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Journal
of General Virology (1997), 78, 785–793. Printed in Great Britain
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Secretion of a murine retroviral Env associated with resistance
to infection
Abdallah Nihrane, Irina Lebedeva, Myung Soo Lyu, Kazunobu Fujita and Jonathan Silver
Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD
20892-0460, USA
Fv4 is an endogenous defective murine leukaemia
virus (MuLV) which expresses high levels of an
envelope protein (Env) closely related to that of the
ecotropic class of MuLVs. Mice bearing the natural
Fv4 gene or a transgenic version are resistant to
infection by ecotropic MuLVs. Fv4 mice secrete the
surface peptide (SU) of the Fv4 Env in their serum
and this secreted Env can block infection of NIH3T3
cells. To study the secretion of Fv4, we metabolically
Introduction
Retroviral envelope glycoproteins have amino-terminal
signal sequences which direct nascent Env peptides to the
lumen of the endoplasmic reticulum (ER) where they are
glycosylated. The Env precursors are then transported to the
Golgi complex where sugars are modified and the Env
precursor is cleaved by a cellular protease to generate the
surface glycoprotein (SU), responsible for binding to the
ecotropic receptor, and the transmembrane anchor (TM),
involved in viral-cell fusion and anchoring SU to virus and cell
membranes (Van Zaane et al., 1976 ; Hunter & Swanstrom,
1990). SU and TM are held together by non-covalent bonds
and possibly also by disulfide bonds (Pinter & Honnen, 1983 ;
Thomas & Roth, 1995).
In the case of murine leukaemia viruses (MuLVs), the initial
Env gene product found in the ER is a molecule with N-linked
oligosaccharides, mostly mannose sugars, with an apparent
molecular mass of 80–90 kDa, depending on virus strain
(Witte & Wirth, 1979). This ‘ gp85 ’ molecule, which is sensitive
to digestion by endoclycosidase H (EndoH) and PNGase F
(peptide : N-glycosidase F), is transported to the Golgi where
the high-mannose sugars are converted to ‘ complex sugars ’,
which are resistant to cleavage by EndoH but sensitive to
PNGase. Also in the Golgi, the Env precursor is cleaved to
form the hydrophilic SU gp70 and hydrophobic TM p15E
Author for correspondence : Jonathan Silver.
Fax ­1 301 402 0226. e-mail jsilver!nih.gov
labelled cells expressing Fv4 Env or Env from
infectious MuLVs and followed synthesis, glycosylation, proteolytic processing and secretion of Env
species. We found no difference in the kinetics of
synthesis or processing of Fv4 Env compared to the
envelopes of infectious MuLVs, but Fv4 Env
associated more weakly with its transmembrane
anchor and was shed from the surface of cells.
moieties (Pinter et al., 1978). During virus budding, the viral
protease cleaves p15E to generate p12E and p2E molecules
(Van Zaane et al., 1976), thereby activating the Env protein for
fusion (Rein et al., 1994).
The Fv4 provirus, located on mouse chromosome 12
(Odaka et al., 1981), resembles the 3« half of a MuLV with an
intact env gene and 3« LTR, but a deletion of the 5« LTR, gag
and most of the pol gene (Ikeda et al., 1985). Transcription of
the Fv4 mRNA is driven by a cellular promoter and this mRNA
encodes an Env protein C 75 % identical (at the nucleotide
level) to ecotropic MuLV Env proteins (Ikeda et al., 1985). The
Fv4 locus was first identified as a gene causing resistance to
Friend murine leukaemia virus (FrMLV) (Suzuki, 1975 ; Gardner
et al., 1980). Mice carrying the dominant allele of Fv4
(corresponding to the defective provirus) resist infection with
ecotropic MuLVs. When transfected with the Fv4 gene,
NIH3T3 cells express the Fv4 Env on the cell surface and, if
they express large amounts of this Env, are resistant to
infection with ecotropic retroviruses (Ikeda & Sugimura, 1989).
Mice that are transgenic for the Fv4 gene are also resistant to
infection, depending on the level of expression of Fv4 Env
(Limjoco et al., 1993). Fv4 resistance can be transferred to nonFv4 mice by bone marrow transplantation (Ikeda & Odaka,
1979 ; Kitagawa et al., 1994 ; Limjoco et al., 1995).
