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Interferon induction by and infectivity of ultraviolet-irradiated double-and multistranded viral RNA's
I95
SHORT COMMUNICATIONS
to o.o2 %); this seems to indicate that the T 7 DNA-S. lutea RNA complex has very httle complementary regions of some extent ~ which escape the action of the nbonuclease 8. This work shows that S. lutea RNA is responsible for the interaction with one on the T 7 coliphage complementary strands; the effect is analogous to, although less important than, that described b y SUMMERS AND SZYBALSKI4,5 with poly G. Surprisingly, no interaction was found between S. lutea denatured DNA and RNA from the same bacterial origin; this is in opposition to the results of 0PARA-KUBINSKA et al?. It is also concluded that it is Important to remove completely the RNA from the preparation before using the S. lutea DNA as a density marker for an unknown denatured DNA. This work was supported b y grants from the Canadian Medical Research Council, the National Cancer Institute of Canada and the Haldina-Fontaine-Hamel Fund. W. G. VERLY M. TUY L. BRAKIER M. JAMES
Ddpartement de Bzoch,mze, Universztd de Montrdal, Montrdal (Canada)
I W G. VERLY AND L. BRAKIER, B*och*m B~ophys. Acta, 174 (1969) 674 2 W. SZYBALSKI AND H ~) MEHNIGMANN, Anal. Bzochem, 3 (1962) 267. 3 Z DISCHE, in E. CHARGAFF AND J. N. I)AVIDSOH, The Nucleic Aczds, Vol I, Academic Press, New York, 1955, p 285 4 W C SUMMERS AND W SZYBALSKI, Vzrology, 34 (I968) 9. 5 W. C SUMMERS AND W. SZYBALSKI, B*ochzm. Bzophys Acta, 166 (1968) 371 6 ]3. W F o x , Intern J Appl. Rad*atzon Isotopes, 19 (1968) 717 7 S K NIYOGI AND C A. THOMAS, Jr., B*ochem B*ophys Res Commun , 26 (1967) 51 . 8 S A YANKOFSKY AND S SPIEGELMAN, Proc Natl Acad Scz U S , 18 (1962) lO69 9 Z OPARA-KUBINSKA, H. KUBINSKI AND W~. SZYBALSKI, Proc Natl Acad Sc~ U S , 52 (1964) 923
Received June i l t h , 197o Bzoch,m B~ophys Acta, 217 (197 o) 192-195 BBA 93532
Interferon induction by and infectivity of ultroviolet-irradioted and multistranded viral RNA's
double-
L cells infected with Mengo virus contain three molecular species of viral coded RNA 1,2 similar to those observed in other small RNA virus systems. The first and predominant species is a single-stranded RNA which is indistinguishable from the RNA isolated from virus particles. The second is a double-stranded RNA consisting of a strand of virus RNA and a complementary strand synthesized after infection. This form has been called the replicative form (RF) ~. The third species, the replicative intermediate (RI), is a hybrid molecule, partially single- and partially double-stranded, apparently composed of a double-stranded core with associated nascent singlestranded branches 4. All three species are infectious. Double-stranded RNA molecules Abbreviations RF, rephcatlve form; RI, rephcatlve intermediate
B,och*m. B~ophys Acta, 217 (197 o) 195-198
196
SHORT COMMUNICATIONS
from n u m e r o u s sources are efficient inducers ot interferon a n d of resistance to v i r a l infection, m wtro a n d i n w v o 5,6. W e h a v e r e c e n t l y d e m o n s t r a t e d 1 t h a t , as expected, t h e Mengo virus R F (14-16 S) is a n effective interferon i n d u c e r when i n o c u l a t e d into mice, while the s i n g l e - s t r a n d e d form is n o t The multmstranded R I also induces interferon a n d this p r o p e r t y is l i n k e d exclusively to its d o u b l e - s t r a n d e d core 2. While the a b i l i t y to infect is t h e expression of a n u n d a m a g e d base sequence, interferon-inducing c a p a c i t y seems to be d e p e n d e n t o n l y on t h e d o u b l e - s t r a n d e d s t r u c t u r e of t h e inducer. I n the present p a p e r the dissociation of these two p r o p e r t i e s of the R F a n d the R I b y u l t r a v i o l e t a c t i o n is r e p o r t e d . R F a n d R I were p r e p a r e d as described elsewhere 