J. gen. Virol. (1984), 65, 1385-1393. Printed in Great Britain
1385
Key words: influenza virus A and B/interference/mechanism
Mechanism of Interference Between Influenza A/WSN and B/Kanagawa
Viruses
By H I I Z U A O K I , * Y U K I H I R O N I S H I Y A M A , T A T S U Y A T S U R U M I ,
M O T O H I R O S H I B A T A , Y A S U H I K O ITO,I- H I S A O S E O , 1 S A I J I Y O S H I I 2
AND K O I C H I R O M A E N O
Laboratory of Virology, Research Institute for Disease Mechanism and Control Nagoya
University School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya, l Department of
Endocrinology and Metabolism, Research Institute of Environmental Medicine, Nagoya
University, Nagoya and 2Chubu National Hospital Ohbu, Aichi, Japan
(Accepted 24 April 1984)
SUMMARY
Simultaneous infection of MDCK cells with influenza viruses A/WSN and
B/Kanagawa resulted in mutual interference with virus protein synthesis and in
significant suppression of A / W S N growth. When infection by one virus preceded the
other by 1 or 2 h, growth of the superinfecting virus was selectively inhibited at the level
of transcription. Interference by the pre-infecting virus was strongly dependent on the
expression of the viral genome but not on haemagglutinin activity. When the
replication of both virus types was restricted to primary transcription by cycloheximide, the only translation products following removal of the drug were those of the preinfecting virus. This result was not affected by blocking secondary transcription by
actinomycin D. These findings suggest that intertypic interference occurs at the level of
primary transcription. This concept was supported further by the observation that a ts
mutant of A/WSN (ts-65) with a defect in primary transcription interfered only with
superinfection by B/Kanagawa at the permissive temperature.
INTRODUCTION
The genomes of both type A and B influenza viruses consist of eight single-stranded RNA
segments and these replicate as distinct units (Desselberger & Palese, 1978 ; Racaniello & Palese,
1979). Mixed infection with two different strains of the same type results in high frequency
recombination by genetic reassortment of genome segments (Sugiura, 1975). In spite of many
similarities in biological properties and structure between type A and type B viruses, intertypic
recombination has not been demonstrated (Sugiura, 1975). However, simultaneous infection
with A and B type viruses results in interference with the multiplication of one or both viruses,
depending on the multiplicity of infection (Gotlieb & Hirst, 1954; Tobita & Ohori, 1979;
Mikheeva & Ghendon, 1982; Kaverin et al., 1983).
Recent studies on the molecular basis of interference between influenza A and B viruses have
suggested that it is exerted at the level of primary transcription (Mikheeva & Ghendon, 1982) or
at later transcriptional events (Kaverin et al., 1983). Rottet al. (1981) have demonstrated that
even homotypic interactions between influenza A viruses differ in outcome, depending on the
time of superinfection as well as the multiplicity of the inocula: simultaneous co-infection with
swine virus and fowl plague virus (FPV) results in interference with swine virus only, while
superinfection by FPV l h after swine virus infection leads to genetic interaction. This
information led us to re-examine the interaction between influenza A and B viruses.
This paper presents evidence that simultaneous infection of M D C K cells with influenza
A/WSN and B/Kanagawa/73 led to the primary transcription of the genomes of both viruses
t Present address : Department of Measles Virus, National Institute of Health of Japan, Murayama Annex,
Gakuen, Musashimurayama, Tokyo, Japan.
0022-1317/84/0000-6046 $02.00
© 1984 SGM
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1386
H. AOKI AND OTHERS
a n d a s i g n i f i c a n t s u p p r e s s i o n o f A / W S N yield. I n c o n t r a s t , w h e n i n f e c t i o n b y o n e v i r u s p r e c e d e d
t h e o t h e r by 1 or 2 h, g r o w t h o f t h e s u p e r i n f e c t i n g v i r u s w a s selectively i n h i b i t e d a t t h e level o f
p r i m a r y t r a n s c r i p t i o n d u r i n g t h e p r i m a r y t r a n s c r i p t i o n a l p r o c e s s o f t h e p r e - i n f e c t i n g virus.
