virions with target cells followed established procedures
(Sarmiento et al., I979; Rauh & Mettenleiter, 199I).
• Animal experiments. The virulence (LD~0) of the gH- fl-gal+ PrV
mutant was established by means of inoculation of 3 pl of 10-fold
dilutions of the virus into the right nostril of female Swiss mice (age 7
weeks) anaesthetized with equithesine (4% chloral hydrate, 16%
pentobarbital). All nasal instillations were made using a Hamilton syringe
connected to a catheter (Lafay et al., 1991). Mice were kept under
observation for 21 days or sacrificed when moribund.
The mice used for histological analysis of virus propagation received
an intranasal inoculation of 3 g[ containing 106 p.f.u, of either complemented gH- fl-gal+ PrV or gH-rescued PrV, to enable a comparison with
the results obtained with gG- fl-gal + PrV after administration of an
equivalent dose (Babic et al., 1994). To determine the kinetics of
propagation of the viruses, five infected mice were sacrificed at 6, 24 and
52 h and three at 76 h after inoculation of complemented gH PrV and
three mice were sacrificed at 52 h p.i. in the case of rescued PrV. The mice
were euthanized with pentobarbital and transcardially perfused (flow
rate: 10 ml/min) with 20 ml PBS (150 mM-NaC1, 7'4 mM-Na2HPO 4,
2"4 mM-KH.aPO4) followed by 4 % paraformaldehyde in PBS (150 ml) and
20 % sucrose in PBS (60 ml). The superior cervical ganglion (SCG) and the
upper half of the spinal cord were dissected out and kept in 20 % sucrose
in PBS for 24 h at 4 °C for cryoprotection. The head was decalcified in
PBS containing 0'1 M-EDTA for 10 days at 4 °C and then kept in 20%
sucrose in PBS for 24 h at 4 °C. All tissues were frozen at -- 70 °C and
cut in transverse serial sections of 30 ,m that were collected in two
parallel series on gelatin-coated slides.
• Detection of infected cells. For detection of fl-gal activity in the
tissues of complemented gH- fl-gal+ PrV-infected mice, one series of
sections was incubated for 4 h in 330 , g / m l X-Gal, 5 mM-K4Fe(CN)a,
5 mM-KaFe(CN)6, 2 mM-MgCIv 0"1% Triton X-100 in PBS (Babic et aL,
1993). After counterstaining with neutral red (0"01%), the sections were
mounted with Entellan (Merck).
For detection of gH-rescued virus which no longer expressed the/~gal marker, tissue sections were permeabilized in PBS/0"1% Triton X-100
for 30 min at room temperature, washed three times in PBS and incubated
overnight at 4 °C with a rabbit anti-PrV serum, diluted 1 : 1000 in PBS.
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After three washes with PBS, the sections were incubated for I h at 4 °C
with a rhodamine-conjugated goat anti-rabbit IgG, diluted 1:300 in PBS.
After three washes, the slides were mounted with Immu-mount
(Shandon).
Results
Role of PrY gH in initiation of infection by free virions
After infection of non-complementing cells with gH PrV,
which had been phenotypically complemented by propagation
on Vero SW78 cells, single infected cells were observed in
accordance with earlier observations by Peeters et al. (1992 b),
demonstrating that gH was essential for direct cell-to-cell
transmission of the virus. However, a few plaques developed
because of the presence of revertants in the viral population at
a frequency of 1"5 x 10 -5. To analyse the ability of the g H PrV mutant to bind to target cells, virions produced in normal
Vero cells were radioactively labelled with [dH]thymidine
(Karger & Mettenleiter, 1993) and incubated with MDBK cells
for 2 h at 0 °C to allow attachment but prevent penetration.
After thorough washing with PBS supplemented by 1% BSA
(PBS-A), the amount of labelled virus which remained bound to
the monolayer was determined as the total binding fraction.
Addition of 50 ~tg/ml heparin during the attachment period
decreased binding by more than 90%. Heparin-resistant
binding was determined by washing with PBS-A containing
50 , g heparin/ml after the 2 h attachment period. As shown in
Table 1. Infection of respiratory epithelium and first
order neurons of trigeminal, sympathetic and olfactory
pathways by the complemented gH- fl-gal + PrV mutant
after intranasal inoculation
Mice (7 weeks old) were infected with an inocutum of 106 p.f.u, of
complemented mutant virus containing 15 p.f.u, of revertant virus.