1,2,*. A f t e r f r a c t i o n a t i o n b y LIC1, further purification was achieved b y c h r o m a t o g r a p h y on cellulose columns 7 a n d stepwxse elution using three different alcohol c o n c e n t r a t i o n s (35 %, 15 %, o °/o ). I r r a d i a t i o n was carried out at 4 ° using a g e r m i c i d a l l a m p (2537 A) a n d a L a t a r j e t m e t e r s for d o s l m e t r y . Doses were corrected for a b s o r p t i o n 9 A f t e r i r r a d i a t i o n , infect i v i t y was m e a s u r e d as p l a q u e - f o r m i n g a b i l i t y on D E A E - d e x t r a n - p r e t r e a t e d L cells. I n t e r f e r o n - i n d u c i n g c a p a c i t y was m e a s u r e d b y i n t r a p e r i t o n e a l inoculation into mice a n d t i t r a t i o n of t h e i n d u c e d circulating interferon on L cells, using vesicular s t o m a t i t I s virus as the challenge virus 100 ~ - ~
-- ~#~-
~- - - -~ "-'---
100
lO ~
10
t~
X
u_
go lOO 1go 26o
' 2~5'% DOSE
(ergs
/ram
a × 10 - 3 )
Fig I Virus and RNA preparations were diluted m phosphate-buffered sahne (pH 7 2) I-ml ahquots Jrradlated m 6o-mm petrl dishes Plaque-forming abthty was measured on L929 cells, pretreated with ioo ffg/ml DEAE-dextran &-/k, Mengo virus, 7 ] ' ' .rN, RI, A - - - A , RF, Interferon-reducing capacity was measured by inoculation into mine (io fig/mouse) mtraperltoneally O" "O, RI, O - O , RF F r o m F i g i , showing the s u r v i v a l curves of purified Mengo virus, R F a n d R I , it can be seen t h a t the p l a q u e - f o r m i n g a b i l i t y of purified R F is more u l t r a v i o l e t r e s i s t a n t t h a n t h a t of the whole virus 1°,11. I n c o n t r a s t w i t h the p h o t o s e n s i t i v i t y of the p l a q u e - f o r m i n g ability, the i n w v o i n t e r f e r o n - i n d u c i n g c a p a c i t y of R F a n d R I is h i g h l y resistant. No change was d e t e c t e d after 50 ooo e r g s / m m 2 to t h e R I or r c o ooo e r g s / m m 2 to t h e R F . A dose of 200 ooo e r g s / m m 2 was necessary to diminish t h e i n t e r f e r o n - i n d u c i n g c a p a c i t y of R F to 75 % of t h e c o n t r o l level. The s a m e phen o m e n o n was seen i n v,tro (Fig. 2). R F solutions ( I o f f g / m l ) were i r r a d i a t e d with various doses of u l t r a v i o l e t h g h t ; D E A E - d e x t r a n - p r e t r e a t e d L cells were i n c u b a t e d w i t h these solutions. The a m o u n t of interferon i n d u c e d b y i r r a d i a t e d R F b e g a n to decrease o n l y after 50 ooo e r g s / m m 2. The i n f e c t i v i t y of R F was m e a s u r e d in the same * The purchase of radlolsotopes was partly supported by the C E A France. Bwch*m Bzophys Acta, 217 (197° ) 195-198
SHORT COMMUNICATIONS
197
experiment b y titration of the Mengo virus produced b y the irradiated RNA's on B H K cells. The slope of the resulting curve is not related to that of interferon production but rather to that of the loss of infectivity of R F due to irradiation (Fig. 2). Ultraviolet action on Interferon-inducing capacity of double-stranded synthetic copolymer poly I-C was also studied , n w v o and , n vitro. Table I summarizes the response of L cells to different doses of poly I. C; IO jug/ml seemed to be the optimal dose for further work Higher doses of inducer were toxic to the cells, a fact whmh TABLE
I
I N T E R F E R O N I N D U C E D *n UCD'O B Y D I F F E R E N T D O S E S OF P O L Y I " C
Poly I . C ~ug/ml)
I n l e r / e r o n (I U / o 5 m l )
250 IOO 25 io i o I o oI
256 lO24 2048 4096 2048 64 Undetectable
could explain the correspondingly lower levels of induced interferon. With the optimal dose of poly I.C, the radioresistance is similar to that of RF and RI: no change in interferon induction could be detected from o to 50 ooo ergs/mm 2. The same results were observed when the induction was measured zn vivo (25#g/mouse) (Fig. 3). ,oo[ . . . . .
