Biol. Chem., Vol. 392, pp. 937–945, October 2011 • Copyright © by Walter de Gruyter • Berlin • Boston. DOI 10.1515/BC.2011.097

Analysis of the autoproteolytic activity of the recombinant helper component proteinase from zucchini yellow mosaic virus

Kajohn Boonrod 1 ,a , Marc W. F ü llgrabe 1 ,a , Gabi Krczal 1 and Michael Wassenegger 1,2, *

1 RLP-AgroScience GmbH , AlPlanta-Institute for Plant Research, Breitenweg 71, D-67435 Neustadt , Germany 2 Centre for Organismal Studies Heidelberg , University of Heidelberg, D-69120 Heidelberg , Germany

* Corresponding authore-mail: michael.wassenegger@agroscience.rlp.de

Abstract

The multifunctional helper component proteinase (HC-Pro) of potyviruses contains an autoproteolytic function that, together with the protein 1 (P1) and NIa proteinase, pro-cesses the polyprotein into mature proteins. In this study, we analysed the autoproteolytic active domain of zucchini yel-low mosaic virus (ZYMV) HC-Pro. Several Escherichia coli -expressed MBP:HC-Pro:GFP mutants containing deletions or point mutations at either the N- or C-terminus of the HC-Pro protein were examined. Our results showed that amino acids essential for the proteolytic activity of ZYMV HC-Pro are dis-tinct from those of the tobacco etch virus HC-Pro, although the amino acid sequences in the proteolytic active domain are conserved among potyviruses.

Keywords: autoproteolytic activity; helper component proteinase; site-directed mutagenesis.

Introduction

The genus Potyvirus contains about 180 members and repre-sents the largest and economically most damaging group of plant viruses for many crops (e.g., cucurbit, tomato, potato and sugarcane). The zucchini yellow mosaic virus (ZYMV) is a member of the genus Potyvirus , and its genome contains one long open reading frame that encodes a 350-kDa poly-protein (Riechmann et al. , 1992, 1995 ). This is processed into ‘ mature ’ proteins by the activity of three viral-encoded proteases. In addition to the NIa protease that catalyses the cleavage of the polyprotein at different sites in trans , includ-ing cleavage of its own C-terminus, two additional proteases, protein 1 (P1) and helper component proteinase (HC-Pro), exhibit autoproteolytic activity at their C-termini and catalyse the cleavage from the polyprotein (Carrington and Dougherty , 1987a,b ; Carrington et al. , 1989a ; Verchot et al. , 1991 ).

a These authors contributed equally to this work.

HC-Pro is a multifunctional protein that covers multiple roles in the viral life cycle. These functions include the trans-mission of the virus by aphids (Thornbury et al., 1985), a protease activity which cleaves the HC-Pro in cis at its own C-terminus of the polyprotein (Oh and Carrington , 1989 ), spreading and replication of the virus (Cronin et al. , 1995 ; Kasschau and Carrington , 1995 ), binding of RNA through two single-stranded RNA-binding domains (Urcuqui -Inchima et al., 2000 ), binding to the coat protein of the virus (Blanc et al. , 1997 ), synergistic infection by other viruses (Shi et al. , 1997 ; Wang et al. , 2002 ) and an RNA silenc-ing suppressor (RSS) activity (Anandalakshmi et al. , 1998 ; Brigneti et al. , 1998 ; Kasschau and Carrington , 1998 ).

Furthermore, multiple interactions of HC-Pro with host proteins were identifi ed, including the rgs-CaM protein of Nicotiana tabacum (Anandalakshmi et al. , 2000 ); the RING-fi nger proteins HIP1 and HIP2 of potato (Guo et al. , 2003 ); three subunits of the 20S proteosomes in Arabidopsis thali-ana (Ballut et al. , 2005 ; Jin et al. , 2007a ); the factor NtMinD, essential for chloroplast division in N. tabacum (Jin et al. , 2007b ); and the regulator of the ATPase of the vacuolar mem-brane (RAV2), a transcription factor regulating the expression of the endogenous silencing suppressor proteins FRY1 and CML38 (Endres et al. , 2010 ).

According to these functions, HC-Pro can be schematically divided into three regions: the N-terminal region, essential for the spread of the virus; the C-terminal region, harbour-ing the protease activity; and a central domain, crucial for the remaining functions. However, some functions overlap or are distributed in different regions of the protein. HC-Pro forms dimers or multimers and is probably active in this form (Thornbury et al., 1985; Wang and Pirone , 1999 ).