Each row corresponds to one infected animal. Infected cells in the
respiratory epithelium, or infected neurons in ganglia and olfactory
epithelium were counted in one section out of two and the number
was doubled to approximate the total.
Time postinoculation
(h)
24
24
24
24
24
52
52
52
52
52
76
76
76
Respiratory
epithelium
I04.
1 0 4*
104*
104*
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2500
310
76
290
150
6
16
78
Trigeminal
ganglion
(TG)
I4
4
I8
38
22
32
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2
6
20
2
0
0
Superior
cervical Olfactory
ganglion epithelium
(SCG)
(OE)
0
0
0
0
4
0
1
0
0
14
0
0
0
0
0
48
0
8
0
0
0
2
0
0
0
0
* This number was only estimated in the respiratory epithelium at
24 h p.i., due to the blurred appearance of labelling.
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Wild-type
gD-
gB-
gH-
Fig. :3. PEG-inducedfusion of gH PrV on Vero cells. Wild-type Pr virions,
as well as virions devoid of gD, gB, or gH were attached to normal, gD-,
gB-, or gH-expressing cells, respectively, for 2 h at 0 °C. Thereafter, they
were either treated with PEG (hatched bars) or left untreated (open bars)
and viral titres were determined under a methylcellulose overlay.
~-28(
Fig. 2, virions devoid of gH attached to target cells as
efficiently as wild-type virions and were fully capable of
secondary heparin-resistant attachment. Therefore, absence of
gH in virions does not alter the attachment phenotype as
compared to wild-type PrV. As expected, rescued virus did not
differ from either wild-type or gH PrV (data not shown).
Since absence of gH did not impair attachment of Pr
virions, we analysed whether a block in penetration might
exist. Generally, a deficiency in fusion between viral and
cellular cytoplasmic membranes can be overcome by treating
attached virions with PEG. Fusion assays were performed on
gH-complementing cells. For comparison, penetration of gBPrV (Rauh & Mettenleiter, 1991) was also assayed on Vero
SW78 cells, which express gB and gH (see above), and gDexpressing cells were used to assay penetration of g D - PrV
(Rauh & Mettenleiter, 1991). As shown in Fig. 3, PEGtreatment enhanced infectivity of gD-, gB- and g H - PrV to a
similar extent (approximately 50-fold). In contrast, wild-type
PrV titres were slightly reduced by PEG treatment. This result
clearly shows that attached Pr virions lacking gH exhibit a
similar deficiency in penetration as virions devoid of either gB
or gD.
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Fig. 4. X-Gal or immunodetection of infected ceils in the nasal cavity of mice 52 h after infection with complemented gH flgal +
PrV (o) or gH-rescued PrV (b, c). (o) Few infected cells were detected in the respiratory epithelium of gH infected mice after
fl-gal-staining (arrow). In contrast, extensive infection of the respiratory epithelium (b) extending to the vomeronasal organ (c)
was observed with the rescued PrV, after immunostaining of the sections with a rabbit anti-PrV serum. Bar markers represent
1O0 l~m.
Glycoprotein H is required for pathogenicity and
penetration in the nervous system of adult mice
The deletion of gH considerably lowered the pathogenic
power of the virus. Only two mice out of five inoculated in the
nasal cavity with 3 ~l containing 106 p.f.u, of complemented
gH PrV died while mice inoculated with lower doses survived.
This result indicates that the LDs0 of complemented gH- PrV
is higher than I06 p.f.u, while that of the parental strain Kaplan
and the wild-type-like gG PrV are 74 p.f.u, and 209 p.f.u.,
respectively (Babic et aI., 1994). In order to study the
neurotropism of complemented gH- PrV, a series of mice was
inoculated intranasally with 106 p.f.u, of either complemented
gH- or gH- rescued PrV. The mice were euthanized 6, 24, 52
or 76 h after inoculation of complemented gH PrV, and at
52 h p.i. in the case of rescued PrV. The head, the superior
cervical ganglion (SCG) and the upper half of the spinal cord
were removed and treated as previously described (Babic et al.,
1994).
