The complete 1,210-amino acid sequence of the human epidermal growth factor (EGF) receptor precursor, deduced from cDNA clones derived from placental and A431 carcinoma cells, reveals close similarity between the entire predicted v-erb-B mRNA oncogene product and the receptor transmembrane and cytoplasmic domains. A single transmembrane region of 23 amino acids separates the extracellular EGF binding and cytoplasmic domains. The receptor gene is amplified and apparently rearranged in A431 cells, generating a truncated 2.8-kilobase mRNA which encodes only the extracellular EGF binding domain.

To identify structural characteristics of the closely related cell surface receptors for insulin and IGF‐I that define their distinct physiological roles, we determined the complete primary structure of the human IGF‐I receptor from cloned cDNA. The deduced sequence predicts a 1367 amino acid receptor precursor, including a 30‐residue signal peptide, which is removed during translocation of the nascent polypeptide chain. The 1337 residue, unmodified proreceptor polypeptide has a predicted Mr of 151,869, which compares with the 180,000 Mr IGF‐I receptor precursor. In analogy with the 152,784 Mr insulin receptor precursor, cleavage of the Arg‐Lys‐Arg‐Arg sequence at position 707 of the IGF‐I receptor precursor will generate alpha (80,423 Mr) and beta (70,866 Mr) subunits, which compare with approximately 135,000 Mr (alpha) and 90,000 Mr (beta) fully glycosylated subunits.

We have recently described the cloning of a portion of the hepatitis E virus (HEV) and confirmed its etiologic association with enterically transmitted (waterborne, epidemic) non-A, non-B hepatitis. The virus consists of a single-stranded, positive-sense RNA genome of approximately 7.5 kb, with a polyadenylated 3' end. We now report on the cloning and nucleotide sequencing of an overlapping, contiguous set of cDNA clones representing the entire genome of the HEV Burma strain [HEV(B)]. The largest open reading frame extends approximately 5 kb from the 5' end and contains the RNA-directed RNA polymerase and nucleoside triphosphate binding motifs. The second major open reading frame (ORF2) begins 37 bp downstream of the first and extends approximately 2 kb to the termination codon present 65 bp from the 3' terminal stretch of poly(A) residues. ORF2 contains a consensus signal peptide sequence at its amino terminus and a capsid-like region with a high content of basic amino acids similar to that seen with other virus capsid proteins. A third open reading frame partially overlaps the first and second and encompasses only 369 bp. In addition to the 7.5-kb full-length genomic transcript, two subgenomic polyadenylated messages of approximately 3.7 and 2.0 kb were detected in infected liver using a probe from the 3' third of the genome. The genomic organization of the virus is consistent with the 5' end encoding nonstructural and the 3' end encoding the viral structural gene(s). The expression strategy of the virus involves the use of three different open reading frames and at least three different transcripts. HEV was previously determined to be a nonenveloped particle with a diameter of 27-34 nm. These findings on the genetic organization and expression strategy of HEV suggest that it is the prototype human pathogen for a new class of RNA virus or perhaps a separate genus within the Caliciviridae family. o tsst Academic PWSS, IIX.

No abstract

Recently, we found that more than 10% of the cases of acute non-A, non-B, non-C hepatitis in Taiwan were caused by a novel strain of hepatitis E virus (HEV). Since none of these patients had a history of travel to areas where HEV is endemic, the source of transmission remains unclear. The recent discovery of a swine HEV in herd pigs in the United States has led us to speculate that HEV may also circulate in herd pigs in Taiwan and may serve as a reservoir for HEV in Taiwan. Of 275 herd pigs obtained from 10 pig farms in Taiwan, 102 (37%) were seropositive for serum anti-HEV immunoglobulin G (IgG). A 185-bp genomic sequence within the ORF-2 of the HEV genome was amplified and cloned from serum samples of an anti-HEV positive pig and subsequently from serum samples of a patient with acute hepatitis E. Sequence comparison revealed that the swine and human isolates of HEV share 97.3% identity. Phylogenetic analyses further showed that the Taiwan swine and human isolates of HEV form a distinct branch divergent from all other known strains of HEV, including the U.S. swine strain. To examine the potential risk of cross-species transmission of swine HEV to humans, the seroprevalences of anti-HEV IgG in 30 swine handlers, 20 pork dealers, and 50 control subjects were assessed and were found to be 26.7, 15, and 8%, respectively (for swine handlers versus controls,P = 0.048). Our findings may help provide an understanding of the modes of HEV transmission and may also raise potential public health concerns for HEV zoonosis.

