Archives of Virology
Archives of Virology 57, 153--159 (1978)
© by Springer-Verlag 1978
Ribonuelease Aeti,jties Associated With Purified Foot and Mouth Disease Virus By C. D. DENOYA, E. A. SCOD]~I~LER, C. VASqUEZ, and J. L. LA TO,RE Centro de Virologfa Animal, Buenos Aires, Argentinia With 3 Figures Accepted December 16, 1977
Summary Ribonuclease activities internally and externally associated with purified footand mouth disease virus were detected. The outer activity was easily :removed b y cesium chloride or b y detergent (Sarkosyl). The inner activity is not removable b y a n y procedure used and could be the enzyme responsible for the heterogeneity normally observed in the extracted FMDV-I~NA. I t is not known at present if both activities are related to the same or to different enzymes.
Introduction Foot-and-mouth disease virus (FMDV) genome is composed of a single stranded R N A molecule, having a molecular weight of 2.70 × 106 daltons. However fragments of lower molecular weight are usually found when the viral RNA is analysed by different procedures (1, 2, 8). Several causes for the genomic I~NA breakdown were postulated, but most of them can be ruled out, since degradation of the viral R N A can be avoided if a shortened procedure is used to obtain purified virions (5). I t has been shown t h a t incubation of FMDV at 37 ° C for different lengths of time, leads to an inactivation of the virus with conservation of its physical and immunological properties. The loss of the infectivity upon the incubation of the virions was correlated with a simultaneous degradation of the 35 S genomie R N A into fragments of smaller size (3); similar results were obtained with rhinovirus type 14 (RV 14) (7) and it has been suggested that inactivation of Picornaviruses, at low temperatures, also invo]ved a mechanism directed against the viral I~NA (6). Since degradation of I~V14 R N A during incubation occurred in situ within the viral particles, GAC~TT (7) postulated the presence of a nuelease localized inside the virions. On the other hand nucleuses associated with purified virions of l%ous sarcoma virus (14), vaccinia virus (18), adenovirus (4), frog virus-3 (9, 16), simian virus (10, 11), and polyoma virus (13, 19) have also been reported.
0304-8608/78/0057/0153/$ 01.40
154
C.D.
DENOYA,
E. A. SCODELLER,
C. VASQUEZ, and J. L. LA ToI~I~E :
D a t a presented herein suggests t h a t there are ribonuclease activities i n t e r n a l l y a n d externally associated with the purified virions of FMDV.
Materials and Methods All buffer solutions were autoclaved and all glassware heated for 3 hours at 180° C to avoid contamination by nucleases.
Cell Cultures and Vir~ts Preparation Foot-and-mouth disease virus type A24 (strain Cruzeiro) was used throughout. Virus replication and labeling of the viral I~NA were performed as previously reported (5). Purification of the labeled virus was carried out by using the shortened procedure, described by DE:~OYA et al. (5).
Preparation o/ Viral and Marker R N A s Viral R N A was extracted from the purified virions in the presence of 1 per cent Sarkosyl NL97 (Geigy) by two rounds of phenol-chloroform treatment as described by PER~Z et al. (17). Rat liver t l N A was obtained as described for chick embryo I~NA (5) b u t the I~NA pellets were extracted twice with cold 3 ~ sodium acetate pt{ 6.0 (12). The ribosomal I~NA was resuspended in N E T buffer (0.1 r~ NaC1; 0.001 ~I E D T A ; 0.05 ~ Tris-ttC1, pH 7.4).
Assay o/Ribonuelease Activity 15,000 cpm of 3H-uridine labeled virus (5--20 ~g) and 3.50.D.~60 n m units of rat liver RNA were diluted up to 1 ml in N E T buffer and incubated at 37 ° or at 0 ° C for 15 hours at p H 7.4. When the incubation was performed with disrupted virions, the pt:[ of the mixture was adjusted to 5.4 at the beginning of the experiment by adding an appropriate amount of I x ]-ICI. After I0 minutes at 37 ° C the pH was restored at 7.4 by adding 40 i~l of I ~ Tris-HCl pH 7.4, and the mixture was further incubated during 15 hours. At the end of the incubation the I%NA were extracted with phenolchloroform and analysed on sucrose gradients.
Radioactivity Counting Fractions from the gradients were mixed with an appropriate amount of toluenebased scintillation cocktail (New England Nuclear) and directly counted in a liquid scintillation counter (Beckman LS 150). Except for Sarkosyl containing gradients, TCA insoluble counts were alternatively assayed; no differences were oserved in the distribution of counts with either method.
