Virus-Specific mRNA Capping Enzyme Encoded by Hepatitis E Virus

Hepatitis E virus (HEV) is an important etiological agent of acute epidemic and sporadic enteric hepatitis affecting millions of people mainly in developing countries. The first confirmed HEV epidemic, due to contamination of drinking water in New Delhi, India, was described in 1955. In addition to large epidemics in India and China, there are annually about 2 million sporadic cases of HEV infections in India alone. The mortality among HEV patients has been 0.5 to 4%, except in the case of pregnant women, for whom the average mortality is 20% (for reviews, see references 22 and 33). Recently, closely related viruses have been isolated from pigs, cows, sheep, goats, and rats, indicating zoonotic HEV infections (13). HEV is a nonenveloped virus with a diameter of 27 to 34 nm (9, 10), which does not replicate in cell cultures (1). The complete nucleotide sequence of the positive-strand RNA genome has been determined for several isolates from different parts of the world (40). (For references, see reference 8.) The HEV genome consists of a 27-nucleotide (nt)-long 5′ noncoding region followed by an open reading frame (ORF) coding for a nonstructural protein of 1,693 aa residues. ORF2 starts 38 nt downstream of the termination codon of ORF1 and codes for the capsid protein of 660 aa. ORF3 between nt 5105 and 5476 overlaps with ORF2 and codes for a 123-aa-long polypeptide with unknown function. The 3′ noncoding region is 65 nt, ending with a 150- to 200-nt-long poly(A) tail. A recent finding indicates that the HEV genome has an m7G cap structure at the 5′ end of the RNA (15). Expression of HEV capsid protein in COS-1 cells revealed that it is a glycoprotein with a size of 88 kDa, which is synthesized as a precursor (ggPORF2), processed to gPORF2 by cleavage of a signal sequence of 22 aa, and transported to the plasma membrane (14, 46). A more stable cytosolic form of capsid protein PORF2 with a size of 78 kDa has been detected in HepG2 cells by using a Semliki Forest virus (SFV) expression vector (41, 42). So far, which form of capsid protein is responsible for the production of infectious HEV is unknown. A truncated 50-kDa form of PORF2 produced in insect cells is able to assemble into hollow particles with an icosahedral symmetry of T=1 (25, 44). Expression of ORF3 in eukaryotic cells revealed that it is a phosphoprotein with affinity for the cytoskeleton through a hydrophobic amino-terminal domain of 32 aa residues (45). Expression of complete ORF1 coding for 1,693 aa residues in vitro, in Escherichia coli and in HepG2 cells, resulted in a large 186-kDa protein that was not autocatalytically processed (7). In another study, prolonged in vivo expression yielded N-terminal 78-kDa and C-terminal 107-kDa fragments (34). Transfection of in vitro-synthesized RNA, consisting of the complete HEV genome, to HepG2 cells resulted in synthesis of ORF1, ORF2, and ORF3 products as well as release of small amounts of infectious virus into the culture medium (29). However, no 186-kDa protein was detected in pulse-chase experiments. Instead, region-specific antisera precipitated smaller 35- to 40-kDa polypeptides. Computer-assisted assignments for the putative functions of HEV ORF1 suggested that the amino-terminal domain from 60 to 240 aa may be a methyltransferase followed by a Y domain with unknown function, a papain-like protease domain (around 440 to 610 aa), a proline-rich spacer region, an X domain of unknown function, a helicase domain (around 960 to 1,200 aa), and an RNA polymerase domain (around 1,200 to 1,700 aa) (17). None of the predicted functions has been experimentally verified. Indeed, it was recently shown that one of the putative protease active site residues is not conserved in different isolates and that mutation of the predicted active site cysteine did not abrogate P186 processing (34). Thus, the predicted protease appears not to exist or have enzymatic activity. Based on the ORF1 sequence, HEV belongs to a large alphavirus-like superfamily of positive-strand RNA viruses with putative methyltransferase, protease, helicase, and RNA polymerase domains (18). The distinctive methyltransferase domain is the hallmark of the alphavirus-like superfamily (28, 35). It contains sequence similarity to cellular S-adenosylmethionine (AdoMet)-dependent methyltransferases (5). In addition to guanine-7-methyltransferase activity (20, 36), the amino-terminal part of alphavirus nonstructural polyprotein, termed nsP1, also posesses guanylyltransferase activity (2). Both activities are needed in the capping of viral mRNAs (3, 5), and thus the conserved domain can more appropriately be designated as the capping enzyme domain. Both nsP1-catalyzed reactions are virus specific (i.e., there are no known host cell enzymes with similar specificities). Methyltransferase catalyzes the transfer of a methyl group from AdoMet to GTP, resulting in m7GTP, which forms the covalent complex nsP1-m7GMP. These reactions can be inhibited by cap analogs (22). In the present paper, we show the first enzymatic activity found for the HEV nonstructural protein. Truncated nonstructural protein P110 derived from HEV ORF1 produced in insect cells has virus-specific methyltransferase and guanylyltransferase activities similar to those of alphavirus replicase proteins. This finding provides a novel approach for development of specific inhibitors against hepatitis E infection.