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Originally published In Press as doi:10.1074/jbc.M504122200 on June 7, 2005

J. Biol. Chem., Vol. 280, Issue 34, 30291-30300, August 26, 2005
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"Natively Unfolded" VPg Is Essential for Sesbania Mosaic Virus Serine Protease Activity*

Panayampalli Subbian Satheshkumar{ddagger}§, Pananghat Gayathri§, Kasaragod Prasad¶, and Handanahal Subbarao Savithri{ddagger}||

From the {ddagger}Department of Biochemistry and the Molecular Biophysics Unit, Indian Institute of Science, Bangalore-560 012, India

Received for publication, April 15, 2005 , and in revised form, June 7, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Polyprotein processing is a major strategy used by many plant and animal viruses to maximize the number of protein products obtainable from a single open reading frame. In Sesbania mosaic virus, open reading frame-2 codes for a polyprotein that is cleaved into different functional proteins in cis by the N-terminal serine protease domain. The soluble protease domain lacking 70-amino-acid residues from the N terminus ({Delta}N70Pro, where Pro is protease) was not active in trans. Interestingly, the protease domain exhibited trans-catalytic activity when VPg (viral protein genome-linked) was present at the C terminus. Bioinformatic analysis of VPg primary structure suggested that it could be a disordered protein. Biophysical studies validated this observation, and VPg resembled "natively unfolded" proteins. CD spectral analysis showed that the {Delta}N70Pro-VPg fusion protein had a characteristic secondary structure with a 230 nm positive CD peak. Mutation of Trp-43 in the VPg domain to phenylalanine abrogated the positive peak with concomitant loss in cis- and trans-proteolytic activity of the {Delta}N70Pro domain. Further, deletion of VPg domain from the polyprotein completely abolished proteolytic processing. The results suggested a novel mechanism of activation of the protease, wherein the interaction between the natively unfolded VPg and the protease domains via aromatic amino acid residues alters the conformation of the individual domains and the active site of the protease. Thus, VPg is an activator of protease in Sesbania mosaic virus, and probably by this mechanism, the polyprotein processing could be regulated in planta.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Sesbania mosaic virus (SeMV)1 belongs to the genus Sobemovirus, which infects Sesbania grandiflora (1). It is an icosahedral virus with T = 3 symmetry, and the capsid is made up of 180 coat protein subunits of molecular mass 29 kDa (2). It is a positive sense RNA virus of genome size 4149 nucleotides having four overlapping open reading frames (1). Open reading frame-2 encodes a polyprotein consisting of N-terminal serine protease (Pro), viral protein genome-linked (VPg), a 10-kDa protein, and C-terminal RNA-dependent RNA polymerase (RdRP) domains (3). The Pro domain is responsible for the proteolytic maturation of the polyprotein and cleaves between E-T and E-S amino acid residues. The active site and cleavage site residues have been mapped and confirmed by mutational analysis (3).

The protease domain was able to carry out cis-cleavage between Pro-VPg precursor, albeit at a slower rate. The cis-proteolytic activity considerably increased when the N-terminal 70-amino-acid residues predicted to form a transmembrane domain were deleted. Further, it was demonstrated that the cleavage site mutant E325A of {Delta}N70Pro-VPg was active in trans (3). However, the protease alone was unable to perform the trans-cleavage function. It has been observed earlier that in some viruses, VPg influences the activity of the protease. For instance, in Tomato ringspot nepovirus (TomRSV), VPg-Pro precursor was shown to be more active in cleavage at one of the sites (4). In cowpea mosaic virus, expression of the protease along with the N-terminal extension corresponding to the VPg sequence enhanced its proteolytic activity (5). However, the mechanism by which VPg activates the proteolytic activity is not known.

VPg is usually a small protein or peptide, which serves as the protein primer for RNA synthesis in many animal and plant viruses. In polioviruses, the VPg is 22 amino acid residues long (6), and in cowpea mosaic virus, it consists of 28 amino acids (5). NMR studies on cowpea mosaic virus VPg suggested that it does not have any ordered structure (7). In Sobemoviruses, the size of VPg varies between 9 and 12 kDa (8), and it is 9 kDa in SeMV, consisting of 77 amino acid residues (1). The presence of VPg linked to the 5' end of the genomic RNA has been confirmed by determining the N-terminal amino acid sequence of VPg in Southern bean mosaic virus, Cocksfoot mottle virus, and SeMV genomic RNA (1, 9, 10). However, no further studies have been reported on the characterization of VPg in Sobemoviruses.

"Natively unfolded" proteins are a unique class of proteins that exhibit their function in the absence of ordered structure. These proteins are believed to adopt a rigid conformation stabilized in vivo upon interaction with natural substrates (11, 12). We wanted to examine whether the recombinant SeMV VPg exists in a natively unfolded state and whether its presence regulates the protease activity.

