Download Intrinsic Interference between Different Enveloped RNA Viruses

J. gen. Virol. 0972), x7, 255-264
255
Printed in Great Britain
Intrinsic Interference between Different Enveloped R N A Viruses
By R. R O T T , C. S C H O L T I S S E K ,
H.-D. KLENKAND G. K A L U Z A
Institut fiir Virologie, Justus Liebig-Universith't, Giessen, Germany
(Accepted 24 July I972 )
SUMMARY
Intrinsic interference is not limited to Newcastle disease virus: vesicular stomatitis (VSV) and orthomyxoviruses can be similarly inhibited at the stage of R N A
synthesis. Detailed investigations of the interference of fowl plague virus (FPV)
by VSV show: (I) adsorption of the interfering virus is not responsible; (2) the
synthesis of all FPV virus components is affected, if the interfering virus is
present during the initial stages of FPV replication and (3) competition of the
interfering R N A polymerase with heterologous templates can be excluded by
experiments carried out in vitro.
INTRODUCTION
Several unrelated viruses have been found to induce an inhibition of the multiplication
o f Newcastle disease virus (NDV) as determined by the haemadsorption-negative plaque
test (Marcus & Carver, 1965, 1967; Beard, 1967, Wainwright & Mims, 1967; Seto & Carver,
I969; Marcus & Zuckerbraun, r97o ). The NDV-refractory state develops only in those
individual cells of a population actually infected by the inducing virus, and was termed
intrinsic interference. Intrinsic interference thus differs from mutual exclusion at the
adsorption step. It is defined as' a viral genome-induced cellular state of resistance to challenge
by high multiplicities of NDV, coexistent with a state of susceptibility to a broad spectrum
of other viruses' (Marcus & Carver, 1967). Unlike interferon-mediated interference, intrinsic
interference does not depend on new cellular R N A synthesis.
We have found that this kind of interference is not confined to NDV. Semliki Forest
virus (SFV) can induce intrinsic interference also of vesicular stomatitis (VSV) and fowl
plague viruses-(FPV). Furthermore, influenza viruses are inhibited by VSV, SFV and NDV.
METHODS
Virus strains. The various influenza strains used were the same as described recently
(Scholtissek & Rott, 1969). Furthermore, the NDV strain ITALIEN,the SFV strain OSTERRIETH,
and the VSV, type Indiana, were investigated.
Tissue cultures and virus multiplication. Primary chick fibroblasts at a density of 2 x io 7
cells in Petri dishes of 9 cm in diameter were infected 46 h after seeding. In a few experiments BHK-cells were used. The cells were infected with an input multiplicity of IO to 5o
p.f.u./cell. At the times after infection indicated, virus yields were determined. Intracellular
virus was assayed after breaking the cells by 3 cycles of freezing and thawing. Since the titres
o f the virus activities in the culture medium were as a rule only about IO ~ or less of that
of the cell-associated ones, only the latter have been listed in the Tables and Figs. Thus
there was no significant interference with virus release.
Virus infectivity was determined by the plaque technique in primary chick fibroblasts. In
order to determine the yield of p.f.u, of either virus after a double infection, one virus was
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256
R. R O T T , C. S C H O L T I S S E K ,
H.-D. K L E N K
A N D G. K A L U Z A
neutralized by mixing samples of the cell extracts with specific antiserum directed against
one of the two viruses used. The extract-antibody mixtures were left at room temperature
for 3o rain and diluted serially for the plaque test.
Haemagglutination tests were performed in the usual manner with chick erythrocytes (for
influenza viruses at room temperature, for NDV at 4 °C). Haemagglutinating units (H.A.U.)
represent the reciprocal of the haemagglutination titre.
Neuraminidase activity was determined as before (Drzeniek, Seto & Rott, I966 ) using
bovine sialolactose as substrate. The amount of neuraminidase which liberated I /~M-sialic
acid from the substrate/min at 37 °C was defined as one enzyme unit (EU).
RNP-antigen was determined by complement fixation (Schmidt & Lennette, I965).
