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Effect of UV irradiation of viral haemorrhagic septicaemia virus on virus-specific intracellular syntheses
ELSEVIER Paris 1985
Ann. Inst. Pasleur/Virol. i985, 136 E, 213-222
EFFECT
OF
OF
VIRAL
ON
VIRUS-SPECIFIC
UV
IRRADIATION
HAEMORRHAGIC
SEPTICAEMIA
INTRACELLULAR
VIRUS
SYNTHESES
by J. Bernard and P. de Kinkelin Laboratoire d' Ichlgopalhologie, I N R A , Groupe de Laboratoires de Palhologie Animale, route de Thiverval, 78850 Thiverval-Grignon (France)
SUMMARY The effect of UV irradiation of virions on subsequent virus-specific intracellular protein synthesis was studied. The order of three genes (N, M1 and G) was determined, but L and M2 could not be mapped. KEY-WORDS: Viral haemorrhagic septicaemia virus, Genome, UV, Polypepfide; Synthesis, EPC cells.
INTRODUCTION Viral haemorrhagic septicaemia (VHS) virus is a member of the Rhabdovirus family and belongs to the Lyssa genus. The mature virus particle contains 5 polypeptides, L, G, N, M1 and Ms, with molecular weights of 190, 80, 38, 25 and 19 • 103, respectively [12, 14] and a negative-stranded RNA genome the S(2o,w) coefficient of which is 38-40 [9]. In the present work, we attempted to locate the different genes on t h a t genome. The order of the genes on a genome can be established by analysing the effect of ultraviolet (UV) irradiation of virions on subsequent intracellular synthesis. The method pioneered by Sauerbier [16] has been well documented for rhabdoviruses [1, 2, 3, 8], but it is valid only if the observed syntheses are the result of primary transcription, so t h a t newly replicated, undamaged genomes could not interfere. Thus, since primary transcription of VHS virus is difficult to observe due to the low level of RNA synthesis, we chose the indirect method and studied the inactivation of protein synthesis in order to determine the time-course of viral syntheses and to then map part of the genome. Manuscrit re~u le 3 mat 1985, accept6 le 3 aofit 1985.
214
J. BEBNAFID AND P. D E K I N K E L I N
MATERIALS
AND
METHODS
Virus and cells. In order to avoid any drift of virulence [6, 4] VHS virus strain 07-71 [11] was reisolated from an infected fish and submitted to only 2 passages at 14 ~ C in Epithetioma papulosum cyprini (EPC) cells [7] as already described [5] without plaque purification. Virus stocks titrating at 1 • 109 plaque-forming units ( P F U ) / m l were frozen at --70 ~ C until use, then concentrated by centrifugation when high multiplicities of infection (MOI) were needed. D E A E - d e x t r a n (50 [xg/ml) was added to the inocula.
UV irradiation. Virus suspensions in duplicate were irradiated in 24-well (Nunc) plastic plates (200 ~xl/well) at a distance of 14 cm from a germicidal lamp (10.6 • 10 .6 Joules/ s/ram 9, 106 ergs/s/mm ~, as determined with a L a t a r j e t dosimeter) and immediately used to infect EPC cells. Portions were sampled to determine the original titre and inactivation rate by means of plaque titration under an agarose overlay. E P C cells seeded in plastic (Corning) Petri dishes 30 m m in diameter (4.5 • 10" cells per dish) were irradiated for 15 s at a distance of 8 cm from the same germicidal lamp, then immediately infected.