Besides its RSS activity, HC-Pro also exhibits protease activity. It has been shown that the conserved Cys-344 and His-418 of tobacco etch virus (TEV) HC-Pro, which are located in the active domain, are essential for the proteolytic activity (Oh and Carrington , 1989 ). Furthermore, the residues at positions 1, 2, 4 and 1 ′ (P1, P2, P4 and P1 ′ ) at the transition of the HC-Pro protein to the protein P3 have a major effect on the processing of the polyprotein by HC-Pro (Carrington and Herndon , 1992 ).

In this work, the proteolytic active domain and essential amino acids for proteolytic activity of ZYMV HC-Pro were investigated by analysing different mutants (deletion and sub-stitution) of the C-terminal part of the HC-Pro coding region. We showed that the autoproteolytic active domain of ZYMV HC-Pro differs from that of TEV HC-Pro. Moreover, the amino acid sequence at the cleavage site was not crucial for cleavage effi ciency.

938 K. Boonrod et al.

Results

Expression of MBP:HA-HC-Pro:GFP in

Escherichia coli leads to autoproteolytic

cleavage of green fl uorescent protein (GFP)

To analyse the autoproteolytic activity of ZYMV HC-Pro, a protein sequence alignment of the C-terminal region of

different potyvirus HC-Pro proteins was performed. The alignment showed that Cys-342 and His-415 as well as the residues at positions P1, P2, P4 and P1 ′ are conserved among the members of potyviruses. Furthermore, the align-ment revealed that Leu-351, Asn-353 and Glu-356 are also highly conserved among HC-Pro proteins of potyviruses (Figure 1 ). It was reported that the autoproteolytic active domain of TEV HC-Pro is located at Cys-344 and His-418

Figure 1 Sequence analysis of the C-terminal conserved amino acids (single-letter code) of the HC-Pro proteins of ZYMV (NP_734184.1), TuMV (NP_734214.1), TEV (NP_734208.1), PVY (NP_734244.1) and lettuce mosaic virus (LMV) (NP_734154.1). (A) Conserved residues are in bold letters and indicated in the consensus sequence by an asterisk. The putative proteolytic active domain is highlighted. In the ZYMV HC-Pro sequence, C-terminal deletion and point mutations are indicated by arrows. (B) Western blot analysis of the soluble fraction extracted from bacteria expressing the recombinant MBP:HA-HC-Pro:GFP protein (Wt) using anti-GFP antibodies. Neg, total soluble protein extraction from non-induced bacteria, as a negative control. GFP, GFP positive control. Proteins were subjected to 9 % SDS-PAGE.

Autoproteolytic activity of the recombinant HC-Pro from zucchini yellow mosaic virus 939

(Oh and Carrington , 1989 ). To analyse the effects of these and additional conserved residues, different deletion mutants of ZYMV HC-Pro were produced and analysed for their role in autoproteolytic activity (Figure 1 A).

ZYMV HC-Pro was successfully expressed as a functional maltose binding (MBP)-fused protein (MBP:HA-HC-Pro) in E. coli (Fuellgrabe et al. , 2010 ). To study the autoproteolytic activity, the GFP was fused at the C-terminus of MBP:HA-HC-Pro by using the pMAL:HA-HC-Pro clone (Fuellgrabe et al. , 2010 ), resulting in the pMAL:HA-HC-Pro:GFP (Wt) fusion protein. The HA-tag at the HC-Pro C-terminus enabled, in addition to the GFP, an alternative for detection or purifi cation of the fusion protein by using anti-HA antibod-ies. After transformation of BL21 + E. coli cells, suspended cells were induced with isopropyl- β - d -thiogalactopyranoid (IPTG) and cultured overnight at 14 ° C. Cells were harvested and extracted before the soluble and insoluble fractions were analysed by gel electrophoresis and Western blotting with anti-GFP antibodies.

Western blot analysis showed that MBP:HA-HC-Pro:GFP was mainly detectable in the cell pellet as a full-length protein with a molecular weight of 126 kDa (see below). However, in the total soluble protein fraction, a band closely correspond-ing to the size of the GFP was detected, suggesting that GFP was effi ciently processed from the fusion protein (Figure 1 B, Wt).

N-terminal sequencing of processed GFP

The cleavage site of HC-Pro was determined by sequencing the N-terminus of the immunoprecipitated cleavage product of the E. coli -expressed MBP:HA-HC-Pro:GFP. The sequence data showed that cleavage occurred in the GFP between an alanine (Ala-37) and a threonine (Thr-38), demonstrating that the cleavage site was outside of the HC-Pro protein sequence (Figure 2 A). Upon cleavage, a truncated GFP (xGFP) with a molecular weight of about 24 kDa was produced. To confi rm this observation, proteins were subjected to SDS-PAGE (17 % ). In a 17 % gel, it should be possible to discriminate the truncated 24 kDa from the 28-kDa full-length GFP protein. SDS-PAGE showed that indeed the xGFP that was cleaved from MBP:HC-Pro:GFP had a smaller size than the GFP (Figure 2 B).

