2006, 80(16):8030. DOI: 10.1128/JVI.00474-06.
Palmarini Chiara Pinoni, Marcelo De las Heras and Massimo
Marco Caporale, Christina Cousens, Patrizia Centorame,
Sufficient To Induce Lung Tumors in Sheep
Retrovirus Envelope Glycoprotein Is
Expression of the Jaagsiekte Sheep
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JOURNAL OF VIROLOGY, Aug. 2006, p. 8030–8037
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 80, No. 16
Expression of the Jaagsiekte Sheep Retrovirus Envelope Glycoprotein
Is Sufficient To Induce Lung Tumors in Sheep
Marco Caporale,1Christina Cousens,2Patrizia Centorame,3Chiara Pinoni,3
Marcelo De las Heras,4and Massimo Palmarini1*
Institute of Comparative Medicine, University of Glasgow Veterinary School, Glasgow, United Kingdom1; Moredun Research Institute,
Edinburgh, United Kingdom2; Istituto Zooprofilattico Sperimentale degli Abruzzi e Molise, Teramo, Italy3; and
Facultad de Veterinaria, Universidad de Zaragoza, Zaragoza, Spain4
Received 7 March 2006/Accepted 30 May 2006
Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma (OPA). The
expression of the JSRV envelope (Env) alone is sufficient to transform a variety of cell lines in vitro and induce
lung cancer in immunodeficient mice. In order to determine the role of the JSRV Env in OPA tumorigenesis
in sheep, we derived a JSRV replication-defective virus (JS-RD) which expresses env under the control of its
own long terminal repeat (LTR). JS-RD was produced by transiently transfecting 293T cells with a two plasmid
system, involving (i) a packaging plasmid, with the putative JSRV packaging signal deleted, expressing the
structural and enzymatic proteins Gag, Pro, and Pol, and (ii) a plasmid which expresses env in trans for JS-RD
particles and provides the genomes necessary to deliver JSRV env upon infection. During the optimization of
the JS-RD system we determined that both R-U5 (in the viral 5? LTR) and the env region are important for
JSRV particle production. Two independent experimental transmission studies were carried out with newborn
lambs. Four of five lambs inoculated with JS-RD showed OPA lesions in the lungs at various times between 4
and 12 months postinoculation. Abundant expression of JSRV Env was detected in tumor cells of JS-RD-
infected animals and PCR assays confirmed the presence of the deleted JS-RD genome. These data strongly
suggest that the JSRV Env functions as a dominant oncoprotein in the natural immunocompetent host and that
JSRV can induce OPA in the absence of viral spread.
Jaagsiekte sheep retrovirus (JSRV) is the causative agent of
ovine pulmonary adenocarcinoma (OPA) (29). OPA is one
of the most common viral diseases of sheep in many regions of
the world (38) and is a unique large-animal model for lung
carcinogenesis (14, 26).
Among oncogenic retroviruses, JSRV appears to employ
unique mechanisms to induce cell transformation. The
JSRV envelope glycoprotein (Env) is an oncoprotein (1, 20,
33) which induces transformation of a variety of primary and
established cell lines in vitro (1, 9, 18–20, 33, 44) via the
activation of the phosphatidylinositol 3-kinase/Akt and
MEK/mitogen-activated protein kinase (MAPK) pathways
by as-yet-uncharacterized mechanisms (18, 19, 27). In addi-
tion, immunodeficient mice inoculated with an adeno-asso-
ciated virus vector expressing the JSRV Env develop lung
adenocarcinoma, indicating that the JSRV Env can also
behave as an oncoprotein in vivo, although the same vector
is not efficient in inducing tumors in immunocompetent
The role of the JSRV Env in OPA tumorigenesis in sheep,
the natural host of JSRV infection, is not completely clear.
