Oligomerization of Hantavirus N protein: C-terminal alpha-helices interact to form a shared hydrophobic space

Abstract

The structure of the nucleocapsid protein of bunyaviruses has not been defined. Earlier we have shown that Tula hantavirus N protein oligomerization is dependent on the C-terminal domains. Of them, the helix-loop-helix motif was found to be an essential structure. Computer modeling predicted that oligomerization occurs via helix protrusions, and the shared hydrophobic space formed by amino acids residues 380-IILLF-384 in the first helix and 413-LI-414 in the second helix is responsible for stabilizing the interaction. The model was validated by two approaches. First, analysis of the oligomerization capacity of the N protein mutants performed with the mammalian two-hybrid system showed that both preservation of the helix structure and formation of the shared hydrophobic space are crucial for the interaction. Second, oligomerization was shown to be a prerequisite for the granular pattern of transiently expressed N protein in transfected cells. N protein trimerization was supported by three-dimensional reconstruction of the N protein by electron microscopy after negative staining. Finally, we discuss how N protein trimerization could occur.

FIG. 1.

(a) Secondary-structure predictions for the C-terminal region of the N proteins of Tula, Sin Nombre, and Hantaan viruses, which represent the three major groups of hantaviruses. (b) Mammalian two-hybrid results for point mutants G389P and G399P. (c) Sequence alignment of regions corresponding to the helix-loop-helix structure in Tula virus N protein and nonhantaviral proteins. The middle line shows identical amino acid residues, and + indicates the positions of homologous amino acids residues in the two proteins.

FIG. 2.

Computer modeling of the C-terminal interaction regions of two N protein molecules (in blue and in yellow). Helix I and helix II of neighboring molecules interact with each other via hydrophobic contacts between amino acid side chains (depicted for one interacting interface).

FIG. 3.

Study of the interaction capacity of N protein mutants in the mammalian two-hybrid system. The indicated mutations were introduced to both DNA-binding domain and activation domain constructs. (A and B) Two-hybrid results in which hydrophobic residues (underlined in the sequence above) were mutated in helix I (A) and in helix II (B). (C) Two-hybrid results of mutated residues (bold in the sequence above) participating in the putative cation-π interaction.

FIG. 3.

Study of the interaction capacity of N protein mutants in the mammalian two-hybrid system. The indicated mutations were introduced to both DNA-binding domain and activation domain constructs. (A and B) Two-hybrid results in which hydrophobic residues (underlined in the sequence above) were mutated in helix I (A) and in helix II (B). (C) Two-hybrid results of mutated residues (bold in the sequence above) participating in the putative cation-π interaction.

FIG. 4.

Localization of transiently expressed N protein mutants in COS7 cells. Immunofluorescence was used to visualize N proteins in cells transfected with a construct expressing full-length N protein (a) or mock transfected (b). Mutant N proteins localizing diffusely (c to e), mutant N proteins localizing in the perinuclear region (f to k) and mutant N proteins localizing both diffusely and in perinuclear region (l to o) are shown.

FIG. 5.

Computer modeling of the C-terminal region of the N protein trimer. The C-terminal helix-loop-helix structure of three N protein molecules (yellow, red, and blue) is represented. The core of each monomer is illustrated with colored ovals. The arrow points towards the N terminus of each monomer. The helix-loop-helix structure is shown as a top view (A) and as a side view (B).

FIG. 6.

Three-dimensional model of an N protein trimer reconstructed from electron micrographs of a negatively stained sample. The recombinant N protein (9) was applied to carbon film-coated 300- or 400-mesh Au grids (Quantifoil) diluted, i.e., floated in sequence in 1% uranyl acetate and negatively stained. Electron microscopic pictures were collected at ×50,000 at 80 kV with a Jeol 1200EX microscope, and negatives were scanned at 4,000 dots per inch (Nikon LS-8000 ED). For picking projections (≈38,000) of proteins from electron micrographs, the random extensive sampling method was used (6, 14) when distinct particles were difficult or impossible to identify. For making the three-dimensional reconstruction, EMAN was used (; EMAN web-site, http://ncmi.bcm.tmc.edu/≈s̈tevel/EMAN/doc/index.htm) (A). The N protein trimer model showing three monomers (in red, in yellow, and in blue) and their interacting helices (cylinders): coiled-coil motifs in the N-terminal region (above) and the helix-loop-helix in the C-terminal region (bottom). Helices of the same color belong to the same monomer (B). The arrow depicts a possible orientation of the viral RNA.