4
64
MURAKAMI ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
are structurally different (cytidine versus uridine), it could be
suggested that PSI-6130 provides a novel mechanism for de-
livering a combination therapy. To date, it has been difficult to
select for replicons with reduced sensitivity to PSI-6130 in
passaging experiments. Therefore, it is enticing to speculate
that in the clinic, the provision of two structurally different,
active metabolites may increase the genetic barrier which the
virus must overcome to achieve resistance, thus preventing or
slowing the emergence of resistant virus. We continue to study
the mechanism of action of PSI-6130 to try to elucidate the
role that this combination of metabolites plays in the activity of
the compound and in the emergence of resistance.
12. Klumpp, K., V. Leveque, S. Le Pogam, H. Ma, W. R. Jiang, H. Kang, C.
Granycome, M. Singer, C. Laxton, J. Q. Hang, K. Sarma, D. B. Smith, D.
Heindl, C. J. Hobbs, J. H. Merrett, J. Symons, N. Cammack, J. A. Martin, R.
Devos, and I. Najera. 2006. The novel nucleoside analog R1479 (4Ј-azido-
cytidine) is a potent inhibitor of NS5B-dependent RNA synthesis and hep-
atitis C virus replication in cell culture. J. Biol. Chem. 281:3793–3799.
1
3. Liou, J. Y., P. Krishnan, C. C. Hsieh, G. E. Dutschman, and Y. C. Cheng.
2003. Assessment of the effect of phosphorylated metabolites of anti-human
immunodeficiency virus and anti-hepatitis B virus pyrimidine analogs on the
behavior of human deoxycytidylate deaminase. Mol. Pharmacol. 63:105–110.
4. Lohmann, V., F. Korner, U. Herian, and R. Bartenschlager. 1997. Biochem-
ical properties of hepatitis C virus NS5B RNA-dependent RNA polymerase
and identification of amino acid sequence motifs essential for enzymatic
activity. J. Virol. 71:8416–8428.
1
15. Lohmann, V., A. Roos, F. Korner, J. O. Koch, and R. Bartenschlager. 1998.
Biochemical and kinetic analyses of NS5B RNA-dependent RNA polymer-
ase of the hepatitis C virus. Virology 249:108–118.
1
6. Ma, H., W. R. Jiang, N. Robledo, V. Leveque, S. Ali, T. Lara-Jaime, M.
Masjedizadeh, D. B. Smith, N. Cammack, K. Klumpp, and J. Symons. 2007.
Characterization of the metabolic activation of hepatitis C virus nucleoside
inhibitor beta-D-2Ј-deoxy-2Ј-fluoro-2Ј-C-methylcytidine (PSI-6130) and
identification of a novel active 5Ј-triphosphate species. J. Biol. Chem. 282:
ACKNOWLEDGMENT
We thank Veronique Zennou for helpful discussions.
REFERENCES
2
9812–29820.
1
2
. Alaoui-Lsmaili, M. H., M. Hamel, L. L’Heureux, O. Nicolas, D. Bilimoria, P.
Labonte, S. Mounir, and R. F. Rando. 2000. The hepatitis C virus NS5B
RNA-dependent RNA polymerase activity and susceptibility to inhibitors is
modulated by metal cations. J. Hum. Virol. 3:306–316.
. Alter, M. J., D. Kruszon-Moran, O. V. Nainan, G. M. McQuillan, F. Gao,
L. A. Moyer, R. A. Kaslow, and H. S. Margolis. 1999. The prevalence of
hepatitis C virus infection in the United States, 1988 through 1994. N. Engl.
J. Med. 341:556–562.
17. Maley, G. F., A. P. Lobo, and F. Maley. 1993. Properties of an affinity-
column-purified human deoxycytidylate deaminase. Biochim. Biophys. Acta
1162:161–170.
18. Migliaccio, G., J. E. Tomassini, S. S. Carroll, L. Tomei, S. Altamura, B.
Bhat, L. Bartholomew, M. R. Bosserman, A. Ceccacci, L. F. Colwell, R.
Cortese, R. De Francesco, A. B. Eldrup, K. L. Getty, X. S. Hou, R. L.
LaFemina, S. W. Ludmerer, M. MacCoss, D. R. McMasters, M. W. Stahlhut,
D. B. Olsen, D. J. Hazuda, and O. A. Flores. 2003. Characterization of
resistance to non-obligate chain-terminating ribonucleoside analogs that in-
hibit hepatitis C virus replication in vitro. J. Biol. Chem. 278:49164–49170.
19. Murakami, E., H. Bao, M. Ramesh, T. R. McBrayer, T. Whitaker, H. M.
Micolochick Steuer, R. F. Schinazi, L. J. Stuyver, A. Obikhod, M. J. Otto,
and P. A. Furman. 2007. Mechanism of activation of -D-2Ј-deoxy-2Ј-fluoro-
2Ј-C-methylcytidine and inhibition of hepatitis C virus NS5B RNA polymer-
ase. Antimicrob. Agents Chemother. 51:503–509.