Labelling, extraction and electrophoresis o/ inlracellular proteins. Irradiated cells were either mock-infected with suspension medium (Stocker's medium supplemented with 10% t r y p t o s e and 2 % foetal calf serum) or infected with the virus suspension (150 [xl per dish: M O I = 8 9 ) and the virus was adsorbed for 1 h at 14 ~ C. The cells were then pretreated for 1 h post-adsorption (PA) with Earle's saline to deplete the intracellular amino acids, and were finally labelled for the indicated time with Earle's saline containing 30 ~Ci/ml of ~SS-methionine (specific activity 800 Ci/mM; Amersham). At the end of the labelling period, the cells were washed 3 times with Earle's saline, then lysed with 300 ~xl per dish of K buffer (0.01 M NaCI; 0.01 M Tris-hydrochloride ( p H 8.0); 0.5% sodium dodecyl sulphate). The viscous e x t r a c t was sonieated (20 s in ice) and the proteins were precipitated at 4 ~ C with acetone. The samples were centrifuged for 15 min at 13,000 g (Hettich Mikroliter centrifuge). The pellets were resuspended in 20 ~zl of K buffer. One volume of dissociating buffer (62.5 mM Tris-hydrochloride ( p H 6.8); 1% sodium dodecyl sulphate; 10% glycerol; 2 % 2-mercaptoethanol; 0.001% bromophenol blue) was added and the samples were maintained for 1.5 min in a boiling water bath, then cooled and loaded in the wells of a discontinuous 10% acrylamide slab gel in L a e m m l i ' s [10] b u f f e r system. Electrophoresis was run at 12 mA for the stacking gel, then 24 mA for the resolving gel, constant eurrenL until the tracer dye had migrated for 7-10 em. The gels were t h e n stained for 15 min with Comassie blue, destained, dried under v a c u u m and autoradiographed for 3-24 h using ~ R P X-OMAT K o d a k ~ film; the autoradiograms were then scanned.
EPC = epithelioma papulosum eyprini. MOI = multiplicity of infection. PA = post-adsorption.
PFU = plaque-forming unit. UV = ultraviolet. VHS = viral haemorrhagic septicaemia.
UV AND VHS VIRAL INTRACELLULAR SYNTHESES
215
RESULTS
Inhibition o[ cellular syntheses. Since VHS virus does not inhibit cellular syntheses, it was necessary to pretreat the cells so t h a t virus-specific syntheses could be observed. W h e n the cells were UV-irradiated for 15 s at 10.6 • 10 .6 J o u l e s / s / m m ~, residual cellular activity still obscured virus-specific polypeptides on the autoradiograms (data not shown). Increasing the UV dose by exposing the cells at 8 em from the lamp almost completely inhibited cellular syntheses, and the viral polypeptides could be easily evidenced, except for the Ms. The G migrated as a sharp band and t h u s was probably not glyeosylated (see below). Plaque counts were impossible because the cells died within 24 h post-irradiation.
Timing of lranscription in irradiated cells. To verify t h a t the irradiated cells could still support viral syntheses, t h e y were infected at a MOI of 89 P F U of non-irradiated virus per cell, t h e n labelled from 1 to 3, 1 to 4 and 1 to 5 h PA. By 3 h, the N and M1 polypeptides appeared on the autoradiograms (fig. 1A, lane 0). B y 4 h, traces of G and Ms could be seen (fig. 1B, lane 0). B y 5 h, the 4 polypeptides were present in significant amounts, b u t the L was not visible (fig. 1C, lane 0). Secondary transcription results in amplification of virus syntheses and its onset is more easily determined in cells infected with irradiated genomes since, in t h a t ease, the inactivation curves as a function of the dose are no longer representative of single-hit events. The virus particles were thus irradiated for various lengths of time to determine the survival curves. For infectivity, the t a r g e t of which is the full-length genome, the 37% P F U survival representative of one hit per t a r g e t was repeatedly obtained at 2-s irradiation (212 ergs/mm 2) (fig. 2). The irradiated virus particles were used to infect irradiated cells at an MOI equivalent to 89 PFU, and the resulting viral protein syntheses were studied from 1 to 3, 1 to 4 and 1 to 5 h as above. By 3 h PA, N was still visible in cells infected with virus irradiated for 15 s (fig. 1A) b u t its 37~o survival dose could not be accurately determined because, due to the low rate of synthesis, standard deviation was high (fig. 2). By 4 h PA, N was again visible in all samples, M1 up to 8 s of irradiation and G up to 6 s (fig. 1B). The survival curves of N and G were bimodal (fig. 2) and t h u s representative of the onset of secondary transcription. M~ could not be quantified due to numerous discrete bands. Those discrete bands were not visible on the control uninfected cells samples (lanes C). By 5 h PA, the synthesis of all 3 polypeptides was amplified and t h e y could be seen in all samples (fig. 1C).
