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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 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 14 May 2017 13:16:24 256 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. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 14 May 2017 13:16:24 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 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 14 May 2017 13:16:24 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. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 14 May 2017 13:16:24 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 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 14 May 2017 13:16:24 260 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 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 14 May 2017 13:16:24 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 . Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 14 May 2017 13:16:24 262 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 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 14 May 2017 13:16:24 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). 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(Received 3o M a y I 9 7 2 ) Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 14 May 2017 13:16:24