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Journal of Virology, January 2003, p. 943-952, Vol. 77, No. 2
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.2.943-952.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

The Multimerization of Hantavirus Nucleocapsid Protein Depends on Type-Specific Epitopes

Kumiko Yoshimatsu,1 Byoung-Hee Lee,1 Koichi Araki,2 Masami Morimatsu,3 Michiko Ogino,1 Hideki Ebihara,1 and Jiro Arikawa1*

Institute for Animal Experimentation, Graduate School of Medicine,1 Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-8638,2 Laboratory of Veterinary Physiology, Faculty of Agriculture, Iwate University, Morioka 020-8550, Japan3

Received 28 June 2002/ Accepted 15 October 2002


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ABSTRACT
 
Multimerization of the Hantaan virus nucleocapsid protein (NP) in Hantaan virus-infected Vero E6 cells was observed in a competitive enzyme-linked immunosorbent assay (ELISA). Recombinant and truncated NPs of Hantaan, Seoul, and Dobrava viruses lacking the N-terminal 49 amino acids were also detected as multimers. Although truncated NPs of Hantaan virus lacking the N-terminal 154 amino acids existed as a monomer, those of Seoul and Dobrava formed multimers. The multimerized truncated NP antigens of Seoul and Dobrava viruses could detect serotype-specific antibodies, whereas the monomeric truncated NP antigen of Hantaan virus lacking the N-terminal 154 amino acids could not, suggesting that a hantavirus serotype-specific epitope on the NP results in multimerization. The NP-NP interaction was also detected by using a yeast two-hybrid assay. Two regions, amino acids 100 to 125 (region 1) and amino acids 404 to 429 (region 2), were essential for the NP-NP interaction in yeast. The NP of Seoul virus in which the tryptophan at amino acid number 119 was replaced by alanine (W119A mutation) did not multimerize in the yeast two-hybrid assay, indicating that tryptophan 119 in region 1 is important for the NP-NP interaction in yeast. However, W119A mutants expressed in mammalian cells were detected as the multimer by using competitive ELISA. Similarly, the truncated NP of Seoul virus expressing amino acids 155 to 429 showed a homologous interaction in a competitive ELISA but not in the yeast two-hybrid assay, indicating that the C-terminal region is important for the multimerization detected by competitive ELISA. Combined, the results indicate that several steps and regions are involved in multimerization of hantavirus NP.


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INTRODUCTION
 
Hantaviruses, which are classified in the family Bunyaviridae, genus Hantavirus, are the causative agents of two rodent-borne viral zoonoses: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) (22, 30). Each hantavirus appears to have a single predominant rodent species that serves as its natural reservoir (31), which is thought to coevolve with its host rodent (25). Consequently, hantaviruses form three large groups according to their host rodents: Murinae-associated, Arvicolinae-associated, and Sigmodontinae-associated hantaviruses.

The Murinae-associated hantaviruses include three viruses that cause HFRS: Hantaan virus (HTNV), Seoul virus (SEOV), and Dobrava virus (DOBV). HTNV is carried by Apodemus agrarius and A. peninsulae, DOBV is carried by A. agrarius and A. flavicollis, and SEOV is carried by Rattus norvegicus and R. rattus. Although HTNV, DOBV, and SEOV have similar antigenicities (12), their reservoir rodents differ. Both HTNV and SEOV occur in Asia, due to the spread of their specific host rodents. Therefore, it is important to differentiate HTNV and SEOV infections in order to control the reservoir rodents.

Hantaviruses have a segmented negative-sense RNA genome; S (small), M (medium), and L (large) segments encode the nucleocapsid protein (NP), two envelope glycoproteins (G1 and G2), and viral polymerase, respectively. Generally, focus reduction neutralization tests are needed to serotype HTNV, SEOV, and DOBV infections. To differentiate HTNV and SEOV infections, we tried to establish serotyping antigens for enzyme-linked immunosorbent assay (ELISA) by using recombinant NP. The NP molecules of HTNV, SEOV, and DOBV consist of 429 amino acid (aa) residues. The major linear epitope common to HTNV, SEOV, and DOBV is located in the 100 aa at the N-terminal end of the NP (13, 39, 40). We tried to develop a serotyping antigen by deleting the major linear epitope common to HTNV and SEOV. Truncated NPs lacking 154 aa at the N-terminal end were generated (155-429 antigen) from the entire cDNA for HTNV and SEOV NP and expressed in a baculovirus system. As we expected, the SEOV 155-429 antigen proved to be a useful ELISA antigen for detecting serotype-specific antibody in the sera of HFRS patients. However, HTNV 155-429 antigen showed extremely low antigenicity, especially in ELISAs (21). Next, we generated truncated NP antigens lacking 49 aa of the N-terminal end (50-429 antigen) for HTNV, SEOV, and DOBV. The 50-429 antigens were useful ELISA antigens for serotyping HTNV, SEOV, and DOBV infections (3). In these trials, it was not clear why the HTNV 155-429 antigen lost its antigenicity. We postulated that the HTNV 155-429 antigen had undergone a structural alteration.