FIG. 1. - - Viral protein synthesis after U V irradiation o/ V H S virus particles. Cellular protein synthesis was depressed b y UV irradiation; cells were t h e n infected w i t h UV-irradiated (2-10 s) or non-irradiated (lane 0) v i r u s at an MOI equivalent to 90 P F U of non-irradiated virus, and labelled f r o m 1 to 3 (A), 1 to 4 (B) or 1 to 5 (C) h o u r s PA. Lanes in C show mock-infected controls. Multiple exposures of the X - r a y film were used so as to be within t h e range of the fihn response.
Fro. 1
UV AND VHS VIRAL INTRACELLULAR SYNTHESES
5(
07Y71
"82 X , \
PFU
219
G
2E
o= o~
I
~ ~~ ~ . 9
~
I
e
M1
~,
10
5 SECONDS
10
t.N
FIG. 2. - - Dose response curves of V H S virus infectivity
and viral protein synthesis as a {unction of U V dose. P F U c u r v e : t h e o r i g i n a l t i t r e w a s 4 • 10 8 P F U / m l ; all c a l c u l a t i o n s w e r e d o n e o n a m e a n of 2 e x p e r i m e n t s a n d a m i n i m u m of 400 p l a q u e s w e r e c o u n t e d f o r e a c h p o i n t o f e a c h e x p e r i m e n t . A l l o t h e r c u r v e s : f r o m d e n s i t o m e t e r t r a c i n g s u s i n g a ,, G e l m a n D C D 16 ,, d e n s i t o m e t e r , of 4 a u t o r a d i o g r a m s s i m i l a r t o t h a t in fig. 1. 9 : 3 h P A ; 9 ~ 4 h P A ; 9 : 5 h P A . A l l o r d i n a t e s a r e o n a l o g scale.
Tentative to map the genome.
Mapping the genome b y means of UV irradiation is valid only during primary transcription, which in our case was 3 h PA. On the other hand, b y t h a t time, numerous discrete bands were seen in infected cells, which could represent any one of the nascent polypeptides and cause an underestimation. Thus, we turned to pulse-chase experiments. The cells were labelled from 0.5 to 3 h PA, then washed and incubated for 3 h more in Stoker's medium so t h a t the labelled polypeptides could be initiated during prim a r y transcription, then terminated and accumulated in t h e absence of the radioactive precursor (fig. 3). In these conditions, when the percentage of survival was plotted against t h e UV dose, t h e values we obtained for G and M1 aligned on straight lines (fig. 4) with 3 7 % survival doses of, respectively, 3.6 and 6 s, b u t L could not be seen and M2 could still not be quantified. DISCUSSION V H S virus does not inhibit cellular synthesis and, since viral synthesis is maintained at a low level, an inhibitory t r e a t m e n t had to be found, Osmotic shock was avoided because it disturbs t h e synthesis of G for other Lyssa viruses [8, 13]. Heat-shock [15] would have been inefficient because V H S virus is more thermosensitive t h a n E P C cells [6, 7]. W e finally irra-
220
J. B E R N A R D
F I 6 . 3. - -
A N D P. D E K I N K E L I N
Pulse-chase labelling of viral proteins.
