Vol.
89,
No.
August
3, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages 830-836
13, 1979
THE BROAD SPECTRUM ANTIVIRAL
AGENT RIBAVIRIN
INHIBITS
CAPPING OF mRNA Biswendu
B. Goswami,
Ernest
Borek
and Opendra
Department of Microbiology, University of Colorado Medical Denver, Colorado 80262
James Fujitaki
and Roberts
Department University Los Angeles, Received
June
19,
K. Sharma
Center,
A. Smith
of Chemistry, of California, California 90024
1979
SUMMARY: Ribavirin (l-B-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) is a broad spectrum antiviral substance active against a wide range of both DNA and RNA viruses. It is, however, virtually inactive against polio virus. Its pharmacological mechanism of action was obscure. A possible common taroet for a chemotherapeutic agent in both DNA and RNA reaction of mRNAs which inter alia involves viruses is the "capping" the formation of a ouanine pvrophosphate structurxtm' terminus by mRNA guanylyl transfera&. tie have observed that Ribavirin triphosphate is a potent competitive inhibitor of the capping guanylation of viral mRNA. This finding could account for the antiviral potency of the drug against both DNA and RNA viruses and its ineffectiveness against a virus in which the mRNAs derived from them are not capped. Ribavirin a broad both
(1-8-D-ribofuranosyl-1,2,4-triazole-3-carboxamide)
spectrum
(2).
Its
almost
certainly
in cell
culture
its
pharmacological
pool
this
--in vivo inhibitor
were
guanine
size
its
sole
is,
mechanism
of action
and Ribavirin
of action
830
but
antiviral
effect
Ribavirin (3).
diminishes
Ribavirin
is
5'-monophosphate
is
a potent
dehydrogenase
(4).
But
5'-phosphate
selectivity.
@ 1979 by Academic Press, Inc. of reproduction in any form reserved.
its
of polio
was obscure,
since
nucleotides
range
against
by guanosine.
OOOS-291X/79/150830-G7$01.00/0 Copyright All rights
a wide
inactive
nucleotides
of guanine
inosine
against
however,
reversed
(4,5), of
active
mechanism
can be totally
intracellular
competitive
It
(1).
involves
phosphorylated
if
substance
DNA and RNA viruses
virus
the
antiviral
is
(6),
it
would
not
account
for
Vol. 89, No. 3, 1979
A possible
BIOCHEMICAL
common target
and RNA viruses
is the
posttranscriptional structure GMP from
of a methyl guanosine and (c)
blocked
group
from
5'
by S-adenosylmethionine: methylation
We report inhibitor large
DNA-containing
cells.
Vaccinia
methionine
that
which
the (8).
and posttranscriptional
modification
are
conditions
as substrate
for
the
and tit-terminal
studying We therefore
modification
7 of the
added
by S-adenosylmethionine:
is a potent
vaccinia
mRNA.
in the
cytoplasm
in the
presence
m7G(5')pppGm needed
experimental
tion
in vitro -___
enzymes
manipulating
(11).
of
replicates
cap structures All
transfer
methyltransferase,
5'-triphosphate
mRNAs synthesized
which
(7).
guanylation
virus
This
of a guanine
mRNA, (b)
to position
nucleoside
Ribavirin
5'-terminal
contain
5'-terminus
of acceptor
methyltransferase
DNA
of mRNAs.
formation
mRNA (guanine-7-)
of a penultimate
here
of the
in vitro
terminus
the
in both
mRNA guanylyltransferase,
S-adenosylmethionine
mRNA nucleoside-2'-O-)
the
the
structure
(a)
enzyme GTP:
RESEARCH COMMUNICATIONS
agent
"cap"
involves:
by the
GTP to
a chemotherapeutic
methylated
modification
pyrophosphate transfers
for
AND BIOPHYSICAL
for
the
present
examined
in the
of vaccinia
effect
of
is a
infected
of S-adenosyl-
synthesis
of blocked the
Vaccinia
and m7G(5')pppAm
mRNA can be isolated
synthesis
competitive
of viral virion which
methylated of Ribavirin
at mRNAs
(9,lO).
By
can serve structures on transcrip-
mRNA.
