Download Expression of Virus Structural Proteins on Murine Cell Surfaces in

J. gen. Virol. (I976), 33, 355-36o
355
Printed in Great Britain
Expression of Virus Structural Proteins on Murine
Cell Surfaces in Association with the Production of
Murine Leukaemia Virus
(Accepted 9 July I976)
SUMMARY
We have used a quantitative radiolabelled antibody procedure to measure the
amount of certain virus structural antigens on the surface of BALB/c R A G cells
producing endogenous B-tropic type C virus. R A G cells expressed group specificities of MuLV p3o on their cell surface but did not express gp7o group specificities. However, type specificities of gp7o were expressed on BALB/c cell lines
infected with Moloney leukaemia virus. The majority of p3o antigens detected on
the R A G cell surface were removed by trypsin and their reappearance was prevented by cycloheximide, even in the presence o f ' conditioned medium' containing
MuLV. Passive adsorption of exogenous MuLV p3o to the surface of virus negative
BALB/c fibroblasts reached a maximum of 2o ~ of the protein detectable on virus
producing R A G cells. These data support the hypothesis that much, but not all, of
the surface p3o is expressed de novo on the cell membrane and not derived from
passive adsorption of p3o released from shed virus or as a by-product of virus
infection of a cell.
Recent work has suggested that certain of the type C virus structural proteins (p3o, gp7o
and plo) can be detected on the external cell surface of MuLV infected cells (Yoshiki,
Mellors & Hardy, 1973; Grant et al. 1974; Yoshiki et al. I974)- The mode of origin of these
virus proteins is unclear to date and could be derived from at least three possible events:
(I) the insertion of these proteins in the membrane as a preliminary event to virus assembly
on the cell membrane; (2) the adsorption of virus proteins to cell surfaces from ruptured
viruses released in the culture milieu; (3) the placement of virus proteins on cell surfaces as
a by-product of virus infection. We have employed a radioisotopic paired label assay for the
study of virus associated surface antigens. We present here a quantitative estimation of p3 o
and gp7o on cell surfaces and evidence that the majority of cell surface p3o appears de novo
in contrast to a smaller amount which is passively adsorbed.
R A G adenocarcinoma cells, a BALB/c derivative, which are resistant to 8-azaguanine
(Klebe, C h e n & Ruddle, I97O), were found to be positive for type C particles by electron
microscopy and by particulate RNA-dependetu D N A polymerase in the culture fluid (IO to
30 pmol deoxymonophosphates incorporated/h/Io 6 cells). We found no evidence of type
A or intracisternal B particles despite the history of the cell being derived from an
adenocarcinoma.
DNA polymerase activity was assayed as described (Wu et al. I972). Particle associated
enzyme activity in R A G culture fluid was linear with time for one hour and with protein
concentration up to a Ioo-fold concentrate of culture fluid. The activity was optimal at
I mm-Mg 2+ (Mn 2+ would not substitute) using rA'dTx2-1s as a template and 3H-thymidine
triphosphate as a substrate. The host range of the R A G virus was determined by passing
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356
Short communications
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Fig. x. (a) Cell titration of R A G cells at a constant concentration of lnq-labelled IgG (20 #g/ml)
from two antisera: 0 ) rat anti-Moloney sarcoma virus (MSV) from a Fischer rat with an MSVderived tumour ( A - - A ) , (2) goat anti-Rauscher leukaemia virus (RLV) p3o ( 0 - - 0 ) . The
sera were provided by D r R. Wilsnack through the Office of Program Resources and Logistics,
Viral Oncology Program, NCI. Monospecific antiserum was prepared by inoculating protein
purified by agarose gel filtration (single band on polyacrylamide gels) into goats. The anti-p3o had
a titre at least 2 logs higher for MuLV p3o than for MuLV gp7o in a competition radioimmunoassay (R. Wilsnack, personal communication). Cell surface antigen was detected by the paired
label assay for cell surface antigens (Boone et aL I97I, I973; O'Brien et al. I976). Immune and
normal IgG from the same species were prepared by ammonium sulphate precipitation and
labelled catalytically with la~I and " I . A mixture of equimolar amounts of the normal and immune
IgG was adsorbed with [o 7 mosquito cells/rag IgG to remove non-specific binding protein. Cells
were incubated with 2o #g immune and normal ]gG in a final volume of ]-o ml for 3o min at
25 °C, washed, and counted in a gamma spectrometer. The amount of 1all minus the 1~5Inormalized
according to the sp. act. of IgG indicated the nanograms of antibody bound on a per cell basis.