METHODS
Virus and cell cultures. Influenza A/WSN/33 (H1N1) and B/Kanagawa/1/73 strains were prepared by
inoculating a 10 6 dilution of stock virus into allantoic sacs of 10-day-old chick embryos, and the virus was
harvested at 40 h after infection. The infectivity and haemagglutination (HA) titre of the virus were 1-5 x
108 p.f.u./ml and 512 HAU/ml for A/WSN, and 2 x 108/ml and 256 HAU/ml for B/Kanagawa. Temperaturesensitive (ts) mutants of A/WSN (ts-53 and ts-65), kindly supplied by Dr A. Sugiura (Institute of Public Health,
Tokyo, Japan), were grown in MDBK cells at 34 °C. HA, neuraminidase (NA) and plaque formation were
measured as described previously (Maeno & Kilbourne, 1970; Shibata et al., 1982). Madin& Darby canine kidney
(MDCK) cells were grown in Eagle's MEM containing 10~ calf serum.
Infection andprotein synthesis. MDCK cell monolayers (l x 106 cells/dish) were infected with virus at an input
multiplicity of 6 p.f.u./cell, unless stated otherwise. Superinfection, when carried out, was at the same m.o.i. After
1 h of adsorption at 35 °C, the cells were washed three times with Hanks' solution and incubated in serum-free
MEM at 35 °C for various times. Cell cultures were frozen and thawed and examined for virus yields. For
radiolabelling, the culture medium was removed and replaced with L-[3sS]methionine (Amersham, 1000 Ci/mmol)
in medium lacking unlabelled methionine. After further incubation at 35 °C for appropriate times, the cells were
harvested and examined for radioactive polypeptides by 10~ SDS-PAGE and autoradiography as described
previously (Maeno et al,, 1979). In some experiments, radioactive polypeptide bands were cut from the dried gel by
using the autoradiogram as a reference template and the radioactivities were determined in a Beckman liquid
scintillation spectrometer in a toluene-based scintillation cocktail.
Preparation of virion RNA (vRNA) and ~2s I-labelled vRNA. After clarification of virus-containing allantoic fluid,
virions were pelleted at 20000 r.p.m, for 1 h and the virus suspension was layered on a 10 to 4 0 ~ (w/v) linear
sucrose density gradient in phosphate-buffered saline (PBS) and centrifuged in a Beckman SW27 rotor for 45 min
at 18000 r.p.m. The visible band was collected, resuspended in PBS, and pelleted at 20000 r.p.m, for 1 h. The
virion RNA (vRNA) was extracted repeatedly with phenol--chloroform (1 : 1), precipitated from the final aqueous
phase with 2 M-LiC1 for 16 h at 4 °C and dissolved in distilled water. The amount of RNA was calculated assuming
that 1 A260 unit is 40 ~tg/ml (Seo et al., 1977). 125I-labelled vRNA was prepared by the thallium chloride procedure
of Tereba & McCarthy (1973).
R N A - R N A hybridization. Extraction of RNA from uninfected cells (1 x 10s cells) was carried out according to
the method of Seo et al. (1977). The extracted RNA preparations were serially diluted in 30 ~tl of distilled water,
mixed with 20 ~tl of 0-1 M-Tris HC1 pH 7.4, containing 0.75 M-NaC1, 5 mM-EDTA, 0.05 ~ SDS, and 125I-labelled
vRNA and held at 68 °C for 96 h. Each mixture was then split into two parts, one being treated with S 1 nuclease at
40 °C for 1 h and the other left at 40 °C. The acid-precipitable radioactivity was determined in an Aloka Auto Well
Gamma System.
U.v, irradiation. Virus-containing allantoic fluid was clarified by centrifugation at 1000 g for 20 min and 2 ml of
the supernatant was placed in a 9 cm Petri dish and exposed to u.v. radiation at a rate of 2.8 J/m2s with occasional
shaking.
Antisera. Antisera against A/WSN and B/Kanagawa were prepared by intravenous injection of purified virions
into rabbits as described previously (Maeno & Kilbourne, 1970). Each antiserum had a haemagglutination
inhibition (HI) titre of 5120 against 4 HA units of homologous virus.
Chemicals. Na 125I (100 mCi/ml) and $1 nuclease (100000 to 200000 units/rag protein) were purchased from
Amersham and Sigma, respectively.
