Molecular characterization of a major outer capsid protein encoded by the Threadfin aquareovirus (TFV) gene segment 10 (S10)
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Arch Virol (2005) 150: 2021–2036 DOI 10.1007/s00705-005-0550-9 Molecular characterization of a major outer capsid protein encoded by the Threadfin aquareovirus (TFV) gene segment 10 (S10) E. K. Seng1 , Q. Fang2 , Y. M. Sin3 , and T. J. Lam1 1 Department of Biological Sciences, National University of Singapore, Singapore 2Wuhan Institute of Virology, CAS, Wuhan, P.R. China 3 Singapore Fish Breeding and Immunization Center, Teo Way Yong & Sons, Pte. Ltd., Singapore Received December 27, 2004; accepted April 4, 2005 Published online June 3, 2005 c Springer-Verlag 2005 Summary. Genome segment 10 (S10) of Threadfin aquareovirus (TFV) was cloned, sequenced, analyzed and found to be 987 bp long encoding a protein of 298 aa with a predicted molecular mass of 32.0 kDa. The TFV S10 gene possesses terminal motifs, (5 GTTTTA and ATTCATC 3 ) which are also conserved in the S6 and S11 TFV gene segments. Sequence comparison revealed that the TFV S10 gene was similar to the Striped bass reovirus (SBR) VP7 outer capsid protein (OCP). A conserved putative zinc-finger motif, CCHC, present in the mammalian reovirus (MRV) δ3 protein, was identified in TFV and other aquareovirus VP7 pro- tein. Phylogenetic analysis of the TFV VP7 protein indicated that TFV is closely related to SBR and Chum salmon reovirus (CSV) and possibly belong to the same species Aquareovirus A as SBR and CSV. The TFV VP7 protein was expressed in E. coli, purified and injected into mice. Serum specific antibodies were generated, however, the serum showed weak neutralizing activity. In contrast, co-incubation of this serum with another serum obtained from mice immunized with another OCP encoded by the TFV S6 gene segment resulted in a highly elevated antibody neutralization titer. Introduction Members of the genus Aquareovirus, a relatively new genus included in the family Reoviridae, contain viruses that infect fish and shellfish [44] and are among The nucleotide sequence data reported in this paper have been deposited to the GenBank database under accession no. AY236219.
2022 E. K. Seng et al. one of the more frequently isolated fish viruses [19]. Like all the members of the family Reoviridae, the genome of aquareovirus is composed of segmented double-stranded RNA (dsRNA). The fish aquareovirus genome has 11 seg- ments of dsRNA (S1–S11), similar to isolates of the genus Rotavirus, family Reoviridae [24]. Using cryoelectron microscopy, Shaw et al. [39] revealed that the aquareovirus genome is contained in a core surrounded by a double-layered icosahedral capsid (coat protein) that physically resembles the capsids of mammalian orthoreovirus (MRV), family Reoviridae. In the past, molecular characterization of aquareoviruses was achieved by comparing the relative mo- bilities of the dsRNA segments of different isolates, reciprocal RNA–RNA hy- bridization and serological tests [5, 6, 17, 18, 34, 41]. These studies classified aquareovirus isolates into six genogroups, Genogroups A–F. Recently, the In- ternational Committee on Taxanomy of Viruses (ICTV) revised the old classifi- cation of aquareovirus isolates from Genogroups A–F to species: Aquareovirus A to F (ARV A–F), and this includes various tentative species [25]. The type species is represented by Striped bass reovirus (SBR), species ARV-A. Other members of the species ARV-A, are Chum salmon reovirus (CSV), Angelfish reovirus (AFR) and many others. The species ARV-B is represented by Coho salmon reovirus (CSR) and six other isolates. Species ARV-C has one member the Golden shiner virus (GSV). Members of the species Aquareovirus D, is represented by Channel catfish reovirus (CRV) while the species Aquareovirus E contains the Turbot reovirus (TRV). The last species, Aquareovirus F, has two members, Chum salmon reovirus (PSR) and Coho salmon reovirus (SSR) [25]. Aquareoviruses grow in various fish cell lines and produce a plaque-like cyto- pathic effect (CPE) during the course of infection. Most aquareoviruses have been isolated from seemingly healthy fish but some have been recovered from diseased fish [19]. The typical clinical symptom of aquareovirus infection is hemorrhage in the abdominal area, followed by lethargic behavior, which eventually results in death [10, 38]. Hence, the diseases caused by such aquareovirus isolates are a potential threat to the aquaculture industry. For example, Grass carp reovirus (GCRV) has been reported to cause severe hemorrhages in fingerlings and yearling grass carp, and can cause up to 80% mortality [16]. In 1998, a reo-like virus was isolated from homogenates of brain, eye, liver, kidney and spleen tissues from diseased threadfin fish (Eleutheronema tetradactylum) collected from a fish farm in Singapore [4]. The virus was propa- gated on bluegill fry fish cell line (BF-2), then further characterized and found to belong to the genus Aquareovirus and was designated as Threadfin aquareovirus (TFV) [38]. Experiments revealed that TFV is able to infect threadfin fingerlings and cause 100% mortality and is able to infect sea bass fingerlings causing 60% mortality [38]. Therefore, physical, biochemical and molecular character- ization of each new aquareovirus isolate is vital in order to quickly identify the virus so that countermeasures can be taken to prevent the spread of the dis- ease. As of today, no commercial vaccine or drug against aquareovirus has been reported.
