J. gen. ViroL (I977), 35, 579-58z
579
Printed in Great Britaht
The Effect of Cycloheximide on the Replication o f Measles Virus (Accepted 7 February I977) SUMMARY
Treatment of measles virus-infected cells with cycloheximide results in a threefold increase of 3H-uridine incorporation into the i2 to 36S m R N A species and in the inhibition of genomic 5oS RNA synthesis. Consistent with these observations was the finding of a build-up of polyribosomes but an absence of nucleocapsids in the infected cells. These results suggest that measles virus R N A replication, but not transcription, is dependent upon active protein synthesis. Infection of Vero cells with measles virus results in the synthesis of virus-specific RNAs which sediment at I2 to 36S and 5oS in aqueous sucrose gradients. The I2 to 36S RNAs contain poly(A) sequences and are thought to be the virus messengers, whereas R N A sedimenting at 5oS is a mixture of virus genome material and double-stranded RNA. It has been shown previously that treatment of paramyxovirus-infected cells with cycloheximide results in an increased production of virus-specific messenger R N A and in the inhibition of 5oS genomic RNA synthesis (see review by Kingsbury, 1974). In contrast to these findings it has been reported that measles virus 5oS RNA synthesis was not inhibited by cycloheximide suggesting that the replication of measles virus may be fundamentally different from that of the classical paramyxoviruses (Kiley & Payne, 1974). The present report re-assesses the effect of cycloheximide on measles virus RNA synthesis. In all experiments Vero cells were infected with the Edmonston stain of measles virus and the methods used have been described (Hall & ter Meulen, 1977)- At ~6 h post-infection (p.i.) actinomycin (Act) D (IO/~g/ml) with or without ioo/~g/ml cycloheximide (Serva Ltd.) in MEM without serum was added to the infected cultures. After incubation for I h at 37 °C, aH-uridine (sp. act. 20 t o 30000 mCi/mmol) at 25/~Ci/ml was added. Cells were incubated for a further 3 h at 37 °C before the isolation of RNA and intracellular structures. R N A was isolated and analysed by sucrose gradient sedimentation as described by Hall & ter Meulen (I977). Intracellular structures were isolated as follows. Infected cells were washed twice with semi-frozen PBS, scraped from the glass into the same buffer and collected by centrifuging at 2ooo rev/min for to min at 4 °C. Pellets were suspended in reticulocyte standard buffer (RSB: o-ot M-tris-HCl, pH 7"4; o-o~ M-KC1; o'ooi5 M-MgC12) swollen on ice for 1o min and disrupted by IO strokes in a glass Dounce homogenizer. Nuclei were removed by centrifuging at Iooo g for 5 min at 4 °C. Supernatants from each preparation were made I ~ in Triton X-Ioo and 0"5 % in sodium deoxycholate and divided into two equal portions. One portion was made o.oI M in EDTA and both were then immediately centrifuged on 15 to 40 ~ (w/w) sucrose gradients in RSB. Centrifugation was for 18o rain in a Beckman SW 27. [ rotor at 25ooo rev/min at 4 °C. Gradients were fractionated into 0"55 ml fractions using an ISCO fraction collector with continous monitoring at 254 nm. Samples (5o #1) of each fraction were then assayed for TCA-precipitable radioactivity. Fig. I shows typical sucrose gradient profiles of 3H-uridine labelled R N A synthesized in the presence of lo/zg/ml of Act D and in the presence or absence of Ioo/zg/ml of cyclo38-2
Short communications
580 I
,
i
i
i
,
~,
I
I 30
~,
_
m r
× e-.
