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J Virol, February 1998, p. 1657-1661, Vol. 72, No. 2
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Visna Virus dUTPase Is Dispensable for
Neuropathogenicity
Gudmundur
Pétursson,1,2,*
Priscilla
Turelli,2
Sigrídur
Matthíasdóttir,1
Gudmundur
Georgsson,1
Ólafur S.
Andrésson,1
Sigurbjörg
Torsteinsdóttir,1
Robert
Vigne,2
Valgerdur
Andrésdóttir,1
Eggert
Gunnarsson,1
Gudrún
Agnarsdóttir,1 and
Gilles
Quérat2
Institute for Experimental Pathology,
University of Iceland, Keldur, 112 Reykjavik,
Iceland,1 and
INSERM U372, Campus de
Luminy, 13276 Marseille, France2
Received 11 June 1997/Accepted 22 October 1997
 |
ABSTRACT |
The major part of the dUTPase-encoding region of the visna virus
genome was deleted. Intracerebral injection of the mutant virus
resulted in a somewhat reduced viral load compared to that resulting
from injection of the wild type, especially in the lungs, but the
neuropathogenic effects were comparable. The dUTPase gene is
dispensable for induction of lesions in the brain.
| |
TEXT |
The enzyme dUTPase (EC 3.6.1.23) has
been demonstrated in various procaryotic and eucaryotic organisms
(5, 6, 31). It hydrolyzes dUTP to dUMP and PPi
and thus provides a substrate for thymidylate synthase in the major
biosynthetic pathway to TTP. Its activity lowers the dUTP/TTP ratio and
leads to decreased misincorporation of uracil into DNA. Cellular
dUTPase activity is reported to be cell cycle regulated, high in
dividing cells but low in terminally differentiated nondividing cells
(9, 10, 16, 21, 23). It may correlate with the pool of
intracellular deoxynucleoside triphosphates, which is reported to be
very low in macrophages (24).
dUTPase activity has been demonstrated in several viruses, including
herpesviruses (4, 32), poxviruses (3), type B and
D retroviruses (11), and in a subset of lentiviruses,
namely, equine infectious anemia virus (EIAV), feline
immunodeficiency virus (FIV), caprine arthritis-encephalitis
virus (CAEV), and visna virus of sheep (7, 14). Human
immunodeficiency virus and other primate lentiviruses do not contain a
dUTPase gene. Two main theories have been proposed to explain the role
of the viral dUTPases. One is that this viral enzyme permits or
facilitates viral DNA synthesis in cells with low levels of
deoxynucleoside triphosphates and thus enables the virus to replicate
in nondividing cells such as neurons or macrophages. Evidence
supporting this theory has been published for the dUTPase-containing
lentiviruses EIAV and FIV (13, 22, 25, 30). The second
theory maintains that the viral dUTPase exerts an antimutator function
by reducing the misincorporation of uracil into viral DNA. Such
misincorporation could lead to perturbation of viral DNA replication
(20) and decreased viral fitness, and uracil residues in
viral DNA might affect the function of the DNA such as binding of
transcription factors (19, 29).
We have previously reported that dUTPase-deficient mutants of CAEV and
visna virus show delayed replication in nondividing goat macrophages
(27). In the present study, we showed that dUTPase-deficient
visna virus is still fully pathogenic after intracerebral infection of
sheep.
Construction and propagation of dUTPase-deficient virus.
We
constructed an in-frame deletion mutant of the dUTPase gene of the
pathogenic proviral molecular clone of the neurovirulent visna virus
strain KV 1772 (1, 2) as previously described (27), which resulted in the deletion of three of the five
conserved domains of the dUTPase, including the central domain with a
tyrosine residue which is thought to be at the active site of the
enzyme (14, 15). The deleted provirus and the wild-type
provirus were transfected into primary goat synovial cells
(27). The activity of the virion-associated dUTPase was
assayed in pelleted virus from clarified supernatants of infected cells
by measuring the relative incorporation of tritium-labeled dUTP and
dTTP into a poly(rA)-oligo(dT) template under the conditions of the
classical reverse transcriptase (RT) assay as described previously
(27). The dUTP/TTP incorporation ratio of the
dUTPase-deficient (DU-2) virus was found to be 0.26, as compared to a
ratio of 0.04 for the wild-type (WT) virus.