Characterization of TFV VP7 capsid protein and neutralization studies 2023 In lieu of the lack of such knowledge on fish Aquareovirus, we have cloned, characterized, analyzed and investigated the antigenicity of the TFV outer coat protein(s) which maybe suitable candidates for vaccine development. Materials and methods Viruses and cells TFV was propagated in BF-2 fish cell line as previously described [38]. Guppy reovirus (GPV) and Grass carp reovirus (GCRV) was propagated and purified as previously described [36]. TFV infectivity was assayed using 96-well microtiter plates (IWAKI) of BF-2 cells. Virus titer was determined using the 50% tissue culture infective dose (TCID50 ml−1 ) assay [31] with endpoints calculated by the method of Reed and Muench [33]. Virus purification and extraction of genomic dsRNA TFV particles were obtained from the supernatant of virus infected BF-2 cells and purified by sucrose gradient centrifugation using the method of Seng et al. [38]. Viral double stranded RNA (dsRNA) was then extracted from TFV particles as previously described [36] and stored at −80◦ C until use. Cloning and sequencing of TFV genome segments Synthesis of cDNA from TFV dsRNA was carried out according to the method of Lambden and Clarke [14] with slight modifications. A detailed protocol is described by Seng et al. [37]. After screening the cDNA generated by EcoRI endonuclease digestion and sequencing the inserts, several clones containing the complete full-length sequence (987 bp) of the TFV S10 gene was identified. The 987 bp TFV S10 gene segment sequences was then submitted to GenBank and was assigned an accession no. AY236219. The complete full-length sequence of another TFV S6 gene (2056 bp) was also submitted to GenBank and was assigned an accession no. AY235428. Analysis of nucleotide and deduced amino acid sequences The NCBI BLAST N program was performed to seek for sequences similar to the TFV sequences. Further, sequence analysis was carried out using two computer programs, DNASTAR (Lasergene, Inc.) and DNASIS ver.2.5 (Hitachi Software Engineering Company, Ltd). Multiple sequence alignment of the TFV S10 gene (AY236219) with other S10 gene sequences from various isolates of aquareoviruses: CSV, GSV, GCRV, SBR (GenBank ac- cession nos. AF418303, AF403407, AF236688, U83396) and Coho salmon aquareovirus (CSR) (GenBank accession no. U90430), and the S4 gene sequence of mammalian orthoreovirus serotype 3 (MRV) (GenBank accession no. NC004276) was performed using CLUSTAL W [42]. Hydropathy plots of the deduced amino acid of the aquareovirus genome segment 10 and MRV S4 genome segment were carried out according to the method of Kyte and Doolittle [12] using the DNASTAR program. Phylogenetic analysis was performed using the maximum parsimony method as implemented in the phylogeny inference package, PHYLIP [8]. The phylogenetic tree was constructed using Treeview 1 [29]. Bootstrap analysis was performed using 1000 data re-samplings. Subcloning of TFV S10 genome segment into expression plasmid The ORF of the S10 genome segment of TFV was amplified from purified plasmid harboring the complete TFV S10 nucleotide sequence using appropriate linker primers and Expand
2024 E. K. Seng et al. High Fidelity Taq DNA polymerase (Roche). The linker primer sequences are: 5 GAC GACGACAAGATGGAGACCAAACCAATTC 3 , upstream (sense) primer, and 5 GAGGA GAAGCCCGGTCACGGCAATGGGTTGGGC AG 3 downstream (antisense) primer. PCR was carried out as follows: one cycle of denaturation (94 ◦ C, 2 min) followed by 30 cycles (94 ◦ C, 30 s); (55 ◦ C, 30 s); (72 ◦ C, 1 min) and one cycle (72 ◦ C, 5 min). Amplified DNA was electrophoresed on agarose gel, correct band was excised and purified using GFX PCR DNA & Gel Band Purification Kit (Amersham), and cloned into the pET-30 Ek/LIC plasmid