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Journal of Virology, December 1999, p. 10536-10539, Vol. 73, No. 12
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Dependence of Echovirus 9 on the Enterovirus RNA Replication
Inhibitor 2-(
-Hydroxybenzyl)-Benzimidazole Maps to Nonstructural
Protein 2C
Dirk
Hadaschik,
Marcus
Klein,
Holger
Zimmermann,
Hans
J.
Eggers, and
Birgit
Nelsen-Salz*
Institut für Virologie der
Universität zu Köln, 50935 Cologne, Germany
Received 16 February 1999/Accepted 8 September 1999
 |
ABSTRACT |
HBB [2-(
-hydroxybenzyl)-benzimidazole] selectively inhibits
RNA synthesis of most enteroviruses. However, isolation of
HBB-dependent variants is possible. Sequence analysis and
characterization of recombinant viruses revealed that HBB dependence
maps to the nonstructural protein 2C. A single point mutation at
position C4782U is sufficient to establish the
HBB-dependent phenotype in our echovirus 9 model.
| |
TEXT |
The antiviral agent
2-(-hydroxybenzyl)-benzimidazole (HBB) was first described in
1958 (10) and was discovered to specifically inhibit the
multiplication of many members of the genus Enterovirinae within the picornavirus family (8). Viruses belonging to
other families were found to be HBB insusceptible (8).
Studies on the mechanism of action of HBB demonstrated that the
substance, at concentrations nontoxic to cells, inhibits the synthesis
of viral RNA, whereas cellular RNA synthesis remains unaffected
(6-8). In the course of this work not only HBB-resistant
but also HBB-dependent virus mutants were discovered (5). In
the present communication, we show that HBB dependence maps to the
nonstructural protein 2C.
Isolation and characterization of an HBB-dependent echovirus 9 variant.
Echovirus 9 Barty MP5 (E9B MP5) (24) was
cultivated under selective pressure of 50 µM, 150 µM, and 220 µM
concentrations of HBB. To ensure that further experiments were
performed with virus stocks as homogeneous as possible, the selected
dependent virus stock was plaque purified with 220 µM HBB, and single
virus plaques were harvested and propagated after 4 days.
Determination of 50% tissue culture infective doses
(TCID50 assays) in the presence or absence of HBB gives a
first indication of the influence of HBB on replication efficiency. Ten
plaque-purified isolates selected in the presence of 220 µM HBB were
investigated (Table 1). Isolate 4 (E9H.dep-4) was chosen for further experiments, since its
TCID50 assays revealed the highest 
value (Table 1). In
order to compare the influence of HBB on replication of E9H.dep-4 and
the original isolate, E9B MP5, 10 TCID50 of the respective virus was incubated under increasing concentrations of HBB, and the
amount of virus-induced cell damage was determined at the indicated
time points. These sensitivity assays clearly exhibit the HBB-dependent
phenotype of E9H.dep-4 (Fig. 1A).
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FIG. 1.
HBB sensitivity assays of the echovirus 9 strain Barty,
the HBB-dependent variant E9H.dep-4, and recombinant viruses. (A)
Echovirus 9 Barty MP5 is sensitive to HBB, and the HBB-dependent
variant E9H.dep-4 and the recombinant virus rE9-H.dep-f1 exhibit
comparable dependence on the antiviral substance. The chimeras exhibit
the sensitive (B) or dependent (C) character, comparable to the
progenitors in panel A. (D) HBB sensitivity assays of viruses derived
from RNA generated by site-directed mutagenesis. The nomenclature is
the same as in Fig. 2. The indicated viruses were cultivated in the
presence of 0 µM (square), 73 µM (rhomb), 146 µM (triangle), or
220 µM (circle) HBB. The extent of virus-induced cell damage
reflecting viral multiplication was estimated by microscopic
examination and is given as a percentage. p.i., postinfection.
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|
Cloning and sequencing of E9H.dep-4.
Isolate E9H.dep-4 was
cloned and sequenced (pE9H.dep-40.8). Comparison to the wild-type
sequence (24) revealed 10 point mutations in the genome of
the HBB-dependent variant, seven of which give rise to amino acid
mutations (Table 2).
Proceeding from clone pE9H.dep-40.8 and the infectious E9 Barty clone
pE9B described earlier (23), a full-length clone containing the complete dependent genome was constructed (pE9-H.dep-f1) and transcribed in vitro. The infectivity of the resulting RNA was shown in
transfection experiments. In sensitivity assays, the resulting viruses
(rE9-H.dep-f1) revealed a level of dependence on HBB which was
quantitatively comparable to that of the original isolate, E9H.dep-4
(Fig. 1A). Since the virus dilution was done freshly for every
experiment, the 24-h delay for the onset of cytopathic effect is in the
range of experimental variation.