Results
Analysis o/the FLUIDV - R ~ A Extracted From Virions Harvested at Two Di]/erent Times The effect of a prolonged h e a t i n g a t 37 ° C d u r i n g virus multiplication, u p o n the i n t e g r i t y of the F M D V - R N A was studied b y h a r v e s t i n g the virus from celt fluids at two different times: 5 a n d 14 hours post infection (3 a n d 12 hours post labeling respectively). Virions were purified from cell fluids a n d the viral I~NA was extracted with phenol-chloroform. The extracted R N A s were t h e n analysed b y m e a n s of two identical sucrose gradients. As can be observed in Figure 1, the I~NA e x t r a c t e d from the virus harvested 14 hours p.i. appears more heterogeneous t h a n t h a t o b t a i n e d from the 5 hours harvest, as 30 to 40 per cent of the t~NA molecules s e d i m e n t e d trailing on the less dense side of the 35S peak. I t is i m p o r t a n t to p o i n t out t h a t the heterogeneity of the t~NA of the t 4 hours h a r v e s t
:Nucleases in Purified FMDV
155
is not very extensive; however this was expected, since infected cell cultures arc constantly producing new virus, and virions produced tater in the growth cycle would have, at the time of harvesting, a more intact R N A than those produced earlier. The possibility that the R N A of the late virus were more heterogeneous than that of the early virus was ruled out, since virions harvested sequentially each 5 hours (0--5; 5--10; and 10--14 hours post infection) (not shown), had intact genomic RNAs. Since isolated viral R N A was found to be very stable even when submitted to drastic denaturation procedures or prolonged incubation at 37°C (5), we decided to study the degradation of the viral R N A by using highly purified virions, suspecting that they contained a nucleolytic activity, responsible for the degradation of the viral genome.
~5
¢0 ).
20
'
:t:
I0
FRACTION
20
30
NUMBER
Fig. 1. Sedimentation analysis of ~H-FMDV-RNA extracted from virions harvested at 5 and 14 hours post infection. The RNA samples were layered over two different 17 ml, 10--30 per cent (w/v) linear sucrose gradients made in NET buffer plus 0.5 per cent Saxkosyt, and centrifuged (Beckman S}V27 rotor, 27,000 rpm, 12 hours, 2° C). (, ®), RNA from virions harvested 5 hours p.i.; (~- . . . . =), RNA from the 14 hours harvest Incubation o/ Puri/ied Virions and Isolated Viral R N A at 37 ° C Incubations of virions at 37°C were carried out with the 5 hours harvest, purified as described in Methods. Virions were mixed with rat liver 28 and 18S ribosomal RNAs and incubated at 0 ° or at 37°C during 15 hours at p H 7.4. A]iquots of the incubated virions were submitted to analysis in sucrose or cesium chloride gradients and compared with the nondncubated virus. We observed that incubation does not affect the physicochemical properties of the virions, a result that agrees with those already reported by Blcow~ and WILD (3). Both viral and ribosomal RNAs were extracted from the incubation mixture and analysed b y means of continuous sucrose gradients. As can be seen in Figure 2 a, both ribosomal and viral RNAs remained intact after incubation of the virions at 0 ° C and p H 7.4 during 15 hours. FMDV-RNA sediments as a single homogeneous peak at 35S. ]~'igure 2 b shows the size distribution of the genomie R N A obtained from virions incubated, this time, at 37 ° C for t5 hours at pm 7.4. As can be seen, FMDV-RNA became partially degraded showing only about 30 per cent of the molecules banding at the 35S position. The behaviour of the ribosomal R N A is quite different, since
156
C.D.
DE~OYA,
E. A. SCODELLEn,
C.
VASQUEZ, and J. L. LA ToiaI~E:
all molecules were degraded during the incubation, sedimenting as small fragments around the position of the 4 S l~NA. The presence of contaminant nucleases in the incubation mixture was ruled out, since isolated molecules of viral and ribosomal R,NAs remained practically intac~ upon incubation at 37 ° C in the same buffer and with the same amount of sucrose (Fig. 2d). On the other hand when virions were disrupted at low p H and then incubated together with ribosomal R N A at 37 ° C and p H 7.4 during 15 hours, the viral and the ribosomal t~NAs appeared completely degraded (Fig. 2c). The experiments shown in Figure 2 are in agreement with the idea of nucleases associated with purified FMDV. The degradation of the ribosomal RNA coincubated with the virions could be an indication that a nucleolytic activity is attached to the outside part of the virions however there is also concomitant degradation of the viral R N A although it is not as extensive as that shown with the ribosomal RNAs. Two possible ~lternatives can be considered to ~5
o
II/ ,~
,o
~.
li
o
o
(c)
.....