Although hantaviruses are classified in the family Bunyaviridae, they are distinct from other members of the Bunyaviridae. Viruses that belong to the genus Hantavirus require a rodent vector, whereas other members of the Bunyaviridae need arthropod vectors (8). The envelope glycoproteins of Black Creek Canal hantavirus are transported to the plasma membrane, where the virus is assembled (26), whereas the envelope glycoproteins of other members of the Bunyaviridae are retained in the Golgi complex and the virus particles are assembled at the Golgi membrane (reviewed in reference 29). Therefore, it has been suggested that hantaviruses have a different mode of replication and assembly from other members of the family Bunyaviridae.

In general, NP is an important viral component in the assembly of viral particles, and it forms the viral nucleocapsid after binding with the viral genome. This step is thought to involve the multimerization of NP with other NP molecules. In viruses in the family Bunyaviridae, it is thought that the nucleocapsid interacts with transmembrane regions of the viral glycoprotein and that both are necessary for the budding process (7).

Recently, several studies for multimerization of Arvicolinae-associated (Tula and Puumala virus) (15) and Sigmodontinae-associated (Sin Nombre virus; SNV) hantaviruses (1) were reported. Alfadhli et al. suggested that Prospect Hill virus (PHV) NP forms a trimer and that the N-terminal region contributed to NP-NP interaction via a coiled-coil structure (2). Kaukinen et al. showed that divalent cations enhanced NP-NP interaction (15).

In the present study, we tried to clarify the relationship between the multimerization and the antigenicity of Murinae-associated hantavirus NP and to determine the regions responsible for multimerization by using competitive-capture ELISA and a yeast two-hybrid assay. These findings should be directly applicable to the design and production of diagnostic antigens. In addition, the results will also further our understanding of the NP-NP interaction involved in hantavirus assembly.


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MATERIALS AND METHODS
 
Viruses and cells. HTNV strain 76-118 (16) was propagated in the Vero cell E6 clone (ATCC c1008 and CRL 1586) grown in Eagle minimal essential medium (Nissui, Tokyo, Japan) supplemented with 5% fetal bovine serum (FBS). Recombinant baculoviruses (Autographa californica nuclear polyhedrosis virus) containing coding information for the NP of HTNV or SEOV and the HTNV envelope glycoprotein were supplied by C. S. Schmaljohn of USAMRIID, Frederick, Md. (4, 32). The recombinant baculoviruses for expressing various truncated nucleocapsid proteins were described previously (21). They were propagated in High Five cells grown in Grace's insect cell culture medium (Grace's medium; Gibco-BRL) supplemented with 10% FBS. 293T cells grown in Dulbecco modified Eagle medium (Nissui) supplemented with 10% FBS were used to prepare the recombinant proteins.

Preparation of ELISA antigens. Monolayers of High Five cells cultured in 75-cm2 flasks were inoculated with 1 ml of recombinant baculovirus culture fluid (2.2 x 108 focus-forming units/ml). Six days later, the cells were pelleted by low-speed centrifugation (1,400 x g for 5 min). The cells were washed with Dulbecco phosphate-buffered saline (PBS) and resuspended in lysis buffer (0.01 M Tris-HCl-2% Triton X-100-5 mM EDTA-0.15 M NaCl-0.6 M KCl (pH 7.8) (18) to 107 cells/ml and stored at -80°C. To prepare authentic antigen, Vero E6 cells were inoculated with Hantaan virus 76-118, clone-1 (11, 36) at a multiplicity of infection of 0.01. At 7 days after inoculation, the cells were dispersed by digestion with 0.1% trypsin (Difco, 1:250), washed with PBS, and resuspended in lysis buffer to 107 cells/ml. To prepare recombinant antigen expressed in mammalian cells, 293T cells transfected with various expression vector plasmids were collected 3 days after transfection, washed with PBS, and resuspended in lysis buffer to 107 cells/ml.