""
"
"
.
I'ooz
/'-,.
1
12
o
34567'1o
50
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o
....
°
|
loo
~.
'
DOSE (er£ s/ mrnaxlO -3)
;o
'
lbo
o
1;o
'
2bo"
DOSE (ergs/mm2xlO -3)
Fig. 2 R F p r e p a r a t i o n s were d i l u t e d in p h o s p h a t e - b u f f e r e d s a l i ne t o a c o n c e n t r a t i o n of i o / z g / m l a n d i - m l a l i q u o t s i r r a d i a t e d C o n f l u e n t CCL I cells, p r e t r e a t e d w i t h D E A E - d e x t r a n , r e c e i v e d 2/~g of R N A . After I h of c o n t a c t , cells were w a s h e d a n d fed w i t h 2. 5 ml of fresh m e d i u m . T h e Mengo v i r u s a n d i n t e r f e r o n p r o d u c e d were m e a s u r e d in t h e s u p e r n a t a n t s 18 h l a t e r. Before t i t r a t l o n s , m t e r f e r o n w a s s u b j e c t e d to p H 2 for 24 h a n d u l t r a c e n t r i f u g e d . • - - - • , Mengo v i r u s ; O" • "O, i n t e r f e r o n F i g 3. I n t e r f e r o n - m d u c m g c a p a c i t y of i r r a d i a t e d p o l y I . C w a s m e a s u r e d *n vzvo ( 2 5 / , g / m o u s e ) or zn v,tro ( 2 5 / ~ g / p e t r l dish of CCLI cells). O " • " 0 , zn v w o , 0 - - - 0 , zn v,tro
The survival curve of R I has an intermediate position between Mengo virus (single-stranded RNA) and R F (double-stranded RNA). These results m a y be a consequence of the particular structure of each species of RNA. It is accepted t h a t enzymatic excision of pyrlmidine dlmers from DNA is followed b y the reconstruction of the twin helix b y repair enzymes, using the intact opposite strand as template 12. The existence of such repair of ultraviolet-damaged double-stranded RNA has also been suggested 11. The sizes of the reparable regions of the R F and the R I would be B,ochzm
B~ophys
A c t a , 217 (197 o) 195-198
198
SHORT COMMUNICATIONS
different: while in the R F the plus infectious strand is hydrogen-bonded to its complementary minus strand, in the R I the plus strand is partially displaced from the association with the complementary template b y the ongoing synthesis of other progeny strands. The plus strand seems to be the only infectious one (J. M. BECHET AND L. MONTAGNIER,personal communication; ref. 13). These facts m a y account for the difference in reparability and, hence, in ultraviolet sensitivity of the plaque-forming abihty of the two double-stranded forms of RNA. The major products of ultraviolet irradiation at the molecular level are the formation of pyrimidine dimers and uracil hydrates. By these or other mechanisms, ultraviolet irradiation probably impairs the transcription of the genetic information involved in virus multiplication. Since the interferon-inducing capacity of the R F is completely resistant to IOO ooo ergs/mm 2, it is likely that this property is independent of the integrity of the genome but is still a function of the double-strandedness of the RNA; no changes in ribonuclease resistance or hyperchromicity due to this high dose of irradiation were detected with RF, suggestmg that the double-stranded structure had not been broken down. The sedimentation propertms in a sucrose density gradient of poly I-C and RF, irradiated at IOOOOOergs/mm 2, were also unaffected. The rephcative forms of RNA viruses are substances which could possibly be used as interferon inducers in human antiviral therapy. The therapeutic assay is handicapped b y their intrinsic infectivity. Ultraviolet irradiation provides a method for abolishing the infectivity without modifying the interferon-inducing capacity. We t h a n k Professor R. Latarjet for advice and information, and Drs. R. D a y and J. Coppey for valuable discussions. The technical assistance of Misses A. De Vomecourt and J. Sanceau is also acknowledged.