OPA is experimentally reproducible when lambs are inocu-
lated intratracheally with concentrated virus preparations ob-
tained from lung secretions (or lung fluid) of OPA-affected
sheep or from supernatant of cells transfected with JSRV in-
fectious molecular clones (12, 29, 35, 37). The incubation pe-
riod, in this experimental model of OPA, can be as short as a
few weeks, suggesting that the viral Env can function as a
dominant oncogene in vivo as well as in vitro. However, nat-
urally occurring OPA is characterized by a very long incubation
period lasting even a few years (6, 39, 40). Diseases with a long
incubation time, in retrovirus-induced tumorigenesis, are often
associated with the classical mechanisms of insertional activa-
tion. This is the case with, for example, mice, chickens, and
cats with leukemias induced by Moloney murine leukemia
virus, avian leukosis virus, and feline leukemia virus, respec-
tively (34). However, common integration sites have not
consistently been found in OPA (7) and tumors appear to be
We have established recently that, under natural conditions,
the majority of JSRV-infected animals do not develop OPA
lesions during their commercial lifespan (6). In addition, it is
relatively difficult to find JSRV in the lungs of infected sheep
with no tumor lesions, suggesting that the target cells for JSRV
transformation might not necessarily be the reservoirs of virus
infection in many cases (6). Thus, many aspects of the patho-
genesis of OPA need further clarification.
In this study, we wanted to investigate if expression of the
JSRV Env was sufficient to induce lung adenocarcinoma in
sheep, the natural host of JSRV infection. To this end, we
derived a JSRV replication-defective virus (JS-RD) that upon
integration expresses only the viral env under the control of the
JSRV long terminal repeat (LTR). JS-RD was able to induce
OPA lesions in 4/5 of the inoculated animals, demonstrating
that the viral envelope is the primary determinant of oncogen-
esis in the natural host of JSRV infection.
* Corresponding author. Mailing address: Institute of Comparative
Medicine, University of Glasgow Veterinary School, 464 Bearsden
Road, Glasgow G61 1QH, Scotland. Phone: 44-(0)141-3302541. Fax:
44-(0)141-3302271. E-mail: email@example.com.
on June 3, 2013 by guest
MATERIALS AND METHODS
Plasmids. Plasmids employed in this study, unless stated otherwise, were
derived by standard molecular biology techniques (2) from the expression plas-
mid of the JSRV21infectious molecular clone, pCMV2JS21 (29). pJS-XE is
derived from pJS21m (23). pJS-XE has part of gag and the entire pro and pol
genes deleted (nucleotides [nt] 1278 to 5269 of JSRV21, GenBank accession
number AF105220); in addition, the first ATG (nt 264 to 266) of gag has been
replaced with a stop codon (TAG) by site-directed mutagenesis. A multiple
cloning site at the end of the truncated gag has also been added in pJS-XE. A
schematic diagram of the various plasmids is shown in Fig. 1. In pJSRVstop1, the
first ATG and the splicing acceptor site of the JSRV env gene have been
disrupted by silent mutagenesis. pJSRV?env has most of env deleted (between nt
5479 and nt 7074). pGPP?3? has the R-U5 region and the untranslated gag
region (nt 129 to 263) deleted. pGPP is derived from pGPP?3?, in which the
JSRV env and 3? LTR have been replaced by the constitutive transport element
(CTE) of Mason-Pfizer monkey virus (MPMV) and the simian virus 40 polyad-
enylation signal; in addition, a Kozak-like sequence has been added in front of
the gag open reading frame. The MPMV CTE was derived from plasmid
pGem7fz(?)MPMV250, a gift from Marie-Louise Hammarskjo ¨ld. pGPP?5? is
identical to pGPP, with the exception of the addition in the former of the R-U5
and untranslated gag regions. pGPP-MX is derived from pGPP?5? by the dele-
tion, from the latter, of the 5? gag untranslated region (between nt 129 and 263).
pCDNA3-HA-Sam68 is an expression plasmid for the RNA binding protein
Sam68 (43) and was a gift from David Shalloway.
Cell culture and virus expression. 293T cells were grown in Dulbecco’s mod-
ified Eagle’s medium supplemented with 10% fetal bovine serum at 37°C, with
5% CO2and 95% humidity. JSRV particles were produced in 293T cells by
transfection with the expression plasmid pCMV2JS21 as already described (29).
