RNA-dependent RNA Polymerase Activity in Murine Coronavirus-infected Cells

Abstract

SUMMARYThe multiplication of murine coronavirus strains A59 or JHM in Sac(-) cells was unaffected by the presence of ~-amanitin at concentrations which inhibited the host cell DNA-dependent RNA polymerase activity. In cells infected with the A59 virus strain, actinomycin D-resistant RNA synthesis could readily be detected by pulselabelling with [3H]uridine; this virus-specific RNA synthesis was not induced in the presence of the protein synthesis inhibitor anisomycin. A new RNA-dependent RNA polymerase activity was detected in the large particle fraction of A59 virus-infected cells. Optimal conditions for enzyme activity in vitro were established. Maximum activity occurred 5 h after infection, coincident with the peak of virus-specific RNA synthesis detected by pulse-labelling in vivo.

Coronaviruses: Structure and Genome Expression

“…Brayton et al (1982Brayton et al ( , 1984 have described two enzymicaUy distinct RNA polymerase activities (acting early and late during infection); the early polymerase was involved in negative-strand RNA synthesis, whereas the late polymerase synthesized positive-stranded RNA. In contrast, only one polymerase activity was detected by Mahy et al (1983) and Compton et al (1987). Inhibition of in vitro RNA transcription by antibody to the N protein suggests a role in coronavirus replication (Compton et al, 1987); whether this inhibition reflects a direct function of N in RNA synthesis or is the result of rapid RNA degradation is not known.…”

“…A. M. van der Zeijst, personal communication). An RNAdependent RNA polymerase activity has been detected in TGEV-and MHV-infected cells (Dennis & Brian, 1982;Brayton et al, 1982;Mahy et al, 1983). Brayton et al (1982Brayton et al ( , 1984 have described two enzymicaUy distinct RNA polymerase activities (acting early and late during infection); the early polymerase was involved in negative-strand RNA synthesis, whereas the late polymerase synthesized positive-stranded RNA.…”

The Biology of Coronaviruses

“…It is unlikely, however, that this 'leader' sequence is produced by a conventional splicing mechanism. The replication of MHV is not impaired in cells treated with actinomycin D or c~-amanitin (Brayton et al, 1981;Lai et al, 1981;Mahy et al, 1983;Spaan et al, 1981) and replication has been reported in enucleated cells (Brayton et al, 1981 ;Wilhelmsen et al, 1981). Also, the synthesis of each mRNA is inactivated by u.v.…”

“…For MHV, the messenger function of these positive-stranded RNA species has been demonstrated in vitro (see Coronavirus-directed protein synthesis). No structural difference between the genome RNA and genome-sized intracellular mRNA has been described (Brayton et al, 1982;Cheley et al, 1981 a;Dennis & Brian, 1982;Jacobs et al, 1981 ;Lai et al, 1981Lai et al, , 1982aLeibowitz et al, 1981 ;Mahy et al, 1983 ;Spaan et al, 1981Spaan et al, , 1982Stern & Kennedy, 1980a, b;Wege et al, 1981b, c;Weiss & Leibowitz, 1982).…”

“…In addition to the proteins described above, virus-specific RNA polymerase activities have been isolated from coronavirus-infected cells, although their polypeptide components have not been identified either in vivo or in vitro (Brayton et al, 1982;Dennis & Brian, 1982;Mahy et al, 1983). Usually, these activities have been isolated late in infection and their membrane location and association with replicative intermediate RNA has been demonstrated.…”