T h e c e l l s w e r e l a b e l l e d f r o m 0.5 t o 3 h P A , t h e n medium.
r
r
10( il
G
maintained
until 6 b PA in non-radioactive
k .l\ :
05C Z
U~
~o
SECONDS FIG. 4. - -
UV
Dose-response curves of viral protein synthesis in pulse-chase experiments.
UV AND VHS VIBAL INTRACELLULAR SYNTHESES
221
diated the cells to death prior to infection. In such conditions, cellular synthesis were drastically reduced (fig. 1, lanes C) and viral polypeptides became visible in the same order as published for actionomyein-D-treated cells [6]. On the other hand, the synthesis of M2 was impaired, as described for rabies virus [8]; G, however, could be identified, which contrasts with rabies virus in irradiated BHK21 cells [8]. Since viral polypeptides became visible in the order N-~M~ ~ G Ms ~ L (fig. 1), V H S virus genes are p r o b a b l y transcribed sequentially with N located at the 3' end of the genome. The results of pulse-chase experiments reinforce the hypothesis t h a t M1 is transcribed before G (37 % survival doses of, respectively, 3.6 and 6 s) and t h a t G is not terminal on the 5' end, since the P F U 37 % survival dose is 2 s. L could not be evidenced before the onset of secondary transcription, which could be due either to a v e r y low b u t continuous rate of transcription or to a delayed expression of the 5' end of the genome.
Bt~SUMt~
SUR
LES
E F F E T DE L ' I R R A D I A T I O N U V DU VIRUS DE LA SEPTICI~MIE tII~MORRAGIQUE VIRALE SYNTHESES INTRACELLULAIRES SPI~CIFIQUES
DU
VIRUS
Nous avons ~tudi~ les syntheses virales apr~s infection de cellules E P C par des partieules irradi6es. L'ordre de 3 g~nes, N, M1 et G, a pu ~tre d6termin6 mais eelui de M2 et L est incertain. MOTS-CLI~S : Septicemic h6morragique p e p t i d e ; Syntheses, Cellules EPC.
virale,
GLnome, UV,
Poly-
ACKNOWLEDGMENTS
We are grateful to M. Bearzotti-Le Berre, who prepared the virus used as an inoculum.
REFERENCES
[1] ABRAHAM, G. & BANERJEE, A. K., Sequential transcription of the genes of vesicular stomatitis virus. Proc. nat. Acad. Sci. (Wash.), 1976, 73, 15041508. [2] BALL, L. A., Transcriptional mapping of vesicular stomatitis virus in vivo. J. Virol., 1977, 21, 411-414. [3] BALL, L. A. & WHITE, C. i . , Order of transcription of genes of vesicular stomatitis virus. Proc. nat. Acad. Sci. (Wash.), 1976, 73, 442-446. [4] BERNARD, J . , BEARZOTTI-LE BERRE, M. & DE KINKELIN, P., Viral haemorrhagic septicaemia in rainbow trout. Tentative to relate interferon production, antibody synthesis and the structure of the virus with the mechanism of virulence. Ann. Insl. Pasleur/Virol., 1985, 136 V., 13-26. Ann. Inst. Pasteur/Virol., 136 E,
n~ 3~ 1985.