O - - O , Virus negative Basc-2 cells with anti-MSV and identical points with goat anti-p3o. (b)
Plot of data from (a) as a function of ng antibody bound/]o 6 cells/ml against ng antibody bound at
increasing cell concentration depicted in (a). Extrapolation of the linear portion of these curves
provides an estimate of the number of antigenic determinants per cell (see text). (c) Blocking of
binding of goat anti-p3o to the R A G cell surface by purified Rauscher P3O. Increasing amounts
of Rauscher MuLV p3o purified by isoelectric focusing were added to ] o/zg of labelled goat antip3o IgG in a paired label mixture in 0"7 ml PBS. After 4 h incubation at 4 °C, o.I ml inactivated
foetal bovine serum and 1.5 x to 7 R A G cells in o.2 ml PBS were added and assayed for bound
anti-p3o as in (a). (d) Cell titration of R A G ( A - - ~ ) , Basc-I ( © - - O ) and LSTRA ( O - - t ) , with
goat anti-RLV gp7o. Increasing cell concentrations were tested for immune IgG binding as in (a).
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357
tissue culture filtrates (0.45/~m) of log phase R A G cells on to test cells for 6 h in the
presence of 2 #g/ml polybrene. After 2 to 4 weeks, the test cells were tested for virus production by assay for particle associated reverse transcriptase in the culture fluid. The host range
of the virus was B-tropic with respect to the Fv-z locus since it infected the murine cell lines
3T3-BALB, SC-I, and Basc-2 (a BALB/c adult fibroblast line), but not 3T3-NIH, VA-2
(human), FCO I2I (cat), SIRC (rabbit) or MS-1 (Chinese hamster). A B-tropic virus has
been previously reported to occur in BALB/c mice tumours and in aged BALB]c mice
(Peters, Spahn & Rabstein, I973; Peters et al. I973).
Titrations of R A G cells at constant immune serum concentration (2o #g/ml) were performed with a broadly reactive rat anti-MSV serum and a monospecific goat anti-Rauscher
MuLV p3o using the paired label assay (Fig. I a; Boone, Irving & Rubenstein, I97I ; Boone,
Gordin & Kawakami, I973 ; O'Brien et al. I976). The R A G cell surface clearly bound large
quantities of both sera relative to normal serum binding and relative to virus negative
Basc-2 cells (adult BALB/c fibroblasts). For a given incubation volume, the amount of
antibody bound per cell is inversely proportional to the total amount of antibody bound in
regions of antigen excess (Fig. I b; Boone et al. I97I, 1973). By extrapolation to the region
of low cell concentration (antibody excess), it is possible to estimate the maximum amount
of IgG which can bind to the cell surface at the indicated antiserum concentration. For the
rat anti-MSV, 39 ng/Io6 cells represents (39 x io -6 x 6.023 x IO23)/(I'5 × 105 × IO9) = I'6 X IOs
bound antibody molecules per cell (Boone et aL I97I). For goat anti-MuLV p3 o, 26 ng/io n
cells represent ~.o× Io 5 bound antibody molecules per cell. The number of antigenic
sites per cell is, therefore, the same as or up to twice these numbers depending upon whether
the IgG binds mono- or bivalently to the cell surface.
