Functional consequences of mutational analysis of norovirus protease

FEBS Letters 585 (2011) 369–374

journal homepage: www.FEBSLetters.org

Functional consequences of mutational analysis of norovirus protease Yuichi Someya ⇑, Naokazu Takeda 1 Department of Virology II, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashi-Murayama, Tokyo 208-0011, Japan

a r t i c l e

i n f o

Article history: Received 24 November 2010 Revised 9 December 2010 Accepted 14 December 2010 Available online 17 December 2010 Edited by Miguel De la Rosa Keywords: Norovirus Protease Mutagenesis Proteolysis

a b s t r a c t Norovirus protease has been subjected to an extensive mutagenesis study. Ala-scanning mutation at 13 different positions (Trp6, Trp19, Thr27, Leu86, Leu95, Leu97, Met101, Gln117, Leu121, Thr134, Tyr143, Val144, and Val167) led to loss of function and/or stability. Considering the crystal structure of the protease, it was revealed that a hydroxyl group of Thr134 and an aromatic ring of Tyr143 were important for substrate recognition along with His157. It was notable that several of the residues identified were in close proximity to each other, suggesting their importance for the integrity and stability of the protease. Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction Noroviruses are a major cause of non-bacterial acute gastroenteritis in humans, and genetically and antigenically diverse strains have been isolated worldwide [1,2]. Norovirus, a member of the family Caliciviridae, has a positive-sense single-stranded RNA genome which is 7.7 kb in length with a poly(A) tail at its 30 -end. Genome-linked viral protein (VPg) is likely to be bound to the 50 -end in place of the cap structure [3]. The genome encodes three open-reading frames (ORFs) [1,2]. The ORF1 product is a polyprotein and is cleaved by its viral 3C-like protease activity into six non-structural proteins including 2C-like nucloside triphosphatase, 3B VPg and 3D RNA-dependent RNA polymerase in addition to 3C-like protease [1,2]. The ORF2 and ORF3 products are a major and a minor structural protein (VP1 and VP2), respectively. The norovirus virion consists of 180 virion structural protein 1 (VP1) molecules with the VPg-linked RNA genome and VP2 molecules inside. The sequence diversity of the P2 domain of VP1 is correlated with the wide variety of antigenicity among noroviruses [4,5] and potentially provides different patterns of binding to histo-blood group antigens [6].

Norovirus 3C-like protease is a central enzyme that is solely responsible for the maturation of ORF1 polyprotein [7,8] and has significant similarity with proteases of other caliciviruses and picornavirus 3C proteases. Norovirus protease is a potent target of medication for the treatment of norovirus gastroenteritis. Our extensive mutagenesis study of 3C-like protease of the Chiba strain revealed that His30 and Cys139 were responsible for proteolysis [9]. The X-ray crystal structure revealed that the norovirus 3C-like protease had a chymotrypsin-like fold and that Glu54 was possibly involved in the catalysis [10,11]. A previous mutagenesis study showed that Arg8, Lys88, Arg89, Asp138, and His157 were sensitive to Ala mutation [9]. Arg89 and Asp138 were salt-bridged with each other, and His157 played a critical role in substrate recognition [9,10]. In order to obtain information on an enzyme–substrate interaction and the mechanism of proteolysis, we here complete our mutagenesis study that includes Alascanning mutagenesis of 106 of 181 residues constituting the protease (Table 1). Thirteen residues are identified as important, and their significance is discussed.

2. Materials and methods Abbreviations: VPg, genome-linked viral protein; VP, virion structural protein; GST, glutathione S-transferase; IPTG, isopropyl-b-D-galactopyranoside; PBS, phosphate-buffered saline ⇑ Corresponding author. Fax: +81 42 561 4729. E-mail address: [email protected] (Y. Someya). 1 Present address: Thailand-Japan Research Collaboration Center on Emerging and Re-emerging Infections, Research Institute for Microbial Diseases, Osaka University, Japan.

2.1. Bacterial strains and plasmids Escherichia coli JM109 and DH5a strains (Nippon Gene Co., Ltd., Tokyo, Japan) were used for plasmid construction. The BL21CodonPlus(DE3)-RIPL strain (Stratagene, La Jolla, CA) was used for the expression of the recombinant proteins.