16
222
J. BERNARD AND P. DE K I N K E L I N
[5] DE KINKELIN, P. & LE BERRE, M., Mass virus production in fish cell system. Develop. biol. Stand., 1978, 42, 99-104. [6] DE KINKELIN, P., BEARZOTTI-LE BERRE, M. & BERNARD, J., Viral haem.orrhagic septicaemia of rainbow trout. Selection of a thermoresistant virus variant and comparison of polypeptide synthesis with the wild type virus strain. J. Virol., 1980, 36, 652-658. [7] EIJAN, N., SULIMANOVIC, D., BEARZOT~fI, M., MUZINIC, D., ZWILLENBERG, L. 0., CHILMONCZYK,S., VAUTHEROT, J. F. & DE KINKELIN, P., Some properties of the Epithelioma papulosum cgprini (EPC) cell line from carp Cgprinus earpio. Ann. Virol. (Inst. Pasteur), 1983, 134 tl, 207-220. [8] FLAMAND,A. & DELAGNEAU, J. F., Transcriptional mapping of rabies virus in vivo. J. Virol., 1978, 28, 518-523. [9] HILL, B. J., UNDERWOOD, B. O., SMALE,C. J. & BROWN, F., Physicochemieal and serological characterization of five rhabdoviruses infecting fish. J. gen. Virol., 1975, 27, 369-378. [10] LAEMMLI,U. K., Cleavage of structural proteins durin~ the assembly of the head of bacteriophage T4. Nature (LoAd.), 1970, 227, 369-378. [11] LE BERRE, M., DE KINKELIN, P. & METZGER, A., Identification s6rologique des rhabdovirus des salmonid6s. Of/. int. Epizoot. (Bull.), 1977, 87, 391-393. [12] LENOm, G. & DE KINKELIN, P., Fish rhabdoviruses: comparative study of protein structure. J. Virol., 1975, 16, 259-266. [13] MADORE,H. P. & ENGLAND,J. M., Rabies virus protein synthesis in infected BHK-21 cells. J. Virol., 1977, 22, 102-112. [14] McALLmTER,P. E. & WAGNER, R. R., Structural proteins of two salmonids rhabdoviruses. J. Virol., 1975, 15, 733-738. [15] NAGY, E. & DOBOS, P., Synthesis of Drosophila X virus proteins in cultured Drosophila cells. Virology, 1984, 134, 358-367. [16] SAUERBIER, W., Radiology applied. Mapping transcriptional organization in pro- and eukaryotes, in (( Proceedings of the Fifth International Congress of Radiation Research ))(p. 651-662). Academic Press, New York, London, 1975.
Ann. Inst. Pasleur/Virol. i985, 136 E, 213-222
EFFECT
OF
OF
VIRAL
ON
VIRUS-SPECIFIC
UV
IRRADIATION
HAEMORRHAGIC
SEPTICAEMIA
INTRACELLULAR
VIRUS
SYNTHESES
by J. Bernard and P. de Kinkelin Laboratoire d' Ichlgopalhologie, I N R A , Groupe de Laboratoires de Palhologie Animale, route de Thiverval, 78850 Thiverval-Grignon (France)
SUMMARY The effect of UV irradiation of virions on subsequent virus-specific intracellular protein synthesis was studied. The order of three genes (N, M1 and G) was determined, but L and M2 could not be mapped. KEY-WORDS: Viral haemorrhagic septicaemia virus, Genome, UV, Polypepfide; Synthesis, EPC cells.
INTRODUCTION Viral haemorrhagic septicaemia (VHS) virus is a member of the Rhabdovirus family and belongs to the Lyssa genus. The mature virus particle contains 5 polypeptides, L, G, N, M1 and Ms, with molecular weights of 190, 80, 38, 25 and 19 • 103, respectively [12, 14] and a negative-stranded RNA genome the S(2o,w) coefficient of which is 38-40 [9]. In the present work, we attempted to locate the different genes on t h a t genome. The order of the genes on a genome can be established by analysing the effect of ultraviolet (UV) irradiation of virions on subsequent intracellular synthesis. The method pioneered by Sauerbier [16] has been well documented for rhabdoviruses [1, 2, 3, 8], but it is valid only if the observed syntheses are the result of primary transcription, so t h a t newly replicated, undamaged genomes could not interfere. Thus, since primary transcription of VHS virus is difficult to observe due to the low level of RNA synthesis, we chose the indirect method and studied the inactivation of protein synthesis in order to determine the time-course of viral syntheses and to then map part of the genome. Manuscrit re~u le 3 mat 1985, accept6 le 3 aofit 1985.
