Coronavirus and Arterivirus replication complexes

Following the infection of cells with positive strand RNA viruses dramatic rearrangements of intracellular membranes can be observed which are generally double membrane vesicles (DMV) and convoluted membranes (CMs) attached to a cellular membrane. These structures contain viral dsRNA intermediates and thus prevent pattern recognition receptors from recognizing dsRNA and thus prevent the induction of an antiviral response. In addition, DMVs sequester viral and cellular enzymes necessary for viral replication as well as providing a scaffold for viral particle assembly and/or means for the newly synthesized viral RNA to enter the cytoplasm.  

Coronaviridae are no exception. In the case of two prototype Coronaviridae, the murine Coronavirus (Mouse Hepatitis Virus, MHV) and human SARS-associated Coronavirus, SARS-CoV, these replication-transcription complexes (RTCs) -another terminology for DMV- consist of the non-structural proteins (nsp) required for RNA synthesis, RNA-dependent RNA Polymerase (RdRp/nsp12), as well as the membrane spanning nsp3, 4, and 6 proteins, although nsp3 and 4 are sufficient for inducing the DMVs and the formation of DMVs is not dependent on active RNA synthesis. 

Non structural proteins involved in Coronavirus replication are encoded within the PP1ab

Whereas nsp12 contains the enzymatic activity for RNA synthesis, the remaining nsps anchor the DMV to the ER membrane. In this context it is interesting to note that most fusion proteins of these nsps derived from MHV or SARS-CoV do not retain the ability of nsp3 and 4 to anchor DMVs to the ER and with the exception of nsps 2 and 6 fail to recruit nsps required for viral replication such as the viral RNA Helicase. The notable exception are fusion proteins in which the large luminal loop between the first and second transmembrane domain of nsp4 derives from the same species as the nsp3, a notion which was confirmed by truncation analysis of nsp4, whereas the luminal loop of nsp3 may have a stabilizing function and be involved in recruitment larger nsps via nsp6.

Localisation of nsp3, nsp4 and nsp4 of MHV and SARS-CoV

In both SARS-CoV and MHV infected cells, dsRNA intermediates -and thus active viral RNA synthesis- co-localising with RdRp/nsp12 with are localized in the interior of the DMV at early timepoints post infection. As the infection progresses however the co-localisation of dsRNA and newly synthesized viral RNA becomes less apparent suggesting that viral RNA transcription ceases. In the case of MHV, it has been demonstrated that the RTCs translocate to the assembly site of new viral particles at 8-16 h p.i. thus obliviating the need of active RNA transcription. At this timepoint the DMVs are not only devoid of dsRNA but also of RNA helicases. 

Replication structures of Arteriviridae: similarities to Coronaviridae

Similar to the Coronaviridae, following infection of cells with Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) or Equine Arterivirus (EAV)  -two of the most commonly studied Arteriviridae- the replicative proteins (with the notable exception of nsp1 and nsp2TF) are assembled in virus induced membrane structures consisting of modified intracellular membranes in the perinuclear region of the infected cell. As it is the case for Coronavirus infected cells, these DMVs are associated with viral replication and transcription and thus contain viral proteins and dsRNA intermediates as well as viral positive and negative sense ssRNA. In the case of the Arteriviridae, the scaffold of the RTCs seems to consist of the putative membrane spanning proteins nsp2, nsp3 and nsp5; similar to the Coronaviridae, these are expressed as part of the orf1a gene which is processed by autoprocessing using viral proteases. In addition, both the nsp2 and nsp3 are sufficient to induce the formation of DMVs (as is the case for the Coronavirus nsp3 and 4) and DMVs can form structures of modified ER which are interconnected by their outer membrane as revealed in tomographic studies. Although host proteins implicated in the initiation of arteriviral RNA synthesis binding either to the 3’ end of the genome or the anti-genome of PRRSV or EAV have been postulated to localize to DMVs in the addition of viral proteins the precise composition of these has not been identified.