A
~H ,.o..,o
,.o
z
x
o_
/i ,
8 ~
o ° 0
9"~
:~,,.,~ (d)3H'i
(b} 0 --
i 8
-
i\i
5
IO
15
20
5
o
IO
15
20
FRACTION N U M B E R
Fig. 2. Sedimentation analysis of .~I-I-FMDV-RNA extracted from vii'ions incubated under different conditions together with ribosomal RNA. The extracted I:~NAs were put on top of 11 ml, 15--30 per cent (w/v) linear sucrose gradients made in NET buffer containing 0.5 per cent SDS. Gradients were centrifuged (Beckman SW41 rotor, 40,000 rpm, 4.75 hours, 20 ° C). (8 --.), Sl-I FMDV-RNA; (o- .... o), unlabeled 28S and 18S rat livex ]%NAs. IgNA extracted from virions incubated for 15 hours at pH 7.4 and at 0 ° C (a), or at 37 ° C (b) ; I~NA obtained from virions disrupted at low pH and then incubated for 15 hours at 37 ° C at pH 7.4 (c), and isolated viral and ribosomal RNAs incubated during 15 hours at 37 ° C, pH 7.4 (d)
Nueleases in Purified FMDV
157
explain the degradation of the genomic R N A : one is that the viral R N A is exposed to an external nuclease only at restricted points. The other is the existence of a nucleolytic activity located inside the viral capsid. Experiments focused on the removal of the nucleolytic activity were designed in order to find out what was the degree of association of the enzymes to the virions. Purified virions were treated with 1 per cent of the sodium salt of N-Lauroyl Sarkosine (Sarkosyl, Geigy), a detergent which under certain conditions can release proteins (i5), without damaging the virus structure; the treatment was carried out at 0°C during i0 minutes and the virions were sampled at the top of a 15---45 per cent (w/v) linear sucrose gradient, made without the detergent; a homogeneous peak was obtained at the end of the run and those fractions containing the virus were pooled and tested for nuclease activity. An aliquot of the treated virus was mixed with ribosomal R N A and held at 0 ° C for 15 hours at p H 7.4; another aliquot was incubated at 37 ° C under the same conditions. As can be seen in Figure 3a, the RNA extracted from the 0 ° C incubated virions (control) and the ribosomal RNAs sedimented as homogeneous peaks. Figure 3 b shows the profile of the viral R N A extracted from Sarkosyl treated virions that were incubated at 37 ° C for 15 hours. As can be appreciated the viral R N A was degraded during the incubation and very few molecules were found sedimenting at the 35S position. The pattern obtained is comparable with that shown in Figure 2b. On the contrary, ribosomal RNA coincubated with the treated virions showed no degradation, indicating that the detergent was able to remove the external nucleolytic activity but not that responsible for the degradation of the genomic RNA. However this activity was not specific for the viral RNA since ribosomal RNA coincubated with p H disrupted virions was also degraded (data not shown). On the other hand non radioactive, Sarkosyl purified virions were also unable to degrade isolated radioactive viral R N A upon incubation at 37 ° C and p H 7.4 during 15 hours (not shown).
.,
35
5
io
~5
" ~o
FRACTION NUMBER
~
28
lo
~8
~5
ao
FRACTION NUMBER
Fig. 3. Sedimentation analysis of 3H-~vIDV-I~NA extracted from Sarkosyl treated
virions tha~ were incubated a~ 0° and 37° C, respectively, together with rat liver ribosomal RNA. RNA samples were layered on 11 rnl, 15--30 per cent (w/v) linear sucrose gradients made in :NET buffer containing 0.5 per cent SDS and centrifuged (Beckman SW41 rotor, 40,000 rpm, 4.5 hours, 20° C. (. - - , ) aH FMDV-RNA; (o. . . . . . . o) unlabeled 28S and 18S R:NAs; R:NA from virions incubated 15 hours at 0° C and pH 7.4 (a), or at 37° C (b)
158
C.D. DENOYA,
E. A. SCO])ELLER, C. VASQU~Z,
and J. L. LA TOiglCE:
Since cesium salts are also known to remove contaminant proteins adsorbed to the virus (20), we decided to use them to confirm the precedent conclusion. An additional cesium chloride gradient was performed with the already purified virions. The fraetions containing the virions (1.42 g/cma peak) were pooled, diluted with N E T buffer and the virus pelleted. The pelleted virions were resuspended in N E T buffer and assayed for nuclease activity. The results obtained were similar to those shown in Figure 3 indicating that cesium chloride is also able to remove the external but not the internal nueleolytie activity.
Diseussion Based on the results presented herein we concluded that there are ribonuclease activities associated with the purified FMDV virions. One is located in the outer part of the virions and its specific activity was variable in each viral preparation tested, suggesting that it is loosely attached and could be lost during the purification procedures. Moreover, it has been shown that this enzymatic activity is easily removable b y detergent or cesium chloride. The other one was found to be constant in all the preparations tested. I t is probably located inside the viral particles, and we think that it could be the enzyme responsible for the genomic FMDV-I~NA breakdown. However we do not know whether these activities represent really two different enzymes or whether they correspond to the same protein with a dual distribution (i. e., it could be possible that during the assembly process a given amount of the same enzyme which is to be incorporated into the virions became attached to the surface of the mature virus). There are m a n y reports concerning the presence of several deoxyribonucleases associated to different viruses (13, 17, 4, 9, 10, 12, 18). However up to now, only two ribonucleases were described. These were tentatively located in the outer capsid of the Frog virus-3 (15, 20). The nuclease reported in this work seems to be the first ribonuelease described to be located within a Pieornavirus. The biological significance and the origin of this enzyme is at present unknown.