Since MuLV p3o is a core protein, we considered that the detection of p3o on the R A G
cell surface could be the result of goat antibodies against small amounts of virus surface
proteins which have contaminated the inoculating p3o antigen. In order to investigate this
question, the ability of purified Rauscher MuLV p3o to interfere with the binding of goat
anti-p3o was examined (Fig. t c). Purified Rauscher p3o was added to a paired label mixture
of goat anti-p3o and normal goat sera at increasing concentrations for 4 h at 4 °C. The
mixture was tested for binding to R A G cells. After an initial rise in binding, probably due
to adsorption of p3 o to the cell surface (see below), increasing amounts of p3 o effectively
block the binding of 60 to 70 % of the measured sites. The failure to block the binding to
the remaining sites may be because the p3o used to raise the antiserum differed in source
and purification from that used to compete for antiserum binding.
Between ~5o and 2oo ng of RLV p3o was required for complete adsorption of Io #g antip3o. From the cell titration (Fig. I b) it is possible to estimate the amount of specific antibodies/mg IgG by dividing the maximum antibody bound to R A G cells by the #g of IgG
in the reaction mixture. Thus the immune serum contains zoo ng binding antibody/2o #g
input IgG. With the requirement of divalent antibody binding for adsorption, the Io #g
IgG used in the adsorption (Fig. I c) would require zoo ng x 2 x 3oooo[I5OOOO = 67 ng
for equivalence of antigen to binding site concentration. Empirically, a threefold excess was
required for complete adsorption.
Since gp7o is a major surface component of the MuLV virion, and therefore a likely
contaminant in the p3o immunogen, we examined the surface of R A G cells with a goat antiRauscher MuLV gp7o (Fig. I d) using a cell titration. The amount of anti-gp7 o bound to
R A G cells and to Basc-I cells (a BALB/c adult skin fibroblast which expresses endogenous
type C virus at high levels) was very low. The possibility that the group-specific region(s) o f
cell membrane bound gp7 o were relatively inaccessible to antibody, while the type-specific
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Fig. z. (a) Passive adsorption of MuLV p3o to cell surface. 1.2 x i@ Basc-z (virus negative adult
BALB/c fibroblasts) were incubated with MuLV-p3o for 2 h at 37 °C. The cells were washed 3
times and tested with goat anti-p3o as in Fig. I (a). i . z x Io 6 R A G cells bound 3I ng/Io n cells
under the same reaction conditions. (b) The effect of cycloheximide and supernatant oncornavirus
on the appearance of p3o on R A G surface following trypsin removal. R A G cells were scraped with a
rubber policeman from near confluent r 5o mm tissue culture dishes (viability = 68 to 73 %), washed
twice and resuspended at 4 x io 6 cells in z ml phosphate buffered saline containing o'625 mg/ml
trypsin for 2o min. The reaction was terminated by the addition of ovamucoid trypsin inhibitor
(2"5 mg). The cell clumps caused by trypsin treatment were dispersed by addition of I oo/zg DNase
for 5 rain following the trypsin treatment. Cells were washed twice, and dispensed into 4 ml
nutrient medium (RPMI-t64o+ so ~ foetal bovine serum ( Q - - O , A - - A ) , or in 'conditioned'
medium ( © - - © , A - -A), which was a z4 h supernatant fluid of a log phase R A G cell culture
(contained I5 to 20 pmol reverse transcriptase activity units/ml culture fluid). The triangles represent cultures supplemented with IOO #g/ml cycloheximide. Circles are controls with no antibiotics.
At the indicated period the cells were washed once and assayed for p3o on their surface by the
paired label assay. Square (D) indicates R A G cells not treated with trypsin. Cycloheximide
treatment failed to affect antibody binding to R A G cells in control experiments.
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359
determinants were accessible, has been suggested (Obata et al. 1975; Tung et al. 1975). Since
we would not expect to detect type specificities of BALB/c endogenous viruses with goat
anti-Rauscher (of the F M R group) gp7o, we examined a BALB/c lymphoma line, LSTRA,
which was infected with Moloney leukaemia virus for surface gP7o (Fig. I d). Any 'type'specific surface components of gp7o shared by Rauscher and Moloney virus would be
detected on the LSTRA cell surface. A considerable amount of antibody of the goat antiRLV gp7o bound to the surface of LSTRA (o'24 × to 5 molecules/cell as a maximum) and
to other cell lines infected with Moloney leukaemia virus (S. J. O'Brien, unpublished data).