Fv4 causes resistance to ecotropic MuLVs by interference,
a virological phenomenon that involves non-virion Env
binding to virus receptor inside cells, during synthesis and
transport of these molecules, or on the cell surface, thereby
blocking receptor-mediated virus uptake. In cells expressing
0001-4263 # 1997 SGM
HIF
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A. Nihrane and others
the ecotropic Env and the ecotropic receptor, glycosylation of
the ecotropic receptor is altered, implying that these molecules
interact intracellularly (Kim & Cunningham, 1993). Fv4 mice
also secrete Fv4 SU in their serum and the secreted SU can
block binding of FrMLV to the ecotropic receptor on other
cells (Kitagawa et al., 1995 ; Nihrane et al., 1996). Thus,
interference ‘ from without ’ may also play a role in resistance.
Fv4 may also alter the immune response to MuLV by
preventing immunosuppression associated with FrMLV infection (Morrison et al., 1986), or by specifically altering the
immune response to viral envelope antigens as a result of prior
exposure to the inherited Fv4 envelope protein (Limjoco et al.,
1995 ; A. Nihrane & J. Silver, unpublished).
Fv4 Env is first synthesized as a gp85 precursor molecule
which is subsequently processed to SU gp70 and TM p15E
moieties (Ikeda & Odaka, 1983, 1984 ; Nihrane et al., 1996). We
previously showed that transgenic Fv4 mice secrete gp70 as a
soluble glycoprotein and that this gp70 is derived largely, but
not exclusively, from haematopoietic cells (Nihrane et al.,
1996). Here, we show that gp70 secretion is quantitatively
much greater for cells expressing Fv4 Env than for cells
expressing Moloney murine leukaemia virus (MoMLV) Env ;
that the Fv4 Env is secreted in the absence of p15E TM ; that
Fv4 Env is synthesized, glycosylated and cleaved with the
same kinetics as other MuLV envelopes ; and that secretion of
Fv4 gp70 can be explained in part by dissociation of Fv4 SU
from the cell surface.
Methods
+ Mice, cells, antibodies, plasmid and reagents. FVB}N.Fv4-2rr
mice have been described previously (Limjoco et al., 1993). All cells were
maintained in Dulbecco’s modified Eagle’s medium (DMEM ;
BioWhittaker) with 10 % foetal calf serum (FCS). NIH3T3 cells were
purchased from ATCC. FrMLV was obtained from Janet Hartley
(National Institutes of Health, Bethesda, Md., USA). Tail cultures from 10
to 15-day-old mice were established as previously described (Nihrane et
al., 1996). Briefly, tails were dipped for 40 s in 70 % alcohol, cut and
minced in collagenase solution [150 U}ml in Hanks balanced salt
solution (HBSS) with the antibiotics penicillin (10 000 U}ml), streptamycin (10 000 µg}ml) and fungizone (250 µg}ml)]. The tissue fragments
were then washed and cultured in DMEM. MAb 372 anti-p15E (Chesebro
et al., 1981) was purchased from ATCC. Polyclonal goat antibody antiRauscher gp70 (catalogue no. 04-0031 ; lot no. 74S000510) was
purchased from Quality Biotech. The MoMLV expression plasmid was a
gift from Tetsuro Matano (Matano et al., 1993). NHS-fluorescein (Nhydroxysuccinimide-fluorescein) and NHS-SS-biotin [sulfosuccinimidyl2-(biotinamido) ethyl-1,3-dithiopropionate] were purchased from Pierce.
NHS-fluorescein was suspended in dimethylsulfoxide at a concentration
of 10 mg}ml. PNGase F and EndoH enzymes were obtained from New
England Biolabs.
+ Establishment of MoMLV envelope cell lines. The MoMLV
envelope expression plasmid DNA (Matano et al., 1993) was transformed
into E. coli DH5α competent cells (Life Technologies), grown overnight
and purified by QIAGEN Plasmid Maxi Kit (Qiagen). One day before the
transfection, NIH3T3 cells were seeded at 10' cells per T25 flask in
DMEM, 10 % FCS. The following day, 20 µg purified plasmid DNA was
HIG
transfected into the cells by calcium phosphate precipitation (Graham &
Van der Eb, 1973) and the cells were incubated overnight at 37 °C in 5 %
CO . Clones were selected in hygromycin (500 µg}ml) and characterized
#
by flow cytometry and immunoprecipitation for envelope expression.