Determination of the influence of the point mutations on HBB
dependence.
In further experiments, we analyzed which of the seven
amino acid mutations gives rise to the HBB-dependent character of the variant. For that purpose, genome regions of the infectious E9 Barty
clone pE9B were replaced by corresponding restriction fragments of
clone pE9H.dep-40.8 containing series of the point mutations detected
in the dependent genome (Fig. 2).
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FIG. 2.
Survey of the genomes of chimeric echovirus 9 Barty
strains. Name and position of the mutations of the dependent variant
are given in the schematic genome above. Genome fragments derived from
the dependent variant are represented by filled bars. Fragments
obtained from the sensitive wild-type echovirus 9 are depicted by open
bars.
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|
Recombinant viral RNA carrying mutations 1 and 2 (pE9-H.dep-C2 and
pE9-H.dep-E1) and 1 to 5 (pE9-H.dep-A1) proved to be infectious only in
the absence of HBB (data not shown). In contrast, the RNA of clones
pE9-H.dep-A2 and pE9-H.dep-F1, both containing mutations 6 and 7, codes
for infective virus particles only in the presence of HBB. Sensitivity
assays using recombinant viruses purified from the supernatant of the
transfection experiments support these results (Fig. 1B and C).
Mutations 1 to 5 do not alter the sensitive phenotype of clone pE9B
(rE9-H.dep-A1, -C2, and -E1), but the introduction of mutations 6 and 7 together leads to a dependent variant (rE9-H.dep-A2 and -F1).
Introduction of mutation 6 or 7 alone by exchanging appropriate
restriction fragments was not possible, since both mutations are
located in close proximity. Hence, they were introduced to pE9B-ic by
site-directed mutagenesis. In each case, three independent clones were
tested in transfection experiments and were discovered to be infectious
depending on HBB concentration. RNA containing mutation 6 gives rise to
infectious virus only in the presence of HBB, whereas mutation 7 in the
genetic background of a sensitive phenotype causes no alteration (data
not shown). Subsequently, one construct was analyzed in each
sensitivity assay (Fig. 1D), and the results of the transfection
experiments were confirmed.
Thus, constructs carrying the mutation C4782T reveal the
same dependent phenotype as the original isolate E9H.dep-4. The six other point mutations detected in the genome of E9H.dep-4 that lead to
an amino acid exchange are not able to alter the growth behavior of the
virus with regard to dependence of HBB.
The genomic regions coding for the 2C proteins of the three plaque
isolates (numbers 3, 6, and 8) which were also shown to be HBB
dependent (Table 1) were amplified by reverse transcription-PCR and
were sequenced. Both mutations C4782U and
U4970C are present in the three HBB-dependent isolates
(data not shown). It is noteworthy that all plaque isolates
investigated in this study were purified from the same virus stock and
propagated under the selective pressure of HBB. Hence, the mutations
C4782U and U4970C may be early events and all
variants are the offspring of the same progenitor.
Discussion.
The data presented suggest that HBB interacts
directly or indirectly with the nonstructural protein 2C, a highly
conserved gene product within the picornavirus family (1).
Comparable results were obtained with guanidine, another substance that
specifically inhibits enterovirus replication in cell culture
(18). Mutations leading to guanidine-resistant or -dependent
poliovirus variants are located within the 2C gene (2, 11, 15-17,
21). The majority of these amino acid exchanges are clustered at
positions 179 and 187, respectively; however, the region between amino
acids 225 and 235 is also a hot spot for mutations (21).
This finding leads us to propose that the two viral inhibitors, HBB and
guanidine, may affect similar 2C functions. However, certain
differences should be noted; e.g., the antiviral spectrum of both
substances is not identical. As shown previously, guanidine is only a
weak inhibitor of echovirus 9 replication, in contrast to its strong effect on polioviruses. Under the selective pressure of a 2 mM concentration of guanidine, the virus becomes resistant, but we were
not able to select a completely dependent variant of echovirus 9, even
after seven passages. Furthermore, whereas mutation C4782T manifests HBB as well as guanidine dependence, variant E9G.dep-12, one
of the typical, slightly guanidine-dependent isolates, remains sensitive to HBB to a significant degree (Fig.
3).
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FIG. 3.
