Mutational Analysis of Plum Pox Potyvirus Polyprotein Processing By the NIa Protease in Escherichia Coli

Abstract

A binary Escherichia coli expression system has been used to study the pathway for proteolytic processing of the plum pox potyvirus (PPV) polyprotein. Trans cleavage at the carboxyl end of the cylindrical inclusion protein occurred, although with lower efficiency than that at the large nuclear inclusion protein-capsid protein junction. No trans cleavage at the carboxyl end of the small nuclear inclusion protein (NIa) was detected. The proteolytic activities at different cleavage sites of several deletion and point mutations of NIa protein have been analysed, The large ASX deletion and two different point mutations at His 239 abolished proteolytic activity at all sites. The effect of other mutations, particularly a Glu substitution for Asp 274, depended on the particular cleavage site analysed. The results obtained with the PPV NIa protein mutants were similar to those reported for comparable mutations in the tobacco etch virus 49K protease, despite differences in the sequences recognized for processing. No evident competitive inhibition of the proteolytic activity of PPV NIa protease by the presence of an excess of the different protease mutants could be demonstrated.

Highlights and prospects of potyvirus molecular biology

“…The results obtained suggest that the recognition and catalytic sites of the NIa proteases are closely interlinked and that the main determinants for substrate specificity lie in the Cterminal one-third of the protease . It has also been suggested that the extreme carboxyl end of the protease could be involved in maintenance of the proper structure that would allow other parts of the protein to interact correctly with the various cleavage sites, since the effect caused by different mutations in this region depends on the cleavage site considered (Carrington & Dougherty, 1987a;Garcia et al, 1990). Finally, it has been shown that the potyviral NIa protease processes a great deal of substrate efficiently (Garcia et al, 1989c).…”

“…NIa protease is catalytically active within the polyprotein, excising itself by cis cleavages at sites D and E in a process that might happen as a primary event (maybe cotranslational) in the proteolytic cascade (Carrington & Dougherty, 1987a, b;Hellmann et al, 1988;Garcia et al, 1990). In summary, NIa-catalysed proteolytic maturation of the potyviral polyprotein is a regulated process involving cis and trans cleavages at sites which are recognized with different efficiencies and processed at different rates.…”

“…The proposed catalytic triad, composed of His, Asp and Cys residues, is located in the C-terminal half of the molecule (Dougherty et al, 1989 b;Garcia et al, 1990;Ghabrial et al, 1990) and, in fact, NIa protease is probably multifunctional as its N-terminal half is not relevant for its proteolytic activity in vitro or in E. coli (Carrington & Dougherty, 1987a; but has been found covalently attached to the genomic RNA (VPg; see below). Interestingly, a substitution of Glu for the catalytic Asp in the TEV and PPV NIa proteases resulted in anomalous proteolytic activities, the effect of these substitutions depending on the particular cleavage site analysed (Dougherty et al, 1989b;Garcia et al, 1990). It could be inferred from these results that there are different structural requirements in the protease, probably similar in TEV and PPV, for processing at the various cleavage sites.…”

Potyviral NIa Proteinase, a Proteinase with Novel Deoxyribonuclease Activity

“…The reaction was terminated after 30 min by addition of 6 times loading dye and was monitored by electrophoresis on 0.7% agarose gel followed by ethidium bromide staining. Lanes 3,5,7,9, and 11 represent the reaction products of His-tagged NIa, NIa C151A, NIa H46A, NIa D81N, and NIa D81G proteinase, respectively; lanes 4,6,8,10, and 12 represent the reaction products of GST-fused NIa, NIa C151A, NIa H46A, NIa D81N, and NIa D81G proteinase, respectively; samples with heat-denatured His-tagged NIa and GST-NIa added as negative control are represented in lanes 1 and 2, respectively. B, phage DNA (50 g) was incubated with purified His-tagged NIa proteinase (5 g) in 20 mM Tris-HCl, pH 8.2, containing 1 mM MgCl 2 at 37°C for different times, and the reaction was stopped by addition of equal volumes of acid lanthanum reagent (20 mM La(NO 3 ) 3 in 0.2 N HCl). The precipitate was removed by centrifugation, and absorbance of the supernatant solution was measured at 260 nm against a blank sample without enzyme.…”

“…Site-specific mutagenesis studies with tobacco etch virus (TEV) (13), Plum pox potyvirus (8), and PVBV NIa proteinases (9) have unequivocally established the role of His-Asp-Cys in forming the catalytic triad responsible for the cleavage by the proteinase. The specificity of these proteinases is determined by the heptapeptide recognition sequence surrounding each cleavage site (10,11).…”