vector (Novagen) and transformed into NovaBlue E. coli (Novagen). Transformed E. coli harboring recombinant plasmids with correct insert was screened by restriction enzyme digestion. Three recombinant plasmids were amplified, purified and the corresponding insert sequences were checked by DNA sequencing. A recombinant plasmid with the correct in frame insert was then transformed into the expression host, E. coli BL-21 (DE3) (Novagen) and transformants were selected by colony PCR. Expression and identification of recombinant TFV VP7 protein Transformed E. coli BL-21 (DE3) containing the insert was grown LB-Kan (50 µg ml−1 ) and protein expression was induced by the addition of IPTG (Promega) to a final concentration of 1 mM. Protein expression was checked using SDS-PAGE gel electrophoresis using 12% acrylamide gel [13] and protein bands were stained using 1% (w/v) Coomassie brilliant blue R-250 and destained using a 40% methanol, 7% acetic acid solution. Solubility of the recombinant protein was determined as detailed in the QIA expressionistTM handbook (Qiagen). Three hours after induction, induced bacterial cells were pelleted by centrifugation at 7000 × g for 20 min, 4 ◦ C and kept at −80 ◦ C until further purification. The TFV recombinant outer capsid protein (rVP7) was purified under denaturing conditions using Ni-NTA beads (Qiagen) followed by serial dialysis in decreasing urea concentrations of 6 M, 4 M, 2 M, 1 M and 0.5 M Urea containing 500 mM NaCl, 20% glycerol and 20 mM Tris-HCL, pH 7.4 to allow proteins to refold. A final dialysis step in 1X PBS was performed to remove remaining traces of urea and the refolded protein was concentrated by dialysis using PEG-30 000 (Sigma). Protein concentration was measured using the BioRad Protein Assay Kit (BioRad). rVP7 was identified by Western blot analysis [43] using a mouse anti-histidine serum (Roche) and an alkaline phosphatase labeled goat anti-mouse IgG (H + L) conjugate (Sigma), with NBT/BCIP (Boehringer, Manheim, GmbH, Germany) as the substrate. In another blot, the primary antibody, mouse anti-rVP7 serum (prepared in this study) was used. Generation of anti-VP7 polyclonal antiserum in mice Nine female adult Swiss Albino mice (20–25 g) were purchased from the Animal Holding Unit (AHU), National University of Singapore. Purified rVP7 (50 µg) was mixed with Freund’s complete adjuvant (FCA) in a 1:1 (v/v) ratio to form a 0.5 ml emulsion. Three mice were each injected intraperitoneally (i.p.) with this protein-adjuvant mixture. The other three mice received only FCA and the remaining three mice did not receive any injection (act as controls). Two weeks later, a booster injection which consist a mixture of purified rVP7 (50 µg) and Freund’s incomplete adjuvant in a 1:1 ratio was similarly administered. A third injection comprising of only purified rVP7 (50 µg) in PBS was administered two weeks after the second injection. The mice were then bled seven days after the last injection. Blood was left to clot overnight at 4 ◦ C and serum was collected after centrifugation at 1000 × g for 10 min, and inactivated at 56 ◦ C for 30 min, and stored at −20 ◦ C until further use. Viral neutralization test Serum neutralization titer was determined using a modified method of Payment and Trudel [31], which assess the neutralization of infectivity i.e., inhibition of cytopathic effect (CPE)