Laborato~re Pasteur, Inst, tut du R a d i i , 26 rue d'Ulm, Paris 5e (France) i 2 3 4 5 6 7 8 9 IO ii 12 13
R. FALCOFF E. FALCOFF L. CATINOT
t{. FALCOFF AND E T FALCOFF, B*ochzm B*ophys. Acta, 182 (1969) 5Ol. R. FALCOFF AND ]~. T. FALCOFF, B,och,m Bwphys. Acta, 199 (197 o) 147. L. MONTAGNIER AND F SANDERS, Nature, 199 (1963) 664. M. FENWlCK, R ERIKSON AND R FRANKLIN, Sczence, 146 (1964) 527 . G. LAMPSON, A TYTELL, A. FIELD, A ~EMES AND M. HILLEMAN, Proc. Natl Acad. Sc*. U S , 58 (1967) 782 E. FALCOFF AND R. FEREZ BERCOFF, Bwchzm Bwphys. Acta, 174 (1969) lO8 R. FRANKLIN, Proc. Natl Acad. Sc, U.S., 55 (1966) 15o 4. R. LATARJET, P. MORENNE AND R BERGER, Ann. Inst. Pasteur, 85 (1953) 174. R. LATARJET, R CRAMEB. AND L. MONTAGNIER, Vzrology, 33 (1967) lO4. J. M BISHOP, N. QUINTRELL AND G. KOCH, J. Mol Bzol, 24 (1967) 125 . Z ZAVADOVA, L GRESLAND AND •. ROSENBERGOVA, Acta Vzrol, i2 (1968) 515. P. HOWARD-FLANDERS, A n n Rev Bwchem., 37 (I968) 175. J. M. BISHOP, G. KOCH, B. EVANS AND ~¢[. MERRIMAN, J. Mol. B w l , 46 (1969) 235.
Received April i5th, 197o B~ochim. B*ophys
Acta, 217 (197 o) 195-198
SHORT COMMUNICATIONS
to o.o2 %); this seems to indicate that the T 7 DNA-S. lutea RNA complex has very httle complementary regions of some extent ~ which escape the action of the nbonuclease 8. This work shows that S. lutea RNA is responsible for the interaction with one on the T 7 coliphage complementary strands; the effect is analogous to, although less important than, that described b y SUMMERS AND SZYBALSKI4,5 with poly G. Surprisingly, no interaction was found between S. lutea denatured DNA and RNA from the same bacterial origin; this is in opposition to the results of 0PARA-KUBINSKA et al?. It is also concluded that it is Important to remove completely the RNA from the preparation before using the S. lutea DNA as a density marker for an unknown denatured DNA. This work was supported b y grants from the Canadian Medical Research Council, the National Cancer Institute of Canada and the Haldina-Fontaine-Hamel Fund. W. G. VERLY M. TUY L. BRAKIER M. JAMES
Ddpartement de Bzoch,mze, Universztd de Montrdal, Montrdal (Canada)
I W G. VERLY AND L. BRAKIER, B*och*m B~ophys. Acta, 174 (1969) 674 2 W. SZYBALSKI AND H ~) MEHNIGMANN, Anal. Bzochem, 3 (1962) 267. 3 Z DISCHE, in E. CHARGAFF AND J. N. I)AVIDSOH, The Nucleic Aczds, Vol I, Academic Press, New York, 1955, p 285 4 W C SUMMERS AND W SZYBALSKI, Vzrology, 34 (I968) 9. 5 W. C SUMMERS AND W. SZYBALSKI, B*ochzm. Bzophys Acta, 166 (1968) 371 6 ]3. W F o x , Intern J Appl. Rad*atzon Isotopes, 19 (1968) 717 7 S K NIYOGI AND C A. THOMAS, Jr., B*ochem B*ophys Res Commun , 26 (1967) 51 . 8 S A YANKOFSKY AND S SPIEGELMAN, Proc Natl Acad Scz U S , 18 (1962) lO69 9 Z OPARA-KUBINSKA, H. KUBINSKI AND W~. SZYBALSKI, Proc Natl Acad Sc~ U S , 52 (1964) 923
Received June i l t h , 197o Bzoch,m B~ophys Acta, 217 (197 o) 192-195 BBA 93532
Interferon induction by and infectivity of ultroviolet-irradioted and multistranded viral RNA's
double-
