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...et al., 1989, 1993; Gorbalenya et al., 1991). Third, the scissile bond that is cleaved to release the carboxyl terminus of p28 was found to be located between Gly-247 and Val-248 (Dong & Baker, 1994; =-=Hughes et al., 1995-=-). In subsequent studies, immunoprecipitation experiments with region-specific antisera revealed that additional proteolytic cleavages within the amino-proximal region of the MHV-A59 replicase polypro...

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... of MHV–A59) (35,76) resulted in the loss of proteinase activity, suggesting that these residues form the catalytic dyad (77). Cleavage to give p28 was at an RGV motif at the G247/V248 dipeptide bond =-=(78,79)-=-, and presumbably occurred in cis (75). Reactions of specific antisera raised against different regions of MHV POL1awith potential cleavage products with apparent molecular weights of 65, 50, 240, and...

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...between residues 1290 and 1310, just downstream of the catalytic His1272. The cleavage sites of most viral papain-like proteases follow amino acids with small side chains, such as glycine and alanine =-=(12, 17)-=-, and this region contained several candidate GG and AA dipeptides. In one experiment, cells infected with a construct in which Tyr-1315 was changed to methionine (dsSIN/NSPro2:Y1315M) (this substitut...

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...d M. D. Ryan Fig. 4. For legend see facing page. BIIE Review: Togavirus proteinases cleavage-site specificity is similar to other viral papain-like proteinases [mouse hepatitis virus PLP-1 – Gly}Val (=-=Hughes et al., 1995-=- ; Bonilla et al., 1997) ; equine arteritis virus – Gly}Gly (Snijder et al., 1994) ; potyvirus HC-Pro – Gly}Gly (Carrington et al., 1989) ; hypovirulence associated virus p29 proteinase – Gly}Gly (Cho...

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...(MHV) PL2pro but not in MHV PL1pro (Fig. 1D). This fact correlates with the available specificity data showing a preference for the large arginine residue at the P2 position in the case of MHV PL1pro =-=(2, 3, 9)-=- and with the observation that the irreversible inhibitor E-64d, which contains a bulky P2 residue (leucine), inhibits MHV PL1pro (5, 11) but not MHV PL2pro (10). Structural similarities to HAUSP sugg...

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...fused ORF (Fig. 1). This region of gene 1a shows a sequence nearly identical to that of the same region of the MHV genome (4) and therefore probably represents the first 96 aa of a homolog of MHV p28 =-=(14)-=-. To test whether the BCV DI ORF plays a functional role in RNA replication, site-specific point mutations were introduced into pDrep1 such that the character of the ORF was changed (Fig. 2), and each...

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...f the determinants of the p28 cleavage sites revealed that the P5 (arginine or lysine), P2 (arginine), and P1 (glycine or alanine) positions are critical for recognition and processing of p28 by PLP1 =-=(13, 24)-=-. The p65 cleavage site showed similar dependence on glycine or alanine in the P1 position and arginine or lysine in the P5 position. Furthermore, the p28 and p65 cleave sites could be interchanged an...

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...ely after translation of the PLP1 domain within p210, and p28 appears to be stable in infected cells for several hours after cleavage (15, 16). Determinants of p28 cleavage have been defined in vitro =-=(16, 20, 21)-=- and recently confirmed by direct mutagenesis of the cleavage site using a reverse genetic approach (11, 40). Interestingly, MHV mutants that are incapable of liberating p28 from the nascent polyprote...

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...ing bovine coronavirus and mouse hepatitis virus (MHV), PLP1 has been shown in vitro to cleave an amino-terminal 28-kDa protein and a 65-kDa protein, referred to as p28 and p65, respectively (Fig. 1) =-=(1, 2, 7, 8, 10, 14)-=-. In contrast, SARS-CoV, as well as the avian coronaviruses (group 3), such as infectious bronchitis virus (IBV), only express active PLP2, which is responsible for cleavage of amino-terminal proteins...

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...n and a 195-kDa protein in IBV-infected cells (23, 24, 26). Similar enzymatic activities were also identified for the first and second papain-like proteinase domains of human and murine coronaviruses =-=(1, 2, 3, 11, 14, 17, 20, 21, 36, 45)-=-. Processing of the 1a and 1a/1b polyproteins at other positions is mediated by the 3C-like proteinase. This proteinase was shown to recognize several conserved Q-S (G and N) dipeptide bonds, resultin...