N-terminal region of ZYMV HC-Pro is not

involved in autoproteolytic activity

Previous studies revealed that the catalytic domain of the tobacco vein mottling virus NIa protease is located at the N-terminal region of the protein (His-46, Asp-81 and Cys-151) (Hwang et al. , 2000 ). To test whether the N-terminal region of ZYMV HC-Pro is essential for autoproteolytic activity, the fi rst 93 residues of the N-terminus of ZYMV HC-Pro were deleted, resulting in the pMAL: ∆ N-HC-Pro:GFP protein (N1). The N1 protein was expressed in E. coli , and the total soluble protein fraction was analysed by Western blotting with anti-GFP antibodies. In addition, Western blot analysis of pellet fractions was conducted to provide experimental evidence for the consistent expression of the N1 protein (and for all other recombinant proteins, see below) (Figure 3 A). In the pellet fraction, generally no processing of the ZYMV HC-Pro fusion proteins was observed. Thus, by using anti-GFP antibodies, the full-length proteins could be detected without interference of autocatalytic processing processes. In the soluble protein fraction, a band corresponding to GFP was detectable, suggest-ing that the autoproteolytic activity of ∆ N-HC-Pro was still present (Figure 3 B). From this result, it can be concluded that the catalytic centre of ZYMV HC-Pro is not in its N-terminal region, at least not in a region comprising residues 1 – 93. This fi nding was in agreement with the suggestion that conserved motifs within the N-terminal region are mainly involved in virus transmission (Dolja et al. , 1993 ).

Mutation of the conserved Gly-456 residue

did not affect the autoproteolytic activity of

ZYMV HC-Pro

The proteolytic activity of TEV HC-Pro requires residues fl anking the C-terminal cleavage site. It was reported that substitutions of the highly conserved residues at P1, P2, P4 or P1 ′ positions, which are localised at the transition of HC-Pro to the fusion protein of the polyprotein, resulted in elimina-tion of the autoproteolytic activity (Carrington and Herndon , 1992 ). Comparison of the homologous sequences of potyvi-ral polyproteins revealed that these conserved residues are also present in ZYMV HC-Pro (Figure 1 A). To investigate

Figure 2 N-terminal sequencing of the MBP:HC-Pro:GFP C-terminal cleavage product and Western blot analysis of the processed product. (A) N-terminal sequencing of the immunoprecipitated MBP:HA-HC-Pro:GFP C-terminal cleavage product (xGFP) revealed that the cleavage site of the MBP:HA-HC-Pro:GFP fusion protein was located between alanine (Ala-37) and threonine (Thr-38) of the GFP protein. The physical map represents the amino acid residues junction of MBP, HA-HC-Pro and GFP. The putative cleavage site is indicated by a solid triangle. At both ends of the protein sequences, amino acid residues are indicated by ‘ x ’ . (B) Western blot analysis of the soluble fraction extracted from bacteria expressing the recombinant MBP:HA-HC-Pro:GFP protein (Wt) using anti-GFP antibodies. Neg, total soluble protein extraction from non-induced bacteria, as a negative control. GFP, GFP positive control. Proteins were subjected to 17 % SDS-PAGE.

940 K. Boonrod et al.

Figure 3 Western blot analysis of the soluble protein fractions extracted from bacteria expressing recombinant MBP:HA-HC-Pro:GFP proteins and the mutants. The induced bacterial cells were harvested and extracted as described. The total insoluble (A and C) and soluble protein (B and D) extracts were subjected to 17 % SDS-PAGE. Proteins were blotted and detected with anti-GFP antibodies. Wt, MBP:HA-HC-Pro:GFP; GFP, GFP positive control; Neg, total soluble protein extraction from non-induced bacteria, as a negative control; N1, N-terminal truncated HC-Pro protein MBP: ∆ N HC-Pro:GFP; C1 – 10, C-terminal deletions or point mutations in the HC-Pro protein; C2 + 3 and C2 + 3 + 8, double and triple mutants as indicated in the text.

whether substitution of these residues in ZYMV HC-Pro would also affect the proteolytic activity, a mutant of ZYMV HC-Pro was generated, where the Gly-456 at P1 was substi-tuted by a lysine (Lys-456), resulting in the pMAL:HA-HC-Pro mut C1:GFP (C1) protein (Figure 1 A). The protein was expressed and analysed by Western blotting with anti-GFP antibodies.

