The genome of Coronaviruses encodes not only structural proteins required for the formation of viral particles, but also for non-structural proteins (nsp’s) which are required for viral replication. Some of the latter are involved in the formation of viral replication centers (RTCs), particularly nsp-3/-4/-6, while others are involved in processing the viral orf1ab polyprotein, interfering with the cellular antiviral response, or with facilitating the replication of the viral RNA.
During viral replication, following the cleavage of the viral orf1ab polyprotein by viral proteases, the viral nsp’s assemble at the ER into a multienzyme complex which is associated with viral RNA and double membrane vesicles that derive from the ER and whose formation is induced by the viral nsp-3, -4, and -6 proteins as described before. As described before, CoV replicate in the cytoplasm of infected cells although an involvement of the nucleus cannot be ruled out. Consequently, the viral positive strand ssRNA genome is transcribed into a negative sense RNA and subsequently into full length and subgenomic RNAs by the viral RNA dependent RNA polymerase (RdRP) in the cytoplasm and thus (1) not protected from degradation by cellular 5’ to 3” exoribonucleases, (2) subject to recognition by cellular proteins such as Toll-like receptors which are part of the innate immune response and (3) are not efficiently translated. Furthermore, due to the lack of RNA helicases, dsRNA intermediates formed would prevent translation of viral subgenomic RNAs. Consequently, a subset of the nsp’s encoded are exhibiting RNA helices, 3’ to 5’ exoribnuclease (ExoN) activity as well as Methyltransferase (MT) activity. Whilst a number of RNA viruses express proteins with ExoN and MT activity are well characterised, in the case of CoV the roles of the viral nsp-14 and -15 proteins only begin to emerge. CoV nsp-14 is a bifunctional protein, where the 3’ to 5’ ExoN activity is localised within the N-terminus and the guanine-N7-methyltransferase (N7-MTase) activity is located with the C-terminal domain; both activities however depend on each other, i.e. mutations rendering the ExoN domain inactive also inhibit the N7-MTase activity. Functionally, nsp-14 has been postulated to remove excise 3′-end mismatched nucleotides from the dsRNA intermediate synthesised by the viral RdRP (nsp-12), which is enhanced by binding of a co-factor, nsp-10, and nsp-10 mutant which do not bind nsp-14 fail to stimulate nsp-14 activity. Aside from the proofreading mechanism, the 3′–5′ ExoN domain is also involved in the degradation of viral dsRNA replication intermediates and thus may inhibit the induction of the cellular type I Interferon response akin to Lassa Fever virus nucleoprotein. In contrast to the ExoN activity, N7-MTase activity is not activity is not affected by nsp-10.
In addition to nsp-14, nsp-10 also binds to nsp-16. In contrast to nsp-14, nsp-16 however is solely involved in RNA capping, more precisely in converting the 7MeGpppN cap (cap 0) generated by the N7-MTase activity of nsp-14 into a cap-1 structure via a 2O-Methyltransferase activity, a step that enhances the translation efficiency of the viral RNA. Indeed, mutations of nsp-10 preventing the interaction with nsp-14 inhibit the ExoN activity of nsp-14 whereas failure to interact with nsp-16 only has moderate effect on viral replication. nsp-14 also interacts with a complex of nsp-7 and -8, thus forming a tripartite complex, which in turn binds to nsp-14 as described above. Functionally, the nsp-7/-8 complex is a primate, i.e. a primer independent RNA Polymerase synthesising primer sequences utilised by the viral RdRP.
|Coronavirus replication complex|
Both nsp-7 and -8 also bind to nsp-12/RdRP and again this tripartite complex interacts with nsp-14, thus leading to the assembly of a complex which not only allows synthesis of the viral RNA but also a proofreading mechanism as well as capping the nascent RNA and thus protecting viral RNA from being degraded and recognised by the cellular pattern recognition receptors.
Consequently, mutations of the conserved D/ExD/E site rendering ExoN inactive result in viable mutant which exhibit a substantial increase in the mutation rate of the viral genome with a decrease in viral titers. Mutations of nsp-16 or nsp-10 likewise lead to a decrease in viral titers (albeit less pronounced than nsp-14 mutants) or non-viable viruses. Furthermore antiviral drugs targeting conserved residues within the proteins that form part of the complex might provide treatment not only during infections with currently circulating Coronaviruses but also for novel and emerging Coronaviruses in humans as well as animals.
Finally, how is the replication complex target to the double membrane vesicles induced by the expression of nsp-3, -4, and-6? In murine DBT cells transfected with both nsp-4 and nsp-8 both proteins localise to the ER and SARS-CoV nsp8 co-localises with p6 in the perinuclear region (presumably the ER). In addition, nsp-4 co-localises with nsp-8 in cells infected with MHV-A59. These results suggest that nsp-8 is recruited to the ER by interacting with nsp-4 and maybe SARS-CoV p6. nsp-8 then might recruit nsp-7, nsp-12, nsp-10, and nsp-14 (and viral RNA) prior to the formation of the RTC; whether viral RNA is required or not - that remains to be seen.
|Recruitment of nsp-7/-8 by nsp-4 and /or p6 initiates the replication of viral RNA|
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