2147
J. gen. Virol. (1983), 64, 2147 2156. Printed in Great Britain
Key words: La Crosse virus/glycoproteins/proteolytic cleavage/antibody neutralization
The Effect of Proteolytic Cleavage of La Crosse Virus G1 Glycoprotein on
Antibody Neutralization
By L A U R A K I N G S F O R D *
AND D O U G L A S
W. H I L L 1
Department of Microbiology, California State University, Long Beach, California 90840, U.S.A.
and 1Department of Cellular, Viral, and Molecular Biology, University of Utah, Salt Lake City,
Utah 84132, U.S.A.
(Accepted 3 June 1983)
SUMMARY
The envelope of the bunyavirus La Crosse contains two glycoproteins, G1 (120000
mol. wt.) and G2 (38000 mol. wt.). When incubated with trypsin or plasmin, the G1
glycoprotein of virus grown in cell culture was cleaved, leaving two different sized
polypeptides in the envelope (67 000 and 95 000 mol. wt.). Chymotrypsin cleaved G 1
leaving polypeptides of 70000 and 100000 mol. wt. G2, however, was not altered by
these enzymes. When used in antibody neutralization studies, these proteolytically
modified viruses were neutralized approximately 1 to 2 log10 units in 60 min while
control virus was neutralized by over 4 log10 units in 20 min. Because antibody to G1,
but not G2, was involved in La Crosse virus neutralization, cleavage of G 1 appeared to
be directly responsible for these altered kinetics of neutralization. Antibody did bind to
the polypeptides remaining associated with the envelope resulting in infectious virusantibody complexes. This indicated that a critical site in terms of antibody
neutralization was removed from G I by proteolytic enzymes.
INTRODUCTION
La Crosse (LAC) virus is a member of the family Bunyaviridae and related to the California
serogroup of viruses within the family (Porterfield et al., 1975/76). The negative-strand genome
of these viruses is composed of three segments of single-stranded R N A (Obijeski et al., 1976;
Ctewley et al., ~977). It has been shown that the medium-sized R N A codes for the two viral
glycoproteins, G 1 and G2 (Gentsch et al., 1980), which become an integral part of the virion
envelope (Obijeski et al., 1976). Apart from this, and the known mol. wt. of 120000 (120K) for
G1 and 34K for G2 (Obijeski et al., 1976), little is known about the structure and function of the
two glycoproteins. Gentsch et al. (1980) have shown that antibody raised against the medium
R N A gene products (G1 and G2) does have neutralizing activity when incubated with LAC
virions. This is consistent with studies of other viruses that indicate that the viral glycoproteins
interact with host cells in the initial steps of virus replication (Fries & Helenius, 1979) and that
when reacted with antibody made specifically to these surface glycoproteins, the virus is
neutralized (Kelley et al., 1972; Dalrymple et al., 1976). The study with antibody to the medium
R N A gene products of LAC, however, does not indicate which of the gene products, G1 or G2
or both, are involved in antibody neutralization. Thus, we report here experiments designed to
learn more about the structure of LAC glycoproteins and which ones are involved in attachment
to host cells and in antibody neutralization. Our results indicate that G 1 is needed for viral
infectivity and only antibody made to G1 results in neutralization of LAC virions. In addition,
sites critical for antibody neutralization on G1 can be removed by certain proteolytic enzymes.
Antibody molecules can bind to the part of G 1 left intact after proteolysis, resulting in a high
proportion of infectious virus-antibody complexes.
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2148
L. KINGSFORD
AND
D. W. HILL
METHODS
Radiochemicals. L-[35S]Methionine (420 Ci/mmol), D-[6-3H(N)]glucosamine, L-[6-3H]fucose and 14C-amino
acids were purchased from New England Nuclear. [3H]Acetic anhydride (2-5 Ci/mmol) was purchased from
Amersham/Searle.
Virus and cells. LAC virus was obtained from Dr L. A. Thomas, Rocky Mountain Laboratory, Hamilton, Mt.,
U.S.A., as a mouse brain suspension at passage level eight. Upon receipt, the virus was passaged in suckling mice
and plaque-purified three times. A pool of virus was made by one more mouse brain passage and used as seed stock
for all subsequent passages into continuous cell lines.