JS-RD particles were produced by transfecting 293T cells with plasmids pGPP-
MX, pJS-XE, and pCDNA3-HA-Sam68. Viral particles were collected from
supernatants of transfected cells, 24 and 48 h posttransfection and virus was
concentrated (200?) by ultracentrifugation as described previously (29). For
analysis of intracellular viral proteins, cells were lysed 48 h posttransfection
following standard techniques (2). Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and Western blotting were performed on cell
lysates (50 ?g of protein extract) and supernatants (10 ?l of concentrated
supernatants) essentially as previously described (29) using ECL Plus (Amer-
sham). Virus expression in cells transfected by various plasmids (or a combina-
tion of plasmids) as described in Results was quantified, when necessary, by
Western blotting by scanning membranes and measuring chemifluorescence with
a Molecular Dynamics Storm 840 imaging system using ImageQuant TL software
(Molecular Dynamics). Results were normalized with respect to JSRV p23 ob-
tained by transfecting cells with pCMV2JS21. Experiments (from transfections to
Western blotting) were performed independently at least three times and are
presented as the mean value for each sample (? standard error); statistical
significance was determined by applying the two-sample t test. A P value of ?0.05
was considered statistically significant. JSRV was detected by using rabbit poly-
clonal sera against JSRV p23 (matrix protein) (23) or the surface domain of Env
Animals. Animal experiments were performed at the Moredun Research In-
stitute (Penicuik, Scotland) and at the Istituto Zooprofilattico Sperimentale
dell’Abruzzo e Molise (Teramo, Italy) in accordance with local approved pro-
tocols regulating experimental use of animals. Two independent studies were
performed using a total of 11 lambs. In study 1, two lambs were inoculated with
JS-RD and two lambs with concentrated supernatants derived from 293T trans-
fected with the JSRV packaging plasmid alone. In study 2, three lambs were
inoculated with JS-RD, two with JSRV, and two with concentrated supernatants
derived from 293T transfected with the JSRV packaging plasmid alone. All
lambs were inoculated intratracheally during the first week after birth. Animals
of study 1 were kept for 12 months before being euthanized. In study 2, animals
were euthanized at 8 months postinoculation with the exception of one of the
lambs injected with JSRV and one of those injected with JS-RD, which were
killed at 7 weeks and 4.5 months postinoculation, respectively, because they were
showing clinical signs of respiratory distress. At the postmortem examination,
tissues were collected from seven distinct anatomical regions of the lungs: the
cranial part of the left cranial lobe, caudal part of left cranial lobe, left diaphrag-
matic lobe, right diaphragmatic lobe, right middle lobe, right cranial lobe, and
accessory lobe. Tissue samples were excised from the organ and cut into two
portions: one was snap-frozen and stored at ?80°C, while the other was fixed in
formalin for subsequent histopathological and immunohistochemical examina-
Histopathology and immunohistochemistry. Four- to six-micrometer lung sec-
tions were stained with hematoxylin and eosin and examined by light microscopy
for tumor lesions. Sections were taken from paraffin blocks of tissues collected
from seven anatomically distinct regions, as described above. Sections were
examined also by immunohistochemistry for the presence of JSRV Env as pre-
viously described, using the EnVision (DAKO) detection system (6, 35). OPA
tumor sections were used as positive controls. The activation of MAPK Erk1/2
signaling pathway was detected in tumors induced by JS-RD and JSRV by
immunohistochemistry employing rabbit polyclonal anti-phospho-Erk1/2 (Cell
Signaling) as described previously (11).
JS-RD- and JSRV-specific PCR assays. The presence of the JS-RD in the
inoculated lambs was assessed by PCR with primers designed to amplify specif-
ically the JS-XE genome or JSRV. Four PCR tests were used: two were JS-XE
specific (PCRs no. 1 and 2) and the other two were JSRV specific (PCRs no. 3
and 4). Both the JS-XE- and JSRV-specific PCRs employed the same forward
primers designed in variable region 1 of JSRV. Reverse primers for the JS-XE-
specific PCRs exploited the presence of a multiple-cloning-site region present
upstream of the env gene only in JS-XE. For the JSRV-specific PCRs (PCRs no.
3 and 4), reverse primers were designed for a portion of gag not present in JS-XE.