+ Pulse-chase labelling and immunoprecipitation. Cells (1–
2¬10') in 60 mm diameter dishes were starved for 10 min at 37 °C in
1 ml methionine- and cysteine-free DMEM medium containing 5 % FCS
and then labelled for 30 min with 200 µCi TRANS$&S label (ICN
Biomedical). The cells were then washed, placed in 1±5 ml fresh DMEM,
10 % FCS, and incubated for various times at 37 °C, after which they were
disrupted in lysis buffer (100 mM NaH PO .H O, 100 mM NaCl, 0±5 %
# % #
sodium deoxycholate, 0±01 % BSA, 0±1 % SDS). The cell lysate and the
corresponding filtered cell supernatants (0±45 µm pore size) were precleared by incubating with protein A beads (Life Technologies) for 1 h at
4 °C. After removing the beads by centrifugation, samples were
incubated for 1 h with 50 µl protein A beads previously treated with 5 µl
polyclonal goat anti-Rauscher gp70 antibody. The beads were then
washed three times with 0±1 % Triton X-100, 300 mM NaCl, 50 mM Tris–
HCl (pH 7±5). The labelled proteins and beads were split into three parts.
One part was eluted at 100 °C in 60 µl reducing sample buffer (Laemmli,
1970) and analysed by SDS–12 % PAGE and autoradiography. The other
two parts were used in deglycosylation experiments.
+ PNGase and EndoH treatment. Labelled proteins were recovered
from the protein A beads by incubating at 100 °C for 10 min in 25 µl
denaturing solution (0±5 % SDS, 1 % β-mercaptoethanol). For PNGase
digestion, samples were incubated for 2 h at 37 °C in the above
denaturing solution plus 3±3 µl 10 % NP40, 3±3 µl 10¬ G7 buffer (New
England Biolabs) and 2000 U PNGase. For EndoH treatment, samples
were incubated for 1 h at 37 °C in the denaturing solution plus 3 µl 10¬
G5 buffer and 2000 U EndoH. Proteins were subsequently characterized
by SDS–PAGE and autoradiography.
+ Cell surface fluoresceination and flow cytometry assay for
supernatant Fv4 Env. Tail cells (1–2¬10') in 60 mm diameter dishes
were incubated with 1 ml of a 0±5 mg}ml solution of NHS-fluorescein at
4 °C for 30 min as described by Vigers et al. (1988). The cells were then
washed once with DMEM and three times with ice-cold PBS containing
calcium and magnesium (PBS}CM), and cultured at 37 °C in 1±5 ml
complete DMEM. Some samples were assessed for viability by staining
with Trypan Blue. Supernatants were recovered at various time points,
filtered and assayed for FITC-labelled Fv4 Env capable of binding the
ecotropic receptor as follows. Approximately 10' trypsinized NIH3T3
cells were incubated for 45 min at 37 °C with 1±5 ml of recovered
supernatants. After washing three times with HBSS, 0±1 % BSA, 0±1 %
sodium azide, the cells were analysed using an EPICS Profile Cytometer
(Coulter). As receptor-negative controls, we used NIH3T3 cells that were
chronically infected with FrMLV or uninfected NIH3T3 cells that were
blocked with 100 µl 20 % Fv4 serum (obtained from FVB}N.Fv4-2
transgenic mice) for 45 min at 37 °C.
+ Cell surface biotinylation and SDS–PAGE analysis for
surface and supernatant Fv4 Env. Cell surface Fv4 Env was
characterized by surface biotinylation of radiolabelled cells (Cole et al.,
1987 ; LeBivic et al., 1989). At the end of a 3 h pulse, Fv4 tail cells were
washed three times with ice-cold PBS}CM, and incubated at 4 °C for
30 min with 1 ml of a 0±5 mg}ml solution (in PBS}CM) of NHS-SSbiotin. Free biotin was then blocked with DMEM and the cells were
washed three times with PBS}CM. The biotinylated cells were returned
to culture in 1±5 ml complete DMEM for an additional 3 or 5 h. At each
time point, cells were disrupted in 1±5 ml lysis buffer. The cell extracts and
supernatants were immunoprecipitated with protein A-agarose beads
(Life Technologies) previously coated with anti-Rauscher gp70 antibody.
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Secretion of murine retroviral Env
After washing, the proteins were eluted from the beads for 10 min at
100 °C in 20 µl 10 % SDS and diluted in 1 ml PBS. The boiled beads were
removed by centrifugation and the supernatants were incubated with
50 µl streptavidin–agarose beads (Life Technologies) for 4 h at 4 °C. The
beads were then washed and the proteins eluted and characterized by
SDS–PAGE and autoradiography.
