Structural features stabilized by divalent cation coordination within hepatitis E virus ORF1 are critical for viral replication
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eLife assessment
The paper aims to provide structural and functional information on the hepatitis E virus replication complex. The study will be of interest to a broad number of people studying at virus replication, since the replication complex are targets for therapeutic interventions.
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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.
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eLife assessment
The paper aims to provide structural and functional information on the hepatitis E virus replication complex. The study will be of interest to a broad number of people studying at virus replication, since the replication complex are targets for therapeutic interventions.
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Joint Public Review:
Hepatitis E virus (HEV) causes over 20 million infections per year. The open reading frame 1 (ORF1) is responsible for genome replication, however very little is known about the structure and functions of several of the components. The author use a diverse a diverse number of techniques (molecular virology, structure prediction using AlphaFold, site directed mutagenesis and biochemistry) to probe ORF1 activity. The work is thorough, well prepared, and discusses the strength and weakness of the structural information. Interestingly, AlphaFold prediction of the papain-like cysteine protease domain did not identify a classic papain-like fold. Lastly, the authors demonstrate the necessity of six conserved cysteines within the putative PCP domain.
The presence and necessity of proteolysis for genome replication or …
Joint Public Review:
Hepatitis E virus (HEV) causes over 20 million infections per year. The open reading frame 1 (ORF1) is responsible for genome replication, however very little is known about the structure and functions of several of the components. The author use a diverse a diverse number of techniques (molecular virology, structure prediction using AlphaFold, site directed mutagenesis and biochemistry) to probe ORF1 activity. The work is thorough, well prepared, and discusses the strength and weakness of the structural information. Interestingly, AlphaFold prediction of the papain-like cysteine protease domain did not identify a classic papain-like fold. Lastly, the authors demonstrate the necessity of six conserved cysteines within the putative PCP domain.
The presence and necessity of proteolysis for genome replication or cleavage of other host factors still remains an uncharacterized problem, which is beyond the scope of this manuscript. My only concern relates to the presence of a zinc ion in ORF1.
The authors use extensive triplet alanine scanning to test for virus replication capacity and in some cases see gains above WT (Figure 3). Do these patterns match natural variation observed in comparisons of HEV sequences un any way?Overall, the study presents an intriguing hypothesis for HEV ORF1 function not involving protease processing as assumed by early bioinformatic analysis. The alternate hypothesis of metal ion coordination is supported by increasingly sophisticated structural modeling tools and related experiments. However, a lack of direct evidence leaves, as the authors note, alternate hypotheses such as disulfide bond coordination or protease functions that occur intramolecularly within ORF1.
The study will likely have an impact on the field, especially if evidence builds in the future directly supporting the mechanism proposed. HEV is an impactful pathogenic virus that is relatively underappreciated. In addition to a major revision in HEV biology, the idea that many proteins initially annotated with canonical functions might instead have different mechanisms is also of high interest beyond the field of virology.
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