In this study, we report that the purified recombinant SeMV VPg is a natively unfolded protein lacking both secondary and tertiary structures. However, when present at the C terminus of the protease domain ({Delta}N70Pro-VPg), VPg modulates both cis- and trans-catalytic activities of the protease by inducing a conformational change in the individual domains. Trp-43 of VPg was identified to be crucial for these interactions. Deletion of the VPg domain from the polyprotein resulted in the complete inhibition of proteolytic processing although the protease domain was intact. The results presented suggested that the natively unfolded VPg regulates the proteolytic maturation of the polyprotein in sobemoviruses.



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  		FIG. 1.
trans-Catalytic activity of {Delta}N70Pro. A, trans-cleavage assay was carried out using purified protein samples in 20 mM Tris, pH 8.0 buffer. {Delta}N70Pro-VPg-S284A was used as the substrate, and cleavage was performed with {Delta}N70Pro (lane 3), {Delta}N70Pro purified from {Delta}N70Pro-VPg precursor (lane 4), and {Delta}N70Pro-VPg-E325A (lane 5). The cleavage assay performed with {Delta}N70Pro in the presence of VPg is shown in lane 8. The expected positions of the different protein products are indicated. * indicates the position of protein products obtained by suboptimal cleavage at Ala-134-Val-135. B, schematic representation of the products of trans-cleavage.

 

   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Expression and Purification of Recombinant Proteins—{Delta}N70Pro, {Delta}N70Pro-VPg-S284A, {Delta}N70Pro-VPg-E325A, {Delta}N70Pro-VPg-W43F, {Delta}N70Pro-VPg-P10-RdRP, {Delta}N70Pro-VPg-P10-RdRP-W43F, and {Delta}N70Pro-P10-RdRP genes were cloned in pRSET A vector (Invitrogen) at NheI and BamHI sites. The cloning strategy results in the addition of 13 amino acids from the vector in the N terminus, including the His6 residues for affinity purification. All the recombinant clones were expressed in Escherichia coli BL21 DE3 pLysS cells and induced with 0.3 mM isopropyl-1-thio-{beta}-D-galactopyranoside at 30 °C for 5 h. After induction, the cells were harvested by centrifugation and sonicated in buffer containing 50 mM Tris, pH 8.0, 200 mM NaCl, 5% glycerol, and 0.2% Triton X-100 (buffer A). The His-tagged proteins were purified using Ni-NTA chromatography (Novagen) according to the manufacturer's protocol. Since the expression of His-tagged VPg was less, it was purified from {Delta}N70Pro-VPg-S284A by cleaving it with {Delta}N70Pro-VPg-E325A. As the His tag is present in the N terminus of the protease, cleaved {Delta}N70Pro and uncleaved {Delta}N70Pro-VPg-E325A will bind to the Ni-NTA column, whereas free VPg is obtained in unbound fraction. The purity of the proteins was checked by SDS-PAGE.



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  		FIG. 2.
Protein folding predictions analysis suggest the unfolded nature of VPg. The protein folding predictions were performed using the software PONDR, Predictors of Natural Disordered Regions. The amino acid sequences of {Delta}N70Pro and VPg were subjected to analysis using the default values. The outputs of prediction analysis are presented. A, {Delta}N70Pro; B, VPg; C, {Delta}N70Pro-VPg.

 
Protease Cleavage Assays—trans-Cleavage reaction was performed in 20 mM Tris, pH 8.0 buffer. 100 µg of {Delta}N70Pro-VPg-S284A (substrate) was incubated with the enzyme ({Delta}N70Pro-VPg-E325A) in the molar ratio of 1:10 at 37 °C for 1 h. The reaction was stopped by the addition of SDS loading dye followed by boiling. The reaction products were separated in 15% SDS-PAGE and stained with Coomassie Brilliant Blue. Since the {Delta}N70Pro was not active in 1:10 dilution, cleavage was carried out with higher concentration (1:3 dilution), and thus, the added protease can be observed in the gel. For determining the cis-catalytic activity of the protease, the respective clones were expressed in BL21 DE3 pLysS cells and induced with 0.3 mM isopropyl-1-thio-{beta}-D-galactopyranoside, and then cells were harvested, sonicated in buffer A, and subjected to 12% SDS-PAGE.

