1983 [Advances in Virus Research] Volume 28 __ The Molecular Biology of Coronaviruses

ADVANCES IN VIRUS RESEARCH, VOL 28 THE MOLECULAR BIOLOGY OF CORONAVIRUSES Lawrence S. Sturman Center for Laboratories and Reseorch, New York State Department of Health Albany, New York Kathryn V. Holmes Deportment of Pathology, Uniformed Services University of the Health Sciences Bethesda, Maryland ................................. B. Emergence of Coronaviruses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Viral Genome . . . . . . . , . . . . . . . . . . . . E. Viral Lipids.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Other Host-Derived Components. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Viral Replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Summary ........................ . . . _ . . . . . _ . _ . . . . . . B. Adsorption, Penetration, and Uncoa C. Characterization of Viral mRNAs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Kinetics of Intracellular Synthesis, Processing, and Transport 111. ......,.......... F. Virion Assembly, Release, and Cytopathic Effects. . . . . . . . . . . . . . . . IV. Structure and Biological Activities of Coronavirus Glycoproteins . . . . . . A. The Peplomeric Glycoprotein, E2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. The Matrix Glycoprotein, E l . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References, . . . . . . . V. Antigenic Relationships among Coronaviruses VI. 36 36 37 39 39 41 43 54 56 57 58 60 60 60 63 68 74 79 83 85 85 87 81 92 94 95 98 Copyright D 1983 by Academic Press, Inc All rights of reproduction In any form reserved ISBN 0-12-039828-1 35 36 LAWRENCE S. STURMAN AND KATHRYN V. HOLMES The image of progress in science as a kind of conquest of an unknown domain with a definite “frontier” and successive ‘breakthroughs’ seems to me more and more to be only a romantic illusion. . . . There are many frontiers and it comes down to the fact that in science one can only sometimes talk of progress. Whenever there is an ad- vance, there is a frontier, not the other way around. George E. Uhlenbeck (1971, p. 449-450)’ Facts are never completely independent of each other . . . every fact reacts upon many others. Every change and every discovery has an effect on a terrain that is virtually limitless. It is characteristic of advanced knowledge, matured into a co- herent system, that each new fact harmoniously-though ever so slightly-changes all earlier facts. Here every discovery is actually a recreation of the whole world as construed by a thought collective. Ludwik Fleck (1979, p. 102)z I. INTRODUCTION A. Dedication This contribution is dedicated to the memory of our colleague and friend, Frederik Bang, who uncovered some of the most intriguing questions about the biology of coronaviruses. Beginning with the dis- covery that mouse hepatitis virus (MHV) could grow in and destroy macrophages in culture (Bang and Warwick, 1959) and that mac- rophage susceptibility in uitro reflected a genetic determinant for sus- ceptibility in the mouse, Bang and Warwick, (1960; Kantoch et al., 1963) constructed a simple model to explain genetic resistance and susceptibility to this virus. For more than two decades, Bang (1978, 1981) and his colleagues explored genetic and environmental aspects of host resistance and susceptibility to MHV, and the effects of MHV on macrophages. At the Wurzburg Symposium on the Biochemistry and Biology of Coronaviruses in 1980, Dr. Bang (1981) recounted the changes in his thinking about mechanisms of host resistance, as many of the conclusions drawn from earlier experiments were reinterpreted in the light of subsequent findings. As always, he spoke with humility and humor, and displayed his distinctive interest in host-parasite in- terrelations. Unfortunately, that was his last opportunity to address his colleagues in this rapidly expanding field. We shall miss him. 1Reprinted by permission from Nature 232, 449-450. Copyright 0 1971 Macmillan Journals Limited. 