key: cord-0713649-9ulw7n16
authors: Hulswit, Ruben J. G.; Paesen, Guido C.; Bowden, Thomas A.; Shi, Xiaohong
title: Recent Advances in Bunyavirus Glycoprotein Research: Precursor Processing, Receptor Binding and Structure
date: 2021-02-23
journal: Viruses
DOI: 10.3390/v13020353
sha: fcb2e45718029722c23326aae1f3ed6fe3703d27
doc_id: 713649
cord_uid: 9ulw7n16

The Bunyavirales order accommodates related viruses (bunyaviruses) with segmented, linear, single-stranded, negative- or ambi-sense RNA genomes. Their glycoproteins form capsomeric projections or spikes on the virion surface and play a crucial role in virus entry, assembly, morphogenesis. Bunyavirus glycoproteins are encoded by a single RNA segment as a polyprotein precursor that is co- and post-translationally cleaved by host cell enzymes to yield two mature glycoproteins, Gn and Gc (or GP1 and GP2 in arenaviruses). These glycoproteins undergo extensive N-linked glycosylation and despite their cleavage, remain associated to the virion to form an integral transmembrane glycoprotein complex. This review summarizes recent advances in our understanding of the molecular biology of bunyavirus glycoproteins, including their processing, structure, and known interactions with host factors that facilitate cell entry.

Bunyaviruses constitute an expanding and extremely diverse group of RNA viruses with linear, segmented, single-stranded, negative-sense or ambisense RNA genomes, even more so since they were recently re-categorized by the International Committee on Taxonomy of Viruses (ICTV) from a family (Bunyaviridae) to an order (Bunyavirales) [1] . The order accommodates more than 480 named species (collectively known as bunyaviruses), and is now classified into 12 families: Arenaviridae, Cruliviridae, Fimoviridae, Hantaviridae, Leishbuviridae, Mypoviridae, Nairoviridae, Peribunyaviridae, Phasmaviridae, Phenuiviridae, Tospoviridae and Wupedeviridae [1, 2] as illustrated by the polymerase-based phylogenetic tree of the representative members of the order (Figure 1 ). In line with their potential to unpredictably emerge and cause severe disease, several viruses in the order are now recognized as priority pathogens by the World Health Organization (WHO) [3] .

The majority of the bunyaviruses are of little renown or consequence beyond their natural arthropod and mammalian host reservoirs. However, a significant number have repeatedly shown the capacity to cross the species barrier and impose a burden upon human health, animal husbandry and agriculture. Renowned examples include Lassa virus (LASV) (belonging to the Arenaviridae family), La Crosse virus (LACV), Schmallenberg virus (SBV) and Oropouche virus (OROV) (all members of the Peribunyaviridae family), Hantaan virus (HTNV), Andes virus (ANDV) and Sin Nombre virus (SNV) (Hantaviridae), Rift Valley fever virus (RVFV) and severe fever with thrombocytopenia syndrome virus (SFTSV) (Phenuiviridae), Crimean-Congo hemorrhagic fever virus (CCHFV) (Nairoviridae) [4] [5] [6] . Tomato spotted wilt virus (TSWV) (Tospoviridae) and rice stripe virus (RSV) (Phenuiviridae) infect hundreds of plant species, causing great economic losses worldwide [7] . The emergence of novel bunyaviruses and the increasing frequency of outbreaks constitute a growing threat to [33] , herbeviruses (Peribunyaviridae) [34] , Uukuviruses (e.g., UUKV) (Phenuiviridae) [35] , and the RNA2 segment of the emaraviruses (Fimoviridae) [36] encode GPCs containing two structural glycoproteins, Gn and Gc. (2) The M segments of orthobunyaviruses (Peribunyaviridae) [11] encode three proteins, with an NSm located between Gn and Gc in the precursor protein. (3) The M segments of phleboviruses [33] , herbeviruses (Peribunyaviridae) [34] , Uukuviruses (e.g., UUKV) (Phenuiviridae) [35] , and the RNA2 segment of the emaraviruses (Fimoviridae) [36] encode GPCs containing two structural glycoproteins, Gn and Gc. (2) The M segments of orthobunyaviruses (Peribunyaviridae) [11] encode three proteins, with an NSm located between Gn and Gc in the precursor protein. (3) The M segments of phleboviruses (e.g., RVFV) (Phenuiviridae) [37] and orthophasmaviruses (e.g., Ferak virus [FRKV] and jonchet virus, [JONV] (Phasmaviridae) encode GPCs containing three proteins: Gn and Gc, and an N-terminal NSm [38, 39] . (4) The M segment of nairoviruses (e.g., CCHFV) (Nairoviridae) encodes a GPC with five proteins: Gn and Gc, and three non-structural proteins; Mucin like protein/domain (MLD), GP38, and NSm [40, 41] . The M segments of other members of the family encode precursors for two to four proteins whose exact nature has yet to be confirmed experimentally [42] . (5) The ambisense M segments of orthotospoviruses (Tospoviridae) [29] and RNA2 segments of tenuiviruses (Phenuiviridae) encode GPCs containing Gn and Gc in the antigenomic sense, and an NSm in the genomic sense [43] . (6) The ambisense S segments of members of the Hartmanivirus, Mammarenavirus and Reptarenavirus genera (Arenaviridae family) encode a so-called stable signal peptide (SSP) and the glycoproteins GP1 and GP2 in the genomic sense, and a nucleocapsid protein in the antigenomic sense [26] . Similarly, the M segment of Wēnlǐng frogfish arenaviruses (Antennavirus genus) encode their GPCs in the genomic sense and an unknown protein in the anti-genomic sense [44] .