Results
Gp70 secretion is peculiar to Fv4
To see if secretion of SU by Fv4-expressing cells was
peculiar to the Fv4 Env, we transfected NIH3T3 cells with a
vector encoding the MoMLV Env (Matano et al., 1993),
isolated three Env-expressing clones and compared the
synthesis and processing of envelope glycoproteins in these
cells to that in tail cells from Fv4-transgenic mice and FrMLVinfected NIH3T3 cells. Cells were metabolically labelled with
[$&S]methionine and [$&S]cysteine for 30 min and ‘ chased ’ with
non-radioactive amino acids for various times, after which the
cells were lysed and cell lysates and supernatants were
immunoprecipitated with a polyclonal goat antibody to MuLV
gp70. An Env precursor of about 85 kDa was detected in the
cell extract immediately after labelling (Fig. 1 a, lane C0). This
precursor was cleaved into species of approximately 70 kDa
(gp70) and 15 kDa (p15E), easily seen at the 2 and 5 h chase
points (Fig. 1, lanes C2 and C5). The small amount of p15E in
the immunoprecipitate of gp70 shows that some p15E remains
associated with gp70 during immunoprecipitation, as has been
noted many times before. In the supernatant of the MoMLV
env cell line, only a faint band corresponding to gp70 was
detected (Fig. 1, lane S5), indicating that only a small amount
of MoMLV envelope was released from the cells. Comparable
data were obtained with the other two MoMLV envelope cell
lines. Several other groups have reported that ecotropic MuLV
SU glycoproteins are only weakly released from envelopeexpressing cells (Heard & Danos, 1991 ; Battini et al., 1995).
In contrast, tail cells from Fv4-transgenic mice released a
large amount of gp70 into the supernatant (Fig. 1 b, lower
panel, lanes S1–S5). We used Fv4 tail cells in these experiments
rather than NIH3T3 cells transduced with Fv4 Env because the
former produce more Fv4 Env (Nihrane et al., 1996). Our
previous work showed that the ratio of secreted SU to cellassociated SU was about 1 : 1 in Fv4-NIH3T3 cells (Nihrane et
al., 1996), as is the case in the Fv4 tail cells examined here (Fig.
1 b, lanes S4–S5 versus lanes C4–C5), compared to less than
1 : 10 in the MoMLV Env cell lines (Fig. 1 a, lane S5 versus C5).
NIH3T3 cells infected with FrMLV also produce a large
amount of gp70 in the supernatant (Fig. 1 b, upper panel), but
this is expected since these cells produce viral particles.
Fv4-gp70 is secreted free of p15E
We previously showed that p15E was detected in immunoprecipitates of gp70 from cell extracts of Fv4 tail cells, whereas
p15E was not cross-immunoprecipitated from the supernatants
of these cells despite the presence of large amounts of gp70
(Nihrane et al., 1996). We interpreted these results as indicating
that Fv4 SU is secreted in the absence of TM. However,
another possibility is that both Fv4 TM and SU are secreted but
for some reason are not associated with one another in the
supernatant. To address this directly, we immunoprecipitated
cell lysates and supernatants with an anti-p15E MAb (Chesebro
et al., 1981). The MAb detected p15E in the cell lysate after 1 h
of chase (Fig. 2 a, lanes C1, C2 and C5). However, no p15E or
p12E (the cleaved form of p15E present in virions) was found
in the supernatant of Fv4 cells showing that it is not secreted
(Fig. 2 a, lanes S0–S5).
The anti-p15E MAb detected the gp85 Env precursor
weakly in cell lysates (Fig. 2 a, lanes C1–C5). The absence of
this band in the C0 lane is believed to be due to a technical
problem with loss of sample since the background smear is also
reduced in this lane ; plenty of gp85 was present in the C0
sample since it was immunoprecipitated with anti-gp70
antiserum (Fig. 2 a, left side, C0 lane). The weakness of the
gp85 signal using the anti-p15E MAb suggests that the
epitope detected by this MAb is partially ‘ buried ’ in the gp85
precursor. It is also of interest that the p15E MAb did not
cross-immunoprecipitate Fv4 gp70 from the cell lysate,
suggesting that binding of the p15E MAb may disrupt the
association of p15E with gp70.
We performed the same studies with chronically infected
NIH3T3 cells and the NIH3T3-MoMLV envelope cell lines. In
the extracts of FrMLV-infected cells, the MAb immunoprecipitated p15E and gp85 as well as some background bands
(Fig. 2 b, left side, lanes C0–C5), whereas in the supernatants of
these cells it immunoprecipitated a p12E species consistent
with cleavage of p15E in viral particles (Fig. 2 b, lanes S1–S5).
The anti-p15E MAb did not cross-immunoprecipitate gp70
from the supernatant, despite its abundance (cf. Fig. 1 b upper
panel). As a negative control, uninfected NIH3T3 cells are
shown on the right side, along with a positive control of
FrMLV-NIH3T3 cells from the same experiment. In the
MoMLV Env cell line, p15E and gp85 were detected in the cell
extracts. No p15E species were detected in the supernatant, as
expected, since this cell line does not secrete viral particles or
Env proteins (Fig. 2 c, lanes C1–C5 and S1–S5).