L cells infected with Mengo virus contain three molecular species of viral coded RNA 1,2 similar to those observed in other small RNA virus systems. The first and predominant species is a single-stranded RNA which is indistinguishable from the RNA isolated from virus particles. The second is a double-stranded RNA consisting of a strand of virus RNA and a complementary strand synthesized after infection. This form has been called the replicative form (RF) ~. The third species, the replicative intermediate (RI), is a hybrid molecule, partially single- and partially double-stranded, apparently composed of a double-stranded core with associated nascent singlestranded branches 4. All three species are infectious. Double-stranded RNA molecules Abbreviations RF, rephcatlve form; RI, rephcatlve intermediate
B,och*m. B~ophys Acta, 217 (197 o) 195-198
196
SHORT COMMUNICATIONS
from n u m e r o u s sources are efficient inducers ot interferon a n d of resistance to v i r a l infection, m wtro a n d i n w v o 5,6. W e h a v e r e c e n t l y d e m o n s t r a t e d 1 t h a t , as expected, t h e Mengo virus R F (14-16 S) is a n effective interferon i n d u c e r when i n o c u l a t e d into mice, while the s i n g l e - s t r a n d e d form is n o t The multmstranded R I also induces interferon a n d this p r o p e r t y is l i n k e d exclusively to its d o u b l e - s t r a n d e d core 2. While the a b i l i t y to infect is t h e expression of a n u n d a m a g e d base sequence, interferon-inducing c a p a c i t y seems to be d e p e n d e n t o n l y on t h e d o u b l e - s t r a n d e d s t r u c t u r e of t h e inducer. I n the present p a p e r the dissociation of these two p r o p e r t i e s of the R F a n d the R I b y u l t r a v i o l e t a c t i o n is r e p o r t e d . R F a n d R I were p r e p a r e d as described elsewhere 1,2,*. A f t e r f r a c t i o n a t i o n b y LIC1, further purification was achieved b y c h r o m a t o g r a p h y on cellulose columns 7 a n d stepwxse elution using three different alcohol c o n c e n t r a t i o n s (35 %, 15 %, o °/o ). I r r a d i a t i o n was carried out at 4 ° using a g e r m i c i d a l l a m p (2537 A) a n d a L a t a r j e t m e t e r s for d o s l m e t r y . Doses were corrected for a b s o r p t i o n 9 A f t e r i r r a d i a t i o n , infect i v i t y was m e a s u r e d as p l a q u e - f o r m i n g a b i l i t y on D E A E - d e x t r a n - p r e t r e a t e d L cells. I n t e r f e r o n - i n d u c i n g c a p a c i t y was m e a s u r e d b y i n t r a p e r i t o n e a l inoculation into mice a n d t i t r a t i o n of t h e i n d u c e d circulating interferon on L cells, using vesicular s t o m a t i t I s virus as the challenge virus 100 ~ - ~
-- ~#~-
~- - - -~ "-'---
100
lO ~
10
t~
X
u_
go lOO 1go 26o
' 2~5'% DOSE
(ergs
/ram
a × 10 - 3 )
Fig I Virus and RNA preparations were diluted m phosphate-buffered sahne (pH 7 2) I-ml ahquots Jrradlated m 6o-mm petrl dishes Plaque-forming abthty was measured on L929 cells, pretreated with ioo ffg/ml DEAE-dextran &-/k, Mengo virus, 7 ] ' ' .rN, RI, A - - - A , RF, Interferon-reducing capacity was measured by inoculation into mine (io fig/mouse) mtraperltoneally O" "O, RI, O - O , RF F r o m F i g i , showing the s u r v i v a l curves of purified Mengo virus, R F a n d R I , it can be seen t h a t the p l a q u e - f o r m i n g a b i l i t y of purified R F is more u l t r a v i o l e t r e s i s t a n t t h a n t h a t of the whole virus 1°,11. I n c o n t r a s t w i t h the p h o t o s e n s i t i v i t y of the p l a q u e - f o r m i n g ability, the i n w v o i n t e r f e r o n - i n d u c i n g c a p a c i t y of R F a n d R I is h i g h l y resistant. No change was d e t e c t e d after 50 ooo e r g s / m