Deletion and Site-directed Mutagenesis—N- and C-terminal truncations were carried out by using PCR with the appropriate primers (Table I) and cloned in pRSET A vector. Site-directed mutagenesis was carried out using a PCR-based sense and antisense primer approach (13) with mutant oligonucleotide primers (Table I). Deletion of VPg domain from the polyprotein was carried out by a PCR-based approach. Antisense primer (VPg{Delta}BglII-a) corresponding to the protease C terminus and sense primer (VPg{Delta}BglII-s) corresponding to that of the N terminus of P10 domain were designed with BglII sites. PCR was carried out with {Delta}N70Pro sense/VPg{Delta}BglII-a (product 1) and RdRP antisense/VPg{Delta}BglII-s (product 2) primers using {Delta}N70Pro-VPg-P10-RdRP-containing clone. The PCR products were eluted from the gel and digested with BglII restriction enzyme. The digested PCR products were ligated using T4 DNA ligase at 16 °C for 8 h. The ligated product was subjected to PCR amplification with {Delta}N70Pro sense/RdRP antisense primers. Therefore, only when products 1 and 2 are ligated would amplification occur with this set of primers. The final PCR product thus obtained lacked the internal VPg domain and was recloned again in pRSET A vector at NheI and BamHI sites to obtain the clone {Delta}N70Pro-P10-RdRP.


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  		TABLE I
List of oligonucleotides used for cloning and mutagenesis

 
Circular Dichroism Spectroscopy—Circular dichroism was carried out using a Jasco-815 spectropolarimeter (Japan Spectroscopic Co., Tokyo, Japan). The ellipticity was monitored from 190 to 250 nm using 0.5 mg/ml protein in a 0.2-cm path length cuvette with a bandwidth of 1 nm and response time of 1 s. The molar ellipticity was calculated from the software by using the molar concentration of protein. The CD spectra of denatured proteins were recorded after incubating the protein with different concentrations of urea for 5 h at 25 °C. All the spectra were corrected with the respective buffer controls.

Fluorescence Spectroscopy—The fluorescence experiments were carried out in a PerkinElmer Life Sciences LS5S luminescence spectrometer. The intrinsic fluorescence spectrum was monitored from 300 to 400 nm upon excitation at 280 nm in a 1-cm path length cuvette. 0.2-0.4 mg/ml of proteins was used in 20 mM Tris buffer, pH 8.0. The stability of {Delta}N70Pro, VPg, and {Delta}N70Pro-VPg-E325A was studied by incubating 0.2 mg/ml protein with 8 M urea for 5 h at 25 °C.

Gel Filtration Analysis—-The oligomeric status of {Delta}N70Pro, VPg, {Delta}N70Pro-VPg E325A, and {Delta}N70Pro-VPg E325A-W43F was analyzed using a Superdex 200 analytical gel filtration column (Amersham Biosciences) precalibrated with standards of known molecular mass (native SeMV, which elutes in void volume, tyroglobulin 669 kDa; apoferitin, 443 kDa; {beta}-amylase, 200 kDa; alcohol dehydrogenase, 150 kDa; bovine serum albumin, 66 kDa; carbonic anhydrase, 29 kDa; cytochrome c, 12 kDa; aprotinin, 6.5 kDa). From the elution volume, the Rf value was calculated, and the molecular weights of the proteins were estimated from the standard graph.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
trans-Catalytic Activity of {Delta}N70Pro and {Delta}N70Pro-VPg-E325A—The trans-catalytic activity was performed with purified {Delta}N70Pro using {Delta}N70Pro-VPg-S284A (an active site mutant, in which protease is inactive due to mutation of active site residue Ser-284 to Ala but cleavage site E-T is retained) as substrate. Under the reaction conditions, {Delta}N70Pro did not cleave the substrate {Delta}N70Pro-VPg-S284A, suggesting that it was not active in trans (Fig. 1, lane 3; the 25-kDa band seen in lanes 3 and 4 is due to the added {Delta}N70Pro). However, {Delta}N70Pro-VPg-E325A fusion protein (cleavage site mutant in which the catalytic triad residues are intact and the cis-cleavage is abolished by the mutation of cleavage site residue Glu-325 to Ala) was able to cleave {Delta}N70Pro-VPg-S284A substrate in trans, resulting in the generation of two domains, {Delta}N70Pro and VPg (Fig. 1, lane 5). To rule out the possibility that the expression of {Delta}N70Pro alone rendered it inactive, cis-active {Delta}N70Pro was purified from {Delta}N70Pro-VPg precursor. Expression of {Delta}N70Pro-VPg in E. coli results in complete cleavage between protease and VPg. Since the His tag was present at the N terminus, the cleaved protease was purified using Ni-NTA chromatography. Even this cis-active {Delta}N70Pro was not able to cleave the substrate (Fig. 1, lane 4). To check whether VPg could act in trans, cleavage reaction was performed with {Delta}N70Pro in the presence of VPg. However, the addition of VPg did not enhance protease activity in trans (Fig. 1, compare lanes 7 and 8; the 25- and 9-kDa bands in lane 8 are due to the added {Delta}N70Pro and VPg), suggesting that only when VPg was present as a C-terminal fusion did the protease exhibit trans-cleavage activity. The additional bands (shown as *) in lanes 4 and 7 are due to another suboptimal cleavage between Ala-134-Val-135.2 A schematic representation of the results of trans-cleavage assay is presented in Fig. 1B. To understand the nature of VPg-mediated interactions in the activation of protease domain, bioinformatic and biophysical analyses of VPg were performed.



