Crystal Structure of the Core Region of Hantavirus Nucleocapsid Protein Reveals the Mechanism for Ribonucleoprotein Complex Formation

Abstract

Hantaviruses, which belong to the genus Hantavirus in the family Bunyaviridae, infect mammals, including humans, causing either hemorrhagic fever with renal syndrome (HFRS) or hantavirus cardiopulmonary syndrome (HCPS) in humans with high mortality. Hantavirus encodes a nucleocapsid protein (NP) to encapsidate the genome and form a ribonucleoprotein complex (RNP) together with viral polymerase. Here, we report the crystal structure of the core domains of NP (NPcore) encoded by Sin Nombre virus (SNV) and Andes virus (ANDV), which are two representative members that cause HCPS in the New World. The constructs of SNV and ANDV NPcore exclude the N- and C-terminal portions of full polypeptide to obtain stable proteins for crystallographic study. The structure features an N lobe and a C lobe to clamp RNA-binding crevice and exhibits two protruding extensions in both lobes. The positively charged residues located in the RNA-binding crevice play a key role in RNA binding and virus replication. We further demonstrated that the C-terminal helix and the linker region connecting the N-terminal coiled-coil domain and NPcore are essential for hantavirus NP oligomerization through contacts made with two adjacent protomers. Moreover, electron microscopy (EM) visualization of native RNPs extracted from the virions revealed that a monomer-sized NP-RNA complex is the building block of viral RNP. This work provides insight into the formation of hantavirus RNP and provides an understanding of the evolutionary connections that exist among bunyaviruses.

Importance: Hantaviruses are distributed across a wide and increasing range of host reservoirs throughout the world. In particular, hantaviruses can be transmitted via aerosols of rodent excreta to humans or from human to human and cause HFRS and HCPS, with mortalities of 15% and 50%, respectively. Hantavirus is therefore listed as a category C pathogen. Hantavirus encodes an NP that plays essential roles both in RNP formation and in multiple biological functions. NP is also the exclusive target for the serological diagnoses. This work reveals the structure of hantavirus NP, furthering the knowledge of hantavirus RNP formation, revealing the relationship between hantavirus NP and serological specificity and raising the potential for the development of new diagnosis and therapeutics targeting hantavirus infection.

FIG 1

Structures of SNV NP and ANDV NPs. (A) Schematic diagram of the construction of SNV NP. Previously suggested NP_NCC, NP_core, and NP_CT structures are in green, blue, and red, respectively. The residue numbers for each end are at the top. (B) Size exclusion chromatogram (SEC) of the construct covering residues E111 to G398, which was run on a Superdex-200 column. The blue and red lines indicate absorbance at 280 and 260 nm, respectively. The inset shows a 12% SDS-PAGE gel of the major peak in SEC. Ribbon representation of SNV (C) and ANDV (E) NPs with rainbow coloring from the N (blue) to the C terminus (red). Each structural portion and secondary structural element is labeled. (D and F) Electrostatic surfaces of SNV and ANDV NPs. Positive and negative charges are in blue and red, respectively. RNA-binding crevices, indicated by large positively charged residues, are noted. (G) Comparison of SNV and ANDV NPcore. The overall structures of SNV and ADNV NP_core are aligned and are shown in two perpendicular views. The SNV and ANDV NP_core molecules are shown as cartoon diagrams in red and green, respectively. Structural elements with obvious differences are highlighted by red frames.

FIG 2

Comparison of hantavirus NP with orthobunyavirus and phlebovirus NPs. (A and B) The structures of BUNV (orthobunyavirus) NP (A) and RVFV (phlebovirus) NP (B) are aligned with that of SNV NP, and they are shown in two perpendicular views. Polypeptides of SNV, BUNV, and RVFV NPs are in red, green, and blue, respectively. The N- and C-terminal ends of each molecule are indicated. (C) Topologies of SNV, BUNV, and RVFV NPs. Helices and strands are, respectively, presented as rectangles and arrows and are rainbow colored, where blue and red indicate N- and C-terminal ends.

FIG 3

Interprotomer interactions. (A) A model of SNV NP oligomerization was built according to its high structural homology with orthobunyavirus NP. Three adjacent SNV NP molecules, including NP_CT and the linker NP_core-NP_NCC, are shown as cartoons in red, yellow, and green. (B and C) Contact of NP_CT of the NP⁰ protomer with the C lobe of the NP⁻¹ protomer and the contact of the linker NP_core-NP_NCC with the N lobe of the NP⁺¹ protomer. (D and E) Pulldown assays to verify the effects of mutations on NP homotypic interactions in the region of NP_CT-NP_core (D) and the linker NP_core-NP_NCC with NP_core (E).

FIG 4

RNA-binding crevice of hantavirus NP. (A) Electric potential surface of the RNA-binding crevice in SNV NP_core. The residues that provide positive charges are labeled. Bound PO₄³⁺ is shown as colored sticks. The deep pocket is indicated. To show the bound PO₄³⁺, the surfaces of residues P182 to E192 were removed. (B) Localization of positively charged residues that orchestrate RNA binding in SNV NP. SNV NP_core is shown as a semitransparent cartoon diagram, in which the N and C lobes are in blue and green, respectively. The basic residues and a bound PO₄³⁺ are displayed as colored sticks. (C) Binding affinities of the recombinant wt and mutational SNV NP_core with probe RNA. (D) Impacts of positively charged residues on SNV replication. The histogram shows the activity of the chloramphenicol acetyltransferase (CAT) reporter gene in SNV NP mutants, which are colored as in panel B and are normalized to wt SNV NP. Each experiment was performed in three replicates, and error bars indicate standard deviations.

FIG 5

EM images of native hantavirus RNPs extracted from virions. (A, B, and C) Most native HNTV RNPs displayed relaxed and linear architectures with widths of approximately 10 nm. (D) In some visualizations, select regions of the native RNPs displayed apparent helical characteristics (inset). Bars, 100 nm (A) and 50 nm (B, C, and D).

FIG 6

Implication of the role of hantavirus NP_NCC in NP oligomerization. (A and B) Comparison of the BUNV NP-RNA tetramer (PDB code 4IJS) and a model of the SNV NP oligomer. For each tetramer, NP0 was covered with an electrostatic surface potential, while the other three protomers are presented as colored cartoon diagrams. (C) Electrostatic surface potential of SNV NPNCC (PDB code 2IC6) in two views. The basic residues on the surface are labeled. (D) Schematic model of the linear hantavirus RNP formed using the monomer-sized NP RNA as a building block.

FIG 7

Epitopes in hantavirus NP_core. (A) Sequence conservation mapping on the surface of hantavirus NPs. Primary sequence homologies of representative hantaviruses are defined, and the structure of SNV NP_core is shown in front, side, and back views, covering its molecular surface. The deep pocket inside the RNA-binding crevice is enlarged for detail. Strictly conserved residues are in red, while conserved and variable residues are in gold and white, respectively. (B) Epitopes related to serotype specificity in hantavirus NP_core. Previously reported epitopes on NP_core that can distinguish SNV and ANDV (64) are highlighted in the structure of SNV NP_core. The two regions with the most serotype specificity (i.e., 244 to 263 and 274 to 286) are in red; the other identified epitopes are in gold.