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Insect BTI-TN-5B1-4 (Tn5) cells have been used extensively with recombinant baculoviruses to express foreign genes. When a recombinant baculovirus containing the hepatitis E virus capsid protein gene was used to infect Tn5 cells, unknown virus particles in addition to the anticipated hepatitis E virus-like particles were produced in the infected cells. The unknown virus particles were 35 nm in diameter and contained RNA that was highly homologous to full-length RNA1 (3,107 bp) and RNA2 (1,383 bp) genomic RNAs of flock house virus. Surprisingly, both RNAs seen in these induced nodavirus particles could be amplified from commercially available Tn5 cells without infection with or induction by a baculovirus. The nucleotide sequences from the purified nodavirus particles and the normal Tn5 cells were identical, demonstrating that the Tn5 cells themselves were latently infected with a nodavirus. However, the generation of nodavirus particles was significantly stimulated by infection with recombinant baculoviruses. Phylogenetic analysis suggested that this new nodavirus belongs to the genus Alphanodavirus in the family Nodaviridae .

Hepatitis E virus (HEV) is a noncultivable virus that causes acute liver failure in humans. The virus's major capsid protein is encoded by an open reading frame 2 (ORF2) gene. When the recombinant protein consisting of amino acid (aa) residues 112 to 660 of ORF2 is expressed with a recombinant baculovirus, the protein self-assembles into virus-like particles (VLPs) (T.-C. Li, Y. Yamakawa, K. Suzuki, M. Tatsumi, M. A. Razak, T. Uchida, N. Takeda, and T. Miyamura, J. Virol. 71:7207-7213, 1997). VLPs can be found in the culture medium of infected Tn5 cells but not in that of Sf9 cells, and the major VLPs have lost the C-terminal 52 aa. To investigate the protein requirement for HEV VLP formation, we prepared 14 baculovirus recombinants to express the capsid proteins truncated at the N terminus, the C terminus, or both. The capsid protein consisting of aa residues 112 to 608 formed VLPs in Sf9 cells, suggesting that particle formation is dependent on the modification process of the ORF2 protein. In the present study, electron cryomicroscopy and image processing of VLPs produced in Sf9 and Tn5 cells indicated that they possess the same configurations and structures. Empty VLPs were found in both Tn5 and Sf9 cells infected with the recombinant containing an N-terminal truncation up to aa residue 125 and C-terminal to aa residue 601, demonstrating that the aa residues 126 to 601 are the essential elements required for the initiation of VLP assembly. The recombinant HEV VLPs are potential mucosal vaccine carrier vehicles for the presentation of foreign antigenic epitopes and may also serve as vectors for the delivery of genes to mucosal tissue for DNA vaccination and gene therapy. The results of the present study provide useful information for constructing recombinant HEV VLPs having novel functions.

In polyomaviruses the pentameric capsomers are interlinked by the long C-terminal arm of the structural protein VP1. The T =7 icosahedral structure of these viruses is possible due to an intriguing adaptability of this linker arm to the different local environments in the capsid. To explore the assembly process, we have compared the structure of two virus-like particles (VLPs) formed, as we found, in a calcium-dependent manner by the VP1 protein of human polyomavirus BK. The structures were determined using electron cryomicroscopy (cryo-EM), and the three-dimensional reconstructions were interpreted by atomic modeling. In the small VP1 particle, 26.4 nm in diameter, the pentameric capsomers form an icosahedral T =1 surface lattice with meeting densities at the threefold axes that interlinked three capsomers. In the larger particle, 50.6 nm in diameter, the capsomers form a T =7 icosahedral shell with three unique contacts. A folding model of the BKV VP1 protein was obtained by alignment with the VP1 protein of simian virus 40 (SV40). The model fitted well into the cryo-EM density of the T =7 particle. However, residues 297 to 362 of the C-terminal arm had to be remodeled to accommodate the higher curvature of the T =1 particle. The loops, before and after the C-terminal short helix, were shown to provide the hinges that allowed curvature variation in the particle shell. The meeting densities seen at the threefold axes in the T =1 particle were consistent with the triple-helix interlinking contact at the local threefold axes in the T =7 structure.

Hepatitis E virus (HEV) is a pathogenic agent that causes fecally-orally transmitted acute hepatitis. The genome, a single-stranded positive-sense RNA, encodes three forward open reading frames (ORFs), in which an approximately 2-kb structural protein is located in the 3' end. To produce HEV-like particles the structural protein, with its N terminus truncated (amino acid residues 112 to 660 of ORF2), was expressed in insect Tn5 cells by a recombinant baculovirus. In addition to the primary translation product with a molecular mass of 58 kDa, a large amount of a further-processed molecule with a molecular mass of 50 kDa was generated and efficiently released into the culture medium. Electron microscopic observation of the culture medium revealed that the 50-kDa protein self-assembled to form empty virus-like particles (VLPs). The buoyant density of the VLPs in CsCl was 1.285 g/cm3 and their diameter was 23.7 nm, a little smaller than the 27 nm of native HEV particles secreted into the bile or stools of experimentally infected monkeys. The yield of the VLPs was 1 mg per 10(7) cells as a purified form. The particles possess antigenicity similar to that of authentic HEV particles and, consequently, they appear to be a good antigen for the sensitive detection of HEV-specific immunoglobulin G (IgG) and IgM antibodies. Furthermore, the VLP may be the most promising candidate yet for an HEV vaccine, owing to its potent immunogenicity.

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.