20. Poynard, T., V. Ratziu, Y. Benhamou, P. Opolon, P. Cacoub, and P. Bedossa.
2000. Natural history of HCV infection. Baillieres Best Pract. Res. Clin.
Gastroenterol. 14:211–228.
21. Poynard, T., M. F. Yuen, V. Ratziu, and C. L. Lai. 2003. Viral hepatitis C.
Lancet 362:2095–2100.
22. Stuyver, L. J., T. R. McBrayer, P. M. Tharnish, J. Clark, L. Hollecker, S.
Lostia, T. Nachman, J. Grier, M. A. Bennett, M. Y. Xie, R. F. Schinazi, J. D.
Morrey, J. L. Julander, P. A. Furman, and M. J. Otto. 2006. Inhibition of
hepatitis C replicon RNA synthesis by beta-D-2Ј-deoxy-2Ј-fluoro-2Ј-C-meth-
ylcytidine: a specific inhibitor of hepatitis C virus replication. Antivir. Chem.
Chemother. 17:79–87.
23. Stuyver, L. J., T. Whitaker, T. R. McBrayer, B. I. Hernandez-Santiago, S.
Lostia, P. M. Tharnish, M. Ramesh, C. K. Chu, R. Jordan, J. Shi, S.
Rachakonda, K. A. Watanabe, M. J. Otto, and R. F. Schinazi. 2003. Ribo-
nucleoside analogue that blocks replication of bovine viral diarrhea and
hepatitis C viruses in culture. Antimicrob. Agents Chemother. 47:244–254.
24. Tong, M. J., N. S. el-Farra, A. R. Reikes, and R. L. Co. 1995. Clinical
outcomes after transfusion-associated hepatitis C. N. Engl. J. Med. 332:
1463–1466.
3
4
. Blight, K. J., A. A. Kolykhalov, and C. M. Rice. 2000. Efficient initiation of
HCV RNA replication in cell culture. Science 290:1972–1974.
. Carroll, S. S., J. E. Tomassini, M. Bosserman, K. Getty, M. W. Stahlhut,
A. B. Eldrup, B. Bhat, D. Hall, A. L. Simcoe, R. LaFemina, C. A. Rutkowski,
B. Wolanski, Z. Yang, G. Migliaccio, R. De Francesco, L. C. Kuo, M.
MacCoss, and D. B. Olsen. 2003. Inhibition of hepatitis C virus RNA rep-
lication by 2Ј-modified nucleoside analogs. J. Biol. Chem. 278:11979–11984.
. Clark, J. L., J. C. Mason, L. Hollecker, L. J. Stuyver, P. M. Tharnish, T. R.
McBrayer, M. J. Otto, P. A. Furman, R. F. Schinazi, and K. A. Watanabe.
5
2
006. Synthesis and antiviral activity of 2Ј-deoxy-2Ј-fluoro-2Ј-C-methyl pu-
rine nucleosides as inhibitors of hepatitis C virus RNA replication. Bioorg.
Med. Chem. Lett. 16:1712–1715.
6
. Collier, J., and R. Chapman. 2001. Combination therapy with interferon-
alpha and ribavirin for hepatitis C: practical treatment issues. BioDrugs
1
5:225–238.
7
. Darby, S. C., D. W. Ewart, P. L. Giangrande, R. J. Spooner, C. R. Rizza,
G. M. Dusheiko, C. A. Lee, C. A. Ludlam, F. E. Preston, et al. 1997. Mortality
from liver cancer and liver disease in haemophilic men and boys in UK given
blood products contaminated with hepatitis C. Lancet 350:1425–1431.
. Di Bisceglie, A. M., J. McHutchison, and C. M. Rice. 2002. New therapeutic
strategies for hepatitis C. Hepatology 35:224–231.
8
9
. Ferrari, E., J. Wright-Minogue, J. W. Fang, B. M. Baroudy, J. Y. Lau, and
Z. Hong. 1999. Characterization of soluble hepatitis C virus RNA-dependent
RNA polymerase expressed in Escherichia coli. J. Virol. 73:1649–1654.
1
1
0. Gribaudo, G., L. Riera, P. Caposio, F. Maley, and S. Landolfo. 2003. Human
cytomegalovirus requires cellular deoxycytidylate deaminase for replication
in quiescent cells. J. Gen. Virol. 84:1437–1441.
25. Yamashita, T., S. Kaneko, Y. Shirota, W. Qin, T. Nomura, K. Kobayashi,
and S. Murakami. 1998. RNA-dependent RNA polymerase activity of the
soluble recombinant hepatitis C virus NS5B protein truncated at the C-
terminal region. J. Biol. Chem. 273:15479–15486.
1. Ishii, K., Y. Tanaka, C. C. Yap, H. Aizaki, Y. Matsuura, and T. Miyamura.
1
999. Expression of hepatitis C virus NS5B protein: characterization of its
RNA polymerase activity and RNA binding. Hepatology 29:1227–1235.