Vol. 62, No. 12
JOURNAL OF VIROLOGY, Dec. 1988, p. 4686-4690
Copyright C) 1988, American Society for Microbiology
The E3 Protein of Bovine Coronavirus Is a Receptor-Destroying
Enzyme with Acetylesterase Activity
REINHARD VLASAK,'t WILLEM LUYTJES,2 JASON LEIDER,' WILLY SPAAN,2 AND PETER PALESE1*
Department ofMicrobiology, Mount Sinai School ofMedicine, New York, New York 10029-6574,' and Department of
Infectious Diseases and Immunology, Division of Virology, State University of Utrecht, Utrecht, The Netherlands2
Received 11 July 1988/Accepted 29 August 1988
In addition to members of the Orthomyxoviridae and Paramyxoviridae, several coronaviruses have been
shown to possess receptor-destroying activities. Purified bovine coronavirus (BCV) preparations have an
esterase activity which inactivates O-acetylsialic acid-containing receptors on erythrocytes. Diisopropyl
fluorophosphate (DFP) completely inhibits this receptor-destroying activity of BCV, suggesting that the viral
enzyme is a serine esterase. Treatment of purified BCV with [3H]DFP and subsequent sodium dodecyl
sulfate-polyacrylamide gel electrophoresis of the proteins revealed that the E3 protein was specifically
phosphorylated. This finding suggests that the esterase/receptor-destroying activity of BCV is associated with
the E3 protein. Furthermore, treatment of BCV with DFP dramatically reduced its infectivity in a plaque
assay. It is assumed that the esterase activity of BCV is required in an early step of virus replication, possibly
during virus entry or uncoating.
Members of at least three families of enveloped RNA
viruses (i.e., Orthomyxoviridae, Paramyxoviridae, and Co-
ronaviridae) bind to cell receptors containing sialic acid as
receptor determinant (25, 27, 28, 34, 36). In addition, virus-
scribed for members ofthese virus families (9, 11, 20, 23, 26,
34). For parainfluenza and for influenza A and B viruses, the
receptor-destroying enzyme is a neuraminidase, which re-
moves sialic acids from cellular receptors. In parainfluenza
viruses, the neuraminidase activity is located on the HN
protein, which also possesses receptor binding/hemagglu-
tinin activity (26). In influenza A and B viruses, the neur-
aminidase is a glycoprotein which is distinct from the hem-
agglutinin protein (for reviews, see references 1 and 17). In
contrast, for influenza C viruses and for bovine coronavirus
(BCV), the receptor-destroying enzyme is not a neuramini-
dase, but an acetylesterase, which removes acetyl groups
from O-acetylated sialic acids (11, 34). The esterase activity
of influenza C virus is associated with the HE protein, which
also possesses receptor-binding and fusion activity (8, 10,
33). However, no direct evidence was available that a
specific BCV protein was associated with the esterase activ-
ity of viral preparations (34).
In the present report, we show that the BCV esterase
activity resides on the E3 glycoprotein, which is one of three
known surface proteins of the virus (6, 14). Enzymatic
activity is inhibited by diisopropyl fluorophosphate (DFP),
indicating that the BCV receptor-destroying enzyme is a
classical serine esterase, such as acetylcholinesterase (5).
Furthermore, inhibition of the BCV acetylesterase by DFP
inhibits viral replication, suggesting that the presence of an
active viral esterase is essential for virus entry into host
activities have been de-
MATERIALS AND METHODS
Viruses and cells. The BCV seed virus was obtained from
Duphar B.V. Weesp (Amsterdam, The Netherlands). BCV
t Present address: Austrian Academy of Sciences, Institute of
Molecular Biology, 5020 Salzburg, Austria.
was grown in Madin-Darby bovine kidney (MDBK) cells and
purified as described previously (34). Influenza A/WSN/33
virus was grown in Madin-Darby canine kidney (MDCK)
cells as described previously (3). Erythrocytes derived from
chickens (strain Rhode Island Red sex-linked chromosome
X) were obtained from Pocono Rabbit Farm (Canadensis,
Acetylesterase assay. Purified BCV preparations (5 ,g)
were incubated at room temperature in 1 ml of phosphate-
buffered saline (PBS) containing 1 mM p-nitrophenylacetate.