MAbs and their purification and biotinylation. Monoclonal antibody (MAb) clone ECO2 directed at the SEOV NP was supplied by C. J. Peters of the Centers for Disease Control and Prevention, Atlanta, Ga. (28). Clones E5/G6 and C24B4, directed at the HTNV NP, were prepared as described previously (40). MAb clones HCO2 and 11E10-2-2 directed at the HTNV envelope glycoprotein G2 were prepared as described previously (5). Each MAb was purified from mouse ascitic fluid by using a Bio-Rad MAPS II kit based on the protein A column system (Bio-Rad, Hercules, Calif.). The purified MAbs were biotinylated by using N-succinyl-A5-biotin (Dojindo, Osaka, Japan) according to the manufacturer's directions.

ELISA. Microtiter plates were coated with purified MAb (2 µg/ml in PBS) overnight at 4°C. After removal of the antibody, the plate was washed three times with PBS containing 0.05% Tween 20 (PBST). The unsaturated protein-binding sites were then blocked with BlockAce (Yukijirushi, Tokyo, Japan) for 30 min at room temperature. After a washing step as described above, the cell lysate diluted with ELISA buffer (PBS containing 0.5% bovine serum albumin and 0.05% Tween 20) was incubated for 1 h at room temperature (50 µl/well) and washed with PBST three times. The biotinylated MAbs diluted with ELISA buffer were added and incubated for 1 h at room temperature (50 µl/well). After a washing step as described above, peroxidase-labeled avidin (Zymed, South San Francisco, Calif.) diluted 1:5,000 was added. After incubation for 30 min at room temperature, the plate was washed as described above. Then, an o-phenylenediamine dihydrochloride tablet (Sigma-Aldrich, Co., St. Louis, Mo.) was added as the peroxidase substrate for a colorimetric reaction, and the absorbance at 450 nm was measured by using a SpectraMax 340 (Molecular Device, Co., Sunnyvale, Calif.)

Plasmid. Yeast-Escherichia coli shuttle plasmids containing the GAL4 DNA-binding domain (pGBT9) and GAL4 activation domain (pGAD424) were from Clontech, Palo Alto, Calif. Plasmids pGBT9/HTNV 1-429 (entire NP), pGBT9/HTNV 1-355, pGBT9/HTNV 155-429, pGBT9/HTNV 320-429, pGBT9/HTNV 50-429, pGAD424/HTNV 1-429, pGAD424/HTNV 1-355, pGAD424/HTNV 155-429, pGAD424/HTNV 320-429, and pGAD424/HTNV 50-429 were generated by fusing the HTNV NP gene (strain 76118; M14626) in frame to the GAL4 binding or activation domain. Plasmid pGAD424/HTNV 1-403 was generated from pGAD424/HTNV 1-429 by using an internal BamHI restriction enzyme site. Similarly, plasmids pGBT9/SEOV 1-429 (entire NP), pGBT9/SEOV 50-429, pGBT9/SEOV 100-429, pGBT9/SEOV 125-429, pGBT9/SEOV 155-429, pGBT9/SEOV 1-420, pGAD424/SEOV 1-429, pGAD424/SEOV 50-429, pGAD424/SEOV 100-429, pGAD424/SEOV 125-429, pGAD424/SEOV 155-429, and pGAD424/SEOV 1-420 were generated from the SEOV NP gene (strain SR-11; M34881) fused in frame to the GAL4 binding or activation domain. Plasmid pGAD424/HTNV 100-412 was generated from pGAD424/SEOV 100-429 by using an internal BglII restriction enzyme site. The mammalian expression vector pCAGGS/MCS (23) was used to express rNP in 293T cells. The complete SEOV and HTNV NP genes were cloned into plasmid pCAGGS/MCS. To produce mutant NP in which the tryptophan residue at 119 was replaced with alanine (W119A mutation), site-directed mutagenesis was induced by using the GeneEditor in vitro site-directed mutagenesis system (Promega, Madison, Wis.). All of the plasmid constructs were sequenced to confirm the mutation or junction between the inserts and vectors.

Yeast two-hybrid assay. To detect the homotypic interaction of HTNV and SEOV NP, the MATCHMAKER two-hybrid system (PT1265-1; Clontech) was used according to the manufacturer's protocol. Briefly, DNA encoding various lengths of the HTNV and SEOV NP was cloned into the yeast GAL4 DNA-binding domain vector pGBT9 and the GAL activation domain vector pGAD424. The yeast (Saccharomyces cerevisiae) strain HF7c was grown in yeast extract-peptone-dextrose medium. Yeast was cotransformed with the GAL4 DNA-binding-domain plasmids and GAL4 activation plasmids by the lithium acetate method and selected for leucine and tryptophan prototrophy. The protein interactions were monitored as ß-galactosidase activity, which was assayed on Hybond N membrane (Amersham Biosciences Corp., Piscataway, N.J.) replicates of yeast transformants. Each membrane was placed in liquid nitrogen for 30 s and incubated for 5 h in buffer containing 4 mM X-Gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside). Blue colonies indicated positive interactions. For the quantitative ß-galactosidase activity assay, the yeast ß-galactosidase assay kit (75768; Pierce, Rockford, Ill.) was used according to the manufacturer's directions.


