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Virus Research xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Virus Research journal homepage: www.elsevier.com/locate/virusres
Production of Norovirus VLPs to size homogeneity
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Yuqi Huo ∗ , Xin Wan, Zejun Wang, Shengli Meng, Shuo Shen Wuhan Institute of Biological Products, Wuhan PR China
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Article history: Received 27 February 2015 Received in revised form 1 April 2015 Accepted 8 April 2015 Available online xxx
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Keywords: VLPs Norovirus Truncated capsid protein Smaller viral particles
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1. Introduction
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Expression of full-length major capsid protein of Noroviruses (NoVs) in sf9 cells using recombinant baculovirus expression system leads to the formation of virus-like particles (VLPs) with two sizes. In our pursuit of VLPs with uniform sizes, we find that N terminal truncated capsid protein formed primary VLPs with an average size of 21 nm. This kind of VLPs showed similar binding patterns to those produced with full-length major capsid protein. HBGA-VLPs binding assay and saliva-VLPs blocking analysis, as well as stability test demonstrate that the smaller 21 nm VLPs might be an excellent candidate for NoVs vaccine. © 2015 Published by Elsevier B.V.
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Noroviruses (NoVs), formerly known as Norwalk-like virus, are a genus of the Caliviviridae family and are further divided into five genogroups designated GI, GII, GIII, GVI and GV; GI and GII primarily infect humans which are further subdivided into 9 and 22 genotypes, respectively [1]. NoVs are small, icosahedral, singlestranded and positive-sensed RNA viruses with a genome size of around 7.5 kb and are currently the leading cause of non-bacterial acute gastroenteritis worldwide and infect people of all ages [2–9]. The NoV genome contains three open reading frames (ORFs), ORF1 encodes a polyprotein that is processed co- or post-translationally by the viral 3C-lile protease to 6 non-structural proteins, p48, NTPase, p22, VPg, 3CLpro, and RdRp, ORF2 encodes the major capsid protein that self-assembles into virus-like particle when expressed in sf9 cells with morphology and immunogenicity similar to native virus, ORF3 encodes a basic protein presumed to stabilize VLPs formed by major capsid proteins [10–12]. Expression of full-length capsid protein gene of Norwalk virus (NV) in eukaryotic cells leads to formation of recombinant Norwalk virus (rNV) VLPs composed of a single protein with two sizes, the full-length protein and a truncated protein [13,14]. In addition, the rNV VLPs formed using above mentioned cell lines also exhibit two sizes, the 38 nm rNV VLPs and the 19–21 nm subviral rNV VLPs. Further analysis showed that the two sizes of VLPs are composed
∗ Corresponding author at: Wuhan Institute of Biological Products, 9 Linjiang Avenue, Wuchang, Wuhan, 430060, PR China. E-mail address: [email protected] (Y. Huo).
of the full-length protein and truncated protein [15]. In this study, we expressed the capsid protein of newly isolated Sydney 2012-like GII.4 variant using recombinant baculovirus expression system and constructed several N-terminal capsid deletion mutants to study the effects of N-terminal amino acids over the morphology of NoV VLPs.
2. Materials and methods Sydney 2012-like GII.4 variant was isolated in Jingzhou, Hubei, China from an inpatient of two years old boy hospitalized with acute gastroenteritis [16]. The full-length capsid protein coding sequence (Genbank accession number, KF306214) was optimized based on codon frequency of Spodoptera (S.) frugiperda cells, synthesized by Sangon Biotech (Shanghai, China) and cloned into pVL1393 baculovirus transfer vector (BD Biosciences, San Diego, CA) flanked by BamHI and NotI sites at its 5 and 3 ends, respectively. The N terminal capsid mutants were constructed based on optimized capsid coding sequence. Baculovirus recombinant containing the VP1 gene in pVL1393 vector was obtained by homologous recombination between the transfer vector and linearized Baculovirus DNA (BD Biosciences, San Diego, CA). In brief, sf9 cells were transferred to a 6-well plate at a density of 2 × 105 /ml and let adhere at room temperature (RT) for 2 h. Purified sterile pVL1393 vector (2 g) containing target sequence and linearized Baculovirus DNA (0.2 g) were mixed with equal volume of Lipofectin (Invitrogen, US). The mixture was vortexed and incubated at RT for 20 min before it was added to a 6-well plate. Cytopathic effects were generally observed within 3–5 days and further amplification of recombinant Baculovirus was performed by infecting more cells in T225
http://dx.doi.org/10.1016/j.virusres.2015.04.009 0168-1702/© 2015 Published by Elsevier B.V.