RESULTS
I n t e r f e r e n c e b e t w e e n influenza A / W S N
and B/Kanagawa
M D C K m o n o l a y e r s w e r e i n f e c t e d i n d i v i d u a l l y or s i m u l t a n e o u s l y w i t h A / W S N a n d
B / K a n a g a w a (m.o.i. o f 6 p.f.u, e a c h ) a n d t h e yield o f e a c h a t 12 h a f t e r i n f e c t i o n w a s m e a s u r e d
b y t h e use o f a n t i s e r u m . T a b l e 1 s h o w s t h a t t h e v i r u s yields o f B / K a n a g a w a w e r e c o m p a r a b l e to
t h o s e in singly i n f e c t e d cultures, b u t A / W S N w a s p r o d u c e d in s i g n i f i c a n t l y r e d u c e d a m o u n t s .
Cells were i n f e c t e d w i t h e i t h e r A / W S N or B / K a n a g a w a a n d 1 to 2 h l a t e r s u p e r i n f e c t e d w i t h t h e
o t h e r v i r u s type. T h e g r o w t h o f t h e s u p e r i n f e c t i n g v i r u s was selectively i n h i b i t e d , i r r e s p e c t i v e o f
t h e v i r u s type ( T a b l e 1). C o - i n f e c t e d cells w e r e l a b e l l e d for 1 h w i t h [ 3 5 S ] m e t h i o n i n e 6 h a f t e r
s i m u l t a n e o u s i n f e c t i o n or s u p e r i n f e c t i o n , in p a r a l l e l w i t h singly i n f e c t e d cultures, a n d e x a m i n e d
for v i r a l p o l y p e p t i d e s b y P A G E a n d a u t o r a d i o g r a p h y (Fig. 1). I n singly i n f e c t e d cells t h e H A ,
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1387
Mechanism of interference in influenza viruses
0
60
120
i i
B
1
AB
2
A
3
min
it
B
4
AB
5
A
6
I
B
7
AB
8
A
9
U
10
A
NP
i,,, tn.
Fig. 1. PAGE analysis of viral proteins of A/WSN and B/Kanagawa in singly or mixedly infected
MDCK cells (m.o.i. of 6 p.f.u, each). Cells were infected with B/Kanagawa, simultaneously (lanes 1, 2,
3), 1 h (lanes 4, 5, 6) or 2 h (lanes 7, 8, 9) before superinfection with A/WSN and incubated for another
6 h. Singly infected cultures were also prepared in parallel as controls. The culture medium was then
removed and the cells were labelled for 1 h with [35S]methionine (2.5 ~tCi/ml). Unlabelled virion
polypeptides were also run in parallel as markers (not shown). Migration is from top to bottom in this
and subsequent figures. B, B/Kanagawa-infected cells; A, WSN-infected cells; AB, mixedly infected
cells, U, uninfected cells.
NP, NS 1 and M of B/Kanagawa, and N P and M + NS1 of A / W S N were clearly visible, but the
H A of A / W S N , and P proteins and N A of both types could not be observed under these
conditions. In simultaneously co-infected cells, these polypeptides of both type A and type B
viruses, except for N S of B/Kanagawa, were synthesized in reduced amounts (lane 2). A similar
observation has been described by K a v e r i n et al. (1983). W h e n N P bands were cut from the gel
and examined for their radioactivities, the results showed that the amounts of N P of A / W S N
and B / K a n a g a w a in simultaneously co-infected cells were 27 ~ and 41 ~ respectively o f those in
singly infected cultures. Immunofluorescent staining revealed the presence of type A and type B
N P antigens in all cells o f the co-infected cultures (data not shown). W h e n B / K a n a g a w a
preceded A / W S N by 1 or 2 h, the protein synthesis of superinfecting A / W S N was selectively
inhibited (lanes 5 and 8). The reciprocal result was obtained when A / W S N preceded
B/Kanagawa. W h e n B / K a n a g a w a preceded A / W S N by 2 h, the amounts of labelled N P o f
A / W S N and B / K a n a g a w a were respectively 9~o and 96~o of those in singly infected cultures.
The molecular basis of this interference was investigated in the following experiments.
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1388
H. A O K I A N D O T H E R S
(a)
I
I
I
(b)
~o
M
×
10
6.~_
.~
?