Sequences of the PCR primers employed in this study are available on request.
The presence of PCR inhibitors or degraded DNA was ruled out for all samples
that were tested by amplifying JSRV-related endogenous retroviruses (enJSRVs)
gag (PCR no. 5) as already described (24).
JS-RD design and development. The main goal of this study
was to determine if expression of the JSRV Env in sheep (the
natural host of JSRV infection) is sufficient to induce ovine
pulmonary adenocarcinoma. Accordingly, we devised a JSRV
replication-defective virus that could faithfully mimic the
unique tropism and expression properties of JSRV (25) and
FIG. 1. Schematic representation of the plasmids used in this study.
JS-RD particles were derived by transfecting 293T cells with pJS-XE
and pGPP-MX. VR1, variable region 1; CMV, cytomegalovirus imme-
diate early promoter; ?, packaging signal; MCS, multiple cloning site.
VOL. 80, 2006 JSRV ONCOGENESIS IN VIVO8031
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that delivered a genome with deletions expressing only the
viral env. We aimed to produce JS-RD particles by transiently
transfecting cells with two plasmids: (i) one expressing the
JSRV structural and enzymatic proteins Gag, Pro, and Pol and
with the putative packaging signal deleted, and (ii) one func-
tioning both to express Env in trans for the JS-RD particles and
to provide the genomes necessary to deliver JSRV env upon
We initially optimized the combination of constructs that
could produce the largest amount of JS-RD virions in vitro. A
schematic representation of the plasmids used is shown in Fig.
1. Because of the lack of a suitable tissue culture system for the
propagation of JSRV, we measured virion production by quan-
tifying virus particles released into the supernatant of trans-
fected cells by Western blotting. The first packaging construct
tried was pGPP, which has a cytomegalovirus promoter driving
expression of JSRV Gag, Pro, and Pol followed by the MPMV
CTE (for optimal RNA export) and a heterologous poly(A)
signal (Fig. 1). However, production of viral particles in cells
transfected by pGPP was approximately 50 times less abundant
than that in cells transfected with the expression plasmid for
JSRV (Fig. 2A and B). Restoration of the 3? end of the JSRV
genome in pGPP?3? did not increase the production of viral
particles, suggesting that the lack of env and 3? LTR in pGPP
was not responsible for the defect shown by this construct.
Indeed, transfection of 293T cells with pGPP?5? produced
quantities of viral particles comparable to JSRV (78% ?
8.1%) as judged by the content of p23 in the supernatants of
transfected cells. We modified pGPP?5? by deleting the un-
translated region upstream of the gag open reading frame in
order to use the resulting plasmid (called pGPP-MX) as a
packaging construct. pGPP-MX RNA would presumably not
be efficiently packaged by JSRV virions, considering the fact
that the untranslated gag region is invariably a major part of a
retroviral packaging signal (32), although this has not been
precisely mapped in JSRV. Cells transfected with pGPP-MX
produced approximately half (47.8% ? 10.5%; P ? 0.03) the
quantity of viral particles produced by JSRV, suggesting that
either the untranslated gag region has some beneficial role for
JSRV expression or the lack of viral genomic RNA negatively
influences virus assembly/exit. The latter seems most likely
because Gag expression in the cell lysates is comparable be-
tween the various constructs. In order to enhance the produc-
tion of viral particles by pGPP-MX, we provided the RNA
binding factor Sam68 in trans, as it was previously shown that
Sam68 increases the cytoplasmic utilization of RNA containing
the MPMV CTE (8). Indeed, cells cotransfected with
pGPP-MX and pCDNA3-HA-Sam68 produced quantities of
viral particles comparable to JSRV (Fig. 2C).
The JSRV env influences viral particle production. We ob-
served that cotransfecting pJS-XE with pGPP-MX increased
viral particle productionrelative
pGPP-MX alone. In order to analyze this observation further,
we constructed and analyzed pJSRV?Env, in which we re-
moved most of the env region of JSRV, and pJSRVstop1, in
which we introduced two mutations that altered the splice
acceptor and the first ATG of env (Fig. 1). Both these con-
structs were able to produce viral particles with no detectable
envelope (Fig. 3A to C). However, cells transfected with
pJSRV?Env and pJSRVstop1 produced, respectively, 16% ?