Western blot analysis of the total soluble protein frac-tion revealed that only xGFP but no residual full-length C1 protein was detectable (Figure 3 B, C1). On the other hand, the full-length C1 protein was detectable in the pellet frac-tion (Figure 3 A, C1). These results suggest that the C1 protein was fully processed, indicating that, at least, one of the C-terminal residues that were essential for the auto proteolytic activity of TEV HC-Pro was not required for ZYMV HC-Pro activity.

ZYMV HC-Pro contains a catalytic domain

different from TEV HC-Pro

TEV HC-Pro was proposed as a cysteine protease and its pro-teolytic active domain contains the conserved Cys-345 and His-418 residues. The substitution of one of these residues by a serine resulted in loss of the autoproteolytic activity (Oh and Carrington , 1989 ). Accordingly, two different mutants were generated in which the conserved Cys-342 and His-415 of ZYMV HC-Pro (Figure 1 A) were substituted by a serine, resulting in pMAL:HA-HC-Pro mut C2(H 415 S):GFP (C2) and pMAL:HA-HC-Pro mut C3(C 342 S):GFP (C3), respectively (Figure 1 A). The proteins were expressed and analysed for their autoproteolytic activity by Western blot analysis with anti-GFP antibodies. The xGFP was detectable in the total soluble protein fraction of both mutants (Figure 3 B, C2 and C3), indicating that GFP was processed from HC-Pro. The substitution of Cys-342 and His-415 did not abolish the auto-proteolytic activity of ZYMV HC-Pro, suggesting that the essential amino acids required for the proteolytic activity of ZYMV HC-Pro differ from those of TEV HC-Pro. These results further demonstrate the diversity of HC-Pro proteins from different potyviruses.

Identifi cation of the proteolytic active

domain of ZYMV HC-Pro

It has been shown that the active domain for autoproteolytic activity of TEV HC-Pro is localised in the C-terminal part of the protein (Plisson et al., 2003). We hypothesised that the proteolytic active domain of ZYMV HC-Pro should also be restricted to this region. Thus, the C-terminal region comprising residues 350 – 456 was deleted from ZYMV HC-Pro, resulting in pMAL:HA-HC-Pro mut C4:GFP (C4) (Figure 1 A).

Western blot analysis of the protein extracted from E. coli expressing C4 showed that the full-length protein with a molecular mass of 111 kDa was detectable in the pellet fraction (Figure 3 A). However, in contrast to the results of mutants N1, C1, C2 and C3, no signal corresponding to the xGFP was detected in the total soluble protein fraction, indi-cating that autoproteolytic activity of the C4 mutant was com-pletely abolished (Figure 3 B, C4). From this result, it can be concluded that the autoproteolytic active domain of ZYMV HC-Pro or a part of it is within the C-terminal region between residues 350 and 456. To further localise the proteolytic active domain, two additional C-terminal deletion mutants were generated. In pMAL:HA-HC-Pro mut C5:GFP (C5), residues 359 – 456, and in pMAL:HA-HC-Pro mut C6:GFP (C6) resi-dues 373 – 456, were deleted (Figure 1 A). Both mutants were expressed in E. coli , and total proteins (soluble and pellet frac-tions) were analysed by Western blotting with anti-GFP anti-bodies. The full-length C5 and C6 proteins accumulated in the pellet fractions (Figure 3 A). However, xGFP was detected in the total soluble fractions of both mutants, indicating that the proteo lytic active domain of ZYMC HC-Pro is localised between residues 350 and 359.

Autoproteolytic activity of the recombinant HC-Pro from zucchini yellow mosaic virus 941

The protein alignment revealed that Leu-351, Asn-353 and Glu-356 are highly conserved among potyvirus HC-Pro proteins (Figure 1 A). Therefore, we hypothesised that these residues could play an important role in the autoproteolytic activity of ZYMV HC-Pro. Thus, Glu-356 and Asn-353 were separately deleted, resulting in pMAL:HA-HC-Pro- ∆ Glu356:GFP (C7) and pMAL:HA-HC-Pro- ∆ Asn353:GFP (C8) (Figure 1 A). Western blot analysis showed that the full-length fusion proteins were detected in the pellet frac-tions, indicating that the C7 and C8 proteins were expressed (Figure 3 C). To investigate if deletion of Glu-356 or Asn-353 affected the autoproteolytic activity of ZYMV HC-Pro, C7 and C8 proteins accumulating in the soluble fractions were applied to Western blot analysis. The intensity of xGFP sig-nal was signifi cantly less than that observed for the Wt pro-tein, indicating that the autoproteolytic activity of C7 and C8 was partially destroyed (Figure 3 B, C7 and C8). The dele-tion of Asn-353 and Glu-356 may change the protein struc-ture, resulting in a strongly reduced activity. Alternatively, these two amino acids could be part of the active centre and would be required for optimal activity. To investigate which of the two options likely applies, Glu-356 and Asn-353 were replaced by glycine [pMAL:HA-HC-Pro-Glu356Gly:GFP (C9)] and by isoleucine [pMAL:HA-HC-Pro-Asn353I:GFP (C10)] instead of deleting them. Western blot analysis of E. coli -expressed proteins showed that the C9 and C10 pro-teins were fully processed. xGFP was detected in the soluble fractions for both proteins (Figure 3 D). This restored proteo-lytic activity indicated that loss of the proteolytic activity of the C7 and C8 proteins was likely due to altered protein struc-tures or protein misfolding.