"N
Z
I
I
I
I
0.25
0.5
0.25
0.5
R N A (gg/gl)
Fig. 2. Annealing of i 25I-labelled v R N A of A / W S N or B / K a n a g a w a to R N A s from co-infected, singly
infected and uninfected cells. M D C K cells were infected with B/Kanagawa, 2 h later superinfected
with A / W S N and incubated for another 2 h. Singly infected cultures were prepared in parallel as
controls. Increasing amounts of the R N A were mixed with 20 gl aliquots of l z s I - v R N A in a total
volume of 50 pl. A 20 pl sample of each mixture was annealed in duplicate for 96 h at 68 °C and each
sample was further divided into equal parts. One was treated with $1 nuclease and the other not, and
RNase-resistant radioactivity was recorded as a proportion of the a m o u n t of R N A used. After
hybridization of 125I-vRNA with over 0.25 l~tg/gl of R N A from B/Kanagawa-infected or A / W S N infected cells, the R N a s e resistance of v R N A from B / K a n a g a w a and A / W S N was 5 0 ~ and 6 0 ~
respectively. Hybridization of 1:s I - v R N A of A / W S N (a) or B / K a n a g a w a (b) with R N A from A / W S N infected cells (O), B/Kanagawa-infected cells (1), co-infected cells (O) and uninfected cells (Fq) is
shown.
T a b l e 1.
Virus growth in MDCK cells mixedly infected with A~WSN and B/Kanagawa
Virus yields*
h
Normal serum
Inoculum
A / W S N alone
B/Kanagawa alone
B/Kanagawa + A / W S N t
0
60
120
A / W S N + B/Kanagawa:~
60
120
Anti-B serum
Anti-A serum
HA
(0.25 ml)
P.f.u.
(1 ml)
HA
(0.25 ml)
P.f.u.
(1 ml)
HA
(0.25 ml)
P.f.u.
(1 ml)
512
256
4.1 x 106
2.7 × 106
256
<4
2.6 × 106
<104
<4
256
<104
1.2 × 106
256
512
256
ND§
5.5 × 105
128
1.0 x 106
4"0 × 104
128
2"0 × 106
ND
8
4
<4
<104
128
1.0 x 106
256
512
ND
ND
128
256
1.3 × 106
2"5 × 106
16
<4
1.0 x 105
<104
ND
* At 12 h after infection with the first virus, cells and culture fluids were frozen and thawed and assayed for virus
yields in the presence or absence of antiserum (1/400 dilution).
t B/Kanagawa-infected cells were superinfected with A / W S N at the indicated times (min) after the first
infection.
Cells were infected with A / W S N 60 or 120 min before superinfection with B/Kanagawa.
§ ND, Not determined.
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1389
Mechanism of interference in influenza viruses
B
1
BA
A
0
40
120
240
2
3
4
5
6
A
mNp
- - M + NS~
Fig. 3. Protein synthesis in MDCK cells mixedly infected with u.v.-inactivated B/Kanagawa and
A/WSN. Cells were infected with B/Kanagawa (lanes 2) or the same preparation u.v.-irradiated for 40 s
(lane 3), 120 s (lane 4) or 240 s (lane 5) 2 h before superinfection with A/WSN (m.o.i. 6 p.f.u, each) and
incubated for another 6 h. The medium was then removed and cells were labelled for 1 h with
[35S]methionine (2.5 ~tCi/ml). Lane 1, B/Kanagawa-infected cells; lane 6, A/WSN-infected cells.
Synthesis of viral RNA in co-infected cells
Cells were superinfected with A / W S N 2 h after infection by B/Kanagawa. After a further
2-5 h incubation, R N A was extracted from each of the singly and mixedly infected cultures as
described in Methods. Increasing amounts of the R N A were hybridized with 125I-labelled
v R N A from B / K a n a g a w a or A / W S N virions and the RNase-resistant radioactivities were
measured. Labelled v R N A from B / K a n a g a w a was protected from R N a s e by hybridization with
increasing amounts of R N A from co-infected and B/Kanagawa-infected cells but not with the
R N A from A / W S N - i n f e c t e d cells (Fig. 2b). In contrast, labelled v R N A from A / W S N was
protected from R N a s e only by hybridization with high concentrations of the R N A from
A / W S N - i n f e c t e d cells (Fig. 2a). These results indicate that pre-infecting B / K a n a g a w a
interferes with c R N A synthesis of the superinfecting A / W S N .