Sera. The following antisera were used for virus neutralization: FPV and NDV specific
antisera were prepared in chicken according to Rott (I965); SFV and VSV specific antisera
from rabbits were obtained by Dr M. Mussgay, Ttibingen. Volumes ofo.I ml of the respective
antisera neutralized at least io 3 p.f.u./ml. Monospecific antisera against RNP-antigen was
prepared in rabbits. Contaminating antibodies against host material and envelope components were removed with solid immunabsorbents (Becht, I97~).
Determination of virus RNA. The RNA polymerase test was performed according to
Scholtissek (1969). Cytoplasmic fractions of four pooled cultures were prepared at the times
after infection as listed in Tables 3 and 5. The RNA labelled during a 20 rain pulse at 36 °C
was isolated and dissolved in 2 ml of 5 mM-tris HC1, pH 7"4, containing I mM-EDTA.
Samples were heated for 5 min at Ioo °C and rapidly chilled. To one sample RNase was
added immediately, to a second one saturating amounts of non-labelled fowl-plague particle
R N A (7 #g/sample) and to a third one an excess of non-labelled fowl-plague complementary
R N A was added prior to hybridization. A fourth sample was self-annealed.
FPV particle and complementary RNA was determined in the presence of newly synthesized cellular R N A by specific hybridization with an excess of non-labelled virus-specific
R N A (Scholtissek & Rott, I97O). For each time point in Table 4 the cells of four culture
plates were pooled. To each culture 25 #Ci [3H]-uridine was added at the times after the
first infection as indicated. The cells were processed I h later and the RNA was isolated
from the cytoplasmic extract. Samples of the dissolved RNA were treated as described above.
The non-labelled particle RNA of FPV used for hybridization contained o'7 mg/ml virus
RNA. The absolute concentration of the complementary RNA is not known. The preparation was titrated before use in order to be sure to work under saturating conditions. In
a preliminary experiment it was found that after swelling of the infected cells in a hypotonic
buffer and homogenization, about 9o ~ of virus RNA could be isolated from the cytoplasmic
extract while only 50 ~ of the radioactive cellular RNA was found in the fraction. In this
way the background of RNase-resistant cellular RNA was negligible. N D V - R N A in the
presence of heterologous virus R N A was determined by specific hybridization with RNA
isolated from NDV particles according to Kingsbury (I966).
Labelling of the proteins of the infected cells. Confluent monolayers were inoculated with
an input multiplicity of Io to 5o p.f.u./cell. After an adsorption period of 3o rain, the inoculum
was removed and replaced by minimal medium (Eagle & Habel, t956). The medium was
removed 6 h after infection and replaced by medium containing radioactive isotopes. These
were used at the following concentrations: [14C]-protein hydrolysate, Io/~Ci/ml; [3H]amino acids, Ioo/~Ci/ml. The medium containing the isotopes was left on the cells for I h.
The monolayer was washed 3 times with cold phosphate buffered saline (PBS), scraped off
in I ml PBS with a rubber policeman, and stored at - 2o °C.
Polyaerylamide gel electrophoresis. The cells were disrupted by ultrasonic vibration.
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Intrinsic interference in R N A viruses
257
Samples of ~oo #1 were used for gel electrophoresis. Proteins were dissociated with SDS
a n d mercaptoethanol. They were separated by electrophoresis o n lo % acrylamide gel as
described (Caliguiri, K l e n k & Choppin, x969). The slicing a n d processing of gels for the
d e t e r m i n a t i o n of radioactivity by liquid scintillation has been reported previously (Klenk,
Caliguiri & Choppin, 197o).
Radioisotopes. Guanosine-5'-triphosphate-8-[ZH] (9 Ci/m-mol) was o b t a i n e d from
Schwarz Bio Research, Orangeburg, N.Y., U.S.A. Uridine-5-[3H] (3o Ci/m-mol), protein
hydrolysate-[14C] (U), L-leucine-4,5-[3H] (I.O Ci/m-mol), L-valine-2,3-[3H] (1"5 Ci/m-mol),
a n d L-tyrosine-3,5-[ZH] 0 " o C i / m - m o l ) were purchased from Radiochemical Centre,
A m e r s h a m , Buckinghamshire, England.