In order to prepare a 100 mM stock solution, p-nitropheny-
lacetate was dissolved in acetonitrile. The final acetonitrile
concentration in the assay was 1%. Hydrolysis of the sub-
strate was monitored at 400 nm with a chart recorder.
Enzyme inhibition assay. DFP, phenylmethylsulfonyl fluo-
ride (PMSF), and N-tosyl-L-phenylalanine chloromethyl ke-
tone (TPCK) were dissolved in isopropanol in order to
prepare 10Ox stock solutions. Purified BCV (5 ,ug) was
preincubated with inhibitor for 10 min at room temperature
in a volume of 50 ,ul. PBS was then added to a final volume
of 990 ,ul. The reaction was started by the addition of 10 ,ul
of substrate, and the activity was monitored at 400 nm.
Hemagglutination assay. A BCV suspension (22.5RI1con-
taining 1,024 hemagglutination units) was mixed with 2.5 ,ul
of 10 mM DFP (in 10% isopropanol), incubated at room
temperature for 10 min, and used in the assay. Alternatively,
DFP-treated BCV was purified over a 20 to 60% sucrose
gradient. After the virus band was collected, the volume was
adjusted to 500 ,u1 with 20% sucrose. Controls were treated
in the same way except that inhibitor was omitted. Hemag-
glutination assays were performed in V-shaped microtiter
plates (Flow Laboratories, Inc., McLean, Va.) as described
Plaque assays. BCV and influenza A/WSN/33 virus were
incubated with 1 mM DFP for 10 min at room temperature,
purified over a 20 to 60% sucrose gradient, and titrated on
MDBK cells by standard procedures (24, 29).
Protein labeling with [3H]DFP. Viral protein (140 p,g) was
labeled in PBS containing 0.1 mM [3H]DFP (4.4 Ci/mmol;
Dupont, NEN Research Products, Boston, Mass.) for 30 min
at room temperature. Labeled virus was purified from unin-
CORONAVIRUS E3, A RECEPTOR-DESTROYING ENZYME
corporated DFP on a 20 to 60% sucrose step gradient and
5-,ug aliquots were analyzed on a sodium dodecyl sulfate-
polyacrylamide gel (7% polyacrylamide) with or without
fixed, soaked in Amplify (Amersham Corp., Arlington
Heights, Ill.), dried, and fluorographed for 2 to 6 days at
-70°C. Lanes of the gel containing molecular weight stan-
dards (Bio-Rad Laboratories, Richmond, Calif.) were silver
Protein labeling with [35S]methionine. Confluent MDBK
monolayers in 60-mm-diameter dishes were infected with
BCV (multiplicity of infection of approximately 2) and
incubated at 37°C with 3T3 medium (Dulbecco modified
Eagle medium supplemented with 10% heat-inactivated fetal
calf serum, 50 U of penicillin G per ml, and 50 jig of
streptomycin sulfate per ml). At 24 h postinfection, mono-
layers were washed with PBS, and protein-labeling medium
(Hanks balanced salt solution supplemented with 0.5%
NaHCO3, 0.004% [wt/vol] phenol red, 50 U of penicillin G
per ml, 50 ,ug of streptomycin sulfate per ml, and 0.2%
glucose) was added to the cells. After incubation at 37°C for
30 min, cells were washed with PBS and incubated at 37°C
with labeling medium
[35S]methionine per ml (1,100 Ci/mmol; Dupont NEN). At 36
h postinfection, culture supernatants were collected, and
labeled virus was purified on a 20 to 60% sucrose step
gradient (see above).
Acetylesterase activity of BCV. In a previous article, we
showed that an acetylesterase activity is associated with
BCV. This enzyme releases acetate from bovine submaxil-
lary mucin at a rate comparable with that ofinfluenza C virus
esterase (34). To allow a more detailed characterization of
the BCV enzyme, the synthetic low-molecular-weight sub-
strate p-nitrophenylacetate was selected. Enzymatic mea-
surements using this substrate involve determination of the
cleavage product p-nitrophenol at 400 nm. This procedure is
much less cumbersome than the cascade assay used for
measuring acetate that is released from bovine submaxillary
mucin. A purified BCV preparation effectively hydrolyzed
this O-acetylester (Fig. 1). For further characterization of
the BCV enzyme, different inhibitors were tested. DFP, a
serine esterase and protease inhibitor, completely inhibited
the BCV esterase, when preincubated with the virus at a 1
mM concentration (Fig. 1). Serine protease inhibitors PMSF
and TPCK partially inhibited the BCV esterase (Table 1).