10.4% and 52% ? 10.9% viral particles compared to cells
transfected with the expression plasmid for JSRV; in both
cases, differences with JSRV were statistically significant (P ?
0.001 and 0.01, respectively). Cotransfection of pJS-XE with
FIG. 2. Optimization of the JSRV packaging construct. Cell lysates
(bottom panel) and virus pellets from supernatants (top panel) of trans-
fected cells were analyzed 48 h posttransfection by SDS-PAGE/Western
blotting employing an antiserum against the JSRV p23 (matrix). (A) 293T
cells were transfected with the plasmids indicated above each lane. Note
JSRV-proximal LTR released viral particles into the supernatant. Note
that the bands below Gag in the cell lysates are likely the product of
partially cleaved Gag (present also in panel C). (B) Quantification of virus
release was done by chemifluorescence on Western blots of viral pellets.
Shown are the means (? standard errors) obtained in three independent
experiments. (C) A positive effect on JSRV particle release was observed
in 293T cells cotransfected with pGPP-MX and increasing amounts (from
0.5 to 14 ?g) of an expression plasmid for Sam68.
8032 CAPORALE ET AL. J. VIROL.
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pJSRVstop1 or pJSRV?Env almost doubled the amount of
viral particles compared to cells transfected by pJSRVstop1 or
pJSRV?Env alone, although in the latter case, differences
were above significance (P ? 0.1). Thus, available data suggest
that the JSRV env facilitates viral particle release in cis and
likely in trans.
Experimental induction of ovine pulmonary adenocarci-
noma with JS-RD. Because of the lack of an in vitro system to
efficiently test and titrate JSRV infectivity, we proceeded di-
rectly to the experimental inoculation of newborn lambs with
JS-RD. Two independent studies were performed, and the
results obtained are summarized in Table 1.
In the first study, two lambs (no. 74 and 77) were inoculated
with JS-RD, while two lambs were used as negative controls
(no. 71 and 73) and inoculated with the supernatant of cells
transfected with the packaging plasmid alone. Lambs were
kept for 12 months and were then euthanized without showing
signs of clinical distress with the exception of one of the JS-
RD-inoculated animals which showed dyspnea (labored res-
piration). At the postmortem examination, neither negative-
FIG. 3. The JSRV env region is important for optimal viral particle production. (A) 293T cells were transfected with the plasmids indicated
above each lane. Virus pellets from cell supernatants (top panel) and cell lysate (50 ?g) were analyzed 48 h posttransfection by SDS-PAGE/
Western blotting. Where indicated, Env was supplied in trans by the JS-XE plasmid. Note that the bands below Gag in the cell lysates are likely
the product of partially cleaved Gag (present also in panel D). (B) Quantification of virus release was performed by chemifluorescence on Western
blots of virus pellets. Shown are the means (? SE) obtained in three independent experiments. (C) 293T cells were transfected with the plasmids
indicated. Forty-eight hours posttransfection supernatants were harvested. Virus pellets were analyzed by SDS-PAGE/Western blotting employing
an antiserum raised against the JSRV Env. As expected, neither JSRV?Env nor JSRVStop1 expresses detectable Env. (D) 293T cells were
transfected with the indicated plasmids and p23/Gag was detected as described above. Note that cotransfection of pGPP-MX, pJS-XE, and
pCDNA3-HA-Sam68 yields JS-RD virus particles in quantities similar to those of replication-competent JSRV.
TABLE 1. Summary of the results obtained in the in vivo studiesa
Result for indicated PCR
a?, positive; ?, negative; ND, not done.
bPresence of histological OPA lesions.
cDetection of JSRV Env by immunohistochemistry (IHC).
