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RNA-dependent RNA Polymerase Assay for Hepatitis E Virus

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Original research article

A brief version of this protocol appeared in:
PLOS Pathogens
Apr 2016

Abstract

RNA-dependent RNA polymerase (RdRp) is essential for the replication of viral RNA for RNA viruses. It synthesizes the complementary strand of viral genomic RNA, which is used subsequently as a template to generate more copies of viral genome. This assay measures activity of the hepatitis E virus (HEV) RdRp. In contrast to protocols available to assay the RdRp activity of many other viruses, this assay utilizes DIG-11-UTP as a nonradioactive alternative to 32P-UTP, thereby increasing the convenience of performing the assay.

Keywords: Hepatitis E virus, RNA virus, RdRp, RdRp assay, Viral replication, Nonradioactive RdRp assay

Background

No assay was available to measure the activity of HEV RdRp. RdRp activity has been measured in few other viruses such as hepatitis C virus using a radiolabeled nucleotide (Behrens et al., 1996). We have adapted the protocol described by Behrens et al. (1996) and modified it to establish a non-radioactive assay protocol, which is dependent on incorporation of DIG-11-UTP into the antisense RNA strand as a measure of the activity of HEV RdRp. This assay utilizes an in vitro synthesized viral RNA fragment as a template to measure the activity of HEV RdRp protein purified from human hepatoma cells using a chemiluminescence-based strategy.

How to cite:  Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Nair, V. P., Anang, S., Srivastava, A. and Surjit, M. (2017). RNA-dependent RNA Polymerase Assay for Hepatitis E Virus. Bio-protocol 7(7): e2199. DOI: 10.21769/BioProtoc.2199.
  2. Nair, V. P., Anang, S., Subramani, C., Madhvi, A., Bakshi, K., Srivastava, A., Shalimar, Nayak, B., Ct, R. K. and Surjit, M. (2016). Endoplasmic reticulum stress induced synthesis of a novel viral factor mediates efficient replication of genotype-1 hepatitis E virus. PLoS Pathog 12(4): e1005521.
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