EDTA had no effect, indicating that divalent cations are
most likely not required for enzymatic activity, and dithio-
threitol actually enhanced the activity.
Specific labeling of the E3 protein of BCV by [3H]DFP.
Since DFP inhibits serine proteases and serine esterases by
binding covalently to the serine on the active site (2, 5), BCV
preparations were radioactively labeled with [3H]DFP by
incubation at room temperature for 30 min. Labeled virus
was purified over a 20 to 60% sucrose step gradient, and viral
proteins were analyzed on a sodium dodecyl sulfate-poly-
acrylamide (7% polyacrylamide) gel with and without beta-
mercaptoethanol. After fluorography, a single labeled pro-
tein was detected, migrating with an apparent M,of 62,000
(62K) under reducing conditions (Fig. 2, lane 2) and with an
apparent M, of approximately 125K under nonreducing
conditions (Fig. 2, lane 3). In both instances, the [3H]DFP-
labeled protein migrated in the position of the [35S]methio-
nine-labeled E3 protein of BCV (Fig. 2, lanes 1 and 4). The
FIG. 1. Hydrolysis of p-nitrophenylacetate by BCV. Purified
virus was incubated in a 1-ml cuvette containing 1 mM p-nitropheny-
lacetate in PBS-1% acetonitrile. For DFP treatment, 5 ,ug of virus
was preincubated with 1 mM DFP for 10 min at room temperature
(see Materials and Methods) and used in the assay. Hydrolysis of
the chromogenic substrate was monitored at 400 nm using a chart
recorder.OD4.,Optical density at 400 nm.
E3 protein of BCV is a homodimer composed of two 62K
subunits, apparently connected by disulfide bridges, and it is
one of the viral proteins recognized as making up the virion
structure (7, 14, 15).
Hemagglutination ofDFP-treated BCV. We then addressed
the question of whether inactivation of the viral esterase
affects the binding of BCV to erythrocyte receptors. BCV
preparations were incubated with 1 mM DFP for 10 min at
room temperature and purified over a 20 to 60% sucrose step
gradient. DFP-treated and mock-treated BCV were col-
lected, adjusted to a volume of 500
receptor-binding activity by using a hemagglutination assay.
No difference between treated and untreated BCV prepara-
tions was detected when hemagglutination
performed at 4°C, indicating that there was no requirement
of an active esterase for binding to cell receptors (Fig. 3A).
However, if the temperature was shifted up to 20°C and then
to 37°C, untreated virus started to elute from erythrocytes as
a result of the presence of the receptor-destroying activity.
In contrast, the hemagglutination pattern of DFP-treated
virus was stable under these conditions (Fig. 3B). The same
result was obtained when BCV was incubated with 1 mM
DFP and used directly in hemagglutination assays without
prior purification over a sucrose gradient. Clearly, inhibition
,lI, and tested for
TABLE 1. Effect of different inhibitors on BCV esterasea
aPurified BCV was preincubated with DFP (diisopropyl fluorophosphate),
chloromethyl ketone), EDTA (ethylenediaminetetraacetic acid), or DFT
(dithiothreitol) at the indicated concentration for 10 min at roomtemperature
in a volume of 50 ,d. After 20-fold dilution with PBS, acetylesterase activity
was determined by using p-nitrophenylacetate (1 mM) as the substrate (see
Materials and Methods).
fluoride), TPCK, (N-tosyl-L-phenylalanine
VOL. 62, 1988
VLASAK ET AL.
