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Antimicrobial Agents and Chemotherapy, August 2007, p. 2920-2928, Vol. 51, No. 8
0066-4804/07/$08.00+0     doi:10.1128/AAC.00186-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Pyrophosphorolytic Excision of Nonobligate Chain Terminators by Hepatitis C Virus NS5B Polymerase{triangledown}

Jérôme Deval,1,{dagger} Megan H. Powdrill,1 Claudia M. D'Abramo,2 Luciano Cellai,3 and Matthias Götte1,2,4*

Departments of Microbiology & Immunology,1 Medicine,2 Biochemistry, McGill University, Montreal, Quebec, Canada,4 Istituto di Cristallografia, Sede di Roma, CNR, Rome, Italy3

Received 7 February 2007/ Returned for modification 16 March 2007/ Accepted 7 May 2007

Nonobligate chain terminators, such as 2'-C-methylated nucleotides, block RNA synthesis by the RNA-dependent RNA polymerase (RdRp) of hepatitis C virus (HCV). Previous studies with related viral polymerases have shown that classical chain terminators lacking the 3'-hydroxyl group can be excised in the presence of pyrophosphate (PPi), which is detrimental to the inhibitory activity of these compounds. Here we demonstrate that the HCV RdRp enzyme is capable of removing both obligate and clinically relevant nonobligate chain terminators. Pyrimidines are more efficiently excised than are purines. The presence of the next complementary templated nucleotide literally blocks the excision of obligate chain terminators through the formation of a dead-end complex (DEC). However, 2'-C-methylated CMP is still cleaved efficiently under these conditions. These findings show that a 2'-methylated primer terminus impedes nucleotide binding. The S282T mutation, associated with resistance to 2'-C-methylated nucleotides, does not affect the excision patterns. Thus, the decreased susceptibility to 2'-C-methylated nucleotides appears to be based solely on improved discrimination between the inhibitor and its natural counterpart. In conclusion, our data suggest that the phosphorolytic excision of nonobligate, pyrimidine-based chain terminators can diminish their potency. The templated nucleotide does not appear to provide protection from excision through DEC formation.

* Corresponding author. Mailing address: Department of Microbiology & Immunology, McGill University, Duff Medical Building, 3775 University Street, Montreal, Quebec, Canada H3A 2B4. Phone: (514) 398-1365. Fax: (514) 398-7052. E-mail: matthias.gotte{at}mcgill.ca

{triangledown} Published ahead of print on 14 May 2007.

{dagger} Present address: Biochemical Pharmacology, Roche, Palo Alto, CA.

Antimicrobial Agents and Chemotherapy, August 2007, p. 2920-2928, Vol. 51, No. 8
0066-4804/07/$08.00+0     doi:10.1128/AAC.00186-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Tchesnokov, E. P., Obikhod, A., Schinazi, R. F., Gotte, M. (2008). Delayed Chain Termination Protects the Anti-hepatitis B Virus Drug Entecavir from Excision by HIV-1 Reverse Transcriptase. J. Biol. Chem. 283: 34218-34228 [Abstract] [Full Text]  


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