214
J. BEBNAFID AND P. D E K I N K E L I N
MATERIALS
AND
METHODS
Virus and cells. In order to avoid any drift of virulence [6, 4] VHS virus strain 07-71 [11] was reisolated from an infected fish and submitted to only 2 passages at 14 ~ C in Epithetioma papulosum cyprini (EPC) cells [7] as already described [5] without plaque purification. Virus stocks titrating at 1 • 109 plaque-forming units ( P F U ) / m l were frozen at --70 ~ C until use, then concentrated by centrifugation when high multiplicities of infection (MOI) were needed. D E A E - d e x t r a n (50 [xg/ml) was added to the inocula.
UV irradiation. Virus suspensions in duplicate were irradiated in 24-well (Nunc) plastic plates (200 ~xl/well) at a distance of 14 cm from a germicidal lamp (10.6 • 10 .6 Joules/ s/ram 9, 106 ergs/s/mm ~, as determined with a L a t a r j e t dosimeter) and immediately used to infect EPC cells. Portions were sampled to determine the original titre and inactivation rate by means of plaque titration under an agarose overlay. E P C cells seeded in plastic (Corning) Petri dishes 30 m m in diameter (4.5 • 10" cells per dish) were irradiated for 15 s at a distance of 8 cm from the same germicidal lamp, then immediately infected.
Labelling, extraction and electrophoresis o/ inlracellular proteins. Irradiated cells were either mock-infected with suspension medium (Stocker's medium supplemented with 10% t r y p t o s e and 2 % foetal calf serum) or infected with the virus suspension (150 [xl per dish: M O I = 8 9 ) and the virus was adsorbed for 1 h at 14 ~ C. The cells were then pretreated for 1 h post-adsorption (PA) with Earle's saline to deplete the intracellular amino acids, and were finally labelled for the indicated time with Earle's saline containing 30 ~Ci/ml of ~SS-methionine (specific activity 800 Ci/mM; Amersham). At the end of the labelling period, the cells were washed 3 times with Earle's saline, then lysed with 300 ~xl per dish of K buffer (0.01 M NaCI; 0.01 M Tris-hydrochloride ( p H 8.0); 0.5% sodium dodecyl sulphate). The viscous e x t r a c t was sonieated (20 s in ice) and the proteins were precipitated at 4 ~ C with acetone. The samples were centrifuged for 15 min at 13,000 g (Hettich Mikroliter centrifuge). The pellets were resuspended in 20 ~zl of K buffer. One volume of dissociating buffer (62.5 mM Tris-hydrochloride ( p H 6.8); 1% sodium dodecyl sulphate; 10% glycerol; 2 % 2-mercaptoethanol; 0.001% bromophenol blue) was added and the samples were maintained for 1.5 min in a boiling water bath, then cooled and loaded in the wells of a discontinuous 10% acrylamide slab gel in L a e m m l i ' s [10] b u f f e r system. Electrophoresis was run at 12 mA for the stacking gel, then 24 mA for the resolving gel, constant eurrenL until the tracer dye had migrated for 7-10 em. The gels were t h e n stained for 15 min with Comassie blue, destained, dried under v a c u u m and autoradiographed for 3-24 h using ~ R P X-OMAT K o d a k ~ film; the autoradiograms were then scanned.
EPC = epithelioma papulosum eyprini. MOI = multiplicity of infection. PA = post-adsorption.
PFU = plaque-forming unit. UV = ultraviolet. VHS = viral haemorrhagic septicaemia.
UV AND VHS VIRAL INTRACELLULAR SYNTHESES
215
RESULTS
Inhibition o[ cellular syntheses. Since VHS virus does not inhibit cellular syntheses, it was necessary to pretreat the cells so t h a t virus-specific syntheses could be observed. W h e n the cells were UV-irradiated for 15 s at 10.6 • 10 .6 J o u l e s / s / m m ~, residual cellular activity still obscured virus-specific polypeptides on the autoradiograms (data not shown). Increasing the UV dose by exposing the cells at 8 em from the lamp almost completely inhibited cellular syntheses, and the viral polypeptides could be easily evidenced, except for the Ms. The G migrated as a sharp band and t h u s was probably not glyeosylated (see below). Plaque counts were impossible because the cells died within 24 h post-irradiation.