VOL. 80, 2006JSRV ONCOGENESIS IN VIVO8033
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control lamb had pulmonary lesions. On the contrary, one of
the JS-RD-inoculated animals (no. 77) had numerous small
whitish nodules (a few millimeters in diameter) which were
well delimitated from the surrounding lungs (Fig. 4A). Few
larger nodules (approximately 1 to 2 cm in diameter) were
also observed (Fig. 4B). Macroscopic lesions were absent
from the second lamb (no. 74) inoculated with JS-RD al-
though the latter had small areas of the lungs with an ab-
normal color and areas of consolidation (increased density
of the pulmonary tissue). Histological examination con-
firmed the presence of OPA lesions in both the JS-RD-
inoculated lambs (see below) (Fig. 4C and D).
In study 2, three animals were inoculated with JS-RD, two
with cell supernatants transfected with the packaging plasmid
alone (negative controls), and two with JSRV (positive con-
trols). One animal inoculated with JSRV was euthanized 7
weeks postinoculation when it showed respiratory distress (no.
B689). Another animal inoculated with JS-RD (no. B683) had
dyspnea 4.5 months postinoculation and was therefore eutha-
nized. All the other animals of study 2, including both negative
controls and the remaining positive control, were euthanized
approximately 8 months postinoculation. At necropsy, the
lungs of one of the animals inoculated with JSRV (no. B685)
showed OPA-like lesions, while the other JSRV-inoculated
lamb (no. B689) had no gross pathology but had multiple
neoplastic foci upon histopathological examination. One of the
JS-RD-inoculated animals (no. B694) showed multiple small
nodular lesions, as observed with lamb no. 77 in study 1. The
other two JS-RD-inoculated lambs (no. B683 and B695)
showed some small lesions that looked comparable to OPA
lesions. Histopathological examination confirmed the presence
of multiple OPA focal lesions in 2/3 JS-RD-inoculated lambs.
No neoplastic lesions were observed with lamb B695. In sum-
mary, in both studies, OPA was induced in 4/5 JS-RD-inocu-
lated lambs and in 2/2 JSRV-inoculated lambs but not in any of
the 4 negative-control animals.
Expression of the JSRV Env in all the experimental animals
was assessed by immunohistochemistry (Fig. 4D to G). As
expected, abundant expression of JSRV Env was detected in
tumor cells of JS-RD- and JSRV-inoculated animals, while no
FIG. 4. Macroscopic and microscopic lesions induced by JS-RD. Three of the five JS-RD-inoculated lambs showed numerous isolated lesions
creamy in color and of hard consistency (A and B). (C) Histology from a lung section of a JS-RD-inoculated lamb shows the presence of a papillary
to acinar expanding nodule that compresses the surrounding normal alveoli. (D and E) Immunohistochemistry in lung sections from a JS-RD-
inoculated lamb shows expression of the JSRV Env in tumor cells (characterized by the intracytoplasmic brown color). Micrographs are shown at
both low (D) and high (E) magnification. (F and G) Immunohistochemistry of lung sections of JSRV-inoculated lamb no. B689. Note the presence
of neoplastic foci of various sizes in close proximity. Micrographs are shown at both low (F) and high (G) magnification. (H and I) Phosphorylated
Erk1/2 is detectable both in JSRV- and JS-RD-induced tumors. Detection of phospho-Erk1/2 was achieved by immunohistochemistry both in lung
sections from JSRV-inoculated lambs (I) and JS-RD-inoculated lambs (H). A positive reaction is characterized by a cytoplasmic brown color. Bars,
100 ?m (panels E, G, H, and I) and 500 ?m (panels C, D, and F).
8034CAPORALE ET AL. J. VIROL.
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JSRV Env-expressing cells were detected in the negative-con-
trol animals (data not shown).
Histopathological appearance of OPA lesions in JSRV- and
JS-RD-inoculated sheep. OPA lesions in JS-RD-infected ani-
mals appeared to be mostly formed by isolated tumor nodules
composed by cuboidal or columnar proliferating cells growing
in an acinar or papilliform pattern and compressing the sur-
rounding normal alveoli (Fig. 4C and D). Larger neoplastic
foci were observed occasionally in lamb no. 77. Occasion-
ally, myxomatous tissue (probably of mesodermal origin)
was also present around the neoplastic lesions (not shown).
The myxomatous component is often seen in naturally oc-
curring OPA, but its origin (neoplastic versus nonneoplas-
tic) is not clear (10).
