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RNA sequences regulating transcription

The transcription process is controlled by transcription-regulating sequences (TRSs) located at the 3′ end of the leader sequence (TRS-L) and preceding each viral gene (TRS-B). TRSs include a conserved core sequence (CS) 6-7 nt in length  and variable 5′ and 3′ flanking sequences (the 5’TRS and 3’TRS , respectively). Because the CS is identical for the genome leader (CS-L) and all mRNA  coding sequences (CS-B), the CS-L could base-pair with the nascent negative strand complementary to each CS-B (cCS-B), allowing for leader-body joining.

Moreover, as noted below, the complement of the leader sequence supports initiation of positive-strand RNA synthesis, making the negative-strand subgenomic RNAs (sgRNAs) a template for further amplification of positive-strand sgmRNAs.

The observation raises the question of whether the presence of the leader sequence in coronavirus sgmRNAs provides any selective advantage to the virus. The presence of the 5′ leader sequence was shown to protect SARS-CoV mRNAs from nsp1-induced endonucleolytic cleavage of capped mRNAs, providing a strategy for the efficient accumulation of viral mRNAs and viral proteins during infection.

The transcription mechanismin coronaviruses is seemingly complicated as compared with the transcription mechanisms in other positive-strand RNA viruses, such as internal initiation and premature termination. In fact, in contrast to coronavirus and arterivirus sgmRNAs, subgenomic transcripts of other Nidovirales, such as toroviruses and roniviruses, do not have a common 5′ leader sequence.

In contrast to replication, coronavirus transcription includes a discontinuous step during the production of sgmRNA. This process, unique among known RNA viruses, is a hallmark of the order Nidovirales and ultimately generates a nested set of sgmRNAs that are 5′ and 3′ coterminal with the virus genome. These sgmRNAs all include at their 5′ end a common leader sequence, whose length ranges from 65 to 98 nt in different coronaviruses. This common leader sequence is present only once at the very 5′ end of the genome, which implies that sgmRNAs are synthesized by the fusion of noncontiguous sequences,  the leader and the 5′ end of each mRNA coding sequence, called the body (B).

Like that of other positive-strand RNA viruses, coronavirus genome replication is a process of continuous synthesis that utilizes a full-length complementary negative-strand RNA as the template for the production of progeny virus genomes. The initiation  of negative-strand synthesis involves access of the RNA-dependent RNA polymerase (RdRp) to the 3’terminus of the genome, promoted by 3′-end RNA sequences and structures. There is evidence that both 5′- and 3′- end RNA elements are required for the production of progeny  positive-strand  RNA from the intermediate negative-strand RNA, suggesting that interactions between the 5′ and 3′ ends of the genome contribute to replication.

Coronavirus RNA-dependent RNA synthesis includes two differentiated processes: genome replication, yielding multiple copies of genomic RNA (gRNA), and transcription of a collection of sgmRNAs that encode the viral structural and accessories proteins.