Hypothetical model of how Arteri- and Coronavirus nsps induce the formation of LC3-I and LC3-II positive particles by stimulating autophagy

As in the case of MERS-CoV and other members of the Coronaviridae, the replication of EAV and PRRSV is sensitive to inhibitors from members of the Cyclophilin family, probably Cyclophilin A. Furthermore, similar to IBV and other Coronaviruses, microtubule-associated protein 1 light chain 3 (LC3) and ER degradation-enhancing α-mannosidase-like1 (EDEM1) proteins associate with DMVs at 16 h p.i. (in the case of MHV) and the depletion of LC3 reduces EAV replication. These results suggest that in both Coronavirus and Arterivirus infected cells autophagy may play a role late in infection similar to Rotavirus infected cells. So far it remains to be seen which viral protein -or combination of of viral proteins- is responsible for the recruitment of the components of the autophagy pathway and to which extent autophagy inhibitors are preventing the spread of viral particles and CPE. In the opinion of the author, the most likely pathway inducing the recruitment of LC3 to DMVs is by inducing ER stress by  inhibition of mTOR via the arteriviral nsp2 and 3 (and Coronavirus nsp3 and 4) proteins - in other words the question which remains to be answered is, if the formation of DMVs and the induction of autophagosome like structures influence viral assembly and if the stabilization of these structures has any effect. Also it remains to be seen if the recruitment of ER chaperones such as EDEM1 by the murine Coronavirus and EAV is required for the correct folding of viral proteins. In this context it is important to note that viral replication itself does not require the presence of autophagy related protein (ATG) 7 and thus the lipidiated form of LC3 (LC3II). It would be of interest to investigate if in ATG7 depleted cells infected with EAV, SARS-CoV or MHV, EDEM1 is stabilized and if the formation of viral particles is affected. Finally, is the recruitment of EDEM1 necessary for the detection and degradation of viral glycoproteins such as the Coronavirus S protein that are misfolded akin to the cellular unfold protein response ? Furthermore, it might be possible that RTC are predominating LC3-II positive whereas the assembly particles containing the coronaviral S, E, M and N proteins as well as EDEM1 are predominantly LC3-I.

The application of Cyclophilin A therefore might lead to an increase in misfolded viral and cellular proteins that are recognised by EDEM1 and degraded in autophagosomes. If the infection of cells with Corona-or Arteriviridae interferes with the ability of cells to respond to aggregated misfolded proteins, this might explain why MERS-CoV infected cells are sensitive to Cyclosporin A. Who is up to the challenge?

Further Reading

Hagemeijer MC, Vonk AM, Monastyrska I, Rottier PJ, & de Haan CA (2012). Visualizing coronavirus RNA synthesis in time by using click chemistry. Journal of virology, 86 (10), 5808-16 PMID: 22438542 

Knoops K, Kikkert M, Worm SH, Zevenhoven-Dobbe JC, van der Meer Y, Koster AJ, Mommaas AM, & Snijder EJ (2008). SARS-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum. PLoS biology, 6 (9) PMID: 18798692 

van den Worm SH, Knoops K, Zevenhoven-Dobbe JC, Beugeling C, van der Meer Y, Mommaas AM, & Snijder EJ (2011). Development and RNA-synthesizing activity of coronavirus replication structures in the absence of protein synthesis. Journal of virology, 85 (11), 5669-73 PMID: 21430047 

Angelini MM, Akhlaghpour M, Neuman BW, & Buchmeier MJ (2013). Severe acute respiratory syndrome coronavirus nonstructural proteins 3, 4, and 6 induce double-membrane vesicles. mBio, 4 (4) PMID: 23943763