RESULTS
Multiplication of various RNA-containing viruses in mixedly infected cells
Table I shows the results of m u t u a l cross-infections of chick fibroblasts with SFV, VSV,
N D V a n d FPV. It can be seen that SFV interferes with the synthesis of all the other viruses
tested. I n addition, VSV prevents the multiplication of FPV. VSV does n o t affect N D V a n d
SFV, N D V does n o t interfere with VSV, F P V a n d SFV, neither does F P V with N D V , VSV
a n d SFV. Essentially the same results were obtained, when the p r i m a r y infected cells were
super-infected 2 h later with the exception that in this case preinfection with NDV, too, caused
Table i. Virus yieM after mixed infections
Viruses assayed
Viruses
SFV
(p.f.u./ml)
VSV
(p.f.u./ml)
--
< lO6
(3'2 X I O 9)
SFV
-
VSV
-
3 x IO 8
(5 X IO 8)
NDV
I x 109
(2 x I09)
FPV
5 x 1o 7
( I "5 X I08)
--
--
NDV
r
~
~
(p.f.u./ml)
H.A.U.
FPV
~
~
(p.f.u./ml)
H.A.U.
1.5 x lO~
(2"5 X I0v)
2× I07
4
(246)
98
3 ×IOG
(I "2 X IOs)
3"5 x 105
< 4
(1024)
< 2
(224)
(1"3 X IO s)
(iO24)
(2 × 107)
1 x 107
--
--
9 x 107
770
(I"7 x to7)
---
--
(4"3 X 107)
(512)
7"5 x 107
(I ' 7 x I08)
2 ' 5 × 107
(5 X I07)
512
--
(770)
--
---
Mixtures were prepared with paired viruses; the cells were infected with this mixture; 8 h after infection
yield of the viruses listed in the upper line was determined by plaque assays employing specific antisera to
suppress growth of the other viruses in the test. Controls in parentheses: p.f.u, or H.A.U. for the respective
virus grown without any second virus.
Table 2. Double infection of FPV and u.v.-inactivated VSV
FPV
-~
H.A.U.
FPV
VSV
IO24
--
VSV (u.v.)
FPV+VSV
FPV+VSV (u.v.)
-<2
256
-,
p.f.u./ml
I "5 × I08
--
-3 x lO~
5 × [07
VSV
(p.f.u./ml)
-i × 108
4 x lO~
5"5 x lO8
--
U.v.-inactivated VSV and infectious FPV were mixed and applied to chicken fibroblasts; 8 h later the virus
yields were determined.
17
VIR
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17
258
R. ROTT, C. SCHOLTISSEK, H.-D. K L E N K AND G. K A L U Z A
8
I
r
r
I
I
I
1
I
z
I
l o o o - ~" 2
7
6
-
4
I
I
x
-x
,._1
~ 500-._"~ 1
-
A
X
50 L~
-Z
I
I
100~
1
1
2
3
4
Addition of VSV after infection
with FPV (h)
0
Fig. I
0
1
2
3
4
Addition of VSV after infection with FPV (h)
Fig. 2
Fig. I. Formation of p.f.u, in the presence of VSV. VSV was added to FPV infected cells at the
time indicated. Infectious FPV was assayed both at the time of addition of VSV ( O - - O ) and
8 h after FPV infection (0--0).
Fig. 2. Formation of neuraminidase, haemagglutinin and RNP-antigen in the presence of VSV.
Cultures were mixedly infected as in Fig. i. Assays and controls were made 8 h after infection. As
a control ( x ) the virus titre without any addition of VSV 8 h after infection of FPV was determined. O - - O , neuraminidase; 11--O, haemagglutinin; A--A, RNP-antigen.
a considerable r e d u c t i o n o f infectious F P V ( a b o u t 2 log units). This delayed effect can be
explained by the fact t h a t in c o m p a r i s o n to S F V a n d VSV, N D V grows m o r e slowly. O n the
o t h e r hand, the r a p i d l y multiplying S F V inhibited the g r o w t h o f the other viruses even when
a d d e d 2 h after the p r i m a r y infection. I n the following, the inhibition o f F P V by VSV was
s t u d i e d in m o r e detail.