METHODS Mouse L929 cells were grown in roller bottles at 37°C in Eagle's minimum essential medium (MEM) containing 5% fetal bovine serum. Confluent monolayers were infected with vaccinia virus in MEM lacking fetal bovine serum. After 30 minutes of absorption, growth medium was added and incuInfected cells were harvested, washed bation continued for 18 hours. twice with balanced salt solution and stored frozen at -70°C. Vaccinia virus was isolated and purified by centrifugation through a sucrose cushion and two sucrose gradient centrifugations (12,13). The final virus preparation contained approximately 70 ug protein per A260 unit. Uncapped vaccinia mRNA as a substrate for guanylyltransferase reaction in vitro was synthesized in the presence of S-adenosylhomocysteine (ll]T-' The-incubation system contained in a total volume of 50-100 ml: 4-5 A /ml of purified virus, 50 mM Tris-HCl (pH 8.5), 10 mM MgCl 10 mM DTT, 6685% Nonidet P-40, 2.5 mM each of ATP, GTP, CTP and UTP, and2i00 J.IM S-adenosylhomocysteine. After 90 minutes of incubation at 37"C, cores
831
Vol.
89,
No.
3,
1979
BIOCHEMICAL
TABLE
1:
Effect
of
Incorporation Transcription Reaction
Experiment
AND
BIOPHYSICAL
Ribavirin of
3[H]
and UMP and
by Vaccinia
Core
Mixture
GTP,
Associated
on the vitro Enzymes Incorporated GMP
1000 CTP
negligible
P-40
+Ribavirin,
negligible 1 ti
+Ribavirin
5'-monophosphate,
+Ribavirin
5'-triphosphate,
1000 1 n+i 1 mM
1000 1000
2*
50
Completea +Ribavirin
5'-triphosphate,
1 mM
Completeb +Ribavirin
50 75
5'-triphosphate,
1 mM
CompleteC +Ribavirin
GMP on --in
COMMUNICATIONS
1
Nonidet
Experiment
Nucleotides
pMole UMP
Complete -ATP,
Its
RESEARCH
72 80
5'-triphosphate,
1 r&i
80
To study the effect of Ribavirin and its phosphorylated derivatives (5) on RNA synthesis by virion RNA polymerase, the reaction mixture contained in 100 ~1: 4-5 A units/ml 50 mM Tris-HCl (pH 8.5), 10 fi'MgC1 of purified virus, 10 mM DTT, 0.05% Nonidet P-40, 2.5 mM each of ATP, CTP*ind GTP, 0.5 mM UTP (sigma) and 2 pCi of [3H] UTP (New England Nuclear, Sp. Act. 14.1 Ci/mMole). After 30 minutes of incubation at 37"C, TCA insoluble radioactivity was collected on glass fiber filters, washed with cold 5% TCA and ethanol, *The RNA polymerase reaction mixture dried and counted. was modified to contain 2.5 mM UTP and all unlabell d GTP was replaced with (a) 5uM[8-3H] GTP; (b) 25 uM [8- 5 HI GTP; and (c) 45 ~.IM [8-3~1 GTP (New England Nuclear, Sp. Act. Reactions were incubated for 10 minutes. 11.2 Ci/mMole). were removed by centrifugation at 30,000 g for 30 min. The supernatant was made 0.1 M in respect to Tris-HCl (pH 8.5) and 0.5% in respect to SDS, and was extracted with water saturated phenol in cold. To the aqueous phase, 2 volumes of ethanol was added and was kept at -20" for several hours. The dense precipitate was dissolved in water and high molecular weight RNA was precipitated with 2 M LiCl. The LiCl precipi-
832
Vol.
89,
No.
3,
1979
BIOCHEMICAL
tation was repeated RNA was precipitated 2 M sodium acetate
twice more. twice with (14,15).
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
The pellet was dissolved in water and 2 volumes of ethanol and 0.1 volume of
Vaccinia "capping" enzyme activities were isolated from purified virus (16). After solubilizinq the virus cores with 0.1% sodium deoxycholate in 0.3 M Tris-HCl (pH 8.4), 50 mM DTT and 0.25 M NaCl and centrifugation at 140,000 g for 60 minutes to remove insoluble material, the supernatant was adjusted to 0.2M NaCl, 10% glycerol, 0.1% Triton X-100 and 1 mM EDTA. It was then passed throu h a DEAE-cellulose column equilibrated with 0.15M Tris-HCl (pH 8.4 7 , 0.1% Triton X-100, 2 mM DTT, 10% glycerol and 1 mM EDTA containing 0.2M NaCl. The flow-through from the column which was devoid of most nucleic acid was used for all enzymatic reactions (9, 10).