(b)
'6 12
0 ~.,x_._._._.l 10
I 20 Fraction number
Fig. I. Effect of cycloheximide on measles virus-specific RNA synthesis. Intracellular RNA was labelled with 3H-uridine in the presence of Act D (a) without or (b) with cycloheximide between I7 and zo h p.i. RNA was isolated and fractionated as described in the text. Samples (o-05 ml) of each fraction were collected, TCA-precipitated and counted in Bray's solution. • ~,, 3Huridine, ct/min. The arrows represent the sedimentation positions of Vero cell 28S and I8S ribosomal RNAs as determined by extinction at 254 nm. heximide. I n cells treated only with A c t D R N A sedimented in the I2 to 26S, 28 to 36S a n d 5oS regions (Fig. I a). T r e a t m e n t with cycloheximide resulted in an a p p r o x i m a t e threefold increase in the i n c o r p o r a t i o n o f r a d i o a c t i v i t y into the t2 to 26S and 28 to 36S R N A s and a complete inhibition o f 5oS R N A synthesis was observed. To localize the R N A present in infected cells, c y t o p l a s m i c extracts were p r e p a r e d a n d f r a c t i o n a t e d by sucrose gradient sedimentation. E a c h c y t o p l a s m i c extract was divided into two equal portions. One p o r t i o n was f r a c t i o n a t e d directly on 15 to 4o ~o sucrose gradients whereas the second was treated with E D T A before sedimentation. Typical r a d i o a c t i v i t y profiles are shown in Fig. 2. It can be seen t h a t infected cells treated only with A c t D c o n t a i n p o l y r i b o s o m e s which sediment up to 3ooS (Fig. 2 a). Release o f the R N A b y E D T A t r e a t m e n t d e m o n s t r a t e d the presence o f a single p e a k o f r a d i o a c t i v i t y at a b o u t 23oS
Short communications
112.
I
I
58I ~z,
8 6 4 2 x 0 -~ 30
(b)
20 15 10 5 0
0
10
20 Fraction number
30
Fig. 2. Effect of cycloheximide on the synthesis of measles vlrus-specific intracellular structures. Intracellular structures were labelled with 3H-uridine in the presence of (a) Act D or (b) with Act D and eycloheximide between 17 and 2o h p.i. Cytoplasmic extracts were prepared as described in the text and divided into two equal portions. One part was made o.oi M with respect to EDTA whereas the other received no treatment. Extracts were then directly centrifuged on linear sucrose gradients and fraetionated as described in the text. Samples (0"05 ml) of each fraction were assayed for TCA-precipitable radioactivity. [] [], 3H-uridine ct/min without EDTA treatment; © ©, with EDTA treatment. The position of the ribosome monomers was determined by extinction at 254 nm and sedimented at the position indicated by the arrow (T). (Fig. 2a). R N A extracted f r o m this m a t e r i a l sedimented at 5oS ( d a t a n o t shown) d e m o n strating t h a t it was newly synthesized virus nucleocapsids. T r e a t m e n t o f cells with cycloheximide resulted in a threefold increase in the r a d i o a c t i v i t y associated with the p o l y ribosomes, a n d in an increase in their s e d i m e n t a t i o n range (Fig. 2b). This finding s u p p o r t s t h e previous o b s e r v a t i o n o f an increased b u i l d up o f virus-specific m R N A s . Analysis o f the E D T A - t r e a t e d extract (Fig. 2b) revealed t h a t no nucleocapsids h a d been synthesized in the c y c l o h e x i m i d e - t r e a t e d cells. The absence o f nucleocapsids is consistent with the finding t h a t measles virus g e n o m e synthesis is inhibited by the drug. The observed a c c u m u l a t i o n o f p o l y r i b o s o m e s in infected cells is to be expected since the d r u g a p p a r e n t l y acts b y showing t h e m o v e m e n t o f r i b o s o m e s along the m R N A s ( G o d c h a u x , A d a m s o n & H e r b e r t , i967;
582
Short communications
Robinson, I97I). It is also possible that the build up of mRNAs could arise from the increased availability of 5oS template which is not being utilized for genome replication. The results we have described suggest that the replication but not transcription of the measles virus genome is dependent upon active protein synthesis. Similar findings have been described for Newcastle disease virus (Clavell & Bratt, I97l; Kaverin & Varich, 197I; Weiss & Bratt, 1975), Sendai virus (Robinson, I97I; Portner & Kingsbury, 1972; Zaides et al. I973), mumps virus (East & Kingsbury, I97I) and the rhabdovirus, VSV (Perlman & Huang, I973). The results are in disagreement with those reported for measles virus by Kiley & Payne (I974). These workers found a similar effect of the drug on mRNA synthesis but reported that the incorporation of 3H-uridine into 5oS RNA and nucleocapsids was unaffected. The reasons for this inconsistency are not understood since the same cells and strain of virus were used in both works. In conclusion the results demonstrate the close similarity of measles RNA transcription and replication to that of the paramyxoviruses. We suggest that the basic mechanisms involved with measles virus are not fundamentally different from the paramyxoviruses. This work was supported by the Deutsche Forschungsgemeinschaft, Schwerpunkt 'Multiple Sklerose und verwandte Erkrankungen', Az. Me 27o/I4 . We are grateful to Ingeborg Euler and Dorothea Miintjes for their expert technical assistance.