Our data suggest that the activity of ZYMV HC-Pro is more dependent on the structure than on the protein sequence. However, we cannot exclude the possibility that additional residues in this region play an important role in the autoprote-olytic activity of the ZYMV HC-Pro protein. TEV HC-Pro was classifi ed as a cysteine protease which requires histidine and cysteine for proteolytic activity (Oh and Carrington , 1989 ). Moreover, a highly conserved asparagine is often found at the active site of cysteine proteases together with a histidine and a cysteine. To prove that the combination of three residues could play a role in proteolytic activity of ZYMV HC-Pro, two additional mutants, the double mutant C2 + 3 (H-415 to S + C-342 to S) and the triple mutant C2 + 3 + 8 (H-415 to S + C-424 to S + Asn-353 to I), were generated. The two proteins were expressed in E. coli , and Western blot analy sis of the soluble protein and pellet fractions showed that the prote-olytic activity of the C2 + 3 protein was retained while it was abolished in the C2 + 3 + 8 protein. This result indicated that the proteolytic activity of ZYMV HC-Pro may require a catalytic triad residue as it is known for papain-like cysteine proteases (Vernet et al. , 1995 ).

Discussion

In previous studies, it was shown that TEV HC-Pro exhib-its an autoproteolytic cleavage activity with its catalytic

domain localised to the C-terminus. TEV HC-Pro was pro-posed as a cysteine-type proteinase (Carrington et al. , 1989b ; Verchot et al. , 1991 ). On the basis of these studies, we con-ducted a genetic analysis of the catalytic domain and of the requirement of residues fl anking the catalytic site of ZYMV HC-Pro.

ZYMV HC-Pro was expressed in E. coli as a fusion pro-tein with an MBP at its N-terminus for increasing the protein solubility and with a GFP at its C-terminus for detecting the autoproteolytic activity by Western blot analysis with anti-GFP antibodies. We showed that E. coli -expressed ZYMV HC-Pro exhibited autoproteolytic activity. Since auto-proteolytic activity was detectable in a virus-free system, cleavage appeared to be exclusively based on the activity of HC-Pro and did not involve additional virus or host fac-tors. However, mutagenic analysis revealed that the auto-proteolytic activi ty of ZYMV HC-Pro and TEV HC-Pro exhibit different characteristics.

The residues at position P1, P2, P4 and P1 ′ in TEV HC-Pro had a major effect on the processing of the poly-protein by HC-Pro. Replacement of these conserved residues resulted in a reduction or complete loss of TEV HC-Pro autoproteolytic activity (Carrington and Herndon , 1992 ). In contrast, substitution of Gly-456 in position P1 by Lys did not eliminate the autoproteolytic activity of ZYMV HC-Pro. Moreover, the glycine – methionine junc-tion between ZYMV HC-Pro and GFP differed from the glycine – glycine junction between the HC-Pro and P3 pro-tein of the viral polyprotein, suggesting that cleavage could be independent of the amino acid sequence of the fusion partner. Therefore, one may speculate that the autoproteo-lytic activity of ZYMV HC-Pro is sequence independent. Similar results were obtained using the TEV NIa protein-ase and the rice yellow mottle virus (RYMV) P1 protein. (Rorrer et al. , 1992 ; Weinheimer et al. , 2010 ). In addition, N-terminal protein sequencing and Western blot analysis of cleavage products in a high-percentage polyacrylamide gel revealed that the cleavage site was within GFP, probably between residues 37 and 38. This result differed from the autoproteolytic activity of the RYMV P1 protein where the cleavage site was mapped directly after the fi rst residue of fused GFP. The MBP:HC-Pro:GFP protein may fold in a way that residues that will become processed are brought close to the proteolytic centre. Folding could be dependent on the N- and C-terminal fusion partner of the HC-Pro. A possible effect of the N-terminal region of ZYMV HC-Pro on its autoproteolytic activity seems unlikely because an N-terminal deletion of 93 amino acids did not affect pro-tease activity. This result demonstrated that the ZYMV HC-Pro protease domain maps to the central or C-terminal part of the protein. Similar results were reported for TEV and wheat streak mosaic virus HC-Pro (Oh and Carrington , 1989 ; Stenger et al. , 2006 ). Moreover, it indicates that a proper folding of ZYMV HC-Pro occurs even in the absence of the entire N-terminal region.

