Assessing Activity Model For MERS Coronavirus Proteases Using Luciferase-Based Biosensors And Its Application

Background The Middle East respiratory syndrome coronavirus(MERS-Co V) is a serious threat to human health, the death rate is up to 40%, which is far higher than that of SARS in 2003. A recent epidemic happened in May last year, which caused 33 people died of 183 infections in South Korea. Coronavirus is a respiratory virus, which is widespread, variety and has high mutation rate. Currently, research in anti-coronavirus drugs is far from enough. One of the key strategies to control the virus replication is to inhibit the activity of replicase complex(RC), which including the proteases(3CLpro or PLpro). The two kinds of protease are encoded in common by small RNA virus families, but there is no similar protease in human, so it’s the suitable target to block viral replication by preventing its catalytic function to effectively process the viral polyprotein. In this study, a biosensor has been designed basing on a luciferase reporter system to simulate the mechanism of Co V proteinases in the cellular level, to detect the cleavage activity of proteinases and to screen protease inhibitors in a high sensitivity and high throughput manner. Further studies have been performed to determine the protease catalytic sites and the cleavage sites with the biosensor model.Finnally some potential protease inhibitors were obtained with the biosensors. These results will be helpful for further studies of protease structure and functions and screening anti-Co V treatments.Objective Establishing a model to detect the activity of Co V protease, and then using the model to study the catalytic sites and conserved sequence of its substrate of human MERS-Co V papain-like protease(PLpro) and trypsin protease(3CLpro). Then, we have compared the processing activity of different Co V proteases and infered the processing mechanism of the proteases. We have screened the antiviral protease inhibitors by using the biosenser, which could provide research direction for the screening of human anti-virus drugs.Methods Firstly, the MERS-Co V PLpro, MERS-Co V 3CLpro and their biosensor substrates such as p Glo-30F-M-nsp1/2, 2/3, 3/4(PLpro), 4/5, 5/6, 6/7, 7/8(3CLpro)have been synthesized. Secondly, PLpro/3CLpro protease catalytic active sites including Cys-His-Asp/Cys-His have been mutated to Ala by PCR-based site-directed mutagenesis in order to obtain these enzyme catalytic active site mutants. At the same time, each corresponding mutant of protease substrates p Glo-30F-M-nsp2/3 and p Glo-30F-M-nsp7/8 has also been constructed. Thirdly, the function of MERS PLpro/3CLpro protease has been detected using dual luciferase reporter assay, the differences of the catalytic activitivies among different coronavirus have been compared with the biosensors, and Western blot has been used to confirm the expression of proteases. Finally, inhibitor of anti-coronavirus proteases have been screened by using the biosensor model based on activity of luciferase.Results1. MERS PLpro protease biosensor has been constructed successfully. The main components include MERS PLpro and the engineered plasmid construct carrying luciferase reporter and the protease recognizing sequence. The principle of the model is that the these plasmid constructs were transferred to HEK293 T cells, then the expressed protease identify and cleave the biosensor substrates, which lead the two luciferase subunits conformational changed, resulting in the activation of the luciferase. So the amount of fluorescence was detected corresponding to the Co V protease activities. The activites of protease was indirectly detected by means of fluorescence detection. The specificity of the model for processing different substrates was verified.2. The biosensor of MERS PLpro has been applied to study the specific processing the substrates protein of MERS PLpro: MERS PLpro completely cleaved substrate nsp2/3and nsp3/4 to produce the fluorescence, but not completely cleaved substrate nsp1/2.The enzymatic activity of MERS PLpro mutants was significantly reduced. In addition,the experimental results show that when the sequence of the sensor substrate p Glo-30F-M-nsp2/3 mutated, recognition of PLpro to LVGG site is weakened obviously.MERS-Co V PLpro recognizes the highly conserved sequence of the protease substrates,which was dependent on the catalytic site of PLpro protease.3. Activity of different coronavirus PLpro has been studied: SARS /MERS PLpro and PEDV/NL63 PLP2 have been compared to recognize and cleave the site of nsp, with the sensor-substrates of MERS-Co V. The results showed that the PLpro of SARS and MERS was similar with processing the substrates, but PLP2 of PEDV has the low efficiency on processing the substrate, which suggest that the significant differences may be related to the different kind host of coronavirus.4. MERS 3CLpro biosensor has been constructed successfully, the components of it include MERS 3CLpro and the engineered plasmid construct carrying luciferasereporter and the protease recognizing sequence. 3CLpro recognized and cleaved the substrates, which verifed that the 3CL protease biosensor model was successfully constructed. The biosensor of MERS 3CLpro has been applied to detect the specific processing the substrates of MERS 3CLpro, MERS 3CLpro completely cleaved substrate nsp5/6 and nsp7/8 to produce the fluorescence, but not completely cleaved substrate nsp6/7. The enzymatic activity of MERS 3CLpro mutants was significantly reduced. In addition, the experimental results show that when the sequence of the sensor substrate p Glo-30F-M-nsp7/8 mutated, recognition of PLpro to LQA site is weakened obviously. MERS-Co V 3CLpro recognized and cleavaged the substrates specifically,which was dependent on the catalytic site of 3CLpro protease.5. Activity of different coronavirus 3CLpro has been studied, the SARS and MERS3 CLpro have been compared to recognize and cleavage the site of nsp, with the sensor substrates of MERS-Co V and SARS-Co V. The results showed that the 3CLpro of SARS and MERS have the same trend on the recgnizing of different substrates, thus suggests that Co V protease was conservative, and the activity of SARS protease is much higher.6. The potential anti-Co V inhibitors targeting 3CLpro were obtained by the Co V protease biosensors from the compounds including previously reported small molecule anti-SARS drugs and the compounds extracted from natural plants. Our results indicated that Cinaserin can inhibit the activity of MERS 3CLpro, which was previously reported to inhibit the activity of SARS 3CLpro.ConclusionThe biosensor models have been constructed based on the luciferase to detect the activity of MERS-Co V proteases. The model has been verified by using the PLpro and3 CLpro proteases with mutanted catalytic sites, and mutanted substrates in the processing sites of MERS PLpro and 3CLpro. We found that the recognition sequence of substrate is conserved. The proteases of different Co V have certain catalytic differences on the substrate processing, which may inferred the differences related to its host species and the infectivity of the coronaviruses. At last, an potential antiviral drug named Cinaserin was obtained with the biosensor model. Overall, the biosensor model will be helpful for further research of structure and functions of Co V proteases and screening the potential anti-Co V inhibitors targeting Co V proteases.