J. gen. Virol. (1984), 65, 1285 1293. Printedin Great Brim&
1285
Key words: Hantaan virus/bunyaviruses/protein characterization
Hantaan Virus: Identification of Virion Proteins
By L U A N N E
H. E L L I O T T , * M I C H A E L P. K I L E Y AND
J O S E P H B. M c C O R M I C K
Centers for Disease Control, Public Health Service, U.S. Department of Health and Human
Services, Atlanta, Georgia 30333, U.S.A.
(Accepted 18 April 1984)
SUMMARY
SDS-PAGE and immunoprecipitation analyses were carried out on the virion and
cell-associated proteins of Hantaan virus, the causative agent of haemorrhagic fever
with renal syndrome (HFRS). Purified virions have a density of 1.17 g/ml in sucrose,
and contain four proteins with molecular weights of 45 000 (45K), 56K, 72K and 200K,
confirming recent evidence that the virus is a member of the family Bunyaviridae.
Detergent treatment of virions indicates that the 45K protein is the virus
nucleoprotein. Both the 72K and the 56K proteins were labelled with [3 H]glucosamine
and were removed from virions by bromelain treatment, indicating that they are
envelope glycoproteins. The 200K protein was found only in [35S]methionine-labelled
preparations. By analogy to prototype viruses of the family Bunyaviridae, these proteins
were designated N, G1, G2, and L respectively. Three virus-specific proteins (N, G1,
G2) were detected in virus-infected cells. These proteins were precipitable by human
convalescent serum and by serum of a Rattus norvegicus trapped in the United States.
No additional virus proteins were detected in infected cells. These results confirm
recent morphological and RNA studies that Hantaan virus is a member of the family
Bunyaviridae. Our results also support the suggestion that Hantaan virus be placed in a
new genus of Bunyaviridae.
INTRODUCTION
The disease haemorrhagic fever with renal syndrome (HFRS) gained attention in 1951 when
many United Nations military personnel contracted the disease in Korea (Smadel, 1953). It was
not until 1978 that a viral agent, Hantaan virus, was isolated from Apodemus agrarius coreae, a
rodent reservoir (Lee et al., 1978). On the basis of virion morphology (McCormick et al., 1982;
White et al., 1982) and virion R N A properties (Schmaljohn & Dalrymple, 1983; Schmaljohn et
al., 1983), it has been proposed that Hantaan virus be classified as a member of the family
Bunyaviridae. We recently adapted Hantaan virus to replicate in the E6 clone of Vero 76 cells
(McCormick et al., 1982). The increased sensitivity and yield of these cells has allowed the
isolation of additional virus strains and more advanced morphological and biochemical studies
(Tsai et al., 1982 a, b; Kitamura et al., 1983; Hung et al., 1983). The virus particles are spherical
with an average diameter of 92.5 nm, with a lipoprotein envelope and capsomeres which give
them a knobby appearance (McCormick et al., 1982). In this paper, we report the
characterization by SDS-PAGE of four proteins which appear to be analogous to the L, G 1, G2,
and N proteins of prototype Bunyaviridae. The molecular weights of these proteins, however,
differ from those of other members of this family.
Hantaan virus represents a significant biohazard and has been assigned to Biosafety Level 4
in the U.S. Public Health Service's guidelines. Our work was conducted in the Maximum
Containment Laboratory (MCL) at the Centers for Disease Control (CDC).
METHODS
Cellcultures. The E6 cellline, a clonedline of Verocells (ATCC VerocloneCRL 1586), was used in these studies
(McCormick et al., 1982; Johnson et al., 1981). Cells were grown in Eagle's minimum essential medium (MEM)
00022-1317/84/0000-6027
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L. H. E L L I O T T , M. P. K I L E Y A N D J. B. M c C O R M I C K
supplemented with 10% heat-inactivated foetal bovine serum (FCS) and an antibiotic mixture of 100 units of
penicillin, 50 ~tg of streptomycin, and 2 ~tg of amphotericin B per ml. Cells were free of mycoplasma. After
inoculation with virus, growth m e d i u m was replaced with maintenance m e d i u m in which the FCS was reduced to
2%.
