Shakuntala Mahilkar
National Institute of Virology, India
Title: Binding of hepatitis E virus RNA dependent RNA polymerase to the cis-acting regulatory elements in the HEV genome
Biography
Biography: Shakuntala Mahilkar
Abstract
Hepatitis E, caused by Hepatitis E virus (HEV), is an important public-health concern in developing as well as developed countries.There is a need to understand virus biology because new aspects of virus infection are emerging such as zoonotic transmission and chronic cases in organ transplant and immune compromised patients also, it shows unexplained high mortality rate in pregnant women. Hepatitis E virus replication is still not completely understood due to lack of efficient cell culture system. The virus genome is positive sense ssRNA of about 7kb having a 5’ cap and a 3’poly A tail. The expression of its three open reading frames is achieved by producing genomic and subgenomic RNAs. The viral genome has 5’ and 3’ non-coding regions (NCR) with conserved stem loop structures known to be important during viral replication. Interaction between RNA dependent RNA polymerase (RdRp) and the viral cis-acting sequences is likely to be an important step in viral RNA replication. We analyzed binding affinity of viral RdRp to these cis-acting elements using Electrophoretic Mobility Shift Assay (EMSA). Purified recombinant RdRp protein, expressed in bacterial system, was incubated with putative regulatory elements in the HEV genome. While, binding affinity of the enzyme with these elements was determined by carrying out competition binding assays. The same assay was used for mapping of subgenomic promoter (sgP) region to find minimal binding region for efficient RdRp binding. The enzyme exhibited high binding affinity with the proposed sgP, between ORF1 and ORF2 junction region and 3’UTR with a poly A tail. While, affinity for the 5’NCR was comparatively less than the sgP and 3’NCR. A competition assay between the 3’NCR and sgP showed a super-shift suggesting RdRp to have either two separate binding motifs/ or co-operative binding with these two cis-acting elements. These studies give us a molecular insight for virus transcription and replication and provide targets for antiviral development.