It’s been well established that in several picornaviruses, em e.g /em . inserted between two versions of modified yellow fluorescent proteins that are capable of F?rster resonance energy transfer (FRET). Cleavage in the linker sequence is accompanied by the concomitant loss of FRET signal. Albeit the potential adaptability in turning this assay into a high-throughput screening vehicle, the utility of this method, however, is also limited as it cannot study the influence of other factors on proteolysis such as putative exosites. The substrate sequence of the 3C cleavage sites in the HAV polyprotein were initially predicted from sequence homology of the HAV genome to the other picornaviral genomes [1]. The location of several cleavage sites has subsequently been confirmed or corrected experimentally [5], [8], [9], [10], [11], [12], [13]. Table 542.1 shows the amino acid sequence of seven HAV 3C cleavage sites in the polyprotein which have been experimentally confirmed. Table 542.1 Hepatitis A virus 3C proteinase cleavage sites in the viral polyprotein determined quantitatively the inhibition of HAV 3C with peptide substrate-derived aldehyde inhibitors in which the basic design of the inhibitor is to have a reactive warhead appended C-terminally to a tetrapeptide analog representing the P4-P3-P2-P1(Qdm) residues of a substrate with P1(Qdm) being glutaminal with its side chain amide dimethylated [21]. This design was followed by other experimentations that saw the replacement of the aldehyde group by halomethyl ketone or phthalhydrazide [6], [16]; in one variant of the latter case, the P1 Gln was also substituted with a 2-oxo-pyrrolidine ring to improve the inhibitory effect [18]. Similarly, Huang form or in complex with various inhibitors [23], [27], [28]. The overall fold and domain structure of the HAV 3C picornain resembles that of the chymotrypsin-like serine proteinases (Clan S1) with the proteolytic active site formed between two anti-parallel -barrel domains (Figure 542.1). Unique features of the HAV 3C picornain are the amino- and carboxyl-terminal helices that pack against the opposite domain, and a long anti-parallel -ribbon that extends from the -barrel of the carboxyl-terminal domain and forms part of the active site (colored green in Figure 542.1A). Cys172, His44 and Asp84 NBI-42902 form the canonical catalytic triad in the active site. In the first refined crystal structure of the active enzyme, an ordered water molecule takes up the place of the carboxylate of a third member of a typical catalytic triad. It was thus suggested that a charged form of the side chain of Tyr143 stabilizes this arrangement NBI-42902 and may be involved in catalysis [28]. However, more recent, higher-resolution crystal structure of HAV 3C in a different crystal form confirmed the existence of the canonical Cys:His:Asp catalytic triad in the enzymes active site, finally laying the dyad proteinase activity assay confirmed that the inhibitory effect is slow-acting requiring hours of pre-incubation of the compound with the enzyme but nevertheless irreversible. It was also derived from this structure that an unusual episulfide cation may be the intermediate molecular species that is formed during the chemical reactions leading to either inhibition or peptide hydrolysis DNAJC15 [27]. Preparation HAV 3C picornain has NBI-42902 been expressed in bacteria [4], [9], [29], cell free transcription-translation systems [10], [30] and eukaryotic cells [12], [13]. For kinetic and structural studies the enzyme has been purified from a bacterial overexpression system as described by Malcolm and systems. Interpretation of these results is further NBI-42902 complicated by the appearance of aberrant initiation and premature termination products [11]. Schultheiss is not clear. Nevertheless, it is becoming increasingly obvious that HAV is distinct from the other members of the viral family in its NBI-42902 polyprotein processing [5], [12], [13]. Most importantly, 3C appears to be the only virally encoded entity that shows proteolytic activity in HAV [5]. This is in sharp contrast to other picornaviruses which typically have two functional viral proteinases. Picornaviral genomes are generally only ~7500 nucleotides long and encode less than a dozen individual viral proteins when fully processed from polyproteins. Such genetic austerity is compensated via several means to enhance the coding capacity of their RNA genomes, one of which is to have individual protein engaged in two or more different tasks. Besides their role in polyprotein processing, 3C proteinases are also involved in viral mRNA translation, viral genomic RNA replication and virus-host antagonism [32]. For example, 3C or its precursor, 3CD,.