For growth of LAC in cell culture, baby hamster kidney (BHK) cells were infected by the addition of mouse
brain-derived virus at an input multiplicity of 1 to 5 p.f.u./cell. After incubation for 24 to 30 h at 37 °C, the virus
suspensions were clarified by centrifugation at 12100 g for 30 min, and stored at - 70 °C. Second passage virus
added to the appropriate cells at an input multiplicity of 1 was used in all studies reported.
To radiolabel virus, [3H]glucosamine (7 ~tCi/ml) or [3H]fucose (7 ItCi/ml) and 1*C-amino acids (1 ~tCi/ml) were
incorporated into the media added at the end of the 1 h adsorption period. [35S]Methionine-labelled virus was
prepared as follows. After adsorption of virus to cells for 1 h at room temperature, 10 ml of medium containing, by
vol., 4 8 ~ Eagle's minimum essential medium (MEM), 5 0 ~ phosphate-buffered saline (PBS), 1~o calf serum and
1 ~ Tryptose (Difco) was added to each 150 cm 2 flask of BHK cells. After incubation for 3 h at 37 °C, this medium
was removed and replaced with 10 ml of methionine-free MEM with 1X calf serum and 8 to 10 ~tCi/ml
[35S]methionine. Virus was harvested at 30 h.
BHK cells were grown in 150 cm 2 plastic tissue culture flasks for virus propagation or in 60 × 15 m m dishes for
plaque assays. All cells were grown in Eagle's M E M with Hanks' salts supplemented with 1 0 ~ calf serum, 0-2~
Tryptose and 0.35 g/1 of N a H C O : . The medium used for overlay in the plaque assay consisted of M E M with 5
calf serum, 0-2~ Tryptose, 0.02 i - T r i s base and 0 . 4 ~ agarose. The medium was adjusted to p H 7-3 with HC1.
Virus purification. Clarified virus (10 ml) was sedimented (2 h at 20000 rev/min at 4 °C in a SW27 rotor) through
2 0 ~ (w/v) sucrose (in 1.0 mM-Tris, 0.1 mM-EDTA and 10~o by vol. of dimethyl sulphoxide) onto a 6 5 ~ sucrose
cushion. Alternatively, virus was centrifuged through a 10 to 3 0 ~ linear sucrose velocity gradient for 90 min at
20 000 rev/min in a SW27 rotor. Fractions from the sucrose-sucrose interphase or peak fractions from the velocity
gradient were pooled and frozen at -70 °C or immediately diluted twofold with buffer and layered onto a 20 to 65
linear sucrose gradient. Following centrifugation for 5 h under the above conditions, 1 ml fractions were collected
and an aliquot assayed for infectivity. Virus was stored frozen at - 70 °C until needed.
Assay ofvirat infectivity. Monolayers of B H K cells were washed once with Dulbecco's PBS and an inoculum of
0-2 ml added to each plate. Virus was allowed to adsorb to cells for 1 h at room temperature before the addition of 5
ml of molten overlay medium. Plates were incubated at 37 °C for 45 to 48 h when 2.0 ml of neutral red solution
(0.02~ in 0-85~ NaC1) was added. Following incubation at 37 °C for 2 h, the excess neutral red solution was
removed. Plates were kept in the dark at room temperature for 10 to 15 h before plaques were counted.
Virus neutralization. The virus neutralization procedure utilized was essentially that described by Habel (1969).
The appropriate virus inoculum was diluted in PBS plus 0-1 ~ bovine serum albumin (PBS-BSA) to contain 5 x
106 p.f.u./ml. All antisera were heated at 56 °C for 30 min and diluted in PBS-BSA. Aliquots of serum and virus
were equilibrated at 37 °C for 5 min and equal volumes mixed at the appropriate times. Zero-time and 60 min
controls for heat inactivation were taken from a mixture of virus and normal rabbit serum. (Under these
conditions, less than 10 ~ of the virus was inactivated by heat.) Samples of 0-1 ml were taken at appropriate times
and diluted immediately into 9.9 ml of cold PBS BSA. The diluted samples were kept in an ice-bath until all had
been taken, further diluted as necessary, and assayed.