m 2 to t h e R I or r c o ooo e r g s / m m 2 to t h e R F . A dose of 200 ooo e r g s / m m 2 was necessary to diminish t h e i n t e r f e r o n - i n d u c i n g c a p a c i t y of R F to 75 % of t h e c o n t r o l level. The s a m e phen o m e n o n was seen i n v,tro (Fig. 2). R F solutions ( I o f f g / m l ) were i r r a d i a t e d with various doses of u l t r a v i o l e t h g h t ; D E A E - d e x t r a n - p r e t r e a t e d L cells were i n c u b a t e d w i t h these solutions. The a m o u n t of interferon i n d u c e d b y i r r a d i a t e d R F b e g a n to decrease o n l y after 50 ooo e r g s / m m 2. The i n f e c t i v i t y of R F was m e a s u r e d in the same * The purchase of radlolsotopes was partly supported by the C E A France. Bwch*m Bzophys Acta, 217 (197° ) 195-198
SHORT COMMUNICATIONS
197
experiment b y titration of the Mengo virus produced b y the irradiated RNA's on B H K cells. The slope of the resulting curve is not related to that of interferon production but rather to that of the loss of infectivity of R F due to irradiation (Fig. 2). Ultraviolet action on Interferon-inducing capacity of double-stranded synthetic copolymer poly I-C was also studied , n w v o and , n vitro. Table I summarizes the response of L cells to different doses of poly I. C; IO jug/ml seemed to be the optimal dose for further work Higher doses of inducer were toxic to the cells, a fact whmh TABLE
I
I N T E R F E R O N I N D U C E D *n UCD'O B Y D I F F E R E N T D O S E S OF P O L Y I " C
Poly I . C ~ug/ml)
I n l e r / e r o n (I U / o 5 m l )
250 IOO 25 io i o I o oI
256 lO24 2048 4096 2048 64 Undetectable
could explain the correspondingly lower levels of induced interferon. With the optimal dose of poly I.C, the radioresistance is similar to that of RF and RI: no change in interferon induction could be detected from o to 50 ooo ergs/mm 2. The same results were observed when the induction was measured zn vivo (25#g/mouse) (Fig. 3). ,oo[ . . . . .
""
"
"
.
I'ooz
/'-,.
1
12
o
34567'1o
50
'°°r-
o
....
°
|
loo
~.
'
DOSE (er£ s/ mrnaxlO -3)
;o
'
lbo
o
1;o
'
2bo"
DOSE (ergs/mm2xlO -3)
Fig. 2 R F p r e p a r a t i o n s were d i l u t e d in p h o s p h a t e - b u f f e r e d s a l i ne t o a c o n c e n t r a t i o n of i o / z g / m l a n d i - m l a l i q u o t s i r r a d i a t e d C o n f l u e n t CCL I cells, p r e t r e a t e d w i t h D E A E - d e x t r a n , r e c e i v e d 2/~g of R N A . After I h of c o n t a c t , cells were w a s h e d a n d fed w i t h 2. 5 ml of fresh m e d i u m . T h e Mengo v i r u s a n d i n t e r f e r o n p r o d u c e d were m e a s u r e d in t h e s u p e r n a t a n t s 18 h l a t e r. Before t i t r a t l o n s , m t e r f e r o n w a s s u b j e c t e d to p H 2 for 24 h a n d u l t r a c e n t r i f u g e d . • - - - • , Mengo v i r u s ; O" • "O, i n t e r f e r o n F i g 3. I n t e r f e r o n - m d u c m g c a p a c i t y of i r r a d i a t e d p o l y I . C w a s m e a s u r e d *n vzvo ( 2 5 / , g / m o u s e ) or zn v,tro ( 2 5 / ~ g / p e t r l dish of CCLI cells). O " • " 0 , zn v w o , 0 - - - 0 , zn v,tro