Institute of Virology and Immunobiology University of Wiirzburg Versbacher Landstrafle 7 87oo W~irzburg West Germany
W . W . HALL D. GENIUS V. TER MEULEN
REFERENCES CLAVELL, L. A. & BRATT, M. A. (197I). Relationship between ribonucleic acid-synthesizing capacity of ultraviolet-irradiated Newcastle disease virus and its ability to induce interferon. Journal of Virology 8, 50o-508. EAST, J. J. & KINCSBURV, O. W. (197I). M u m p s virus replication in chick embryo lung cells: properties o f ribonucleic acids in virions and infected cells. Journal of Virology 8, I6I-I73. GODCHAUX, W., III, ADAMSON,S. D. & HERBERT, E. (I967). Effects o f cycloheximide on polyribosome function in reitculocytes. Journal of Molecular Biology 27, 57-72. HALt, W. W. & TER MEtJLEN, V. (1977). Polyadenylic acid [poly (A)] sequences associated with measles virus intracellular ribonucleic acid (RNA) species. Journal of General Virology 35, 497-5IO. KAVERIN, N. V. & VARICH, N. I-. (I97I). Virus-specific R N A formed in Newcastle disease virus infected cells after suppression o f protein synthesis by cycloheximide. Archiv fiir die gesamte Viru~forschung 35, 378-384. KILEY, M.P. & PAYNE, F. E. (I974). Replication of measles virus: continued synthesis o f nucleocapsid R N A and increased synthesis o f m R N A in the presence o f cycloheximide. Journal of Virology I4, 758-764. KINGSBURY, D.W. (1974)- The molecular biology o f the paramyxoviruses. Medical Microbiology and Immunology i6o, 73-83. PERLMAN, S. M. & HUANG, A. S. (1973). RNA synthesis o f vesicular stomatitis virus. V. Interactions betweer~ transcription and replication. Journal Of Virology xz, I395-I4OO. PORTNER, A. & KINGSBURY, D.W. (I972). Identification o f transcriptive and replicative intermediates in Sendai virus-infected cells. Virology 47, 7I 1-725. ROBINSON, W. S. (I97I). Sendai virus R N A synthesis and nucleocapsid formation in the presence o f cycloheximide. Virology 44, 494-5o2. WElSS, S. R. & BRATT, M. A. (1975). Effect o f cordycepin (3'-deoxyadenosine) on virus-specific R N A species synthesized in Newcastle disease virus infected cells. Journal of Virology 16, I575-I583. ZAIDES, V.M., NIKOLAYEVA, O.G., SELIKOVA, L. M. & BUKRINSKAYA, G. 0973). Effect o f protein synthesis inhibitors on the replication o f virus-induced R N A in Sendai virus-infected cells. Acta Virologica 17, 355-358.