2Reprinted from “Genesis and Development of a Scientific Fact” by Ludwig Fleck by permission of The University of Chicago Press. Copyright 0 1979 by The University of Chicago Press. MOLECULAR BIOLOGY OF CORONAVIRUSES 37 B. Emergence of Coronaviruses Coronaviruses have been known, although not by that name, for almost five decades. This previously unrecognized group emerged dur- ing the 1960s in the aftermath of the discovery of several new human respiratory pathogens. Avian infectious bronchitis virus (IBV), mouse hepatitis virus, and some newly described human respiratory viruses were noted to have a similar appearance (Almeida and Tyrrell, 1967; McIntosh et al., 196713; Becker et al., 1967). In contrast to myxoviruses, with which they had been previously compared (Berry et al., 1964; Mallucci, 1965), these viruses displayed a characteristic fringe of large, distinctive, petal-shaped peplomers or spikes which resembled a crown, like the corona spinarum in religious art; hence the name coro- naviruses (Fig. 1A; Tyrrell et al., 1968). In addition to their mor- phological similarities, some of the human coronaviruses (HCV) were noted to be antigenically related to MHV (Tyrrell et al., 1968; McIn- tosh et al., 1969; Bradburne, 1970). Between 1968 and 1974, research on coronaviruses emphasized morphologic and immunologic relation- ships and comparative biology. Several new viruses were added to the coronavirus group: porcine transmissible gastroenteritis virus (TGEV), porcine hemagglutinating encephalomyelitis virus (HEW, rat coronavirus (RCV), sialodacryoadenitis virus of rats (SDAV), tur- key bluecomb disease virus (TCV), and neonatal bovine diarrhea coro- navirus (BCV). A comprehensive review by McIntosh (1974) provides an excellent overview of early coronavirus research. In 1975, the International Committee on the Taxonomy of Viruses approved the creation of a new family, Coronaviridae, with one genus, Coronavirus (Tyrrell et al., 1975). Additional species were added later including canine coronavirus (CCV), feline infectious peritonitis virus (FIPV), and human enteric coronaviruses (HECV) (Tyrrell et al., 1978), rabbit coronaviruses (Small et al., 1979; LaPierre et al., 1980), and several others (reviewed by Wege et al., 1982). Table I shows the coronaviruses with their natural hosts and the major diseases which they induce. Clearly the coronaviruses are important pathogens of man and domestic animals. Timely reviews of several aspects of the biology and pathogenesis of coronaviruses have been published by Vir- elizier (19811, Macnaughton and Davies (1981), and Wege et al. (1982). During the past several years, substantial progress has been made in understanding the structure and replication of coronaviruses. The Symposium on the Biochemistry and Biology of Coronaviruses held in Wurzburg in October, 1980 dramatized the emergence of exciting fron- tiers in coronavirus research (ter Meulen et al., 1981). Siddell et al. (1982) have written an excellent review of the structure and replica- 38 LAWRENCE S. STURMAN AND KATHRYN V. HOLMES TABLE I MEMBERS OF THE CORONAVIRUS GROUP Natural Common name of virus Designation host Avian infectious bronchitis Bluecomb disease virus Mouse hepatitis virus virus Rat coronavirus Sialodacryoadenitis Transmissible gastroen- Hemagglutinating encepha- teritis virus lomyelitis virus Porcine virus CV777 Neonatal calf diarrhea Human coronavirus Human enteric coronavirus Canine coronavirus Feline infectious peritonitis coronavirus virus Feline enteric coronavirus Pleural effusion disease Rabbit enteric coronavirus virus IBV TCV MHV RCV SDAV TGEV HEV PCV BCV HCV HECV ccv FIPV FECV RbCV RbECV Chicken Turkey Mouse Rat Rat Pig Pig Pig Bovine Human Human Dog Cat Cat Diseases Tracheobronchitis, nephritis, Enteritis Hepatitis, encephalomyelitis, oviduct hypoplasia enteritis, interstitial pneumonia Pneumonia, rhinotracheitis Sialodacryoadenitis, ker- atoconjunctivitis, rhinotracheitis Gastroenteritis Encephalomyelitis, gastroen- teritis (“vomiting and wasting disease”) Enteritis Gastroenteritis Common cold Gastroenteritis (?I Gastroenteritis