Large epidemic outbreaks of enterically transmitted non-A, non-B viral hepatitis (ET-NANBH) have been documented in developing countries. A molecular clone derived from the causative agent, the hepatitis E virus (HEV), has recently been described (G.R. Reyes, M.A. Purdy, J.P. Kim, K.-C. Luk, L.M. Young, K.E. Fry, and D. Bradley, Science 247:1335-1339, 1990). We now report the isolation, by serologic screening, of two cDNA clones derived from a fecal sample collected during a 1986 outbreak of ET-NANBH in Telixtac, Mexico. The cDNA clones encode epitopes that specifically reacted with acute- and convalescent-phase sera collected during five different ET-NANBH epidemics and represent the initial cloning of the Mexico strain of HEV. Recombinant fusion proteins expressed from these clones were also recognized by antibodies from cynomolgus macaques experimentally infected with HEV. The cDNA clones were shown to be derived from HEV by their specific hybridization to the previously recognized full-length genomic RNA transcript of approximately 7.5 kb. In addition, however, subgenomic polyadenylated transcripts of approximately 2.0 and approximately 3.7 kb were also identified in HEV-infected cynomolgus monkey liver. Sequences homologous to the epitope clones were isolated from the Burma strain of the virus, and these demonstrated reactivity comparable to that seen with the Mexico strain epitopes. When compared with the available full-length sequence of the Burma strain of HEV, it was discovered that the cDNA clones were encoded in different open reading frames (ORFs). The comparison between Mexico and Burma HEV strains indicated amino acid homologies of 90.5 and 73.5% for these epitope-encoding clones derived from ORF2 and ORF3, respectively. The identification of these clones not only has provided insight into the expression strategy of HEV but has also resulted in a source of recombinant protein useful in the diagnosis of HEV-induced hepatitis.

Hepatitis E virus (HEV) is an unclassified, plus-strand RNA virus whose genome contains three open reading frames (ORFs). ORF1, the 5' proximal ORF of HEV, encodes nonstructural proteins involved in RNA replication which share homology with the products of the corresponding ORF of members of the alphavirus-like superfamily of plus-strand RNA viruses. Among animal virus members of this superfamily (the alphavirus and rubivirus genera of the family Togaviridae), the product of this ORF is a nonstructural polyprotein (NSP) that is cleaved by a papain-like cysteine protease (PCP) within the NSP. To determine if the NSP of HEV is similarly processed, ORF1 was introduced into a plasmid vector which allowed for expression both in vitro using a coupled transcription/translation system and in vivo using a vaccinia virus-driven transient expression system. A recombinant vaccinia virus expressing ORF1 was also constructed. Both in vitro and in vivo expression under standard conditions yielded only the full-length 185 kDa polyprotein. Addition of co-factors in vitro, such as divalent cations and microsomes which have been shown to activate other viral proteases, failed to change this expression pattern. However, in vivo following extended incubations (24-36 hours), two potential processing products of 107 kDa and 78 kDa were observed. N- and C-terminus-specific immunoprecipitation and deletion mutagenesis were used to determine that the order of these products within the NSP is NH2-78 kDa-107 kDa-COOH. However, site-specific mutagenesis of Cys483, predicted by computer alignment to be one member of the catalytic dyad of a PCP within the NSP, failed to abolish this cleavage. Additionally, sequence alignment across HEV strains revealed that the other member of the proposed catalytic dyad of this PCP, His590, was not conserved. Thus, the cleavage of the NSP observed following prolonged in vivo expression was not mediated by this protease and it is doubtful that a functional PCP exists within the NSP. Attempts to detect NSP expression and processing in HEV-infected primary monkey hepatocytes were not successful and therefore this proteolytic cleavage could not be authenticated. Overall, the results of this study indicate that either the HEV NSP is not processed or that it is cleaved at one site by a virally-encoded protease novel among alpha-like superfamily viruses or a cellular protease.

The hypothesis that activation of apoptosis and DNA fragmentation is involved in TNF-mediated cytolysis of U937 tumor cells was investigated. Morphological, biochemical, and kinetic criteria established that TNF activates apoptosis as opposed to necrosis. Within 2-3 h of exposure to TNF, U937 underwent the morphological alterations characteristic of apoptosis. This was accompanied by cleavage of DNA into multiples of nucleosome size fragments. Both of these events occurred 1-2 h prior to cell death as defined by trypan blue exclusion or 51Cr release. DNA fragmentation was not a non-specific result of cell death since U937 cells lysed under hypotonic conditions did not release DNA fragments. The percentage of cells undergoing apoptosis depended on the concentration of TNF and was augmented by the addition of cycloheximide. A TNF-resistant variant derived from U937 did not undergo apoptosis in response to TNF, even in the presence of cycloheximide. Furthermore, TNF could still activate NFkB in this variant, suggesting that this pathway is not involved in TNF-mediated cytotoxicity. Two agents known to inhibit TNF-mediated cytotoxicity, ZnSO4 and 3-aminobenzamide, were shown to inhibit TNF-induced apoptosis. Taken altogether, these data support the hypothesis that activation of apoptosis is at least one essential step in the TNF lytic pathway in the U937 model system.