Inactivation of interference by u.v. light
Exposure of B / K a n a g a w a to u.v. radiation resulted in selective inactivation of its infectivity
without any loss of H A and N A activities (Table 2). W h e n inactivated with u.v. light to a
survival level of less than 10-5 (Table 2), B / K a n a g a w a lost its ability to inhibit H A yields of
A / W S N . U n d e r these conditions, B/Kanagawa-specific proteins were barely detectable and,
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H. AOKI AND OTHERS
1390
T a b l e 2. U.v. inactivation of biological activities of B/Kanagawa virus*
Viral activities
~k
r
HA yields in mixedly
infected cells (0.25 ml)
A
Irradiation
time (s)
0
40
120
240
HA
(0.25 ml)
256
256
256
256
NA t (A549)
(0-1 ml)
0.233
0"270
0"210
0,249
P.f.u.
(1 ml)
2 x l0 s
5 × 104
<1 × 103
<1 x 103
Anti-B/Kanagawa
+
<4
<4
32
128
serum:~
_
128
128
128
128
* See also legend to Fig. 3.
i" Neuraminidase assay was performed with a fetuin substrate by a modification of Warren's thiobarbituric acid
method and neuraminidase activity was measured by Asa 9 readings.
:[:Irradiated B/Kanagawa plus A/WSN. Yield at 12 h post-infection was measured in the presence of antiB/Kanagawa serum (+) or normal serum ( - ) ,
T a b l e 3. HA yield of superinfecting B/Kanagawa after mixed injections with A~ WSN (wild-type
or ts mutants) and B/Kanagawa*
HA titres (0.25 ml)
A
Inoculum virus
WSN wild-type
+
B/Kanagawa
Ts-65
+
B/Kanagawa
Ts-53
+
B/Kanagawa
Incubation temperature
(°C)
39.5 + 34
_
_
Anti-A/WSN serum
+
<4
128
34 + 34
39-5 + 34
<4
128
128
128
34 + 34
39.5 + 34
4
4
128
128
34 + 34
8
128
* MDCK cells were infected with A/WSN (wild-type, ts-65 or ts-53) and incubated at 34 °C or 39.5 °C for 2 h.
The cells were then superinfected with B/Kanagawa and incubated at 34 °C for another 12 h. The cultures were
frozen and thawed and assayed for HA activity in the presence of anti-A/WSN serum (+) or normal serum ( - ).
instead, N P and M + N S 1 of A / W S N a p p e a r e d in a p p r e c i a b l e a m o u n t s (Fig. 3, lanes 4 and 5).
T h e s e results indicate that the expression of the interfering virus g e n o m e but not H A activity is
essential for the interference effect.
Interference occurs at the level of primary transcription
Cells were infected with B / K a n a g a w a 2 h before s u p e r i n f e c t i o n by A / W S N (m.o.i. of 30 p.f.u.
each) and i n c u b a t e d for a n o t h e r 4 h. C y c l o h e x i m i d e (100 ~tg/ml) was a d d e d to the cultures
30 m i n before the p r i m a r y infection to restrict virus replication to p r i m a r y t r a n s c r i p t i o n
(Scholtissek & Rott, 1970; Pons, 1973). At the end of infection, the drug was r e m o v e d and the
cells were labelled for 15 m i n with [35S]methionine (200 ~tCi/ml) (Fig. 4).
S i m u l t a n e o u s co-infection led to the synthesis of N P polypeptides of b o t h types (lane 4). In
contrast, w h e n B / K a n a g a w a p r e c e d e d A / W S N , viral protein synthesis was confined to the N P
o f the pre-infecting B / K a n a g a w a (lane 2). T h i s can be interpreted as i n d i c a t i n g t h a t the
replication of A / W S N is interfered w i t h at the level o f p r i m a r y t r a n s c r i p t i o n or at an earlier step.
A c t i n o m y c i n D (1 p~g/ml), w h i c h inhibits the t r a n s c r i p t i o n o f the influenza virus g e n o m e (Barry
et al., 1962; Scholtissek & Rott, 1970; Ports, 1973), was added to the cultures 30 rain before the
r e m o v a l o f c y c l o h e x i m i d e to i n h i b i t secondary transcription, but this t r e a t m e n t had little effect
on viral protein synthesis (data not shown), indicating that the viral proteins synthesized u n d e r
the a b o v e conditions are the translation products of the p r i m a r y transcripts.
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Mechanism o f interference in influenza viruses
B
1
2
3
4
5
6
1391
A
Fig. 4. Induction of interference in the presence of cycloheximide. MDCK cells were infected with
B/Kanagawa (m.o.i. 30 p.f.u.), 2 h later superinfected with A/WSN (m.o.i. 30 p.f.u.), and incubated for
another 4 h. Cycloheximide (100 ~tg/ml) was added to the cultures from 30 min before the primary
infection. The cells were washed with chilled MEM and pulse-labelledfor 15 min with [35S]methionine
(200 ~tCi/ml)in prewarmed methionine-free MEM, followed by PAGE and autoradiography (lane 2).