TABLE 2. Plaque formation of DFP-treated BCV preparationsa
Expt no. and virus
BCV + DFP
BCV + DFP
BCV + DFP
A/WSN/33 + DFP
A/WSNI33 + DFP
2.6 x 107
6.5 x 104
2.5 x 106
3.0 x 104
5.4 x 107
1.3 x 105
1.5 x 107
1.3 x 107
6.2 x 107
5.6 x 107
aBCV and influenza AIWSN/33 virus were incubated with and without 1
mM DFP and purified over a 20 to 60% sucrose gradient (experiment 1 and 2).
Alternatively, DFP-treated virus was used without further purification (exper-
b Reciprocal of the highest dilution of virus giving full hemagglutination
after 60 min at 4'C.
"Measured with 1 mM p-nitrophenylacetate. NA, Not applicable.
-a ." K
in 10-fold serial dilutions for plaque assays. DFP-treated
BCV had approximately 100- to 400-fold-lower infectivity
titers than mock-treated BCV (Table 2). To exclude the
possibility of unspecific effects caused by DFP, assays with
influenza A/WSN/33 virus were done in parallel. This virus
does not possess an esterase activity, and DFP treatment
does not result in phosphorylation of a viral protein. No
difference was found between the titer of DFP-treated and
strongly suggest that a functional BCV esterase iS reqUired
for virus replication.
Previously we have shown that human coronavirus OC43
and BCV bind to sialic acid-containing cell receptors (34).
Since the influenza C virus receptor-destroying enzyme, an
O-acetylesterase (11), removed coronavirus receptors,
was concluded that OC43 and BCV recognize receptors
similar to those of influenza C virus. In addition, a receptor-
destroying or acetylesterase activity was found to be asso-
ciated with BCV (34).
In order to determine whether the latter activity could be
attributed to a viral protein, experiments were directed at
analyzing the specificity and catalytic mechanism of the
was conveniently measured with the low-molecular-weight
substrate p-nitrophenylacetate (33), we also used this assay
for monitoring the BCV enzyme. Of several inhibitors
tested, only DFP, a serine protease and esterase inhibitor,
completely inhibited the BCV esterase. Serine protease
PMSF and TPCK gave only partial inhibition, and
no effect was detected by preincubation with EDTA, sug-
gesting that divalent cations are not required for catalytic
activity. Since the BCV esterase was quantitatively inhibited
by DFP, it is suggested that the enzyme is aserineesterase.
Similar findings were reported for other esterases(5,19). By
affinity labeling with the site-specific reagent DFP and anal-
ysis of the BCV protein on polyacrylamide gels, the E3
protein was identified as containing the viral esterase.
No difference in hemagglutination titers was observed
between mock-treated and DFP-treated BCV in hemaggluti-
FIG. 2. Analysis of [3H]DFP-labeled BCV proteins. [3H]DFP-
labeled and [35S]methionine-labeled BCV was electrophoresed in a
7% polyacrylamide gel after solubilization in sample loading buffer
with (lanes 1 and 2) or without (lanes 3 and 4) beta-mercaptoethanol.
Lanes 2 and 3, [3H]DFP-labeled BCV; lanes 1 and 4, [35S]methio-
nine-labeled BCV. Positions of viral proteins are indicated by
arrows between lanes 2 and 3; positions of molecular weight
markers (in thousands [K]) are shown on the right of both gels by
of the viral esterase activity stabilized binding of BCV to
Esterase activity required for BCV replication. In order to
explore the biological significance of the BCV esterase
during virus replication,
formed with DFP-treated BCV.
treated and mock-treated BCV were tested for acetyleste-
rase activity and hemagglutination titers and then were used
titrations were per-
enzyme. Since the influenza C virus acetylesterase activity
FIG. 3. Hemagglutination pattern of DFP-treated BCV. BCV
(1,024 hemagglutination units) was incubated with 1 mM DFP for 10
min at room temperature. Hemagglutination was performed by using
chicken erythrocytes and BCV preparations at serial 1:2 dilutions.
