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Expression of the coronavirus gene 1-encoded polyproteins, pp1a and pp1ab, is linked to a series of proteolytic events involving virus-encoded proteinases. In this study, we used transfection and immunoprecipitation assays to show that the human coronavirus 229E-encoded papain-like cysteine proteinase, PCP1, is responsible for the release of an amino-terminal protein, p9, from the gene 1-encoded polyproteins. The same protein, p9, has also been identified in virus-infected cells. Furthermore, using an in vitro trans-cleavage assay, we defined the proteolytic cleavage site at the carboxyl terminus of p9 as pp1a-pp1ab amino acids Gly-111 and Asn-112. These results and a comparative sequence analysis suggest that substrate positions P1 and P5 seem to be the major determinants of the PCP1 cleavage site and that the latter can occupy a variable position at the amino terminus of the coronavirus ppla and pplab polyproteins. By combining the trans-cleavage assay with deletion mutagenesis, we were also able to locate the boundaries of the active PCP1 domain between pp1a-pp1ab amino acids Gly-861-Glu-975 and Asn-1209-Gln-1285. Finally, codon mutagenesis was used to show that Cys-1054 and His-1205 are essential for PCP1 proteolytic activity, suggesting that these amino acids most likely have a catalytic function.

The sequence of the HCV 229E gene 1 has been determined and compared with the homologous sequences of the murine hepatitis virus and the avian infectious bronchitis virus. The coding sequence of gene 1 is 20,273 nucleotides in length. Within this coding region are two large open reading frames, ORF 1a (4,086 codons) and ORF 1b (2,687 codons) which overlap by 40 nucleotides. In the overlapping region, the genomic RNA can be folded into a pseudoknot structure, an element which is known to mediate -1 ribosomal frame-shifting in other coronaviruses. Assuming that -1 frame-shifting occurs at the HCV sequence UUUAAAC (nucleotides 12,514-12,520), the ORF 1a - ORF 1b product is predicted to be 6,758 amino acids in length. Our sequence analysis of the HCV 229E gene 1 has revealed a high degree of similarity within the ORF 1b of HCV, MHV and IBV, whereas ORF 1a is much less conserved. Elements which are believed to be necessary for specific (e.g. frame-shifting) and general (e.g. NTP-binding/helicase) transcriptional functions have been identified. This study completes the genomic sequence of HCV 229E which is 27.27 kb long and one of the largest known RNA genomes.

The gene encoding the spike glycoprotein of the human coronavirus HCV 229E has been cloned and sequenced. This analysis predicts an S polypeptide of 1173 amino acids with an Mr of 128,600. The polypeptide has 30 potential N-glycosylation sites. A number of structural features typical of coronavirus S proteins can be recognized, including a signal sequence, a membrane anchor, heptad repeat structures and a carboxy-terminal cysteine cluster. A detailed, computer-aided comparison with the S proteins of infectious bronchitis virus, feline infectious peritonitis virus, transmissible gastroenteritis virus and murine hepatitis virus, strain JHM is presented. We have also done a Northern blot analysis of viral RNAs in HCV 229E-infected cells using synthetic oligonucleotides. On the basis of this analysis, and by analogy to the replication strategy of other coronaviruses, we are able to propose a model for the organization and expression of the HCV 229E genome.

The sequence of the gene encoding the membrane protein of human coronavirus 229 E (HCV 229 E) has been determined. The primary translation product, deduced from the DNA sequence, is a polypeptide of 225 amino acids with a predicted molecular weight of 26,000. The polypeptide has 3 potential N-glycosylation sites. Many structural similarities with the membrane proteins of other coronaviruses can be recognized.

Positive-stranded genomic RNA of coronavirus MHV and its six subgenomic mRNAs are synthesized in the cytoplasm of the host cell. The mRNAs are composed of leader and body sequences which are non-contiguous on the genome and are fused together in the cytoplasm by a mechanism which appears to involve an unusual and specific 'polymerase jumping' event.

Protein synthesis in the murine hepatitis virus JHM-infected cells was temporarily inhibited by hypertonic shock. When the cells were returned to isotonic medium the synthesis of six virus-specific polypeptides, 150K, 65K, 60K, 30K, 23K and 14K was reinitiated simultaneously. Polyadenylated RNA isolated from the cytoplasm or polysomes of infected cells was translated in vitro and the products included polypeptides with molecular weights (mol. wt.) of 120,000, 60,000, 30,000, 23,000 and 14,000. Immunoprecipitation and fingerprinting of [35S]methionine-containing tryptic peptides showed that the 60,000 and 23,000 mol. wt. products were identical to the 60K and 23K polypeptides found in infected cells; the 120,000 mol. wt. product showed identity with the 150K intracellular polypeptide and a virus-specific 120K polypeptide synthesized in tunicamycin-treated cells. Two-dimensional polyacrylamide gel electrophoresis strongly suggested that the 30,000 and 14,000 mol. wt. products are equivalent to virus-specific 30K and 14K intracellular polypeptides. [3H]Uridine-labelled polyadenylated virus RNA was isolated from infected cells and sedimented in sucrose gradients containing formamide. The distribution in the gradient of each of the previously identified virus RNAs was determined by gel electrophoresis and gradient fractions enriched for each RNA were translated in vitro. The 120,000, 60,000, 30,000, 23,000 and 14,000 mol. wt. polypeptides were found to be encoded by mRNAs 3, 7, 2, 6, and 4 or 5 respectively. These results demonstrate that the virus-specific polypeptides in JHM-infected cells are encoded in separate subgenomic mRNAs and are translated independently. The assignment of coding functions and the known sequence relationships of JHM RNAs permitted a gene order to be deduced.

Sequences encoding the surface projection glycoprotein of the coronavirus, murine hepatitis virus (MHV), strain JHM, have been cloned into pAT153 using cDNA produced by priming with specific oligonucleotides on infected cell RNA. The regions of three clones pJMS1010, pJS112 and pJS92, which together encompass the surface protein gene have been sequenced by the chain termination method. The sequence of the primary translation product, deduced from the DNA sequence, predicts a polypeptide of 1,235 amino acids with a molecular weight of 136,600. This polypeptide displays the features characteristic of a group 1 membrane protein; an amino-terminal signal sequence and carboxy-terminal membrane and cytoplasmic domains. There are 21 potential glycosylation sites in the polypeptide and a cysteine-rich region in the vicinity of the transmembrane domain. During maturation proteolytic processing of the polypeptide occurs and at positions 624 to 628 the sequence Arg-Arg-Ala-Arg-Arg is found, which is similar to a number of basic sequences involved in the cleavage of enveloped RNA virus glycoproteins. The fusogenic properties of the MHV surface protein do not appear to correlate with a strongly hydrophobic region at the putative amino terminus of the carboxy-terminal cleavage product.