First Crystal Structure of a Non-Structural Hepatitis E Viral Protein Identifies a Putative Novel Zinc-Binding Protein

Abstract

Hepatitis E virus (HEV) is a 7.2 kb positive-sense, single-stranded RNA virus containing three partially overlapping reading frames, ORF 1-3. All non-structural proteins required for viral replication are encoded by ORF1 and are transcribed as a single transcript. Computational analysis of the complete ORF1 polyprotein identified a previously uncharacterized region of predicted secondary structure bordered by two disordered regions coinciding partially with a region predicted as a putative cysteine protease. Following successful cloning, expression and purification of this region, the crystal structure of the identified protein was determined and identified to have considerable structural homology to a fatty acid binding domain. Further analysis of the structure revealed a metal binding site, shown unambiguously to specifically bind zinc via a non-classical, potentially catalytic zinc-binding motif. We present analysis for the first time of this identified non-structural protein, expanding the knowledge and understanding of the complex mechanisms of HEV biology. ImportanceHepatitis E virus (HEV) is an emerging virus found predominately in developing countries causing an estimated 20 million infections, which result in approximately 57,000 deaths a year. Although it is known that the non-structural proteins of the HEV ORF1 are expressed as a single transcript, there is debate as to whether ORF1 functions as a single polyprotein or if it is processed into separate domains via a viral or endogenous cellular protease. In the following paper, we present the first structural and biophysical characterization of a HEV non-structural protein using a construct that has partially overlapping boundaries with the predicted putative cysteine protease. Based on the structural homology of the HEV protein with known structures, along with the presence of a catalytic zinc-binding motif, it is possible that the identified protein corresponds to the HEV protease, which could require activation or repression through the binding of a fatty acid. This represents a significant step forward in the characterization and the understanding of the molecular mechanisms of the HEV genome.