0014-5793/$36.00 Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2010.12.018

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disrupted by vigorous vortexing in the presence of glass beads (0.1 mm diameter). After centrifugation at 15,000g for 10 min, the supernatant was transferred to a new tube. Proteins were determined with a BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL) using BSA as a standard. Extracted proteins were separated by SDS–PAGE, followed by Coomassie Brilliant Blue staining or Western blotting with rabbit anti-glutathione S-transferase (anti-GST) antibody (Abcam, Cambridge, MA), rat anti-VPg or mouse anti-protease antisera [13]. For several mutants, transformed E. coli cells were also grown on 4 ml of 2 YT broth at 37 °C until OD at 610 nm reached around 0.5. After the addition of isopropyl-b-D-galactopyranoside (IPTG) to a final concentration of 0.1 mM, protein expression was induced for 2 h. Cells were resuspended in 100 ll of PBS containing 1.0% (w/v) Triton X-100. Proteins were extracted in the same manner as described above.

To assess the effects of mutations on proteolytic activity in E. coli cells, an expression plasmid pGEX-2TK-3aBC (Fig. 1) [12] was used. All mutations were introduced into pGEX-2TK-3aBC using mutagenic oligonucleotides (Supplementary Tables S1 and S2). All mutations were first detected based on the appearance or disappearance of restriction sites, and then verified by DNA sequencing. 2.2. Expression of the recombinant proteins in E. coli cells and SDS– PAGE analysis E. coli BL21-CodonPlus(DE3)-RIPL cells harboring each of the pGEX-2TK-3aBC plasmids were grown on 4 ml of MagicMedia E. coli Expression Medium (Invitrogen, Carlsbad, CA) at 37 °C for 16 h, harvested and resuspended in 600 ll of phosphate buffered saline (PBS) containing 1.0% (v/v) Triton X-100. Cells were

2.3. GST binding assay Table 1 Target amino acid residues in the 3C-like protease of the Chiba strain for Ala-scanning mutagenesis. Amino acids

Completely conserveda

Highly conservedb

Less conservedc

Pro Val

3, 66, 96 9, 24, 72, 81, 99, 152, 168 94

22, 43, 78 10, 36, 64, 114, 144, 171 5, 68, 95, 113, 135

Ile Phe Tyr Trp Ser Thr

2, 33, 136, 142 20, 31, 82, 153, 156, 167 73, 86, 97, 121, 122, 132, 180 26 40, 55, 60 143 6, 16, 19 7, 21 27, 123, 134, 166

49, 87, 109 12, 18, 25

14, 84, 91 161

Cys Met Asn Gln

139d 101, 120 126, 165 117

77, 169 71, 130 149 110

32, 44, 47, 85, 104 39, 58 145 151 46, 61, 106, 118, 163 4, 23, 28, 29, 34, 56, 69, 178, 179 83, 154 107 148 57, 172

Leu

Cell extract containing 500 lg of protein was applied to a Glutathione Sepharose 4B MicroSpin column (GE Healthcare, Piscataway, NJ). Glutathione-resin-bound proteins were eluted with 50 ll of 50 mM Tris–HCl (pH 8.0) containing 10 mM reduced glutathione. A portion (5 ll) of the eluates was analyzed by SDS–PAGE. 3. Results 3.1. Ala-scanning mutagenesis In this study, 106 of 181 amino acid residues constituting the 3C-like protease of the Chiba strain were subjected to Ala-scanning mutagenesis as targets (Table 1). Initially, transformed E. coli cells were grown in MagicMedia. As a result, Ala mutation at six positions (Trp6, Leu95, Leu97, Leu121, Thr134 and Val167) affected the protease activity (Fig. 2). The W6A, T134A and V167A mutants had absolutely no activity, whereas the L95A, L97A and L121A mutants retained very slight activity since very faint amounts of GST3a and GST-3aB could be observed (Fig. 2). The amount of the V167A mutant fusion was extremely low, and the mutation might make the protease unstable. In Fig. 2 (and also Fig. 3), some very faint bands could be discerned in some samples. The bands of about 48 kDa were identified as the GST-3aB intermediate, but other faint bands were not detected by Western blotting with anti-GST antibody (data not shown). Therefore, these might be non-specifically eluted bacterial proteins and were not subjected to further analysis.