Hagemeijer, M., Monastyrska, I., Griffith, J., van der Sluijs, P., Voortman, J., van Bergen en Henegouwen, P., Vonk, A., Rottier, P., Reggiori, F., & de Haan, C. (2014). Membrane rearrangements mediated by coronavirus nonstructural proteins 3 and 4 Virology, 458-459, 125-135 DOI: 10.1016/j.virol.2014.04.027 

Nal, B. (2005). Differential maturation and subcellular localization of severe acute respiratory syndrome coronavirus surface proteins S, M and E Journal of General Virology, 86 (5), 1423-1434 DOI: 10.1099/vir.0.80671-0 

Bost AG, Prentice E, & Denison MR (2001). Mouse hepatitis virus replicase protein complexes are translocated to sites of M protein accumulation in the ERGIC at late times of infection. Virology, 285 (1), 21-9 PMID: 11414802

Lontok E, Corse E, & Machamer CE (2004). Intracellular targeting signals contribute to localization of coronavirus spike proteins near the virus assembly site. Journal of virology, 78 (11), 5913-22 PMID: 15140989 

Snijder, E., Kikkert, M., & Fang, Y. (2013). Arterivirus molecular biology and pathogenesis Journal of General Virology, 94 (Pt_10), 2141-2163 DOI: 10.1099/vir.0.056341-0 

Monastyrska I, Ulasli M, Rottier PJ, Guan JL, Reggiori F, & de Haan CA (2013). An autophagy-independent role for LC3 in equine arteritis virus replication. Autophagy, 9 (2), 164-74 PMID: 23182945 

Bernasconi R, Noack J, & Molinari M (2012). Unconventional roles of nonlipidated LC3 in ERAD tuning and coronavirus infection. Autophagy, 8 (10), 1534-6 PMID: 22895348

de Wilde AH, Raj VS, Oudshoorn D, Bestebroer TM, van Nieuwkoop S, Limpens RW, Posthuma CC, van der Meer Y, Bárcena M, Haagmans BL, Snijder EJ, & van den Hoogen BG (2013). MERS-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin A or interferon-α treatment. The Journal of general virology, 94 (Pt 8), 1749-60 PMID: 23620378 

Ciechomska IA, Gabrusiewicz K, Szczepankiewicz AA, & Kaminska B (2013). Endoplasmic reticulum stress triggers autophagy in malignant glioma cells undergoing cyclosporine a-induced cell death. Oncogene, 32 (12), 1518-29 PMID: 22580614

Reggiori F, de Haan CA, & Molinari M (2011). Unconventional use of LC3 by coronaviruses through the alleged subversion of the ERAD tuning pathway. Viruses, 3 (9), 1610-23 PMID: 21994798 

Reggiori, F., Monastyrska, I., Verheije, M., Calì, T., Ulasli, M., Bianchi, S., Bernasconi, R., de Haan, C., & Molinari, M. (2010). Coronaviruses Hijack the LC3-I-Positive EDEMosomes, ER-Derived Vesicles Exporting Short-Lived ERAD Regulators, for Replication Cell Host & Microbe, 7 (6), 500-508 DOI: 10.1016/j.chom.2010.05.013 

de Haan CA, Molinari M, & Reggiori F (2010). Autophagy-independent LC3 function in vesicular traffic. Autophagy, 6 (7), 994-6 PMID: 20814233 Bernasconi R, Galli C, Noack J, Bianchi S, de Haan CA, Reggiori F, & Molinari M (2012). Role of the SEL1L:LC3-I complex as an ERAD tuning receptor in the mammalian ER. Molecular cell, 46 (6), 809-19 PMID: 22633958 

Arnoldi F, De Lorenzo G, Mano M, Schraner EM, Wild P, Eichwald C, & Burrone OR (2014). Rotavirus increases levels of lipidated LC3 supporting accumulation of infectious progeny virus without inducing autophagosome formation. PloS one, 9 (4) PMID: 24736649 

