Expression of potyviral polyproteins in transgenic plants reveals three proteolytic activities required for complete processing.

Abstract

All proteins encoded by the plant potyvirus, tobacco etch virus (TEV), arise by proteolytic processing of a single polyprotein. Two virus‐encoded proteinases (NIa and HC‐Pro) that catalyze most of the proteolytic events have been characterized previously. The two proteins that are derived from the N‐terminal 87 kd region of the viral polyprotein are a 35 kd protein and HC‐Pro (52 kd). It is demonstrated in this study that a third proteolytic activity is required to process the junction between these proteins. Proteolysis at the HC‐Pro N terminus to separate these proteins occurred poorly, if at all, after in vitro synthesis of a 97 kd polyprotein, whereas cleavage of the HC‐Pro C terminus occurred efficiently by an autoprocessing mechanism. Synthesis of the same polyprotein in transgenic tobacco plants, however, resulted in complete and accurate proteolysis at both termini of HC‐Pro. A point mutation affecting an amino acid residue essential for the proteolytic activity of HC‐Pro had no effect on N‐terminal processing. Expression in transgenic plants of a construct with a large deletion in the 35 kd protein coding region resulted in partial inhibition of HC‐Pro N‐terminal cleavage, suggesting that the 35 kd protein may affect the proteolytic event but not in a catalytic role. We speculate that this cleavage event is catalyzed by either a cryptic potyviral proteinase that requires a host factor or subcellular environment for activation, or possibly a host proteinase.

Nuclear Transport of Plant Potyviral Proteins

“…Monospecific antisera were raised in New Zealand White rabbits against Nla, Nlb, and HC-Pro proteins that were overproduced in Escherichia coli and against CI and capsid proteins isolated from infected plants (Dougherty and Hiebert, 1980a) and virions, respectively, using the method of Harlow and Lane (1 988). Anti-HC-Pro serum has been described (Carrington et al, 1990).…”

“…A Dral-Dral DNA fragment encoding a 52-kD segment of Nlb was isolated from pTL-5495 (Carrington and Dougherty, 1987) and inserted into Ncol-cut, Klenow-treated pKK233-2. The Nla and Nlb proteins were isolated in the form of insoluble granules from cultures (3 hr post-induction) containing the recombinant plasmids as described previously (Carrington et al, 1990). The granules were dissolved in SDS (0.1 70) before emulsification with adjuvant and injection into rabbits.…”

“…The TEV 5'-nontranslated region (NTR) has been incorporated into pTL7SN.3, resulting in cap-independent enhancement of translation of transcripts in vitro and in vivo (Carrington and Freed, 1990). Each plasmid (pTL7SN.3-Nla, pTL7SN.3-Nlb, and pTL7SN.3-GUS) was used to produce two mutagenized derivatives-one that resulted in insertion of a Bglll restriction enzyme site at the 5' end of the open reading frame and one that resulted in a Bglll site at the 3' end.…”

“…Total SDS-soluble protein extracts were prepared from leaf tissue (0.1 g) after O days to 7 days post-inoculation as described (Carrington et al, 1990), except that the samples were first frozen in liquid nitrogen and ground to a fine powder with a mortar and pestle. The concentration of protein in each extract was measured using the Bio-Rad Protein Assay Kit with BSA as a concentration standard.…”

“…Methods for transcription by SP6 RNA polymerase and translation in a rabbit reticulocyte lysate have been described previously N. tabacum cv Xanthi nc protoplasts were isolated as described (Carrington and Freed, 1990). Plasmid DNA (20 pg) was introduced into protoplasts (3 to 5 X 105) for transient expression using the polyethylene glycol-mediated transfection procedure (Negrutiu et al, 1987).…”