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 ofHantaviruses, 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 B...

Oligomerization of Hantavirus N Protein: C-Terminal α-Helices Interact To Form a Shared Hydrophobic Space

“…One can conclude either that the residue alone is crucial or that the mutation disrupts the folding of the C-terminal region and destroys the interaction. Interestingly, W119 of Seoul virus N protein was shown to have a similar effect on N protein interaction in the yeast two-hybrid system (34). These data suggest that the tryptophan helps to maintain the overall structure of the N protein.…”

“…Previously published data suggest the N-terminal region as one of the candidates: (i) the N-terminal regions of Tula virus N protein (residues 1 to 43) and Sin Nombre virus N proteins (residues 1 to 39) were shown to participate in the homotypic interaction in the two-hybrid assays (1,10), (ii) the N-terminal residues of hantavirus N proteins were predicted to form coiled coils (1), and (iii) oligomerization studies of N protein peptides corresponding to residues 3 to 75 assembled into trimeric coiled coils (2). Other regions, e.g., the one that includes amino acid residues 100 to 125, may also be essential for the interaction (34). As soon as the C-terminal regions of three N protein monomers are brought closer to each other, this will allow helix II-helix I contact (as described in this paper), which occurs in a cyclic manner between neighboring N protein molecules, completing trimer formation.…”

“…Previously published data have shown that the N-terminal and C-terminal regions are involved in the hantavirus N protein interaction (1,9,10,34). The presence of coiled-coil motifs has been demonstrated in the N-terminal region (1, 2).…”

Essential Amino Acids of the Hantaan Virus N Protein in Its Interaction with RNA

“…For example, in a study by Alfadhli et al, the C-terminal regions of the Sin Nombre virus and Prospect Hill virus N proteins were required for N protein-N protein interactions in a yeast two-hybrid assay (2). Furthermore, in a recent study by Yoshimatsu et al, HTNV, Seoul virus, and Dobrava virus N protein multimerization was observed in a competitive enzyme-linked immunosorbent assay even when 49 amino acids were deleted from the N-terminal region (38). Additionally, using a yeast two-hybrid assay, they found that amino acids 100 to 125 and 404 to 429 were required for N protein multimerization.…”

Cell Fusion Activities of Hantaan Virus Envelope Glycoproteins

“…The cDNAs that contain the coding sequences for the HTNV GPs G1 and G 2 and the NP were subcloned into the expression plasmid pCAGGS/MCS (15) and designated pCHTNM (16) and pCHTNS (27), respectively. Vero E6 cell monolayers at 80% confluency were prepared in eightchamber LabTek Chamber Slides (Nunc, Inc., Naperville, Ill.) 1 day before transfection.…”