In vitro replication of DU-2 and WT visna viruses.
Replication
of DU-2 and WT visna viruses was tested in sheep monocyte-derived
macrophages in culture. Sheep monocyte-derived macrophages were
prepared from heparinized blood by centrifugation on Histopaque-1077 as
described previously (26), washed repeatedly, and seeded in
plastic tissue culture vessels. After 24 h, adherent cells were
washed repeatedly to remove unattached cells and incubated for 7 days
in Dulbecco modified Eagle medium supplemented with 5 × 10
5 M mercaptoethanol and 10% lamb serum before they
were used for infection. Viral replication was measured by RT assay as
described previously (27). The replication of the DU-2
mutant virus in sheep macrophages was slightly delayed compared to that
of the wild type when measured by this assay (Fig.
1). This difference, although slight, was
consistently reproducible in repeated tests, whereas no significant
difference in growth curves could be demonstrated when the infectivity
of the macrophage-derived viruses was titrated in sheep choroid plexus
cell cultures (Fig. 2).
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FIG. 1.
Growth curves of DU-2 and WT visna viruses in sheep
monocyte-derived macrophages as measured by RT activity (kcpm/0.5
ml). p.i., postinfection.
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FIG. 2.
Growth curves of DU-2 and WT visna viruses in sheep
monocyte-derived macrophages as measured by infectivity titration in
sheep choroid plexus cells. Titration end points were calculated by the
Reed-Muench method. p.i., postinfection.
|
|
Animal experiments.
To test the pathogenic potential of the
DU-2 mutant, we inoculated five Icelandic 10-month-old sheep
intracerebrally in the left hemisphere with 0.4 ml of DU-2 virus
(titer, 107.5 tissue culture infective doses/ml) which had
been grown in sheep choroid plexus cells; five sheep were also infected
with the same dose of WT virus grown in the same way. The sheep were
bled at regular intervals to test for serum antibodies to the virus by an enzyme-linked immunosorbent assay (ELISA) (28) and virus neutralization by standard methods, and circulating virus was tested by
cocultivation of buffy coat cells with sheep choroid plexus cells.
Samples of spinal fluid for cell counts and virus isolation were
obtained at 4, 12, and 25 weeks after infection. All sheep were
sacrificed at 25 weeks after infection, and virus isolations from the
following tissues were attempted: plexus choroideus, cerebellum,
medulla oblongata, spinal cord (cervical, thoracic, and lumbar parts),
cervical, mediastinal, and mesenteric lymph nodes, spleen, bone marrow,
and lungs. The histopathological lesions of the central nervous system
were graded on a scale of 0 to 6 as described previously
(17).
The frequency of virus isolation from sheep infected with DU-2 virus
was somewhat lower than that of sheep inoculated with
the WT, as shown
in Table
1. The difference between the
groups
was statistically significant for determinations for the lungs
and for all tissues taken together, but the frequencies of virus
isolations from the central nervous systems of both groups were
equal.
The deletion of mutant virus reisolated from infected sheep
was
confirmed by PCR and sequencing (data not shown).
Virus-specific serum antibodies against whole virus antigen measured by
an ELISA (
26) were found to increase in titer with
time
after infection in both groups. The geometric means of the
antibody
titers were consistently lower in the sheep infected
with the DU-2
virus than in the WT virus-infected sheep, although
the range was
rather wide (Table
2). As shown in Table
3, the
virus-neutralizing titers were
similar for both groups.
