Institut für Virologie der Universität zu Köln, Fürst-Pückler-Str. 56, 50935 Köln, Germany1
Author for correspondence: Birgit Nelsen-Salz. Fax +49 221 4783902. e-mail birgit.nelsen-salz{at}medizin.uni-koeln.de
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Abstract |
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Introduction |
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Many investigations on poliovirus-1 have revealed that guanidine targets the non-structural protein 2C (Baltera & Tershak, 1989 ; Pincus et al., 1986
; Pincus & Wimmer, 1986
) and recently, the determinant for dependence on HBB was found to be located within the 2C region of echovirus-9 (E9) (Hadaschik et al., 1999
). Therefore, inhibitors like HBB or guanidine may serve as important tools to extend our knowledge of protein 2C and, in consequence, picornavirus replication.
Protein 2C is one of the most conserved polyproteins of picornaviruses (Argos et al., 1984 ). However, the function of 2C is still not well understood and seems to be complex (Rueckert, 1996
; Wimmer et al., 1993
). It has been shown that this protein plays an important role in RNA replication, and the presence of 2C in enterovirus replication complexes comprised of membrane particles, viral RNA and other virus-encoded proteins has been demonstrated (Bienz et al., 1990
). Functions like membrane binding, proteinprotein interactions, NTPase activity and RNA binding have been revealed for poliovirus 2C by different in vitro techniques (Cuconati et al., 1998
; Echeverri & Dasgupta, 1995
; Mirzayan & Wimmer, 1994
; Pfister & Wimmer, 1999
; Rodriguez & Carrasco, 1993
, 1995
). NTPase activity was also observed in E9 (Klein et al., 1999
).
In this report, resistance to HBB and guanidine was defined at the genetic level, and the influence of these antiviral substances on known functions of E9 2C was investigated.
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Methods |
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Sensitivity assay.
GMK monolayers were infected with the respective virus and the virus-induced cell damage (CPE) was estimated by microscopic examination (Eggers & Tamm, 1961 ). The average of at least two independent values, differing by not more than 50%, was taken. Infecting virus doses were standardized to approximately 100 TCID50 values (median 50% tissue culture infective doses).
Northern blot analysis.
Total RNA of single-cycle infected GMK cells was isolated using the guanidium thiocyanate protocol (Ausubel et al., 1994 ). After spectrophotometric measurement of the total RNA concentrations, 10 µg portions were subjected to denaturing gel electrophoresis (1% polyacrylamide), and blotted onto an uncharged nylon membrane (Amersham) using standard techniques (Ausubel et al., 1994
). In vitro-transcribed full-length echovirus RNAs of negative and positive polarity were included as positive controls. Fragment sizes were determined by an RNA mass marker stained separately with ethidium bromide.
After KpnI digestion of the E9B full-length clone rE9B-fl.20 (Zimmermann et al., 1996 ), the 353 bp fragment comprising nucleotides 28443196 was cloned into a KpnI-linearized pT7/T3
-18 vector (BRL), transcribed in vitro and purified according to standard protocols (Ausubel et al., 1994
). These uniformly [32P]CTP-labelled RNAs of both orientations were used as probes in the Northern blotting experiments. For an internal standard, a labelled RNA transcript from a mouse
-actin cDNA clone (Stratagene) was prepared in the same way. Finally, the hybridized membranes were autoradiographed.
NTPase assay.
Echovirus 2C wild-type and mutants were purified as GST fusion proteins and the ATPase reaction was carried out as previously described (Klein et al., 1999 ). In brief, approximately 1 µg protein was incubated for 60 min at 37 °C in reaction buffer [20 mM HEPESKOH (pH 7·5), 5 mM MgCl2, 2·5 mM DTT, 0·1% BSA, 0·05% Triton X-100, 50 µM ATP and 10 µCi/ml [
-32P]ATP (3000 Ci/mmol; Amersham)]. For detection, one tenth of the reaction volume was subjected to TLC followed by autoradiography.
Non-denaturing purification of His-tagged 2C.