(A) The microtiter plate was incubated at 4°C for 1 h and photo-
graphed. (B) The same plate was incubated overnight at room
temperature, followed by incubation at 37°C for 30 min, and
rephotographed. Symbols: +, BCV incubated with DFP; -, mock-
CORONAVIRUS E3, A RECEPTOR-DESTROYING ENZYME
FIG. 4. Schematic representation of
glycoproteins of orthomyxoviruses, par
viruses. Sendai virus and BCV are uses
xoviruses and coronaviruses, respecti
NA, neuraminidase, HE, hemagglutinin
HN, hemagglutinin-neuraminidase; E2,
navirus hemagglutinin-esterase. Boxes
functions have been determined.
nation assays under standard cond
when the incubation temperature v
BCV eluted from chicken erythri
inactivated BCV retained its hema
stroying activity in BCV actually i
virus-receptor interactions. Simila
tained earlier when the receptor
influenza viruses were inhibited (4,
To investigate the role of the B(
replication, plaque assay experimei
lowing inactivation of the esteras
virus was titrated in tissue culture
treated BCV, virus with an inac
approximately 100- to 400-fold redu
infectivity of DFP-treated influenzz
ished. Thus, data obtained from
indicate a direct involvement of tI
phases of BCV replication. Since I
acid-containing receptors was not
vation of the esterase, it appears t
replication, binding to cellular recc
the presence or absence of a rece
We thus speculate that an active e
for either endocytosis and/or unc4
subsequent release of viral RNA ir
There are now three RNA vir
viruses which have been found
stroying activities (Fig. 4). Neurai
with influenza A and B viruses (1, 1
viruses (26). Esterases have bee
influenza C virus (8, 10, 11, 19,
coronavirus family, such as BC'
destroying activities are either loca
(neuraminidases in influenza A and
a multifunctional protein as in the (
influenza C virus. Similarly, the
may be localized in a separate pr
influenza A and B viruses) or it n
multifunctional protein (the HN an
xoviruses and influenza C virus). Fi
again be associated with a unique
fluenza subgroup (27), or at the oth
HE protein (10, 21), which possest
Taking into account that BCV ar
closelyrelatedtomousehepatitis virus (12), yet appearto
have an additional E3 surface glycoprotein, no biological
function can be attributed to this protein by direct analogy.
Since the receptor-binding and fusion activity in mouse
hepatitisvirus are located on the E2protein (18, 31, 32, 35),
it might be hypothesized that the E2 proteins of BCV and
OC43 have homologous activities. But what, then, is the
precise function of the E3 protein? Earlier studies revealed
that the E3proteinof BCV is the viralhemagglutinin (15).
Thus, is the E3 protein an additional receptor-binding pro-
tein or is it the only receptor-binding protein, with the E2
protein alone possessing fusion activity? The present study
suggests that the E3 protein of BCV has an acetylesterase
activity necessary for virus replication. Thus, the interesting
possibility arises that this protein has receptor-binding as
well as receptor-destroying activities. Although a great deal
has already been learned about the molecular biology of
coronaviruses (16, 30), identification of the precise roles of
all proteins during coronavirus replication awaits further
analysis. Additional experimentsshould also show whether
all or only some coronavirusespossess receptor-binding/re-
ceptor-destroying activities and whether different strategies
of attachment and uncoating prevail for different coronavi-
the functions of the surface
ramyxoviruses, and corona-
d as prototypes for paramy-
ively. HA, Hemagglutinin;
-esterase; F, fusion protein;
peplomer protein; E3, coro-
indicate proteins for which
litions at 4°C. However,
vas raised, mock-treated
*ocytes, and only DFP-
tgglutination titer. These
ion of the receptor-de-
increases the stability of
r results had been ob-
-destroying activities of
2V esterase during virus
nts were performed. Fol-
;e with DFP, infectious
Compared with mock-
tivated esterase had an
Iced titer. In contrast, the
a A virus was not dimin-
these experiments could
he actylesterase in early
binding of BCV to sialic
impaired by DFP inacti-
hat the first step in viral
eptors, is independent of
sterase may be required
oating of the virus with
ito the cytoplasm.
us families that include
to possess receptor-de-
rninidases are associated
7) and with parainfluenza
~n shown to be part of
33) an members
V (34). These receptor-
ited in a separate protein
B viruses) or arepartof
case of the HE proteinof
rotein (hemagglutinins of
nay be associated with a
d HE proteinsofparamy-
fusion activity may
nally, fusion activity may
protein, as in the parain-
ier extreme bepartof the
ses several functions.