Please cite this article in press as: Huo, Y., et al., Production of Norovirus VLPs to size homogeneity. Virus Res. (2015), http://dx.doi.org/10.1016/j.virusres.2015.04.009
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culture flasks. The medium was harvested 5–7 days post infection and frozen at −80 ◦ C as viral stocks. Mouse anti-Baculovirus envelope GP64 (eBioscience, US) monoclonal antibody (MAb) was used for the determination of recombinant baculovirus titers by using IFA method. The sf9 cells cultured in Sf-900 III SFM in monolayer and suspension at a cell density of 2 × 106 /ml were infected with appropriate recombinant baculovirus at a multiplicity of infection of 10. Cells in suspension were cultured in 500 ml vented flasks (Corning, US) at 27 ◦ C in a temperature controllable shaking incubator at 140 rpm/min. The cell medium was harvested 5–7 days post infection. The harvested cell medium was clarified at 3000 × g to remove cell debris followed by ultracentrifugation at 141,000 × g for 3 h at 8 ◦ C in a SW 28 rotor to pellet VLPs. Pellets were resuspended in PBS (pH 7.2–7.4) prefiltered with 0.22 m membrane. VLPs in PBS were mixed with equal volume of CsCl (1.6 g/ml) and centrifuged at 288,000 × g for 24 h at 4 ◦ C in a Beckman SW 41 Ti rotor. Visible bands were collected and analyzed by western blotting using rabbit anti-GII.4 VP1 specific hyperimmune serum and presence of VLPs was confirmed by electron microscopy (EM) observation after negative staining with phosphotungstic acid. The amino-terminal 10 amino acids of capsid protein and mutants were determined by Edman degradation method. HBGAVLPs binding assay and saliva-VLPs blocking assay were performed as described by others [17]. For HBGA-VLPs binding assay, eight synthetic oligosaccharides–PAA–biotin conjugates (GlycoTech, US) at 5 g/ml in carbonate buffer, pH 9.6, were added to 96-well plates (100 l/well) precoated with avidin and incubated at 37 ◦ C overnight, followed by blocking with PBS containing 0.5% Tween20 (PBS-T) and 1% BSA overnight at 4 ◦ C. Purified VLPs at 2 g/ml were added to above wells in triplicates (100 l/well), followed by an incubation time of 1 h at 37 ◦ C. After washing for five times with PBS-T, guinea pig anti-VLP serum (Prepared by immunizing guinea pigs with purified VLPs) at 1:2000 was added (100 l/well) and incubated at 37 ◦ C for 30 min. The plate was washed for five times with PBS-T before HRP-conjugated goat anti-guinea pig IgG at 1:20,000 was added (100 l/well), followed by an incubation period of 30 min at 37 ◦ C. The plates were washed for five times with PBS-T and reaction was developed with 50 l of substrate A containing tetramethylbenzidine (TMB) and 50 l of substrate B containing urea peroxide for 15 min, and color development was stopped by the addition of an equal volume of 2 M phosphoric acid. Absorbance was measured at 450 nm using a Multiskan MK3 plate reader (Thermo Scientific, US). For saliva-VLPs blocking assay, secretor positive, blood type AB saliva was used. Briefly, saliva with known ABO antigens was boiled for 10 min to inactivate native antibodies. The boiled saliva was diluted at 1:2000 in carbonate buffer, pH 9.6, added into 96-well plates (100 l/well) and incubated at 37 ◦ C overnight. After washing five times with PBS-T, the wells of each plate were blocked with 150 l PBS-T containing 1% BSA by incubating at 37 ◦ C for two hours. Then the plate was washed five times with PBS-T, dried, sealed and stored at 4 ◦ C. Before conducting saliva-VLP blocking assay using mouse sera, all reagents, sera dilutions and reaction times were optimized. PBS-T containing VLPs at 0.5 g/ml was used to dilute serum samples at 1:200 in EP tubes. VLPs in serum-free PBS-T was selected as binding positive control and serum-free PBS-T as binding inhibition control. The diluted sera were vortexed and incubated at 37 ◦ C for 30 min, and then transferred to wells pre-coated with saliva (100 l/well), followed by an incubation for 1 h at 37 ◦ C. Procedures after addition of guinea pig anti-VLP serum and HRP-conjugated goat anti-guinea pig IgG were the same as mentioned above. The blocking index was calculated in % as (mean OD without sera − sample mean OD with sera/mean OD without sera) × 100%. The reactivity of mouse sera with full-length, N26 and N38 truncated capsid protein was determined by Western blot. In brief,
the purified proteins separated by SDS-PAGE were transferred to nitrocellulose membrane and detected using corresponding mouse anti-GII.4 VLPs serum at a dilution of 1:500 in PBS-T. The membrane was washed with PBS-T for three times to remove unbound antibodies and then HRP conjugated goat anti-mouse IgG polyclonal antibody was added at 1:1000 in PBS-T. The membrane was developed after another three times wash using PBS-T with DAB color-developing agent following the manufacture’s instruction.