Requirement of active VSV for the inhibition of FPV
VSV inactivated with u.v. to a survival level o f 4 x I@ p.f.u./ml was c o m p a r e d with active
virus (I x 10 8 p.f.u./ml) in its capacity to prevent F P V multiplication. The results are presented in Table 2. As can be seen, the infectivity o f VSV particles is essential to inhibit the
g r o w t h o f F P V . This shows t h a t a d s o r p t i o n o f the interfering virus c a n n o t be responsible
for the effect.
Influence of VSV on various stages of FPV multiplication
In o r d e r to o b t a i n some i n f o r m a t i o n on the intracellular effect o f VSV on F P V replication, VSV was a d d e d at different stages o f the infectious cycle. The p r o d u c t i o n o f R N P antigen, haemagglutinin, n e u r a m i n i d a s e a n d p.f.u, was examined.
A s shown in Figs. I a n d 2, VSV b l o c k e d the f o r m a t i o n o f p.f.u., n e u r a m i n i d a s e a n d
h a e m a g g l u t i n i n completely only when a d d e d simultaneously with F P V . U n d e r these conditions small a m o u n t s o f R N P - a n t i g e n were still detectable. W h e n VSV was a d d e d 2 h before
F P V infection, R N P - a n t i g e n synthesis was c o m p l e t e l y blocked. The yield o f virus m a t e r i a l
increased as the p e r i o d between F P V infection a n d superinfection with the interfering VSV
was extended until a m i n i m u m i n h i b i t o r y effect was reached at 4 h. It s h o u l d be stressed t h a t
when VSV was a d d e d I h after p r i m a r y infection, all F P V c o m p o n e n t s were detectable
a l t h o u g h in highly r e d u c e d amounts.
The replication cycle o f VSV was not influenced in mixedly infected cells.
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Intrinsic interference in R N A viruses
I
'
' G ' N NS
25000
[
I
S
I
I
259
!
I
6000
20000
15000
4000
VSV
10000
200O
5000
"~
P
iqP
~HA ; H&
18000
HAz M
~ INS
+i, 4
¢
,L
FPV
12000
'i/~/
I0000
6000
",
10
20
5000
N
30
40 50 60 70
Fraction number
80
90
Fig. 3. Polyacrylamide gel electrophoresis of VSV- and FPV-specific proteins in simultaneously
infected cells.
Upper panel. Co-electrophoresis of doubly infected cells labelled with a mixture of [a4C]-amino
acids ( 0 - - 0 ) and of VSV-infected cells labelled with a mixture of [aH]-amino acids (O - - - O ) .
The arrows indicate the virus-specific polypeptide peaks in VSV-infected cells. In fractions io to
5o the difference between the curve of the doubly infected cells and the VSV-infected cells has been
determined. The resulting curve (A--A) has a single peak corresponding to the nucleocapsid
protein (NP) of FPV.
Lower panel. Co-electrophoresis of doubly infected cells labelled with a mixture of [14C]-amino
acids (O---O) and of FPV-infected cells labelled with a mixture of [aH]-amino acids ( O - - O).
The arrows indicate the virus-specific polypeptide peaks in FPV-infected cells. In each case cells
were labelled 4 h after infection by pulse with p4C]-amino acids 0o/,Ci/ml) or 13H]-amino acids
(IO0 #Ci/ml) for i h.
Polypeptide pattern of cells doubly infected with FPV and VSV
Fig. 3 shows t h a t cells simultaneously infected with VSV a n d F P V c o n t a i n e d 4 distinct
p o l y p e p t i d e peaks. The p a t t e r n c o r r e s p o n d s well to t h a t o f cells infected only with VSV
(Wagner, Snyder & Y a m a z a k i , I97O). It reveals the VSV-specific envelope g l y c o p r o t e i n G,
the nucleocapsid p r o t e i n N, the n o n - s t r u c t u r a l p r o t e i n NS, a n d the c a r b o h y d r a t e - f r e e surface
p r o t e i n S. In addition, a c o m p a r i s o n o f FPV-specific proteins with the p o l y p e p t i d e s o f
d o u b l y infected cells shows t h a t most F P V - p r o t e i n s were absent. The internal p r o t e i n P, the
h a e m a g g l u t i n i n proteins HA1 a n d HA2, the c a r b o h y d r a t e - f r e e envelope p r o t e i n M, a n d the
n o n - s t r u c t u r a l proteins H A a n d N S o f F P V could n o t be detected in d o u b l y infected cells.