Uninfected (lane 6), B/Kanagawa-infected (lanes 1,5), A/WSN-infected (lane 3), and simultanouslycoinfected cultures (lane 4) were also prepared in parallel as controls. B/Kanagawa-infected (lane B) and
A/WSN-infected cells (lane A) were pulse-labelledfor 1 h with pS]methionine 6 h after infection and
run in parallel.
A / W S N ts-65 has no capacity to initiate primary transcription at the non-permissive
temperature (39.5 °C), while ts-53 fails to induce v R N A synthesis subsequent to primary
transcription at 39 -5 °C (Sugiura et al., 1975 ; Krug et al., 1975). Using these mutants, we further
examined the interaction between A / W S N and B/Kanagawa. Cells were infected with one ts
mutant of A / W S N and incubated for 2 h at the permissive (34 °C) or non-permissive
temperature before superinfection with B/Kanagawa. After a further 10 h incubation at the
permissive temperature, the cultures were examined for H A activities in the presence or absence
of anti-A/WSN serum. As shown in Table 3, wild-type and ts-53 both inhibited the H A yield of
B/Kanagawa even at 39.5 °C, but ts-65 failed to interfere with B/Kanagawa only at the nonpermissive temperature. These results suggest that intertypic interference between A / W S N and
B/Kanagawa occurs at the level of the primary transcription.
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H. A O K I A N D O T H E R S
DISCUSSION
When infection by influenza virus type A or B preceded infection by virus of the other type by
1 or 2 h, the pre-infecting virus completely inhibited the synthesis of cRNA as well as of proteins
of the superinfecting virus. U.v. irradiation provided evidence that this interference by the preinfecting virus depends on the expression of the virus genome but not the HA activity. The
initial event in the expression of the influenza virus genome is primary transcription induced by
virion-associated RNA polymerases. When virus replication in co-infected cells was restricted
by cycloheximide to primary transcription, after removal of the drug only protein synthesis by
the pre-infecting virus was detected. This viral protein synthesis did not appear to be affected by
blocking secondary transcription by the addition of actinomycin D. These results suggest that
primary transcription by the pre-infecting virus is responsible for interference with the
replication of superinfecting virus at the level of the latter's primary transcription or at an earlier
step. This concept was supported by experiments on the interaction between B/Kanagawa and
ts mutants of A/WSN. Mutant is-53, with a ts-defect in v R N A synthesis, interfered with the
growth of superinfecting B/Kanagawa at both the permissive and non-permissive temperatures,
while ts-65, having a ts defect in the primary transcription, failed to do so only at the nonpermissive temperature. These results provide further evidence that intertypic interference
between A/WSN and B/Kanagawa occurs at the level of primary transcription and not at some
earlier stage such as uncoating.
Simultaneous infection of M D C K cells with influenza A/WSN and B/Kanagawa led to
significant suppression of the multiplication of A/WSN, but A/WSN protein synthesis
appeared to be less suppressed than virus yield. Similar results were also described by Tobita &
Ohori (1979), and preferential suppression of the expression of the HA and NP genes of
influenza A virus has recently been reported by Kaverin et al. (1983). We could not determine
the inhibitory effect of B/Kanagawa on the synthesis of the A/WSN HA polypeptide, because of
its poor incorporation of radioactive methionine. Thus, it is possible that the drastic suppression
of A/WSN yield in simultaneously co-infected cells could be due to preferential inhibition of
transcription of the HA gene.
Mikheeva & Ghendon (1982) have recently reported that, on simultaneous infection with
influenza A and B type viruses, one interferes with the replication of the other at the level of
primary transcription, although which type of virus is inhibited varies from experiment to
experiment. This finding contrasts with our data. When virus replication in simultaneously coinfected cells was restricted to primary transcription by cycloheximide, the translation products
of both virus types were clearly detected, suggesting that simultaneous co-infection induces
primary transcription of both type A and type B virus genomes, although the possibility of
mutual interference at the level of primary transcription remains to be tested. More recent
evidence has been presented by Kaverin et al. (1983), suggesting that intertypic interference
occurs not at the level of primary transcription but at subsequent transcription. Taken together,
the interference between influenza A and B viruses at the ievel of transcription may explain in
part why there is no genetic interaction between influenza A and B type viruses (Sugiura, 1975).