3. Results EM demonstrated that culture conditions could significantly influence the size of VLPs with the ratio of 38 nm:21 nm VLPs being higher in monolayer culture than in suspension culture (Fig. 1). SDS-PAGE analysis showed two protein bands for both preparations and truncated protein dominated in monolayer preparation. Sequence analysis showed an in-frame ATG codon right downstream of N terminus. We initially assumed that the observed truncated protein might be due to internal translation initiation at this site or degradation or cleavage product of thus formed unstable protein. To prove our hypothesis, site-directed mutagenesis was performed and re-expression of this mutated capsid protein (M27G) led to successful self-assembly into VLPs in two sizes and SDS-PAGE analysis still revealed the presence of two protein bands. So the truncated protein is a degradation or cleavage product of the full-length capsid protein. Results from literature indicate that the smaller and larger VLPs are composed of full-length and degraded capsid protein. In order to determine what role the degraded or cleaved capsid protein plays in the formation of 21 nm and 38 nm particles, we studied the expression of two N-terminal deletion mutants, N26 and N38 (with 26 and 38 amino acid residues, respectively, deleted from the N-terminus). The N38 deletion mutant was based on our sequencing result of degraded product of full-length capsid protein, as in which 38 residues were removed from the N terminus (Fig. 2B). Expression of the two deletion mutants in Sf9 cells led to formation of VLPs with an average size of 21 nm. SDS-PAGE analysis of the two deletion mutants of capsid protein showed a single band of similar molecular size (Fig. 2A). It is noteworthy that 38 nm particles were present, even though in very low ratio (less than 5% based on counting of several EM images, data not shown), in N26 deletion mutant formed VLPs, but they were not observed in N38 deletion mutant formed VLPs. This finding can shed new light on the assembly pathway of NoV capsid proteins. Based on our results, we proposed that N-terminal degradation or cleavage of capsid protein was initiated after VLPs assembly. Firstly, N-terminal sequencing of expressed full-length, N26 and N38 deletion mutants of capsid protein showed the same N-terminal ending (Fig. 2B). If the assembly of VLPs started right after degradation or cleavage completion, then uniform VLPs should be observed. However, 38 nm VLPs were also observed in expressed N26 mutant capsid protein preparation. Secondly, monolayer culture led to formation of primarily 38 nm VLPs, but SDS-PAGE analysis showed dominance of degraded or cleaved capsid protein (Fig. 1C). If degradation or cleavage started before assembly, then the majority of observed VLPs should be in the size Q3 of 21 nm (Fig. 3). To fully characterize the VLPs formed by N26 and N38 deletion mutants of capsid protein, HBGA-VLPs binding assay and saliva-VLPs binding blocking assay were performed. As shown in Fig. 4A, VLPs formed by N26 and N38 capsid mutants exhibited similar binding patterns compared with that of full-length capsid protein. The observed higher binding of the mutants (Fig. 4B) may have to do with differences in structure rather than in deletions. The receptor-coated wells of the microtiter plate may bind more smaller than larger VLPs. The saliva-VLPs blocking assay (Fig. 4B)
Please cite this article in press as: Huo, Y., et al., Production of Norovirus VLPs to size homogeneity. Virus Res. (2015), http://dx.doi.org/10.1016/j.virusres.2015.04.009
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Fig. 1. Culture conditions influence the sizes of NoV VLPs purified from recombinant baculovirus infected Sf9 insect cells. (A) Suspension culture, (B) monolayer culture, (C) SDS-PAGE analysis of CsCl purified VLPs from monolayer culture (a) and suspension culture (b). Bar, 100 nm.
Fig. 2. (A) SDS-PAGE analysis on a 4–12% polyacrylamide gel of 8 g of CsCl purified VLPs from full-length (FL), N26 and N38 deletion mutants of capsid protein. (B) Amino acid sequences of expressed proteins from panel A. The predicted and actual amino acid sequences of the N-terminal are indicated.