Numbers indicate positions in the amino acid sequence of the 3C-like protease of the Chiba strain. The positions whose Ala mutation led to a loss of activity and/or a decreased level of expression are indicated by bold letters. a Amino acid residues conserved in all noroviruses from genogroup I (human), II (human), III (bovine) and V (murine), whose nucleotide sequences are available in public databases. b One of two kinds of amino acid residues is located at the positions indicated. c Over three kinds of amino acid residues are found. d The Cys139 residue has previously been shown to be essential for proteolysis [9].

Ptac

GST

‘3A

3B

3C

(GST)...lvprgsrrasvgsCETEEEGTSEEMNVELPTATSE/GKNK...(3B)...EKLSFE/APPT...(3C)...TTLE* C-term. 22 AA of 3A 3B, 136 AA 3C, 181 AA 3C

3C GST-3aBC

(64.7 kDa)

GST-3aB

(45.3 kDa)

GST-3a

(29.2 kDa) 3BC

(35.5 kDa)

3B

(16.1 kDa) 3C (19.4 kDa)

Fig. 1. Plasmid construction of pGEX-2TK-3aBC. The gene fragments encoding the 22 C-terminal amino acid residues of 3A and the entire 3B VPg and 3C-like protease were fused in-frame to the GST gene of the pGEX-2TK vector. The fusion protein, GST-3aBC, has two protease recognition sites indicated by arrows. The calculated molecular weight of the parental protein and possible intermediates along with the final products are shown.

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Thr27 WT

Ala Ser Val

Thr134

Tyr143

Trp6

Trp19

Ala Ser Val

Ala Phe His Ser

Ala Phe His Leu

Ala Phe His Leu

255 150 102 76

GST-3aBC

52 38 31

GST-3a

24 17 12

Met101 Ala Leu Gln Val

Gln117 Phe Ile

Ala Glu Leu Asn

Val167

Val144 His Val

Ala Leu Met Gln

Thr

Ala Leu Met Gln

Thr

255 150 102 76

GST-3aBC

52

GST-3aB

38 31

GST-3a

24 17 12

L eu 86 Ala Met Gln Val

L eu9 5 Phe Ile

Ala Met Gln Val

L eu 1 21

L eu 97 Phe Ile

Ala Met Gln Val

Phe Ile

Ala Met Gln Val

Phe Ile

255 150 102 76

GST-3aBC

52

GST-3aB

38 31

GST-3a

24 17 12 Fig. 2. GST binding assay using the lysates of E. coli cells cultured in MagicMedia. The wild-type (WT) construct was used as a positive control. The GST-containing proteins were indicated.

Thr27

Tyr143

Trp6

Trp19

Ala Ala His Ser His Leu Ala Leu

Met101

Gln117

Ala Gln Val Phe Ile Ala Glu Asn His

Val144 Val167 Leu86

Leu121

Ala Gln Gln Ala Gln Val Met

255 150 102 76

GST-3aBC

52

GST-3aB

38 31

GST-3a

24 17 Fig. 3. GST binding assay using the lysates of E. coli cells induced with IPTG. Mutants whose products were not detected in experiments in Fig. 2 were cultured in 2 x YT media, and the protein expression was induced by 0.1 mM IPTG for 2 hr.

On the other hand, Ala mutation at seven positions (Trp19, Thr27, Leu86, Met101, Gln117, Tyr143 and Val144) drastically decreased the expression level (Fig. 2). This might be because fu-

sions containing mutated protease moiety were unstable, and therefore the proteins were not tolerant under the prolonged induction condition (16 h) in MagicMedia. The expression of

C139A

V167L

L121Q

L121F

L121A

L97Q

L97A

L95A

L86V

L86A

Wild-type

C139A

Q117A

M101L

M101A

T27A

W6L

W6H

A

W6A

Y. Someya, N. Takeda / FEBS Letters 585 (2011) 369–374

Wild-type

372

215 150 100 GST-3aBC

60 50 35 25

3C

C139A

V167L

L121Q

L121F

L121A

L97Q

L97A

L95A

L86V

L86A

Wild-type

C139A

Q117A

M101L

M101A

T27A

W6L

W6H

B

W6A

Wild-type

16

215 150 100 60 50

GST-3aBC

35 25 3B 16 Fig. 4. Western blotting analysis with mouse anti-protease (A) and rat anti-VPg (B). The wild type and the C139A mutant were used as a positive and negative control, respectively. The bands indicated by arrows are of specifically reacted proteins. The GST-3aB (in panel B) and 3BC (in panels A and B) intermediates are expected to be detected, but they are not specified because of interference by non-specifically reacted bands. In the T27A, L86A, L86 V, M101A, M101L and Q117A mutants, both 3C protease and 3B VPg were detected as well as the parental GST-3aBC. In the W6A, W6H, W6L, L95A, L97A, L97Q, L121A, L121F and L121Q mutants, neither 3C nor 3B was detected. Although the V167L mutant had partial activity (see Fig. 2), the GST-3aB intermediate was hardly identified.