RESULTS AND DISCUSSION
vivo
Ribavirin
is
(17,18).
However,
mouse lymphoma (3).
cell
We studied
by vaccinia lated
the
did
to rule
activity
incubation
and its
nucleotides
The effect synthesized
did of
of
Ribavirin
Rat liver removed 5'-terminal
2).
these
terminal by the
7-methylguanosine
in the
TCA similar of
of
RNA
GTP in
presence
conditions,
guanylation soluble
5'-triphosphate
enzyme
of
Ribavirin
of -___ in vitro isolated
from
was a potent
competi-
(Figure
1).
The Km
constant
(Ki)
GTP was 22 uM and an inhibitor
5'-triphosphate
by oxidation
UMP into
activity.
mRNA: guanylyltransferase for
phosphory-
presence
out
RNA polymerase 5'
RNA synthesis
any inhibition
by the
under
--in vitro
conformation
that
-in either
or its
[3H]
was carried even
on the
poly(A)-containing
chemically
of
was overcome
inhibit
inhibit
Ribavirin
possibility
Ribavirin
vaccinia
guanylyltransferase
of 32 UM for
the
mRNA was studied
(Table
inhibitor
not
1).
has molecular
However,
Ribavirin
uncapped cores
Ribavirin
not
RNA polymerase
incorporation
RNA synthesis
of GTP.
does
on DNA-dependent
(Table
by Ribavirin
mixture,
amounts
of Ribavirin
out
RNA and DNA synthesis
or mouse liver
inhibit
(19),
limiting
for
not
both
5'-phosphate
polymerase
Since
polymerase
tive
effect
material.
to guanosine
of
DNA polymerase
derivatives
vaccinia
inhibitor Ribavirin
associated
insoluble
the
a potent
was found. RNA from which
with
5'-capped
NaI04,
followed
by B-elimination
(20)
833
structure
by excision served
was of
the
as a substrate
Vol.
89,
No.
3, 1979
BIOCHEMICAL
TABLE
2:
Effect
of
--in
of
vitro
AND
Ribavirin
RESEARCH
5'-Triphosphate
Synthesized
Vaccinia
Vaccinia Reaction
BIOPHYSICAL
COMMUNICATIONS
on 5'-Guanylation mRNA by GTP:
mRNA
Guanylyltransferase 3[H]
Mixture
GMP Incorporated*
Complete
(100)
+Ribavirin,
100
1 mM
+Ribavirin
5'-monophosphate,
+Ribavirin
5'-triphosphate
100
1 mM
50 pM
48
75 pM
45
mRNA guanylyltransferase assay mixture contained in 100 ul: 50 mM Tris-HCl (pH 7.8), 2.5 mM MgC12, 1 mM DTT, 14 ug of synthesized uncapped vaccinia mRNA, 10 pg of --in vitro enzyme and 20 UM [8-31-i] GTP (Sp. Act. 11.2 Ci/mMole); 10,000 cpm/p mole) (9). After 30 minutes of incubation at 37'C, TCA precipitated material was collected on glass fiber filters (GF/C), washed with 5% TCA and ethanol. The filters were dried and counted in a toluene based scintillation fluid. *Expressed as percentage of incorporation by the complete system, which in this experiment was 12 p moles. for
guanylyltransferase.
mRNA was also
5'-Guanylation
competitively
of chemically
inhibited
modified
by Ribavirin
rat
liver
5'-triphosphate
(Km for
GTP = 33 pM, and Ki = 37 PM). Experiments is
are
incorporated
at
Ribavirin methylation absence ively
in progress the
vaccinia
of GTP.
(11).
of mRNAs by Ribavirin could
would account
Ribavirin
concentrations reaction
vaccinia
5'-phosphate
the
out
antiviral
834
in the
much more effect-
(21).
of proper
to accumulation for
was carried
in mRNAs is
Inhibition lead
(1 mM) inhibited
mRNA is inhibited
antibiotic
7-methylguanosine
translation
This
higher
an antifungal
The 5'-terminal
synthesis.
at
of
whether
of mRNAs.
mRNA, when the
Methylation
by sinefungin,
efficient
5'-terminus
5'-triphosphate of
to determine
required
for
modification
of
of mRNAs inert potency
of
their
this
5'-terminus
in protein drug
Vol.
89,
No.
3, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
O.l-
Fig.1 Linfeweaver-Burk plots showing competitive inhibition of viral guanylyltransferase by Ribavirin 5'-triphosphate. The incubation mixture in a total volume of 100 ul contained: 50 mM Tris-HCl (oH 7.8). 1 mM OTT. 2.5 mM MglX12, satur ting amounts (30 ug) of RNA substrate, 5 ug'of enzymeprotein, 5-100 uM [ 9 H] GTP (11.2 Ci/mMole) and 100 pM Ribavirin 5'-triphosphate, where indicated. The reactions were incubated at 37°C for 30 minutes and stopped by the addition of 1 ml ice cold 10% TCA. After 15 minutes in ice, the precipitates were collected on glass fiber filter discs washed with 5% TCA and ethanol, dried and counted. The lines were fitted to the points by the method of least squares using a Smith-Corona Marchant 1016 computer with an IOTA-l programmer. ti no Ribavirin 5'-triphosphate; 100 pM Ribavirin 5'-triphosphate: H¶ against
bo,th DNA and RNA viruses
in which
mRNAs derived
Impai'rment a test
system
work
them are
for
designing We thank
has been supported
capped
antiviral
Sylvia
J.
by grants
REFERENCES Witkowski, J.T., Robins, R.K., 1. J. Med. Chem., E, 1150-1154.
from
Sidwell,
835
against
such as polio
modification
potential Dr.
ineffectiveness
not
of posttranscriptional
ACKNOWLEDGEMENTS: This
from
and its
a virus virus.
of mRNA may provide agents.
Kerr
for
the
her
National
helpful
suggestions.
Cancer
R.W. and Simon,
L.N.
Institute.
(1972)
Vol. 89, No. 3, 1979
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. ;43: 15. 16. 17. 18. 19. ;::
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Sidwell, R.W., Huffman, J.H., Khare, G.P., Allen, L.B., Witkowski, J.T. and Robins, R.K. (1972) Science, 177, 705-706. Muller, W.E., Maidhof, A., Taschner, H. and Zahn, R.K. (1977) Biochem. Pharmacol., 26, 1071-1075. Streeter, D.G., Witkowski, J.T., Khare, G.P., Sidwell, R.W., Barren, R.J., Robins, R.K. and Simon, L.N. (1973) Proc. Natl. Acad. Sci., 70, 1174-1178. Kller, J.P., Kigwana, L.J., Streeter, D.G., Robins, R.K., Simon, L.N., Roboz, J. (1977) Ann. N.Y. Acad. Sci., 284, 211-229. Scholtissek, C. (1976) Archiv. Virol., 50, 349-352. Moss, B., Martin, S.A., Ensinger, M., Boone, R.F. and Wei, C. (1976) Progress in Nucleic Acid Res. Mol. Biol., 19, 63-81. Wei, C. and Moss, B. (1975) Proc. Natl. Acad. Sci., 72, 318-322. Ensinger, M.J., Martin, S.A., Peoletti, E. and Moss, B. (1975) Proc. Natl. Acad. Sci., 72-, 2525-2529. Martin, S.A., Paoletti, E., Moss, B. (1975) J. Biol. Chem., 250, 93229329. Muthukrishnan, S., Moss, B., Cooper, J.A., Maxwell, E.S. (1978) J. Biol. Chem., 253, 1710-1715. Joklik, W.K. (1962) Virology, 18, 9-18. Nevins, J.R. and Joklik, W.K. fl977) J. Biol. Chem., 252, 6930-6938. Nevins, J.R. and Joklik, W.K. (1975) Virology, 63, 1-14. Villa-Komaroff, L., McDowell, M., Baltimore, D. and Lodish, H.F. (1974) Methods Enzymology, m, 709-723. Paoletti, E., Rosemond-Hornbeak, H. and Moss, B. (1974) J. Biol. Chem., 249, 3273-3280. Streeter, D.G., Khare, G.P., Sidwell, R.W. and Simon, L.N. (1972) Fed. Proc., 31, 576. Khare, G.P., Streeter, D.G., Sidwell, R.W., Simon, L.N. and Robins, R.K. (1972) Abst. 72nd Meeting Am. Sot. Microbial., 227. Prusiner, P. and Sundaralingam, M. (1973) Nature New Biol.,z, 116-118. Levin, K.H. and Samuel, C.E. (1977) Virology, 77, 245-259. Borchardt, R.T. and Stone, H.O. (1978) J. Biol. Chem., Pugh, C.S.G., 253, 4075-4077.