The coding region of echovirus 2C was amplified by PCR using the primers ATG GCT GAG CTC GAG AGT AAT GGC TGG CTC AAG (positions 40814113) and GTA GAC CTC GAG TTA TTG GAA CAG AGC TTC AGT AAG (positions 50965061), which contain manually inserted XhoI restriction sites (in bold). After digestion with XhoI, the PCR product was cloned into a XhoI-linearized pET-14b (Novagen) expression vector. Proper orientation and ligation were controlled by sequence analysis, and the plasmid was transformed into E. coli BL21 (DE3). Expression of His2C in LB medium was started after induction with 1 mM IPTG at an A600 of 1. After an incubation time of 2 h at 25 °C, the bacteria were harvested and resuspended in lysis buffer [0·5 M NaCl, 20 mM TrisHCl (pH 7·9), 10 mM imidazole, 2 mM -mercaptoethanol, 0·1 mM PMSF, 10% glycerol, 1 µg/ml pepstatin A, 1 µg/ml leupeptin, and 0·2% each of Tween 20, Nonidet P-40 and Triton X-100]. Cells were lysed by three freezethaw cycles followed by mild sonication. Further purification steps were performed by Ni2+ affinity chromatography according to the manufacturers recommendations (Qiagen). After step-wise washing and elution, 2C-containing fractions were analysed by SDSPAGE, concentrated with spin columns (Amicon), and dialysed against 20 mM HEPESKOH (pH 7·5), 100 mM KCl, 5 mM DTT and 25% glycerol.
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Results |
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Cloning and sequencing of E9-H.res and E9-G.res
Viral RNA of E9-H.res was prepared and transcribed and the longest fragment [position 3893 to the 3' poly(A) tail] was cloned into the infectious full-length wild-type clone rE9B-fl.20 (Zimmermann et al., 1996 ), giving rise to the recombinant clone rE9-H.res (Fig. 3b
). Sequence comparison of the HBB-resistant cDNA clones and the wild-type clone revealed amino acid exchanges: A4775C and C4782U. Both mutations are located in the coding region of 2C (Table 1
).
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Influence of the point mutations on the replication of E9
After transcription of the plasmids rE9-H.res and rE9-G.res in vitro, transfection experiments were performed using the DEAE method (Ausubel et al., 1994 ), and the resulting viruses were propagated in GMK cells. Sensitivity assays of these recombinants revealed a resistant phenotype as found for the original isolates, E9-H.res and E9-G.res (Fig. 4a
). In both cases, virus replication was possible even at high inhibitor concentrations, indicating that the detected mutations in the 2C gene are relevant for drug resistance.
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Like the originally isolated HBB-resistant variant, the recombinant mutants derived from E9-H.res were able to multiply well in the presence of guanidine (Fig. 4). On the other hand, the mutants derived from isolate E9-G.res were only partially resistant to HBB.
Effect of HBB on viral RNA replication
To investigate whether the mutations in protein 2C directly influence RNA replication, both positive- and negative-strand RNA synthesis by the virus variants were analysed using single-cycle infection experiments followed by Northern blotting. When using the HBB-sensitive E9 wild-type, no positive-strand RNA was detectable in the presence of HBB after a replication time of 4 or 6 h (Fig. 5a). Also, in contrast to the uninhibited samples, negative-strand RNA could not be detected at these time-points (Fig. 5b
). In an analogous study, the replication assay was carried out using the recombinant viruses. The resistant mutants rE9-H.res and rE9-A4775C were able to replicate the RNA in the absence and presence of HBB, whereas for the dependent mutant rE9-C4782U, positive-strand RNA was detectable only in the presence of the inhibitor (Fig. 5c
).
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Discussion |
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We have demonstrated that the picornavirus replication inhibitors HBB and guanidine seem to differ in terms of their precise mechanism of antiviral action. The limited or, more precisely, unidirectional cross-resistance of the two drugs and the fact that HBB and guanidine exhibit a synergistic action allow us to propose that the inhibitors act in a similar, but not identical way.
To elucidate the mechanisms of antiviral action of both substances at the molecular level, a panel of experiments was performed using drug-resistant E9B variants. For poliovirus, it is known that mutations crucial for resistance against or dependence on picornavirus replication inhibitors map to the coding region of 2C (Pincus et al., 1986 ; Pincus & Wimmer, 1986
). In addition, previous work in our laboratory revealed that the only determinant for HBB dependence of E9B is located within the 2C gene (Hadaschik et al., 1999
). Consequently, we focused our interest on this region of the viral genome. Indeed, two mutations were detected in the 2C coding region of each resistant variant and two of them are sufficient to induce a phenotype identical to the original resistant isolates. For guanidine-resistant or -dependent poliovirus, it has been revealed that most of the responsible amino acid substitutions are located at position 179 or 187, respectively (Baltera & Tershak, 1989
; Tolskaya et al., 1994
). However, in the case of E9, the crucial amino acid exchanges conferring resistance are located at different sites, i.e. position 133 (guanidine) and 227 (HBB), and may suggest different ways of drug inhibition (Table 1
).