these seven mutants could be observed when induced with 0.1 mM IPTG for 2 h, followed by isolation of GST-containing species (Fig. 3). The W19A, Y143A and V144A mutant proteases were completely inactive. The T27A and L86A proteases had slightly lower activity than the wild type since trace amounts of the parental GST-3aBC and the GST-3aB intermediate were also present along with the final product of GST-3a. The M101A and Q117A mutations decreased the proteolytic activity markedly because the parental GST-3aBC was a main species despite the production of the GST-3a final species. The fact that the T27A, L86A, M101A and Q117A mutant proteases were active was confirmed by Western blotting (Fig. 4). These results suggested that Trp6, Trp19, Leu95, Leu97, Leu121, Thr134, Tyr143, Val144 and Val167 were important for the activity and/or stability of the protease, and that Thr27, Leu86, Met101 and Gln117 were involved in stability. Ninety-three residual Ala mutants behaved in virtually the same manner as the wild type (data not shown). 3.2. Site-directed mutagenesis To further investigate the amino acid requirement at 13 positions, several site-directed mutants were constructed and analyzed (Figs. 2–4). The results are described below in the order in which the positions appeared in Fig. 2. Thr27 – The Ser and Val mutants also had protease activity, confirming that Thr27 was not essential for proteolysis.

Thr134 – In the crystal structure, the OH group of Thr134, together with an imidazole ring of His157, interacted directly with the P1 position (Glu or Gln) of the substrate [10]. Only the Ser mutant had the activity in spite of the slightly lower expression level, indicating that an OH group was required at this position. Tyr143 – The Phe mutant was stable and active, whereas the His and Ser mutations like the Ala mutation led to a loss of both activity and stability. In the crystal structure, the OH group of Tyr143 also hydrogen-bonded with an imidazole ring of His157 [10], but the result suggested that a stack of two aromatic rings from His157 and Tyr143 stabilized the protease. Trp6 – Replacement with Phe was acceptable, but the His and Leu mutations completely inactivated the protease as did the Ala mutation. A neutral aromatic residue is required at this position. Trp19 – Compared with Trp6, the His mutation was also acceptable. Aromaticity, independent of charge, is an important property. Met101 – All mutations tested caused extreme decreases in the expression level. However, since the Ala and Leu mutants had significant activity, Met101 was not essential for proteolysis, but was likely to be important for protein stability. Gln117 – The Leu and Val mutants showed wild-type activity. On the other hand, the Glu, Asn or His mutations decreased the expression level and inactivated the enzyme. It was suggested that the presence of charge (both negative and positive) was not preferred, but that the side chain volume was an important determinant. It was notable that Met101 and Gln117 were in close

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Val and Ile mutants showed wild-type activity. A large, aliphatic amino acid is required for the activity.

proximity to each other [10]. The interaction between these two residues may be important for stability. Val144 – The Leu, Met and Thr mutations retained their activity, but the Gln mutation did not. It was suggested that high hydrophilicity should be avoided. Val167 – Compared with Val144, the amino acid requirement was strict. Only the Thr mutant whose side-chain volume was close to that of Val exhibited high activity. The Leu mutant produced the GST-3aB intermediate, but did not produce a significant amount of GST-3a, suggesting that this mutation affected the substrate specificity. In fact, Val167 is one of the residues constituting the S1 specificity pocket that accommodates the P1 residue of the substrate [10]. Leu86 – This residue could be replaced with Met, Phe or Ile, their protease activities being indistinguishable from that of the wild type. Even Ala and Val mutants had slight but significant activity, although stability seemed to be impaired, as shown by the fact that they were expressed by IPTG induction (Fig. 3). The Gln mutation impaired both activity and stability. These results suggested that a large, hydrophobic amino acid was preferred at this position. Leu95 – No mutations except the Ala mutation affected the protease expression and activity, indicating that Leu95 itself was not essential for proteolysis and stability. Leu97 – As with Leu95, most mutations did not affect the protease activity. The Ala and Gln mutations drastically decreased the activity, and very slight amounts of GST-3a and GST-3aB (Fig. 2) and 3BC (Fig. 4) were produced. The hydrophobicity of the side chain seems important. Leu121 – The Met mutant could not be expressed even by IPTG induction, suggesting that this mutant was unstable. Besides the Ala mutant, the Gln and Phe mutants had no activity, while the