Antisera to the virus were obtained from young adult rabbits immunized with purified virus which had been
grown in BHK cells or in suckling mouse brain. Animals were bled to obtain 'normal' serum samples, injected with
2 ml of purified virus (109 p.f.u, from a sucrose cushion) intramuscularly at days 0, 8, 43 and 84 and bled for serum
by cardiac puncture at days 52 and 95. The 'hyperimmune' sera obtained at day 95 were used for the experiments
described in this paper.
To obtain antibody specific for LAC proteins (G 1, G2 and N), purified BHK-derived virus was electrophoresed
in a 10 ~ SDS-polyacrylamide gel. The G 1, G2 and N protein bands were 3 to 5 cm apart, so that each one could be
cut without any possible cross-contamination. A vertical strip was cut from each gel and stained to determine the
location of the virus proteins. The rest of the gel was kept at 4 °C overnight. By staining a strip of a gel which had
been refrigerated overnight, it was determined that the proteins did not diffuse. With allowance for swelling of the
stained gel strip, the position of each protein in the unstained gel was determined by relative migration. A 3 to 4
mm horizontal strip containing each protein was cut out and the gel ground with a mortar and pestle and mixed
with PBS. Young female rabbits received weekly intramuscular injections of one of the LAC proteins. Each
injection contained purified G1, G2 or N from approximately 2 x 1010 p.f.u, of virus. Rabbits were bled after six
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Proteolytic cleavage o f L A C glycoprotein
2149
weekly injections and sera collected and frozen at - 2 0 °C. Antibodies to LAC and G1 were also obtained in
ascitic fluids in mice using the procedure of Qureshi & Trent (1973). Each mouse received 104 p.f.u, on day 1 and
l06 p.f.u, on days 14 and 35, or G1 cut from gels (see above) on days 1, 14, 25 and 35. Injections were given
intraperitoneally except those on day 35 which were given by the intravenous (LAC) or subcutaneous (G1) routes.
Sarcoma 180 ceils were injected intraperitoneally on day 28. Ascites fluids collected on days 40 to 45 were
centrifuged to remove cells and clots and stored frozen at - 2 0 °C.
Polyacrylamide gel electrophoresis. Proteins were electrophoresed in 1-5 mm slab gels consisting of a 10%
acrylamide resolving gel and a 3-5% acrylamide stacking gel (O'Farrell et aL, 1973). The SDS gel and buffer
solutions used are those described by Laemmli (1970). Electrophoresis was carried out at 30 V until the
bromophenol blue dye reached the resolving gel, then at 30 mA until the dye reached the bottom of the gel. The gel
was stained in 0.2% Coomassie Brilliant Blue in 50% methanol and 7% acetic acid for 2 to 4 h and then destained
with repeated washings of a solution containing 7 % acetic acid and 5 % methanol. Gels were dried by vacuum and
heat in an apparatus designed by Maizel (1971) and autoradiographed on Kodak RP Royal O-Mat medical X-ray
film. Fluorography (Bonner & Laskey, 1974) was used to detect the radioactivity of gels containing proteins with
low amounts of 3sS label.
LAC proteins were also electrophoresed in cylindrical phosphate~rea gels and analysed for radioactive content
as previously described (Kingsford et al., 1980).
Proteolytic cleavage of LAC virus glycoproteins. A stock solution of trypsin (Sigma, type III) containing 400 ~tg/ml
was made in Tris-buffered saline (TBS) with CaC12 (0.1 M-NaC1, 0.01 M-Tris-HC1 pH 7.5, 0.01 M-CaClz) and
stored at - 70 °C. The enzymic activity of this trypsin was equal to 0.68 units/gg of protein. The trypsin was
diluted to 20 gg/ml in TBS-CaC12 and mixed with an equal volume of virus also diluted in TBS-CaC12 . The virustrypsin mixture was incubated for 90 min at 35 °C. Calf serum was added to the virus-trypsin mixture to a final
concentration of 10 %. The addition of heated calf serum stopped the trypsin activity as well or better than soybean
trypsin inhibitor. Control virus was treated the same way with the exception that TBS-CaC12 was used instead of
the trypsin solution.