We thank E. Iwata and T. Tsuruguchi for their excellent technical assistance. This work was supported by a
research grant from the Ministry of Education, Science and Culture of Japan and in part from The Ishida
Foundation research grant for 1982.
REFERENCES
BARRY, R. D., IRES, D. S. & CRUICKSHANK,J. G. (1962). Participation of deoxyribonucleic acid in the multiplication
of influenza virus. Nature, London 194, 1139-1140.
DESSELBEROER, U. & PALESE, P. 0978). Molecular weight of R N A segments of influenza A and B viruses. Virology
88, 394-399.
GOTLIEB, T. & HmST, G. K. 0954). The experimental production of combination forms of virus. III. The formation
of doubly antigenic particles from influenza A and B virus and study of the ability of individual particles to
yield two separate strains. Journal of Experimental Medicine 99, 307-320.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 06:54:23
Mechanism o f interference in influenza viruses
1393
KAVERIN, N. V., VARICH, N. L., SKLYANSKAYA, E. I., AMVROSIEVA,T. V., PETRIK, J. & VOVK, T. C. (1983). Studies on
heterotypic interference between influenza A and B viruses: a differential inhibition of the synthesis of viral
proteins and R N A s . Journal of General Virology 64, 2139-2146.
KRUG, g. M., UEDA, M. & PALESE, P. (1975). Temperature-sensitive m u t a n t s of influenza W S N virus defective in
virus-specific R N A synthesis. Journal of Virology 16, 790-796.
MAENO, K. & KILBOURNE, E. D. (1970). Developmental sequence and intracellular sites of synthesis of three
structural antigens of influenza A2 virus. Journal of Virology 5, 153-164.
MAENO, K., YOSHII, S., MITA, K., ttAMAGUCHI, M., YOSHIDA,T., IINUMA, M., NAGAI, Y. & MATSUMOTO,T. (1979). Analysis
of the inhibitory effect of canavanine on the replication of influenza RI/5 + virus. I. Inhibition of assembly of
R N P . Virology 94, 128-137.
MIKHEEVA,A. & GHENDON, e. z. (1982). Intrinsic interference between influenza A and B virus. Archives of Virology
73, 287-294.
PONS, M. (1973). The inhibition of influenza virus R N A synthesis by actinomycin D and cycloheximide. Virology
51, 120-128.
RACAYIELLO,V. R. & PALESE, P. (1979). Influenza B genome: assignment of viral polypeptides to R N A segments.
Journal of Virology 29, 361-373.
ROTT, R., ORLICH, M. & SCHOLTISSEK, C. (1981). Intrinsic interference between swine influenza and fowl plague
virus. Archives of Virology 69, 25-32.
SCHOLTISSEK, C. & ROTT, R. (1970). Synthesis in vitro of influenza virus plus and m i n u s strand R N A and its
preferential inhibition by antibodies. Virology 40, 989-996.
SEO, H., V~SAgT, G., BROCaS, H. & REFETOFF, S. (1977). Triiodothreonine stimulates specifically growth hormone
m R N A in rat pituitary tumor cells. Proceedings of the National Academy of Sciences, U.S.A. 74, 2054-2058.
SHmATA, M., MAENO, K., ISOMURA,S., TSURUMX,T., AOKI, a. & SUZUKI, S. (1982). Plaque formation by influenza B
virus in a porcine kidney cell line. Microbiology and Immunology 26, 441-444.
suGIupo~, A. (1975). In The Influenza Viruses and Influenza, pp. 171-213. Edited by E. D. Kilbourne. N e w York:
Academic Press.
SUGIURA, A., UEDA, M., TOBITA, K. & ENOMOTO, C. (1975). Further isolation and characterization of temperaturesensitive mutants of influenza virus. Virology 65, 363-373.
TEREBA, A. & McCARTHY,B. J. (1973). Hybridization of 125I-labelled ribonucleic acid. Biochemistry 12, 4675-4679.
TOBITA, K. & OHORI, K. (1979). Heterotypic interference between A/Aichi/2/68 and B/Massachusetts/I/71. Acta
virologiea 23, 263-266.
(Received 6 January 1984)
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 06:54:23