Shenkman M, Groisman B, Ron E, Avezov E, Hendershot LM, & Lederkremer GZ (2013). A shared endoplasmic reticulum-associated degradation pathway involving the EDEM1 protein for glycosylated and nonglycosylated proteins. The Journal of biological chemistry, 288 (4), 2167-78 PMID: 23233672

Park S, Jang I, Zuber C, Lee Y, Cho JW, Matsuo I, Ito Y, & Roth J (2014). ERADication of EDEM1 occurs by selective autophagy and requires deglycosylation by cytoplasmic peptide N-glycanase. Histochemistry and cell biology PMID: 24664425 

 Zuber C, Cormier JH, Guhl B, Santimaria R, Hebert DN, & Roth J (2007). EDEM1 reveals a quality control vesicular transport pathway out of the endoplasmic reticulum not involving the COPII exit sites. Proceedings of the National Academy of Sciences of the United States of America, 104 (11), 4407-12 PMID: 17360537

Role of Sialic acid binding in Coronavirus attachment and entry

Binding of the viral particle is a crucial step in the establishment of viral infection and subsequent viral replication. In the case of Coronaviridae, the binding of the virus is mediated viral spike protein, a homotrimer composed of subunits that are about 150 kDa in size each. The spike protein itself is composed of two subunits, S1 and S2, the former sufficient for receptor binding and the latter required for the fusion and entry of the virus particle. During the viral replication the S protein is synthesized as a precursor protein and co-translationally glycosylated in the Golgi followed by a cleavage generating the S1 and S2 subunits at a dibasic cleavage site (BBXBB). The S1 subunit contains the receptor-binding site (RBD) followed (in the case of MHV) by a hypervariable region, whereas the S2 subunit contains two heptad repeats (HR1 and 2) as well as the transmembrane region.

Domains of a prototype Coronavirus S potein

Of particular interest is the RBD since blocking peptides or neutralizing antibodies designed to bind the RBD might be used in treating Coronavirus caused diseases, not only in humans (such as SARS or MERS) but also in animals. On the other hand, based on experiments done using the murine Coronavirus (MHV) the heptad repeat domains as well as the putative fusion peptide located within the S2 subdomain may play an important role in the formation of syncytia and thus may contribute to the CPE. Furthermore, the HR might also play a role in the interaction of the RBD with the cellular receptor during viral entry, probably by stabilizing the receptor-RBD complex not only in the case of MHV but also SARS-CoV.

In the past years, however a number of Coronaviruses have been shown to not only contain one but two RBD, one located at the C-terminal end of S1 which is responsible for binding the cellular receptor and an additional one located at the N-terminal end of S1 binding sialic acid. In general, the consensus is that binding to sialic acid by the S1 subunit allows Coronavirus’ to bind to epithelial target cells of the respiratory tract as well the intestine which are normally covered by mucus and thus not readily accessible. This is particular true for members of the Alpha-, Beta-, and Gammacoronaviridae which bind to ciliated intestine and respiratory cells, such as the porcine TGEV and PEDV as well as the enteric feline Coronavirus (FECV) but also for the bovine Coronavirus (BCoV) and the human Coronavirus OC43 (HCoV-OC43) as well as the avian Infectious Bronchitis Virus (IBV). In contrast, MERS-CoV generally does bind and infect primarily non-ciliated bronchial epithelial and alveolar cells of the lower lung and thus might not need sialic acid to bind to DPP4 (although hDPP4 does have sialic acid residues).

                        Feline Enteric Coronavirus (FECV)

Feline intestinal epithelial cells derived from the Ileum and the Colon (illenocytes and coloncytes respectively) pretreated with neuroaminidase exhibit an increase in the efficiency of FECV infection, suggesting that sialic acid might inhibit viral entry. Based on results showing that the pretreatment of porcine TGEV strain Perdue and PEDV with neuroaminidase can unmask the viral sialic acid binding activity, similar experiments confirmed these results for FECV. The application of α2-6-sialyllactose binds and reduces the infectivity of pretreated FECV, demonstrating FECV can bind α2-6-sialic acid. Desialylated cells however were resistant to inhibition of inhibition by α2-6-sialyllactose treatment. FECV therefore does have

a sialic acid binding capacity, which during the passage of the virus through the stomach may be partially masked by virus-associated sialic acids. In the absence of viral enzymes removing virus-associated sialic acids, enzymes within the mucus might remove the sialic acid thus allowing FECV to bind its cellular receptor and thus requiring sialidases for efficient enterocyte infections.