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RESULTS
 
Detection of multimerization of authentic or recombinant HTNV NP in cell lysate. Hantaan virus NP and envelope glycoproteins produced in Vero E6 cells by actual infection were detected by using competitive ELISA. Figure 1A shows the detection of HTNV envelope glycoprotein. HTNV- or mock-infected Vero E6 cell lysate was treated with MAb 11E10-2-2. The envelope proteins captured with 11E10-2-2 were then detected by using the biotinylated MAbs HCO2 or 11E10-2-2. HTNV-infected cell lysate reacted with biotinylated HCO2 but not with 11E10-2-2. The biotinylated 11E10-2-2 MAb was believed to show competitive capture with MAb 11E10-2-2. The control cell lysate did not show a reaction with either biotinylated HCO2 or 11E10-2-2. Envelope glycoprotein G2 was detected as a monomer under the ELISA conditions. Figure 1B shows the detection of authentic NP expressed in HTNV-infected Vero E6 cells and recombinant HTNV NP expressed in insect High Five cells. As control antigens, mock-infected Vero E6 cells and recombinant envelope glycoprotein expressed in High Five cells were used. The antigens were captured with ECO2 and detected with biotinylated MAb E5/G6. Figure 1C shows the detection of multimerized NP in cell lysate by competitive ELISA with the same antigens as in Fig. 1B and detected with biotinylated MAb ECO2. The authentic and recombinant NP antigens were also captured and detected with MAb E5/G6 (data not shown). This assay detected authentic and recombinant NP antigens as a multimer. These results show that the competitive ELISA system with capture/detector antibodies was useful for differentiating monomeric antigen from multimerized antigen. In addition, the recombinant NP expressed in insect cells by the baculovirus vector also formed a multimer, like the authentic antigen.



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  		FIG. 1. Detection of the multimerization of authentic and recombinant NP of HTNV in Vero E6 cells and High Five cells. (A) Detection of HTNV envelope glycoprotein G2. Envelope protein G2 captured with 11E10-2-2 was detected with biotinylated MAbs HCO2 or 11E10-2-2. HTNV-infected cell lysate was detected with biotinylated HCO2 (Vero/HTNV/HCO2b [•]) but not with 11E10-2-2 (Vero/HTNV/11E10b [{blacksquare}]). The control cell lysate did not react with either biotinylated HCO2 (Vero/HCO2b [{circ}]) or 11E10-2-2 (Vero/11E10b [{square}]). (B) Detection of authentic NP expressed in VeroE6 cells with HTNV infection (Vero/HTNV [•]) and recombinant HTNV NP expressed in insect High Five cells (rNP/High Five [{blacksquare}]). The control antigens were uninfected Vero E6 cells (Vero [{circ}]) and recombinant envelope glycoprotein expressed in High Five cells (rEnv/High Five [{square}]). Antigens were captured with ECO2 and detected with biotinylated MAb E5/G6. (C) Detection of multimerized NPs in authentic and recombinant HTNV NP. The symbols are the as same as in (B). Antigens were captured and detected with MAb E5/G6. Authentic and recombinant NP antigens were detected as multimers.

Comparison of the reactivity of recombinant nucleocapsid protein with rabbit immune sera and multimerization antigens. The reactivity of rabbit immune sera directed to HTNV, SEOV, and DOBV against various recombinant and truncated antigens is summarized in Table 1. The complete NP antigens, consisting of aa 1 to 429 of HTNV, SEOV, and DOBV, showed strong cross-reactivity to heterologous antisera. The SEOV 155-429 and DOBV 155-429 antigens, which lacked major linear epitopes (aa 1 to 100), showed only a type-specific reaction. However, HTNV 155-429 antigen lost its type-specific reactivity to HTNV-infected rabbit sera. In addition, the truncated NPs of the 50-429 antigens of HTNV, SEOV, and DOBV possessed type-specific reactivities. In Fig. 2, multimerization of these antigens was tested by using competitive ELISA. Figure 2D shows the schema for competitive ELISA and illustrates the binding regions of the capture/detector antibodies. Figure 2A and B shows the antigens produced by each recombinant. In Fig. 2A (upper panel) recombinant HTNV NP antigens 1-429, 1-335, and 1-244 showed sufficient antigenicity. In Fig. 2C (upper panel) the multimerization of each recombinant HTNV antigen was tested by competitive ELISA. Of these HTNV recombinants, the entire and 50-429 antigens were detected as multimers, whereas the 1-335, 1-244, and 155-429 antigens were detected as monomers. Of the SEOV and DOBV recombinants, the 1-429 (entire), 155-429, and 50-429 antigens were detected as multimers (Fig. 2C, middle and lower panels). These results are summarized in Table 1 in comparison with the serotype-specific antigenicity. Of the 155-429 antigens, only the HTNV 155-429 antigen lost its antigenicity and its ability to form a multimer. In contrast, the HTNV 50-429 antigen showed serotype-specific antigenicity and multimer formation. These results suggest that a serotype-specific epitope on the hantavirus NP is related to the multimerization of NP molecules.


