Hepatitis: Deval J

Deval J. · Roche Palo Alto LLC, Palo Alto, California 94304, USA. · Drugs. · Pubmed #19228073 No free full text.

Abstract: Over the past 20 years, nucleoside analogues have constituted an arsenal of choice in the fight against HIV, hepatitis B and C viruses, and herpesviruses. Classical antiviral nucleosides such as zidovudine act as obligate chain terminators. Once incorporated as monophosphates into the viral nucleic acid, they immediately block the progression of the polymerase as a result of their lack of a reactive 3'-hydroxyl (3'-OH) group. This review explores beyond the paradigm of obligate chain termination, from a structural and a mechanistic perspective, the strategy of inhibiting viral polymerases (RNA- and DNA-dependant) with nucleoside analogues containing a 3'-OH group. Depending on their mechanism of action, these molecules typically fall into the following three categories: (i) delayed chain terminators; (ii) pseudo-obligate chain terminators; or (iii) mutagenic nucleosides. Delayed chain terminators (i.e. penciclovir, cidofovir and entecavir) block the polymerase at an internal position within the viral nucleic acid, whereas R7128 and the 4'C substituted nucleosides do not permit subsequent incorporation events. Ribavirin, 5-hydroxydeoxycytidine and KP1461 are not chain terminators. Instead, they inhibit viral replication after mispairing with the template base, resulting in random mutations that are often lethal. Finally, brivudine, clevudine and other L-nucleosides have unique or yet to be defined mechanisms of inhibition.

Deval J, Powdrill MH, D'Abramo CM, Cellai L, Götte M. · Department of Microbiology & Immunology, McGill University, Duff Medical Building, Montreal, Quebec, Canada. · Antimicrob Agents Chemother. · Pubmed #17502402 links to  free full text

Abstract: 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 (PP(i)), 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.

Deval J, D'Abramo CM, Zhao Z, McCormick S, Coutsinos D, Hess S, Kvaratskhelia M, Götte M. · Department of Microbiology, McGill University, Montreal, Quebec H3A 2B4, Canada. · J Biol Chem. · Pubmed #17449464 links to  free full text

Abstract: The nucleic acid binding channel of the hepatitis C virus RNA polymerase remains to be defined. Here we employed complementary footprinting techniques and show that the enzyme binds to a newly synthesized duplex of approximately seven to eight base pairs. Comparative analysis of surface topologies of free enzyme versus the nucleoprotein complex revealed certain lysines and arginines that are protected from chemical modification upon RNA binding. The protection pattern helps to define the trajectory of the nucleic acid substrate. Lys(81), Lys(98), Lys(100), Lys(106), Arg(158), Arg(386), and Arg(394) probably interact with the bound RNA. The selective protection of amino acids of the arginine-rich region in helix T points to RNA-induced conformational rearrangements. Together, these findings suggest that RNA-protein interaction through the entire substrate binding channel can modulate intradomain contacts at the C terminus.

D'Abramo CM, Deval J, Cameron CE, Cellai L, Götte M. · Department of Microbiology & Immunology, and Department of Medicine, McGill University, Montréal, Québec H3A 2B4, Canada. · J Biol Chem. · Pubmed #16831816 links to  free full text

Abstract: The RNA-dependent RNA polymerase of the hepatitis C virus and the bovine viral diarrhea virus(BVDV)is able to initiate RNA synthesis denovo in the absence of a primer. Previous crystallographic data have pointed to the existence of a GTP-specific binding site (G-site) that is located in the vicinity of the active site of the BVDV enzyme. Here we have studied the functional role of the G-site and present evidence to show that specific GTP binding affects the positioning of the template during de novo initiation. Following the formation of the first phosphodiester bond, the polymerase translocates relative to the newly synthesized dinucleotide, which brings the 5'-end of the primer into the G-site, releasing the previously bound GTP. At this stage, the 3'-end of the template can remain opposite to the 5'-end of the primer or be repositioned to its original location before RNA synthesis proceeds. We show that the template can freely move between the two locations, and both complexes can isomerize to equilibrium. These data suggest that the bound GTP can stabilize the interaction between the 3'-end of the template and the priming nucleotide, preventing the template to overshoot and extend beyond the active site during de novo initiation. The hepatitis C virus enzyme utilizes a dinucleotide primer exclusively from the blunt end; the existence of a functionally equivalent G-site is therefore uncertain. For the BVDV polymerase we showed that de novo initiation is severely compromised by the T320A mutant that likely affects hydrogen bonding between the G-site and the guanine base. Dinucleotide-primed reactions are not influenced by this mutation, which supports the notion that the G-site is located in close proximity but not at the active site of the enzyme.