This chapter discusses the manipulation of clones of coronavirus and of complementary DNAs (cDNAs) of defective-interfering (DI) RNAs to study coronavirus RNA replication, transcription, recombination, processing and transport of proteins, virion assembly, identification of cell receptors for coronaviruses, and processing of the polymerase. The nature of the coronavirus genome is nonsegmented, single-stranded, and positive-sense RNA. Its size ranges from 27 to 32 kb, which is significantly larger when compared with other RNA viruses. The gene encoding the large surface glycoprotein is up to 4.4 kb, encoding an imposing trimeric, highly glycosylated protein. This soars some 20 nm above the virion envelope, giving the virus the appearance-with a little imagination-of a crown or coronet. Coronavirus research has contributed to the understanding of many aspects of molecular biology in general, such as the mechanism of RNA synthesis, translational control, and protein transport and processing. It remains a treasure capable of generating unexpected insights.

The molecular biology of coronaviruses

The nucleotide sequence determination of the genome of the Beaudette strain of the coronavirus avian infectious bronchitis virus (IBV) has been completed. The complete sequence has been obtained from 17 overlapping cDNA clones, the 5'-most of which contains the leader sequence (as determined by direct sequencing of the genome) and the 3'-most of which contains the poly(A) tail. Approximately 8 kilobases at the 3' end of this sequence have already been published. These contain the sequences of mRNAs A to E within which are the genes for the spike, the membrane and the nucleocapsid polypeptides: the main structural components of the virion. The remainder of the sequence, equivalent to the 'unique' region of mRNA F, is some 20 kilobases in length and is thought to code for a polymerase or polymerases which are involved in the replication of the genome and the production of the subgenomic messenger RNAs. This sequence contains two large open reading frames, potentially coding for polypeptides of molecular weights 441,000 and 300,000. Unlike other large open reading frames in the virus, the 300,000 open reading frame appears to have no subgenomic RNA associated with it which would allow it to be at the 5' end of an mRNA species. Because of this, and because of the characteristics of the sequence in the region immediately upstream of its start codon, other mechanisms of translation, such as ribosome slippage, must be postulated.

Completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus.

Introduction. The Coronaviridae is a monogeneric family comprising 11 viruses which infect vertebrates. Members of the group are responsible for diseases of clinical and economic importance, in particular respiratory and gastrointestinal disorders (Table 1). The group was originally recognized on the basis of a characteristic virion morphology (Tyrrell et al., 1968), but can now be defined by biological and molecular criteria. Various aspects of coronavirus biology have been dealt with in recent reviews (Robb & Bond, 1979; Siddell et al., 1982; Wege et al., 1982).
Structure. Morphology. Coronavirions are pleomorphic, although generally spherical, 60 to 220 nm in diameter and bear widely spaced, club-shaped surface projections about 20 nm in length. Complete virions have a density in sucrose of about 1.18 g/ml. In thin sections the virion envelope may be visualized as inner and outer shells separated by a translucent space. In negatively stained preparations of avian infectious bronchitis virus (IBV) an inner tongue-shaped membrane is visible (Bingham & Almeida, 1977; Fig. 1).

The biology of coronaviruses.

Publisher Summary Coronaviruses have recently emerged as an important group of animal and human pathogens that share a distinctive replicative cycle. Some of the unique characteristics in the replication of coronaviruses include generation of a 3' coterminal-nested set of five or six subgenomic mRNAs, each of which appears to direct the synthesis of one protein. Two virus-specific RNA polymerase activities have been identified. Many of the distinctive features of coronavirus infection and coronavirus-induced diseases may result from the properties of the two coronavirus glycoproteins. The intracellular budding site, which may be important in the establishment and maintenance of persistent infections, appears to be due to the restricted intracytoplasmic migration of the E1 glycoprotein, which acts as a matrix-like transmembrane glycoprotein. E1 also exhibits distinctive behavior by self-aggregating on heating at 100°C in sodium dodecyl sulfate (SDS) and by its interaction with RNA in the viral nucleocapsid. The E1 of mouse hepatitis virus (MHV) is an O-linked glycoprotein, unlike most other viral glycoproteins. Thus, the coronavirus system may be a useful model for the study of synthesis, glycosylation, and transport of O-linked cellular glycoproteins.

The genome of the porcine transmissible gastroenteritis coronavirus is a plus-strand, polyadenylylated, infectious RNA molecule of approximately 20 kilobases. During virus replication, seven subgenomic mRNAs are generated by what is thought to be a leader-priming mechanism to form a 3'-coterminal nested set. By using radiolabeled, strand-specific, synthetic oligodeoxynucleotide probes in RNA blot hybridization analyses, we have found a minus-strand counterpart for the genome and for each subgenomic mRNA species in the cytoplasm of infected cells. Subgenomic minus strands were found to be components of double-stranded replicative forms and in numbers that surpass full-length antigenome. We propose that subgenomic mRNA replication, in addition to leader-primed transcription, is a significant mechanism of mRNA synthesis and that it functions to amplify mRNAs. It is a mechanism of amplification that has not been described for any other group of RNA viruses. Subgenomic replicons may also function in a manner similar to genomes of defective interfering viruses to lead to the establishment of persistent infections, a universal property of coronaviruses.

Coronavirus subgenomic minus-strand RNAs and the potential for mRNA replicons

Avian infectious bronchitis virus, a coronavirus, directed the synthesis of six major single-stranded polyadenylated RNA species in infected chicken embryo kidney cells. These RNAs include the intracellular form of the genome (RNA F) and five smaller RNA species (RNAs A, B, C, D, and E). Species A, B, C, and D are subgenomic RNAs and together with the genome form a nested sequence set, with the sequences of each RNA contained within every larger RNA species (D. F. Stern and S. I. T. Kennedy, J. Virol 34:665-674, 1980). In the present paper we show by RNase T1 oligonucleotide fingerprinting that RNA E is also a member of the nested set. Partial alkaline fragmentation of the genome followed by sucrose fractionation, oligodeoxythymidylate-cellulose chromatography, and RNase T1 fingerprinting gave a partial 3'-to-5' oligonucleotide spot order. A comparison of the oligonucleotides of each of the five subgenomic RNAs with this spot order established that all of the RNAs are comprised of nucleotide sequences inward from the 3' end of the genome. This result is discussed in relation to the multiplication strategy both of coronaviruses and of other RNA-containing viruses.

/pdf/coronavirus-multiplication-strategy-ii-mapping-the-avian-1ewm3ybpg9.pdf

D F Stern

Papers

Coronavirus multiplication strategy. II. Mapping the avian infectious bronchitis virus intracellular RNA species to the genome.