Meningoencephalitis, pan- ophthalmitis, peritonitis, pleuritis, pneumonia, wasting disease, vasculitis (“immune complex dis- ease”), disseminated gran- ulomatous disease Enteritis Rabbit (?) Pleuritis, myocarditis Rabbit Enteritis tion of coronaviruses, summarizing the state of knowledge shortly after the Wurzburg Symposium. The purpose of this article is to describe the development of our present understanding of the molecular biology of coronaviruses. We have taken an historical approach to the subject, using figures from some of the key papers to illustrate the development of our present concepts. We are grateful to our colleagues for making these figures and their unpublished data available to us. We hope that this contribu- tion will convey the sense of excitement and cooperation which has characterized this period in coronavirus research. MOLECULAR BIOLOGY O F CORONAVIRUSES 39 11, STRUCTURE AND ORGANIZATION OF VIRIONS A. Viral Structure Negative stains of coronaviruses from eggs, clinical specimens or media over tissue, or organ cultures infected with coronaviruses re- vealed the characteristic viral structure shown in Fig. 1A (Berry et al., 1964; Tyrrell and Almeida, 1967; McIntosh et al., 1967a; Almeida and Tyrrell, 1967; Apostolov et al., 1970; Oshiro et al., 1971; Kennedy and Johnson-Lussenberg, 1975-1976). The virions were spherical, enve- loped particles ranging from 80 nm to 160 nm in diameter. They showed some pleiomorphism, and frequently had a shallow central hollow containing some negative stain. Indeed, on the basis of nega- tively stained images of IBV, Bingham and Almeida (1977) suggested that the morphology of coronavirus virions might resemble a punched- in sphere. Whether this represents the true morphology of the virions or is a deformation resulting from drying and negative staining re- mains to be determined. Coronavirus morphology has been reviewed by McIntosh (1974), Oshiro (19731, Pensaert and Callebaut (1978), and Robb and Bond (1979a). Most coronaviruses appear to have only one morphologic type of surface projection or peplorner. The peplomers of coronaviruses are large and roughly club shaped. For MHV, each peplomer is about 20 nm long by 7 nm wide at the tip (Sturman et al., 1980). There are approximately 200 peplomers per virion. It is not known how many glycoprotein molecules form each peplomer. The peplomers of different coronaviruses have somewhat different appearances in negatively stained preparations (Caul and Egglestone, 1977; Davies and Mac- naughton, 1979). For BCV, Bridger et al. (1978) have suggested that there may be two morphologically distinct types of peplomers. Analysis of the structural proteins of BCV (Section IIC) also suggested that there might be an additional species of glycoprotein in the virion (King and Brian, 1982). Possibly this additional glycoprotein forms the second type of peplo- mer. Two types of peplomers have been observed rarely on HEV and MHV virions (Greig et al., 1971; Sugiyama and Amano, 1981). It is not clear to what extent the morphological differences between peplomers of coronaviruses reflect differences in amino acid sequence, glycosyla- tion, proteolytic cleavage, or reduction of disulfide bonds (see Sections II,C and IV,A). Many preparations of cornaviruses contain some virus particles which partially or completely lack peplomers. Storage of virions may lead to detachment of peplomers, and under some conditions virions lacking peplomers can be formed in infected cells (Holmes et al., 40 LAWRENCE S. STURMAN AND KATHRYN V. HOLMES FIG. 1. Ultrastructure of coronavirus virions and viral components. (A) Virions of the avian coronavirus IBV in negatively stained preparation. Characteristic petal-shaped peplomers project from the viral envelope and a central hollow contains a shallow pool of negative stain. x 120,000. (Courtesy of J. Almeida.) (B) Nucleocapsid released from 2293 virions and isolated by sucrose density gradient ultracentrifugation after the method in Caul et al., (1979). The helical structure and the hollow central core of the necleocapsid are evident. ~150,000. (Courtesy of E. 0. Caul.) (C) The membrane glycoprotein El of mouse hepatitis virus (MHV) purified by density gradient ultracentrifugation following detergent disruption ofpurified virions after the method in Sturman etal. (1980). The E l glycoprotein forms aggregates of irregular sizes x 100.000. (D) The peplomeric glycopro- tein E2 of MHV purified by the same technique forms rosettes or single peplomers. Arrows indicate rosettes. x 100,000. (C and D from K. V. Holmes.) 