Acknowledgments We wish to thank Carmen Ricarte and Elisa Yampolsky for technical assistance. This investigation was supported by the Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET) and Fundacion para la Educaeion la Ciencia y la Cultura (FECIC).
References i. A~RLINGHAUS, ic~. B., POLATNICK, J., VANDE WOUDE, C. F. : Studies on Foot-andMouth disease virus ribonucleic acid synthesis. Virology 30, 541--550 (1966). 2. BIgEESE, S. S., Jig. : A comparison of molecular weights of foot-and-mouth disease virus I~]NA fragments determined from lengths and r-rates. J. gem Viro]. 31, I--8 (1976). 3. BigowN, F., WILD, T. F.: The effect of heat on the structure of foot-and-mouth disease virus and the viral ribonucleie acid. Bioehim. biophys. Acta 119, 301--308 (1966). 4. BUigLINCmAM, B. T., DOEigFLER, W., PETTEigSSON, U., PmLIPSON, L. : Adenovirus endonuclease: association with the penton of adenovirus type 2. J. tool. Biol. 60,
45--64 (t971).
Nucleases in Purified FMDV
159
5. DENOYA, C. D., SCODELLER, E. A., GIMENEZ, B. i., V±SQUEZ, C., LA TORRE, J. L. : Foot-and-mouth disease virus. I. Stability of its ribonueleic acid. Virology (in press). 6. I)IMMOCK, IN. J. : Differences between the thermal inactivation of Picornaviruses at "high" and "low" temperatures. Virology 31, 338--353 (1967). 7. GATJN~:T, C. J. : Fragmentation of I~NA in virus particles of l~hinovirus type 14. J. Virol. 13, 762--764 (1974). 8. HARalS, T. J. 1%., BROWN, F. : Biochemical analysis of a virulent and an avirulent strains of foot-and-mouth disease virus. J. gen. Virol. 34, 87--105 (1977). 9. K~NG, H. S., McAvsLAN, B. 1%. : Virus-associated nucleases : location and properties of deoxyribonucleases and ribonucleases in purified Frog virus 3. J. Virol. lff. 202--210 (1972). 10. KAPLA~, J. C., WILBERT, S. M., BLACK, P. H. : Endonuclease activity associated with purified simian virus 40 virions. J. Virol. 9, 800--803 (1972). 1 i. KIDWELL, W. I~., SAI~AI,, 1%., MARTIN, 1%. G., 0ZEI~, H. L. : Characterization of an endonuclease associated with simian virus 40 virions. J. Virol. I0,410--416 (1972). 12. KIEB¥, K. S. : Isolation of nucleic acids with phenolic solvents. In: CoLowlcK, S., KAI)LAN, :N. O. (eds.), Methods in enzymology, Vol. XII, par~ B, 87--98. :New York: Academic Press Inc. 1968. 13. McMILLEN, J., CENTER, M. S., CONSlGLI, R. A. : Origin of the polyoma virusassociated endonuclease. J. Virol. 17, 127--131 (1976). 14. MIz~ANI, S., TEMI~, H. M., KODA~IA, M., WELLS, R. T.: I)iNA tigase and exonuclease activities in virions of l%ous sarcoma virus. :Nature (:New Biol.) 230, 232--235 (1971). 15. :NELL, H., E ~ A ~ D , S. : The use of sodium and lithium dodecyl sulphate in nucleic acid i~olation. I n : Cor.owIcx, S., KAPLAN, N. O. (eds.), Methods in enzymology, Vol. X I I , part B, 129--155. New York: Academic Press Inc. 1968. t6. P~ESE, P., KocH, G. : I)egradation of single- and double-stranded R N A b y Frog virus 3. Prec. :Nat. Ac. Sci. U.S.A. 69, 698--701 (1972). 17. P E ~ ¥ , R. P., LA T O ~ E , J. L., KEL:LE¥, D. E., GaEEZ~BERG,J. t~. : On the lability of poly (A) sequences during extraction of messenger RI~A from polyribosomes. Biochim. biophys. Acta 262, 220--226 (1972). 18. PoGo, B. G. T., I)AT.ES,S. : Two deoxyribonuclease activities within purified vaccinia virus. Prec. :Nat. Ae. Sci. U.S.A. 63, 820--827 (1969). 19. ROUGET, P., PAI~ODI, A., BLANGY, I)., CUZX~, F. : Origin of polyoma virus-associated endonuclease. J. Virol. 20, 9.~--13 (1976). 20. SANGAR, D. V., ROWLA~DS, I). J., CAVANAGtt,D., BJ_~ow~, F. : Characterization of the minor polypeptides in the foot-and-mouth disease particle. J. gen. Virol. ~1, 35--46 (1976).
