Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Sun, 25 Nov 2018 20:30:17 Short Communication Deubiquitination activity associated with hepatitis E virus putative papain-like cysteine protease Yogesh A. Karpe and Kavita S. Lole Correspondence Kavita S. Lole lolekavita37@yahoo.com Received 28 April 2011 Accepted 30 May 2011 Hepatitis Division, National Institute of Virology, Microbial Containment Complex, Sus Road, Pashan, Pune 411021, India Hepatitis E virus (HEV) ORF1 protein (pORF1) contains methyltransferase (MetT), papain-like cysteine protease (PCP), RNA helicase (Hel) and RNA-dependent RNA polymerase (RdRp) domains. ORF1 sequence analysis showed two consensus LXGG cleavage sites at 664 and 1205. LXGG sequence is recognized by viral and cellular deubiquitinating enzymes. The protein encompassing the predicted MetT-PCP domains of HEV ORF1 was tested for deubiquitinating activity using fluorogenic substrates – ubiquitin-7-amino-4-methylcoumarin (AMC), IFN-stimulated gene 15 (ISG15)-AMC, Nedd8-AMC and SUMO-AMC. MetT-PCP cleaved all four substrates but processing of ISG15-AMC was more robust. There was no processing of the Hel and RdRp domains having the conserved (1205) LXGG site by the protein. MetT-PCP carried out deISGylation of the ISG15-conjugated cellular proteins, suggesting a possible role in combating cellular antiviral pathways. Positive-sense viral genomes can be translated after entering into the host cells. Viruses use several strategies for efficient synthesis of viral proteins by using the host cell machinery (Gale et al., 2000). Viral polyprotein processing is usually required for enzymic activities, interactions with other proteins, subcellular localization, assembly, etc. Hepatitis E virus (HEV), (genus Hepevirus, family Hepe- viridae) has a positive-sense ssRNA genome (7.2 kb) encoding non-structural protein from the 59 ORF called ORF1 and the capsid protein from the 39 ORF known as ORF2 and an auxiliary protein encoding ORF3 overlapping with ORF2 (Tam et al., 1991). Translation of ORF2 and ORF3 requires synthesis of subgenomic mRNA (Graff et al., 2006). Based on the sequence homology, HEV ORF1 protein (pORF1) is proposed to contain putative domains for methyltransferase (MetT), papain-like cysteine protease (PCP), RNA helicase (Hel), RNA-dependent RNA poly- merase (RdRp) and domains of unknown function – X and Y (Koonin et al., 1992). Among these, functional activities of RdRp (Agrawal et al., 2001), Hel (Karpe & Lole, 2010a, b) and MetT (Magden et al., 2001) have been experiment- ally verified. Protease function still remains an unsolved puzzle in HEV biology. At the amino acid level, HEV predicted PCP shows very less homology with the other viral PCPs (Koonin et al., 1992). It is not entirely clear whether the pORF1 is processed into biochemically dis- tinct units. When expressed using a prokaryotic system (Escherichia coli) or using in vitro coupled transcription and translation system, no processing was observed (Ansari et al., 2000). However, ORF1 expressed using recombinant vaccinia virus showed ~186 kDa protein with shorter incubation periods and extended incubations for 24–36 h resulted in two bands of ~107 and ~78 kDa proteins (Ropp et al., 2000). HepG2 cells transfected with HEV genomic RNA produced in vitro from cDNA showed distinct processed products. Anti-MetT, anti-Hel and anti-RdRp antibodies detected specific products of y35, y38 and y36 kDa, respectively (Ansari et al., 2000). ORF1 protein expressed in insect cells using baculovirus system was processed into smaller fragments, and the processing could be inhibited by E-64d, a cell-permeable cysteine-protease inhibitor. But it was not clear whether processing occurred due to protease activity from the host or from the virus (Sehgal et al., 2006). Thus, extensive attempts have been made by various groups to study pORF1 processing documenting contradictory results. We carried out HEV ORF1 amino acid sequence analysis to search for PCP cleavage site(s) reported in different positive-sense RNA viruses. A total of 168 ORF1 sequences representing all HEV genotypes were aligned and analysed. Two LXGG sites were identified in ORF1 between (i) PCP and X domains, at aa 664 (128/168 sequences had LXGG and 27/168 had LXGN), none of the avian (n58), rabbit (n52) or rat (n51) sequences had this site; (ii) Hel and RdRp domains, at aa 1205 (160/168 sequences had LXGG), none of the avian isolates had this site (Fig. 1a). Ubiquitination is the tagging of proteins with ubiquitin (Ub) for selective destruction in proteasomes. IFN- stimulated gene 15 (ISG15), SUMO, ATG8 and Nedd8 are small Ub-like molecules (UBLs) expressed in eukary- otes that are conjugated to target proteins, often resulting Journal of General Virology (2011), 92, 2088–2092 DOI 10.1099/vir.0.033738-0 2088 033738 G 2011 SGM Printed in Great Britain