Coronavirus JHM: Cell-Free Synthesis of Structural Protein p60

Abstract

Sac(-) cells infected with murine coronavirus strain JHM shut off host cell protein synthesis and synthesized polypeptides with molecular weights of 150,000, 60,000, and 23,000. The 60,000-and 23,000-molecular-weight polypeptides comigrated with virion structural proteins p60 and p23, and the 60,000-molecularweight protein was identified as p60 by tryptic peptide fingerprinting. Polyadenylate-containing RNA [poly(A) RNA] extracted from the cytoplasm of infected cells directed the synthesis of both 60,000-and 23,000-molecular-weight polypeptides in messenger-dependent cell-free systems derived from mouse L-cells and rabbit reticulocytes. The reticulocyte system also synthesized a 120,000-molecular-weight polypeptide that was specifically immunoprecipitated by antiserum raised against JHM virions. The identity of the 60,000-and 23,000-molecularweight in vitro products was established by comigration with virion proteins, immunoprecipitation, and in the case of p60, tryptic peptide fingerprinting. The cytoplasmic poly(A) RNAs which encoded p60 and p23 sedimented in sucroseformamide gradients at 17S and 19S, respectively, and were clearly separable. These RNAs were among the major poly(A) RNA species synthesized in the cytoplasm of actinomycin D-treated cells late in infection, and the in vitro translation of size-fractionated RNA released from polysomes confirmed that they represent physiological mRNA's. These results suggest that the expression of the coronavirus JHM genome involves more than one subgenomic mRNA. Coronaviruses cause a variety of diseases in both animals and humans. They are associated with respiratory and enteric diseases in humans, bronchitis in birds, transmissible gastroenteritis and encephalitis in pigs, and demyelinating encephalitis and hepatitis in rodents (11, 20; J. A.

The virus-specific intracellular RNA species of two murine coronaviruses: MHV-A59 and MHV-JHM

“…They are polyadenylated, as expected of mRNAs, and Robb and Bond (1979) have shown that multiple MHV-specific RNA species spanning the size range of the subgenomic RNAs 2-7 are present on polysomes of infected cells. Furthermore, Siddell et al (1980) have recently demonstrated that polyadenylated RNA isolated from JHMV-infected cells can be translated in vitro. Using sucrose gradients they were able to partially resolve 1'7 and 19 S JHMV-specific RNA species.…”

“…In contrast to the data obtained on coronavirus virion RNA, few data have been published on intracellular coronavirusspecific RNA. Mishra and Ryan (1973) reported that porcine kidney cells infected with transmissible gastroenteritis virus (TGEV) contained actinomycin D-resis-tent RNA species which sedimented between 18 and 28 S. Robb and Bond (1979b) have studied murine hepatitis virus (MHV)-infected cells and found that deproteinized RNA from virus-specific polysomes sedimented between 10 and 28 S. RNA in this size range was recently demonstrated to code for two MHV structural proteins (Siddell et al, 1980). Stern and Kennedy (1980a,b) have identified six virus-specific RNA species in cells infected with avian infectious bronchitis virus (IBV).…”

Viral and Cellular mRNA Translation in Coronavirus-Infected Cells

“…and peaks at 6 h p.i. (Siddell et al, 1980(Siddell et al, , 1981a. An increased number of free 80S ribosomes and a shift to lighter polysomes during MHV infection suggest a potential suppression at the translation initiation phase (Anderson and Kedersha, 2009;Siddell et al, 1981b).…”

“…Being obligate intracellular parasites, viruses heavily depend on host cell structures and functions to complete their life cycle, and they also use the translational apparatus of the infected cell to express their proteins. In several cases, viruses have been shown to affect and/or modulate the status of the host translational machinery to achieve efficient viral protein synthesis and replication, while cellular mRNA translation is inhibited (Hilton et al, 1986;Narayanan et al, 2008a;Siddell et al, 1980Siddell et al, , 1981a. In eukaryotic cells, translation occurs in the cytoplasm and essentially involves four steps: initiation, elongation, termination, and recycling (Kapp and Lorsch, 2004).…”

Sequencing of coronavirus IBV genomic RNA: a 195-base open reading frame encoded by mRNA B

“…Both IBV and MHV contain three main structural polypeptides: the nucleocapsid, the membrane (El), and the spike or peplomer (E2) polypeptides (Cavanagh, 1981;Siddell et al, 1983). In both systems the smallest RNA codes for the nucleocapsid protein but in MHV the next smallest RNA codes for the membrane polypeptide, whereas in IBV the membrane polypeptide is coded for by the third smallest RNA (Siddell et al, 1980;Siddell, 1983;Rottier et al, 1981;Stern et al, 1982;Stem and Sefton, 1984). These differences are summarised in Fig.…”