4. Discussion Ala-scanning mutagenesis of 106 residues resulted in the discovery that 13 Ala mutants showed a loss of activity and/or loss of stability. The 11 residues other than Leu95 and Val144 are absolutely conserved in all 3C-like proteases from genogroups I, II, III and V. Although genogroup I (GI) proteases including the Chiba strain and GV (murine noroviruses) have Leu at position 95, GII proteases have Met or Ile and GIII (bovine viruses) counterparts have Val, Leu or Ile. As for position 144, it is occupied by Val in GI and GV proteases, but by Ile or Val in GII and by Phe in GIII. Low conservation at these two positions seems to be consistent with the result of mutagenesis that various residues could substitute for the two residues. It is interesting that 10 of 13 residues (Leu86, Leu95, Leu97, Met101, Gln117, Leu121, Thr134, Tyr143, Val144 and Val167) were mapped on the C-terminal domain, and only 3 of 13 (Trp6, Trp19 and Thr27) on the N-terminal domain (Fig. 5). Surprisingly, among these residues, only Thr134 directly interacted with a substrate. Thr134 together with His157 recognizes the P1 position of a substrate [10], which is consistent with the result showing that a hydroxyl group at position 134 was critical for proteolysis. Leu97, Leu121, Tyr143 and Val167 are residues constituting the S1 specificity pocket [10] which make a band forming a circle around the barrel in the C-terminal domain (Fig. 5). No other residues important for an enzyme-substrate interaction could be found in this mutagenesis study. These findings suggest that the substrate is held via its P1 position by the protease.

Cys139 32 12

His30

15

aI

29

20

7

52

55

fI

Thr27

Trp19

Arg8

Glu54

cI

bI

58

Trp6

gI

48 47

23

45 44

2

37

eI

42 107

N

39

dI

bII 114

C

146

150

81

C

dII

Met101

Gln117

Val144

fII

Tyr143

aII

Val167 His157

160

cII Leu97

Lys88

140

Cys139

eII

170

Leu86

Arg89

Leu121

fII

Val167

122

Leu95

165

Asp138 136

93

Thr134

Fig. 5. Map of amino acid residues important for the proteolytic activity and/or the stability. Important residues identified in this report and the previous report [9] are mapped on the topological view of the 3C-like protease. A dashed line between two residues indicates that the two residues are in close proximity to each other. A double dashed line indicates an ionic interaction. The triplet Cys139-His30-Glu54 constitutes a catalytic triad for proteolysis [9,12] although Glu54 is not essential [12,13]. Arg89 was salt-bridged with Asp138 [10]. For clarity, the Cys139 residue and the fII beta sheet are represented in two places.

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It is also notable that residues found in the C-terminal domain form a network in which side chains are in close proximity and possibly interact with each other (Fig. 5). The network, most of which is based on hydrophobic interaction, is suggested to be important for the stability and integrity of the protease since mutations, especially those of Met101 and Gln117, often lead to a decreased level of expression. The N-terminal domain was in marked contrast to the C-terminal domain since the Ala mutation at only 3 positions affected its activity and/or stability. The crystal structure indicates that Trp19 is located on the top of an aromatic residue cluster in the N-terminal domain [10]. Since aromaticity at position 19 was essential for the activity, an aromatic-aromatic interaction would be important, but its precise role is unknown. On the other hand, Trp6 is sandwiched between Leu86 and Lys88, thus representing the only interaction between two domains that was identified in this work. Previous crystallographic analysis revealed two ionic interactions between two domains, Arg11-Asp90 and Val9 carbonyl-Lys88. Since only Lys88 among these four residues was important [9], a Val9-Lys88 interaction is likely the more important interaction. In any case, there seem to be few interactions between the N-terminal domain and the C-terminal domain. It is suggested that this results in the flexibility of the protease during proteolysis. Acknowledgement This work was supported partly by a Grant-in-aid from the Ministry of Health, Labour and Welfare, Japan. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.febslet.2010.12.018.

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