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  		TABLE 1. Detection of the multimerization of truncated nucleocapsid proteins and their reactivities with rabbit immune sera



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  		FIG. 2. Multimerization of recombinant and truncated NP of HTNV, SEOV, and DOBV. Recombinant envelope glycoprotein expressed in High Five cells by using a baculovirus vector was used as a negative control (HTNV rEnv). (A) Detection of various recombinant NPs expressed in High Five cells. Antigens were captured with ECO2 and detected with biotinylated MAb E5/G6. (B) Detection of various recombinant HTNV NPs expressed in High Five cells. Antigens were captured with E5/G6 and detected with biotinylated MAb C24B4. (C) Detection of multimerized NP with recombinant NPs. Antigens were captured and detected with MAb E5/G6. (D) The competitive ELISA used to detect monomeric or multimerized NP is illustrated schematically.

Yeast two-hybrid assay of the homotypic and heterotypic interactions of hantavirus NP. To characterize the hantavirus NP-NP interaction, a yeast two-hybrid assay was performed (Table 2). Homotypic interactions of the NP of HTNV and SEOV were confirmed. In addition, heterotypic interactions between HTNV and SEOV were confirmed. These results suggest that the mechanism for the NP-NP interaction is common to HTNV and SEOV.


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  		TABLE 2. NP-NP interaction between HTNV and SEOV in the yeast two-hybrid assaya

Yeast two-hybrid assay of truncated HTNV NP. To clarify which region is responsible for the HTNV NP-NP interaction, truncated gene products were tested (Table 3). Truncated NP 50-429 interacted, whereas NP 155-429 did not. These results indicate that aa 50 to 155 are important for the NP-NP interaction. Since HTNV 1-403 antigen lost it reactivity in this assay, the C-terminal region consisting of aa 404 to 429 is also essential for the NP-NP interaction. These results indicate that two regions, from aa 50 to 155 and from aa 404 to 429, are both required for the HTNV NP-NP interaction.


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  		TABLE 3. Yeast two-hybrid assay for HTNV NP

Yeast two-hybrid assay of truncated SEOV NP. To clarify which region is responsible for the SEOV NP-NP interaction, truncated gene products were tested (Table 4). Although the entire NP, and truncated aa 50 to 429 and aa 100 to 429 NPs showed homotypic interactions, SEOV 125-429 did not interact at all. These results suggest that the essential region for the homotypic interaction is located between aa 100 and 125 on the SEOV NP. Of the N-terminal SEOV truncates, aa 100 to 429 had the strongest ß-galactosidase activity, which was higher than that of the intact SEOV NP or truncated NP from aa 50 to 429. This suggests that the N-terminal region inhibits the NP-NP interaction in the yeast two-hybrid assay. Next, the C-terminal truncates were tested. Truncated NP (from aa 1 to 420) interacted with the intact NP and the N-terminal truncates from aa 50 to 429, aa 100 to 429, and aa 1 to 420. Although truncated NP from aa 100 to 412 and the intact SEOV NP interacted, the region from aa 100 to 412 did not interact with the region from aa 1 to 420, indicating that the region from aa 413 to 420 is important for the SEOV NP-NP interaction. These results indicate that two regions, aa 100 to 125 and aa 413 to 420, are both required for the SEOV NP-NP interaction.


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  		TABLE 4. Yeast two-hybrid assay for SEOV NP

Comparison of regions 1 and 2 among the various hantavirus strains. In the yeast two-hybrid assay of the HTNV and SEOV NP-NP interactions, both NPs required two regions for the interaction. The consensus regions were defined as region 1 (essential N-terminal region) and region 2 (essential C-terminal region). To compare the amino acid sequences of regions 1 and 2 with those of other hantaviruses belonging to different serotypes, the amino acid sequences of regions 1 and 2 were aligned (Fig. 3). Both regions were conserved, and a GQTAD/NW sequence in region 1 was highly conserved among 20 species of hantaviruses listed in Virus Taxonomy (37), including Thottapalayam virus (TPMV). TPMV is an Insectivora-derived hantavirus isolated from Suncus murinus (9) and is the most distinct hantavirus from Rodentia-derived hantaviruses (20). These results suggest that the GQTAD/NW sequence in region 1 plays an important role in a function common to hantavirus NPs, such as the NP-NP interaction.