The exchange C4782U leads to an HBB-dependent phenotype (Fig. 4). It is noteworthy that the identical exchange has been determined in an independent project with the aim of isolating an HBB-dependent E9B variant (Hadaschik et al., 1999
). Whether this constitutes mere coincidence an unlikely possibility or the only molecular mechanism for HBB dependence can only be elucidated by investigation of a larger number of dependent E9B isolates.
Three of the four mutations characterized in this project are located in the direct vicinity of two of three predicted NTP binding and splitting domains of 2C (Table 1). The NTPase activity of these domains was proven in previous work (Klein et al., 1999
). However, 2C fusion proteins of both E9B wild-type and resistant mutants did not exhibit altered NTPase activity in the absence or presence of physiologically active inhibitor concentrations (Fig. 6
). The weak inhibitory effect of 20 mM guanidine is probably due to the initiation of protein denaturation. Therefore, the NTPase activity of E9 2C does not appear to be a target for HBB or guanidine antiviral activity on E9. Recently, Pfister & Wimmer (1999)
demonstrated that the NTPase function of poliovirus wild-type fusion protein GST2C is inhibited in the presence of low guanidine levels, whereas the respective activity of a protein derived from a resistant mutant was unimpaired. These results may be a further hint that guanidine inhibits echoviruses and polioviruses in a different way.
Preliminary experiments allow the assumption that echovirus 2C binds ssRNA unspecifically, and again this 2C function does not seem to be influenced by HBB or guanidine (data not shown). Taken together, since neither the NTPase nor the RNA binding capacity of 2C could be affected by the inhibitors in vitro, our results support the conjecture that there may be an additional function of 2C acted upon by the drugs. A helicase activity as well as involvement in packaging have been discussed for poliovirus (Gorbalenya et al., 1990 ; Kadaré & Haenni, 1997
; Li & Baltimore, 1990
; Vance et al., 1997
). Because inhibition of RNA synthesis of E9 was proven at least for HBB (Fig. 5a
, b
), it is more likely that the helicase function is affected by the antiviral drugs, if such an activity is actually true for protein 2C.
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Acknowledgments |
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Footnotes |
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References |
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Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidmann, J. G., Smith, J. A. & Struhl, K. (1994). Current Protocols in Molecular Biology. New York: John Wiley.
Baltera, J. F.Jr & Tershak, D. R. (1989). Guanidine-resistant mutants of poliovirus have distinct mutations in polypeptide 2C. Journal of Virology 63, 4441-4444.[Medline]
Bienz, K., Egger, D., Troxler, M. & Pasamontes, L. (1990). Structural organization of poliovirus RNA replication is mediated by viral proteins of the P2 genomic region. Journal of Virology 64, 1156-1163.[Medline]
Caliguiri, L. A. & Tamm, I. (1973). Guanidine and 2-(-hydroxybenzyl)-benzimidazole (HBB): selective inhibitors of picornavirus multiplication. In Selective Inhibitors of Viral Function, pp. 257-293. Edited by W. A. Carter. Florida: CRC Press.
Cuconati, A., Xiang, W., Lahser, F., Pfister, T. & Wimmer, E. (1998). A protein linkage map of the P2-nonstructural proteins of poliovirus. Journal of Virology 72, 1297-1307.
Echeverri, A. C. & Dasgupta, A. (1995). Amino terminal regions of poliovirus 2C protein mediate membrane binding. Virology 208, 540-553.[Medline]
Eggers, H. J. (1977). Selective inhibition of uncoating of echovirus 12 by rhodanine. Virology 78, 241-252.[Medline]
Eggers, H. J. (1982). Benzimidazoles. Selective inhibitors of picornavirus replication in cell culture and in the organism. In Chemotherapy of Viral Infections, pp. 377-417. Edited by P. E. Came & L. A. Caliguiri. Berlin, Heidelberg, New York: Springer Verlag.