The protease domain of TEV HC-Pro was identifi ed, and the highly conserved residues Cys-344 and His-417 were found to be essential for autoproteolytic activity (Oh

942 K. Boonrod et al.

and Carrington , 1989 ). However, substitutions of these conserved residues (single and double mutants) in ZYMV HC-Pro did not abolish activity. This result demonstrated that the properties of the protease domains of ZYMV HC-Pro and TEV HC-Pro differ. Due to the diversity of the amino acid sequences, the two proteins probably fold into different secondary structures which could involve the requirement of differentially modulated protease domains. To roughly map the proteolytic active domain of ZYMV HC-Pro, a set of deletion mutants were generated and analysed. Autoproteolytic activity was completely abolished when the C-terminal region comprising residues 350 – 456 was deleted. However, it was retained when only residues 359 – 456 were removed, indicating that the catalytic active domain mediat-ing the autoproteolytic activity of ZYMV HC-Pro is between residues 350 and 358. Amino acid alignment data revealed that in this region, only three amino acids, Leu-351, Asn-353 and Glu-356, are conserved among potyvirus HC-Pro proteins (Figure 1 A). Deletion of Asn-353 (C8) or Glu-356 (C7) almost fully eliminated the autoproteolytic activity, but it was maintained when Asn-353 (C10) or Glu-356 (C9) were not deleted but substituted. We suggest that these two resi-dues play a crucial role in the formation of the mature protein structure or that they interfere with the protein folding pro-cess. The highly conserved asparagine is often found at the active site of cysteine proteases representing, together with histidine and cysteine, a catalytic triad which is typical for papain-like cysteine proteases (Vernet et al. , 1995 ). In con-trast to TEV HC-Pro, the conserved cysteine and histidine appeared to be not essential for the autoproteolytic activity of ZYMV HC-Pro. However, it cannot be excluded that a cata-lytic triad is also required for the activity of ZYMV HC-Pro. This assumption was supported by the fi nding that protease activity was heavily affected in the C2 + 3 + 8 triple mutant. It should also be noted that we did not rule out that additional residues in this region could play an important role in the catalytic activity of ZYMV HC-Pro.

Differences between TEV and ZYMV HC-Pro were also reported for their RSS activity. It was shown that in the highly conserved FRNK box of ZYMV HC-Pro, substitution of Arg-180 to Ile-180 did not compromise RSS activity. In contrast, the same mutation in TEV, turnip mosaic virus (TuMV) and potato virus Y (PVY) HC-Pro caused a complete loss of the RSS activity (Shiboleth et al. , 2007 ). Taken together, the results show that the HC-Pro of ZYMV possesses a proteo-lytic active domain and silencing suppressor functions dis-tinct from TEV HC-Pro.

Materials and methods

Plasmids

The ZYMV HC-Pro coding region was amplifi ed by PCR from the pUC57:HC-Pro plasmid (Shiboleth et al. , 2007 ) by using HC-Pro Nco I_fwd and HC-Pro Nco I_rev primers (Table 1 ). PCR products were digested with Nco I and cloned into the pBin:GFP binary vec-tor (Boonrod et al. , 2004 ), resulting in pBin:HC-Pro:GFP. Using the pBin-HA-HC-Pro Kpn I_fwd and pBin-GFP Xba I_fwd primers (Table 1 ), the HC-Pro:GFP coding region was amplifi ed from the pBin:HC-Pro:GFP plasmid, generating a HA-tag coding region upstream of HC-Pro:GFP. PCR products were cloned into the pJET1.2/blunt vector (Fermentas, http://www.fermentas.de), resulting in pJET:HA-HC-Pro:GFP. For cloning of HA-HC-Pro:GFP into the pMAL.c2X vector (NEB, http://www neb.com), Xba I restriction sites were in-troduced by PCR using the primer pair HA-HC-Pro Xba I_fwd/pBin-GFP Xba I_rev (Table 1 ) and pJET:HA-HC-Pro:GFP as template. The resulting HA-HC-Pro:GFP was cleaved with Xba I and the insert was cloned into the corresponding sites of the pMAL.c2X vector, produc-ing pMAL:HA-HC-Pro:GFP.

