As described in past posts of this blog, an outbreak of more than 650 cases of a severe pneumonia with a high percentage of mortality (up to 50%) has been associated with a novel zoonotic Coronavirus, MERS-CoV or Middle East Respiratory Syndrome Virus. To date, no effective antiviral therapy let alone vaccine to treat MERS-CoV infected patients. Whilst a vaccine would protect individuals from infection with MERS-CoV, antiviral therapy focuses on treating infected individuals and reduces viral spread and mortality associated with MERS-CoV. The window of opportunity in the latter case is thus limited to the period between infection and the onset of symptoms - in the case of MERS-CoV mediated infections occurs generally with median incubation time of about 5.2-5.5 days, whereas the peak of the viral load can be measured at around day 11 (in the lower respiratory tract) and day 13 (in the urine).
The emergence of of SARS-CoV in 2002/2003 lead to the identification of a number of potential antivirals which centered around the viral proteases that are required for viral replication. As outlined earlier, the orf1ab of the Coronavirus genome is divided into two large open reading frames, orf1a (which overlaps with orf1b) and orf1b. Both are separated by a ribosome frame shifting sequence (RFS) that allows the translation of two large polyproteins, PP1a (approx.. 500 kDa) and PP1ab (approx. 800 kDa). Not only are these relatively large proteins in size, but in addition they can be further processed by proteolytic auto processing via either the papain-like cysteine protease (PLPRO) or the main Coronavirus 3C chymotrypsin like protease (MPRO or 3CLPRO ), a Poliovirus 3C-like protease which is encoded within orf1a/orf1ab of all members of the Coronaviridae. The proteolytic autoprocessing leads to formation of a number of non-structural proteins which varies among different members of the Coronaviridae.
Besides its function in generation multiple non-structural proteins, both the PLPRO and MPRO of SARS-CoV as well as MERS-CoV antagonize the Interferon response by blocking the nuclear translocation and phosphorylation of IFN regulatory factor (IFR-3) thus suppressing the expression of Interferon-β, CCL5, and CXCL10. Furthermore, both SARS-CoV and MERS-CoV PLPRO act as viral deubiquitinating enzymes, reversing both K48 and K63 linked ubiquitination and ISG15-linked ISGylation and thus downregulate the innate immune response and increase the susceptibility of infected individuals to secondary infections in addition to succumbing to Coronavirus mediated disease. Since all human (and animal) representatives of the Coronaviridae express these proteases, they have been proposed as a potential target for antiviral drugs and compounds targeting SARS-CoV derived PLPRO and 3CLPRO have been developed. Unfortunately however these compounds -although effective in limiting SARS-CoV replication- are not effective against the MERS-CoV derived PLPRO protease due to a amino acid change in the drug binding site. A small-molecule inhibitor however that blocks replication of SARS-CoV (and the murine MHV) by inhibiting 3CLPRO also inhibits the activity of MERS-CoV derived 3CLPRO. It is however important to note that so far these results reflect only the capability of this inhibitor to inhibit MERS-CoV derived 3CLPRO in an experimental system -as experimental studies involving the impact of this inhibitor on viral replication or restoring the antiviral response nor on the pathogenesis of MERS in an animal model have not been conducted yet. These studies, using a replicon based system to determine the individual contribution of various antiviral proteins for instance, need time and are hampered by current limitations set by the absence of a fully working animal model. Also, it should be noted that MERS-CoV expressed additional proteins which have been implicated in the suppression of the antiviral response such as orf4a, orf4b, and orf5. Any successful antiviral treatment therefore needs to be targeted against different proteins. Interestingly, MERS-CoV -in contrast to SARS-CoV- seems to target specifically the Interferon-β mediated immune response, and a combination of Interferon-β and Mycophenolic acid has been shown to reduce MERS-CoV viral titres in vitro whereas the treatment with Ribavirin and Interferon-α is not effective.
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| Processing of PP1ab into non structural proteins |
Besides its function in generation multiple non-structural proteins, both the PLPRO and MPRO of SARS-CoV as well as MERS-CoV antagonize the Interferon response by blocking the nuclear translocation and phosphorylation of IFN regulatory factor (IFR-3) thus suppressing the expression of Interferon-β, CCL5, and CXCL10. Furthermore, both SARS-CoV and MERS-CoV PLPRO act as viral deubiquitinating enzymes, reversing both K48 and K63 linked ubiquitination and ISG15-linked ISGylation and thus downregulate the innate immune response and increase the susceptibility of infected individuals to secondary infections in addition to succumbing to Coronavirus mediated disease. Since all human (and animal) representatives of the Coronaviridae express these proteases, they have been proposed as a potential target for antiviral drugs and compounds targeting SARS-CoV derived PLPRO and 3CLPRO have been developed. Unfortunately however these compounds -although effective in limiting SARS-CoV replication- are not effective against the MERS-CoV derived PLPRO protease due to a amino acid change in the drug binding site. A small-molecule inhibitor however that blocks replication of SARS-CoV (and the murine MHV) by inhibiting 3CLPRO also inhibits the activity of MERS-CoV derived 3CLPRO. It is however important to note that so far these results reflect only the capability of this inhibitor to inhibit MERS-CoV derived 3CLPRO in an experimental system -as experimental studies involving the impact of this inhibitor on viral replication or restoring the antiviral response nor on the pathogenesis of MERS in an animal model have not been conducted yet. These studies, using a replicon based system to determine the individual contribution of various antiviral proteins for instance, need time and are hampered by current limitations set by the absence of a fully working animal model. Also, it should be noted that MERS-CoV expressed additional proteins which have been implicated in the suppression of the antiviral response such as orf4a, orf4b, and orf5. Any successful antiviral treatment therefore needs to be targeted against different proteins. Interestingly, MERS-CoV -in contrast to SARS-CoV- seems to target specifically the Interferon-β mediated immune response, and a combination of Interferon-β and Mycophenolic acid has been shown to reduce MERS-CoV viral titres in vitro whereas the treatment with Ribavirin and Interferon-α is not effective.