Fig. 3. Electron micrograph of CsCl purified VLPs from capsid mutants. (A) VLPs formed from N26 protein. (B) VLPs formed from N38 protein and (C) VLPs formed from VP1(M27G). Bar, 100 nm.
Fig. 4. Binding of VLPs to synthetic oligosaccharides containing human HBGA epitopes (A) and saliva-VLPs blocking assay and Western blot using mouse sera immunized with full-length capsid protein, N26 and N38 assembled VLPs (B). (A) Blood type A antigen (trisaccharide), (B) blood type B antigen (Trisaccharide). Anti-sera were produced by immunizing mice with indicated VLPs at 10 g/dose for two immunizations at an interval of three weeks in the presence of Al(OH)3 .
Please cite this article in press as: Huo, Y., et al., Production of Norovirus VLPs to size homogeneity. Virus Res. (2015), http://dx.doi.org/10.1016/j.virusres.2015.04.009
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exhibited similar blocking efficiencies for sera from mouse immunized with the same amounts of VLPs made of full-length VP1 or the N26 and N38 deletion mutants.
VLPs to 200 g/ml, which is also optimal concentration for negative staining. Another advantage of the N26 and N38 deletion mutants formed VLPs as vaccine is its convenience for quality control and quantitation (enzyme linked immunosorbant assay) during production process.
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4. Discussion It has been known for some time that expression of NoV capsid proteins in sf9 cells, as well as other eukaryotic cells, leads to formation of VLPs of two sizes. In our study, we find culture conditions can significantly influence the ratio of smaller and larger VLPs formed. Oxygen, pH, capsid protein expression level and cell density might be factors affecting the final yields of certain size of VLPs. Oxygen partial pressure can affect cell proliferation and virus replication which might have certain effects over the production of VLPs. It has been shown that in vitro capsid-capsid protein interaction facilitating VLPs formation is pH dependent and the precise control of pH, as well as other factors in aerobic culture using bioreactor might provide insight into the in vivo assembly mechanism of VLPs [18]. The smaller particle has a diameter of 21 nm in this study while it is reported to be 23 nm by others [15]. This difference might be caused by different major capsid proteins (from GII.4 in this study) used and varied cleavage sites on major capsid proteins from different genogroups as 44 aa was cleaved from the N-terminus of the major capsid protein of a GI.2 norovirus (Genbank accession number, KF306212, data not shown). The dominating or completely smaller VLPs formed by N26 and N38 deletion mutants in our study indicate that the N terminal of NoV capsid protein contain key element(s) determining the sizes of VLPs assembled. Besides, Based on our results, it is presumed that assembling of 38 nm VLPs requires the presence of full-length or close to full-length capsid proteins and further degradation or cleavage does not affect the particle sizes [19]. Further evidence in support of our hypothesis is based on the finding that VLPs purified from monolayer culture lead to different results from batch to batch relating to the ratio of full-length capsid protein and degraded or cleaved capsid protein even larger VLPs are dominating in all the preparations (data not shown). X-ray crystal structure analysis showed that the N-terminus of capsid protein is buried inside the particle and contact with proteases is avoided [20,21]. VLPs in cells cultured in the presence of proteinase inhibitor cocktail also showed doublet bands, indicating cleavage of capsid protein before release of VLPs into culture medium (data not shown). It has been shown that HBGA-VLPs binding are genotype specific and dependent of structures in the loop regions that significantly alter the surface topography and electrostatic landscape of the P domain [22]. Results from HBGA-VLPs binding assay indicate conservation of structural integrity of receptor binding regions of VLPs formed by N26 and N38 deletion mutants. Similar blocking efficiencies using sera from mice immunized with the same amounts of VLPs made of full-length VP1 or the N26 and N38 deletion mutants indicate similar immunogenicity of the three types of VLPs. Based on above results, the N26 and N38 deletion mutants formed VLPs might be excellent candidates for vaccine production. Besides, Stability tests indicate that 21 nm VLPs formed by N26 and N38 deletion mutants are stable for at least one months at 4 ◦ C in PBS (0.01 M, pH 7.2), which is comparable to that of 38 nm VLPs, as no significant difference in particle intactness was observed. It has been reported that N-terminal undergoes cleavage more readily in 23-nm recombinant Norwalk virus VLPs and this is true for VLPs formed by N38 deletion mutant as a 45 kDa product was observed after storage at 4 ◦ C in PBS for two weeks (data not shown). VLPs in higher concentrations tend to aggregate which is consistent with other report [23]. This phenomenon can be delayed by diluting
Acknowledgement I would like to thank Zhang Pei from The Core Facility and Technical Support, Wuhan Institute of Virology, for her help with producing EM micrographs.