Sensitivity assays of the recombinant rE9-H.dep-6 and a
guanidine-dependent variant, E9G.dep-12. The indicated viruses were
cultivated either in the presence of 73 µM (rhomb), 146 µM
(triangle), or 220 µM (circle) HBB or under 1 mM (cross) or 2 mM
(star) guanidine. The virus control (square) was grown without an
antiviral drug. Results of sensitivity assays of rE9-H.dep-6 in the
presence of HBB are given in Fig. 1. The extent of virus-induced cell
damage reflecting viral multiplication was estimated by microscopic
examination and is given as a percentage. p.i., postinfection.
|
|
The conclusion that the mutation crucial for the dependence on HBB
targets the 2C protein is plausible, considering the significance of
the protein for RNA synthesis. It is shown that poliovirus 2C plays a
significant role in viral RNA replication (3, 13, 14) and
possibly also in virion assembly (12) or encapsidation (22). This explains the observation that removal of HBB from the medium during the period exhibiting the highest RNA polymerase activity abolishes the production of infectious particles of
HBB-dependent virus variants in less than 15 min (4).
Among others, a nucleoside triphosphate-binding motif composed of three
conserved regions (A, B, and C) typical for nucleoside triphosphate-binding proteins has been identified within 2C of enteroviruses (9). Functional activity of regions A and B
(i.e., ATP and GTP binding and splitting) could be demonstrated
(14, 19, 20). Region C is suggested to act as a helicase;
however, the experimental evidence for this function is still lacking. The Ala-to-Val mutation at position 229 (mutation 6) of the
HBB-dependent variant is located in close proximity to the C motif.
Whether the proposed helicase activity of the protein, if it exists at all, is influenced by HBB remains to be clarified.
| |
ACKNOWLEDGMENTS |
We thank Elke Feldmann and Eva Heimes for skillful technical
assistance and Herbert Pfister for critical examination of the manuscript.
This project is supported by the Deutsche Forschungsgmeinschaft (NE
586/2-2). B.N.-S. maintained a grant from the
Lise-Meitner-Stiftung, and H.Z. was supported by the
Förderverein zur Bekämpfung von Viruskrankheiten e.V. (DVV).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institut
für Virologie der Universität zu Köln,
Fürst-Pückler-Str. 56, 50935 Cologne, Germany. Phone: (49)
221-4783924. Fax: (49) 221-4783902. E-mail: birgit.nelsen-salz{at}medizin.uni-koeln.de.
Present address: Institute of Molecular and Cell Biology, Singapore
117609, Republic of Singapore.
| |
REFERENCES |
| 1.
|
Argos, P.,
G. Kamer,
M. J. H. Nicklin, and E. Wimmer.
1984.
Similarity in the gene organization and homology between proteins of animal picornaviruses and a plant comovirus suggest common ancestry of these virus families.
Nucleic Acids Res.
12:7251-7267[Abstract/Free Full Text].
|
| 2.
|
Baltera, R. F., Jr., and D. R. Tershak.
1989.
Guanidine-resistant mutants of poliovirus have distinct mutations in peptide 2C.
J. Virol.
63:4441-4444[Abstract/Free Full Text].
|
| 3.
|
Barton, D. J., and J. B. Flanegan.
1997.
Synchronous replication of poliovirus RNA: initiation of negative-strand RNA synthesis requires the guanidine-inhibited activity of protein 2C.
J. Virol.
71:8482-8489[Abstract].
|
| 4.
|
Eggers, H. J.
1982.
Benzimidazoles. Selective inhibitors of picornavirus replication in cell culture and in the organism, p. 377-417.
In
P. E. Came, and L. A. Caliguiri (ed.), Chemotherapy of viral infections. Springer-Verlag, Berlin, Germany
|
| 5.
|
Eggers, H. J., and I. Tamm.
1963.
Drug dependence of enteroviruses: variants of coxsackie A9 and echo 13 viruses that require 2-(-hydroxybenzyl)-benzimidazole for growth.
Virology
20:62-74.
|
| 6.
|
Eggers, H. J., and I. Tamm.
1963.
Inhibition of enterovirus ribonucleic acid synthesis by 2-(-hydroxybenzyl)-benzimidazole.
Nature
197:1327-1328.
|
| 7.
|
Eggers, H. J., and I. Tamm.
1962.
On the mechanism of selective inhibition of enterovirus multiplication by 2-(-hydroxybenzyl)-benzimidazole.
Virology
18:426-438.
|
| 8.
|
Eggers, H. J., and I. Tamm.