Eggers, H. J. & Sabin, A. B. (1959). Factors determining pathogenicity of variants of ECHO 9 virus for newborn mice. Journal of Experimental Medicine 110, 951-967.
Eggers, H. J. & Tamm, I. (1961). Spectrum and characteristics of the virus inhibitory action of 2-(-hydroxybenzyl)-benzimidazole. Journal of Experimental Medicine 113, 657-682.
Eggers, H. J. & Tamm, I. (1963a). Inhibition of enterovirus ribonucleic acid synthesis by 2-(-hydroxybenzyl)-benzimidazole. Nature 197, 1327-1328.
Eggers, H. J. & Tamm, I. (1963b). Synergistic effect of 2-(-hydroxybenzyl)-benzimidazole and guanidine on picornavirus reproduction. Nature 199, 513-514.
Gorbalenya, A. E., Koonin, E. V. & Wolf, Y. I. (1990). A new superfamily of putative NTP-binding domains encoded by genomes of small DNA and RNA viruses. FEBS Letters 262, 145-148.[Medline]
Hadaschik, D., Klein, M., Zimmermann, H., Eggers, H. J. & Nelsen-Salz, B. (1999). Dependence of echovirus 9 on the enteroviral replication inhibitor 2-(-hydroxybenzyl)-benzimidazole (HBB) maps to the nonstructural protein 2C. Journal of Virology 73, 10536-10539.
Kadaré, G. & Haenni, A. L. (1997). Virus-encoded RNA helicases. Journal of Virology 71, 2583-2590.
Klein, M., Eggers, H. J. & Nelsen-Salz, B. (1999). Echovirus 9 strain Barty nonstructural protein 2C has NTPase activity. Virus Research 65, 155-160.[Medline]
Li, J. P. & Baltimore, D. (1990). An intragenic revertant of a poliovirus 2C mutant has an uncoating defect. Journal of Virology 64, 1102-1107.[Medline]
Mirzayan, C. & Wimmer, E. (1994). Biochemical studies on poliovirus polypeptide 2C: evidence for ATPase activity. Virology 199, 176-187.[Medline]
Pfister, T. & Wimmer, E. (1999). Characterization of the nucleoside triphosphatase activity of poliovirus protein 2C reveals a mechanism by which guanidine inhibits poliovirus replication. Journal of Biological Chemistry 274, 6992-7001.
Pincus, S. E. & Wimmer, E. (1986). Production of guanidine-resistant and -dependent poliovirus mutants from cloned cDNA: mutations in polypeptide 2C are directly responsible for altered guanidine sensitivity. Journal of Virology 60, 793-796.[Medline]
Pincus, S. E., Diamond, D. C., Emini, E. A. & Wimmer, E. (1986). Guanidine-selected mutants of poliovirus: mapping of point mutations to polypeptide 2C. Journal of Virology 57, 638-646.[Medline]
Rodriguez, P. L. & Carrasco, L. (1993). Poliovirus protein 2C has ATPase and GTPase activities. Journal of Biological Chemistry 268, 8105-8110.
Rodriguez, P. L. & Carrasco, L. (1995). Poliovirus protein 2C contains two regions involved in RNA binding activity. Journal of Biological Chemistry 270, 10105-10112.
Rueckert, R. (1996). Picornaviridae: the viruses and their replication. In Virology, pp. 609-654. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. New York: Raven Press.
Tolskaya, E. A., Romanova, L. I., Kolesnikova, M. S., Gmyl, A. P., Gorbalenya, A. P. & Agol, V. I. (1994). Genetic studies on the poliovirus 2C protein, an NTPase. A plausible mechanism of guanidine effect on the 2C function and evidence for the importance of 2C oligomerisation. Journal of Molecular Biology 236, 1310-1323.[Medline]
Vance, L. M., Moscufo, N., Chow, M. & Heinz, B. A. (1997). Poliovirus 2C region functions during encapsidation of viral RNA. Journal of Virology 71, 8759-8765.[Abstract]
Wimmer, E., Hellen, C. U. T. & Cao, X. (1993). Genetics of poliovirus. Annual Reviews in Genetics 27, 353-436.
Zimmermann, H., Eggers, H. J. & Nelsen-Salz, B. (1996). Molecular cloning and sequence determination of the complete genome of the virulent echovirus 9 strain Barty. Virus Genes 12, 149-154.[Medline]
Received 26 October 1999;
accepted 15 December 1999.