                     Infectious Bronchitis Virus (IBV)

Although the receptor for the avian Infectious Bronchitis Virus is unknown it is known that the treatment of Vero, BHK (Baby Hamster Kidney) as well as primer chicken kidney cells with neuroaminidase -an enzyme which cleaves sialic acid- renders cell lines resistant to infection with IBV strains Beaudette and M41. Moreover, IBV is more sensitive than Sendai or Influenza A virus to pretreatment of cells with neuroaminidase suggesting that IBV requires a higher amount of sialic acid than Influenza A or Sendai and indeed it has been shown that IBV preferentially recognizes α2,3-linked sialic acid as indicated by reacting with lectin. Indeed the infection of the tracheal organ cultures can be inhibited by pretreatment with neuroaminidase. The binding of α2,3-linked sialic acid might be only required for the initial binding of IBV preceding binding to the receptor although the sialic acid binding activity of IBV S protein seems to be more important for viral entry than the sialic acid binding activity of TGEV S protein. This is reflected by the abundance of α2-3 linked sialic acid on susceptible epithelial cells.  

In general, the masking of the viral sialic acid binding site might protect the enteric Coronavirus particles from degradation in the stomach or by gastric mucins. Bacterial and host derived sialidases unmasking these binding site then would allow the virus to attach to the mucin and infect cells of the intestinal tract. In the avian respiratory tract α2-3 linked sialic acid is a common receptor for respiratory viruses such as avian Influenza A. 

So finally what has this to do with emerging Coronaviruses? As I mentioned above so far there is no indication that MERS-CoV S has sialic acid binding activity nor that the primary target cells necessitate this activity. The novel Coronavirus identified in dromedaries however seems to be an enteric Coronavirus and thus the S protein might bind sialic acid.  However, once the genome of DcCoV UAE-HKU23 has been sequenced, we should know more.  One final word about the potential use of neuroaminidase inhibitors which are quite effective in treating Influenza A virus infections: they are not effective against Coronavirus induced infections since Coronaviridae are not dependent on the sialic acid binding to its cognate receptor. 

Further reading

Vlasak R, Luytjes W, Spaan W, & Palese P (1988). Human and bovine coronaviruses recognize sialic acid-containing receptors similar to those of influenza C viruses. Proceedings of the National Academy of Sciences of the United States of America, 85 (12), 4526-9 PMID: 3380803 

Shahwan K, Hesse M, Mork AK, Herrler G, & Winter C (2013). Sialic acid binding properties of soluble coronavirus spike (S1) proteins: differences between infectious bronchitis virus and transmissible gastroenteritis virus. Viruses, 5 (8), 1924-33 PMID: 23896748 

Winter C, Herrler G, & Neumann U (2008). Infection of the tracheal epithelium by infectious bronchitis virus is sialic acid dependent. Microbes and infection / Institut Pasteur, 10 (4), 367-73 PMID: 18396435 

Schmauser B, Kilian C, Reutter W, & Tauber R (1999). Sialoforms of dipeptidylpeptidase IV from rat kidney and liver. Glycobiology, 9 (12), 1295-305 PMID: 10561454

Krempl C, Schultze B, Laude H, & Herrler G (1997). Point mutations in the S protein connect the sialic acid binding activity with the enteropathogenicity of transmissible gastroenteritis coronavirus. Journal of virology, 71 (4), 3285-7 PMID: 9060696 