Lesions in lambs experimentally infected with JSRV were
mostly formed by neoplastic foci with characteristics similar to
those induced by JS-RD. However, neoplastic foci of various
sizes were surrounded by satellite often coalescing nodules
(Fig. 4F). The histopathological features and appearance of
neoplastic cells were essentially the same in JSRV- and JS-
RD-induced tumors, as can be appreciated by the high-mag-
nification micrographs shown in Fig. 4E and G.
Involvement of the H/N-Ras-MEK-MAPK pathway has
been shown in JSRV-induced cell transformation in vitro and
in vivo (11, 19). In order to test whether virus replication and
spread could determine differences in MAPK (Erk1/2) activa-
tion, we performed immunohistochemistry in tissues from both
JS-RD- and JSRV-inoculated animals. Lung sections were
stained with an antibody specific for pErk1/2. Tumor cells in
neoplasms induced by both JS-RD and JSRV showed activa-
tion of the MAPK pathway (Fig. 4H and I).
JS-XE is detectable in JS-RD-inoculated lambs. Finally, we
investigated whether we could detect JS-XE or JSRV in the
lungs of JS-RD-inoculated animals and in the positive- and
negative-control animals by developing two PCR assays that
specifically amplified the viral genome with deletions (JS-XE)
(PCRs no. 1 and 2) and two that detected full-length JSRV
(PCRs no. 3 and 4). Given the lack of obvious macroscopic
OPA lesions in some of the experimentally infected lambs, we
collected tissues from seven distinct anatomical regions in the
lungs. Animals were considered positive for JS-RD or JSRV
when at least one of seven aliquots was positive with either
JS-XE- or JSRV-specific PCR. The results are summarized in
Table 1, and representative data are shown in Fig. 5.
From all sets of positive and negative controls we obtained
the expected results. We detected JSRV in the tumor tissues of
the JSRV-inoculated positive-control animals, while we found
neither JS-XE nor full-length JSRV in any of the lung tissues
collected from the negative controls. In five of five JS-RD-
inoculated animals, we detected the viral genome with dele-
tions and did not detect full-length JSRV, confirming that Env
expression alone was responsible for the induction of OPA.
The integrity of DNA samples was assessed by amplifying
enJSRV sequences which are present in approximately 20 cop-
ies in the sheep genome (28).
In this study, we have shown that expression of the JSRV
Env alone is sufficient to induce OPA in sheep. Thus, the JSRV
Env is a powerful oncoprotein in vivo, in the natural host of
JSRV infection, and viral spread is not necessary for the onset
of lung adenocarcinoma.
JS-RD induced OPA in a high percentage of inoculated
animals, comparable to experimental JSRV infection (29). The
size of the lesions induced by JS-RD was reduced compared to
those observed in lambs experimentally infected with JSRV.
OPA lesions in JS-RD-inoculated animals appeared usually as
well-isolated neoplastic foci compressing the surrounding nor-
mal alveoli. In lambs experimentally infected with JSRV (in
this and previous studies) (12, 29, 35, 37), neoplastic foci of
different sizes were closely together (often coalescing) and
lesions gave the impression of being more invasive. We at-
tribute the histopathological differences noted above to the
ability of JSRV, upon infection of target cells, to produce new
infectious virus that can then infect (and consequently trans-
form) new target cells compromising the respiratory functions
more rapidly. On the contrary, JS-RD, being replication de-
FIG. 5. Detection of JS-RD in experimentally inoculated lambs. Representative examples of PCRs from lambs inoculated with JS-RD, JSRV
(positive control), or packaging construct only (mock). DNA was extracted from seven anatomical regions (indicated with letters A to G and
corresponding, respectively, to the cranial part of the left cranial lobe, caudal part of left cranial lobe, left diaphragmatic lobe, right diaphragmatic
lobe, right middle lobe, right cranial lobe, and accessory lobe) and tested by PCR specific for either JS-XE (no. 1 and 2) or JSRV (no. 3 and 4)
as indicated in Materials and Methods. Specific PCR products for JS-XE are found only in JS-RD-infected lambs (no. 77 and 74). JSRV is
amplified only by PCRs no. 3 and 4 in DNA extracted from tumor tissue of lamb no. 685 (lanes indicated with ?). PCR for enJSRV DNA was
used to control for the quality of the DNA preparations.
