Roche Scientists Uncover Unexpected Enzyme Activity in HCV RNA Polymerase
“This new activity that we uncovered was quite unexpected,” said Klaus Klumpp, Ph.D., Vice President of Virology Discovery, at Roche. “We were very surprised by the finding that a viral RNA polymerase could so efficiently repair a blocked RNA molecule. The reverse transcriptase of HIV has shown the ability to respond to drug pressure by enhancing a similar mechanism and become resistant to nucleoside analogs such as AZT. As a result, we now have to look at viral RNA polymerases and investigate if such a pathway to nucleoside drug resistance could also be possible.”
The scientists developed a novel method to generate a stable elongation complex of HCV polymerase, which was published earlier in the Journal of Biological Chemistry2 , and were able to then characterize the enzyme activities of HCV polymerase. They found that HCV polymerase can remove nucleotides from its produced RNA strand using nucleotide triphosphates (NTP) such as ATP, as an excision mediator. Also, HCV polymerase can remove incorporated nucleoside analogs that block RNA replication, and thus rescue its RNA replication. ATP, which is abundant in cells, may mediate this rescue reaction in HCV replicating cells treated with nucleoside analogs. Another interesting finding from the research is that the byproducts of the reaction are Np4N molecules, which can modulate cell function.
“We were looking at the ability of HCV polymerase to make errors by incorporating the wrong nucleoside into the replicating RNA, but found that in many cases the enzyme started to remove nucleosides from the RNA,” said Zhinan Jin, Ph.D., who performed this work as a Postdoctoral Fellow at Roche. “I was particularly surprised by the efficiency of this excision reaction in the elongation complex, which was substantially higher than that reported previously for the HIV reverse transcriptase. Although it indicates the potential for HCV to develop resistance to certain nucleoside analog drugs, we believe this new knowledge will help scientists to develop drugs that circumvent resistance as well as new strategies to treat patients with better combination therapy.”
Nucleoside analogues are key components for the treatment of a large number of viral diseases, and may also become a cornerstone of future treatment of HCV infection. While nucleoside analogs have shown a high barrier to resistance in clinical trials to date, a few cases have been reported with viral load rebounds or relapse in the absence of known resistance mutations – the reason for treatment failure in these cases remained unexplained. These new findings raise the possibility of an RNA repair pathway to nucleoside resistance for consideration, as previously observed in HIV.
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1. Jin, Z., Leveque, V., Ma, H., Johnson, K. A. & Klumpp, K. NTP-mediated nucleotide excision activity of hepatitis C virus RNA-dependent RNA polymerase. Proc. Natl. Acad. Sci. U.S.A online Early Edition on January 10, 2013
2. Jin, Z., Leveque, V., Ma, H., Johnson, K. A. & Klumpp, K. Assembly, Purification, and Pre- Steady-State Kinetic Analysis of Active RNA-Dependent RNA Polymerase Elongation Complex. J. Biol. Chem. 287, 10674–10683 (2012)