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.
Materials and Reagents
Equipment
Procedure
Data analysis
While setting up the RdRp assay, appropriate controls should be included in order to interpret the data and rule out non-specific signal. Must have negative controls such as omission of nucleotides from the reaction mixture and inclusion of RNase A in the reaction mixture. No bands should be obtained in both the cases. A titration experiment using increasing quantities of RdRp protein in the assay should also be performed to rule out non-specific signals. Assay using an unrelated protein instead of RdRp will also rule out the possible non-specific signal. A sample assay with all controls has been illustrated in Figure 1. The effect of other factors or compounds on RdRp activity may be evaluated by adding them to the reaction mixture. In our experience, template itself does not give any false signal, as the assay is dependent on detection of DIG, which is incorporated into the RNA only as DIG-UTP. Free DIG-UTP is removed from the reaction during migration of the sample in agarose gel. Moreover, the labeled RNA migrates at ~800 base pairs size whereas template RNA size is 380 base pairs. Monitoring the size of the band also allows one to be confident of the output of the assay. Figure 1. Assay of HEV RNA-dependent RNA polymerase activity. NTPs: nucleotide triphosphates.
Recipes
Acknowledgments
The work was funded by Ramalingaswamy fellowship and THSTI core grant to MS. VN is supported by a grant from the Department of Science and Technology, Government of India. The protocol has been adapted from Behrens et al. (1996).
Competing interests
The authors declare no conflict of interest or competing interest.
References
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