Characterization of TFV VP7 capsid protein and neutralization studies 2025 by serum. Serial two-fold diluted samples of mice anti-rVP7, mice anti-rVP6 serum (obtained from mice immunized with protein encoded by the TFV S6 gene segment) [37] were incubated with an equal volume of TFV (103 TCID50 ml−1 ) at 25 ◦ C followed by overnight incubation at 4 ◦ C. This antiserum: virus mixture was then inoculated onto BF-2 cells grown in 96- well microplates and incubated at 25 ◦ C and examined daily for CPE. In all the experiments, positive control: 103 TCID50 ml−1 of TFV, and negative control: serum from control mice was included. Neutralizing antibody (NeuAb) titers were expressed as the reciprocal value of the highest serum dilution showing a 50% reduction of CPE. Results and discussion TFV S10 gene sequence analysis The complete nucleotide sequence and deduced amino acid sequence of the S10 genome segment of TFV is shown in Fig. 1. The nucleotide sequence was derived from three independent recombinant plasmids and represents the consensus se- quence. The S10 gene of TFV is 987 bp long with an open reading frame (ORF) encoding a protein of 298 amino acids in length. The first initiation codon, ATG Fig. 1. Complete nucleotide sequence (presented in cDNA form) and deduced amino acid sequence of the S10 RNA segment of Threadfin aquareovirus (TFV). The conserved 5 and 3 -terminal nucleotide sequences are boxed and the inverted repeats are underlined. In the amino acid sequence, the putative zinc-finger motifs, CCHC (aa 50–71) is in italics with a grey background. Nucleotide and amino acid positions are numbered on the left and right
2026 E. K. Seng et al. (aa 28–30), is consistent with the optimal consensus initiation sequence (A/G)NNATG identified by Kozak [11]. The first stop codon, TAG, is located at position 922–924 leaving 63 untranslated nucleotides (UTR) at the 3 end. The calculated molecular mass of the deduced protein is 32.0 kDa and is in close agreement to the molecular mass of a protein (∼33 kDa) that we previously described in the TFV virion [38]. The terminal ends of the TFV S10 gene seg- ment display the nucleotide sequences, 5 GTTTTA and ATTCATC 3 , motifs which are also conserved in the TFV genome segment 6 (GenBank accession no. AY235428) and segment 11 (GenBank accession no. AF524892), both cloned and sequenced by our group. Further analysis revealed that the 3 -end terminal sequence, TTCATC, is conserved in other aquareovirus S10 genome segments such as in SBR, CSV, GCRV and GSV, and is also identical to those of the mammalian reovirus serotype 3 (MRV), genus Orthoreovirus, another member of the family Reoviridae. Adjacent to the 5 end terminal of the S10 TFV gene, nucleotides CACGCC at position 12–17, was found to be complementary to its 3 end inverted repeat sequence, GGCGTG at position 971–976 (Fig. 1). The presence of conserved terminal sequences and inverted repeats adjacent to the end terminals have been well established to be a common feature of the genome segments of members of the family Reoviridae [24, 25]. BLAST search [1] of the TFV S10 gene sequence showed a high sequence similarity to the S10 genome segment sequence of Striped bass reovirus (SBR) and Chum salmon reovirus (CSV), which codes for the major outer capsid protein (VP7) of the virion [2, 20, 23, 27, 40]. Hence, we deduced that the S10 gene of TFV most likely represents the major outer capsid protein, VP7 of the TFV virion. Sequence comparison between TFV, SBR and CSV S10 genome segment sequences revealed that these three isolates exhibited comparable properties in Table 1. Nucleotide and amino acid identities of the gene segment 10 of TFV, SBR, CSV, CSR, GCRV, GSV and gene segment S4 of mammalian orthoreovirus, MRV Identity (%) Virus isolate TFV SBR CSV CSR GCRV GSV MRV TFV 75.7 64.6 36.5 23.3 24.5 21.0 SBR 86.3 68.8 36.1 22.7 23.4 20.5 CSV 78.9 80.6 34.5 22.3 24.3 21.0 CSR 32.7 32.3 30.3 24.0 23.1 20.3 GCRV 13.7 13.4 12.6 15.5 90.6 21.7 GSV 13.4 13.4 13.4 15.2 95.7 21.0 MRV 11.7 10.4 11.0 13.9 12.6 12.6 Deduced nucleotide sequence identities are in the upper right portion. Deduced amino acid sequence identities are in the lower left portion. Identities are in percentage (%). Percentage identity values were determined using the CLUSTAL software in DNASTAR (Lasergene Inc.).Abbreviations: TFV, Threadfin reovirus; SBR, Striped bass reovirus; CSV, Chum salmon reovirus; CSR, Coho salmon reovirus; GCRV, Grass carp hemorrhage reovirus; GSV, Golden shiner reovirus, MRV, mammalian orthoreovirus serotype 3