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fective, can infect and transform only single cells that expand
by cell division, and these growing lesions have fewer chances
to coalesce with other lesions unless cells in close proximity are
initially infected. Alternatively, it is possible that the neoplastic
cells induced by replication competent JSRV have a more
malignant phenotype than the JS-RD counterpart. We do not
favor the latter scenario, since the histopathological features of
the neoplastic cells per se are the same in JSRV- and JS-RD-
induced tumors. However, we cannot exclude the possibility
that in some instances, proto-oncogenes activated by JSRV by
insertional mutagenesis participate in the process of transfor-
mation initiated by the viral Env (7).
The histopathologies of field cases of OPA differ, with neo-
plastic lesions appearing sometimes as isolated nodules and
sometimes as large neoplastic foci with smaller satellite foci
around them; both types of lesions can be observed in the same
We believe that the age of the animal at the time of infection
and/or the physiological state of the lungs (e.g., concurrent
bacterial or parasitic infection) influences the histopathologi-
cal pattern shown in naturally occurring OPA. Most retrovi-
ruses, with the notable exception of lentiviruses, require ac-
tively replicating cells in order to allow the viral preintegration
complex to pass through the nuclear membrane (4). Trans-
formed cells in OPA are derived from the differentiated epi-
thelial cells of the distal lungs, type II pneumocytes, and Clara
cells, which in general have been believed to give rise to lung
adenocarcinoma in sheep and other species, including humans
(10, 21, 31). However, in adult animals, type II pneumocytes
and Clara cells have a very low replicative index, and conse-
quently, they would not be easily infectible by JSRV. In con-
trast, type II pneumocytes/Clara cells divide in the very young
lamb or in the adult where the epithelium has been injured in
order to repair the lesion; in both cases, these dividing cells
would be permissive to JSRV infection.
Recently, bronchioalveolar stem cells (BASCs) have been
proposed to maintain the bronchiolar Clara cells and pneumo-
cytes and to be the true origin of lung adenocarcinomas (3, 17).
If present also in sheep, BASCs might thus be the true target
for JSRV replication in the lungs. It is likely that BASCs are
most abundant in the young animal or following a lung injury
and thus BASC infection by JSRV would fit the model of OPA
development as proposed above.
The development of the JS-RD system has allowed us also to
further understand the molecular biology of JSRV. The R-U5
region of the viral 5? LTR is necessary for optimal JSRV
expression/particle formation, possibly by interacting with host
proteins similarly to the 5?-terminal RNA elements of other
retroviruses or by allowing proper Gag assembly (5, 15). Also,
our results suggest that for viral particle production, the env
region is important both in cis and in trans. Currently, we are
trying to determine if a CTE-like structure (13) is present
within env of JSRV, as there is in MPMV, and/or whether a
Rev-like export protein (and/or a Rev-responsive element) is
present within env analogously to mouse mammary tumor
virus (16, 22) and HERV-K (42). Additionally, it is likely
that the JSRV Env facilitates viral particle exit in the same
way as has been shown for MPMV (another betaretrovirus-
like JSRV), for which capsid transport is mediated by Env/
Gag interactions (36).
We thank Os Jarrett, Vincenzo Caporale, and the anonymous re-
viewers for suggestions that improved the manuscript. We thank
Marie-Louise Hammarskjo ¨ld and David Shalloway for generously pro-
viding reagents and Mark Dagleish for histopathology of the lambs in
Study 2. We are grateful to Emanuela Rossi, Patricia Dewar, and
clinical staff of the IZS Abruzzi e Molise and MRI for excellent animal
care. We thank Matt Golder and colleagues in the Viral Pathogenesis
Laboratory for continuous discussions and assistance.
This work was supported by grant CA95706-01 from the National
Cancer Institute of the National Institutes of Health, a grant from the
Italian Ministry of Health, and grant FF66/01 from the Scottish Exec-
utive Environment and Rural Affairs Department. M.P. is a Wolfson-
Royal Society research merit awardee.
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