1981a,b; see Section 111,D). Macnaughton and Davies (1980) isolated noninfectious, empty IBV particles. Such particles have not yet been identified for other coronaviruses. The virions of coronaviruses are rather fragile and tend to disrupt upon storage and/or during the negative-staining procedure (Apos- MOLECULAR BIOLOGY OF CORONAVIRUSES 41 tolov et al., 1970). This can be prevented by fixation of virions with glutaraldehyde prior to negative staining. In spontaneously disrupted preparations of coronaviruses, fragments of viral envelopes are fre- quently seen. However, the internal component of coronaviruses has been more difficult to visualize in negatively stained preparations. Thin sections of infected cells or virions demonstrated a flexible, cylin- drical nucleocapsid which was probably helical (see Section 111,F). Kennedy and Johnson-Lussenberg ( 1975-1976) showed that thread- like nucleocapsids, 8-9 nm in diameter, were released from dis- rupted HCV-229E virions. These were very pleiomorphic and may rep- resent strands which had uncoiled from helical nucleocapsids. The most tightly coiled helical nucleocapsids were obtained from virions which were spontaneously disrupted by storage at 25°C overnight. Macnaughton et al. (1978) found hollow, helical nucleocapsids, 14-16 nm in diameter and up to 0.32 pm long, released from purified prepa- rations of HCV-229E and MHV-3 virions. The unit length of coro- navirus nucleocapsids has not yet been determined, although nu- cleocapsids up to 6 pm long have been observed (Davies et al., 1981). Caul et al. (1979) purified helical nucleocapsids from HCV-229E (Fig. 1B). The nucleocapsid was about 9-11 nm in diameter. Coronavirus nucleocapsids appeared to be more flexible and easier to uncoil than paramyxovirus nucleocapsids. Recent studies of helical nucleocapsids of negative-stranded RNA viruses (Heggeness et al., 1980, 1982) showed that these properties depended upon the ionic strength and cation composition of the buffers. It appears likely that the appropriate conditions for further characterization of the structure of coronavirus nucleocapsids will be identified in the near future. In addition to permitting analysis of the viral nucleocapsid, disrup- tion of coronaviruses with nonionic detergents such as NP-40 or Triton X-100 has permitted the isolation of the envelope glycoproteins of coro- naviruses (Section 11,G). The morphology of the two envelope glycopro- teins of MHV isolated from detergent-disrupted virions on sucrose den- sity gradients is shown in Fig. 1C and 1D (Sturman et al., 1980). B. Virus Growth and Purification Early studies of coronaviruses were hampered by limited virus growth and difficulties with virus purification. Coronaviruses exhib- ited restricted host ranges in cell culture, low virus yields were usually obtained, and the viruses were highly labile. These difficulties have now been overcome for many coronaviruses (Siddell et al., 1982). Suit- able permissive cell types and virus strains have been identified and conditions have been described which result in greater virus stability. Many human coronaviruses were first identified by their growth and 42 LAWRENCE S . STURMAN AND KATHRYN V. HOLMES cytopathogenicity in human embryonic tracheal and nasal organ cul- tures (Tyrrell and Bynoe, 1965; Almeida and Tyrrell, 1967; McIntosh et al., 1967a). Some of these viruses have not yet been grown in contin- uous cell lines, and great difficulties are still encountered with prima- ry isolation of HCVs. Although permissive cell hosts have been de- scribed for HCV-229E (Hamre and Procknow, 1966; Hamre