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  		FIG. 3. Multiple alignment of the regions essential for the NP-NP interaction in the yeast two-hybrid assay. The reactivities of the truncated NPs indicated that two regions are essential for the NP-NP interaction. Region 1 consists of aa 100 to 125, and region 2 consists of aa 404 to 429 in HTNV and SEOV. Both regions and homologous regions from various hantaviruses were compared. The deduced amino acid sequences were predicted from the following published nucleotide sequences: HTNV, HTNV strain 76118 (M14626); SEOV, SEOV strain SR-11 (M34881); DOBV, DOBV strain Saaremaa (AJ009773); PUUV, PUUV strain Sotkamo (X61035); KHAV, Khabarovsk virus (U35255); TULV, Tula virus no. 175Ma (Z30941); PHV, Prospect Hill virus 1 (M34011); SNV, Sim Nombre virus (L25784); NYV, New York virus (U09488); BCCV, Black Creek Canal virus (L39950); and ANDV, Andes virus (AF004660). TPMV is the Insectivora-derived hantavirus strain Thottapalayam isolated from S. murinus (9); the sequence of the S segment for TPMV was provided by C. Schmaljohn.

Interactions of W119A mutants of HTNV and SEOV in the yeast two-hybrid assay and competitive ELISA. We introduced a point mutation that changed the tryptophan (W) at position 119 in region 1 to alanine (A) (W119A mutant). The entire W119A mutant NP of SEOV was used in the yeast two-hybrid assay (Table 4). This mutant did not interact with any of the recombinants, indicating that the W at amino acid 119 is important in the multimerization of hantavirus NP. The same mutation was introduced to the NPs of HTNV and SEOV and expressed in 293T cells, and their multimerization was examined by using competitive ELISA. Both the wild-type and the W119A mutant NPs of HTNV expressed in 293T cells were detected as the multimer by using competitive ELISA (Fig. 4B), as were the wild-type and W119A mutant NPs of SEOV (data not shown). Although the W119A mutation was critical for the NP-NP interaction in the yeast two-hybrid assay, the same mutation did not affect the multimerization of NP in competitive ELISA.



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  		FIG. 4. Homotypic interactions of wild-type and W119A mutant NPs of HTNV and SEOV. (A) Summary of the multimerization of the wild-type NP, truncated NP aa 155 to 429, and the W119A mutant of HTNV and SEOV. (B) Detection of wild-type HTNV NP expressed in 293T cells by transfection (rNP-HTNV/293T [•]) and HTNV W119A mutant NP expressed in 293T cells (rNP-HTNV W119A/293T [{circ}]). The control antigen consisted of untreated 293T cells (293T [{blacksquare}]). In panel 1, the antigens were captured with ECO2 and detected with biotinylated MAb E5/G6. In panel 2, the antigens were captured with ECO2 and detected with biotinylated MAb ECO1 to detect multimerization.


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DISCUSSION
 
The mechanisms of viral NP-NP interactions and NP-viral genome interactions have been reported as they relate to the formation of the nucleocapsid, followed by assembly of the virus particle. Homotypic interactions of viral NP have been reported in several viruses, including hepatitis C virus core protein (19, 24) and the gag protein of human immunodeficiency virus type 1 (17). Recently, two studies have reported on the hantavirus NP-NP interaction; one examined two American hantaviruses, SNV and PHV (1), and the other examined the European hantaviruses Tula virus, PUUV, and DOBV (15). We examined the NP-NP interaction of two Asian hantaviruses, HTNV and SEOV, by two methods: a yeast two-hybrid assay and competitive-capture ELISA with recombinant and truncated NP expressed by baculovirus. Betenbaugh et al. reported that recombinant hantavirus NP expressed in insect cells by using baculovirus vector forms a nucleocapsid-like structure similar to the authentic viral nucleocapsid (6). This suggests that the NP multimer in insect cells is a functional multimer rather than an aggregation of NPs. In addition, recombinant NP antigen expressed in insect cells conserves its antigenicity more than rNP antigen expressed in E. coli (40). In addition, an rNP probe prepared in E. coli was not functional in the far-Western assay (data not shown). For these reasons, we selected a baculovirus vector system and not an E. coli expression system to prepare recombinant hantavirus NP.