The olfactory epithelium of adult mice was poorly
permissive to complemented gH- PrV. At 24 h p.i., only a few
foci of infected cells were observed in only 2 out of 5 animals.
At 52 h p.i., only two positive cells were observed in 1 out of
5 animals and at 76 h p.i. virus-infected cells could no longer be
detected in any of the mice (Table 1). The main olfactory bulb
(MOB), that receives input from the olfactory epithelium,
did not contain positive cells at any time point. It should
be noted that only a few infected neurons could be observed in
the MOB of mice dying of infection with rescued virus (at
52 h), thus confirming our previous findings that the olfactory
route is not very permissive to PrV in the mouse model (Babic
et aI., 1994). In the respiratory epithelium, fl-gal-positive cells
were observed as early as 6 h p.i. At that time, the number of
infected cells varied from less than ten to several hundred
depending on the animal. It reached several thousand at 24 h
and then decreased to a few hundred and then to a few dozen
or less at 52 and 76 h respectively (Table I and Fig. 4). Infected
cells were distributed in clusters in two areas facing each other
in the anterior part of the nasal cavity, on the inoculated side.
The infection did not spread to the vomeronasal organ, the
blood vessel wails or the mucus-secreting glands. On the
contrary, these structures were massively infected at 52 h p.i.
in mice inoculated with rescued virus (Fig. 4). At that time, the
extent of infection was indistinguishable from what was
regularly observed in mice infected with wild-type Kaplan, gErescued or gG- PrV (Babic et a]., 1994, 1996). Therefore, there
appeared to be no amplification of the infection in the nasal
cavity following the first cycle of viral replication, which was
initiated by the complemented gH- PrV (phenotypically
identical to wild-type PrV).
Besides the olfactory neurons, the nasal cavity contains
nerve endings of trigeminal, sympathetic and parasympathetic
neurons. The cell bodies of the corresponding first order
neurons are located in the ipsilateral trigeminal ganglion (TG),
the SCG and the pterygopalatine ganglion (PG) (Babic et al.,
1994). The first two ganglia can be easily dissected but the last
one is very small and difficult to identify in the mouse. First
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order neurons in these three pathways project to well-defined
areas of brain or spinal cord. Consequently, we studied the
penetration of gH- and rescued PrV in first order neurons of
trigeminal and sympathetic, but not parasympathetic pathways, and the transneuronal transfer in all three pathways. At
24 and 52 h after inoculation with complemented gH- PrV, the
ipsilateral TG was infected in all mice, but the number of
infected neurons was very low (few dozen at most) (Table 1
and Fig. 5 a). In comparison, the TG of mice infected with
rescued virus contained several thousand infected neurons at
52 h p.i. (Fig. 5 c), similar to what is obtained with gG- or wildtype PrV (Babic et aL, I994). In the 10 gH- PrV-infected mice
examined at 24 or 52 h p.i., seven SCG did not show any sign
of infection and only three SCG contained a few infected
neurons (Table 1, Fig. 5 b). The infection of the TG and SCG
was probably limited to the neurons which were directly
infected by complemented virus in the nasal cavity. The
finding that the number of infected neurons in the TG and SCG
did not increase with time, suggests that the gH- PrV produced
after one cycle of multiplication in the respiratory epithelium
was unable to infect first order neurons in these pathways, and
therefore that gH is also essential for penetration into nerve
endings. Transneuronal transfer in trigeminal, sympathetic and
parasympathetic pathways was never observed in mice
inoculated with the mutant and was normal for rescued virus
(Fig. 5 e-g) (Babic et al., 1994). Local transfer was observed
exclusively after inoculation of rescued virus.
Discussion
Our results show that the lack of infectivity of virions
devoid of gH is not due to a defect in attachment, since gHPr virions are capable of mediating primary and secondary
attachment to target cells with an efficiency similar to that of
wild-type virus. However, attached virions are unable to enter
cells, a defect which could partially be corrected by experimentally induced membrane fusion using PEG. These
results provide evidence that PrV gH, like HSV-1 gH, is
required for virion penetration in addition to gB and gD
(Peeters et at., 199Za; Rauh & Mettenleiter, I991). Therefore,
the structurally homologous HSV-1 and PrV gH glycoproteins
are involved in similar processes.