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Contents lists available at ScienceDirect
Virus Research journal homepage: www.elsevier.com/locate/virusres
Production of Norovirus VLPs to size homogeneity
1
Q1
2
Yuqi Huo ∗ , Xin Wan, Zejun Wang, Shengli Meng, Shuo Shen Wuhan Institute of Biological Products, Wuhan PR China
3 4
5 18
a r t i c l e
i n f o
a b s t r a c t
6 7 8 9 10 11
Article history: Received 27 February 2015 Received in revised form 1 April 2015 Accepted 8 April 2015 Available online xxx
12
17
Keywords: VLPs Norovirus Truncated capsid protein Smaller viral particles
19
1. Introduction
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Expression of full-length major capsid protein of Noroviruses (NoVs) in sf9 cells using recombinant baculovirus expression system leads to the formation of virus-like particles (VLPs) with two sizes. In our pursuit of VLPs with uniform sizes, we find that N terminal truncated capsid protein formed primary VLPs with an average size of 21 nm. This kind of VLPs showed similar binding patterns to those produced with full-length major capsid protein. HBGA-VLPs binding assay and saliva-VLPs blocking analysis, as well as stability test demonstrate that the smaller 21 nm VLPs might be an excellent candidate for NoVs vaccine. © 2015 Published by Elsevier B.V.
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Noroviruses (NoVs), formerly known as Norwalk-like virus, are a genus of the Caliviviridae family and are further divided into five genogroups designated GI, GII, GIII, GVI and GV; GI and GII primarily infect humans which are further subdivided into 9 and 22 genotypes, respectively [1]. NoVs are small, icosahedral, singlestranded and positive-sensed RNA viruses with a genome size of around 7.5 kb and are currently the leading cause of non-bacterial acute gastroenteritis worldwide and infect people of all ages [2–9]. The NoV genome contains three open reading frames (ORFs), ORF1 encodes a polyprotein that is processed co- or post-translationally by the viral 3C-lile protease to 6 non-structural proteins, p48, NTPase, p22, VPg, 3CLpro, and RdRp, ORF2 encodes the major capsid protein that self-assembles into virus-like particle when expressed in sf9 cells with morphology and immunogenicity similar to native virus, ORF3 encodes a basic protein presumed to stabilize VLPs formed by major capsid proteins [10–12]. Expression of full-length capsid protein gene of Norwalk virus (NV) in eukaryotic cells leads to formation of recombinant Norwalk virus (rNV) VLPs composed of a single protein with two sizes, the full-length protein and a truncated protein [13,14]. In addition, the rNV VLPs formed using above mentioned cell lines also exhibit two sizes, the 38 nm rNV VLPs and the 19–21 nm subviral rNV VLPs. Further analysis showed that the two sizes of VLPs are composed
∗ Corresponding author at: Wuhan Institute of Biological Products, 9 Linjiang Avenue, Wuchang, Wuhan, 430060, PR China. E-mail address: [email protected] (Y. Huo).
of the full-length protein and truncated protein [15]. In this study, we expressed the capsid protein of newly isolated Sydney 2012-like GII.4 variant using recombinant baculovirus expression system and constructed several N-terminal capsid deletion mutants to study the effects of N-terminal amino acids over the morphology of NoV VLPs.
2. Materials and methods Sydney 2012-like GII.4 variant was isolated in Jingzhou, Hubei, China from an inpatient of two years old boy hospitalized with acute gastroenteritis [16]. The full-length capsid protein coding sequence (Genbank accession number, KF306214) was optimized based on codon frequency of Spodoptera (S.) frugiperda cells, synthesized by Sangon Biotech (Shanghai, China) and cloned into pVL1393 baculovirus transfer vector (BD Biosciences, San Diego, CA) flanked by BamHI and NotI sites at its 5 and 3 ends, respectively. The N terminal capsid mutants were constructed based on optimized capsid coding sequence. Baculovirus recombinant containing the VP1 gene in pVL1393 vector was obtained by homologous recombination between the transfer vector and linearized Baculovirus DNA (BD Biosciences, San Diego, CA). In brief, sf9 cells were transferred to a 6-well plate at a density of 2 × 105 /ml and let adhere at room temperature (RT) for 2 h. Purified sterile pVL1393 vector (2 g) containing target sequence and linearized Baculovirus DNA (0.2 g) were mixed with equal volume of Lipofectin (Invitrogen, US). The mixture was vortexed and incubated at RT for 20 min before it was added to a 6-well plate. Cytopathic effects were generally observed within 3–5 days and further amplification of recombinant Baculovirus was performed by infecting more cells in T225
http://dx.doi.org/10.1016/j.virusres.2015.04.009 0168-1702/© 2015 Published by Elsevier B.V.