Timing of lranscription in irradiated cells. To verify t h a t the irradiated cells could still support viral syntheses, t h e y were infected at a MOI of 89 P F U of non-irradiated virus per cell, t h e n labelled from 1 to 3, 1 to 4 and 1 to 5 h PA. By 3 h, the N and M1 polypeptides appeared on the autoradiograms (fig. 1A, lane 0). B y 4 h, traces of G and Ms could be seen (fig. 1B, lane 0). B y 5 h, the 4 polypeptides were present in significant amounts, b u t the L was not visible (fig. 1C, lane 0). Secondary transcription results in amplification of virus syntheses and its onset is more easily determined in cells infected with irradiated genomes since, in t h a t ease, the inactivation curves as a function of the dose are no longer representative of single-hit events. The virus particles were thus irradiated for various lengths of time to determine the survival curves. For infectivity, the t a r g e t of which is the full-length genome, the 37% P F U survival representative of one hit per t a r g e t was repeatedly obtained at 2-s irradiation (212 ergs/mm 2) (fig. 2). The irradiated virus particles were used to infect irradiated cells at an MOI equivalent to 89 PFU, and the resulting viral protein syntheses were studied from 1 to 3, 1 to 4 and 1 to 5 h as above. By 3 h PA, N was still visible in cells infected with virus irradiated for 15 s (fig. 1A) b u t its 37~o survival dose could not be accurately determined because, due to the low rate of synthesis, standard deviation was high (fig. 2). By 4 h PA, N was again visible in all samples, M1 up to 8 s of irradiation and G up to 6 s (fig. 1B). The survival curves of N and G were bimodal (fig. 2) and t h u s representative of the onset of secondary transcription. M~ could not be quantified due to numerous discrete bands. Those discrete bands were not visible on the control uninfected cells samples (lanes C). By 5 h PA, the synthesis of all 3 polypeptides was amplified and t h e y could be seen in all samples (fig. 1C).
FIG. 1. - - Viral protein synthesis after U V irradiation o/ V H S virus particles. Cellular protein synthesis was depressed b y UV irradiation; cells were t h e n infected w i t h UV-irradiated (2-10 s) or non-irradiated (lane 0) v i r u s at an MOI equivalent to 90 P F U of non-irradiated virus, and labelled f r o m 1 to 3 (A), 1 to 4 (B) or 1 to 5 (C) h o u r s PA. Lanes in C show mock-infected controls. Multiple exposures of the X - r a y film were used so as to be within t h e range of the fihn response.
Fro. 1
UV AND VHS VIRAL INTRACELLULAR SYNTHESES
5(
07Y71
"82 X , \
PFU
219
G
2E
o= o~
I
~ ~~ ~ . 9
~
I
e
M1
~,
10
5 SECONDS
10
t.N
FIG. 2. - - Dose response curves of V H S virus infectivity
and viral protein synthesis as a {unction of U V dose. P F U c u r v e : t h e o r i g i n a l t i t r e w a s 4 • 10 8 P F U / m l ; all c a l c u l a t i o n s w e r e d o n e o n a m e a n of 2 e x p e r i m e n t s a n d a m i n i m u m of 400 p l a q u e s w e r e c o u n t e d f o r e a c h p o i n t o f e a c h e x p e r i m e n t . A l l o t h e r c u r v e s : f r o m d e n s i t o m e t e r t r a c i n g s u s i n g a ,, G e l m a n D C D 16 ,, d e n s i t o m e t e r , of 4 a u t o r a d i o g r a m s s i m i l a r t o t h a t in fig. 1. 9 : 3 h P A ; 9 ~ 4 h P A ; 9 : 5 h P A . A l l o r d i n a t e s a r e o n a l o g scale.