Spinal fluid samples were obtained from the infected sheep at 4, 12, and 25 weeks postinfection. There was a brisk increase
of mononuclear
cells 1 month after infection (Table
4),
as frequently
observed in intracerebrally infected sheep
(
17). Although the
group infected with the mutant virus
showed more pleocytosis at
this time point than the WT virus-infected
group, the difference
was statistically not quite significant
(
P = 0.11 with the Welch
test); after 12 weeks and at
the time of sacrifice, the sheep
infected with the WT virus had a
somewhat higher number of cells,
but the difference was not
significant.
At the time of sacrifice, histopathological changes were observed only
in the brain (Fig.
3). They were, as
shown in Fig.
3, in all respects like the classical lesions of visna
virus described
previously (
8,
17), consisting of
perivascular and periventricular
infiltrates of mononuclear
inflammatory cells (macrophages, lymphocytes,
and some plasma cells).
As shown in Table
5, there was no
significant
difference in the severity of lesions between sheep
inoculated
with the DU-2 mutant virus and those inoculated with the WT.
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FIG. 3.
Photomicrographs of brain lesions in sheep infected with
WT (A, B, and C) and DU-2 (D, E, and F) visna viruses. (A and D)
Periventricular inflammation and discrete perivascular infiltrates in
adjacent white matter are visible. (B and E) Almost confluent
inflammation of the white matter is evident. (C and F) Inflammation of
the choroid plexus with formation of lymph follicles is visible. Stain,
hematoxylin-eosin. Magnification, ×90.
|
|
These results show that visna virus lacking a functional dUTPase
gene can replicate rather well in sheep macrophages in culture,
unlike
EIAV and FIV (
13,
22,
25,
30), where deletion of
the dUTPase
gene practically abolished virus replication in equine
and feline
macrophages, respectively. CAEV seems to be intermediate
in this
respect, since dUTPase-negative mutants will replicate
in goat
macrophage cultures but at a lower rate than that of wild-type
viruses
(
27). Ponies infected with dUTPase-negative EIAV and
cats
injected with FIV with the dUTPase-encoding gene deleted
showed reduced
viral loads (
12,
13). We have found similar
evidence of a
reduced viral load in the lungs and possibly in
the blood, lymphoid
tissue, and bone marrow of sheep injected
with the DU-2 visna virus.
Recently, it has been found that intra-articular
inoculation of
dUTPase-negative mutants of CAEV resulted in systemic
infection and
dissemination of virus in a manner similar to that
of the WT virus
(
28). The WT virus was pathogenic both locally
in the
injected joint and in the contralateral joint, whereas
the lesions
produced by the mutant were restricted to the inoculated
joint and were
somewhat less severe. In our experiments, lesions
were found only in
the central nervous system, which is in accordance
with our previous
experience with sheep inoculated intracerebrally,
where lung lesions
are rarely observed. The pathogenic potential
of visna virus is
unaffected by the loss of the viral dUTPase
function, at least at the
site of injection. The gene coding for
dUTPase must, however, have some
evolutionary advantage for the
virus since it has been conserved, and
CAEV with a point mutation
in the gene coding for dUTPase has been
shown to revert to the
WT in an infected goat (
28). This
advantage seems to be too
subtle to be detected by our experiment,
possibly because of the
rather high virus dose and the route of
infection. The low frequency
of virus isolation from the lungs of the
sheep infected with the
DU-2 virus may indicate that such mutants would
be less readily
transmissible in the course of natural infection, which
is believed
to be either by the respiratory route or lactogenic
(
18).
| |
ACKNOWLEDGMENTS |
This study was supported by the Agence Nationale de Recherches sur
le SIDA, Paris, France, the Icelandic Research Council Science Fund,
and the University of Iceland Research Fund.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute for
Experimental Pathology, University of Iceland, Keldur v/Vesturlandsveg, IS-112 Reykjavik, Iceland. Phone: 354-5674700. Fax: 354-5673979. E-mail: gpet{at}rhi.hi.is.
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J Virol, February 1998, p. 1657-1661, Vol. 72, No. 2
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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