Coronavirus Transcription Early in Infection

Abstract

We studied the accumulation kinetics of murine coronavirus mouse hepatitis virus (MHV) RNAs early in infection by using cloned MHV defective interfering (DI) RNA that contained an intergenic sequence from which subgenomic DI RNA is synthesized in MHV-infected cells. Genomic DI RNA and subgenomic DI RNA accumulated at a constant ratio from 3 to 11 h postinfection (p.i.) in the cells infected with MHV-containing DI particles. Earlier, at 1 h p.i., this ratio was not constant; only genomic DI RNA accumulated, indicating that MHV RNA replication, but not MHV RNA transcription, was active during the first hour of MHV infection. Negative-strand genomic DI RNA and negative-strand subgenomic DI RNA were first detectable at 1 and 3 h p.i., respectively, and the amounts of both RNAs increased gradually until 6 h p.i. These data showed that at 2 h p.i., subgenomic DI RNA was undergoing synthesis in the cells in which negative-strand subgenomic DI RNA was undetectable. These data, therefore, signify that negative-strand genomic DI RNA, but not negative-strand subgenomic DI RNA, was an active template for subgenomic DI RNA synthesis early in infection.

Nascent Synthesis of Leader Sequence-Containing Subgenomic mRNAs in Coronavirus Genome-Length Replicative Intermediate RNA

“…Although the present study and the previous study (16) established that negative-strand genomic RNA is a template for MHV subgenomic mRNA synthesis throughout the infection, these studies do not eliminate the possibility that MHV subgenomic mRNA is also synthesized from subgenomic negative-strand template RNA late in infection. A number of studies aimed to test whether positive-strand RNA synthesis occurs using subgenomic negative-strand template RNA in cells infected with coronaviruses (8,(12)(13)(14) and with arterivirus (15), yet did not provide direct evidence of positive-strand RNA elongation from the subgenomic-length negative-strand template.…”

“…Although the previous study using the column-purified genome-length RI (11) and the present study used different experimental approaches to examine the nascent RNAs in the genome-length RI, the data from these two independent studies indicated that MHV genome-length negative-strand RNA is the template RNA for subgenomic mRNA synthesis. We showed that negative-strand genomic RNA is the template for subgenomic mRNA synthesis early in infection (16). Our new data strongly support the idea that negative-strand genomic RNA is a template for subgenomic mRNA synthesis throughout the infection.…”

“…One suggests that a genomelength negative-strand RNA serves as a template for all the mRNAs (11), while the other model proposes that each negative-strand subgenomic RNA is a unique template for synthesis of the correspondingly sized subgenomic mRNA (8,(12)(13)(14)(15). A study using mouse hepatitis virus (MHV), a prototypic coronavirus, demonstrated that early in infection negative-strand genomic RNA, and not negative-strand subgenomic RNA, is the template for subgenomic mRNA synthesis (16); this study did not determine the template late in infection.…”

Importance of coronavirus negative-strand genomic RNA synthesis prior to subgenomic RNA transcription

“…In the control group, the DBT cells were coinfected with helper MHV-A59 and UV-irradiated P2 sample. MHV RNA replication activities, but not transcription activities, were active very early in infection (An et al, 1998) therefore, we expected that MIGCAT genomic DI RNA replication, but not transcription, would start immediately after coinfection in the control group; we expected to see similar susceptibility to UV-light irradiation of MIGCAT subgenomic and genomic DI RNAs, because subgenomic-size MIGCAT DI RNA should be synthesized after replication of genomic MIGCAT DI RNA. Intracellular RNAs were extracted 7 h p.i.…”

Murine Coronavirus Replication-Induced p38 Mitogen-Activated Protein Kinase Activation Promotes Interleukin-6 Production and Virus Replication in Cultured Cells

“…Mouse hepatitis virus (MHV) is one of the most well-characterized coronaviruses. After MHV infection, MHV RNA synthesis, which involves synthesis of a genome-length, negative-strand RNA template (2,4,42) and subsequent synthesis of seven, mature mRNA species (31), takes place in the cytoplasm. MHV particles, which contain three envelope proteins (S, M, and E) and an internal helical nucleocapsid, which consists of N protein and genomic RNA, buds from internal cellular membranes (25,37,61).…”