Growth and purification o f the virus. H a n t a a n virus strain 76-118 was used in these studies (Lee et al., 1978;
McCormick et al., 1982; French et al., 1981). Virus stocks were prepared from a plaque-purified seed virus
preparation by infection of Vero E6 cells at an m.o.i, of 0-1 p.f.u./cell. After 7 days, the supernatant fluid was
clarified by centrifugation at 2000 r.p.m, for 15 min. Bovine serum albumin (BSA) was added to 1-5% before
freezing at - 7 0 °C. Virus preparations had an infectivity titre of 1 x 107 p.f.u./ml.
For production of purified virus, supernatant fluids from twenty 150 cm 2 flasks, infected for 7 days, were
clarified by centrifugation at 3000 r.p.m, for 15 m i n and layered onto a 20 % sucrose cushion for centrifugation in a
Spinco SW27 rotor at 22000 r.p.m, for 1 h. Larger volumes could be pelleted in the Beckman J-21 centrifuge at
8000 r.p.m, overnight (H. Lindsey-Regnery, personal communication). Pellets were suspended in sterile Tris
N a C 1 - E D T A (TNE) buffer, layered on a 20 to 70% sucrose gradient and centrifuged to equilibrium for 18 h at
30000 r.p.m, in the Spinco SW40 rotor. The virus material appeared as an opalescent band at a density of
1.17 g/ml. This band was harvested, diluted in T N E and pelleted at 35000 r.p.m, for 1 h in the SW40 rotor. The
pellet was suspended in 200 p.l of T N E and frozen at - 7 0 °C.
If the virus was to be radioactively labelled, the maintenance medium was replaced on day 4 with labelling
m e d i u m consisting of 20% M E M , 2% dialysed FCS, 80% specific nutrient-deficient M E M to which radioactive
nutrients were added as follows: 2 ~tCi/ml of 14C-amino acids, 20 ktCi/ml of D-[6-3H]glucosamine, 10 ktCi/ml of
[3H]leucine, or 20 ktCi/ml of [3SS]methionine (all from New England Nuclear). Supernatant fluids were collected
on day 7 and purified as described above.
Immunoprecipitation of viral proteins. The radioimmunoprecipitation (RIP) procedure used was essentially that
described by Kessler (1975). E6 cells in 25 cm 2 flasks were infected with H a n t a a n virus at an m.o.i, of 1. After an
adsorption period of 30 min, maintenance m e d i u m was added and the ceils incubated for the indicated times at
37 °C. Three h before harvesting cells, the m a i n t e n a n c e m e d i u m was replaced with methionine-free M E M .
One h later this medium was removed and replaced with methionine-free M E M containing 50~tCi/ml of
[3sS]methionine. After a 2 h labelling period, m e d i u m was removed, cells were washed twice with cold, sterile
phosphate-buffered saline (PBS), and then 1 ml of RIP buffer per 25 cm z flask was added. R I P buffer consisted of
N E T buffer (50 mM-Tris-HC1, 0.15 M-NaCI, 5 mM-EDTA, 0.02% NAN3) with 1% Nonidet P40 (NP40), 0.5%
deoxycholate (DOC), and 0-1% SDS. Just before use, 1 mM-phenylmethylsulphonyl fluoride (PMSF) was added.
After 15 rain at 4 °C, the infected cell extract was placed in a 1.5 ml Eppendorf centrifuge tube, mixed well to shear
the D N A , and then centrifuged for 2 m i n in a Beckman Microfuge. 100 ~tl of the resultant cell extract was mixed
with 20 to 50 ~tl of i m m u n e serum and incubated at room temperature for 2 h. Unless otherwise indicated, our
standard antiserum was mouse ascites fluid (MAF) no. 701469 which was prepared in our laboratory against
H a n t a a n strain 76-118 and has an indirect fluorescent antibody (IFA) titre of I :2048.100 ~tl of a 10 % suspension
of staphylococcal Protein A-Bacterial Adsorbent (SPA, Mfles-Yeda, Ltd.) was then added, a n d this mixture was
incubated at room temperature for 15 to 30 min with frequent vortex-mixing. The S P A - a n t i g e n antibody
complex was pelleted, washed twice with cold N E T buffer containing 0.1% NP40 and 0.1% DOC, and pelleted
proteins were dissociated by boiling for 2 min in 100 ~tl of N E T buffer containing 2 % SDS, 2 % 2-mercaptoethanol,
50% sucrose and bromophenol blue (BPB).