Schwegmann-Weßels, C., Bauer, S., Winter, C., Enjuanes, L., Laude, H., & Herrler, G. (2011). The sialic acid binding activity of the S protein facilitates infection by porcine transmissible gastroenteritis coronavirus Virology Journal, 8 (1) DOI: 10.1186/1743-422X-8-435 

Desmarets, L., Theuns, S., Roukaerts, I., Acar, D., & Nauwynck, H. (2014). The role of sialic acids in feline enteric coronavirus infections Journal of General Virology DOI: 10.1099/vir.0.064717-0

Are broad spectrum antivirals for Coronavirus infections are just around the corner?

The emergence of a new highly pathogenic virus in animal as well as human populations presents a unique challenge for both veterinarians and physicians alike since vaccines more often than not are not readily available, leaving antiviral treatments the only option to contain an outbreak. Pharmaceuticals however take time to be developed and tested thus the only option available is to identify the antiviral pathways targeted by viral proteins in the hope that existing drugs are available and effective in activating these pathways and thus suppress viral replication. In the meantime, clinicians can only offer supportive care and use serum from convalescent patients - often a scarce commodity and not readily available. As an alternative however pharmaceuticals used and approved for the treatment of other viral diseases or indeed for other diseases might be repurposed.

The recent emergence of both the SARS-CoV in 2002 and MERS-CoV in 2012 have lead to substantial increase in Coronaviruses as a potential human pathogen. Following the emergence of MERS-CoV, the International Respiratory and Emerging Infection Consortium (ISARIC) compiled a list of pharmaceuticals available to physicians based on the experience gained during the SARS-CoV epidemic in 2002/2003, with the most promising drugs being Interferon and Ribavirin, which had been used in combination as well as separate to treat SARS-CoV and pandemic Influenza A/2009 patients. Indeed, both drugs are effective to prevent MERS-CoV replication in a rhesus macaque model but failed to be effective in patients with a severe infection. A screen of chemical library of 1280 pharmaceuticals known to be effective against Influenza A was also assessed for their ability to reduce viral yield and prevent the cytopathic effect following the infection of cells with MERS-CoV confirmed that at least under laboratory conditions MERS-CoV is sensitive to Interferon as well as to two antiretroviral drugs, nefinavir and lopinavir. At first it may seem surprising that two antiretroviral drugs can prevent the replication of a Coronavirus. Both drugs were developed to prevent the replication of HIV by targeting the HIV protease. As discussed before however, the Coronavirus genome encodes for a protease, 3CL^pro which is required for the processing of the orf1ab polyprotein and has been shown sensitive to nefinavir and lopinavir due to the inhibition of the viral 3CL^pro    protease.  Both drugs are non-specific for MERS-CoV and also effective in treating SARS-CoV related infections.

Other targets of antiviral therapy most certainly include preventing viral entry. As discussed in a previous post, monoclonal antibodies against the viral S protein and small molecules binding to the receptor-binding site of the S protein have been shown to be effective to neutralize viral particles. Another possibility is to target the release of the viral genome into the cytoplasm of the cell, which is dependent on a low pH within the endosome. The application of a lysosomotropic agent such as Chloroquine/Hydroxychloroquine (the protonated form of Chloroquine) (an antimalarial drug) or NH4Cl might therefore prevent the fusion of the virus with the endosome by raising the pH. Indeed the application of low doses of Chloroquine to cells infected with SARS-CoV or MERS-CoV as well as Influenza A have shown to prevent viral replication. In addition to prevent the fusion of the viral particle with the endosome, Chloroquine might also prevent the glycosylation of ACE2, the receptor for SARS-CoV and thus prevent binding of the SARS-CoV S1 subunit to its receptor (it remains to be seen if this is the case with DPP4, the receptor for MERS-CoV). The glycosylation of proteins is targeted by inhibiting glycosyltransferases, namely quinone reductase 2, which is involved in the biosynthesis of sialic acid, a component of cellular receptors. Sialic acid moieties are also present within the glycoproteins of HIV-1 glycoproteins, the SARS-CoV receptor ACE2, the MERS-CoV receptor DPP4/CD26, Coronavirus S proteins as well in the receptors for Influenza A thus explaining the broad spectrum activity of Chloroquine. Quinone reductase 2 inhibitors therefore reduce the glycosylation of SARS S proteins although it seems that the reduction has no effect on viral infectivity (or only a marginal effect).