Authors' address: Dr. J. L. L& TORI~E, Centre de Virologfa Animal, Serrano 661, 1414 CapitM Federal, b u e n o s Aires, Argentina. Received :November 4, 1977
Archives of Virology 57, 153--159 (1978)
© by Springer-Verlag 1978
Ribonuelease Aeti,jties Associated With Purified Foot and Mouth Disease Virus By C. D. DENOYA, E. A. SCOD]~I~LER, C. VASqUEZ, and J. L. LA TO,RE Centro de Virologfa Animal, Buenos Aires, Argentinia With 3 Figures Accepted December 16, 1977
Summary Ribonuclease activities internally and externally associated with purified footand mouth disease virus were detected. The outer activity was easily :removed b y cesium chloride or b y detergent (Sarkosyl). The inner activity is not removable b y a n y procedure used and could be the enzyme responsible for the heterogeneity normally observed in the extracted FMDV-I~NA. I t is not known at present if both activities are related to the same or to different enzymes.
Introduction Foot-and-mouth disease virus (FMDV) genome is composed of a single stranded R N A molecule, having a molecular weight of 2.70 × 106 daltons. However fragments of lower molecular weight are usually found when the viral RNA is analysed by different procedures (1, 2, 8). Several causes for the genomic I~NA breakdown were postulated, but most of them can be ruled out, since degradation of the viral R N A can be avoided if a shortened procedure is used to obtain purified virions (5). I t has been shown t h a t incubation of FMDV at 37 ° C for different lengths of time, leads to an inactivation of the virus with conservation of its physical and immunological properties. The loss of the infectivity upon the incubation of the virions was correlated with a simultaneous degradation of the 35 S genomie R N A into fragments of smaller size (3); similar results were obtained with rhinovirus type 14 (RV 14) (7) and it has been suggested that inactivation of Picornaviruses, at low temperatures, also invo]ved a mechanism directed against the viral I~NA (6). Since degradation of I~V14 R N A during incubation occurred in situ within the viral particles, GAC~TT (7) postulated the presence of a nuelease localized inside the virions. On the other hand nucleuses associated with purified virions of l%ous sarcoma virus (14), vaccinia virus (18), adenovirus (4), frog virus-3 (9, 16), simian virus (10, 11), and polyoma virus (13, 19) have also been reported.
0304-8608/78/0057/0153/$ 01.40
154
C.D.
DENOYA,
E. A. SCODELLER,
C. VASQUEZ, and J. L. LA ToI~I~E :
D a t a presented herein suggests t h a t there are ribonuclease activities i n t e r n a l l y a n d externally associated with the purified virions of FMDV.
Materials and Methods All buffer solutions were autoclaved and all glassware heated for 3 hours at 180° C to avoid contamination by nucleases.
Cell Cultures and Vir~ts Preparation Foot-and-mouth disease virus type A24 (strain Cruzeiro) was used throughout. Virus replication and labeling of the viral I~NA were performed as previously reported (5). Purification of the labeled virus was carried out by using the shortened procedure, described by DE:~OYA et al. (5).
Preparation o/ Viral and Marker R N A s Viral R N A was extracted from the purified virions in the presence of 1 per cent Sarkosyl NL97 (Geigy) by two rounds of phenol-chloroform treatment as described by PER~Z et al. (17). Rat liver t l N A was obtained as described for chick embryo I~NA (5) b u t the I~NA pellets were extracted twice with cold 3 ~ sodium acetate pt{ 6.0 (12). The ribosomal I~NA was resuspended in N E T buffer (0.1 r~ NaC1; 0.001 ~I E D T A ; 0.05 ~ Tris-ttC1, pH 7.4).
Assay o/Ribonuelease Activity 15,000 cpm of 3H-uridine labeled virus (5--20 ~g) and 3.50.D.~60 n m units of rat liver RNA were diluted up to 1 ml in N E T buffer and incubated at 37 ° or at 0 ° C for 15 hours at p H 7.4. When the incubation was performed with disrupted virions, the pt:[ of the mixture was adjusted to 5.4 at the beginning of the experiment by adding an appropriate amount of I x ]-ICI. After I0 minutes at 37 ° C the pH was restored at 7.4 by adding 40 i~l of I ~ Tris-HCl pH 7.4, and the mixture was further incubated during 15 hours. At the end of the incubation the I%NA were extracted with phenolchloroform and analysed on sucrose gradients.
Radioactivity Counting Fractions from the gradients were mixed with an appropriate amount of toluenebased scintillation cocktail (New England Nuclear) and directly counted in a liquid scintillation counter (Beckman LS 150). Except for Sarkosyl containing gradients, TCA insoluble counts were alternatively assayed; no differences were oserved in the distribution of counts with either method.