SDS-PAGE. The procedure used was that described by Laemmli (1970) except that 2-5 M-urea was incorporated
into the gels. The separating gels contained 1 5 ~ or 12.5% acrylamide and the stacking gels contained 3%
acrylamide. Slab gels measured 1.5 m m x 14 cm x 20 cm and were electrophoresed at a constant voltage of 50 V
for 16 h. Gels were fixed in 10% acetic acid for 1 h, placed in E N 3 H A N C E (New England Nuclear) for 1 h, rinsed
in water for 30 min, dried under vacuum and exposed to X-ray film (Kodak X-Omatic or DuPont Cronex 4) at
- 70 °C. Appropriate molecular weight standards were included. 12-5 % acrylamide cylindrical gels were also run
at 50 V for 16 h. After electrophoresis, gels were extruded into 10% acetic acid for 1 h, then frozen and sliced into
1 m m sections. Slices were placed in 6 ml of 4a20 (Research Products International, Elk Grove Village, Ill.,
U.S.A.) scintillation fluid containing 6 % TS-1 tissue stabilizer and incubated at 37 °C overnight. Radioactivity of
each sample was determined in a Beckman scintillation counter.
RESULTS
SDS-PAGE analysis of purified virion proteins
Three Hantaan virion proteins with estimated molecular weights of 45 000 (45K), 56K and
72K were detected by SDS-PAGE analysis with Coomassie Brilliant Blue staining and
radioactive labelling (Fig. 1). When 35S-labelled virion proteins were electrophoresed, a fourth
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Hantaan virus proteins
(a)
(b)
(c)
(d)
200K
92.5K
68K
IG1
~1 G2
43K
25.7K
Fig. 1. Electrophoretic analysis of Hantaan virion proteins. Hantaan virus grown in the presence of
14C.amino acids was purified, disrupted and proteins were separated in a 12.5% polyacrylamide gel. (a)
Prestained mol. wt. standards (Bethesda Research Laboratories) consisting of, in order of decreasing
size, myosin (H chain), phosphorylase b, bovine serum albumin, ovalbumin and ~-chymotrypsinogen;
(b) extracellular virus protein after pelleting through a 20~ sucrose cushion, Coomassie Brilliant Blue
stain; (c) virus from (b) following further purification in a 20 to 70~ sucrose gradient, Coomassie
Brilliant Blue stain; (d) autoradiograph of preparation used in (c).
protein with a mol. wt. of approximately 200K was observed (Fig. 2a, c and Fig. 6). These four
proteins correspond, respectively, to the N, G1, G2, and L proteins reported for prototype
member viruses of the family Bunyaviridae (Obijeski & Murphy, 1977).
Virion glyeoproteins
When the virus was grown in the presence of [3 H]glucosamine the 72K and 56K proteins were
labelled (Fig. 2b). These are analogous to the G 1 and G2 glycoproteins of members of the family
Bunyaviridae. To further characterize these glycoproteins, purified 35S-labelled particles were
treated with bromelain (Obijeski et al., 1976), and then pelleted at 35000 r.p.m, for 2 h in an
SW50.1 rotor. Fig. 2(e) shows that the glycoproteins were removed by this treatment and that
only the L and N proteins remained associated with virions.
Identification of the virion nucleocapsid protein
If Hantaan virus is a member of the family Bunyaviridae, then one of its proteins should be
associated with a helical nucleocapsid (Obijeski & Murphy, 1977). To test this premise, virusinfected cells were double-labelled with [3 H]glucosamine and 14C-amino acids 5 to 7 days postinfection. Virus was partially purified through a 2 0 ~ sucrose cushion, then treated with N E T
plus 1 ~ NP40 for 15 min at 4 °C and re-pelleted (Lesnaw & Dickson, 1978). Because the virus
was only partially purified by this procedure, R I P was used to concentrate and further purify
virus proteins. When this was done, only the 45K protein was associated with the pelleted virion
nucleocapsid fraction (Fig. 3). Normal M A F precipitated only non-viral, cellular proteins.
