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

In addition to the SARS coronavirus (treated separately elsewhere in this volume), the complete genome sequences of six species in the coronavirus genus of the coronavirus family [avian infectious bronchitis virus-Beaudette strain (IBV-Beaudette), bovine coronavirus-ENT strain (BCoV-ENT), human coronavirus-229E strain (HCoV-229E), murine hepatitis virus-A59 strain (MHV-A59), porcine transmissible gastroenteritis-Purdue 115 strain (TGEV-Purdue 115), and porcine epidemic diarrhea virus-CV777 strain (PEDV-CV777)] have now been reported. Their lengths range from 27,317 nt for HCoV-229E to 31,357 nt for the murine hepatitis virus-A59, establishing the coronavirus genome as the largest known among RNA viruses. The basic organization of the coronavirus genome is shared with other members of the Nidovirus order (the torovirus genus, also in the family Coronaviridae, and members of the family Arteriviridae) in that the nonstructural proteins involved in proteolytic processing, genome replication, and subgenomic mRNA synthesis (transcription) (an estimated 14–16 end products for coronaviruses) are encoded within the 5′-proximal two-thirds of the genome on gene 1 and the (mostly) structural proteins are encoded within the 3′-proximal one-third of the genome (8–9 genes for coronaviruses). Genes for the major structural proteins in all coronaviruses occur in the 5′ to 3′ order as S, E, M, and N. The precise strategy used by coronaviruses for genome replication is not yet known, but many features have been established. This chapter focuses on some of the known features and presents some current questions regarding genome replication strategy, the cis-acting elements necessary for genome replication [as inferred from defective interfering (DI) RNA molecules], the minimum sequence requirements for autonomous replication of an RNA replicon, and the importance of gene order in genome replication.

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Coronavirus genome structure and replication

RNA recombination in animal and plant viruses.

Introduction. Progress in coronavirology is illustrated by the number of workshops convened and reviews written. International meetings have been held in Germany (1980), the Netherlands (1983) and the U.S.A. (1986), and the Fourth Coronavirus Symposium will be organized by one of us (D.C.) in Cambridge, U.K. in July 1989. In addition, reviews have appeared which highlighted particularly interesting characteristics of the family, e.g. the replication strategy (Lai, 1986) and the glycoproteins (Sturman & Holmes, 1985). As the last general accounts were published some 5 years ago (Siddell et al., 1983; Sturman & Holmes, 1983) an update is timely. The present article is based on the large amount of sequence data accumulated in these years and focuses on the viral nucleic acids and proteins and their function.
Coronaviruses cause infections in man, other mammals and birds. Most experimental data have been obtained from studies of mouse hepatitis virus (MHV) and infectious bronchitis virus of chickens (IBV).

Coronaviruses: structure and genome expression.

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.

The Purdue strain of transmissible gastroenteritis virus, a porcine coronavirus, was grown to titers of greater than 10(8) PFU/ml in a swine testicle cell line, and the RNA was isotopically labeled with [3H]uridine. The RNA was extracted from purified virus and was found to have the following properties. (i) It consisted primarily of a homogeneous large-molecular-weight species which electrophoretically migrated with an apparent molecular weight of 6.8 X 10(6) under denaturing conditions. (ii) It migrated electrophoretically at the same rate on nondenaturing gels before and after heat denaturation, suggesting that it does not consist of subunits. (iii) It was susceptible to pancreatic RNase A digestion in high (0.3 M) NaCl. (iv) It was polyadenylated to the extent that greater than 60% of the native RNA bound to oligodeoxythymidilic acid-cellulose under conditions of high (0.5 M) NaCl. RNA extracted from virions was infectious. This coronavirus can therefore be characterized as a positive-strand RNA virus.

Genome of porcine transmissible gastroenteritis virus.

An enzymatic activity which incorporates [3H]UMP into acid-precipitable material in the presence of endogenous template was found in the cytoplasm of porcine cells infected with the transmissible gastroenteritis virus of swine. This activity was not found in uninfected control cells, nor was it found in purified virus. The activity was associated with the mitochondrial fraction of infected cells, suggesting that the enzyme is membrane bound. The activity required the presence of all three ribonucleoside triphosphates in addition to [3H]UTP, and it was not inhibited by actinomycin D. The heated product was digested by RNase but not by DNase. Mg2+ was required for enzymatic activity, and its optimal concentration was approximately 5 mM. The size of the in vitro products was compared by electrophoresis with that of in vivo-synthesized virus-specified RNA to confirm the viral specificity of the polymerase activity. Virus-specified RNA from infected cells consisted of 10 species of single-stranded, polyadenylated RNA with molecular weights of 6.8 X 10(6), 6.2 X 10(6), 3.15 X 10(6), 1.40 X 10(6), 1.05 X 10(6), 0.94 X 10(6), 0.66 X 10(6), 0.39 X 10(6), 0.34 X 10(6), and 0.24 X 10(6). In vitro synthesized RNA consisted of a high-molecular-weight species, of apparently higher molecular weight than genomic RNA, and two single-stranded species that electrophoretically comigrated with the species of 1.40 X 10(6) and 0.66 X 10(6) molecular weight made in vivo.

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D. E. Dennis

Papers

Genome of porcine transmissible gastroenteritis virus.

RNA-dependent RNA polymerase activity in coronavirus- infected cells.