id OC43 are antigenically
This work was supported in part by Public Health Service grants
Al-11823 andAl18998from the National Institutes of Health. R.V.
was supported in part by a Max Kade postdoctoral fellowship. W.L.
was supported by a grant from Duphar B.V. Weesp (Amsterdam,
The Netherlands). J.M.L. was supported by Medical Scientist
training grant GM07280 from the National Institutes of General
1. Air, G. M., and R. W. Compans. 1983. Influenza B and C
viruses, p. 281-304. In P. Palese and D. W. Kingsbury (ed.),
Genetics of influenza viruses. Springer-Verlag, Vienna.
2. Bender,M.L., and F. J. Kezdy. 1965. Mechanism of action of
proteolytic enzymes. Annu. Rev. Biochem. 34:49-76.
3. Brand, C., and P. Palese. 1980. Sequential passage of influenza
virus in embryonated eggs or tissue culture: emergence of
mutants. Virology 107:424-433.
4.Bucher, D.,and P. Palese.1975.Thebiologicallyactiveproteins
of influenza virus: neuraminidase, p. 83-123. In E. D. Kilbourne
(ed.), The influenza virus and influenza. Academic Press, Inc.,
5. Cohen, J. A., R. A. Oosterbaan, and F. Berends. 1967. Organo-
phosphorous compounds.Methods Enzymol.11:686-702.
6. Deregt, D., and L. A. Babiuk. 1987. Monoclonal antibodies to
bovine coronavirus: characteristics and topographical mapping
of neutralizing epitopes on the E2 and E3 glycoproteins. Virol-
7. Deregt, D., M. Sabara, and L. A. Babiuk. 1987. Structural
proteins of bovine coronavirus and their intracellular process-
ing. J. Gen. Virol. 68:2863-2877.
8. Formanowski, F., and H. Meier-Ewert. 1988. Isolation of the
influenza C virus glycoprotein in a soluble form by bromelain
digestion.Virus Res. 10:177-192.
9. Gottschalk, A. 1957. The specific enzyme of influenza virus and
Vibrio cholerae. Biochim. Biophys. Acta 23:645-646.
lo.Herrier, G., I. Durkop, H. Becht, and H.-D. Klenk. 1988. The
glycoprotein of influenza C virus is the haemagglutinin, esterase
and fusion factor. J. Gen. Virol. 69:839-846.
11. Herrler, G., R. Rott, H.-D. Klenk, H.-P. Muller, A. K. Shukla,
and R. Schauer. 1985. The receptor destroying enzyme of
influenza C virus is neuraminate-O-acetylesterase. EMBO J. 4:
12. Hogue, B. A., B. King, and D. A. Brian. 1984. Antigenic
VOL. 62, 1988
VLASAK ET AL.
relationship among proteins of bovine coronavirus, human
respiratory coronavirus OC43, and mouse hepatitis coronavirus
A59. J. Virol. 51:384-388.
13. Homma, M., and M. Ohuchi. 1973. Trypsin action on the growth
of Sendai virus in tissue culture cells. III. Structural difference
of Sendai virus grown in eggs and tissue culture cells. J. Virol.
14. King, B., and D. A. Brian. 1982. Bovine coronavirus structural
proteins. J. Virol. 42:700-707.
15. King, B., B. J. Potts, and D. A. Brian. 1985. Bovine coronavirus
hemagglutinin protein. Virus Res. 2:53-59.
16. Lai, M. M. 1986. Replication of coronavirus RNA, p. 115-136.
In J. J. Holland, P. Ahlquist, and E. Domingo (ed.), RNA
genetics, vol. 1: RNA-directed virus replication. CRC Press,
Inc., Boca Raton, Fla.
17. Lamb, R. A. 1983. The influenza virus RNA segments and their
encoded proteins, p. 21-69. In P. Palese and D. W. Kingsbury
(ed.), Genetics of influenza viruses. Springer-Verlag, Vienna.