Studies in this laboratory using monoclonal antibodies (unpublished results) have confirmed
that the 45K protein is the nucleocapsid protein.
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L. H. ELLIOTT, M. P. KILEY AND I. B. McCORMICK
10[
I
|
I
I
I
I
(a)
N
I
I
I
G2
L
(a)
(b)
(c)
(d)
Gl
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20
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3
.~
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40
60
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Fraction no.
Fig. 2
100
Fig. 3
Fig. 2. Identification of Hantaan virus proteins by SDS-PAGE. (a) [35S]Methionine-labelled Hantaan
virus, purified through a 2 0 ~ sucrose cushion and a 20 to 7 0 ~ sucrose gradient. The pellet was
resuspended in NET buffer with 2 ~ SDS, 2 ~ 2-mercaptoethanol, 5 0 ~ sucrose, and BPB, boiled 2 min
and electrophoresed on a 12.5 ~ acrylamide tube gel. The gel was cut into 1 mm slices and radioactivity
was estimated. (b) One 150 cm 2 flask of E6 cells was infected for 7 days and labelled for 72 h with
50 ~tCi/ml of [3H]glucosamine. The supernatant was centrifuged through a 20 ~ sucrose cushion and the
pellet processed as in (a). (c) 3sS-labelled Hantaan virus treated with bromelain at 37 °C for 30 min,
pelleted at 35000 r.p.m, for 2 h, and electrophoresed in a tube gel as in (a).
Fig. 3. Identificationofvirion nucleocapsidproteins. Virus was double-labelled with[3H]glucosamine
and laC-amino acids 5 to 7 days post-infection and partially purified through 2 0 ~ sucrose. Samples in
(a) and (b) were treated with NET buffer with 1~ NP40 only. Samples in (c) and (d) were treated with
RIP buffer. (a) Pellet from NP40-treated sample, RIP with MAF 701469; (b) supernatant from this. (c)
Sample treated with RIP buffer and RIP with MAF 701469; (d) same sample as (c) but RIP with
normal MAF.
Intracellular proteins
I n t h e e x p e r i m e n t d e p i c t e d in Fig. 4, cells w e r e l a b e l l e d for 2 h w i t h [ 3 5 S ] m e t h i o n i n e a t 24, 48,
a n d 72 h p o s t - i n f e c t i o n . S e r a used for i m m u n o p r e c i p i t a t i o n w e r e c o n v a l e s c e n t h u m a n sera f r o m
s u r v i v o r s o f H F R S (no. 7 0 0 0 4 7 a n d 126462). B o t h sera h a d I F A t i t r e s o f 1:1024. T h e r o d e n t
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Hantaan virus proteins
(a)
(b)
(c)
(d)
(e)
(f)
(g)
1289
(h)
(0
(J)
(k)
(l)
Fig. 4. Time course of protein synthesis. Radioimmunoprecipitation of Hantaan virus-infected E6
ceils, labelled for 2 h with [35S]methionineand run on 15% acrylamide gels without urea. (a to c) 24, 48,
72 h cell extracts RIP with Baltimore rat no. 5 serum; (d to]) 24, 48, 72 h cell extracts RIP with human
serum no. 700047; (g to i) 24, 48, 72 h cell extracts RIP with human serum no. 126462; (j) 72 h cell
extract RIP with normal human serum; (k) uninfected E6 cell extract RIP with 700047; (/) uninfected
E6 cell extract RIP with Baltimore rat no. 5 serum.