(Received 28 July I976)
579
Printed in Great Britaht
The Effect of Cycloheximide on the Replication o f Measles Virus (Accepted 7 February I977) SUMMARY
Treatment of measles virus-infected cells with cycloheximide results in a threefold increase of 3H-uridine incorporation into the i2 to 36S m R N A species and in the inhibition of genomic 5oS RNA synthesis. Consistent with these observations was the finding of a build-up of polyribosomes but an absence of nucleocapsids in the infected cells. These results suggest that measles virus R N A replication, but not transcription, is dependent upon active protein synthesis. Infection of Vero cells with measles virus results in the synthesis of virus-specific RNAs which sediment at I2 to 36S and 5oS in aqueous sucrose gradients. The I2 to 36S RNAs contain poly(A) sequences and are thought to be the virus messengers, whereas R N A sedimenting at 5oS is a mixture of virus genome material and double-stranded RNA. It has been shown previously that treatment of paramyxovirus-infected cells with cycloheximide results in an increased production of virus-specific messenger R N A and in the inhibition of 5oS genomic RNA synthesis (see review by Kingsbury, 1974). In contrast to these findings it has been reported that measles virus 5oS RNA synthesis was not inhibited by cycloheximide suggesting that the replication of measles virus may be fundamentally different from that of the classical paramyxoviruses (Kiley & Payne, 1974). The present report re-assesses the effect of cycloheximide on measles virus RNA synthesis. In all experiments Vero cells were infected with the Edmonston stain of measles virus and the methods used have been described (Hall & ter Meulen, 1977)- At ~6 h post-infection (p.i.) actinomycin (Act) D (IO/~g/ml) with or without ioo/~g/ml cycloheximide (Serva Ltd.) in MEM without serum was added to the infected cultures. After incubation for I h at 37 °C, aH-uridine (sp. act. 20 t o 30000 mCi/mmol) at 25/~Ci/ml was added. Cells were incubated for a further 3 h at 37 °C before the isolation of RNA and intracellular structures. R N A was isolated and analysed by sucrose gradient sedimentation as described by Hall & ter Meulen (I977). Intracellular structures were isolated as follows. Infected cells were washed twice with semi-frozen PBS, scraped from the glass into the same buffer and collected by centrifuging at 2ooo rev/min for to min at 4 °C. Pellets were suspended in reticulocyte standard buffer (RSB: o-ot M-tris-HCl, pH 7"4; o-o~ M-KC1; o'ooi5 M-MgC12) swollen on ice for 1o min and disrupted by IO strokes in a glass Dounce homogenizer. Nuclei were removed by centrifuging at Iooo g for 5 min at 4 °C. Supernatants from each preparation were made I ~ in Triton X-Ioo and 0"5 % in sodium deoxycholate and divided into two equal portions. One portion was made o.oI M in EDTA and both were then immediately centrifuged on 15 to 40 ~ (w/w) sucrose gradients in RSB. Centrifugation was for 18o rain in a Beckman SW 27. [ rotor at 25ooo rev/min at 4 °C. Gradients were fractionated into 0"55 ml fractions using an ISCO fraction collector with continous monitoring at 254 nm. Samples (5o #1) of each fraction were then assayed for TCA-precipitable radioactivity. Fig. I shows typical sucrose gradient profiles of 3H-uridine labelled R N A synthesized in the presence of lo/zg/ml of Act D and in the presence or absence of Ioo/zg/ml of cyclo38-2
Short communications
580 I
,
i
i
i
,
~,
I
I 30
~,
_
m r
× e-.