So far however its effectiveness has not been demonstrated in the animal model of MERS and studies with Influenza A have shown that Chloroquine -although effective in cell lines- is not effective in humans thus adding some caution. Apart from being a potential pharmaceutical against a variety of human Coronaviruses, Chloroquine is well tolerated and better known in treating in Malaria patients at therapeutic doses in micro molar concentrations.  Pharmaceuticals effective specifically against MERS-CoV, two pharmaceuticals emerged recently, mycophenolic acid (MPA) and IFN-β, both of which have been discussed previously.      

As outlined previously, the polyprotein 1ab is processed further by auto proteolysis that generates a number of nonstructural proteins varying among the Coronaviridae, which includes not the RNA dependent RNA Polymerase (RdRp) but also an NTPase/Helicase known as nsp 12 and 13 respectively.  In simian Vero E6 cells infected with SARS-CoV these are located within perinuclear double membrane bound vesicles representing replication-transcription complexes containing nascent viral subgenomic RNAs, RdRp as well as viral positive strand RNA and dsRNA intermediates which are resolved by the viral Helicase. Although the precise mechanism and specific function of the Coronavirus Helicase is not known, the replication of SARS-CoV, MERS-CoV, and the murine MHV can effectively inhibited by a small compound, SSYA10-001, targeting the Helicase at amino acid residues K508, R507, and Y277 respectively, thus offering a potential broad spectrum inhibitor of Coronavirus mediated infections and highlighting the importance of the Coronavirus Helicase for viral replication since inhibition of the SARS-CoV Helicase by Bismuth has been shown to inhibit SARS-CoV replication in the past. In addition to its wide spectrum of antiviral activity, SSYA10-001 exhibits only minimal cytotoxicity if applied to cells. The viral RdRp itself can be inhibited by combination of Ribavirin and 5-Flourouracil, the latter being mutagenic and thus sensitizing infected cells to Ribavirin treatment (Ribavirin itself being ineffective). 

Overview of potential and existing antiviral strategies to treat Coronavirus infections

In conclusion, whilst future outbreaks of novel respiratory viruses cannot prevented, pharmaceuticals which are already available might be used in the treatment during a pandemic or an epidemic whilst bioinformatics in conjunction with the identification of ways that viral proteins interact with the host cell might identify effective broad spectrum inhibitors which target highly conserved proteins. A recent screen of potential antiviral pharmaceuticals revealed that even antipsychotic drugs can have an antiviral effect against MERS-CoV, revealing the hidden potential of many drugs already approved.

Further reading

Dyall J, Coleman CM, Hart BJ, Venkataraman T, Holbrook MR, Kindrachuk J, Johnson RF, Olinger GG Jr, Jahrling PB, Laidlaw M, Johansen LM, Lear CM, Glass PJ, Hensley LE, & Frieman MB (2014). Repurposing of clinically developed drugs for treatment of Middle East Respiratory Coronavirus Infection. Antimicrobial agents and chemotherapy PMID: 24841273

Falzarano D, de Wit E, Rasmussen AL, Feldmann F, Okumura A, Scott DP, Brining D, Bushmaker T, Martellaro C, Baseler L, Benecke AG, Katze MG, Munster VJ, & Feldmann H (2013). Treatment with interferon-α2b and ribavirin improves outcome in MERS-CoV-infected rhesus macaques. Nature medicine, 19 (10), 1313-7 PMID: 24013700

