In this task, you're given a paragraph from the research paper and your task is to generate a suitable title for the research paper based on the given paper. Under 100 words is a good title length.

Q: The production of functional nidovirus replication-transcription complexes involves extensive proteolytic processing by virus-encoded proteases. In this study, we characterized the viral main protease (M pro ) of the type species, White bream virus (WBV), of the newly established genus Bafinivirus (order Nidovirales, family Coronaviridae, subfamily Torovirinae). Comparative sequence analysis and mutagenesis data confirmed that the WBV M pro is a picornavirus 3C-like serine protease that uses a Ser-His-Asp catalytic triad embedded in a predicted two-␤-barrel fold, which is extended by a third domain at its C terminus. Bacterially expressed WBV M pro autocatalytically released itself from flanking sequences and was able to mediate proteolytic processing in trans. Using N-terminal sequencing of autoproteolytic processing products we tentatively identified Gln2(Ala, Thr) as a substrate consensus sequence. Mutagenesis data provided evidence to suggest that two conserved His and Thr residues are part of the S1 subsite of the enzyme's substrate-binding pocket. Interestingly, we observed two N-proximal and two C-proximal autoprocessing sites in the bacterial expression system. The detection of two major forms of M pro , resulting from processing at two different N-proximal and one C-proximal site, in WBV-infected epithelioma papulosum cyprini cells confirmed the biological relevance of the biochemical data obtained in heterologous expression systems. To our knowledge, the use of alternative M pro autoprocessing sites has not been described previously for other nidovirus M pro domains. The data presented in this study lend further support to our previous conclusion that bafiniviruses represent a distinct group of viruses that significantly diverged from other phylogenetic clusters of the order Nidovirales. Cell culture and propagation of virus. Epithelioma papulosum cyprini (EPC) cells (RIE173) were obtained from the cell culture collection of the Friedrich-Loeffler-Institut (Greifswald-Insel Riems, Germany). They were grown in minimum essential medium with Earle's salts, 25 mM HEPES, GlutaMAX I (Invitrogen, catalog no. 42360-024) supplemented with 1 mM sodium pyruvate (Invitrogen, catalog no. 11360-039) and 10% fetal bovine serum (PAA Laboratories) at 27°C in a 5% CO 2 atmosphere. WBV isolate DF24/00 (23) was propagated on EPC cells at 20°C. Construction and mutagenesis of expression plasmids. Plasmids were generated by using standard cloning procedures. All PCR products were generated by using Pfu polymerase (Promega) according to the manufacturer's instructions. The correct sequences of the inserts were verified by sequencing using a BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems). To generate pMal-WBV-3424-3725-GST and pMal-WBV-3424-3725_S3589A-GST, the coding sequence for the WBV pp1a/pp1ab amino acid residues Gln-3424 to Met-3725 was amplified by using the primers MalE24 (5Ј-GGTCGTCAGACTGTCGATGAA GCC-3Ј) and M pro _BbsI (5Ј-AATAAGAAGACTGTTGTTAACTGGTGATG TGAGTGT-3Ј) and pMal-WBV-3CL_559-560 or pMal-WBV-3CL_S3589A (38) as a template, while the Schistosoma japonicum glutathione S-transferase (GST) coding sequence was amplified from pCITE-GST (44) by using the primers GSTup_BsaI (5Ј-AAAAGGTCTCTACAACATGAGCCCTATACTAGGTTA TTGG-3Ј) and GSTdn_BamHI (5Ј-AAAGGATCCTTAACGCGGAACCAGA TCCGATTTTGG-3Ј). The resulting PCR products were digested with BbsI and BsaI, respectively, and subsequently ligated by using T4 DNA ligase (New England Biolabs [NEB]). The resulting pp1a-3424-3725-GST fusion sequence was amplified by using MalE24 and GSTdn_BamHI, digested with SacI and BamHI, and ligated with SacI/BamHI-digested pMal-c2X plasmid DNA (NEB). To construct pMal-WBV-M pro -CHis, the coding region for pp1a/pp1ab amino acids Gln-3424 to Thr-3707 was amplified from pMal-pp1a-3424-3725-GST by using the primers JZ559 (5Ј-TCAGCATCAGAATGCATTCTGTAT-3Ј) and RU32 (5Ј-TTTGGATCCTTAGTGATGGTGATGGTGATGTGTTACTGGT GCTACTGGGAG-3Ј). The resulting PCR product was phosphorylated by using T4 polynucleotide kinase (PNK; NEB), digested with EcoRI and ligated with XmnI/EcoRI-digested pMal-c2X plasmid DNA (NEB). The inserts for plasmids pMal-WBV-N1-C1, pMal-WBV-N1-C2, pMal-WBV-N2-C1, and pMal-WBV-N2-C2 were amplified from pMal-WBV-3424-3725_S3589A-GST with the primers RU36 (5Ј-GCCCTCCAACGTATCAGAC AAGC-3Ј) in combination with RU177 (5Ј-ATAGAATTCTTATTGTCTGGT GATGGTGTTAC-3Ј), RU36 in combination with RU191 (5Ј-AAAGGATCCT TATTGTCCTACTGTGACAGATGT-3Ј), RU37 (5Ј-GCAGTCACAGTTGTT GTAACAC-3Ј) in combination with RU177, and RU37 in combination with RU191, respectively. The 5Ј termini of the resulting PCR products were phosphorylated by using PNK. The inserts for pMal-WBV-N1-C1 and pMal-WBV-N2-C1 were digested with EcoRI and then ligated with XmnI/EcoRI-digested pMal-c2X plasmid DNA. The inserts for pMal-WBV-N1-C2 and pMal-WBV-N2-C2 were digested with BamHI and ligated with XmnI/BamHI-digested pMal-c2X plasmid DNA. Site-directed mutagenesis of plasmid constructs was carried out by PCR-based methods essentially as described previously (28, 55) . Sequences of oligonucleotides used for mutagenesis are available on request. Expression of fusion proteins. Escherichia coli TB1 (NEB) cells transformed with pMal-pp1a-3424-3725-GST or pMal-pp1a-3424-3725_S3589A-GST were grown in Luria-Bertani (LB) medium containing ampicillin at a concentration of 100 g per ml until the culture reached an optical density at 600 nm (OD 600 ) of

