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About: Abstract The hepatitis E virus (HEV) ORF1 gene encodes the non-structural polyprotein wherein the ‘X-domain’ still remains poorly defined. Cellular X-domain associated macrodomain protein/ADP-ribose-1″-monophosphatase (Appr-1″-pase) activities are also reported in coronaviruses (CoV), including identification of its homologs in alpha and rubella viruses. The present study investigated the role(s) of X-domain residues in HEV replication cycle. In silico analysis showed a high degree of evolutionary conservation of X-domain (a.a. 785–942) a.a. positions wherein the N-terminus residues ‘Asn806, Asn809, His812, Gly815, Gly816, and Gly817’ formed a potential catalytic-site homolog of CoVAppr-1″-pase. To experimentally test this prediction, X-domain ‘active-site’ residues were subjected to mutational analysis using the HEV-SAR55 replicon (pSK-GFP). FACS analysis of mutant RNA transfected S10-3 cells showed that Gly816Ala and Gly817Ala constructs completely abrogated HEV replication, similar to their Gly816Val and Gly817Val counterparts. However, ‘Gly815Ala’ mutant replicated very poorly in contrast to ‘Gly815Val’ that completely abolished GFP synthesis. Furthermore, while ‘Asn806Ala’ mutant retained RNA replication, the ‘Asn809Ala’ and His812Leu mutants showed non-viability. Notably, in a sequential-nucleotide mutation analysis, the dispensability of X-domain in HEV replication at transcriptional level has already been demonstrated (Parvez, 2013b). Taken together, the present data strongly argue for an essential role of X-domain residues (Asn809, His812, Gly816 and Gly817) at post-translational level, indicating its involvement in viral replication. In conclusion, the speculated regulatory role of ORF1 X-domain in HEV replication cycle critically depends on the ‘Asn, Asn, His, Gly, Gly, Gly’ segment/secondary structure. Nevertheless, further biochemical or biophysical characterizations of HEV X-domain associated Appr-1″-pase activity would only confirm its biological significance in virus or host-pathogenesis.

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