Falzarano D, de Wit E, Martellaro C, Callison J, Munster VJ, & Feldmann H (2013). Inhibition of novel β coronavirus replication by a combination of interferon-α2b and ribavirin. Scientific reports, 3 PMID: 23594967 

Chan, J., Chan, K., Kao, R., To, K., Zheng, B., Li, C., Li, P., Dai, J., Mok, F., Chen, H., Hayden, F., & Yuen, K. (2013). Broad-spectrum antivirals for the emerging Middle East respiratory syndrome coronavirus Journal of Infection, 67 (6), 606-616 DOI: 10.1016/j.jinf.2013.09.029

Kilianski A, & Baker SC (2014). Cell-based antiviral screening against coronaviruses: developing virus-specific and broad-spectrum inhibitors. Antiviral research, 101, 105-12 PMID: 24269477

Al-Tawfiq JA, Momattin H, Dib J, & Memish ZA (2014). Ribavirin and interferon therapy in patients infected with the Middle East respiratory syndrome coronavirus: an observational study. International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases, 20, 42-6 PMID: 24406736

Smith EC, Blanc H, Vignuzzi M, & Denison MR (2013). Coronaviruses lacking exoribonuclease activity are susceptible to lethal mutagenesis: evidence for proofreading and potential therapeutics. PLoS pathogens, 9 (8) PMID: 23966862 


Hart BJ, Dyall J, Postnikova E, Zhou H, Kindrachuk J, Johnson RF, Olinger GG Jr, Frieman MB, Holbrook MR, Jahrling PB, & Hensley L (2014). Interferon-β and mycophenolic acid are potent inhibitors of Middle East respiratory syndrome coronavirus in cell-based assays. The Journal of general virology, 95 (Pt 3), 571-7 PMID: 24323636

Coleman CM, Liu YV, Mu H, Taylor JK, Massare M, Flyer DC, Glenn GM, Smith GE, & Frieman MB (2014). Purified coronavirus spike protein nanoparticles induce coronavirus neutralizing antibodies in mice. Vaccine, 32 (26), 3169-74 PMID: 24736006

Keyaerts E, Li S, Vijgen L, Rysman E, Verbeeck J, Van Ranst M, & Maes P (2009). Antiviral activity of chloroquine against human coronavirus OC43 infection in newborn mice. Antimicrobial agents and chemotherapy, 53 (8), 3416-21 PMID: 19506054

Savarino A, Di Trani L, Donatelli I, Cauda R, & Cassone A (2006). New insights into the antiviral effects of chloroquine. The Lancet infectious diseases, 6 (2), 67-9 PMID: 16439323

Vincent MJ, Bergeron E, Benjannet S, Erickson BR, Rollin PE, Ksiazek TG, Seidah NG, & Nichol ST (2005). Chloroquine is a potent inhibitor of SARS coronavirus infection and spread. Virology journal, 2 PMID: 16115318

van Hemert, M., van den Worm, S., Knoops, K., Mommaas, A., Gorbalenya, A., & Snijder, E. (2008). SARS-Coronavirus Replication/Transcription Complexes Are Membrane-Protected and Need a Host Factor for Activity In Vitro PLoS Pathogens, 4 (5) DOI: 10.1371/journal.ppat.1000054

Adedeji AO, Singh K, Kassim A, Coleman CM, Elliott R, Weiss SR, Frieman MB, & Sarafianos SG (2014). Evaluation of SSYA10-001 as a Replication Inhibitor of SARS, MHV and MERS Coronaviruses. Antimicrobial agents and chemotherapy PMID: 24841268 

Yang N, Tanner JA, Wang Z, Huang JD, Zheng BJ, Zhu N, & Sun H (2007). Inhibition of SARS coronavirus helicase by bismuth complexes. Chemical communications (Cambridge, England) (42), 4413-5 PMID: 17957304