Characterization of TFV VP7 capsid protein and neutralization studies 2027 terms of total length of the gene segment and the protein products it encodes for, a protein of ∼32 kDa in molecular mass. Comparison of the nucleotide sequence and predicted amino acid sequence of the TFV S10 with other reported isolates of aquareovirus and the mammalian reovirus (MRV) δ3 protein revealed that the TFV VP7 protein showed high amino acid sequence identity to VP7 protein of SBR (86.3%), CSV (78.9%), moderate identity to the CSR VP7 protein (32.8%), relatively low identity to GCRV, GSV VP7 protein, and MRV δ3 protein (encoded by the MRV S4 gene segment) with values of only 13.7%, 13.4%, and 11.7% respectively (Table 1). Although the deduced protein sequence from CSR, GCRV and GSV S10 gene and MRV S4, showed moderate to low homology to the deduced protein sequence of TFV S10 (Table 1; Fig. 2), hydrophathy plots (data not presented) calculated using the method of Kyte and Doolittle [12], clearly showed that the predicted proteins encoded by all the Aquareovirus isolates, TFV, SBR, CSV, GCRV, and GSV, as well as the δ3 protein of mammalian reovirus (MRV) showed very similar profiles especially in the N-terminal part of the protein, with four domains in the order hydrophilic-hydrophobic-hydrophilic- hydrophobic (data not presented). Hence, it is highly suggestive that the VP7 protein from these aquareoviruses maybe analogues to the δ3 protein of MRV. This observation is in agreement to Attoui et al.’s [2] finding which showed that the SBR and CSV gene segment 10 (S10), encoding the VP7 protein is most likely the equivalent to the MRV δ3 protein encoded by the S4 gene segment of MRV. Additional confirmation of the relatedness of the protein encoded by TFV S10 genome segment to the MRV δ3 protein was gained from the discovery of a zinc-finger motif found in both proteins (Fig. 2). Analysis of the deduced amino acid sequence of TFV segment 10 revealed the presence of a CX2 CX15 HX1 C motif (Fig. 1, shaded; Fig. 2) within the N-terminal, residues 50–71, analogous to the zinc-finger motif (CCHC), CX2 CX15 HHX1 C (residue 51–73) identified by Mabrouk and Lemay [22] in the mammalian reovirus (MRV) δ3 protein. The CX2 CX15 HX1 C motif identified in TFV was also found at the same location in the deduced SBR and CSV VP7 protein with the exception that in the CSV VP7 protein, the supposed cysteine (C) residue at position 53 was substituted for a tyrosine (Y) instead (Fig. 2). This discrepancy could be unchecked errors encountered during the sequencing of CSV. Further analysis revealed that the deduced VP7 protein encoded by the GCRV, GSV and CSR S10 gene also posses a putative zinc-finger motif, CX2 CX16 HX1 C. By analogy, the CCHC zinc-finger motif, found in all the aquareovirus isolates as well as in the MRV protein, is similar to that reported in retroviral nucleocapsid proteins, which proposed function was to direct the specific packaging of retroviral RNA [7, 9]. Recently, modifications of zinc-binding residues inside the conserved CCHC motif of human immunodeficiency virus Type 1 NCp7 into CCHH, induced a complete loss of infectivity [32]. Studies conducted by Mabrouk and Lemay [22] indicated that the zinc-finger motif in MRV δ3 protein is important in conferring stability to the protein. So far, the exact function(s) of the CX2 CX15−16 HX1 C motif found in all the aquareovirus isolates is unknown and its significance in viral replication or pathogenesis remains to be elucidated.