Please cite this article in press as: Huo, Y., et al., Production of Norovirus VLPs to size homogeneity. Virus Res. (2015), http://dx.doi.org/10.1016/j.virusres.2015.04.009
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culture flasks. The medium was harvested 5–7 days post infection and frozen at −80 ◦ C as viral stocks. Mouse anti-Baculovirus envelope GP64 (eBioscience, US) monoclonal antibody (MAb) was used for the determination of recombinant baculovirus titers by using IFA method. The sf9 cells cultured in Sf-900 III SFM in monolayer and suspension at a cell density of 2 × 106 /ml were infected with appropriate recombinant baculovirus at a multiplicity of infection of 10. Cells in suspension were cultured in 500 ml vented flasks (Corning, US) at 27 ◦ C in a temperature controllable shaking incubator at 140 rpm/min. The cell medium was harvested 5–7 days post infection. The harvested cell medium was clarified at 3000 × g to remove cell debris followed by ultracentrifugation at 141,000 × g for 3 h at 8 ◦ C in a SW 28 rotor to pellet VLPs. Pellets were resuspended in PBS (pH 7.2–7.4) prefiltered with 0.22 m membrane. VLPs in PBS were mixed with equal volume of CsCl (1.6 g/ml) and centrifuged at 288,000 × g for 24 h at 4 ◦ C in a Beckman SW 41 Ti rotor. Visible bands were collected and analyzed by western blotting using rabbit anti-GII.4 VP1 specific hyperimmune serum and presence of VLPs was confirmed by electron microscopy (EM) observation after negative staining with phosphotungstic acid. The amino-terminal 10 amino acids of capsid protein and mutants were determined by Edman degradation method. HBGAVLPs binding assay and saliva-VLPs blocking assay were performed as described by others [17]. For HBGA-VLPs binding assay, eight synthetic oligosaccharides–PAA–biotin conjugates (GlycoTech, US) at 5 g/ml in carbonate buffer, pH 9.6, were added to 96-well plates (100 l/well) precoated with avidin and incubated at 37 ◦ C overnight, followed by blocking with PBS containing 0.5% Tween20 (PBS-T) and 1% BSA overnight at 4 ◦ C. Purified VLPs at 2 g/ml were added to above wells in triplicates (100 l/well), followed by an incubation time of 1 h at 37 ◦ C. After washing for five times with PBS-T, guinea pig anti-VLP serum (Prepared by immunizing guinea pigs with purified VLPs) at 1:2000 was added (100 l/well) and incubated at 37 ◦ C for 30 min. The plate was washed for five times with PBS-T before HRP-conjugated goat anti-guinea pig IgG at 1:20,000 was added (100 l/well), followed by an incubation period of 30 min at 37 ◦ C. The plates were washed for five times with PBS-T and reaction was developed with 50 l of substrate A containing tetramethylbenzidine (TMB) and 50 l of substrate B containing urea peroxide for 15 min, and color development was stopped by the addition of an equal volume of 2 M phosphoric acid. Absorbance was measured at 450 nm using a Multiskan MK3 plate reader (Thermo Scientific, US). For saliva-VLPs blocking assay, secretor positive, blood type AB saliva was used. Briefly, saliva with known ABO antigens was boiled for 10 min to inactivate native antibodies. The boiled saliva was diluted at 1:2000 in carbonate buffer, pH 9.6, added into 96-well plates (100 l/well) and incubated at 37 ◦ C overnight. After washing five times with PBS-T, the wells of each plate were blocked with 150 l PBS-T containing 1% BSA by incubating at 37 ◦ C for two hours. Then the plate was washed five times with PBS-T, dried, sealed and stored at 4 ◦ C. Before conducting saliva-VLP blocking assay using mouse sera, all reagents, sera dilutions and reaction times were optimized. PBS-T containing VLPs at 0.5 g/ml was used to dilute serum samples at 1:200 in EP tubes. VLPs in serum-free PBS-T was selected as binding positive control and serum-free PBS-T as binding inhibition control. The diluted sera were vortexed and incubated at 37 ◦ C for 30 min, and then transferred to wells pre-coated with saliva (100 l/well), followed by an incubation for 1 h at 37 ◦ C. Procedures after addition of guinea pig anti-VLP serum and HRP-conjugated goat anti-guinea pig IgG were the same as mentioned above. The blocking index was calculated in % as (mean OD without sera − sample mean OD with sera/mean OD without sera) × 100%. The reactivity of mouse sera with full-length, N26 and N38 truncated capsid protein was determined by Western blot. In brief,
the purified proteins separated by SDS-PAGE were transferred to nitrocellulose membrane and detected using corresponding mouse anti-GII.4 VLPs serum at a dilution of 1:500 in PBS-T. The membrane was washed with PBS-T for three times to remove unbound antibodies and then HRP conjugated goat anti-mouse IgG polyclonal antibody was added at 1:1000 in PBS-T. The membrane was developed after another three times wash using PBS-T with DAB color-developing agent following the manufacture’s instruction.
