Coronaviruses (CoVs) are important pathogens for human and vertebrates. They can infect respiratory, gastrointestinal, hepatic, and central nervous system of human, livestock, birds, bat, mouse and many other wild animals. The outbreaks of the severe acute respiratory syndrome (SARS) in 2002/2003 and the Middle East respiratory syndrome (MERS) in 2012 have demonstrated the possibility of animal-to-human and human-to-human transmission of newly emerging CoVs. An outbreak of mystery pneumonia in Wuhan since Desember 2019 has been drawing tremendous attention around the world.
- In vitro reconstitution of the RTC core complex will allow the study of the coronavirus proofreading mechanism, the temporal or spatial regulation of proofreading and capping activities, which share several viral components, and the role of N protein RNA chaperone activity
- Understanding the interaction of cell and viral protein within the nucleus, and of nuclear proteins traveling to the cytoplasm to interact with viral factors, may provide novel avenues to clarify coronavirus replication
- It is still unknown whether replication and transcription are simultaneous or sequential processes
- Further research is required on cis-acting elements involved in replication and transcription, and on the viral and cellular proteins that bind them
- The function and dynamics of DMVs and CMs and the precise localization of the sites of active viral RNA synthesis remain unresolved questions
- The contribution of cytoplasmic RNA-protein complexes containing viral RNAs, such as stress granules, to the regulation of coronavirus RNA expression requires further research
- Limited information is available on the temporal regulation of viral translation, replication, and transcription over the course of infection and on how switching between these processes occurs
- Coronavirus cis-acting RNA elements involved in RNA synthesis are mainly located in the highly structured 5′ and 3’UTRs
- The replicase proteins nsp7, nsp8, nsp12, and nsp14 may constitute an RTC core complex
- Coronaviruses encode a proofreading machinery, unique among the RNA viruses, to ensure the maintenance of their large genome size. The ExoN activity of nsp14 is a key element of the proofreading system
- Coronavirus RNA synthesis is associated with extensive modification of intracellulr membranes, including DMVs and CMs
- The requirement of the nucleus for coronavirus replication is variable, but optimum levels of progeny are obtained only in its presence. Several coronavirus proteins involved in RNA synthesis travel to the nucleus. Conversely, many nuclear proteins are transported to the cytoplasm to facilitate coronavirus RNA synthesis.
- Coronavirus express their 3′-proximal ORFs through a collection of overlapping, nested sgmRNAs generated by a mechanism of discontinuous transcription unique among RNA viruses. This process includes a template switch during the synthesis of negative-strand sgRNAs to add a copy of the leader sequence
- Coronavirus transcription is regulated by multiple factors, including the extent of base-pairing between the complement of the TRS-B in the nascent negative strand and the TRS-L as well as protein RNA and RNA-RNA interactions. Moreover, coronavirus N protein RNA chaperone activities essential for efficient transcription.
It is speculated that the encapsidated RTC could act as a starting replication machinery, with a round of genome amplification before translation leading to improved efficiency of virus infection. Further studies are required to investigate whether other viral and cellular components of the RTC are also encapsidated and what biological role they play in the coronavirus life cycle.
Encapsidation of the coronavirus replication-transcription complex
It is currently accepted that, unlike that in negative-strand RNA viruses, the RTC in positive-strand RNA viruses generally is not incorporated into viral particles. However, recent studies based on proteomic, biochemical, and immunoelectron microscopy assays reported the presence of RdRp, nsp2, nsp3, and nsp8 in TGEV particles and nsp2, nsp3, and nsp5 in SARS-CoV particles. These data suggest that the RTC might be encapsidated in coronaviruses.
Interestingly, whereas nsp10 binding has no effect on nsp14 N7-MTase activity, nsp10 required for nsp16 2-0-MTase activity. These data, in conjunction with those in the preceding section, highlight the importance of nsp10 as modulator of two different activities in the coronavirus proofreading and capping machinery.
Coronavirus RNA capping pathway
Capping of viral RNAs by conventional or unconventional pathways leads to 5′-end cap structures that allow efficient viral protein synthesis and, in many cases, escape from the innate immune system. Coronaviruses follow the canonical capping pathway, which consists of four sequential enzymatic reactions:
- RTPase, encoded by the nsp13 helicase, hydrolyzing the γ-phosphate of the mRNA
- an as-yet-unidentified guanyltransferase (GTase) adding GMP to the 5′-diphosphate RNA
- nsp14 N7-MTase methylating the guanosine, leading to a cap-0 structure that is essential for efficient translation initiation
- nsp16 2′-0-methyltransferase (2′-0-MTase) carrying out further methylations, leading to cap-1 and cap-2 structures, which are required to efficiently escape the nonself RNA recognition system of the host cell.