Fig. 2. Multiple alignment of deduced amino acid sequence of protein encoded by S10 RNA segment of Threadfin aquareovirus (TFV) with deduced proteins from other aquareovirus S10 RNA segment; Striped bass reovirus (SBR), Chum salmon reovirus (CSV), Coho salmon reovirus (CSR), Golden shiner reovirus (GSV), grass carp reovirus (GCRV) and the δ3 protein from mammalian reovirus Dearing strain (serotype 3) (MRV). See Methods section for accession numbers of each isolate. Amino acid positions for each individual sequence are numbered on the right. Identical amino acids are indicated by an asterisk. The putative zinc- finger domain (CCHC form), identified by Mabrouk and Lemay [22] within the mammalian reovirus δ3 protein, is boxed with a continuous line (amino acids 51–73) and the corresponding motif within the Threadfin aquareovirus protein is boxed with a dotted line (amino acids 50–71). Potential N-linked glycosylation sites, Nx1 S/T, where x represent any amino acid residue except proline (P), are underlined and can be found in all aquareovirus isolates except CSR. No glycosylation sites are present in MRV δ3 protein
E. K. Seng et al.: Characterization of TFV VP7 capsid protein 2029 Fig. 3. Phylogenetic tree of the gene segment 10 of TFV, SBR, CSV, CSR, GCRV, GSV and gene segment S4 of mammalian orthoreovirus (MRV). Phylogeny was inferred from the complete amino acid sequences of the indicated viruses using the PHYLIP suite program [8]. Bootstrap confidence levels following 1000 replicates are listed next to each branch node. The scale bar is proportional to genetic distance. ARV – Aquareovirus species The presence of the putative zinc-finger motif in all deduced protein of the S10 segment of all the aquareovirus isolates and MRV δ3 protein illustrates that the VP7 protein coded by the S10 genome segment of aquareoviruses may be functionally and structurally conserved. Hence, we concluded that the TFV S10 gene segment, encodes a viral protein, VP7, which is alike the δ3 protein of MRV that also represent the outermost virus capsid protein. The conservation of the 3 ATTCATC gene sequence between TFV S10 and MRV S4 partly implied that both viruses might have evolved from a common ancestral precursor. A phylogenetic analysis we conducted supported this claim (Fig. 3) as all aquareovirus isolates were segregated into three separate clusters with MRV forming the fourth cluster. The separation of MRV could stem from the fact that the MRV genome consist of only 10 dsRNA genome segments as oppose to 11 in aquareoviruses. Among the aquareoviruses, the TFV VP7 protein is more closely related to SBR and CSV VP7 protein, both belonging to the genogroup A (species, ARV-A). CSR of genogroup B (ARV-B) formed an individual clade while GCRV and GSV, both of genogroup C (ARV-C) formed the other clade. This observation is well received as earlier classification methods of aquareovirus isolates by way of reciprocal RNA–RNA hybridization and nucleotide analysis revealed a similar structure of grouping. Thus, TFV should be placed into genogroup A (species ARV-A) due to its close relationship in terms of the VP7 protein sequence of SBR and CSV. Additional evidence for the placement of TFV into the same species as SBR and CSV was
2030 E. K. Seng et al.: Characterization of TFV VP7 capsid protein gained in our previous results that showed that the protein encoded by the TFV S6 gene segment showed high sequence identity to the VP4 protein encoded by CSV S6 gene segment [37]. Further thorough sequence analysis of the TFV VP7 protein sequence revealed that there are three potential N- linked glycosylation sites located at aa residues 245–247 (NQS), 269–271 (NLS) and 276–278 (NKS) (Fig. 2). In SBR and CSV, similar potential N-linked glycosylation sites were discovered. However, in GCRV and GSV, only one potential N-linked glycosylation (aa 244–246) site was found while none was found in CSR (Fig. 2). Generally, the carbohydrate groups of glycoproteins have been known to facilitate proper protein folding, confer protease resistance, maintain structural integrity and conformational stability and affect surface charges and water binding capacity [30]. In recent years, much interest has cumulated investigations into the importance of oligosaccharide chains of many glycoproteins in biological recog- nition, protein trafficking, host-pathogen, and cell–cell interaction. In Rotavirus, the VP7 protein, an outer capsid protein, analogous to the SBR VP7 and the TFV VP7 protein, has been shown to be glycosylated [26]. The major function of carbohydrates on the rotavirus VP7 is to facilitate correct disulfide bond formation and protein folding [26]. In addition, glycosylation of the rotavirus VP7 has also been shown to affect the antigenic specificity