Although data are not presented in this paper, trypsin preparations from several sources were used with the
same results: trypsin, types III and XII (Sigma), acetylated trypsin type V (Sigma), trypsin 1:250 and 1:300
(Nutritional Biochemicals), diphenylcarbamyl chloride-treated trypsin (Sigma), and TPCK-trypsin
(Worthington).
Using the same protocol as for trypsin, incubations of LAC virus were also performed with equal volumes of
each of the following enzymes: 20 ~tg/ml alpha-chymotrypsin (52.9 units/mg, Worthington), 10 ~tg/ml bromelain
(420 units/g solid with 50% activity, Sigma), 10 ~tg/ml thermolysin (Calbiochem-Behring), 10 ~tg/ml Pronase (B
grade, Calbiochem-Behring), 50 ~tg/ml plasminogen, 500 units/ml streptokinase, 500 units/ml plasminogen
activator, or plasmin which was prepared by mixing equal volumes of plasminogen (0.1 mg/ml) with streptokinase
(1000 units/ml in 0.01% gelatin in PBS minus Ca 2÷ and Mg 2÷ ions). Plasminogen, plasmin and streptokinase were
kindly supplied by Dr R. W. Snyder, University of Utah. Chymotrypsin was diluted in TBS-CaCI2 ; the other
enzymes were diluted in TNE buffer (0.1 M-NaC1, 0.05 M-Tris-HC1 pH 7-5, 0-5 mM-EDTA).
Inactivation of trypsin with enzyme inhibitor. One-tenth ml of 0.1 M-phenylmethylsulphonylfluoride (PMSF) in dry
isopropanol was mixed with 2 ml of 0.1 M-trypsin (type XII, Sigma). This mixture was kept in an ice-bath for 3 h
then desalted on a Sephadex G-25 column equilibrated with TBS-CaC12 at pH 7.7. The peak fractions were
determined by absorbance at 280 nm, pooled and frozen in aliquots at - 70 °C. PMSF-inactivated trypsin had a
residual enzymic activity of 7.5 × 10-5 units/ml as compared with stock trypsin with 4-05 units/ml used at a final
concentration of 10 ~tg/ml. Enzyme activity was measured by the spectrophotometric method of Hummel (1959).
Enzyme-immunosorbent assay (EIA). Purified LAC virus or trypsinized LAC virus was diluted in 50 mMcarbonate-bicarbonate buffer pH 9.5, to give the equivalent of 109 p.f.u./ml. One-tenth ml of this antigen was
added to each well of a 96-well microtitre plate (Dynatech, Immulon I) and incubated at 4 °C overnight. To block
non-specific adsorption, antigen was removed and 0.15 ml of 2% ovalbumin (Sigma) in the bicarbonate buffer
added to each well and incubated 30 min at room temperature. After the coated wells were washed with PBS with
0.05% Tween-20 (PBS-T), 0-1 ml of diluted antibody was added to each well and incubated for 2 h at 37 °C. After
removal of unbound antibody by washing with PBS-T, bound antibody was detected by peroxidase-conjugated
antiserum in PBS-T (2 h incubation at 37 °C) and a substrate-indicator consisting of 0-03~o H202 and 0.03%
tetramethylbenzidine in 0.1 M-citrate buffer pH 4.5. The assay was read by eye 30 min after addition of substrate.
Controls with normal mouse serum and known mouse anti-LAC incubated on LAC- or BSA-coated wells were used
to determine the appearance of positive and negative wells.
Haemagglutination inhibition assay. The procedure for the haemagglutination inhibition assay was essentially
that of Clarke & Casals (1958) except that formalin-treated erythrocytes (Ito & Iwasa, 1980) from day-old male
chicks were used in microtitre U-well plates (Dynatech, Immulon I). Antigen was sucrose cushion-purified virus
with an approximate titre of 101° p.f.u./ml which was diluted to give the appropriate haemagglutinating units.
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2150
L.
KINGSFORD
(a)
AND
O.
(b)
II
W.