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  		FIG. 3.
Protein folding predictions analysis suggest the unfolded nature of VPg. The primary amino acid sequences of {Delta}N70Pro and VPg were analyzed using the software FoldIndex. The results of the predictions for {Delta}N70Pro, VPg, and {Delta}N70Pro-VPg are shown in A, B, and C, respectively.

 
Bioinformatic Analysis of Protease and VPg—The protein folding predictions were carried out using two different software programs. The software PONDR®, Predictors of Natural Disordered Regions, is available through a branch of Molecular Kinetics Inc. (14). The unstructured region prediction analysis was performed using default set parameters for {Delta}N70Pro, VPg, and protease-VPg fusion protein amino acid sequences. The result obtained showed that protease and protease-VPg (Fig. 2, A and C) fall in the category of ordered proteins, whereas VPg (Fig. 2B) was grouped with disordered proteins.

Another software program used for unfolded protein prediction is FoldIndex©, available at the Weizmann Institute of Science. The program is based on the algorithm proposed by Uversky et al. (15). In this method, based on the mean net charge and hydrophobicity, a folding index is derived for the folded/unfolded state of the protein. The FoldIndex© analysis of {Delta}N70Pro, VPg, and {Delta}N70Pro-VPg amino acid sequence was performed using default values. The results indicate that the entire sequence of protease occurs in the folded state (Fig. 3A). The results corresponding to VPg clearly showed that it is unlikely to be a folded protein as most of its residues belonged to the unfolded region (Fig. 3B). When the {Delta}N70Pro-VPg sequence was analyzed, only the VPg domain showed a disordered region similar to that of VPg (Fig. 3C). These results suggested that VPg might exist in a natively unfolded conformation. To confirm this prediction, we have carried out secondary and tertiary structural studies on recombinant VPg in the absence/presence of {Delta}N70Pro.



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  		FIG. 4.
CD spectral analysis shows unfolded nature of VPg and 230 nm positive peak in {Delta}N70Pro-VPg-E325A fusion protein. The CD spectra were recorded using 0.5 mg/ml of purified proteins from 190 to 250 nm. The CD spectra of {Delta}N70Pro, VPg, and {Delta}N70Pro-VPg-E325A are shown in A, B, and C, respectively. D, the CD spectra of denatured {Delta}N70Pro-VPg-E325A were recorded upon treatment with 1-4 M urea.

 
CD Spectral Analysis—Far-UV CD spectrum provides information on the folded nature of the proteins. The CD spectrum of purified {Delta}N70Pro showed a minimum at 216 nm, indicating that the protein is folded with a high content of {beta}-sheet conformation (Fig. 4A). On the other hand, the VPg CD spectrum revealed maximum negative ellipticity at 200 nm with negligible ellipticity at 222 nm (Fig. 4B), a feature characteristic of random coil structures (16), consistent with the folding predictions for VPg. Interestingly, the far-UV CD spectrum of {Delta}N70Pro-VPg-E325A showed a positive peak at 230 nm and a negative peak at 210 nm (Fig. 4C). The intensity of the positive peak was very significant, and it was not present in either the protease or the VPg CD spectrum. The presence of a 230 nm peak only in the fusion protein suggested a conformational change in the fusion protein. Further, treatment of {Delta}N70Pro-VPg-E325A with 2 M urea resulted in almost 50% reduction in the 230 nm peak, and incubation with 3 and 4 M urea completely eliminated the 230 nm positive peak (Fig. 4D).

Fluorescence Spectroscopic Analysis—Fluorescence spectrum of {Delta}N70Pro showed maximum emission at 340 nm upon excitation at 280 nm, typical of a folded protein. The emission maximum showed a red shift to 359 nm upon the addition of 8 M urea due to the exposure of the aromatic residues to the solvent (Fig. 5A). On the other hand, the fluorescence spectrum of VPg showed a maximum emission at 357 nm even in the native state. The emission maximum remained the same upon the addition of 8 M urea, indicating that VPg lacks tertiary structure (Fig. 5B). The fusion protein ({Delta}N70Pro-VPg-E325A) behaved like {Delta}N70Pro, with maximum emission at 342 nm, which upon denaturation increased to 356 nm (Fig. 5C).