The survival curve of R I has an intermediate position between Mengo virus (single-stranded RNA) and R F (double-stranded RNA). These results m a y be a consequence of the particular structure of each species of RNA. It is accepted t h a t enzymatic excision of pyrlmidine dlmers from DNA is followed b y the reconstruction of the twin helix b y repair enzymes, using the intact opposite strand as template 12. The existence of such repair of ultraviolet-damaged double-stranded RNA has also been suggested 11. The sizes of the reparable regions of the R F and the R I would be B,ochzm
B~ophys
A c t a , 217 (197 o) 195-198
198
SHORT COMMUNICATIONS
different: while in the R F the plus infectious strand is hydrogen-bonded to its complementary minus strand, in the R I the plus strand is partially displaced from the association with the complementary template b y the ongoing synthesis of other progeny strands. The plus strand seems to be the only infectious one (J. M. BECHET AND L. MONTAGNIER,personal communication; ref. 13). These facts m a y account for the difference in reparability and, hence, in ultraviolet sensitivity of the plaque-forming abihty of the two double-stranded forms of RNA. The major products of ultraviolet irradiation at the molecular level are the formation of pyrimidine dimers and uracil hydrates. By these or other mechanisms, ultraviolet irradiation probably impairs the transcription of the genetic information involved in virus multiplication. Since the interferon-inducing capacity of the R F is completely resistant to IOO ooo ergs/mm 2, it is likely that this property is independent of the integrity of the genome but is still a function of the double-strandedness of the RNA; no changes in ribonuclease resistance or hyperchromicity due to this high dose of irradiation were detected with RF, suggestmg that the double-stranded structure had not been broken down. The sedimentation propertms in a sucrose density gradient of poly I-C and RF, irradiated at IOOOOOergs/mm 2, were also unaffected. The rephcative forms of RNA viruses are substances which could possibly be used as interferon inducers in human antiviral therapy. The therapeutic assay is handicapped b y their intrinsic infectivity. Ultraviolet irradiation provides a method for abolishing the infectivity without modifying the interferon-inducing capacity. We t h a n k Professor R. Latarjet for advice and information, and Drs. R. D a y and J. Coppey for valuable discussions. The technical assistance of Misses A. De Vomecourt and J. Sanceau is also acknowledged.
Laborato~re Pasteur, Inst, tut du R a d i i , 26 rue d'Ulm, Paris 5e (France) i 2 3 4 5 6 7 8 9 IO ii 12 13
R. FALCOFF E. FALCOFF L. CATINOT
t{. FALCOFF AND E T FALCOFF, B*ochzm B*ophys. Acta, 182 (1969) 5Ol. R. FALCOFF AND ]~. T. FALCOFF, B,och,m Bwphys. Acta, 199 (197 o) 147. L. MONTAGNIER AND F SANDERS, Nature, 199 (1963) 664. M. FENWlCK, R ERIKSON AND R FRANKLIN, Sczence, 146 (1964) 527 . G. LAMPSON, A TYTELL, A. FIELD, A ~EMES AND M. HILLEMAN, Proc. Natl Acad. Sc*. U S , 58 (1967) 782 E. FALCOFF AND R. FEREZ BERCOFF, Bwchzm Bwphys. Acta, 174 (1969) lO8 R. FRANKLIN, Proc. Natl Acad. Sc, U.S., 55 (1966) 15o 4. R. LATARJET, P. MORENNE AND R BERGER, Ann. Inst. Pasteur, 85 (1953) 174. R. LATARJET, R CRAMEB. AND L. MONTAGNIER, Vzrology, 33 (1967) lO4. J. M BISHOP, N. QUINTRELL AND G. KOCH, J. Mol Bzol, 24 (1967) 125 . Z ZAVADOVA, L GRESLAND AND •. ROSENBERGOVA, Acta Vzrol, i2 (1968) 515. P. HOWARD-FLANDERS, A n n Rev Bwchem., 37 (I968) 175. J. M. BISHOP, G. KOCH, B. EVANS AND ~¢[. MERRIMAN, J. Mol. B w l , 46 (1969) 235.
Received April i5th, 197o B~ochim. B*ophys
Acta, 217 (197 o) 195-198