HILL
(c)
II
--L
mG1
--
100K
--95K
70K
M67K
--
--G2
--N
Fig. 1. Polyacrylamide gel electrophoresis of LAC virions after treatment with trypsin or
chymotrypsin. Autoradiograph of a gel containing (a) structural proteins of LAC virions or cleavage
products of virions incubated for 90 min at 35 °C with (b) 10 gg/ml trypsin or (c) 20 ~tg/mlchymotrypsin
is shown.
RESULTS
Effect o f trypsin and chymotrypsin on L A C virus
Trypsin and chymotrypsin were initially chosen to determine their effects on the infectivity
and structure of L A C virus. Purified L A C virions were incubated with 10 ~g/ml trypsin or 20 ~tg/
ml chymotrypsin at 35 °C for 90 min. This protease treatment resulted in a consistent 10-fold loss
of infectivity.
To determine what effect trypsin might have on the envelope glycoproteins G1 and G2,
purified [35S]methionine-labelled L A C virus was incubated with trypsin or chymotrypsin,
immediately boiled for 2 rain in gel sample buffer and electrophoresed in 10% S D S polyacrylamide gels along with untreated virus. As seen in Fig. 1, L A C virus has four structural
proteins: L (170K), G1 (120K), G2 (38K) and N (23K). These mol. wt. values are in good
agreement with those assigned to proteins of bunyaviruses by Obijeski et al. (1976). With trypsin
treatment, the majority of the G 1 glycoprotein had disappeared and two major polypeptides of
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Proteolytic cleavage of LA C glycoprotein
2151
95K and 67K were apparent (Fig. 1). Small peptides below N and at the dye front were also
observed. When trypsinized LAC was pelleted or centrifuged through a gradient before
electrophoresis, the only G1 cleavage products remaining with the virions were the 67K and
95K polypeptides. Cleavage products of chymotrypsin-treated virus were similar to those seen
for trypsin, except that both of the peptides were larger: 100K and 70K. The other structural
proteins, L, G2 and N, were not affected by either enzyme. It thus appeared that trypsin and
chymotrypsin cleaved only the G1 glycoprotein in situ and this cleavage resulted in two smaller
major components.
Distribution of oligosaccharides on G1
It has been reported that both the G 1 and G2 glycoproteins of LAC are of the type 'A' serum
glycoproteins containing glucosamine, galactose, fucose, mannose and neuraminic acid
(Vorndam & Trent, 1979). As part of G 1 is proteolytically removed by trypsin, it was of interest
to determine whether all of the sugar moieties would be found on the trypsin-sensitive part or
distributed along the G 1 glycoprotein. Thus, control and trypsinized [3H]glucosamine- and 14Camino acid-labelled LAC were electrophoresed in phosphate-urea gels. As shown in Fig. 2 (a),
the 3H label migrated with the G1 and G2 glycoproteins as expected. When LAC was
trypsinized, only 55 to 60% of the label originally migrating with G1 remained, but was now
found migrating where the 67K and 95K polypeptides would be expected (Fig. 2 b). The amount
of 3H label migrating with G2 was not altered by trypsinization. When this experiment was
repeated using [3H]fucose instead of [3H]glucosamine, the relative amounts of label in G1, G2
and the G1 cleavage products were the same as shown for the [3H]glucosamine-labelled virus.
Proteolytic cleavage of LAC glycoproteins by other proteases
Because trypsin and chymotrypsin produced such unique cleavage products of the G1
glycoprotein, it was of interest to determine what effect other proteolytic enzymes might have on
the glycoproteins. Thus, 35S-labelled LAC was incubated with various proteases and
electrophoresed in a 10% SDS-polyacrylamide gel. Each virus sample was also assayed for
residual infectivity. As seen in Fig. 3, three patterns of proteolytic action on the G 1 glycoprotein
were observed, i.e. complete cleavage, no cleavage or partial cleavage. Bromelain and Pronase
completely digested G1 and reduced the infectivity of 108 p.f.u, by over 6 log10 units.
Thermolysin had no visible proteolytic effect on the glycoproteins or on infectivity. Although
not shown, plasminogen, plasminogen activator and streptokinase, like thermolysin, had no
effect. Yet these enzymes were active as determined by appropriate assays. Plasmin, trypsin and
chymotrypsin only partially cleaved the G1 glycoprotein, resulting in relatively little loss in
infectivity (approx. 1 log10). Like trypsin, plasmin cleaved G1 to give two polypeptides of 95K
and 67K. As shown before, chymotrypsin cleaved G1 resulting in polypeptides of 100K and
70K. Because these protease-treated samples contained all of the degradation products and not
just those remaining with the virions, additional peptides are present in the gel.