Results
Analysis o/the FLUIDV - R ~ A Extracted From Virions Harvested at Two Di]/erent Times The effect of a prolonged h e a t i n g a t 37 ° C d u r i n g virus multiplication, u p o n the i n t e g r i t y of the F M D V - R N A was studied b y h a r v e s t i n g the virus from celt fluids at two different times: 5 a n d 14 hours post infection (3 a n d 12 hours post labeling respectively). Virions were purified from cell fluids a n d the viral I~NA was extracted with phenol-chloroform. The extracted R N A s were t h e n analysed b y m e a n s of two identical sucrose gradients. As can be observed in Figure 1, the I~NA e x t r a c t e d from the virus harvested 14 hours p.i. appears more heterogeneous t h a n t h a t o b t a i n e d from the 5 hours harvest, as 30 to 40 per cent of the t~NA molecules s e d i m e n t e d trailing on the less dense side of the 35S peak. I t is i m p o r t a n t to p o i n t out t h a t the heterogeneity of the t~NA of the t 4 hours h a r v e s t
:Nucleases in Purified FMDV
155
is not very extensive; however this was expected, since infected cell cultures arc constantly producing new virus, and virions produced tater in the growth cycle would have, at the time of harvesting, a more intact R N A than those produced earlier. The possibility that the R N A of the late virus were more heterogeneous than that of the early virus was ruled out, since virions harvested sequentially each 5 hours (0--5; 5--10; and 10--14 hours post infection) (not shown), had intact genomic RNAs. Since isolated viral R N A was found to be very stable even when submitted to drastic denaturation procedures or prolonged incubation at 37°C (5), we decided to study the degradation of the viral R N A by using highly purified virions, suspecting that they contained a nucleolytic activity, responsible for the degradation of the viral genome.
~5
¢0 ).
20
'
:t:
I0
FRACTION
20
30
NUMBER
Fig. 1. Sedimentation analysis of ~H-FMDV-RNA extracted from virions harvested at 5 and 14 hours post infection. The RNA samples were layered over two different 17 ml, 10--30 per cent (w/v) linear sucrose gradients made in NET buffer plus 0.5 per cent Saxkosyt, and centrifuged (Beckman S}V27 rotor, 27,000 rpm, 12 hours, 2° C). (, ®), RNA from virions harvested 5 hours p.i.; (~- . . . . =), RNA from the 14 hours harvest Incubation o/ Puri/ied Virions and Isolated Viral R N A at 37 ° C Incubations of virions at 37°C were carried out with the 5 hours harvest, purified as described in Methods. Virions were mixed with rat liver 28 and 18S ribosomal RNAs and incubated at 0 ° or at 37°C during 15 hours at p H 7.4. A]iquots of the incubated virions were submitted to analysis in sucrose or cesium chloride gradients and compared with the nondncubated virus. We observed that incubation does not affect the physicochemical properties of the virions, a result that agrees with those already reported by Blcow~ and WILD (3). Both viral and ribosomal RNAs were extracted from the incubation mixture and analysed b y means of continuous sucrose gradients. As can be seen in Figure 2 a, both ribosomal and viral RNAs remained intact after incubation of the virions at 0 ° C and p H 7.4 during 15 hours. FMDV-RNA sediments as a single homogeneous peak at 35S. ]~'igure 2 b shows the size distribution of the genomie R N A obtained from virions incubated, this time, at 37 ° C for t5 hours at pm 7.4. As can be seen, FMDV-RNA became partially degraded showing only about 30 per cent of the molecules banding at the 35S position. The behaviour of the ribosomal R N A is quite different, since
156
C.D.
DE~OYA,
E. A. SCODELLEn,
C.
VASQUEZ, and J. L. LA ToiaI~E:
all molecules were degraded during the incubation, sedimenting as small fragments around the position of the 4 S l~NA. The presence of contaminant nucleases in the incubation mixture was ruled out, since isolated molecules of viral and ribosomal R,NAs remained practically intac~ upon incubation at 37 ° C in the same buffer and with the same amount of sucrose (Fig. 2d). On the other hand when virions were disrupted at low p H and then incubated together with ribosomal R N A at 37 ° C and p H 7.4 during 15 hours, the viral and the ribosomal t~NAs appeared completely degraded (Fig. 2c). The experiments shown in Figure 2 are in agreement with the idea of nucleases associated with purified FMDV. The degradation of the ribosomal RNA coincubated with the virions could be an indication that a nucleolytic activity is attached to the outside part of the virions however there is also concomitant degradation of the viral R N A although it is not as extensive as that shown with the ribosomal RNAs. Two possible ~lternatives can be considered to ~5
o
II/ ,~
,o
~.
li
o
o
(c)
.....