Fv4 tail cells, FrMLV-infected NIH3T3 cells and
NIH3T3-MoMLV Env cells show no difference in the
kinetics of synthesis, glycosylation or cleavage of
envelope glycoproteins
The fact that Fv4 Env was peculiar in its propensity to
secrete SU raised the possibility that differences might exist in
the kinetics of synthesis, glycosylation or cleavage of Fv4 Env
compared to FrMLV and MoMLV Env. To address this
question, we analysed metabolically labelled cells at short
times after a 30 min pulse. The envelope glycoproteins were
immunoprecipitated with goat anti-gp70 antibody and
digested with PNGase or EndoH enzymes to evaluate their
glycosylation state. PNGase is believed to cleave all N-linked
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A. Nihrane and others
(a)
(b)
Fig. 1. Comparative pulse-chase and immunoprecipitation of Env
glycoproteins synthesized in Fv4 tail cells, NIH3T3 cells infected with
FrMLV and NIH3T3 cells transfected with a MoMLV Env expression vector.
Cells were metabolically labelled for 30 min with [35S]methionine and
[35S]cysteine and grown for various times in the presence of cold
methionine and cysteine. Cell lysates and supernatants were precipitated
with goat anti-Rauscher gp70 antibody and analysed by SDS–PAGE under
reducing conditions. Chase time in hours is given after the designations
‘ C ’ (for cell lysate) or ‘ S ’ (for supernatant).
oligosaccharides. EndoH removes N-linked oligosaccharides
from high-mannose glycoproteins, but it does not cleave
complex oligosaccharide side-chains. As such, resistance to
EndoH serves as a marker for transport to the Golgi complex
where high-mannose sugars are converted to complex oligosaccharides.
In the Fv4 system, the gp85 Env precursor was sensitive to
PNGase and EndoH since it was entirely converted by these
enzymes to a species of approximately 60 kDa. This is most
easily seen immediately after pulse labelling when no other
bands are present (Fig, 3 a, b, lanes C0«), but it is also true at
later time points (lanes C15«–C60«). This indicates that gp85
does not contain complex sugars.
After 15 min chase, a small amount of gp70 can be seen in
the cell lysate, and this species is easily detectable in the cell
extract and supernatant after 35 min chase (Fig. 3 a, top and
bottom panels, lane C35«). The gp70 moiety was completely
converted by PNGase to a species with a molecular mass of
about 50 kDa. In contrast, digestion with EndoH had little
effect on gp70. This is most clearly seen in the supernatant
HII
where no other bands are present (Fig. 3 b, lower panel), but it
is also true for the gp70 species in the cell lysate (Fig. 3 b, top
panel). EndoH may remove some sugars from some of the
gp70 molecules since it slightly reduced the intensity of the
gp70 band and made it appear somewhat ‘ fuzzy ’ (Fig. 3 b,
lower panel). The fact that essentially all of the gp85 molecules
were cleaved by EndoH while most of the gp70 molecules
were resistant implies that cleavage of gp85 to gp70 occurs
after the high-mannose sugars have been modified in the Golgi
complex. This pattern has been observed for other MuLV
envelopes (Pinter & Honnen, 1983, 1984).
In the MoMLV Env cell line, the gp85 precursor was
similarly sensitive to digestion with PNGase and EndoH and
was reduced to about 60 kDa by both enzymes (Fig. 3 c).
Interestingly, the mobility of the PNGase-deglycosylated form
of gp85 appears to be slightly retarded after longer chase times
(compare the mobility of the C 60 kDa band in the PNGase
lanes at 60« and 35« versus 15« and 0«). Careful inspection of
Fig. 3 (a) shows the same phenomenon for the Fv4 Env. The
mobility of the EndoH deglycosylated form of gp85 also
appears to shift up with increasing chase times. A likely
explanation for these slight mobility shifts is O-glycosylation
since PNGase and EndoH do not remove O-linked sugars.
Gp85 is known to be O-glycosylated, and the mobility
difference attributed to O-glycosylation is about 1 kDa (Pinter
& Honnen, 1988). Our data are therefore consistent with Oglycosylation occurring 30–60 min after synthesis.
As in the case of the Fv4 tail cells, the MoMLV gp70
species was detected after 15 min chase, and was reduced to
about 50 kDa by PNGase (Fig. 3 c). EndoH had a subtle effect
on gp70, increasing its mobility slightly and making it appear
somewhat diffuse. To a first approximation, the kinetics of
glycosylation and processing of Env in the MoMLV cell
lysates was the same as in the Fv4 tail cells.