Gel Filtration Analysis—Gel filtration analysis was carried out using an analytical column, Superdex S200. {Delta}N70Pro eluted as a single peak with the expected molecular mass of 25 kDa of the monomer (Fig. 6A). As expected, VPg did not elute at a position corresponding to its size of 9 kDa. It gave a peak corresponding to the size of 120 kDa, further confirming its open and random conformation (Fig. 6B). Interestingly, {Delta}N70Pro-VPg-E325A gave three peaks, a major peak corresponding to the size of 400 kDa (peak 2), two minor peaks corresponding to 130 kDa (peak 3), and that in the void volume (peak 1) (Fig. 6C). The gel filtration analysis suggests a change in the oligomeric status of the protease when present in fusion with VPg. The protease activities of different oligomeric fractions were checked by trans-catalytic assay with {Delta}N70Pro-VPg-S284A with equal concentrations of {Delta}N70Pro-VPg-E325A from each peak fraction. {Delta}N70Pro-VPg-E325A eluted in peaks 2 and 3 retained protease activity, whereas the higher aggregate eluted in void volume (peak 1) was not active (Fig. 6D). However, the nature of the oligomeric states was difficult to assess because of the abnormal behavior of VPg.



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  		FIG. 5.
Fluorescence spectrum of VPg demonstrates absence of tertiary structure under native condition. The intrinsic fluorescence spectra were recorded between 300 and 400 nm after exciting the protein at 280 nm under native and denatured conditions. The protein was denatured by treatment with 8 M urea. A, B, and C correspond to {Delta}N70Pro, VPg, and {Delta}N70Pro-VPg-E325A fusion proteins.

 
Mutational Analysis of VPg—Comparison of the VPg amino acid sequence between Sobemo- and Poleroviruses revealed a conserved W(A/G)D motif followed by a stretch of acidic amino acid residues (10). In addition to this conserved motif, the amino acid sequence of VPg in SeMV, Southern cowpea mosaic virus, and Southern bean mosaic virus-Ark contains a proline-rich sequence toward the C terminus. Since polyprolineII-like helix gives a positive CD peak at 230 nm (17), we have examined whether proline-rich and acidic domains can form the polyproline II helix. Mutational analysis of VPg was carried out by the deletion of 18 (removal of a proline-rich sequence) and 25 (removal of a acidic and proline-rich stretch) residues from the C terminus (Fig. 7A). The CD spectral analysis of these truncated proteins ({Delta}N70Pro-VPg-S284A-C{Delta}18 and {Delta}N70Pro-VPg-S284A-C{Delta}25) also showed a 230 nm positive peak, ruling out the potential involvement of the C-terminal 25 residues in contributing to the positive CD band at 230 nm (Fig. 7B). Further, the results suggested that poly-proline like helix is not responsible for the positive CD at 230 nm in this case. Therefore, the role of aromatic residues in VPg was examined.

Trp-43 of VPg Contributes for Positive CD Band—The positive CD peak at 230 nm has been attributed to the contribution from aromatic amino acid residues, mainly tyrosine and tryptophan (18). VPg has two tyrosine and three tryptophan residues in its amino acid sequence (Fig. 7A). Deletion of 25 amino acids from the VPg C terminus resulted in removal of one of the tryptophan residues (Trp-72), ruling out its role in the positive CD band at 230 nm. Another tryptophan residue is present in the WAD motif Trp-51, which is preceded by Tyr-50. To check whether these residues contribute for the 230 nm peak, C-terminal deletion was carried out using a unique ScaI restriction enzyme site present in the VPg sequence. Restriction digestion of the PCR product of {Delta}N70Pro-VPg with ScaI enzyme followed by cloning of this PCR product resulted in the removal of 28 residues from the C terminus of VPg (Fig. 7A). CD profile of this mutant {Delta}N70Pro-VPg-S284A-C{Delta}28 also showed the 230 nm peak (Fig. 7C), suggesting that Tyr-50 and Trp-51 do not contribute to the observed change in the CD spectrum. Two more residues present in the VPg sequence, Tyr-21 and Trp-43, were subjected to site-directed mutagenesis (Fig. 7A). Mutation of Tyr-21 to phenylalanine ({Delta}N70Pro-VPg-E325A-Y21F) did not affect the CD profile, and the mutant retained the positive peak (Fig. 7D). Mutation of Trp-43 to Phe ({Delta}N70Pro-VPg-E325A-W43F) completely abolished the positive CD peak (Fig. 7E). It is interesting to note that only one aromatic residue, Trp-43, contributes significantly to the 230 nm CD band when the VPg is present in fusion with protease. Thus, the stacking interaction of Trp-43 of VPg with other aromatic residues of the protease domain could be responsible for the conformational change in the fusion protein.