These results are consistent with the proposition that some gp7 o 'type' specificities are
accessible to antibody on cells making the gtycoprotein, but intraspecies group specificifies
are not. Nevertheless, whether gp7 o type specificities are expressed on R A G cell surfaces or
not, the antigen detected by the goat anti-RLV p3o is clearly not gp7o because monospecific
anti-gp7 o prepared from the same virus strain (Rauscher) as the anti-p3o has a negligible
binding activity.
We have mentioned that the mechanism by which p3o arises at the cell surface might be
through passive adsorption of the protein from viruses in the culture fluid. Virus negative
Basc-2 cells were incubated with increasing amounts of purified exogenous MuLV p3o for
2 h and tested for surface-associated p3o (Fig. 2a). MuLV p3o increasingly binds to the
surface of the virus negative cells resulting in specific binding of anti-RLV p3o to the cell
surface in agreement with the report of Grant et al. (1974). The amount of p3o bound, however, reaches a plateau corresponding to 4 to 5 ng anti-p3o binding lO6 cells. This value is
less than 2o ~ of the amount of p3o detected on the R A G cell surface.
A second experimental approach to this question involved monitoring p3o reappearance
on the cell surface following its removal with trypsin (Fig. 2b). Cells treated with sufficient
trypsin to remove p3o were incubated in nutrient medium for up to 5 h in the presence of
cycloheximide. Cells were similarly treated with trypsin and incubated in 'conditioned'
medium which was taken from a log phase R A G cell culture after 27 h of virus production.
If the p3o on the surface were derived from adsorption of supernatant p3o, or as a byproduct of virus infection, then cycloheximide would not be expected to block the posttrypsin appearance of p3o on the cell surface. However, if the p3o is synthesized by the cell
and inserted on the cell's membrane, then cessation of protein synthesis by cycloheximide
would block the reappearance of the antigen. Fig. z (b) demonstrates that antigen reappearance was effectively blocked by cycloheximide both in fresh or 'conditioned' (virus containing) medium. The reappearance of p3o on the R A G cell surface is complete in approx.
4h.
Our results are compatible with the hypothesis that a considerable amount of MuLV-p3o
is found on the cell surface, and that although some of it can be explained by passive absorption of culture fluid p3o, the majority of it cannot. The simplest interpretation is that it is
placed on the surface rapidly from within, after it is synthesized. An alternative, but less
likely possibility, is that trypsin treatment also removes a receptor for exogenous p3o
adsorption, the synthesis of which is also blocked by cycloheximide. This explanation,
however, fails to account for the low binding of exogenous p3o to virus negative Basc-2 cells
(Fig. 2 a). Furthermore, the re-growth of a receptor might be detected by differential kinetics
of reappearance of cell surface associated MuLV-p3o in the presence and absence of
exogenous virus. The results in Fig. 2 (b) failed to reveal any differences in antigen growth
which were affected by exogenous virus.
The expression of cell surface p3o on m3SR and BALB/3T3 cells which produce no
detectable MuLV (Grant et al. 1974) further suggests that p3o may arise on the cell surface
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i n a p r o c e s s o t h e r t h a n v i r u s i n f e c t i o n o r p a s s i v e a d s o r p t i o n . T h e r e l a t i o n s h i p o f cell s u r f a c e
p 3 o a n d g p 7 o t o v i r u s a s s e m b l y a n d t o i m m u n e r e c o g n i t i o n o f cells e x p r e s s i n g t h e s e a n t i g e n s
is a t p r e s e n t u n c l e a r a n d r e q u i r e s f u r t h e r e x a m i n a t i o n .
Cell Biology Section
Laboratory o f Viral Carcinogenesis
National Cancer Institute
National Institutes o f Health
Bethesda, Maryland, U.S.A.
O'BRIEN
JANICE M . SIMONSON
C. W . BOONE
S . J.
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(Received 3I March I 9 7 6 )
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