3. Results EM demonstrated that culture conditions could significantly influence the size of VLPs with the ratio of 38 nm:21 nm VLPs being higher in monolayer culture than in suspension culture (Fig. 1). SDS-PAGE analysis showed two protein bands for both preparations and truncated protein dominated in monolayer preparation. Sequence analysis showed an in-frame ATG codon right downstream of N terminus. We initially assumed that the observed truncated protein might be due to internal translation initiation at this site or degradation or cleavage product of thus formed unstable protein. To prove our hypothesis, site-directed mutagenesis was performed and re-expression of this mutated capsid protein (M27G) led to successful self-assembly into VLPs in two sizes and SDS-PAGE analysis still revealed the presence of two protein bands. So the truncated protein is a degradation or cleavage product of the full-length capsid protein. Results from literature indicate that the smaller and larger VLPs are composed of full-length and degraded capsid protein. In order to determine what role the degraded or cleaved capsid protein plays in the formation of 21 nm and 38 nm particles, we studied the expression of two N-terminal deletion mutants, N26 and N38 (with 26 and 38 amino acid residues, respectively, deleted from the N-terminus). The N38 deletion mutant was based on our sequencing result of degraded product of full-length capsid protein, as in which 38 residues were removed from the N terminus (Fig. 2B). Expression of the two deletion mutants in Sf9 cells led to formation of VLPs with an average size of 21 nm. SDS-PAGE analysis of the two deletion mutants of capsid protein showed a single band of similar molecular size (Fig. 2A). It is noteworthy that 38 nm particles were present, even though in very low ratio (less than 5% based on counting of several EM images, data not shown), in N26 deletion mutant formed VLPs, but they were not observed in N38 deletion mutant formed VLPs. This finding can shed new light on the assembly pathway of NoV capsid proteins. Based on our results, we proposed that N-terminal degradation or cleavage of capsid protein was initiated after VLPs assembly. Firstly, N-terminal sequencing of expressed full-length, N26 and N38 deletion mutants of capsid protein showed the same N-terminal ending (Fig. 2B). If the assembly of VLPs started right after degradation or cleavage completion, then uniform VLPs should be observed. However, 38 nm VLPs were also observed in expressed N26 mutant capsid protein preparation. Secondly, monolayer culture led to formation of primarily 38 nm VLPs, but SDS-PAGE analysis showed dominance of degraded or cleaved capsid protein (Fig. 1C). If degradation or cleavage started before assembly, then the majority of observed VLPs should be in the size Q3 of 21 nm (Fig. 3). To fully characterize the VLPs formed by N26 and N38 deletion mutants of capsid protein, HBGA-VLPs binding assay and saliva-VLPs binding blocking assay were performed. As shown in Fig. 4A, VLPs formed by N26 and N38 capsid mutants exhibited similar binding patterns compared with that of full-length capsid protein. The observed higher binding of the mutants (Fig. 4B) may have to do with differences in structure rather than in deletions. The receptor-coated wells of the microtiter plate may bind more smaller than larger VLPs. The saliva-VLPs blocking assay (Fig. 4B)
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Fig. 1. Culture conditions influence the sizes of NoV VLPs purified from recombinant baculovirus infected Sf9 insect cells. (A) Suspension culture, (B) monolayer culture, (C) SDS-PAGE analysis of CsCl purified VLPs from monolayer culture (a) and suspension culture (b). Bar, 100 nm.
Fig. 2. (A) SDS-PAGE analysis on a 4–12% polyacrylamide gel of 8 g of CsCl purified VLPs from full-length (FL), N26 and N38 deletion mutants of capsid protein. (B) Amino acid sequences of expressed proteins from panel A. The predicted and actual amino acid sequences of the N-terminal are indicated.