The results with FrMLV-infected NIH3T3 cells were similar
except that these cells have duplicated species of ‘ gp85 ’,
‘ gp70 ’ and their deglycosylation products (Fig. 3 d, e). These
cells were derived by infecting NIH3T3 cells with a spleen
extract from a FrMLV-infected mouse. Such extracts are
known to contain recombinant mink cell focus viruses which
can have Env proteins with slightly different mobilities
(Famulari & Cieplensky, 1984). The kinetics of glycosylation
and cleavage of the Env species in these cells are grossly the
same as for the Fv4 and MoMLV Env cells.
Secretion of Fv4 gp70 can be explained in part by its
dissociation from the cell surface
To investigate whether Fv4 gp70 was released from the
surface of cells, we performed two types of surface-labelling. In
the first, we reacted cells with NHS-fluorescein, a reagent that
attaches fluorescein groups to free amines on the cell surface
(Vigers et al., 1988). We collected supernatant at various times
after fluoresceination and assayed the supernatant for Fv4 Env
using an ecotropic receptor-binding assay as follows. Super-
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Secretion of murine retroviral Env
(a)
(c)
(b)
Fig. 2. Comparative analysis of p15E transmembrane proteins in Fv4 tail cells, NIH3T3 cells infected by FrMLV and NIH3T3
cells transfected with a MoMLV Env expression vector. Cells were metabolically labelled for 30 min with [35S]methionine and
[35S]cysteine and grown for various times in the presence of cold methionine and cysteine. Cell lysates and supernatants were
precipitated with MAb anti-p15E and analysed by SDS–PAGE under reducing conditions. Chase time in hours is given after the
designations ‘ C ’ (for cell lysate) or ‘ S ’ (for supernatant). (a) Fv4 tail cells. For comparison, polyclonal antibody anti-gp70 was
used with cell lysate directly after pulse (C0) or with supernatant 5 h after chase (S5). Arrow indicates position where p12E
species would be expected if it were present. (b) NIH3T3-FrMLV cells. For comparison, uninfected NIH3T3 cells are shown in
the right hand two lanes, along with a positive control of FrMLV-NIH3T3 cells from the same experiment. (c) NIH3T3-MoMLV
Env cell line.
natants were incubated with NIH3T3 cells (which express the
ecotropic receptor on their surface) or, as negative controls,
with NIH3T3 cells chronically infected with FrMLV or preincubated with Fv4 mouse serum, in which cases the ecotropic
receptor is blocked. We previously showed, using MAb to Fv4
Env, that Fv4 Env binds to plain NIH3T3 cells but not to
NIH3T3 cells chronically infected with FrMLV or preincubated with Fv4 mouse serum (Nihrane et al., 1996). In the
current experiments we used flow cytometry to analyse
NIH3T3 cells that were incubated with supernatants from
NHS-fluorescein labelled Fv4 tail cells. Fig. 4 shows a plot of
the ratio of the mean fluorescence of NIH3T3 cells exposed to
such supernatants divided by the mean fluorescence of NIH3T3
cells not exposed to such supernatants. The amount of
fluoresceinated material in Fv4 tail cell supernatants that could
bind to NIH3T3 cells rose steadily over the 6 h observation
period leading to a peak ratio of about 3 (Fig. 4, upper curve).
Essentially no fluoresceinated material bound to chronically
infected NIH3T3 cells or NIH3T3 cells pre-incubated with Fv4
mouse serum (Fig. 4, lower two curves). Similar results were
obtained in a repeat experiment. As a positive control for this
experiment we performed the same fluorescein surfacelabelling of NIH3T3 cells producing FrMLV. One hour after
labelling, supernatants of these cells gave a ratio of 7 in the
ecotropic receptor binding assay (data not shown), implying
that chronically infected cells secrete about twice as much Env
as the Fv4 tail cells.
To characterize biochemically the Fv4 Env released from
the surface of cells, Fv4 tail cells were metabolically labelled
with [$&S]methionine and [$&S]cysteine, chased for 3 h to allow
$&S-tagged Env to reach the cell surface and then surfacelabelled with NHS-SS-biotin (Cole et al., 1987). At various
times thereafter, cell extracts and supernatants were immunoprecipitated with goat anti-gp70 and then the immunoprecipitates were re-precipitated with streptavidin beads to
isolate the biotinylated portion of envelope molecules. As
shown in Fig. 5, radiolabelled and biotinylated gp70 was
present in cell extracts immediately after biotinylation and 3 h
later, but was mostly gone by 5 h. Conversely, biotinylated
gp70 accumulated in the culture medium during the 5 h
incubation. These results corroborate that gp70 molecules are
shed from the surface of Fv4 tail cells with a half-life of less than
5 h.