1961.
Spectrum and characteristics of the virus inhibitory action of 2-(-hydroxybenzyl)-benzimidazole.
J. Exp. Med.
113:657-682[Abstract].
|
| 9.
|
Gorbalenya, A. E.,
E. V. Koonin, and Y. I. Wolf.
1990.
A new superfamily of putative NTP-binding domains encoded by genomes of small DNA and RNA viruses.
FEBS Lett.
262:145-148[Medline].
|
| 10.
|
Hollinshead, A. C., and P. K. Smith.
1958.
Effects of certain purines and related compounds on virus propagation.
J. Pharmacol. Exp. Ther.
123:54-62[Abstract/Free Full Text].
|
| 11.
|
Kirkegaard, K., and D. Baltimore.
1986.
The mechanism of RNA recombination in poliovirus.
Cell
47:433-443[Medline].
|
| 12.
|
Li, J. P., and D. Baltimore.
1990.
An intragenic revertant of a poliovirus 2C mutant has an uncoating defect.
J. Virol.
64:1102-1107[Abstract/Free Full Text].
|
| 13.
|
Li, J. P., and D. Baltimore.
1988.
Isolation of poliovirus 2C mutants defective in viral RNA synthesis.
J. Virol.
62:4016-4021[Abstract/Free Full Text].
|
| 14.
|
Mirzayan, C., and E. Wimmer.
1992.
Genetic analysis of an NTP-binding motif in poliovirus polypeptide 2C.
Virology
189:547-555[Medline].
|
| 15.
|
Pincus, S. E.,
D. C. Diamond,
E. A. Emini, and E. Wimmer.
1986.
Guanidine-selected mutants of poliovirus: mapping of point mutations to polypeptide 2C.
J. Virol.
57:638-646[Abstract/Free Full Text].
|
| 16.
|
Pincus, S. E.,
H. Rohl, and E. Wimmer.
1987.
Guanidine-dependent mutants of poliovirus: identification of three classes with different growth requirements.
Virology
157:83-88[Medline].
|
| 17.
|
Pincus, S. E., and E. Wimmer.
1986.
Production of guanidine-resistant and -dependent poliovirus mutants from cloned cDNA: mutations in polypeptide 2C are directly responsible for altered guanidine sensitivity.
J. Virol.
60:793-796[Abstract/Free Full Text].
|
| 18.
|
Rightsel, W. A.,
J. R. Dice,
R. J. McAlpine,
E. A. Timm,
I. W. McLean, Jr.,
G. L. Dixon, and F. M. Schnabel, Jr.
1961.
Antiviral effect of guanidine.
Science
134:558-559[Abstract/Free Full Text].
|
| 19.
|
Rodriguez, P. L., and L. Carrasco.
1993.
Poliovirus protein 2C has ATPase and GTPase activities.
J. Biol. Chem.
268:8105-8110[Abstract/Free Full Text].
|
| 20.
|
Teterina, N. L.,
K. M. Kean,
A. E. Gorbalenya,
V. I. Agol, and M. Girard.
1992.
Analysis of the functional significance of amino acid residues in the putative NTP-binding pattern of the poliovirus 2C protein.
J. Gen. Virol.
73:1977-1986[Abstract/Free Full Text].
|
| 21.
|
Tolskaya, E. A.,
L. I. Romanova,
M. S. Kolesnikova,
A. P. Gmyl,
A. E. Gorbalenya, and V. I. Agol.
1994.
Genetic studies on the poliovirus 2C protein, an NTPase. A plausible mechanism of the guanidine effect on the 2C function and evidence for the importance of 2C oligomerization.
J. Mol. Biol.
236:1310-1323[Medline].
|
| 22.
|
Vance, L. M.,
N. Moscufo,
M. Chow, and B. A. Heinz.
1997.
Poliovirus 2C region functions during encapsidation of viral RNA.
J. Virol.
71:8759-8765[Abstract].
|
| 23.
|
Zimmermann, H.,
H. J. Eggers, and B. Nelsen-Salz.
1997.
Cell attachment and mouse virulence of echovirus 9 correlate with an RGD motif in the capsid protein VP1.
Virology
233:149-156[Medline].
|
| 24.
|
Zimmermann, H.,
H. J. Eggers, and B. Nelsen-Salz.
1996.
Molecular cloning and sequence determination of the complete genome of the virulent echovirus 9 strain Barty.
Virus Genes
12:149-154[Medline].
|
Journal of Virology, December 1999, p. 10536-10539, Vol. 73, No. 12
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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