Table 1. Comparison of Hantaan virion-associated proteins with those of four prototype viruses of
the family Bunyaviridae
Virus
Congo
Rift Valley fever
Anhembi
La Crosse
Uukuniemi
Hantaan
Genus
Nairovirus
Phlebovirus
Bunyavirus
Bunyavirus
Uukuvirus
Ungrouped
G1
85-95K
65K
83-85K
85K
75K
72K
G2
56K
30-33K
45K
65K
56K
N
48~0K
25K
20-23K
26K
25K
45K
Reference
Bishopet al. (1980)
Rice et al. (1980)
Obijeski & Murphy (1977)'
Ohijeski & Murphy (1977)
Obijeski & Murphy (1977)
serum used was from a Rattus norvegicus collected during a 1982 serosurvey of rodents in
Baltimore (Tsai et al., 1982a). All three antisera precipitated the G1, G2 and N proteins of
H a n t a a n virus. The L protein was not precipitated under these conditions. Normal h u m a n
serum did not precipitate these viral proteins, nor were they precipitated from uninfected E6 cell
extracts reacted with the h u m a n and rat antisera.
Comparison with other viruses of the family Bunyaviridae
We compared the electrophoretic profile on SDS-polyacrylamide gels of H a n t a a n virus
proteins with those of several other prototype members of the Bunyaviridae (Fig. 5a, b).
Cell extracts were also prepared from these infected cultures and immunoprecipitated with
homologous and heterologous sera and MAFs. The results confirmed the serological result,
reported by Tsai et al. (1982a), that the a n t i - A n h e m b i M A F showed a weak one-way crossreaction with the nucleocapsid protein in the H a n t a a n cell extract. None of the a n t i - H a n t a a n
sera or M A F s immunoprecipitated A n h e m b i cell extracts. There were no other crossreactions.
The molecular weights of the proteins of four recognized genera of the family Bunyaviridae are
different from those of H a n t a a n virus. The nucleocapsid proteins of La Crosse, Rift Valley
fever, U u k u n i e m i and A n h e m b i viruses are substantially smaller than that of H a n t a a n virus.
The nucleocapsid protein of Congo virus is closer in size to that of H a n t a a n than to those of the
other three viruses (Table 1).
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L. H. ELLIOTT, M. P. KILEY AND J. B. McCORMICK
(a)
2
..... ~'i :i~•
3
:/.•
ill
~ •
(b)
1
5
2
3
:;::: •
....
: • :
4
•
:::i•
•••
¸
•
Fig. 5. Comparison of polypeptides of Bunyaviridae on 12-5~ acrylamide gels. (a) Four members of the
35 S, partially purified through a sucrose cushion and electrophoresed
on 12.5~ acrylamide gels. Viruses were grown in E6 cells except for La Crosse which was grown in
BHK ceils. The arrow indicates the location of the 42K cellular protein. Viruses, with their times of
harvest, are in the following lanes: (1) Anhembi, 48 h; (2) Congo, 7 days; (3) Hantaan, 7 days; (4) La
Crosse, 48 h; (5) Rift Valley fever, 48 h. (b) Viruses labelled with [aH]leucine, purified on a 20 to 70~
sucrose gradient and electrophoresed on 12.5~ acrylamide gels. Viruses were grown in E6 cells, except
Uukuniemi which was grown in BHK cells. Viruses, with their times of harvest, are in the following
lanes: (1) Congo, 6 days; (2) Hantaan, 6 days; (3) Rift Valley fever, 48 h; (4) Uukuniemi, 6 days.
Bunyaviridae were labelled with
Studies are u n d e r w a y to c o m p a r e the proteins of several newly isolated strains o f H F R S
viruses. Fig. 6 shows the results of S D S - P A G E analysis of SR-11 ( K i t a m u r a et al., 1983) and
T c h o u p a t o u l a s (T. F. Tsai, S. B. Bauer, J. B. M c C o r m i c k & T. K u r a t a , u n p u b l i s h e d U.SI
isolation) c o m p a r e d with H a n t a a n 76-118. T h e y v a r y only in a slight tool. wt. d i f f e r e n c e in the
G l protein.