Effect of VPg W43F Mutation on cis- and trans-catalytic Activities of Protease—To check whether the {Delta}N70Pro-VPg-E325A-W43F mutant that lacks the 230 nm positive CD peak retains the trans-catalytic activity, a cleavage assay was carried out using {Delta}N70Pro-VPg-S284A as substrate. Interestingly, the protease activity in trans was affected considerably in this mutant ({Delta}N70Pro-VPg-E325A-W43F, Fig. 8A, lane 3) when compared with that of {Delta}N70Pro-VPg-E325A (Fig. 8A, lane 1). The result demonstrated a novel and interesting observation that mutation of only one aromatic residue Trp-43 in the VPg domain significantly alters the catalytic activity of the protease domain. Further, to check the role of Trp-43 residue of VPg domain in the cis-catalytic activity of {Delta}N70Pro, Trp-43 was mutated to Phe in {Delta}N70Pro-VPg. When compared with wild type {Delta}N70Pro-VPg (Fig. 8B, lane 1), the cis-catalytic activity decreased drastically in {Delta}N70Pro-VPg-W43F mutant (Fig. 8B, lane 2). Similarly, the Trp-43 to Phe mutation was carried out in polyprotein ({Delta}N70Pro-VPg-P10-RdRP), and the proteolytic processing was monitored. The wild type polyprotein underwent complete processing, resulting in the generation of {Delta}N70Pro and RdRP bands (Fig. 8C, lane 1). On the contrary, proteolytic processing of {Delta}N70Pro-VPg-P10-RdRP-W43F was affected considerably and resulted in the accumulation of full-length polyprotein. Only partial cleavage of the polyprotein was observed (Fig. 8C, lane 2). Thus, these results showed that VPg Trp-43 mediates structural alteration in the protease domain required for both the cis- and the trans-catalytic function.

Role of VPg in Protease Activity—The results presented thus far demonstrated that the VPg domain is essential for protease function. The next obvious question was to check the effect of the deletion of VPg domain from the polyprotein. Thus, a deletion mutant of polyprotein lacking the VPg domain was constructed (Fig. 9A). This results in the generation of a new cleavage site, made up of the C-terminal residue (Glu) of {Delta}N70Pro and the N-terminal residue (Thr) of P10. The {Delta}N70Pro-VPg-P10-RdRP polyprotein as well as the {Delta}N70Pro-P10-RdRP were expressed in E. coli from the corresponding clones. The cis-proteolytic activity was monitored by SDS-PAGE analysis. The wild type polyprotein ({Delta}N70Pro-VPg-P10-RdRP) showed complete processing as observed by the appearance of {Delta}N70Pro and RdRP bands (Fig. 9B, lane 2). However, {Delta}N70Pro-P10-RdRP polyprotein was not proteolytically active, and only the band corresponding to that of the full-length polyprotein was observed (Fig. 9B, lane 1). The results suggested that the VPg domain is essential for the activity of protease in cis. Thus, the presence of VPg downstream of the protease domain is essential for activation of both cis- and trans-catalytic activity of protease.



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  		FIG. 6.
{Delta}N70Pro-VPg-E325A exists in multiple aggregation states. A, gel filtration chromatographic profile of {Delta}N70Pro monitored at 280 nm in a Superdex S200 analytical column precalibrated with known molecular weight standards. AU, absorbance units. B and C, gel filtration profiles of VPg and {Delta}N70Pro-VPg-E325A. The elution was monitored at both 280 nm (left Y axis) and 260 nm (right Y axis). D, trans-cleavage assay performed with three peak fractions of {Delta}N70Pro-VPg-E325A eluted from the gel filtration column. The positions corresponding to the different protein bands are indicated.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Both animal and plant viruses use the strategy of polyprotein expression and processing to obtain functionally different proteins from the same polypeptide chain. For efficient and specific processing, viruses generally encode protease(s) as one of the domains of the polyprotein. It is observed that all the sites within the polyprotein are not cleaved at the same time and to the same extent. In Poty- (19), Como- (5), and Nepoviruses (20), which have the domain arrangement of VPg-protease-RdRP, the cleavage between VPg and protease was shown to occur at a slower rate than between protease-RdRP, and stable VPg-protease precursor could be obtained. Genomes of Sobemo-, Polero-, and Enamoviruses have a different domain arrangement (protease-VPg-RdRP), wherein protease is present before the VPg domain. Even here, for example in SeMV (sobemoviruses), stable protease-VPg precursor was detected (3). These observations suggest that VPg-protease or protease-VPg precursors could be of physiological significance.