et al., 1967) as well as for tissue culture-“adapted” HCV-OC43 (Bradburne and Tyrrell, 1969; Kapikian et al., 1969; Bruckova et al., 1970; Gerna et al., 1979; Macnaughton et al., 1980), virus yields of lo8 PFU per millili- ter. Two additional transformed cell lines have also been found to produce high yields of MHV: DBT, a Schmidt-Ruppin Rous sarcoma virus-induced mouse tumor cell line (Hirano et al., 1974, 1976, 1978), and Sac (-1, a Maloney sarcoma virus-transformed mouse cell line which is defective in retrovirus production (Spaan et al., 1981). In contrast to HCV and MHV, no continuous cell line is available which produces large amounts of IBV. Limited growth of a few strains of IBV has been obtained in VERO and BHK-21 cells (Cunningham et al., 1972; Coria and Ritchie, 1973; Otsuki et al., 1979). However, high yields of some strains of IBV have been obtained in primary chick embryo kidney cells (Otsuki et al., 1979; Stern and Kennedy, 1980a). Another development which facilitated progress in this field was the recognition that coronaviruses are most stable between pH 6.0 and 6.5 (Pocock and Garwes, 1975; Alexander and Collins, 1975; Sturman, 1981) (Fig. 2). At pH 6.0, the half-life of MHV infectivity at 37°C in the presence of 10% fetal bovine serum was 24 hours, whereas at pH 8.0, a 50% loss in virus infectivity occurred in less than 1 hour. The rapid loss MOLECULAR BIOLOGY OF CORONAVIRUSES 43 0 001 3 4 5 6 7 8 PH FIG. 2. pH dependennce of thermal inactivation of MHV. Purified virions of MHV were diluted in buffer at different pHs and incubated for 24 hours at 4" (0) or 37°C (0). Stability of viral infectivity is shown as the ratio of viral titer at 24 hoursititer at 0 time, x100. At 4°C the virus is quite stable from pH 4 to 10. However, at 37°C the virus exhibits marked thermolahility at pHs c4.5 and >6.5. (Reproduced from Sturman, 1981, with permission.) of infectivity at pH 8.0 was associated with aggregation of the peplo- meric glycoprotein (Sturman, 1981). C. Structural Proteins Several unique features of the coronavirus glycoproteins, as well as the lack of suitable permissive cell types for some coronaviruses and residual host-cell contamination of other coronaviruses, delayed recog- nition that coronaviruses all possess a similar pattern of structural proteins. Recently, the general organization of coronavirus structural proteins has become apparent. Figure 3 illustrates a model of the structure of MHV-A59. We will use this model and the nomenclature developed during our studies of the structural proteins of MHV for the following discussion of coronavirus structural proteins and their organization. 44 LAWRENCE S. STURMAN AND KATHRYN V. HOLMES A UNTREATED VIRUS B PRONASE- OR BROMELAIN-TREATED VIRUS I C VIRUS FROM TUNICAMVCIN-TREATED CELLS FIG. 3. Model for the structure of the coronavirus MHV. This model is based on studies of the proteins and nucleic acid of MHV virions. (A) The envelope of the intact virions contains two envelope glycoproteins, E l and E2, in a lipid bilayer. The helical nucleocapsid is composed of a single long strand of message sense, genomic RNA with the nucleocapsid protein, N. Glycosaminoglycan is associated with the viral envelope. (B) Treatment of the virions with Pronase or bromelain removed the bulk of E2, a 5K glycosylated portion of E 1, and the glycosaminoglycan, but leaves the nucleocapsid intact. (C) Virions released from cells treated with tunicamycin lack E2 but contain normal amounts of glycosylated E l and nucleocapsid. 1. Mammalian Coronaviruses In 1975, Garwes and Pocock characterized the structural polypep- tides of TGEV, a porcine coronavirus. Although analysis of coro- navirus polypeptides had been attempted earlier, those studies were less definitive since they utilized virus produced in animals or eggs which may have been contaminated with some host-cell components (Hierholzer et al., 1972; Bingham, 1975). Working with radiolabeled TGEV produced in cell culture, Garwes and Pocock identified four major polypeptide peaks on sodium dodecyl sulfate (SDS)-polyacryl-