Discussion
Our data show clearly that compared to FrMLV and
MoMLV, the Fv4 Env is characterized by weak association
between SU and TM. Thus, while the two molecules are
associated inside cells, SU is secreted from Fv4 cells without
TM, whereas it is not released from MoMLV Env cells and is
released from FrMLV-infected cells in association with TM on
viral particles. Two types of interactions have been proposed
to hold SU and TM together, interchain disulfide bonds and
non-covalent interactions. The literature concerning interchain
disulfide bonds is controversial, probably because the
formation or disruption of these bonds depends on experimental conditions (Pinter et al., 1978 ; Pinter & Honnen,
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A. Nihrane and others
(a)
(c)
(d)
(b)
(e)
Fig. 3. Comparative analysis of synthesis, cleavage and transport of Env glycoproteins in Fv4 tail fibroblasts, NIH3T3-FrMLV
cells and NIH3T3 cells expressing the MoMLV Env. Cells were metabolically labelled for 30 min with [35S]methionine and
[35S]cysteine and grown for various times in the presence of cold methionine and cysteine. Cell lysates and supernatants were
HJA
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Secretion of murine retroviral Env
Ratio of fluorescence intensity
of sample to control cells
3·5
+
3·0
2·5
+
+
+
2·0
1·5+
1·0 E
_
E
_
E
_
E
_
E
_
4
6
0·5
0
0
1
2
Time (hours)
Fig. 5. Pulse-chase analysis of surface-labelled Env glycoproteins. Fv4 tail
cells were metabolically labelled for 30 min with [35S]methionine and
[35S]cysteine and grown for 3 h in the presence of cold methionine and
cysteine. The cells were then surface-labelled with NHS-SS-biotin for
30 min at 4 °C, washed and cultured. At different times after biotinylation,
cell lysates and supernatants were immunoprecipitated with goat antiRauscher gp70 antibody and the immunoprecipitates were purified with
streptavidin beads and analysed by SDS–PAGE under reducing conditions.
1984 ; Gliniak et al., 1991 ; Linder et al., 1992, 1994 ; Thomas &
Roth, 1995). While the sequences of Fv4 and other ecotropic
env genes are about 70 % identical at the amino acid level, all of
the 24 cysteines in SU and TM are conserved (Masuda &
Yoshikura, 1990). Thus, if disulfide bonds were important in
vivo, one might expect Fv4 SU to remain attached to TM and
not be secreted alone. The failure of the anti-p15E MAb to
cross immunoprecipitate gp70 also argues against disulfide
linkage in vivo, although it should be pointed out that a
different anti-p15E MAb was reported to immunoprecipitate a
gp70–p15E complex (Lostrom et al., 1979). The simplest
Fig. 4. Flow cytometry analysis of Fv4
Env binding to NIH3T3 cells. Fv4 tail cells
were labelled for 30 min with NHSfluorescein, washed and at various times
thereafter supernatants from these cells
were incubated with NIH3T3 cells for
40 min at 37 °C. After extensive washing,
the NIH3T3 cells were analysed by flow
cytometry. y-axis : ratio of fluorescence
intensity of treated NIH3T3 cells to
fluorescence intensity of plain NIH3T3
cells ; x-axis : time in hours after
fluorescein labelling. +, NIH3T3 cells
incubated with supernatant from
fluoresceinated Fv4 tail cells. E, NIH3T3
cells treated with serum from Fv4-2 mice
and then incubated with supernatant from
fluoresceinated Fv4 tail cells. _, NIH3T3FrMLV cells incubated with supernatant
from fluoresceinated Fv4 tail cells.
interpretation of the results is that the SU–TM interaction is
non-covalent and weak, especially for Fv4 SU–TM, and that it
is destabilized by the anti-p15E MAb 372.
Site-specific mutagenesis has shown that mutations in the
carboxy-terminal half of SU including the proline-rich ‘ hinge ’
region weaken the association of SU with TM (Lobel & Goff,
1984 ; Gray & Roth, 1993 ; Thomas & Roth, 1995). Substitution
of the carboxy-terminal half of Fv4 for the equivalent segment
of MoMLV led to a 1000-fold drop in virus titre (Masuda &
Yoshikura, 1990), suggesting that Fv4 is defective in this
region. Fv4 has 11 amino acid differences in the carboxyterminal half of SU when compared to sequenced infectious
MuLV clones (Masuda & Yoshikura, 1990). At only three of
these sites are the amino acid changes non-conservative : a
lysine (versus isoleucine, glutamic acid or proline in other
viruses) at position 328, a lysine (versus serine or asparagine)
at position 382, and an arginine (versus glycine or alanine) at
position 393. These amino acid differences are good candidates
for the cause of weaker association of Fv4 SU with TM.