(b)
'6 12
0 ~.,x_._._._.l 10
I 20 Fraction number
Fig. I. Effect of cycloheximide on measles virus-specific RNA synthesis. Intracellular RNA was labelled with 3H-uridine in the presence of Act D (a) without or (b) with cycloheximide between I7 and zo h p.i. RNA was isolated and fractionated as described in the text. Samples (o-05 ml) of each fraction were collected, TCA-precipitated and counted in Bray's solution. • ~,, 3Huridine, ct/min. The arrows represent the sedimentation positions of Vero cell 28S and I8S ribosomal RNAs as determined by extinction at 254 nm. heximide. I n cells treated only with A c t D R N A sedimented in the I2 to 26S, 28 to 36S a n d 5oS regions (Fig. I a). T r e a t m e n t with cycloheximide resulted in an a p p r o x i m a t e threefold increase in the i n c o r p o r a t i o n o f r a d i o a c t i v i t y into the t2 to 26S and 28 to 36S R N A s and a complete inhibition o f 5oS R N A synthesis was observed. To localize the R N A present in infected cells, c y t o p l a s m i c extracts were p r e p a r e d a n d f r a c t i o n a t e d by sucrose gradient sedimentation. E a c h c y t o p l a s m i c extract was divided into two equal portions. One p o r t i o n was f r a c t i o n a t e d directly on 15 to 4o ~o sucrose gradients whereas the second was treated with E D T A before sedimentation. Typical r a d i o a c t i v i t y profiles are shown in Fig. 2. It can be seen t h a t infected cells treated only with A c t D c o n t a i n p o l y r i b o s o m e s which sediment up to 3ooS (Fig. 2 a). Release o f the R N A b y E D T A t r e a t m e n t d e m o n s t r a t e d the presence o f a single p e a k o f r a d i o a c t i v i t y at a b o u t 23oS
Short communications
112.
I
I
58I ~z,
8 6 4 2 x 0 -~ 30
(b)
20 15 10 5 0
0
10
20 Fraction number
30
Fig. 2. Effect of cycloheximide on the synthesis of measles vlrus-specific intracellular structures. Intracellular structures were labelled with 3H-uridine in the presence of (a) Act D or (b) with Act D and eycloheximide between 17 and 2o h p.i. Cytoplasmic extracts were prepared as described in the text and divided into two equal portions. One part was made o.oi M with respect to EDTA whereas the other received no treatment. Extracts were then directly centrifuged on linear sucrose gradients and fraetionated as described in the text. Samples (0"05 ml) of each fraction were assayed for TCA-precipitable radioactivity. [] [], 3H-uridine ct/min without EDTA treatment; © ©, with EDTA treatment. The position of the ribosome monomers was determined by extinction at 254 nm and sedimented at the position indicated by the arrow (T). (Fig. 2a). R N A extracted f r o m this m a t e r i a l sedimented at 5oS ( d a t a n o t shown) d e m o n strating t h a t it was newly synthesized virus nucleocapsids. T r e a t m e n t o f cells with cycloheximide resulted in a threefold increase in the r a d i o a c t i v i t y associated with the p o l y ribosomes, a n d in an increase in their s e d i m e n t a t i o n range (Fig. 2b). This finding s u p p o r t s t h e previous o b s e r v a t i o n o f an increased b u i l d up o f virus-specific m R N A s . Analysis o f the E D T A - t r e a t e d extract (Fig. 2b) revealed t h a t no nucleocapsids h a d been synthesized in the c y c l o h e x i m i d e - t r e a t e d cells. The absence o f nucleocapsids is consistent with the finding t h a t measles virus g e n o m e synthesis is inhibited by the drug. The observed a c c u m u l a t i o n o f p o l y r i b o s o m e s in infected cells is to be expected since the d r u g a p p a r e n t l y acts b y showing t h e m o v e m e n t o f r i b o s o m e s along the m R N A s ( G o d c h a u x , A d a m s o n & H e r b e r t , i967;
582
Short communications
Robinson, I97I). It is also possible that the build up of mRNAs could arise from the increased availability of 5oS template which is not being utilized for genome replication. The results we have described suggest that the replication but not transcription of the measles virus genome is dependent upon active protein synthesis. Similar findings have been described for Newcastle disease virus (Clavell & Bratt, I97l; Kaverin & Varich, 197I; Weiss & Bratt, 1975), Sendai virus (Robinson, I97I; Portner & Kingsbury, 1972; Zaides et al. I973), mumps virus (East & Kingsbury, I97I) and the rhabdovirus, VSV (Perlman & Huang, I973). The results are in disagreement with those reported for measles virus by Kiley & Payne (I974). These workers found a similar effect of the drug on mRNA synthesis but reported that the incorporation of 3H-uridine into 5oS RNA and nucleocapsids was unaffected. The reasons for this inconsistency are not understood since the same cells and strain of virus were used in both works. In conclusion the results demonstrate the close similarity of measles RNA transcription and replication to that of the paramyxoviruses. We suggest that the basic mechanisms involved with measles virus are not fundamentally different from the paramyxoviruses. This work was supported by the Deutsche Forschungsgemeinschaft, Schwerpunkt 'Multiple Sklerose und verwandte Erkrankungen', Az. Me 27o/I4 . We are grateful to Ingeborg Euler and Dorothea Miintjes for their expert technical assistance.