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  		FIG. 7.
Identification of amino acid residue in VPg responsible for 230 nm positive peak in CD spectrum. A, the positions of amino acid residues Trp and Tyr and the sites of C-terminal deletions carried out in VPg are indicated. The CD spectral profiles of {Delta}N70Pro-VPg-S284A-C{Delta}18 and {Delta}N70Pro-VPg-S284A-C{Delta}25 are depicted in B; {Delta}N70Pro-VPg-S284A-C{Delta}28 is depicted in C; site-directed mutant {Delta}N70Pro-VPg-E325A-Y21F is detected in D; and {Delta}N70Pro-VPg-E325A-W43F is depicted in E.

 
An interesting result was obtained when the trans-proteolytic activity of SeMV protease was carried out. Protease alone was not able to cleave the substrate in trans; however, efficient cleavage was observed when the protease was expressed as a fusion protein with VPg (Fig. 1, lane 5). VPg is a relatively small protein made up of only 77 amino acid residues. The results presented in this study, bioinformatic analysis (Figs 2B and 3B), CD spectroscopy (Fig. 4B), fluorescence spectroscopy (Fig. 5B), and gel filtration analysis (Fig. 6B), establish that SeMV VPg does not have a definite folded structure. Thus, the absence of structure is an intrinsic property of VPg, similar to those of natively unfolded proteins.

The CD spectral profile of {Delta}N70Pro-VPg-E325A was considerably different from the sum of the contributions of individual protein domains and was characterized by the presence of a positive peak at 230 nm (Fig. 4C). Denaturation of the {Delta}N70Pro-VPg-E325A fusion protein with urea demonstrated that in parallel with the loss of secondary structure, the positive CD at 230 nm also disappeared (Fig. 4D). To examine the effect of secondary and tertiary structural changes in protease-VPg on the quaternary association of subunits, the oligomeric status was analyzed. Gel filtration analysis of protease revealed a single peak corresponding to the size of a monomer (Fig. 6A), whereas VPg showed abnormal elution behavior (Fig. 6B), probably because of a lack of structure. Protease-VPg fusion protein eluted as a heterogeneous mixture of proteins in different oligomeric states. However, because of the lack of structure in VPg, the exact nature of the oligomer could not be deciphered. It is possible that the 130-kDa peak (peak 3) represents the monomeric fusion protein. CD spectral analysis of peaks 2 and 3 showed that both of them had the 230 nm positive CD band (data not shown). To check whether the oligomerization is necessary for the protease function, equal concentrations of the three peak fractions were used for trans-cleavage assay. The result showed that only the aggregated protein that eluted in void volume was not proteolytically active, whereas the other two peak fractions showed proteolytic activity (Fig. 6D).



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  		FIG. 8.
Mutation of Trp-43 in VPg affects the protease activity both in cis and in trans. A, trans-cleavage assay carried out with {Delta}N70Pro-VPg-E325A and {Delta}N70Pro-VPg-E325A-W43F using {Delta}N70Pro-VPg-S284A as substrate. cis-Catalytic activity of {Delta}N70Pro-VPg-W43F mutant observed by 15% SDS-PAGE is shown in B, and that of {Delta}N70Pro-VPg-P10-RdRP-W43F mutant monitored by 12% SDS-PAGE is shown in C. The expected sizes of protein products are indicated by the arrows.

 
The presence of a positive CD band at 230 nm has been attributed to polyproline II-like helix (17) or contribution by aromatic amino acids, mainly tryptophan and tyrosine (18, 21). The deletion and mutation analyses presented in this study (Fig. 7, B-E) demonstrate that the positive CD band at 230 nm in the fusion protein could arise from the stacking interactions between Trp-43 of VPg and other aromatic residue(s) from the protease domain. Mutation of Trp-43 to Phe reduced the cis-cleavage efficiency of {Delta}N70Pro-VPg and {Delta}N70Pro-VPg-P10-RdRP, as well as the trans-catalytic activity of {Delta}N70Pro-VPg-E325A (Fig. 8, A and B). Further, deletion of the VPg domain from the polyprotein rendered the protease inactive, suggesting that the VPg domain is essential for both cis- and trans-catalytic activities of the protease (Fig. 9B). These results confirm that the presence of the natively unfolded VPg causes structural alterations in the polyprotein that are mandatory for the activation of the protease function. However, the exact mechanism by which the VPg domain brings about the activation of protease remains to be established. It is possible that the interaction of VPg with the protease domain might help in the proper positioning of the cleavage site residues in the active site cleft of the protease.