18. Luytjes, W., L. S. Sturman, P. J. Bredenbeek, J. Charite,
B. A. M. van der Zeijst, M. C. Horzinek, and W. J. M. Spaan.
1987. Primary structure of the glycoprotein E2 of coronavirus
MHV A59 and identification of the trypsin cleavage site. Virol-
19. Muchmore, E. A., and A. Varki. 1987. Selective inactivation of
influenza C virus esterase: a probe for detecting 9-0-acetylated
sialic acids. Science 236:1293-1295.
20. Nagai, Y., H.-D. Klenk, and R. Rott. 1976. Proteolytic cleavage
of the viral glycoproteins and its significance for the virulence of
NDV by proteolytic cleavage. Virology 77:125-134.
21. Ohuchi, M., R. Ohuchi, and K. Mifune. 1982. Demonstration of
hemolytic and fusion activities of influenza C virus. J. Virol. 42:
22. Palese, P., and J. L. Schulman. 1974. Isolation and characteri-
zation of influenza virus recombinants with high and low neur-
neuraminic acid to identify cloned populations. Virology 57:
23. Palese, P., K. Tobita, M. Ueda, and R. W. Compans. 1974.
Characterization of temperature sensitive influenza A virus
mutants defective in neuraminidase. Virology 61:397-410.
24. Parvin, J. D., A. Moscona, W. T. Pan, J. M. Leider, and P.
Palese. 1986. Measurement of the mutation rates of animal
viruses: influenza A virus and poliovirus type 1. J. Virol. 59:
25. Rogers, G. N., G. Herrler, J. C. Paulson, and H.-D. Klenk. 1986.
Influenza C virus uses 9-O-acetyl-N-neuraminic acid as high
affinity receptor determinant for attachment to cells. J. Biol.
26. Scheid, A., L. A. Caliguiri, R. W. Compans, and P. W. Choppin.
1972. Isolation of paramyxovirus glycoproteins. Association of
both hemagglutinating and neuraminidase activities with the
larger SV5 glycoprotein. Virology 50:640-651.
27. Scheid, A., and P. W. Choppin. 1974. Identification of biological
activities of paramyxovirus glycoproteins: activation of cell
fusion, hemolysis, and infectivity by proteolytic cleavage of an
inactive precursor protein of Sendai virus. Virology 57:475-490.
28. Scheid, A., and P. W. Choppin. 1974. The hemagglutinating and
neuraminidase protein of a paramyxovirus: interaction with
neuraminic acid in affinity chromatography. Virology 62:125-
29. Spaan, W. J. M., P. J. M. Rottier, M. C. Horzinek, and B. A. M.
van der Zeijst. 1981. Isolation and identification of virus specific
mRNAs in cells infected with mouse hepatitis virus (MHV-
A59). Virology 108:424-434.
30. Sturman, L. S., and K. V. Holmes. 1983. The molecular biology
of coronaviruses. Adv. Virus Res. 28:35-112.
31. Sturman, L. S., C. S. Ricard, and K. V. Holmes. 1985. Proteo-
lytic cleavage of the E2 glycoprotein of murine coronavirus:
activation of cell fusing activity of virions by trypsin and
separation oftwo different 90K cleavage fragments. J. Virol. 56:
32. Talbot, P. J., A. A. Salmi, R. L. Knobler, and M. J. Buchmeier.
1984. Topographical mapping of epitopes on the glycoproteins
of murine hepatitis virus-4 (strain JHM): correlation with bio-
logical activities. Virology 132:250-260.
33. Vlasak, R., M. Krystal, M. Nacht, and P. Palese. 1987. The
influenza C virus glycoprotein (HE) exhibits receptor binding
(hemagglutinin) and receptor destroying (esterase) activities.
34. Vlasak, R., W. Luytjes, W. Spaan, and P. Palese. 1988. Human
and bovine coronaviruses recognize sialic acid containing recep-
tors similar to those of influenza C viruses. Proc. Natl. Acad.
Sci. USA 85:4526-4529.
35. Wege, H., H. Wege, K. Nagashima, and V. ter Meulen. 1979.
Structural polypeptides of the murine coronavirus JHM. J. Gen.
36. Wiley, D. C., and J. J. Skehel. 1987. The structure and function
of hemagglutinin membrane glycoprotein of influenza virus.
Annu. Rev. Biochem. 56:365-394.