A
~H ,.o..,o
,.o
z
x
o_
/i ,
8 ~
o ° 0
9"~
:~,,.,~ (d)3H'i
(b} 0 --
i 8
-
i\i
5
IO
15
20
5
o
IO
15
20
FRACTION N U M B E R
Fig. 2. Sedimentation analysis of .~I-I-FMDV-RNA extracted from vii'ions incubated under different conditions together with ribosomal RNA. The extracted I:~NAs were put on top of 11 ml, 15--30 per cent (w/v) linear sucrose gradients made in NET buffer containing 0.5 per cent SDS. Gradients were centrifuged (Beckman SW41 rotor, 40,000 rpm, 4.75 hours, 20 ° C). (8 --.), Sl-I FMDV-RNA; (o- .... o), unlabeled 28S and 18S rat livex ]%NAs. IgNA extracted from virions incubated for 15 hours at pH 7.4 and at 0 ° C (a), or at 37 ° C (b) ; I~NA obtained from virions disrupted at low pH and then incubated for 15 hours at 37 ° C at pH 7.4 (c), and isolated viral and ribosomal RNAs incubated during 15 hours at 37 ° C, pH 7.4 (d)
Nueleases in Purified FMDV
157
explain the degradation of the genomic R N A : one is that the viral R N A is exposed to an external nuclease only at restricted points. The other is the existence of a nucleolytic activity located inside the viral capsid. Experiments focused on the removal of the nucleolytic activity were designed in order to find out what was the degree of association of the enzymes to the virions. Purified virions were treated with 1 per cent of the sodium salt of N-Lauroyl Sarkosine (Sarkosyl, Geigy), a detergent which under certain conditions can release proteins (i5), without damaging the virus structure; the treatment was carried out at 0°C during i0 minutes and the virions were sampled at the top of a 15---45 per cent (w/v) linear sucrose gradient, made without the detergent; a homogeneous peak was obtained at the end of the run and those fractions containing the virus were pooled and tested for nuclease activity. An aliquot of the treated virus was mixed with ribosomal R N A and held at 0 ° C for 15 hours at p H 7.4; another aliquot was incubated at 37 ° C under the same conditions. As can be seen in Figure 3a, the RNA extracted from the 0 ° C incubated virions (control) and the ribosomal RNAs sedimented as homogeneous peaks. Figure 3 b shows the profile of the viral R N A extracted from Sarkosyl treated virions that were incubated at 37 ° C for 15 hours. As can be appreciated the viral R N A was degraded during the incubation and very few molecules were found sedimenting at the 35S position. The pattern obtained is comparable with that shown in Figure 2b. On the contrary, ribosomal RNA coincubated with the treated virions showed no degradation, indicating that the detergent was able to remove the external nucleolytic activity but not that responsible for the degradation of the genomic RNA. However this activity was not specific for the viral RNA since ribosomal RNA coincubated with p H disrupted virions was also degraded (data not shown). On the other hand non radioactive, Sarkosyl purified virions were also unable to degrade isolated radioactive viral R N A upon incubation at 37 ° C and p H 7.4 during 15 hours (not shown).
.,
35
5
io
~5
" ~o
FRACTION NUMBER
~
28
lo
~8
~5
ao
FRACTION NUMBER
Fig. 3. Sedimentation analysis of 3H-~vIDV-I~NA extracted from Sarkosyl treated
virions tha~ were incubated a~ 0° and 37° C, respectively, together with rat liver ribosomal RNA. RNA samples were layered on 11 rnl, 15--30 per cent (w/v) linear sucrose gradients made in :NET buffer containing 0.5 per cent SDS and centrifuged (Beckman SW41 rotor, 40,000 rpm, 4.5 hours, 20° C. (. - - , ) aH FMDV-RNA; (o. . . . . . . o) unlabeled 28S and 18S R:NAs; R:NA from virions incubated 15 hours at 0° C and pH 7.4 (a), or at 37° C (b)
158
C.D. DENOYA,
E. A. SCO])ELLER, C. VASQU~Z,
and J. L. LA TOiglCE:
Since cesium salts are also known to remove contaminant proteins adsorbed to the virus (20), we decided to use them to confirm the precedent conclusion. An additional cesium chloride gradient was performed with the already purified virions. The fraetions containing the virions (1.42 g/cma peak) were pooled, diluted with N E T buffer and the virus pelleted. The pelleted virions were resuspended in N E T buffer and assayed for nuclease activity. The results obtained were similar to those shown in Figure 3 indicating that cesium chloride is also able to remove the external but not the internal nueleolytie activity.
Diseussion Based on the results presented herein we concluded that there are ribonuclease activities associated with the purified FMDV virions. One is located in the outer part of the virions and its specific activity was variable in each viral preparation tested, suggesting that it is loosely attached and could be lost during the purification procedures. Moreover, it has been shown that this enzymatic activity is easily removable b y detergent or cesium chloride. The other one was found to be constant in all the preparations tested. I t is probably located inside the viral particles, and we think that it could be the enzyme responsible for the genomic FMDV-I~NA breakdown. However we do not know whether these activities represent really two different enzymes or whether they correspond to the same protein with a dual distribution (i. e., it could be possible that during the assembly process a given amount of the same enzyme which is to be incorporated into the virions became attached to the surface of the mature virus). There are m a n y reports concerning the presence of several deoxyribonucleases associated to different viruses (13, 17, 4, 9, 10, 12, 18). However up to now, only two ribonucleases were described. These were tentatively located in the outer capsid of the Frog virus-3 (15, 20). The nuclease reported in this work seems to be the first ribonuelease described to be located within a Pieornavirus. The biological significance and the origin of this enzyme is at present unknown.