DISCUSSION
T h e isolation a n d description o f four virion p r o t e i n s analogous to those o f established
m e m b e r s of the family Bunyaviridae adds n e w e v i d e n c e in support o f the inclusion o f H a n t a a n
virus in this family. W e found the smallest o f these proteins to be the nucleoprotcin, w h i c h is
consistent w i t h all prototypes of the four g e n e r a o f the Bunyaviridae, w h e r e the smallest virion
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Hantaan virus proteins
(a)
(b)
(c)
(d)
L
G1
G2
N
Fig. 6. Comparison of HFRS viruses. Supernant fluid from one 75 cm 2 flask of E6 cells per virus was
clarified at 2000 r.p.m, for 10 min, then pelleted at 24000 r.p.m, for 1 h through a sucrose cushion.
Harvests were made on day 6 of infection, the cultures having been labelled with [35S]methionine on
days 4 to 6. SDS-PAGE analysis was on 12.5% acrylamide gels: (a) normal E6 cell supernatant; (b)
Hantaan virus; (c) Tchoupatoulas, and (d) SR-11.
proteins are nucleocapsid-associated (Obijeski & Murphy, 1977). A study by Foulke et al. (1981)
has described two proteins, 42K and 30K, both smaller than the 45K nucleocapsid protein of
H a z a r a virus as structural proteins. However, studies of viral proteins in cell extracts often
demonstrate the presence of actin in the 42K range ( N a i t o & Matsumoto, 1978); a 42K protein is
evident in our Fig. 4 and 5(a) and is clearly a cellular protein. In addition, our studies using
Congo virus (Matin strain) confirm the work of others (Bishop et al., 1980; K u i s m a n e n et aL,
1982; Struthers & Swanepoel, 1982) that p30 is a non-structural protein. Results in our
laboratory confirm the report of Bishop et al. (1980) of the presence of a glycoprotein of Congo
virus in the range 85K to 95K (Fig. 5a, b).
Additional analogy with established Bunyaviridae was shown by the presence of two
glycosylated proteins of intermediate molecular weights and a larger L protein. The fact that
the molecular weights of the proteins of H a n t a a n virus differ from those of members of the
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L.H.
ELLIOTT, M. P. KILEY AND J. B. McCORMICK
family, supports the suggestion that Hantaan virus may belong to a new genus within the family
(Schmaljohn & Dalrymple, 1983). This notion is supported by three other observations: (i)
further electron micrograph studies show that Hantaan virus has a unique structure unlike other
members of the Bunyaviridae (M. L. Martin et al., unpublished results); (ii) the 3' end sequence
of Hantaan virus is different from those of prototype viruses of the four current genera
(Schmaljohn & Dalrymple, 1983); and (iii) no serological crossreactions have yet been found
between Hantaan virus and many members of the four current genera (McCormick et al., 1982;
Tsai et al., 1982a).
A key to our study was the ability to grow Hantaan virus to high titres. Passage in E6 cells
following a harvest from suckling mouse brain increased virus yields from 10- to 100-fold over
other culture substrates (Tsai et al., 1982b). In addition, we found that the use of polyethylene
glycol or potassium tartrate gradients destroyed much of the virus; instead, we pelleted virus in
the J-21 centrifuge and used sucrose gradients.
We did not see significant differences between proteins immunoprecipitated from the
infected cell and virion-related proteins. The same proteins were immunoprecipitated both by
neutralizing convalescent serum from two patients recovered from haemorrhagic fever with
renal syndrome, as well as with serum from a rodent trapped in Baltimore which also had
neutralizing antibody to Hantaan virus (Tsai et al., 1982a). The observation that the virion
structural proteins are immunoprecipitated by human convalesent sera from patients who
acquired HFRS during the Korean War and rodent serum from a rat captured in Baltimore 30
years later suggests a very close relationship between Hantaan-like viruses isolated in the U.S.
with those from Korea. Comparisons of the proteins of additional newly isolated strains from
different parts of the world, and current studies using antisera raised against specific isolates,
will further our understanding of the antigenic diversity of HFRS viruses.
W e gratefully acknowledge the helpful suggestions of D r Helen Lindsey-Regnery and Dr Frederick A. Murphy,
and the excellent photography of A. R a y Simons.
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