of the protein [3, 15]. As such, we can only speculate that due to the close percentage similarity between the TFV VP7 and SBR VP7 protein, in which the latter has been demonstrated to be glycosylated [35] it is most likely that the TFV VP7 is also glycosylated. However, we have yet to provide evidence for the presence of oligosaccharides in the TFV VP7 protein. It will be interesting to see whether the TFV and SBR VP7 protein show similar functional properties as the rotavirus VP7 protein in future experiments. TFV VP7 protein expression and immunogenicity Thus far, sequence determination and analysis of genome segment 10 (S10) of TFV only revealed that this gene codes for an outer capsid protein of the virus. In order to verify that the S10 gene codes for a protein present on the TFV virion, the ORF of the S10 TFV gene was cloned into the pET-30 expression vector and a double tagged recombinant outer capsid protein, rVP7, harboring histidine (His) and S-Tag sequences was expressed in E. coli. SDS-PAGE analysis revealed that 1 h and 3 h post induction, the rVP7 protein was overexpressed (Fig. 4A). Overexpressed rVP7 protein accumulated in inclusion bodies and the yield of the rVP7 was 40.0 mg L−1 (∼32% of total bacterial cellular protein content). Im- munoblot analysis using monoclonal mouse anti-His antiserum (Fig. 4B) showed that the rVP7 protein expressed has a molecular mass of ∼36 kDa, much larger than the native TFV VP7 protein (∼33 kDa), but is consistent with the expected fusion protein. The higher molecular mass of rVP7 protein was attributed by the extraneous polypeptide contributed by His and S-tags of the vector. To study the authenticity and antigenicity of the rVP7 protein produced in E.coli, antiserum against the rVP7 was raised in mice and its reactivity with the
Fig. 4. A) SDS-PAGE analysis of recombinant TFV VP7 outer capsid protein expressed in E.coli. Lane 1, control (no IPTG); 2, 1 h after IPTG induction; 3, 3 h after IPTG induction; M, molecular weight marker (BioRad). The rVP7 protein is indicated by an arrow. B) Western blot analysis of purified TFV rVP7 with mouse anti-HIS serum. 1, purified TFV rVP7 protein; M, molecular weight marker (BioRad). Arrowhead indicates the position of the TFV rVP7 protein. C) Western blot analysis of purified TFV, GPV, GCRV and E.coli expressed TFV rVP7 with mouse anti-rVP7 serum. Preparations of purified viruses were separated on SDS- PAGE and transferred to a nitrocellulose membrane. The membrane was then treated with mouse anti-rVP7 serum generated as described in this manuscript. 1, purified TFV virus; 2, purified GPV virus; 3, purified GCRV virus; 4, purified and dialyzed rVP7; M, molecular weight marker (BioRad). Arrowhead indicates the position of the VP7 outer capsid protein of TFV virion. Arrow indicates the position of the purified full-length product of the TFV rVP7 protein. Unfilled arrow indicates a probable dimer form of the rVP7 protein
2032 E. K. Seng et al. TFV, Guppy reovirus (GPV) and Grass carp reovirus (GCRV) VP7 protein was assayed by immunoblotting. As shown in Fig. 4C, both the native TFV VP7 and rVP7 protein ( , ; in Fig. 4C, respectively) was detected by the mice antiserum. In addition, a weak band at ∼72 kDa ( , in Fig. 4C) was also detected. This product could possibly be a dimer form of the rVP7 protein. Dimer formation has been observed in the δ3 protein of MRV [28]. The dimer of the MRV δ3 protein was proposed by Olland and colleagues [28] to be the likely protein form that is involved in binding dsRNA. To our knowledge, this is the first report of the possible formation of a ‘δ3-like’ dimer in an aquareovirus protein but its function remains to be investigated. In Fig. 4C, it was observed that the mice anti-rVP7 serum also reacted to the GPV VP7 protein (Lane 2), but did not react with any of the proteins of the GCRV virus. This clearly showed that TFV, a seawater isolate, is antigenically related to GPV, a freshwater isolate but is not related to GCRV, another freshwater isolate. We next investigated the immunogenicity of the mice anti-rVP7 serum that we generated by conducting viral neutralization tests. A low TFV neutralizing antibody (NeuAb) titer: 16, 32, 32 was recorded from serum recovered from the three mice injected with rVP7 as compared to the NeuAb titer in control mice (≤8, ≤8, ≤8). This indicated that TFV was ineffectively neutralized. This result is in agreement with Lupiani et al.’s [21] observation whereby antiserum from rabbit immunized with recombinant SBR VP7 outer capsid protein expressed in a baculovirus system also showed no neutralizing activity. Collectively, these two independent results suggest that the outer capsid protein, VP7, encoded by TFV S10 genome segment or other aquareoviruses