The G2 glycoprotein was resistant to degradation by all the proteolytie enzymes tested except
bromelain. It should be noted that Pronase completely cleaved the G1 molecule, but left G2
intact (see Fig. 3). Yet this virus was non-infectious. This implicates G1 in attachment of LAC
virions to host cell receptors and/or penetration into the cell. This also indicates that G2 alone is
not capable of this function.
Neutralization of LAC virions
The neutralizing capacity of antibody made to infectious LAC or to G 1, G2 and N proteins
was demonstrated in an assay of the kinetics of neutralization. As seen in Fig. 4, anti-LAC and
anti-G1 gave similar degrees and rates of neutralization while antibody to G2 or N did not
neutralize the virus. The presence of specific antibody molecules in all four antisera was
demonstrated in an immunofluorescence assay using whole BHK cells infected with LAC virus.
The titres of anti-LAC, anti-G1, anti-G2 and anti-N sera were 1:64, 1:16, 1:16 and 1:16,
respectively. Titres of normal sera obtained by pre-bleeding the same rabbits were all less than
1:4. In addition, both anti-G1 and anti-G2 sera had haemagglutination inhibition titres of 1:40
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2152
L. KINGSFORD AND D. W. HILL
I
I
(a)
I
~ '!
16
I
I
I
I
I
I
G1
I
8
N
12
6
8
~
4
,
G2
,
g~
, ',.
x
e-
,
,
~/ %%
t
I
I
I
1
I
I
4
-
2;
x
r~
(b)
g {6
._~
m
m
12
Y
~
(
95K
67K
I
L
G1 ¢ ~
20
i
G2
t
q-.
/~/~0~
40
80
100
60
Gel slice (mm)
Fig. 2. Polyacrylamide gel electrophoresis of [3H]glucosamine- and '4C-amino acid-labelled virus.
LAC virus was grown in BHK cells with 7 pCi/ml [3H]glucosamine (©) and 1 ~tCi/m114C-amino acids
( 0 ) and then purified. Untreated virus (a) or virus incubated with trypsin (b) as described in the legend
of Fig. l was electrophoresed in a 10~ phosphate-urea cylindrical gel. Each gel was cut into 1 mm
fractions and analysed for radioactivity by liquid scintillation counting.
while anti-N serum was negative. In the neutralization assay shown in Fig. 4, the anti-LAC
serum was diluted 1:200 and the other three antisera diluted 1:10. G2 antiserum did not
neutralize the virus even when used undiluted. Anti-LAC and anti-G1 antibodies obtained in
mouse ascitic fluid gave the same results as the rabbit antisera. These data indicate that only the
G1 glycoprotein contains sites which react with antibody to result in neutralization.
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2153
Proteolytic cleavage of LAC glycoprotein
(a)
(b)
(c)
(d)
(e)
(f)
(g)
L m
G1--
G2 m
N
m
Fig. 3. Polyacrylamide gel electrophoresis of LAC virions after incubation with various proteolytic
enzymes. The autoradiograph is of a 10~oSDS gel containing (a) structural proteins of LAC virus or
cleavage products of virions incubated at 37 °C for 60 min with (b) 10 ~tg/ml trypsin, (c) 20 ~tg/ml
chymotrypsin, (d) 10 ~tg/ml bromelain, (e) 10 ~tg/ml thermolysin, (f) 10 ktg/ml Pronase or (g) 5 ~tg/ml
plasmin.
Effect of trypsin and chymotrypsin treatment on neutralization kinetics of LAC virus
After 5 x 107 p.f.u, of L A C had been incubated with 10 ~tg/ml (4.05 units/ml) trypsin or 20 ~tg/
ml chymotrypsin for 90 min at 35 °C, calf serum was added to stop the enzymic activity. The titre
of each virus preparation was consistently 1 loglo lower at this point. Enzyme-treated virus or
5 x 106 p.f.u, of untreated L A C were equilibrated at 37 °C for 5 rain and anti-LAC antibody
(rabbit or mouse) immediately added for a neutralization assay. Results shown in Fig. 5 indicate
that proteolytic treatment of virus resulted in virions that reacted very differently with antibody.
