The Postfusion Structure of the Heartland Virus Gc Glycoprotein Supports Taxonomic Separation of the Bunyaviral Families Phenuiviridae and Hantaviridae

Abstract

Heartland virus (HRTV) is an emerging human pathogen that belongs to the newly defined family Phenuiviridae, order Bunyavirales Gn and Gc are two viral surface glycoproteins encoded by the M segment and are required for early events during infection. HRTV delivers its genome into the cytoplasm by fusion of the viral envelope and endosomal membranes under low-pH conditions. Here, we describe the crystal structure of HRTV Gc in its postfusion conformation. The structure shows that Gc displays a typical class II fusion protein conformation, and the overall structure is identical to severe fever with thrombocytopenia syndrome virus (SFTSV) Gc, which also belongs to the Phenuiviridae family. However, our structural analysis indicates that the hantavirus Gc presents distinct features in the aspects of subdomain orientation, N-linked glycosylation, the interaction pattern between protomers, and the fusion loop conformation. This suggests their family-specific subunit arrangement during the fusogenic process and supports the recent taxonomic revision of bunyaviruses. Our results provide insights into the comprehensive comparison of class II membrane fusion proteins in two bunyavirus families, yielding valuable information for treatments against these human pathogens.IMPORTANCE HRTV is an insect-borne virus found in America that can infect humans. It belongs to the newly defined family Phenuiviridae, order Bunyavirales HRTV contains three single-stranded RNA segments (L, M, and S). The M segment of the virus encodes a polyprotein precursor that is cleaved into two glycoproteins, Gn and Gc. Gc is a fusion protein facilitating virus entry into host cells. Here, we report the crystal structure of the HRTV Gc protein. The structure displays a typical class II fusion protein conformation. Comparison of HRTV Gc with a recently solved structure of another bunyavirus Gc revealed that these Gc structures display a newly defined family specificity, supporting the recent International Committee on Taxonomy of Viruses reclassification of the bunyaviruses. Our results expand the knowledge of bunyavirus fusion proteins and help us to understand bunyavirus characterizations. This study provides useful information to improve protection against and therapies for bunyavirus infections.

Keywords: Heartland virus; bunyavirus; glycoprotein C (Gc); postfusion.

FIG 1

Overall structure of HRTV Gc in the postfusion conformation. (A) Schematic diagram of HRTV Gc with the crystallized ectodomain colored by domains (domain I in red, domain II in yellow, and domain III in blue). The stem region is colored light gray, and the transmembrane domain (TM) is colored dark gray. The cytoplasmic domain is medium gray. Fusion loops are displayed in orange. Three predicted glycosylation sites are colored green. (B) Structure of HRTV Gc in the postfusion conformation. The trimer is shown with one protomer displayed in the same color scheme as that for panel A in cartoon representation. The other two protomers are indicated in white surface representation. Glycans observed in the structure are shown as green sticks.

FIG 2

Orientation of domain III displays family specificity in phenuiviruses and hantaviruses. (A) Superimposition of HRTV (blue) and SFTSV (chartreuse) Gc postfusion structures. (B) Superimposition of HTNV (magenta) and PUUV (yellow) Gc postfusion structures. (C) Superimposition of HTNV (magenta) and HRTV (blue) Gc postfusion structures. (D) On the left, the structures of HRTV (blue) postfusion Gc and RVFV (cyan) prefusion Gc were superimposed on domain I. On the right, domain II pivots 25.9° toward the domain I/II junction. The angle between the two domain IIIs rotates 75.1°. (E) Overlapping of HTNV Gc in prefusion (white) and postfusion (magenta) conformation in two views. This shows a 132.5° hinge of reorientation between domain III on the domain I/III junction and 26° of hinge reorientation of domain II about the domain I/II junction. All of the structures are displayed in cartoon representation. Glycans are indicated as green sticks. The RMSDs are labeled below each panel.

FIG 3

Topology of four bunyaviruses. (A) HRTV; (B) SFTSV; (C) HTNV; (D) PUUV. The color scheme is identical to that described for Fig. 1. Disulfide bonds are labeled as SS and linked with dashes. Glycans are shown as gray hexagons.

FIG 4

N-linked glycosylation sites on four bunyaviruses. (A) HRTV; (B) SFTSV; (C) HTNV; (D) PUUV. Gc structures are shown in cartoon representation. Domains in the same subunit are shown in the same color, while domains in the different subunits are shown in different colors. Glycans are labeled as green sticks. The hydrophobic bonds are indicated as black dashes.

FIG 5

Comparison of the interprotomer interactions among class II fusion proteins. (A to H) Bottom views of fusion protein trimers from eight different viruses. The three protomers in Gc trimers are shown in white, cyan, and violet, respectively. (A) HRTV; (B) SFTSV; (C) HTNV; (D) PUUV; (E) DENV; (F) TBEV; (G) SFV; (H) RuV. The detailed interactions of the four viruses are shown in panels I to L, and the colors are identical to those described for panels A to H. (I) HRTV; (J) SFV; (K) HTNV; (L) RuV.

FIG 6

Comparison of fusion loops in phleboviruses and hantaviruses. The tip of domain II of the postfusion Gc from HRTV (A), SFTSV (B), PUUV (D), and HTNV (E) and the prefusion Gc from RVFV (C) and HTNV (F) are displayed in cartoon representation. The bc loop and cd loop are shown in orange. The disulfide bonds are displayed as green sticks. (G) The amino acid sequence alignment of the fusion loops among 13 phleboviruses. Database sequence accession numbers: HRTV, gb, AFP33394.1; SFTSV, gb, AGM33042.1; RVFV, gb, DQ380193.1; UUKV, gb, M17417.1; SFTV, gb, NC_015411; PPV, gb, HM566179; Palma virus (PALV), gb, JQ956380; Bhanja virus (BHAV), gb, JQ956377; TOSV, gb, AMY16462; CDUV, gb, YP_004347992; PTV, gb, ABD92923; CHGV, gb, AEL29641; and SFNV, gb, AEL29667. (H) The amino acid sequence alignment of the fusion loops among seven hantaviruses. Database sequence accession numbers: HTNV, gb, NP_941978.1; PUUV, gb, BAF49040.1; Seoul virus (SEOV), gb, P28729.1; Andes virus (ANDV), gb, AAK14322.1; Sin Nombre virus (SNV), gb, NP_941974.1; Adler hantavirus (ADLV), gb, AIY68300.1; and Prospect Hill virus (PHV), gb, P27315.1. Disulfide bonds are labeled in green below the sequences. The secondary structure elements are displayed above the sequences. Strictly conserved and highly similar residues are highlighted in red.