The multimerization of hantavirus nucleocapsid protein depends on type-specific epitopes

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.

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 [▪]). The control cell lysate did not react with either biotinylated HCO2 (Vero/HCO2b [○]) or 11E10-2-2 (Vero/11E10b [□]). (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 [▪]). The control antigens were uninfected Vero E6 cells (Vero [○]) and recombinant envelope glycoprotein expressed in High Five cells (rEnv/High Five [□]). 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.

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.

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.

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 [○]). The control antigen consisted of untreated 293T cells (293T [▪]). 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.

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.