黑料网

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.

Biography

Shakuntala Mahilkar is a senior research fellow at National Institute of Virology (NIV), University of Pune, Maharastra, India. Currently she is writing her Ph.D. thesis
entitled “Identification and characterization of regulatory elements on Hepatitis E virus Genome” under the supervision of Dr Kavita S. Lole. My Ph.D and the work
is to explore the Hepatitis E Virus (HEV) genome for the regulatory elements involved in replication and transcription. HEV is a single stranded positive sense RNA
virus which utilizes RNA Dependent RNA Polymerase (RdRp) for its replication and transcription. Identification of binding region for RdRp will give us insight of how
HEV maintain its replication as well as transcription and translation within a small genome, and also an idea about the virus life cycle. This study will further help in
designing the antiviral therapies for HEV infection. Earlier in the year 2006-2007 she worked as a Junior Research Fellow at School of Biotechnology on the project
entitled “Metabolic engineering of E. coli to remove the bottlenecks in recombinant protein expression” sponsored by Department of Biotechnology (DBT). She
had Qualified All India Combined Entrance Test for admission in M.Sc. Biotechnology programme in 2003 conducted by the JNU, New Delhi, and completed her
Masters in Biotechnology from Himachal Pradesh University, Shimla in the year 2005, during that period she did a project work on the topic “Studies on microbial
tyrosinase and b-tyrosinase.” Also she completed winter training at National Institute of Plant Genome Research (NIPGR), New Delhi, on the topic “Cloning of
WRKY gene from Oryza Sativa.”