First Crystal Structure of a Nonstructural 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, ORF1 to ORF3. All nonstructural 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 nonclassical, potentially catalytic zinc-binding motif. 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. We present analysis for the first time of this identified nonstructural protein, expanding the knowledge and understanding of the complex mechanisms of HEV biology. IMPORTANCE Hepatitis E virus (HEV) is an emerging virus found predominately in developing countries; it causes an estimated 20 million infections, which result in approximately 57,000 deaths a year. Although it is known that the nonstructural proteins of 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. Here we present the first structural and biophysical characterization of an HEV nonstructural protein using a construct that has partially overlapping boundaries with the predicted putative cysteine protease.

Processing and Subcellular Localization of the Hepatitis E Virus Replicase: Identification of Candidate Viral Factories

“…Although several domains such as methyltransferase (Met), helicase (Hel) and RNA-dependent RNA polymerase (RdRp) have been reported to be enzymatically active, the function of the Y and X-domains as well as the highly disordered hypervariable region (HVR) remain elusive ( Wang and Meng, 2021 ; Figure 1C ). In addition, the precise location of the protease region (known as papain-like cysteine protease, PCP) and its enzymatic activity are still a matter of debate ( LeDesma et al, 2019 ; Proudfoot et al, 2019 ). Whether or not the HEV polyprotein gets processed by the PCP or cellular proteases remains a difficult question to address considering the low expression level of the polyprotein in HEV cell culture systems as well as the scarcity of functional specific antibodies ( Debing et al, 2016 ; Lenggenhager et al, 2017 ; Nimgaonkar et al, 2018 ; Kenney and Meng, 2019 ).…”

Hepatitis E virus RNA‐dependent RNA polymerase is involved in RNA replication and infectious particle production

“…A recent crystal structure of the putative papain‐like cysteine protease domain revealed a high structural homology to fatty acid binding domains. [ 19 ] Although an enzymatic activity of this domain has not yet been proven, the structure provides a framework for future functional studies. Amphipathic α‐helix 1628–1644, described here, is only the second structurally solved segment of the ORF1 protein.…”

“…This article is protected by copyright. All rights reserved structural homology to fatty acid binding domains (19). While an enzymatic activity of this domain has not yet been proven, the structure provides a framework for future functional studies.…”

Current Trends of Essential Trace Elements in Patients with Chronic Liver Diseases

“…It was recently elucidated that Zn salts (zinc sulfate and zinc acetate) directly inhibited the activity of viral RNA-dependent RNA polymerase, leading to the inhibition of an HEV replication [ 22 ]. In addition, a nonstructural HEV protein was currently identified as a putative Zn-binding protein [ 39 ].…”