Tentative to map the genome.
Mapping the genome b y means of UV irradiation is valid only during primary transcription, which in our case was 3 h PA. On the other hand, b y t h a t time, numerous discrete bands were seen in infected cells, which could represent any one of the nascent polypeptides and cause an underestimation. Thus, we turned to pulse-chase experiments. The cells were labelled from 0.5 to 3 h PA, then washed and incubated for 3 h more in Stoker's medium so t h a t the labelled polypeptides could be initiated during prim a r y transcription, then terminated and accumulated in t h e absence of the radioactive precursor (fig. 3). In these conditions, when the percentage of survival was plotted against t h e UV dose, t h e values we obtained for G and M1 aligned on straight lines (fig. 4) with 3 7 % survival doses of, respectively, 3.6 and 6 s, b u t L could not be seen and M2 could still not be quantified. DISCUSSION V H S virus does not inhibit cellular synthesis and, since viral synthesis is maintained at a low level, an inhibitory t r e a t m e n t had to be found, Osmotic shock was avoided because it disturbs t h e synthesis of G for other Lyssa viruses [8, 13]. Heat-shock [15] would have been inefficient because V H S virus is more thermosensitive t h a n E P C cells [6, 7]. W e finally irra-
220
J. B E R N A R D
F I 6 . 3. - -
A N D P. D E K I N K E L I N
Pulse-chase labelling of viral proteins.
T h e c e l l s w e r e l a b e l l e d f r o m 0.5 t o 3 h P A , t h e n medium.
r
r
10( il
G
maintained
until 6 b PA in non-radioactive
k .l\ :
05C Z
U~
~o
SECONDS FIG. 4. - -
UV
Dose-response curves of viral protein synthesis in pulse-chase experiments.
UV AND VHS VIBAL INTRACELLULAR SYNTHESES
221
diated the cells to death prior to infection. In such conditions, cellular synthesis were drastically reduced (fig. 1, lanes C) and viral polypeptides became visible in the same order as published for actionomyein-D-treated cells [6]. On the other hand, the synthesis of M2 was impaired, as described for rabies virus [8]; G, however, could be identified, which contrasts with rabies virus in irradiated BHK21 cells [8]. Since viral polypeptides became visible in the order N-~M~ ~ G Ms ~ L (fig. 1), V H S virus genes are p r o b a b l y transcribed sequentially with N located at the 3' end of the genome. The results of pulse-chase experiments reinforce the hypothesis t h a t M1 is transcribed before G (37 % survival doses of, respectively, 3.6 and 6 s) and t h a t G is not terminal on the 5' end, since the P F U 37 % survival dose is 2 s. L could not be evidenced before the onset of secondary transcription, which could be due either to a v e r y low b u t continuous rate of transcription or to a delayed expression of the 5' end of the genome.
Bt~SUMt~
SUR
LES
E F F E T DE L ' I R R A D I A T I O N U V DU VIRUS DE LA SEPTICI~MIE tII~MORRAGIQUE VIRALE SYNTHESES INTRACELLULAIRES SPI~CIFIQUES
DU
VIRUS
Nous avons ~tudi~ les syntheses virales apr~s infection de cellules E P C par des partieules irradi6es. L'ordre de 3 g~nes, N, M1 et G, a pu ~tre d6termin6 mais eelui de M2 et L est incertain. MOTS-CLI~S : Septicemic h6morragique p e p t i d e ; Syntheses, Cellules EPC.
virale,
GLnome, UV,
Poly-
ACKNOWLEDGMENTS
We are grateful to M. Bearzotti-Le Berre, who prepared the virus used as an inoculum.
REFERENCES
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J. BERNARD AND P. DE K I N K E L I N
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