may not be the major neutralizing antigen. In this study, we also investigated the neutralizing activity of antiserum ob- tained from mice immunized with another TFV outer capsid protein, VP4, en- coded by the S6 genome segment, which our group recently cloned, sequenced and expressed in E. coli [37]. Molecular analysis revealed that the TFV S6 gene sequence codes for a protein analogous to the VP4 protein of SBR and CSV and the MRV µ1 protein [2, 37]. In this study we found that antiserum recovered from three mice injected with recombinant VP4 protein (rVP4) also showed low neutralizing activity, NeuAb titer: 16, 16, 32. Two possible reasons for the low neutralization activities of the mice antiserum against rVP7 and rVP4 could be due to the loss of important neutralizing epitopes as a result of incorrect protein folding during our protein purification procedure or the ab- sence of glycosylation as we used E. coli derived proteins for the production of antibodies. On the basis of the low neutralizing titer of both mice antiserum, we ra- tionalized that if we combined both the antiserum against rVP7 and rVP4, the neutralization titer of the antibodies should be in the range of 32 to 64. However, when antiserum from mice injected with rVP7 and antiserum from mice injected with rVP4 were mixed in a 1:1 vol:vol ratio, a highly elevated NeuAb titer (128, ≥256; ≥256) was observed. To our knowledge, this is the first report showing that antibodies raised against outer capsid proteins of an aquareovirus isolate did not possess any neutralizing properties when applied individually, but
Characterization of TFV VP7 capsid protein and neutralization studies 2033 exhibited a high NeuAb activity when used in concert. This unique phenomenon of synergism between two antibodies generated from two different viral outer capsid proteins that resulted in neutralization of virus certainly warrants further detailed investigation. A probable mechanism to explain our observations could be indirectly gained from previous studies using monoclonal antibodies (mAb) directed against the mammalian orthoreovirus serotype 3 (MRV) capsid proteins, δ3 and µ1, which showed that some antibodies are non-neutralizing but yet protective, i.e., able to hinder viral replication [45]. Virgin et al. [45] showed that non-neutralizing monoclonal antibody (mAb) directed against the MRV δ3 outer capsid protein inhibits intracellular proteolytic uncoating of the reovirus outer capsid while mAb directed against MRV µ1 outer capsid protein inhibit membrane penetra- tion by infectious subviral particles (ISVP) and as a result block MRV repli- cation. Perhaps, antiserum against the TFV VP7 and VP4 outer coat protein may employ similar mechanisms but must complement each other in order to totally curb TFV infection. Nonetheless, the exact mechanism of how antibody-mediated inhibition of TFV replication remains to be verified in future experiments. In summary, we have cloned, sequence and analyzed the TFV gene segment 10 (S10) which codes for the TFV outer viral coat protein (VP7). Based on amino acid sequence similarities and phylogenetic analysis we demonstrated that TFV likely belongs to the species, ARV-A, of which SBR and CSV are representative members. In addition, we demonstrated that the serum obtained from mice injected with recombinant TFV VP7 protein had a low neutralizing antibody titer. In contrast, by combining the mice anti-VP7 and VP4 serum, a highly elevated neutralizing antibody titer was achieved. A direct implication for this observation is that multiple antibodies directed against several viral coat proteins are required to effectively neutralize the TFV virus. This unique observation, of synergism between two antibodies to neutralize a virus would definitely be interesting to investigate in future experiments. While considerable fundamental and applied research has been carried out on human isolates from the genus Orthoreovirus and Rotavirus, very little progressive research has been done on members of the genus Aquareovirus. As a result, it has lead to the lack of basic fundamental knowledge of aquareovirus genetics, although much can be inferred from works already done on mammalian reoviruses. Hence, our work on cloning and expression of recombinant aquareovirus outer capsid protein will hopefully provide reagents for future works like structural function related studies, production of monoclonal antibodies, determination of epitopes responsible for virus neutralization and possibly explore the mechanisms of antibody mediated virus neutralization. Acknowledgments This work was supported by a research grant (R-154-000-068-112) from the National University of Singapore, Singapore.
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