However, the p e a k with the slowest m i g r a t i o n rate in d o u b l y infected cells was l o c a t e d between F P V - p r o t e i n P a n d F P V - p r o t e i n NP. This suggests t h a t this p e a k in d o u b l y i n f e c t e d cells
x7-2
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260
R. ROTT, C. SCHOLTISSEK, H.-D. K L E N K AND G. K A L U Z A
P HA
It
NP HA1
I
,
i
HA2
'M NS
I
I
I
I
10000
- 10000
8000
FPV+VSV
8O00
6000
6000
4000
4000
rr
2 000
'~'
!
;
I
I
I
10
20
30
I
I
'
"
I
40
50
60
Fraction number
2000
I
I
I
70
80
90
Fig. 4. Polyacrylamide gel electrophoresis of virus-specific proteins in cells infected with FPV
prior to infection with VSV. Co-electrophoresis of doubly infected cells labelled with a mixture of
[14C]-amino acids ( 0 - - 0 ) and of FPV-infected cells labelled with a mixture of [3H]-amino acids.
The arrows indicate the virus-specific proteins in FPV-infected cells. The VSV-specific proteins G,
N and S co-migrate with FPV proteins NP, HA1 and M, respectively; VSV protein NS corresponds
to the peak between FPV proteins HA~ and HA2. FPV-infected cells were labelled 4 h after infection
by a pulse with radioactive amino acids for I h. Doubly infected cells were inoculated with VSV 2 h
after the infection with FPV. The cells were labelled 4 h later by a pulse with radioactive amino acids
f o r I h.
c o n t a i n e d b o t h the VSV a n d the F P V protein. This finding was further substantiated when
the V S V - p o l y p e p t i d e p a t t e r n was s u b t r a c t e d f r o m the p a t t e r n o f d o u b l y infected cells. The
differential p a t t e r n clearly showed that d o u b l y infected cells c o n t a i n e d in a d d i t i o n to the
VSV-proteins some n u c l e o c a p s i d protein N P as the only F P V - p r o t e i n .
W h e n the cells were infected with F P V z h before the infection with VSV, all structural
proteins o f F P V as well as o f VSV were p r o d u c e d (Fig. 4). This is in a c c o r d a n c e with the
biological d a t a a n d shows once m o r e t h a t u n d e r these c o n d i t i o n s b o t h viruses d i d n o t
interfere with each other.
F P V - R N A p o l y m e r a s e and virus R N A synthesis in doubly infected cells
Table 3 presents d a t a which show t h a t only a trace a m o u n t o f F P V - R N A p o l y m e r a s e was
p r o d u c e d in d o u b l y infected cells. I f VSV was a d d e d 2 h after the p r i m a r y infection, the
p r o d u c t i o n o f F P V - R N A p o l y m e r a s e activity was n o t significantly inhibited. T h e activity
o f F P V - R N A p o l y m e r a s e could be d e t e r m i n e d in the presence o f t h a t o f V S V - R N A p o l y merase by specific h y b r i d i z a t i o n o f the radioactive in vitro p r o d u c t with an excess o f n o n labelled F P V particle or c o m p l e m e n t a r y R N A , respectively, because self-annealing o f the
V S V - R N A was relatively low. M o s t o f the in vitro synthesized F P V - R N A h a d a base
sequence c o m p l e m e n t a r y to particle R N A .
After double-infection, F P V - R N A synthesis in vivo was also a l m o s t c o m p l e t e l y inhibited,
while superinfection 2 h after FPV-infection h a d r a t h e r little effect on F P V - R N A p r o d u c t i o n
(Table 4). V S V - R N A synthesis interfered in this k i n d o f experiment m o r e severely with the
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Intrinsic interference in RATA viruses
T a b l e 3.