In Dengue virus, the NS3 serine protease domain involved in polyprotein processing is shown to be active only in the presence of the co-factor NS2B (22, 23). NS3 alone is not active in both cis and trans. The mechanism of activation of NS3 by NS2B is not clearly understood, but it is believed that the interaction of NS2B could cause conformational changes in NS3 that are necessary for efficient binding of substrate amino acid side chains for cleavage (24, 25). Functionally active NS3 protease in fusion with 40-amino-acid residues from the NS2B domain exhibited various aggregation states (26) similar to that of {Delta}N70Pro-VPg-E325A fusion, and one of the residues, Trp-62 of NS2B, was shown to be essential for the activation of NS3 protease (22). Interestingly, NS2B can function in trans, and co-expression of NS2B restores the proteolytic activity of NS3 (23). Similarly, in the case of hepatitis C virus protease (NS3), NS4A was shown to act as the co-factor (27). Even here, binding of NS4A was shown to activate NS3 by causing a conformational change (28). In SeMV, VPg acts as an activator of protease by bringing about conformational changes necessary for catalysis, and in this case, it does not activate protease in trans.



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  		FIG. 9.
Deletion of VPg domain from the polyprotein completely inhibits protease activity. A, schematic representation of polyprotein showing the domain arrangement and the VPg deletion mutant. B, the cis-catalytic activity of the polyprotein lacking the VPg domain was monitored by 12% SDS-PAGE analysis. The expected sizes of protein products are indicated by the arrows.

 
What could be the physiological relevance of the interaction of natively unfolded VPg with the protease? The differential proteolytic activities exhibited by protease and protease precursors might offer temporal regulation for polyprotein processing. Thus, the release of different polyprotein precursors at appropriate stages of viral life cycle could be an important strategy employed by viruses for their efficient multiplication and survival within the host cell. Another possibility is that the conformational changes of VPg in the presence of the protease domain might be necessary for its biological function. The function of VPg is to act as a primer for RNA synthesis. An amino acid side chain hydroxyl group (from tyrosine, serine, or threonine) mimics the 3'-OH group of a ribonucleotide. The residue that acts as the primer is highly specific; for example, it is tyrosine 63 in tobacco etch virus (29). In the genomic RNA isolated from native virus, both the processed and the unprocessed forms (in fusion with nuclear inclusion-a protease) of VPg are linked to the 5' end of the genome (19). If a protein is unfolded, then the probability of the same amino acid acting as a primer would be highly unlikely as the protein can assume multiple conformations. Thus, it is possible that the association of VPg with protease might alter the conformation of VPg such that the hydroxyl group of a specific amino acid can act as the primer. Recently, yet another function for VPg has been assigned in viral RNA translation. It is proposed that VPg might function in a way similar to the 5' 7-methyl guanosine cap structure present at the 5' end of eukaryotic mRNAs in recruiting the translation initiation factors (IFs). Norwalk virus VPg was shown to interact with eIF3 (30). In turnip mosaic virus, VPg was shown to interact with eIF4 and poly(A)-binding protein only in the form of 6K2-VPg-Pro or VPg-Pro polyprotein precursors in planta. Based on this result, a possible role of VPg-Pro in the formation of the assembly of the translation initiation complex has been proposed (31). Thus, by using the strategy of polyprotein processing, viruses can generate several proteins and their precursors, which could have multiple functions. The results presented in this study clearly established that the natively unfolded VPg can act as an activator of protease and thereby regulate polyprotein processing in sobemoviruses.


   FOOTNOTES
 
* This work was supported by the Council of Scientific and Industrial Research and the Department of Biotechnology, New Delhi, India. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Recipients of Council of Scientific and Industrial Research senior research fellowships. Back

|| To whom correspondence should be addressed. Tel.: 91-80-23601561; Fax: 91-80-23600814; E-mail: bchss{at}biochem.iisc.ernet.in.

1 The abbreviations used are: SeMV, Sesbania mosaic virus; VPg, viral protein genome-linked; Pro, protease; Ni-NTA, nickel-nitrilotriacetic acid; RdRP, RNA-dependent RNA polymerase; IF, initiation factor. Back

2 P. S. Satheshkumar, P. Gayathri, K. Prasad, and H. S. Savithri, unpublished observation. Back


   ACKNOWLEDGMENTS
 
We thank Mr. V. Saravanan for the technical assistance. Prof. M. R. N. Murthy, Dr. B. Gopal, Prof. S. K. Podder, Prof. P. Balaram, and Dr. G. L. Lokesh are acknowledged for helpful discussions.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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R. Grzela, E. Szolajska, C. Ebel, D. Madern, A. Favier, I. Wojtal, W. Zagorski, and J. Chroboczek
Virulence Factor of Potato Virus Y, Genome-attached Terminal Protein VPg, Is a Highly Disordered Protein
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