Spleen diesterase action on polynucleotide and ribonucleic acid infectivit

Fraenkel-Conrat, H.

Sherwood, M.

Singer, B.

English

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STUDlcES OF NUCLECFIDE S ~ ~ l ~ C E S  IN ~-~~ * . 11. rn ACTION OF smm DIESTIEUSE* 
B. Singer, M. Shemood and H. Fraenkel-Conrat 
Virus Laboratory and Space Sciences Uhorntory, Universtty of California 
Berkeley, Calif crnia 94720 
It has been reported that limited treatment of W-RNA with 
v e n a  phosphodiesterase (SV-PDX) causes the release of a few nucleotidee 
fraa the 5*-li&ed ("right") ead of the chain without marked lose of 
in fec t iv i ty  (1). Spleen diesterase has been sham t o  attack polynucleo- 
t ides  from the 3'-linked ("left") eod of the chain (2) and t h i s  enzyme 
was therefore used t o  obtain information on the nucleotide sequence at 
that end, and its essent ia l i ty  for the infect ivi ty  of the RTZA. 
Xethods and Materials.- p - l a b e l l e d  EW-NJA was prepared as usual -
( 3 , k ) .  
chraatography according t o  (5 ) . About 0.15-0.5 ug 0," the RITA was treated 
i n  pH 5.2 acetate and Hg2' with 0.02-0.L2 units  of enzyme for various time 
Worthington Spleen PDE was f u r t h e r  ixurlfied by phosphocellulose 
periods at temperatures rangin[: fro31 15-37O. The Kill was then precipitated 
with 2.5 volmes 02 ethanol, generally in  the presence of EMlA. 
supernate was concentrated and two-dimensiocally chrmtographed (6) after 
addition of knua mounts of the four 3'(2')-cucleotides and the corresponding 
2 '-3 ' cyclic p h o q k t e s ,  c.8 w e l l  as occa aionally of the 3 O ,  5 -~ucleosiBe 
diphoslpha-ccs. The mlio3,ctlvPt.y associated with each 02 these a r k e r s  
represented a measwe c2 the extent of digestion. Fbdiosutocp-aghy was 
used t o  detect  other ,reaction products for which markers had not been addled. 
The 
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Eiis iavesfigatiorr m e  supported by GB-3107 from the National Science 
hundation and grant I?& 479 from the Ntrtional Aeronautics and Space 
Mairriatmtion. 
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# The RFJA precipitate was redissolved in $0 and tested for infectl~ty 
after reconstitution, as well as for its macromolecular integrity by ultra- 
centrifugal analysis (Nodel E Spinco, equipped with U.V. optics). 
Results and Discussion.- As illustrated by a typicel exporiment on 
v-
U" 
h 
Table I, limited digestion'!IXV-Rii causod t'ne release 02 a consistent 
pattern of nucleotides (A = U > G > C ) .  
nucleotlcks or cii' riuclcooidc 3 ' ,5 '-dlphosphjatc; or other ~ro~3,icto WCTO 
detected. 
with first-order kinetics. The proportion of 30 S material remained 
approximately unchanged until after about 10-20 nucleotides had been 
released. At Oo, in contrast to SV-PDE and polynucleotide phosphorylase 
(?), spleen diesterase caused little digestion and erratic results. 
1Jo appreciable amounts of 2',3'-cycUc 
The infectivity of such treated preparations decreased rapidly 
Prolonged treatment or greater amounts of enzyme caused disappearance 
of the 30 S naterial and the appcarance of appreciable amounts of the 
cyclic 21,3t-nucleotides and of other distinct products detected on 
radioautograms. These observations indicate the need for further 
purification and characterization of spleen phosphodiesterase. 
indiscriminate use of this enzyiiie, even after the further step of 
purification, m y  yield data which are difficult to Interpret. 
Obvlmly ,  
The question whether the enzyme Iri abie to attack a ~'-phosphox-ylate& 
terminal nucieotiae is in disgute. 
pLosphodiesterase releases small molecular prodwts, with or without prior 
Ce?hosphoryPation with phosphomonoesterase, these have not beea identified 
(8,g). 
without the appearance of a 3t,5'-diphouphate confirms previous conclusions 
that !l!XV-I"?A carries no 5l-terminal phosphate (10). 
progressive nucleotide release is in accord with the results of complete 
digestion by SV-PDE which shawed that A was the 3'-lInked tennfnd residue 
(n), 
I n  :;he case of Is-RNA, although spleen 
I C  
Our Pinung that nucleotides arc released by exonuclease action 
J 
The pattern of 
A sequence such 88  ApUp'JpApOpC * c d d  be tentatively gPoPOsed On . 
the basis of the data paentea. 
L . * 
# The interpretation that the releamd nucleotides cane at leaet in part 
d' Pras the infective molecule, rather thari from fragmento or contaminants, l e  
justified in view of the lOS6 of ieectivity without appreciable loss of 
30 S msterial which results f rom minimal spleen diesterase action. It 
appecss that one lethal hit ( 3 6  residurd infectivity) corresponds to a 
release of 2.3 nucleotides, up to 1.3 of which could bearived f r a  
fragments. 
end of the chain the release of 4-5 nucleotides corresponds to one lethal 
hit, and these 51-aucleotides are not likely to came frcm fragments whose 
Tn contrast, when snake venom diesterase acts on the other 
predominant nature (ApBp.. . .YpZp)Jrendero thein resistant to SV-PDE (s )  
%le importance of the integrity of the 3'-linked, 
S*-linked, chain terminus of TKV-RI?A for it3 infectivity may be significant 
in view of recent eviiknce that the code is read starting at the 314.inked 
(foe., 5'-OX) end of the chain ( $ 3 ) .  
as contrasted to the 
RETEREEES 
1. Singer, B., and Fraenkel-Conrat, R., Biochim. Biophys. Acta, 72, 
534 (1963). - 
3.  Gordon, H. P O ,  Singer, B., an& Fraenlcel-Conrat, E., J. B i o l .  Chen., 2 3  
1014 (1960). 
4. Singer, B., and Fraenkel-Conrat, H., Virology, a, 59 (lsl). 
5 .  Richazdson, C. C., and Kornberg, A. ,  3. Biol. Chem., 2 3  2k (1964) . 
6 .  Felix, F., Potter, 5. L., and TAsharski, M., Y. Biol. Chem., 
1150 (1960). 
7. Singer, B., and Fraenkel-Conrat, R., Federation & No. 2, 
6@ (l%S). 
3 
c - '  
L '  
l ( 7 -  Fraenkel-Conrat, He, Singer, B., Biochemistry, L, 120 (1962). 
l L  Su@ymm, T., and Fraenkel-Conrat, He, Biochemistry 2, 332 (ls3). 
/ 
Id. Singer, Be, and Fraenkel-Conrat, R. ,  Biocblm. Blophye. Acta, '& 
143 (1%3)* 
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Studies of nucleotide sequences in TMV-RNA. II - The action of spleen diesterase

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