261
Activity of FPV- and VSV-RNA polymerase in doubly infected cells
d.p.m, in R N A
Harvest after first infection
Total R N A
Immediately RNase
Self-annealed
Particle R N A added
Complementary R N A added
FPV
zh
FPV
4h
VSV
4h
FPV/VSV
mixed
4h
FPV/'¢SV
2 h later
4h
16800
400
5 40o
13 ooo
z 900
60 ooo
3 600
36 ooo
45 800
I 1700
80 ooo
1 200
61 oo
5 700
6050
78 ooo
1 560
8 ooo
I o 60o
6 6o0
52 ooo
2400
29 800
42 ooo
8 80o
Synthesis of fowl-plague RNA in doubly infected cells
T a b l e 4.
d.p.m, in R N A
r
Start of the pulse
Total R N A
Immediately RNase
Self-annealed
I o f~l particle R N A
25 #1 particle R N A
50/zl complementary R N A
I oo #1 complementary R N A
T a b l e 5-
FPV
3"5 h
VSV
1.5 h
VSV
3'5 h
FPV/VSV
mixed
3"5 h
FPV/VSV
2 h later
3"5 h
305 ooo
9 500
78 ooo
34 ooo
3z ooo
106 ooo
103 ooo
146 ooo
3 ooo
6 900
6 zoo
6 ooo
61 oo
6 ooo
12o ooo
3 700
z9 ooo
30 ooo
31 ooo
29 400
29 ooo
z i o ooo
4 400
76 ooo
71 ooo
70 ooo
73 ooo
76 ooo
150 ooo
2 900
39 400
23 ooo
236oo
54 ooo
56 ooo
Interaction of VSV- and FPV-RNA polymerase in vitro
d.p.m, in R N A
c
Total R N A
Immediately RNase
Self-annealed
Particle R N A added
Complementary R N A added
FPV
VSV
FPV/VSV
mixed
80000
zooo
44 400
63000
8 700
40000
zoo
4 ooo
440o
3900
96000
900
4 ~200
52 ooo
7 500
For each sample five cultures were pooled and cytoplasmic fractions were prepared 4 h after infection.
In the mixed incubation an equal volume of each cytoplasmic fraction was used, while in the single incubation
the cytoplasmic fraction was mixed with an equal volume of minimal buffer (Schottissek, ~969) before
incubation.
d e t e c t i o n o f F P V - R N A , since the s e l f - a n n e a l i n g o f V S V - R N A 4 h a f t e r i n f e c t i o n was q u i t e
high. O n l y t h e failure t o influence this s e l f - a n n e a l i n g by m i x i n g w i t h an excess o f n o n l a b e l l e d F P V p a r t i c l e o r c o m p l e m e n t a r y R N A g a v e an i n d i c a t i o n t h a t in m i x e d l y i n f e c t e d
cells n o F P V - R N A w a s b e i n g synthesized. W h e n V S V was a d d e d P 5 h b e f o r e the [~H]u r i d i n e pulse was s t a r t e d t h e r e was v e r y little V S V - R N A d e t e c t a b l e in a n y case. T h u s , the
R N A as d e t e r m i n e d a f t e r s u p e r i n f e c t i o n w i t h V S V 2 h a f t e r the p r i m a r y i n f e c t i o n b y
h y b r i d i z a t i o n w i t h n o n - l a b e l l e d F P V - s p e c i f i c R N A m u s t be exclusively F P V - R N A .
C o r r e s p o n d i n g results as s h o w n in T a b l e 4 w e r e o b t a i n e d , w h e n S F V was u s e d f o r m i x e d
i n f e c t i o n o r w h e n cells w e r e p r e i n f e c t e d w i t h N D V . I n this case also n o F P V - s p e c i f i c R N A
w a s detectable. I n cells m i x e d l y infected w i t h S F V a n d N D V , S F V - R N A w a s b e i n g synt h e s i z e d n o r m a l l y , w h i l e n o N D V - R N A c o u l d be d e t e c t e d by specific h y b r i d i z a t i o n w i t h a n
excess o f n o n - l a b e l l e d p a r t i c l e N D V - R N A .
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262
R. R O T T , C. S C H O L T I S S E K , H.-D. K L E N K
A N D G. K A L U Z A
Interaction of VSV- and F P V - R N A polymerase in vitro
It has been suggested that intrinsic interference might occur because the polymerase o f
one virus competes with the polymerase of the second virus for the R N A template of the
latter (Marcus & Zuckerbraun, t97o). Therefore an in vitro experiment was performed in
which the RNA polymerase preparations of VSV and FPV were mixed before incubation
with the radioactively labelled nucleoside triphosphate precursor. As shown in Table 5,
the VSV-RNA polymerase did not not influence the synthesis of FPV complementary RNA
in vitro.