The rate of neutralization was greatly decreased, resulting in a much larger persistent fraction as
compared with untreated virus. Increasing the concentration of antibody added to virus which
had been pre-incubated with trypsin or chymotrypsin did not increase the amount of neutralized
virus.
Controls showed that calf serum effectively inhibited the effect of trypsin on the virus, as no
further reduction in viral infectivity occurred over a 2 h period. In addition, antibody preincubated with 10 ~tg/ml of trypsin for 60 min at 37 °C retained its full activity as demonstrated
by its ability to neutralize over 4 log10 units of L A C virus in 20 min. This indicated that any
residual trypsin would not be deleterious for the antibody molecules during the kinetics of
neutralization assay.
To ensure that the trypsin degradation products freed from G I or trypsin itself did not
interfere with the antibody neutralization reaction, L A C virus was incubated as usual with
[3H]acetylated trypsin that had been labelled with [3H]acetic anhydride by the method of
Montelaro & Rueckert (1975). The virus-enzyme mixture was then layered onto a 20 to 6 5 ~
sucrose gradient and centrifuged to equilibrium. All of the 3H-labelled trypsin remained at the
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2154
L. KINGSFORD AND D. W. HILL
100 ~ ~ z : z ~
1
11
0
0
~
I
I0
-
a
0
0.01
.
1
~
0.1
0.01
30
60
0 3 6 912
30
60
Time (rain)
Time (min)
Fig. 4
Fig. 5
Fig. 4. Neutralization kinetics of LAC virus. Aliquots of purified LAC virions cor~taining 5 x 106
p.f.u, were mixed with equal volumes of diluted antisera containing antibodies to LAC virus (0) or to
polyacrylamide gel-purified G1 (O), G2 (A) or N (z~x)proteins. Samples were taken and assayed as
described in Methods.
0 36912
Fig. 5. Neutralization kinetics of virus preincubated with trypsin, PMSF-inactivated trypsin or
chymotrypsin. Virus was preincubated with a final concentration of 10 Bg/ml PMSF-trypsin (equal in
activity to 7.5 × 10-s units/ml) (L~), 10 ~tg/mltrypsin (4-05 units/ml) ~k) or 20 Ixg/mlchymotrypsin (O)
for 90 min at 35 °C. After addition of 10~ calf serum, antiserum containing anti-LAC antibody was
added for studies of the kinetics of neutralization. Untreated LAC (0) was used in the neutralization
reaction as a control.
top of the gradient while the virus banded at its density. Electrophoretic analysis of this virus
indicated that G1 had been cleaved in situ to give 67K and 95K polypeptides that remained
associated with the virions. Small peptides or free amino acids resulting from the trypsinization
were not present in the virus preparation. When this virus was used in an antibody
neutralization assay, the degree and rate of neutralization were identical to those of the
trypsinized virus shown in Fig. 5.
Trypsin inactivated with PMSF was used as a control to determine whether the cleavage of
G1 was directly related to the altered neutralization. An equivalent of 10 ~tg/ml PMSF-trypsin
contained 7.5 x 10-s units/ml of activity. Virus was first reacted with this inactivated enzyme
preparation, then pelleted and electrophoresed in polyacrylamide gels to determine whether the
G1 glycoprotein was cleaved. This preparation of PMSF-trypsin left most of the G 1 intact, but
some cleavage products could be seen.
Because the PMSF-trypsin-treated virus did have some intact G1 glycoprotein left, as
observed in gels, treatment of virus with this 'inactivated' enzyme should give greater levels of
neutralization than trypsin-treated virus. As seen in Fig. 5, neutralization of PMSF-trypsintreated virus gave neutralization kinetics more like the control virus.
These experiments indicate that trypsin does not interfere with neutralization by binding to
virus or by hydrolysing antibody molecules. They also suggest that the degree of neutralization of
LAC virus depends on the amount of intact G1 present in the envelope.