There are several reports on the RNA-binding properties of hantavirus NP. Gott et al. indicated that 100 amino acids of the C-terminal of HTNV and PUUV NP are responsible for RNA-binding without sequence-specificity (14). Recently, Xu et al. reported another RNA-binding region mapped to a central, conserved region 175 to 217 (38). Sevenson et al. examined the RNA-binding activity of the NPs of HTNV and SNV and reported that there was sequence-specific RNA binding between the NP and viral RNA (33, 34). In addition, the NP-RNA interaction for tomato spotted wilt disease virus, which belongs to the Bunyaviridae, has also been reported (27). Despite these studies, the RNA-binding motif of hantaviruses remains unknown.

Recombinant NP expressed in insect cells or yeast cells was considered to be bound with cellular RNA. The role of RNA on NP-NP interaction was not clear. Preliminary experiment indicated that rNP-rNP interaction in insect cells was not disrupted by treatment of RNase (data not shown). However, further study on the relationship of RNA binding and multimerization of NP is needed to understand assembly of hantavirus virion.

Alfadhli et al. reported the hantavirus NP-NP interaction for SNV and PHV (1). These authors detected an NP oligomer in sucrose gradient fractionation and a yeast two-hybrid assay. In addition, they found an NP trimer in the PHV virion. They showed that the C-terminal half of the NP are needed for NP-NP interaction by yeast two-hybrid analysis, and their results in the yeast two-hybrid assay were basically the same as ours (Tables 3 and 4). We found that two regions were needed for the NP-NP interaction in the yeast two-hybrid assay, the C-terminal (aa 404 to 429) and central (aa 100 to 125) regions of the NP. Alfadhli et al. also determined the importance of the N-terminal from a coiled-coil domain analysis (1). In addition, they proposed the conformational switching model from intramolecular to intermolecular coiled coil (2). On the other hand, it is seemed that the N-terminal region of NP of HTNV and SEOV did not contribute to NP-NP interaction in the present study. In their coiled-coil analysis, the possibility scores for HTNV and SEOV were lower than for PHV and SNV. Our finding of the importance of the N-terminal region might result from differences in the hantaviruses studied. We demonstrated a heterotypic NP-NP interaction between HTNV and SEOV (Table 2), and Kaukinen et al. showed similar interactions for Tula virus and PUUV or DOBV (15). These results suggest that the basic mechanisms of the NP-NP interaction are common to all hantaviruses. In contrast, we found that the 155-429 antigens of HTNV, SEOV, and DOBV underwent different homotypic interactions (Table 1). These results suggest that a type-specific region (aa 240 to 300) contributes to the NP-NP interaction.

In ELISA, the C-terminal half (aa 155 to 429) of SEOV was sufficient to form the multimer. In contrast, in the yeast two-hybrid assay, the same region did not interact. Similar reaction patterns were obtained with the W119A mutants (Fig. 4A). We speculated that the differences were caused by differences in the maturation stage of the nucleocapsid. In the yeast two-hybrid assay system, the bait role interaction occurs in nuclei under reducing conditions; therefore, NP-NP interaction detected by yeast two-hybrid assay occurs in nuclei. In contrast, in ELISA the rNP molecules accumulate in the cytoplasm and then the NP-NP interaction is detected after solubilization of NP under nonreducing conditions after treatment with high-ionic-strength medium and detergent. For the authentic NP, the NP is first produced in the cytoplasm under reducing conditions and then forms a ribonucleoprotein complex (RNP). The two-hybrid assay reflects this initial phase. As the viral nucleocapsid matures, the RNP is altered to an insoluble structure for assembly. The results of the ELISA reflect this late phase.

The combined results of ELISA and the two-hybrid assay indicate that the C-terminal region is sufficient to maintain the multimer; under reducing conditions in the cytoplasm in the initial phase, region 1 (aa 100 to 125) is important for the NP-NP interaction in HTNV, SEOV, and DOBV. HTNV NP contains five tryptophan (W) residues that are conserved in Rodentia-associated hantaviruses: HTNV, SEOV, PUUV, PHV, and SNV. Generally, W is an uncommon amino acid residue in proteins. W has the largest side chain among the 20 essential amino acids and frequently plays a role in retaining protein conformation. In the present study, replacing the W at position 119 located in region 1 with alanine (W119A mutation) disrupted the NP-NP interaction in the two-hybrid assay (Table 4 and Fig. 4). This only indicates that the conformation determined by this W is related to the NP-NP interaction and is not direct evidence that W119 participates in multimer formation. To differentiate direct or indirect effect induced by mutation was difficult. We have to discuss results and structure altogether. For further analysis using mutation, we first need more information about the conformation of NP, at least regarding its secondary structure.