In vitro mutagenesis

Mutations were introduced by site-directed mutagenesis (Laible and Boonrod , 2009 ). Briefl y, PCR was performed using the Pfu -Polymerase (Fermentas) and complementary primers (Table 2 ) car-rying the mutation in the central region. The PCR reaction samples were incubated in a thermocycler according to the following pro-gram: an initial denaturation step at 95 ° C for 1 min, followed by 18 cycles of denaturation at 95 ° C for 10 s, annealing at 55 ° C for 1 min, amplifi cation at 72 ° C for 1 min/kb plasmid and a fi nal ex-tension step at 72 ° C for 10 min. PCR products were digested with Dpn I (Fermentas) followed by transformation into competent E. coli cells. A positive clone was selected and sequenced (4base lab, http://www.4base-lab.de).

To delete 93 residues at the N-terminus of the HC-Pro protein, a Sac I restriction site was introduced via site-directed mutagenesis into the coding region at position 3087 additionally to the original Sac I site at position 2709 in the pMAL:HA-HC-Pro:GFP plasmid. A posi-tive clone was digested with Sac I. The purifi ed restricted plasmid was re-ligated and transformed into E. coli resulting in pMAL: ∆ N HC-Pro:GFP (N1).

HC-Pro mutants lacking C-terminal fragments were generated by introducing Nco I restriction sites into the HC-Pro coding region at positions 3866 (C4), 3935 (C5) and 3986 (C6) by PCR addition-ally to the original Nco I site at position 4190 in the pMAL:HA-HC-Pro:GFP construct by using primers as indicated in Table 2 . After Nco I restriction and re-ligation, the following residues were deleted: ∆ 350 – 456 (C4), ∆ 373 – 456 (C5) and ∆ 359 – 456 (C6). The names of the mutated plasmids are indicated in Table 2 .

Table 1 Oligonucleotides used for cloning.

Primer Sequence (5 ′ → 3 ′ )

HA-HC-Pro XbaI_fwd GGT GCT CTA GAG CAA TGT ATC CAT ATG ATG TTC CHC-Pro Nco I_fwd CAT GCC ATG GGG TCG TCA CAA CCG GAA GTT CHC-Pro NcoI_rev CAT GCC ATG GCG CCA ACT CTG TAA TGT TTCpBin-GFP XbaI_rev GCT CTA GAG GAT CCT TAT TTG TAT AGT TCA TCCpBin-HA-HcPro KpnI_fwd GGG GTA CCC CATG TAT CCA TAT GAT GTT CCG GAT TAC GCG TCG TCA CAA CCG GAA GTT C

Autoproteolytic activity of the recombinant HC-Pro from zucchini yellow mosaic virus 943

Table 2 Oligonucleotides used for mutagenesis.

Template Primer Sequence (5 ′ → 3 ′ ) Plasmid after mutagenesis

pMAL:HA-HC-Pro:GFP HC-Pro mut C1_fwd

AAC ATT ACA GAG TTA AGG CCA TGG GTA AAG

pMAL:HA-HC-Pro mut C1:GFP (C1)

HC-Pro mut C1_rev

CTT TAC CCA TGG CCT TAA CTC TGT AAT GTT

pMAL:HA-HC-Pro:GFP HC-Pro mut C2_fwd

CAA ACC ATG TCT GTC ATT GAT TCT TAT GG pMAL:HA-HC-Pro mut C4(H 415 S):GFP (C2)

HC-Pro mut C2_rev

CCA TAA GAA TCA ATG ACA GAC ATG GTT TG

pMAL:HA-HC-Pro:GFP HC-Pro mut C3_fwd

GAA GGT TAT TCC TAT CTC AAT ATT TTC C pMAL:HA-HC-Pro mut C5(C 342 S):GFP(C3)

HC-Pro mut C3_rev

GGA AAA TAT TGA GAT AGG AAT AAC CTT C

pMAL:HA-HC-Pro:GFP HC-Pro mut C4_fwd

CTC AAT ATT TTC CTC GCC ATG GTT GTG AAT G

pMAL:HA-HC-Pro mut C7:GFP (C4)

HC-Pro mut C4_rev

CAT TCA CAA CCA TGG CGA GGA AAA TAT TGA G

pMAL:HA-HC-Pro:GFP HC-Pro mut C5_fwd

GAT CCC CAT GGT TGG GCA GTG G pMAL:HA-HC-Pro mut C8:GFP (C5)

HC-Pro mut C5_rev

CCA CTG CCC AAC CAT GGG GAT C

pMAL:HA-HC-Pro:GFP HC-Pro mut C6_fwd

GAG AAC GAA GCC ATG GAT TTC ACC AAA ATG

pMAL:HA-HC-Pro mut C9:GFP (C6)