Changes in TM could also contribute to weak association.
TM sequences are highly conserved with only four differences
between Fv4 and other viruses, two of which are conservative
(an arginine for lysine at position 552 and an aspartic acid for
glutamic acid at position 686). The two non-conservative
changes in TM are an arginine for glycine at position 525 and
a glutamic acid for lysine at position 545. The former is in the
highly conserved hydrophobic amino terminus of TM, a
region where non-hydrophobic amino acid substitutions in
other viruses block virus-induced fusion (Freed et al., 1992).
The inability of Fv4 Env to cause fusion in the XC assay and the
loss of infectivity and XC fusogenicity when the Fv4 TM is
substituted for the MoMLV TM (Masuda & Yoshikura, 1990)
precipitated with polyclonal antibody anti-gp70, digested or not with PNGase or EndoH, and analysed by SDS–PAGE under
reducing conditions. (a) Fv4 tail cells : cell extracts and supernatants with and without PNGase treatment. (b) Fv4 tail cells : cell
extracts and supernatants with and without EndoH treatment. (c) NIH3T3-MoMLV Env cell line : cell extracts untreated or
treated with PNGase or EndoH. (d) NIH3T3-FrMLV cells : cell extracts untreated or treated with PNGase or EndoH. (e) NIH3T3FrMLV cells : supernatants untreated or treated with PNGase or EndoH.
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A. Nihrane and others
is probably a consequence of the arginine to glycine change at
position 525 in the TM. The second non-conservative TM
difference between Fv4 and other viruses (position 545) may
contribute to the poor association between Fv4 SU and TM.
Given the lack of tight association between Fv4 SU and
TM, we felt it was important to characterize the kinetics of
synthesis and processing of Fv4 Env to see if it was atypical in
any other way. Our experiments showed no significant
difference between Fv4 Env and the Env of other MuLVs in
terms of glycosylation or cleavage. As in the case of other
MuLVs, the Fv4 Env precursor contains EndoH-sensitive
sugars, which are converted to EndoH-resistant sugars at the
time the precursor is cleaved into SU and TM (Van Zanne et al.,
1976 ; Pinter & Honnen, 1988 ; Hunter & Swanstrom, 1990).
These glycosylation changes and cleavage are detected within
15–30 min of synthesis (Fig. 3).
Our studies with fluorescein and biotin labelling of surface
Fv4 SU imply that a portion of the secreted SU is membranebound prior to secretion. Our studies do not rule out that some
SU is secreted directly and it will be of interest to try to make
the surface-labelling studies more quantitative by determining
the fraction of secreted Fv4 SU that is membrane-associated
prior to secretion. The membrane form of Fv4 SU is presumably
associated with TM, albeit weakly, although some could also
be bound to the ecotropic receptor.
The fact that Fv4 SU dissociates easily from TM could
contribute to the potency of the interference phenomenon in
vivo by blocking viral receptor on the outside of cells. In an in
vitro system, secreted Fv4 Env binds to receptor on NIH3T3
cells (Kitagawa et al., 1995) and at high concentration can block
infection of these cells (Nihrane et al., 1996). Secreted forms of
amino-terminal portions of other SU molecules have also been
shown to block infection in vitro (Heard & Danos, 1991 ; Battini
et al., 1995). Secreted Fv4 Env may protect non-Fv4 cells from
infection in vivo in bone marrow chimeras containing a mixture
of Fv4 and non-Fv4 cells (Kitagawa et al., 1994 ; Limjoco et al. ;
1995). Experiments with HIV show that soluble CD4 can block
infection in vitro but this strategy fails in vivo because the
amount of soluble CD4 necessary to block infection is too high
(Deen et al., 1988 ; Fisher et al., 1988 ; Spouge, 1994).
Quantitative studies of the amount of Fv4 Env necessary to
block MuLV infection are needed to better evaluate the
significance of serum Fv4 Env. Serum Fv4 Env could also play
a role in modulating the immune response to MuLV (Kearny et
al., 1994).
We are very grateful to Icy Davies and Carrie Ward from the animal
facility of the NIAID, and to David Stephany and Carol Henry from the
FACS facility of the NIAID, for their help.
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