Virology tidbits

Virology tidbits

Tuesday, 7 October 2014

Coronavirus antivirals: RNA replication machinery as an antiviral target?

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 2􏰃O-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

Further reading 

Prentice E, McAuliffe J, Lu X, Subbarao K, & Denison MR (2004). Identification and characterization of severe acute respiratory syndrome coronavirus replicase proteins. Journal of virology, 78 (18), 9977-86 PMID: 15331731

Jin X, Chen Y, Sun Y, Zeng C, Wang Y, Tao J, Wu A, Yu X, Zhang Z, Tian J, & Guo D (2013). Characterization of the guanine-N7 methyltransferase activity of coronavirus nsp14 on nucleotide GTP. Virus research, 176 (1-2), 45-52 PMID: 23702198 

Bouvet M, Lugari A, Posthuma CC, Zevenhoven JC, Bernard S, Betzi S, Imbert I, Canard B, Guillemot JC, Lécine P, Pfefferle S, Drosten C, Snijder EJ, Decroly E, & Morelli X (2014). Coronavirus Nsp10, a Critical Co-factor for Activation of Multiple Replicative Enzymes. The Journal of biological chemistry, 289 (37), 25783-96 PMID: 25074927 

Bouvet M, Imbert I, Subissi L, Gluais L, Canard B, & Decroly E (2012). RNA 3'-end mismatch excision by the severe acute respiratory syndrome coronavirus nonstructural protein nsp10/nsp14 exoribonuclease complex. Proceedings of the National Academy of Sciences of the United States of America, 109 (24), 9372-7 PMID: 22635272 

Bouvet M, Debarnot C, Imbert I, Selisko B, Snijder EJ, Canard B, & Decroly E (2010). In vitro reconstitution of SARS-coronavirus mRNA cap methylation. PLoS pathogens, 6 (4) PMID: 20421945 

Decroly E, Imbert I, Coutard B, Bouvet M, Selisko B, Alvarez K, Gorbalenya AE, Snijder EJ, & Canard B (2008). Coronavirus nonstructural protein 16 is a cap-0 binding enzyme possessing (nucleoside-2'O)-methyltransferase activity. Journal of virology, 82 (16), 8071-84 PMID: 18417574

Zhou H, & Perlman S (2007). Mouse hepatitis virus does not induce Beta interferon synthesis and does not inhibit its induction by double-stranded RNA. Journal of virology, 81 (2), 568-74 PMID: 17079305 

Menachery VD, Debbink K, & Baric RS (2014). Coronavirus Non-Structural Protein 16: Evasion, Attenuation, and Possible Treatments. Virus research PMID: 25278144 

Hastie KM, Kimberlin CR, Zandonatti MA, MacRae IJ, & Saphire EO (2011). Structure of the Lassa virus nucleoprotein reveals a dsRNA-specific 3' to 5' exonuclease activity essential for immune suppression. Proceedings of the National Academy of Sciences of the United States of America, 108 (6), 2396-401 PMID: 21262835 

Qi X, Lan S, Wang W, Schelde LM, Dong H, Wallat GD, Ly H, Liang Y, & Dong C (2010). Cap binding and immune evasion revealed by Lassa nucleoprotein structure. Nature, 468 (7325), 779-83 PMID: 21085117 

Martínez-Sobrido L, Giannakas P, Cubitt B, García-Sastre A, & de la Torre JC (2007). Differential inhibition of type I interferon induction by arenavirus nucleoproteins. Journal of virology, 81 (22), 12696-703 PMID: 17804508 

Xiao, Y., Ma, Q., Restle, T., Shang, W., Svergun, D., Ponnusamy, R., Sczakiel, G., & Hilgenfeld, R. (2012). Nonstructural Proteins 7 and 8 of Feline Coronavirus Form a 2:1 Heterotrimer That Exhibits Primer-Independent RNA Polymerase Activity Journal of Virology, 86 (8), 4444-4454 DOI: 10.1128/JVI.06635-11 

Li S, Zhao Q, Zhang Y, Zhang Y, Bartlam M, Li X, & Rao Z (2010). New nsp8 isoform suggests mechanism for tuning viral RNA synthesis. Protein & cell, 1 (2), 198-204 PMID: 21203988 

Lundin A, Dijkman R, Bergström T, Kann N, Adamiak B, Hannoun C, Kindler E, Jónsdóttir HR, Muth D, Kint J, Forlenza M, Müller MA, Drosten C, Thiel V, & Trybala E (2014). Targeting membrane-bound viral RNA synthesis reveals potent inhibition of diverse coronaviruses including the middle East respiratory syndrome virus. PLoS pathogens, 10 (5) PMID: 24874215

Subissi L, Posthuma CC, Collet A, Zevenhoven-Dobbe JC, Gorbalenya AE, Decroly E, Snijder EJ, Canard B, & Imbert I (2014). One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities. Proceedings of the National Academy of Sciences of the United States of America, 111 (37) PMID: 25197083 

te Velthuis AJ, van den Worm SH, & Snijder EJ (2012). The SARS-coronavirus nsp7+nsp8 complex is a unique multimeric RNA polymerase capable of both de novo initiation and primer extension. Nucleic acids research, 40 (4), 1737-47 PMID: 22039154 

Eckerle LD, Lu X, Sperry SM, Choi L, & Denison MR (2007). High fidelity of murine hepatitis virus replication is decreased in nsp14 exoribonuclease mutants. Journal of virology, 81 (22), 12135-44 PMID: 17804504 

Eckerle LD, Becker MM, Halpin RA, Li K, Venter E, Lu X, Scherbakova S, Graham RL, Baric RS, Stockwell TB, Spiro DJ, & Denison MR (2010). Infidelity of SARS-CoV Nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing. PLoS pathogens, 6 (5) PMID: 20463816 

Beachboard DC, Lu X, Baker SC, & Denison MR (2013). Murine hepatitis virus nsp4 N258T mutants are not temperature-sensitive. Virology, 435 (2), 210-3 PMID: 23099203 

Oostra M, te Lintelo EG, Deijs M, Verheije MH, Rottier PJ, & de Haan CA (2007). Localization and membrane topology of coronavirus nonstructural protein 4: involvement of the early secretory pathway in replication. Journal of virology, 81 (22), 12323-36 PMID: 17855519 

Kumar P, Gunalan V, Liu B, Chow VT, Druce J, Birch C, Catton M, Fielding BC, Tan YJ, & Lal SK (2007). The nonstructural protein 8 (nsp8) of the SARS coronavirus interacts with its ORF6 accessory protein. Virology, 366 (2), 293-303 PMID: 17532020

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