Fig. 3. Electron micrograph of CsCl purified VLPs from capsid mutants. (A) VLPs formed from N26 protein. (B) VLPs formed from N38 protein and (C) VLPs formed from VP1(M27G). Bar, 100 nm.
Fig. 4. Binding of VLPs to synthetic oligosaccharides containing human HBGA epitopes (A) and saliva-VLPs blocking assay and Western blot using mouse sera immunized with full-length capsid protein, N26 and N38 assembled VLPs (B). (A) Blood type A antigen (trisaccharide), (B) blood type B antigen (Trisaccharide). Anti-sera were produced by immunizing mice with indicated VLPs at 10 g/dose for two immunizations at an interval of three weeks in the presence of Al(OH)3 .
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exhibited similar blocking efficiencies for sera from mouse immunized with the same amounts of VLPs made of full-length VP1 or the N26 and N38 deletion mutants.
VLPs to 200 g/ml, which is also optimal concentration for negative staining. Another advantage of the N26 and N38 deletion mutants formed VLPs as vaccine is its convenience for quality control and quantitation (enzyme linked immunosorbant assay) during production process.
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4. Discussion It has been known for some time that expression of NoV capsid proteins in sf9 cells, as well as other eukaryotic cells, leads to formation of VLPs of two sizes. In our study, we find culture conditions can significantly influence the ratio of smaller and larger VLPs formed. Oxygen, pH, capsid protein expression level and cell density might be factors affecting the final yields of certain size of VLPs. Oxygen partial pressure can affect cell proliferation and virus replication which might have certain effects over the production of VLPs. It has been shown that in vitro capsid-capsid protein interaction facilitating VLPs formation is pH dependent and the precise control of pH, as well as other factors in aerobic culture using bioreactor might provide insight into the in vivo assembly mechanism of VLPs [18]. The smaller particle has a diameter of 21 nm in this study while it is reported to be 23 nm by others [15]. This difference might be caused by different major capsid proteins (from GII.4 in this study) used and varied cleavage sites on major capsid proteins from different genogroups as 44 aa was cleaved from the N-terminus of the major capsid protein of a GI.2 norovirus (Genbank accession number, KF306212, data not shown). The dominating or completely smaller VLPs formed by N26 and N38 deletion mutants in our study indicate that the N terminal of NoV capsid protein contain key element(s) determining the sizes of VLPs assembled. Besides, Based on our results, it is presumed that assembling of 38 nm VLPs requires the presence of full-length or close to full-length capsid proteins and further degradation or cleavage does not affect the particle sizes [19]. Further evidence in support of our hypothesis is based on the finding that VLPs purified from monolayer culture lead to different results from batch to batch relating to the ratio of full-length capsid protein and degraded or cleaved capsid protein even larger VLPs are dominating in all the preparations (data not shown). X-ray crystal structure analysis showed that the N-terminus of capsid protein is buried inside the particle and contact with proteases is avoided [20,21]. VLPs in cells cultured in the presence of proteinase inhibitor cocktail also showed doublet bands, indicating cleavage of capsid protein before release of VLPs into culture medium (data not shown). It has been shown that HBGA-VLPs binding are genotype specific and dependent of structures in the loop regions that significantly alter the surface topography and electrostatic landscape of the P domain [22]. Results from HBGA-VLPs binding assay indicate conservation of structural integrity of receptor binding regions of VLPs formed by N26 and N38 deletion mutants. Similar blocking efficiencies using sera from mice immunized with the same amounts of VLPs made of full-length VP1 or the N26 and N38 deletion mutants indicate similar immunogenicity of the three types of VLPs. Based on above results, the N26 and N38 deletion mutants formed VLPs might be excellent candidates for vaccine production. Besides, Stability tests indicate that 21 nm VLPs formed by N26 and N38 deletion mutants are stable for at least one months at 4 ◦ C in PBS (0.01 M, pH 7.2), which is comparable to that of 38 nm VLPs, as no significant difference in particle intactness was observed. It has been reported that N-terminal undergoes cleavage more readily in 23-nm recombinant Norwalk virus VLPs and this is true for VLPs formed by N38 deletion mutant as a 45 kDa product was observed after storage at 4 ◦ C in PBS for two weeks (data not shown). VLPs in higher concentrations tend to aggregate which is consistent with other report [23]. This phenomenon can be delayed by diluting
Acknowledgement I would like to thank Zhang Pei from The Core Facility and Technical Support, Wuhan Institute of Virology, for her help with producing EM micrographs.
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