Binding of antioG1 antibody to tryps&ized virus
To determine whether antibody binding sites were still present on the trypsin-resistant part of
G1, mouse anti-LAC and anti-Gl were titrated by EIA using LAC and trypsinized LAC as
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Proteolytic cleavage of L A C glycoprotein
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antigens. Anti-G 1 and anti-LAC antibody did bind to trypsinized virus. In fact, the titre of each
antiserum was 1:800 on both untreated and trypsin-treated LAC antigen.
DISCUSSION
Incubation of LAC virions with trypsin, plasmin or chymotrypsin resulted in the loss of
approximately 1 log10 unit of infectivity and the cleavage of the G1 glycoprotein to two
polypeptides. This unique cleavage of G1 but not of G2, together with the high degree of
infectivity remaining, allowed us to learn certain things about the structure and biological
properties of these two glycoproteins. The infectivity of LAC virus seems to be dependent upon
the presence of the GI glycoprotein. When it was removed by Pronase, leaving only G2 intact,
the virions were non-infectious. Thus, G1 is probably the glycoprotein needed for attachment to
host cell receptor sites and/or for penetration and uncoating.
Antibody raised against the G2 glycoprotein did exhibit haemagglutination inhibition
activity. This is probably due, however, to a steric blocking of haemagglutinin sites on G1 rather
than the presence of receptor sites for erythrocytes on G2. A similar situation has been described
for influenza virus by Webster et al. (1982), who proposed that the anti-haemagglutination
activity of antibody to the neuraminidase glycoprotein was due to a steric blocking of sites on
the haemagglutinin glycoprotein.
The neutralization data together with the infectivity of trypsinized virus indicate that there
are multiple recognition sites for host cell receptors and multiple antibody-binding sites on G 1.
When 25K to 53K mol. wt. of protein are removed, a significant amount of infectivity remains,
indicating the presence of cell receptor sites. Anti-G 1 antibody binds to the remaining 67K and
95K polypeptides but does not neutralize the virus. In addition, as anti-G2 and anti-N
antibodies do not neutralize the virus, the critical sites for antibody neutralization as well as
some host cell receptor sites must be on the part(s) of G1 removed by trypsin. The presence of
multiple antibody-binding sites as well as non-neutralizing antibody-binding sites has been
shown for other viruses such as Sindbis (Chanas et al., 1982), measles (ter Meulen et al., 1981)
and mouse mammary tumour (Massey & Schochetman, 1981) viruses by the use of monoclonal
antibodies.
The study with proteolytic enzymes raises questions about how G 1 and G2 are oriented in the
lipid bilayer of the envelope. At first it was thought that trypsin might have cleaved a single G 1
glycoprotein molecule into two pieces (67K and 95K). However, the total mol. wt. of the two
peptides would be equal to 162000, much higher than that of G1. These two cleavage products
migrated similarly in reducing or non-reducing gels (L. Kingsford & D. W. Hill, unpublished
data), indicating they are not disulphide-linked. Thus, cleavage of G1 does not appear to be
analogous to the cleavage of glycoproteins of viruses such as influenza (Skehel & Waterfield,
1975), mouse mammary tumour (Sheffield et al., 1976), and Sendai (Homma & Ohuchi, 1973)
viruses. When LAC virus was treated with chymotrypsin, two cleavage products were also
found, both larger than the polypeptides resulting from trypsin treatment. In addition, trypsin
and chymotrypsin degrade part of G 1 into small peptides which are found between N and the
dye front, or at the dye front (Fig. 1 and 2b). These data indicate that cleavage of two different
G1 molecules has to occur. Perhaps two G1 molecules or G1 and G2 interact in such a way that
one of two trypsin- (or chymotrypsin-) susceptible sites are exposed on different G1 molecules.
G2 was highly resistant to proteolytic enzymes either because of its association with and
protection by G1 and/or its association with the lipid bilayer.
This study was supported in part by Grant AI17931 from the National Institute of Allergy and Infectious
Diseases, United States Public Health Service, Department of Health, Education and Welfare. We wish to thank
Louis C. Cullman, Microbiology Reference Laboratory, Long Beach, California, U.S.A. for performing the
immunofluorescence assays on the rabbit antisera.
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