836
89,
No.
August
3, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages 830-836
13, 1979
THE BROAD SPECTRUM ANTIVIRAL
AGENT RIBAVIRIN
INHIBITS
CAPPING OF mRNA Biswendu
B. Goswami,
Ernest
Borek
and Opendra
Department of Microbiology, University of Colorado Medical Denver, Colorado 80262
James Fujitaki
and Roberts
Department University Los Angeles, Received
June
19,
K. Sharma
Center,
A. Smith
of Chemistry, of California, California 90024
1979
SUMMARY: Ribavirin (l-B-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) is a broad spectrum antiviral substance active against a wide range of both DNA and RNA viruses. It is, however, virtually inactive against polio virus. Its pharmacological mechanism of action was obscure. A possible common taroet for a chemotherapeutic agent in both DNA and RNA reaction of mRNAs which inter alia involves viruses is the "capping" the formation of a ouanine pvrophosphate structurxtm' terminus by mRNA guanylyl transfera&. tie have observed that Ribavirin triphosphate is a potent competitive inhibitor of the capping guanylation of viral mRNA. This finding could account for the antiviral potency of the drug against both DNA and RNA viruses and its ineffectiveness against a virus in which the mRNAs derived from them are not capped. Ribavirin a broad both
(1-8-D-ribofuranosyl-1,2,4-triazole-3-carboxamide)
spectrum
(2).
Its
almost
certainly
in cell
culture
its
pharmacological
pool
this
--in vivo inhibitor
were
guanine
size
its
sole
is,
mechanism
of action
and Ribavirin
of action
830
but
antiviral
effect
Ribavirin (3).
diminishes
Ribavirin
is
5'-monophosphate
is
a potent
dehydrogenase
(4).
But
5'-phosphate
selectivity.
@ 1979 by Academic Press, Inc. of reproduction in any form reserved.
its
of polio
was obscure,
since
nucleotides
range
against
by guanosine.
OOOS-291X/79/150830-G7$01.00/0 Copyright All rights
a wide
inactive
nucleotides
of guanine
inosine
against
however,
reversed
(4,5), of
active
mechanism
can be totally
intracellular
competitive
It
(1).
involves
phosphorylated
if
substance
DNA and RNA viruses
virus
the
antiviral
is
(6),
it
would
not
account
for
Vol. 89, No. 3, 1979
A possible
BIOCHEMICAL
common target
and RNA viruses
is the
posttranscriptional structure GMP from
of a methyl guanosine and (c)
blocked
group
from
5'
by S-adenosylmethionine: methylation
We report inhibitor large
DNA-containing
cells.
Vaccinia
methionine
that
which
the (8).
and posttranscriptional
modification
are
conditions
as substrate
for
the
and tit-terminal
studying We therefore
modification
7 of the
added
by S-adenosylmethionine:
is a potent
vaccinia
mRNA.
in the
cytoplasm
in the
presence
m7G(5')pppGm needed
experimental
tion
in vitro -___
enzymes
manipulating
(11).
of
replicates
cap structures All
transfer
methyltransferase,
5'-triphosphate
mRNAs synthesized
which
(7).
guanylation
virus
This
of a guanine
mRNA, (b)
to position
nucleoside
Ribavirin
5'-terminal
contain
5'-terminus
of acceptor
methyltransferase
DNA
of mRNAs.
formation
mRNA (guanine-7-)
of a penultimate
here
of the
in vitro
terminus
the
in both
mRNA guanylyltransferase,
S-adenosylmethionine
mRNA nucleoside-2'-O-)
the
the
structure
(a)
enzyme GTP:
RESEARCH COMMUNICATIONS
agent
"cap"
involves:
by the
GTP to
a chemotherapeutic
methylated
modification
pyrophosphate transfers
for
AND BIOPHYSICAL
for
the
present
examined
in the
of vaccinia
effect
of
is a
infected
of S-adenosyl-
synthesis
of blocked the
Vaccinia
and m7G(5')pppAm
mRNA can be isolated
synthesis
competitive
of viral virion which
methylated of Ribavirin
at mRNAs
(9,lO).
By
can serve structures on transcrip-
mRNA.
