Structural features stabilized by divalent cation coordination within hepatitis E virus ORF1 are critical for viral replication

Abstract

Hepatitis E virus (HEV) is an RNA virus responsible for over 20 million infections annually. HEV’s open reading frame (ORF)1 polyprotein is essential for genome replication, though it is unknown how the different subdomains function within a structural context. Our data show that ORF1 operates as a multifunctional protein, which is not subject to proteolytic processing. Supporting this model, scanning mutagenesis performed on the putative papain-like cysteine protease (pPCP) domain revealed six cysteines essential for viral replication. Our data are consistent with their role in divalent metal ion coordination, which governs local and interdomain interactions that are critical for the overall structure of ORF1; furthermore, the ‘pPCP’ domain can only rescue viral genome replication in trans when expressed in the context of the full-length ORF1 protein but not as an individual subdomain. Taken together, our work provides a comprehensive model of the structure and function of HEV ORF1.

Thrombin cleavage of the hepatitis E virus polyprotein at multiple conserved locations is required for genome replication

“…Here, we found that the pORF1 polyprotein was unable to be processed auto-catalytically using a well-established in vitro transcription and translation system, in agreement with studies suggesting pORF1 cannot undergo proteolysis [ 17 , 46 ]. We, and others, have used this system to study the processing of many positive-sense RNA viruses.…”

The Protease Domain in HEV pORF1 Mediates the Replicase’s Localization to Multivesicular Bodies and Its Exosomal Release

“…Generally, our approach of separating domains with the support of a structural prediction of pORF1 resulted in a pattern largely fitting to what is described in literature. 24 , 38 , 39 The final separation took hinge regions separating well-defined regions into account. Overall, the HVR presented itself as the only domain lacking a proper secondary structure, which can be explained by repeated polyproline stretches flanking the visible, α-helical structure.…”

Predicted Membrane-Associated Domains in Proteins Encoded by Novel Monopartite Plant RNA Viruses Related to Members of the Family Benyviridae

“…Our analysis revealed that a number Reclovirids encode non-replicative proteins with other types of putative zinc-finger motifs. In particular, the ORF2 protein of Atriplex prostrata VLRA (Supplementary Table S1) contains the hexa-cysteine motif C(X6)C(X)C(XX)CXC(X12)C, which resembles unconventional hexa-cysteine motifs like those found in proteins of Hepatitis virus E and viruses of the genus Pestivirus [16,17], and in the small protein encoded by the ORF preceding TCMB in Colobanthus quitensis VLRA [7].…”