Other influenza viruses and other cells
It was found that inhibition by VSV was not restricted to FPV, but also held equally well
for other influenza A virus strains like Asian A2 (SINCAVORE), AI-FMI, AO-VR8, A SWINE
and A EQU~Z.
If BHK-cells were used instead of chick fibroblasts, VSV also inhibited multiplication of
FPV. Thus this effect was not restricted to chick cells.
DISCUSSION
In this paper it is shown that in cells doubly infected with a variety of enveloped R N A
viruses some are excluded from multiplication. Thus, SFV interferes with the formation of
infectious NDV, VSV and FPV, while its own synthesis is not inhibited. On the other hand,
the production of FPV can be prevented by all the viruses tested. It is a very early step which
does not function in the replication cycle of those viruses which are inhibited. The virusspecific RNA polymerases are not formed and virus RNA is not produced in measurable
amounts. This is not inconsistent with the small amount of FPV-RNP antigen found in
doubly infected cells, since even in the presence of actinomycin D the inhibition of the
production of RNP-antigen was somewhat leaky (Rott & Scholtissek, 1964).
In the VSV-FPV system, which was studied in more detail, the adsorption of FPV was
not influenced by VSV, since u.v.-inactivated VSV had no inhibitory effect. Furthermore,
VSV influenced influenza multiplication, even when added I h after the primary infection.
Induction of interferon by VSV and its action on influenza production cannot be responsible for this effect for the following reasons: (I) interferon production is a rather slow
process; (2) SFV, which is very sensitive to interferon, multiplies normally under analogous
conditions; and (3) u.v.-inactivated VSV inhibits interferon synthesis as efficiently as
infectious VSV (Wagner & Huang, I966).
Because of these considerations, the phenomenon described above is in agreement with
the intrinsic interference as defined by Marcus & Carver (I967) for NDV.
The mechanism of intrinsic interference is not clear. It has been suggested that the R N A
polymerase of the interfering virus might compete for the RNA template of NDV (Marcus
& Zuckerbraun, I97o). In vitro experiments with the RNA polymerases of VSV and FPV
have shown that such a competition does not occur in this system (Table 5). An alternative
explanation would be that interference occurs at the level of translation of the virus mRNA.
Evidence for this has been given by Hattman & Hofschneider (~967, I968) and Hattman
(I 97o). They showed that the exclusion of the multiplication of M 12 phage by the T4 phage
in Escherichia coil is due to inhibition of translation of M I2 RNA on polysomes. Similarly,
Saxton & Stevens (I972) found that poliovirus infection prevents the translation of herpes
simplex virus specific RNA. Thus, the phenomenon of intrinsic interference seems to be a
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Intrinsic interference in R N A viruses
263
more general phenomenon, although exceptions have been described (e.g. for the paramyxoviruses SV5, Choppin & Holmes, t967).
An additional explanation has to be considered for intrinsic interference with orthomyxovirus replication. A cellular function seems to be required early after infection, since
it is known that the multiplication of orthomyxoviruses can be abolished by interference
with cellular DNA-dependent RNA synthesis (Barry, Ives & Cruickshank, I962; Rott,
Saber & Scholtissek, I965; Scholtissek, Becht & MacPherson, I97O; Rott & Scholtissek,
I97O). It is also known that all the interfering viruses block cellular protein synthesis
(Wagner & Huang, 1966; Wilson, I968; Wagner et al. ~97o; Kang & Prevec, I97I; C.
Scholtissek, unpublished results).
Therefore the inhibition of the multiplication of orthomyxoviruses could be explained by
the assumption that the induction of a certain cellular protein, necessary for their multiplication, is prevented. Experiments to clarify this alternative explanation are in progress.
The excellent assistance of Miss M. Orlich and Miss B. Piontek is gratefully acknowledged. This study was supported by the Sonderforschungsbereich 47 (Virologie).
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