Acknowledgments We wish to thank Carmen Ricarte and Elisa Yampolsky for technical assistance. This investigation was supported by the Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET) and Fundacion para la Educaeion la Ciencia y la Cultura (FECIC).
References i. A~RLINGHAUS, ic~. B., POLATNICK, J., VANDE WOUDE, C. F. : Studies on Foot-andMouth disease virus ribonucleic acid synthesis. Virology 30, 541--550 (1966). 2. BIgEESE, S. S., Jig. : A comparison of molecular weights of foot-and-mouth disease virus I~]NA fragments determined from lengths and r-rates. J. gem Viro]. 31, I--8 (1976). 3. BigowN, F., WILD, T. F.: The effect of heat on the structure of foot-and-mouth disease virus and the viral ribonucleie acid. Bioehim. biophys. Acta 119, 301--308 (1966). 4. BUigLINCmAM, B. T., DOEigFLER, W., PETTEigSSON, U., PmLIPSON, L. : Adenovirus endonuclease: association with the penton of adenovirus type 2. J. tool. Biol. 60,
45--64 (t971).
Nucleases in Purified FMDV
159
5. DENOYA, C. D., SCODELLER, E. A., GIMENEZ, B. i., V±SQUEZ, C., LA TORRE, J. L. : Foot-and-mouth disease virus. I. Stability of its ribonueleic acid. Virology (in press). 6. I)IMMOCK, IN. J. : Differences between the thermal inactivation of Picornaviruses at "high" and "low" temperatures. Virology 31, 338--353 (1967). 7. GATJN~:T, C. J. : Fragmentation of I~NA in virus particles of l~hinovirus type 14. J. Virol. 13, 762--764 (1974). 8. HARalS, T. J. 1%., BROWN, F. : Biochemical analysis of a virulent and an avirulent strains of foot-and-mouth disease virus. J. gen. Virol. 34, 87--105 (1977). 9. K~NG, H. S., McAvsLAN, B. 1%. : Virus-associated nucleases : location and properties of deoxyribonucleases and ribonucleases in purified Frog virus 3. J. Virol. lff. 202--210 (1972). 10. KAPLA~, J. C., WILBERT, S. M., BLACK, P. H. : Endonuclease activity associated with purified simian virus 40 virions. J. Virol. 9, 800--803 (1972). 1 i. KIDWELL, W. I~., SAI~AI,, 1%., MARTIN, 1%. G., 0ZEI~, H. L. : Characterization of an endonuclease associated with simian virus 40 virions. J. Virol. I0,410--416 (1972). 12. KIEB¥, K. S. : Isolation of nucleic acids with phenolic solvents. In: CoLowlcK, S., KAI)LAN, :N. O. (eds.), Methods in enzymology, Vol. XII, par~ B, 87--98. :New York: Academic Press Inc. 1968. 13. McMILLEN, J., CENTER, M. S., CONSlGLI, R. A. : Origin of the polyoma virusassociated endonuclease. J. Virol. 17, 127--131 (1976). 14. MIz~ANI, S., TEMI~, H. M., KODA~IA, M., WELLS, R. T.: I)iNA tigase and exonuclease activities in virions of l%ous sarcoma virus. :Nature (:New Biol.) 230, 232--235 (1971). 15. :NELL, H., E ~ A ~ D , S. : The use of sodium and lithium dodecyl sulphate in nucleic acid i~olation. I n : Cor.owIcx, S., KAPLAN, N. O. (eds.), Methods in enzymology, Vol. X I I , part B, 129--155. New York: Academic Press Inc. 1968. t6. P~ESE, P., KocH, G. : I)egradation of single- and double-stranded R N A b y Frog virus 3. Prec. :Nat. Ac. Sci. U.S.A. 69, 698--701 (1972). 17. P E ~ ¥ , R. P., LA T O ~ E , J. L., KEL:LE¥, D. E., GaEEZ~BERG,J. t~. : On the lability of poly (A) sequences during extraction of messenger RI~A from polyribosomes. Biochim. biophys. Acta 262, 220--226 (1972). 18. PoGo, B. G. T., I)AT.ES,S. : Two deoxyribonuclease activities within purified vaccinia virus. Prec. :Nat. Ae. Sci. U.S.A. 63, 820--827 (1969). 19. ROUGET, P., PAI~ODI, A., BLANGY, I)., CUZX~, F. : Origin of polyoma virus-associated endonuclease. J. Virol. 20, 9.~--13 (1976). 20. SANGAR, D. V., ROWLA~DS, I). J., CAVANAGtt,D., BJ_~ow~, F. : Characterization of the minor polypeptides in the foot-and-mouth disease particle. J. gen. Virol. ~1, 35--46 (1976).
Authors' address: Dr. J. L. L& TORI~E, Centre de Virologfa Animal, Serrano 661, 1414 CapitM Federal, b u e n o s Aires, Argentina. Received :November 4, 1977