The results also show a dramatic reduction in the virulence
of complemented gH- PrV after intranasal inoculation. Similarly, complemented gH- HSV-1 has only restricted replicative
ability in animals and has a potential as a biologically safe
vaccine (Farrell et al., 1994). It is likely that disabled PrV
vaccines can be designed following similar lines. We have
compared the multiplication of gH- and rescued PrV in the
nasal cavity and in the nervous system of adult mice. The
presence of the mutant was shown by fl-gal staining of infected
tissues while that of the rescued virus was detected by
immunostaining with anti-PrV polyclonal sera. Both methods
of detection have been extensively compared during previous
experiments and have been found to give very similar results
(Babic et al., 1994, 1996; N. Babic, unpublished results). The
first cycle of replication of phenotypically complemented gHPrV in the respiratory and olfactory epithelia was similar to
that of the wild-type Kaplan or its gG- derivative (Babic et aL,
1994), but the infection did not progress in the nasal cavity, as
it did with rescued virus. Since the virus produced during the
first cycle of multiplication is devoid of gH, this suggests that
this glycoprotein is essential for propagation in the respiratory
epithelium, vomeronasal organ and mucus-secreting glands.
In previous studies, we have shown that a few first order
neurons of trigeminal pathways are infected by direct entry of
inoculated virus into their termini in the nasal cavity (Babic et
aI., 1994). The few neurons infected in the TG at 24, 52 and
76 h after intranasal inoculation of complemented gH PrV
confirm this finding. Direct penetration into sympathetic nerve
endings also explains infection of a few first order neurons in
the SCG. This direct penetration is not efficient since it could
be detected in only 3 out of 13 animals. This probably explains
why it was not observed in previous experiments using a gBPrV mutant (Babic eta]., 1994). The number of infected neurons
did not increase in the TG and SCG between 24 and 52 h p.i.
and seemed to decrease later, suggesting that trigeminal and
sympathetic neurons in these ganglia could not be infected by
non-complemented gH- PrV produced by the first round of
replication in the nasal mucosa or by local spread of the virus
from adjacent neurons (Babic eta]., 1993, 1994; Strack &
Loewy, 1990). In animals infected with rescued virus, the
extent of the infection closely resembled what is observed with
the Kaplan strain, the gE- rescued or gG- PrV (Babic et aL,
1994, 1996), indicating that penetration into first order
neurons, transneuronal and local transfer of the virus were
normal. Therefore the absence of gH and/or gL was indeed
responsible for the phenotype of the gH- PrV mutant.
In conclusion, the present study demonstrates that, besides
its role in cell-to-cell spread (Peeters et al., 1992 b), gH is not
required for stable attachment of PrV to target cells but is
necessary for entry. In the animal, gH is also essential for
penetration into first order neurons as well as local spread and
transneuronal transfer to second order neurons of trigeminal,
sympathetic (f) and parasympathetic (g) pathways (immunostaining of infected cells with a rabbit anti-PrV serum), (e) infection
in the caudal part of the spinal trigeminal nucleus (SpS). (f) upper thoracic segments of the spinal cord: the infection involves
the IML (large arrow), as well as more medial sympathetic cell groups (small arrows). (g) Rostral medulla oblongata: infected
neurons in the superior salivatory nucleus (SSN) (large arrow), Some neurons are also infected in the reticularis magnocellularis
nucleus (small arrow). These structures were not infected by gH- PrV, Bar markers represent 1 O0 lam.
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sympathetic and parasympathetic p a t h w a y s innervating the
nasal mucosa.
We thank G. Ugolini for careful reading of the manuscript. N. Babic
is a predoctoral fellow of the Minist6re de la Recherche et de la
Technologie. We thank G. Payen for excellent technical assistance. This
work was supported by the EU (grant ERB4002PL910264), the C.N.R.S.
(UPR 02431), and the DFG (Me 854/3).
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Received 15 February 1996; Accepted 30 April 1996
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