METHODS Mouse L929 cells were grown in roller bottles at 37°C in Eagle's minimum essential medium (MEM) containing 5% fetal bovine serum. Confluent monolayers were infected with vaccinia virus in MEM lacking fetal bovine serum. After 30 minutes of absorption, growth medium was added and incuInfected cells were harvested, washed bation continued for 18 hours. twice with balanced salt solution and stored frozen at -70°C. Vaccinia virus was isolated and purified by centrifugation through a sucrose cushion and two sucrose gradient centrifugations (12,13). The final virus preparation contained approximately 70 ug protein per A260 unit. Uncapped vaccinia mRNA as a substrate for guanylyltransferase reaction in vitro was synthesized in the presence of S-adenosylhomocysteine (ll]T-' The-incubation system contained in a total volume of 50-100 ml: 4-5 A /ml of purified virus, 50 mM Tris-HCl (pH 8.5), 10 mM MgCl 10 mM DTT, 6685% Nonidet P-40, 2.5 mM each of ATP, GTP, CTP and UTP, and2i00 J.IM S-adenosylhomocysteine. After 90 minutes of incubation at 37"C, cores
831
Vol.
89,
No.
3,
1979
BIOCHEMICAL
TABLE
1:
Effect
of
Incorporation Transcription Reaction
Experiment
AND
BIOPHYSICAL
Ribavirin of
3[H]
and UMP and
by Vaccinia
Core
Mixture
GTP,
Associated
on the vitro Enzymes Incorporated GMP
1000 CTP
negligible
P-40
+Ribavirin,
negligible 1 ti
+Ribavirin
5'-monophosphate,
+Ribavirin
5'-triphosphate,
1000 1 n+i 1 mM
1000 1000
2*
50
Completea +Ribavirin
5'-triphosphate,
1 mM
Completeb +Ribavirin
50 75
5'-triphosphate,
1 mM
CompleteC +Ribavirin
GMP on --in
COMMUNICATIONS
1
Nonidet
Experiment
Nucleotides
pMole UMP
Complete -ATP,
Its
RESEARCH
72 80
5'-triphosphate,
1 r&i
80
To study the effect of Ribavirin and its phosphorylated derivatives (5) on RNA synthesis by virion RNA polymerase, the reaction mixture contained in 100 ~1: 4-5 A units/ml 50 mM Tris-HCl (pH 8.5), 10 fi'MgC1 of purified virus, 10 mM DTT, 0.05% Nonidet P-40, 2.5 mM each of ATP, CTP*ind GTP, 0.5 mM UTP (sigma) and 2 pCi of [3H] UTP (New England Nuclear, Sp. Act. 14.1 Ci/mMole). After 30 minutes of incubation at 37"C, TCA insoluble radioactivity was collected on glass fiber filters, washed with cold 5% TCA and ethanol, *The RNA polymerase reaction mixture dried and counted. was modified to contain 2.5 mM UTP and all unlabell d GTP was replaced with (a) 5uM[8-3H] GTP; (b) 25 uM [8- 5 HI GTP; and (c) 45 ~.IM [8-3~1 GTP (New England Nuclear, Sp. Act. Reactions were incubated for 10 minutes. 11.2 Ci/mMole). were removed by centrifugation at 30,000 g for 30 min. The supernatant was made 0.1 M in respect to Tris-HCl (pH 8.5) and 0.5% in respect to SDS, and was extracted with water saturated phenol in cold. To the aqueous phase, 2 volumes of ethanol was added and was kept at -20" for several hours. The dense precipitate was dissolved in water and high molecular weight RNA was precipitated with 2 M LiCl. The LiCl precipi-
832
Vol.
89,
No.
3,
1979
BIOCHEMICAL
tation was repeated RNA was precipitated 2 M sodium acetate
twice more. twice with (14,15).
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
The pellet was dissolved in water and 2 volumes of ethanol and 0.1 volume of
Vaccinia "capping" enzyme activities were isolated from purified virus (16). After solubilizinq the virus cores with 0.1% sodium deoxycholate in 0.3 M Tris-HCl (pH 8.4), 50 mM DTT and 0.25 M NaCl and centrifugation at 140,000 g for 60 minutes to remove insoluble material, the supernatant was adjusted to 0.2M NaCl, 10% glycerol, 0.1% Triton X-100 and 1 mM EDTA. It was then passed throu h a DEAE-cellulose column equilibrated with 0.15M Tris-HCl (pH 8.4 7 , 0.1% Triton X-100, 2 mM DTT, 10% glycerol and 1 mM EDTA containing 0.2M NaCl. The flow-through from the column which was devoid of most nucleic acid was used for all enzymatic reactions (9, 10).
