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.

FIG. 1

Schematic diagram of the structure of genomic MIGCAT RNA and subgenomic MIGCAT RNA. The locations of leader sequences, an intergenic sequence, and CAT sequences are indicated. Oligonucleotides used for RT-PCR and the preparation of riboprobe 1, competitor A, and competitor B are shown by open arrowheads. T7 Pr, the T7 promoter sequence present at the 5′ ends of oligonucleotides 10319 and 10404. The short boldface line between the open arrowhead and the T7 promoter sequence in oligonucleotide 10319 represents a unique sequence that does not hybridize with either MIGCAT or MHV RNA sequences. Structures of probes used in the present study are also shown; the positions of riboprobe 1 and competitor A relative to genomic MIGCAT RNA and the positions of DNA probe 1 and competitor B relative to subgenomic MIGCAT RNA are shown. Riboprobe 1, competitor A, and competitor B are complementary to positive-strand MIGCAT-specific RNAs. Nucleotide numbers shown for these probes start from the beginning of the CAT sequence.

FIG. 2

Accumulation of positive-strand MIGCAT-specific RNAs. (A) Northern blot analysis of MIGCAT-specific RNAs. Total intracellular RNAs were extracted from MIGCAT DI particle-infected cells at the times shown above the gel. The sample at 0 h p.i. represents intracellular RNAs that were extracted immediately after 30 min of virus adsorption at 0°C. ³²P-labeled DNA probe 1 was used as a probe. (B) RNase protection assay of genomic MIGCAT RNA and subgenomic MIGCAT RNA. Heat-denatured intracellular RNA and radiolabeled riboprobe 1 (see Fig. 1) were hybridized and then treated with RNase A and RNase T₁. Riboprobe 1 fragments that were protected from RNase digestion were detected by separating the sample on 6% sequencing gels. Lanes 1 and 2 represent intracellular RNAs from non-MHV-infected and MHV-infected cells, respectively. Lane 8 is the same as lane 4, except that RNase treatment was omitted. G, genomic MIGCAT RNA; SG, subgenomic MIGCAT RNA.

FIG. 3

Accumulation of negative-strand MIGCAT-specific RNAs. (A) Southern blot analysis of RT-PCR of negative-strand genomic MIGCAT RNA. Total intracellular RNAs were extracted from MIGCAT DI particle-infected cells at the times shown above the gel. Oligonucleotide 10124 was used for cDNA synthesis, and PCR amplification was performed by using oligonucleotides 10124 and 10403. PCR products were separated by agarose gel electrophoresis, and Southern blot analysis was performed by using ³²P-labeled CAT-specific probe DNA probe 1. For lane 13, RNA was extracted at 9 h p.i. from MHV-infected cells that did not contain MIGCAT DI RNA; for lane 14, RNA was extracted at 9 h p.i. from mock-infected cells. G, negative-strand genomic MIGCAT RNA-specific PCR products. (B) Southern blot analysis of RT-PCR of negative-strand subgenomic MIGCAT RNA. Total intracellular RNAs were extracted from MIGCAT DI particle-infected cells at the times shown above the gel. Oligonucleotide 10066 was used for cDNA synthesis, and PCR amplification was performed by using oligonucleotides 10066 and 10403. PCR products were separated by agarose gel electrophoresis, and Southern blot analysis was performed by using ³²P-labeled DNA probe 1. For lane 13, RNA was extracted at 9 h p.i. from MHV-infected cells that did not contain MIGCAT DI RNA; from lane 14, RNA was extracted at 9 h p.i. from mock-infected cells. SG, negative-strand subgenomic MIGCAT RNA-specific PCR products. (C) Southern blot analysis of RT-PCR of negative-strand subgenomic MIGCAT RNA with serial dilutions. Total intracellular RNAs were extracted from MIGCAT DI particle-infected cells at 3 h p.i. Total intracellular RNA was diluted 10-, 25-, or 100-fold and mixed with a constant amount of intracellular RNA extracted from MHV-infected cells at 3 h p.i. Oligonucleotide 10066 was used for cDNA synthesis, and PCR amplification was performed by using oligonucleotides 10066 and 10403. PCR products were separated by agarose gel electrophoresis, and Southern blot analysis was performed by using ³²P-labeled DNA probe 1. SG, negative-strand subgenomic MIGCAT RNA-specific PCR products.

FIG. 4

Comparative RT-PCR of negative-strand MIGCAT-specific RNAs. (A) Total intracellar RNAs that were extracted from MIGCAT DI particle-infected cells at 2 (lanes 1 to 6) or 3 (lanes 8 to 13) h p.i. were mixed with serially diluted competitor A RNA. RT-PCR was performed to coamplify negative-strand genomic MIGCAT RNA and competitor A RNA. The accumulation of RT-PCR products was demonstrated by Southern blot analysis using ³²P-labeled DNA probe 1. Lanes 1 and 8 lack competitor A RNA, while lanes 7 and 14 lack intracellular RNA. (B) Total intracellular RNAs that were extracted from MIGCAT DI particle-infected cells at 3 h p.i. were mixed with serially diluted competitor B RNA. RT-PCR was performed to coamplify negative-strand subgenomic MIGCAT RNA and competitor B RNA. The accumulation of RT-PCR products was demonstrated by Southern blot analysis using ³²P-labeled DNA probe 1. Lane 1 and lane 7 lack competitor B RNA and intracellular RNA, respectively. The amounts of competitor A and competitor B RNA in the undiluted samples in panel A (lanes 7 and 14) and panel B (lane 7) were identical.