Coronavirus mRNA transcription: UV light transcriptional mapping studies suggest an early requirement for a...

Mouse hepatitis virus (MHV) synthesizes seven to eight mRNAs, each of which contains a leader RNA derived from the 5' end of the genome. To understand the mechanism of synthesis of these mRNAs, we studied how the synthesis of each mRNA was affected by UV irradiation at different time points after infection. When MHV-infected cells were UV irradiated at a late time in infection (5 h postinfection), the syntheses of the various mRNAs were inhibited to different extents in proportion to the sizes of the mRNAs. Analysis of the UV inactivation kinetics revealed that the UV target size of each mRNA was equivalent to its own physical size. In contrast, when cells were irradiated at 2.5 or 3 h postinfection, there appeared to be two different kinetics of inhibition of mRNA synthesis: the synthesis of every mRNA was inhibited to the same extent by a small UV dose, but the remaining mRNA synthesis was inhibited by additional UV doses at different rates for different mRNAs in proportion to RNA size. The analysis of the UV inactivation kinetics indicated that the UV target sizes for the majority of mRNAs were equivalent to that of the genomic-size RNA early in the infection. These results suggest that MHV mRNA synthesis requires the presence of a genomic-length RNA template at least early in the infection. In contrast, later in the infection, the sizes of the templates used for mRNA synthesis were equivalent to the physical sizes of each mRNA. The possibility that the genomic-length RNA required early in the infection was used only for the synthesis of a polymerase rather than as a template for mRNA synthesis was ruled out by examining the UV sensitivity of a defective interfering (DI) RNA. We found that the UV target size for the DI RNA early in infection was much smaller than that for mRNAs 6 and 7, which are approximately equal to or smaller in size than the DI RNA. This result indicates that even though DI RNA and viral mRNAs are synthesized by the same polymerase, mRNAs are synthesized from a larger (genomic-length) template. We conclude that a genomic-length RNA template is required for MHV subgenomic mRNA synthesis at least early in infection. Several transcription models are proposed.