RESULTS AND DISCUSSION
vivo
Ribavirin
is
(17,18).
However,
mouse lymphoma (3).
cell
We studied
by vaccinia lated
the
did
to rule
activity
incubation
and its
nucleotides
The effect synthesized
did of
of
Ribavirin
Rat liver removed 5'-terminal
2).
these
terminal by the
7-methylguanosine
in the
TCA similar of
of
RNA
GTP in
presence
conditions,
guanylation soluble
5'-triphosphate
enzyme
of
Ribavirin
of -___ in vitro isolated
from
was a potent
competi-
(Figure
1).
The Km
constant
(Ki)
GTP was 22 uM and an inhibitor
5'-triphosphate
by oxidation
UMP into
activity.
mRNA: guanylyltransferase for
phosphory-
presence
out
RNA polymerase 5'
RNA synthesis
any inhibition
by the
under
--in vitro
conformation
that
-in either
or its
[3H]
was carried even
on the
poly(A)-containing
chemically
of
was overcome
inhibit
inhibit
Ribavirin
possibility
Ribavirin
vaccinia
guanylyltransferase
of 32 UM for
the
mRNA was studied
(Table
inhibitor
not
1).
has molecular
However,
Ribavirin
uncapped cores
Ribavirin
not
RNA polymerase
incorporation
RNA synthesis
of GTP.
does
on DNA-dependent
(Table
by Ribavirin
mixture,
amounts
of Ribavirin
out
RNA and DNA synthesis
or mouse liver
inhibit
(19),
limiting
for
not
both
5'-phosphate
polymerase
Since
polymerase
tive
effect
material.
to guanosine
of
DNA polymerase
derivatives
vaccinia
inhibitor Ribavirin
associated
insoluble
the
a potent
was found. RNA from which
with
5'-capped
NaI04,
followed
by B-elimination
(20)
833
structure
by excision served
was of
the
as a substrate
Vol.
89,
No.
3, 1979
BIOCHEMICAL
TABLE
2:
Effect
of
--in
of
vitro
AND
Ribavirin
RESEARCH
5'-Triphosphate
Synthesized
Vaccinia
Vaccinia Reaction
BIOPHYSICAL
COMMUNICATIONS
on 5'-Guanylation mRNA by GTP:
mRNA
Guanylyltransferase 3[H]
Mixture
GMP Incorporated*
Complete
(100)
+Ribavirin,
100
1 mM
+Ribavirin
5'-monophosphate,
+Ribavirin
5'-triphosphate
100
1 mM
50 pM
48
75 pM
45
mRNA guanylyltransferase assay mixture contained in 100 ul: 50 mM Tris-HCl (pH 7.8), 2.5 mM MgC12, 1 mM DTT, 14 ug of synthesized uncapped vaccinia mRNA, 10 pg of --in vitro enzyme and 20 UM [8-31-i] GTP (Sp. Act. 11.2 Ci/mMole); 10,000 cpm/p mole) (9). After 30 minutes of incubation at 37'C, TCA precipitated material was collected on glass fiber filters (GF/C), washed with 5% TCA and ethanol. The filters were dried and counted in a toluene based scintillation fluid. *Expressed as percentage of incorporation by the complete system, which in this experiment was 12 p moles. for
guanylyltransferase.
mRNA was also
5'-Guanylation
competitively
of chemically
inhibited
modified
by Ribavirin
rat
liver
5'-triphosphate
(Km for
GTP = 33 pM, and Ki = 37 PM). Experiments is
are
incorporated
at
Ribavirin methylation absence ively
in progress the
vaccinia
of GTP.
(11).
of mRNAs by Ribavirin could
would account
Ribavirin
concentrations reaction
vaccinia
5'-phosphate
the
out
antiviral
834
in the
much more effect-
(21).
of proper
to accumulation for
was carried
in mRNAs is
Inhibition lead
(1 mM) inhibited
mRNA is inhibited
antibiotic
7-methylguanosine
translation
This
higher
an antifungal
The 5'-terminal
synthesis.
at
of
whether
of mRNAs.
mRNA, when the
Methylation
by sinefungin,
efficient
5'-terminus
5'-triphosphate of
to determine
required
for
modification
of
of mRNAs inert potency
of
their
this
5'-terminus
in protein drug
Vol.
89,
No.
3, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
O.l-
Fig.1 Linfeweaver-Burk plots showing competitive inhibition of viral guanylyltransferase by Ribavirin 5'-triphosphate. The incubation mixture in a total volume of 100 ul contained: 50 mM Tris-HCl (oH 7.8). 1 mM OTT. 2.5 mM MglX12, satur ting amounts (30 ug) of RNA substrate, 5 ug'of enzymeprotein, 5-100 uM [ 9 H] GTP (11.2 Ci/mMole) and 100 pM Ribavirin 5'-triphosphate, where indicated. The reactions were incubated at 37°C for 30 minutes and stopped by the addition of 1 ml ice cold 10% TCA. After 15 minutes in ice, the precipitates were collected on glass fiber filter discs washed with 5% TCA and ethanol, dried and counted. The lines were fitted to the points by the method of least squares using a Smith-Corona Marchant 1016 computer with an IOTA-l programmer. ti no Ribavirin 5'-triphosphate; 100 pM Ribavirin 5'-triphosphate: H¶ against
bo,th DNA and RNA viruses
in which
mRNAs derived
Impai'rment a test
system
work
them are
for
designing We thank
has been supported
capped
antiviral
Sylvia
J.
by grants
REFERENCES Witkowski, J.T., Robins, R.K., 1. J. Med. Chem., E, 1150-1154.
from
Sidwell,
835
against
such as polio
modification
potential Dr.
ineffectiveness
not
of posttranscriptional
ACKNOWLEDGEMENTS: This
from
and its
a virus virus.
of mRNA may provide agents.
Kerr
for
the
her
National
helpful
suggestions.
Cancer
R.W. and Simon,
L.N.
Institute.
(1972)
Vol. 89, No. 3, 1979
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. ;43: 15. 16. 17. 18. 19. ;::
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Sidwell, R.W., Huffman, J.H., Khare, G.P., Allen, L.B., Witkowski, J.T. and Robins, R.K. (1972) Science, 177, 705-706. Muller, W.E., Maidhof, A., Taschner, H. and Zahn, R.K. (1977) Biochem. Pharmacol., 26, 1071-1075. Streeter, D.G., Witkowski, J.T., Khare, G.P., Sidwell, R.W., Barren, R.J., Robins, R.K. and Simon, L.N. (1973) Proc. Natl. Acad. Sci., 70, 1174-1178. Kller, J.P., Kigwana, L.J., Streeter, D.G., Robins, R.K., Simon, L.N., Roboz, J. (1977) Ann. N.Y. Acad. Sci., 284, 211-229. Scholtissek, C. (1976) Archiv. Virol., 50, 349-352. Moss, B., Martin, S.A., Ensinger, M., Boone, R.F. and Wei, C. (1976) Progress in Nucleic Acid Res. Mol. Biol., 19, 63-81. Wei, C. and Moss, B. (1975) Proc. Natl. Acad. Sci., 72, 318-322. Ensinger, M.J., Martin, S.A., Peoletti, E. and Moss, B. (1975) Proc. Natl. Acad. Sci., 72-, 2525-2529. Martin, S.A., Paoletti, E., Moss, B. (1975) J. Biol. Chem., 250, 93229329. Muthukrishnan, S., Moss, B., Cooper, J.A., Maxwell, E.S. (1978) J. Biol. Chem., 253, 1710-1715. Joklik, W.K. (1962) Virology, 18, 9-18. Nevins, J.R. and Joklik, W.K. fl977) J. Biol. Chem., 252, 6930-6938. Nevins, J.R. and Joklik, W.K. (1975) Virology, 63, 1-14. Villa-Komaroff, L., McDowell, M., Baltimore, D. and Lodish, H.F. (1974) Methods Enzymology, m, 709-723. Paoletti, E., Rosemond-Hornbeak, H. and Moss, B. (1974) J. Biol. Chem., 249, 3273-3280. Streeter, D.G., Khare, G.P., Sidwell, R.W. and Simon, L.N. (1972) Fed. Proc., 31, 576. Khare, G.P., Streeter, D.G., Sidwell, R.W., Simon, L.N. and Robins, R.K. (1972) Abst. 72nd Meeting Am. Sot. Microbial., 227. Prusiner, P. and Sundaralingam, M. (1973) Nature New Biol.,z, 116-118. Levin, K.H. and Samuel, C.E. (1977) Virology, 77, 245-259. Borchardt, R.T. and Stone, H.O. (1978) J. Biol. Chem., Pugh, C.S.G., 253, 4075-4077.
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