Institute of Virology and Immunobiology University of Wiirzburg Versbacher Landstrafle 7 87oo W~irzburg West Germany
W . W . HALL D. GENIUS V. TER MEULEN
REFERENCES CLAVELL, L. A. & BRATT, M. A. (197I). Relationship between ribonucleic acid-synthesizing capacity of ultraviolet-irradiated Newcastle disease virus and its ability to induce interferon. Journal of Virology 8, 50o-508. EAST, J. J. & KINCSBURV, O. W. (197I). M u m p s virus replication in chick embryo lung cells: properties o f ribonucleic acids in virions and infected cells. Journal of Virology 8, I6I-I73. GODCHAUX, W., III, ADAMSON,S. D. & HERBERT, E. (I967). Effects o f cycloheximide on polyribosome function in reitculocytes. Journal of Molecular Biology 27, 57-72. HALt, W. W. & TER MEtJLEN, V. (1977). Polyadenylic acid [poly (A)] sequences associated with measles virus intracellular ribonucleic acid (RNA) species. Journal of General Virology 35, 497-5IO. KAVERIN, N. V. & VARICH, N. I-. (I97I). Virus-specific R N A formed in Newcastle disease virus infected cells after suppression o f protein synthesis by cycloheximide. Archiv fiir die gesamte Viru~forschung 35, 378-384. KILEY, M.P. & PAYNE, F. E. (I974). Replication of measles virus: continued synthesis o f nucleocapsid R N A and increased synthesis o f m R N A in the presence o f cycloheximide. Journal of Virology I4, 758-764. KINGSBURY, D.W. (1974)- The molecular biology o f the paramyxoviruses. Medical Microbiology and Immunology i6o, 73-83. PERLMAN, S. M. & HUANG, A. S. (1973). RNA synthesis o f vesicular stomatitis virus. V. Interactions betweer~ transcription and replication. Journal Of Virology xz, I395-I4OO. PORTNER, A. & KINGSBURY, D.W. (I972). Identification o f transcriptive and replicative intermediates in Sendai virus-infected cells. Virology 47, 7I 1-725. ROBINSON, W. S. (I97I). Sendai virus R N A synthesis and nucleocapsid formation in the presence o f cycloheximide. Virology 44, 494-5o2. WElSS, S. R. & BRATT, M. A. (1975). Effect o f cordycepin (3'-deoxyadenosine) on virus-specific R N A species synthesized in Newcastle disease virus infected cells. Journal of Virology 16, I575-I583. ZAIDES, V.M., NIKOLAYEVA, O.G., SELIKOVA, L. M. & BUKRINSKAYA, G. 0973). Effect o f protein synthesis inhibitors on the replication o f virus-induced R N A in Sendai virus-infected cells. Acta Virologica 17, 355-358.
(Received 28 July I976)