A: Characterization of Bafinivirus Main Protease Autoprocessing Activities ᰔ Downloaded from
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Q: Chemical denaturant sensitivity of the dimeric main protease from severe acute respiratory syndrome (SARS) coronavirus to guanidinium chloride was examined in terms of fluorescence spectroscopy, circular dichroism, analytical ultracentrifuge, and enzyme activity change. The dimeric enzyme dissociated at guanidinium chloride concentration of ,0.4 M, at which the enzymatic activity loss showed close correlation with the subunit dissociation. Further increase in guanidinium chloride induced a reversible biphasic unfolding of the enzyme. The unfolding of the C-terminal domain-truncated enzyme, on the other hand, followed a monophasic unfolding curve. Different mutants of the full-length protease (W31 and W207/W218), with tryptophanyl residue(s) mutated to phenylalanine at the C-terminal or N-terminal domain, respectively, were constructed. Unfolding curves of these mutants were monophasic but corresponded to the first and second phases of the protease, respectively. The unfolding intermediate of the protease thus represented a folded C-terminal domain but an unfolded N-terminal domain, which is enzymatically inactive due to loss of regulatory properties. The various enzyme forms were characterized in terms of hydrophobicity and size-and-shape distributions. We provide direct evidence for the functional role of C-terminal domain in stabilization of the catalytic N-terminal domain of SARS coronavirus main protease.

A: Reversible Unfolding of the Severe Acute Respiratory Syndrome Coronavirus Main Protease in Guanidinium Chloride
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Q: Viral diseases such as influenza, which are easily transferable from person to person or even country to country, pose one of the biggest threats to health today. Viruses such as avian influenza viruses (N1H5 and H9N1) have been reported to spread in the present decade and, very recently, the novel coronavirus that has caused many life-threatening illnesses and deaths all around the world has received much attention. To prevent these highly contagious viral infections, we have proposed the combination of IMODä and Arbidol to increase their immunomodulatory effects as a novel medicine to prevent and cure influenza and some other infectious diseases such as hepatitis B and C. On the one hand, IMODä within the last few years has been proven to safely and effectively increase the life expectancy for human immunodeficiency virus (HIV)-infected individuals by increasing CD4 lymphocytes. On the other hand, Arbidol, an antiviral agent has been used safely and effectively in the past two decades to prevent and cure all types of influenza and flu. Therefore, the combination of both in a single dosage to further increase CD4 lymphocytes and interferon gamma (IFN-c) could be a better choice for treatment of viral infections. This proposal tries to provide enough support and background for approval of a randomized clinical trial by a relevant team of investigators. ª 2014 Published

A:
Combination of IMODä and Arbidol to increase their immunomodulatory effects as a novel medicine to prevent and cure influenza and some other infectious diseases Journal of Medical Hypotheses and Ideas
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