For the NP-NP interaction of SNV, coiled-coil domain in N-terminal was important (2). On the other hand, the roles of two discontinuous determinant (regions 1 and 2) defined in this assay was not clear. The secondary structure of region 1 was difficult to predict. The analysis software indicated the GQTAD/NW sequence in region 1 to form a beta sheet; however, the possibility score was not high. In addition, region 2 contains conserved acidic and basic amino acids, suggesting ionic interaction. However, to better understand the structure and function of regions 1 and 2 in multimerization, further studies are needed that are based on an approach other than truncation or mutation.

Moreover, we must consider two kinds of interactions among NPs. Kaukinen et al. reported on the NP interactions in Tula hantavirus and concluded that NP molecules first trimerize and then the trimers gradually assemble into a longer multimer (15). Distinguishing between these two interactions is important for understanding the maturation of the hantavirus nucleocapsid. In addition, these authors demonstrated that divalent cations, such as Ca2+, enhance NP-NP interaction. However, a typical metal-binding motif is not found in NP. Further studies are needed to characterize each interaction.

The SEOV and DOBV 155-429 antigens were detected as multimers and bound to hantavirus-infected animal and human sera in an ELISA (3). These results indicate that the C-terminal half is sufficient for maintaining the multimer and that the C-terminal half must interact with the C-terminal half. Therefore, the NP-NP interaction involves a tail-to-tail binding model. Considering a head-to-head and a tail-to-tail model, the antigenic structure of hantavirus NP is summarized in Fig. 5. This model explains the previous antigenic analysis of NP (40). We found that the three antigenic domains (I, II, and III) on the HTNV NP overlap. Antigenic domains I and III are the major antigenic domains in an actual infection. Domain I corresponds to ca. 1 to 100 amino acids at the N-terminal end, and domain II includes the MAb E5/G6 binding site (aa 166 to 175). The region responsible for antigenic domain III had not been determined because it only contains discontinuous epitopes. In Fig. 5, antigenic domains I to III are located along the trimer sequentially from the N-terminal end to the C-terminal end. Therefore, antigenic domain III is considered to be responsible for multimerization-dependent structures on the NP. Sjölander et al. produced a recombinant diagnostic antigen for PUUV infection and discussed the importance of a polymerization-dependent epitope on the NP; however, they could not demonstrate it experimentally (35).



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  		FIG. 5. Antigenic structure of HTNV NP. (A) Schematic diagram of HTNV NP showing the MAb ECO2 and E5/G6 binding sites (40), the major linear epitopes of the N-terminal region (13, 39), the minimum binding regions for MAb C24B4 (20), the RNA-binding region (14, 38), and the regions essential for the homotypic interaction determined in the present study (regions 1 and 2). The region highly conserved in hantaviruses (black ovals) and the region conserved in HTNV, SEOV, and DOBV (gray ovals) are also described. The RNA-binding region of the C-terminal (14) and central (38) regions are hatched. (B) Schematic head-to-head and tail-to-tail model of the HTNV NP trimer. Three antigenic domains (I, II, and III) are described (40). In this diagram, the white region (type-specific region) was located in antigenic domain III and was retained by trimerization.

Based on the results of the present and previous studies (3, 21, 40), the antigenic domain III contains only conformational dependent epitopes and it was multimerization dependent. Finally, type-specific epitopes (HTNV, SEOV, and DOBV specific) were contained in multimerization-dependent epitopes. However, we investigated only Murinae-associated hantavirus, HTNV, SEOV, and DOBV. Further studies are needed for other species of hantavirus.

There have been few studies of the interaction between hantavirus NP and cellular protein. Human immunodeficiency virus gag protein and cellular protein cyclophilin A are reported to interact. In this interaction, dimerization of the gag protein is necessary (10). As in this interaction, a multimerized hantavirus NP might recruit new interactions with viral and cellular proteins. The most plausible interaction would be between the multimerized NP and the transmembrane region of the envelope glycoproteins, which results in the assembly of virus particles. To determine the precise role of hantavirus NP in viral assembly and pathogenicity, a functional probe is needed. For this purpose, the homotypic interaction is an important characteristic for evaluating the potential of prepared NPs as a probe.


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ACKNOWLEDGMENTS
 
We thank C. S. Schmaljohn for providing the entire S genome segment sequence of TPMV. Textcheck (English consultants) revised the English in the final draft of the manuscript.

This study was partially supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institute for Animal Experimentation, Graduate School of Medicine, Hokkaido University, Sapporo 060-8638, Japan. Phone: 81-11-706-6906. Fax: 81-11-706-7879. E-mail: j_arika{at}med.hokudai.ac.jp. Back


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Journal of Virology, January 2003, p. 943-952, Vol. 77, No. 2
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.2.943-952.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.




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