Further reading
Al-Abdallat MM, Payne DC, Alqasrawi S, Rha B, Tohme RA, Abedi GR, Al Nsour M, Iblan I, Jarour N, Farag NH, Haddadin A, Al-Sanouri T, Tamin A, Harcourt JL, Kuhar DT, Swerdlow DL, Erdman DD, Pallansch MA, Haynes LM, Gerber SI, & the Jordan MERS-CoV Investigation Team (2014). Hospital-associated outbreak of Middle East Respiratory Syndrome Coronavirus: A serologic, epidemiologic, and clinical description. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America PMID: 24829216
Memish ZA, Al-Tawfiq JA, Makhdoom HQ, Assiri A, Alhakeem RF, Albarrak A, Alsubaie S, Al-Rabeeah AA, Hajomar WH, Hussain R, Kheyami AM, Almutairi A, Azhar EI, Drosten C, Watson SJ, Kellam P, Cotten M, & Zumla A (2014). Respiratory Tract Samples, Viral Load and Genome Fraction Yield in patients with Middle East Respiratory Syndrome. The Journal of infectious diseases PMID: 24837403
Drosten C, Seilmaier M, Corman VM, Hartmann W, Scheible G, Sack S, Guggemos W, Kallies R, Muth D, Junglen S, Müller MA, Haas W, Guberina H, Röhnisch T, Schmid-Wendtner M, Aldabbagh S, Dittmer U, Gold H, Graf P, Bonin F, Rambaut A, & Wendtner CM (2013). Clinical features and virological analysis of a case of Middle East respiratory syndrome coronavirus infection. The Lancet infectious diseases, 13 (9), 745-51 PMID: 23782859
Cauchemez S, Fraser C, Van Kerkhove MD, Donnelly CA, Riley S, Rambaut A, Enouf V, van der Werf S, & Ferguson NM (2014). Middle East respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility. The Lancet infectious diseases, 14 (1), 50-6 PMID: 24239323
Perlman S, & Netland J (2009). Coronaviruses post-SARS: update on replication and pathogenesis. Nature reviews. Microbiology, 7 (6), 439-50 PMID: 19430490
Ramajayam, R., Tan, K., & Liang, P. (2011). Recent development of 3C and 3CL protease inhibitors for anti-coronavirus and anti-picornavirus drug discovery Biochemical Society Transactions, 39 (5), 1371-1375 DOI: 10.1042/BST0391371
Zhang, D., & Zhang, D. (2011). Interferon-Stimulated Gene 15 and the Protein ISGylation System Journal of Interferon & Cytokine Research, 31 (1), 119-130 DOI: 10.1089/jir.2010.0110
Yang, X., Chen, X., Bian, G., Tu, J., Xing, Y., Wang, Y., & Chen, Z. (2013). Proteolytic processing, deubiquitinase and interferon antagonist activities of Middle East respiratory syndrome coronavirus papain-like protease Journal of General Virology, 95 (Pt_3), 614-626 DOI: 10.1099/vir.0.059014-0
Kilianski, A., Mielech, A., Deng, X., & Baker, S. (2013). Assessing Activity and Inhibition of Middle East Respiratory Syndrome Coronavirus Papain-Like and 3C-Like Proteases Using Luciferase-Based Biosensors Journal of Virology, 87 (21), 11955-11962 DOI: 10.1128/JVI.02105-13
Lau, S., Lau, C., Chan, K., Li, C., Chen, H., Jin, D., Chan, J., Woo, P., & Yuen, K. (2013). Delayed induction of proinflammatory cytokines and suppression of innate antiviral response by the novel Middle East respiratory syndrome coronavirus: implications for pathogenesis and treatment Journal of General Virology, 94 (Pt_12), 2679-2690 DOI: 10.1099/vir.0.055533-0
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
Al-Tawfiq, J., Momattin, H., Dib, J., & Memish, Z. (2014). Ribavirin and interferon therapy in patients infected with the Middle East respiratory syndrome coronavirus: an observational study International Journal of Infectious Diseases, 20, 42-46 DOI: 10.1016/j.ijid.2013.12.003
Yang Y, Zhang L, Geng H, Deng Y, Huang B, Guo Y, Zhao Z, & Tan W (2013). The structural and accessory proteins M, ORF 4a, ORF 4b, and ORF 5 of Middle East respiratory syndrome coronavirus (MERS-CoV) are potent interferon antagonists. Protein & cell, 4 (12), 951-61 PMID: 24318862
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