HC-Pro mut C6_rev

CAT TTT GGT GAA ATC CAT GGC TTC GTT CTC

pMAL:HA-HC-Pro:GFP HC-Pro mut C7_fwd

CTT GTG AAT GTT AAT AAC GAA GCA AAG pMAL-HA-HC-Pro- ∆ Gln356:GFP(C7)

HC-Pro mut C7_rev

CTT TGC TTC GTT ATT AAC ATT CAC AAG

pMAL:HA-HC-Pro:GFP HC-Pro mut C8_fwd

GCA ATG CTT GTG GTT AAT GAG AAC G pMAL-HA-HC-Pro- ∆ Asn353:GFP(C8)

HC-Pro mut C8_rev

CGT TCT CAT TAA CCA CAA GCA TTG C

pMAL:HA-HC-Pro:GFP HC-Pro mut C9_fwd

GAA TGT TAA TGG GAA CGA AGC AAA G pMAL:HA-HC-Pro mut C9(E 356 G):GFP (C9)

HC-Pro mut C9_rev

CTT TGC TTC GTT CCC ATT AAC ATT C

pMAL:HA-HC-Pro:GFP HC-Pro mut C10_fwd

CAA TGC TTG TGA TTG TTA ATG AG pMAL:HA-HC-Pro mut C10(N 353 I):GFP (C10)

HC-Pro mut C10 rev

CTC ATT AAC AAT CAC AAG CAT TG

pMAL:HA-HC-Pro:GFP HC-Pro mut N1_fwd

GAT AGT TTC AAG GAG AGC TCT CAT TAC G pMAL- ∆ NHC-Pro:GFP

HC-Pro mut N1_rev

CGT AAT GAG AGC TCT CCT TGA AAC TAT C HC-Pro-GFP (N1)

Protein expression and extraction

BL21 (DE3) CodonPlus (Agilent, http://www.Agilent.com) cells were transformed with the corresponding expression plasmid. Cells were grown in LB medium containing corresponding anti-biotics until an OD 600 of 0.6 was reached. Protein expression was induced by adding 1 m m IPTG. After incubation overnight at 14 ° C, cells were harvested and resuspended in BugBuster Protein Extraction Reagent (Novagen, http://www.emdchemicals.com; 10 ml/g of cells) containing 5 µ l benzonase (25 U/ µ l), 10 m m DTT and one tablet of complete protease inhibitor, EDTA-free (Roche, http://www.roche-applied-science.com). The resuspended cells were incubated for 1 h at 4 ° C under agitation. The lysate was cen-trifuged at 9000 × g for 10 min and the supernatant was analysed by SDS-PAGE.

SDS-PAGE, Western blotting and antibodies

Western blot analysis of the fusion proteins was performed using standard methods with 9 % polyacrylamide pre-cast gels (Anamed, http://www.anamed-gele.com). After separation of the proteins, fragments were electro-blotted onto polyvinylidene difl uoride membranes (Sigma-Aldrich, http://www.sigmaaldrich.com) at 350 mA in transfer buffer containing 30 m m Tris-HCl, 240 m m gly-cine and 20 % SDS. Immunodetection was performed by using polyclonal anti-GFP antibodies (1:500) (Santa Cruz, http://www.scbt.com) and a secondary antibody coupled with horseradish per-oxidase (HRP) anti-rabbit-HRP (1:1000) (Santa Cruz). Membranes were incubated with enhanced chemiluminescence reaction reagent (Pierce, http://www.piercenet.com) and exposed to a detection fi lm (Lumi Film, Chemiluminescent Detection Film, Roche). Detection

944 K. Boonrod et al.

was performed according the manufacturer ’ s protocol (Kodak, http://www.kodak.de).

N-terminal amino acid sequencing

The pMAL:HA-HC-Pro:GFP plasmid was expressed as described. One millilitre of total soluble protein containing processed GFP was incubated with 50 µ l of anti-GFP magnetic beads and the pro-teins were purifi ed as recommended by manufacturer ( http://www.miltenyibiotec.com ). Immunoprecipitated proteins were applied to SDS-PAGE for separation. Electro-blotting and staining were performed according to the instructions of Proteome Factory AG (Berlin, Germany) to perform sequencing of the C-terminal cleav-age product.

Acknowledgments

We thank Mirko Moser for technical support and Peter Wilton for critical reading of the manuscript. This research was supported by the German Research Council ’ s trilateral (German-Israeli-Palestinian) project and the European Union, Project LSHG-CT-2006-037900 (SIROCCO).

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Received July 19, 2011; accepted July 20, 2011; previously published online August 29, 2011

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