Bunyavirus GPCs are co-and post-translationally cleaved into two mature glycoproteins by a number of specific host cell proteases. The precursors studied so far contain N-terminal and internal signal peptides (SP), except for the arenaviruses, which have a single signal sequence (the above-mentioned SSP) at the N-terminus of the GP1 [45] [46] [47] [48] [49] . The SPs are essential for endoplasmic reticulum (ER) membrane translocation and facilitate protein cleavage and maturation. Binding of the nascent precursor SP by signal recognition particles guides the ribosome towards the ER, where the SP becomes inserted into the membrane, directing the remainder of the polypeptide into the ER lumen as it emerges from the ribosome. The SP is co-translationally cleaved from the polypeptide chain by cellular signal peptidases (SPase) [48] . In addition to SPase, several other host proteases that target specific peptide motifs, including signal peptide peptidase (SPP) [50] , subtilisin kexin/isozyme-1 (SKI-1/S1P) and furin-like proteases have been shown to process bunyavirus GPCs [40, 41, 46, 51, 52] .

In line with the extensive genetic diversity within the Bunyavirales order, varied enzymatic pathways are followed for GPC processing. In the following section, we discuss these pathways for representative viruses of the Hantaviridae, Peribunyaviridae, Phenuiviridae, Nairoviridae and Arenaviridae families ( Figure 3 ). Although GPC processing has been described for only a few viruses, we believe that in many cases, the mechanisms are shared amongst many members of the same genus or family.

Bunyamwera virus (BUNV) is the prototype member for both the Orthobunyavirus genus and the Peribunyaviridae family. The Gn/NSm/Gc coding pattern of BUNV GPC is shared by the viruses of three genera (Orthobunyavirus, Pacuvirus and Shangavirus) in the family with the exception of arthropod-specific herbeviruses (Genus Herbevirus), which do not encode NSm [34, 53] . We recently described the processing of BUNV GPC by host SPase and signal peptide peptidase (SPP) to generate Gn, Gc and NSm ( Figure 3A ) [46] . BUNV GPC (Gn/NSm/Gc) contains three SPs that precede the N-termini of each mature product. Co-translational SPase cleavage at the SP cleavage sites in the ER lumen generates three products: a pre-Gn, mature NSm and Gc. Upon SPase cleavage of NSm SP (SP NSm ; previously known as domain I of NSm [54] ), the SP NSm remains linked to the C-terminus of the pre-Gn until the SP is further released by SPP to generate mature Gn. Interestingly, the SP Gc (previously known as domain V of NSm [54] ) is not cleaved from the NSm cytoplasmic region (domain IV) and remains an integral part of the protein, rendering the mature NSm domain IV a cytoplasmic loop structure [46] . Whilst the GPC processing details for all other viruses in the family remain to be elucidated, the mechanism of BUNV GPC is likely shared by viruses with a similar coding pattern.

The M segment genomes of viruses within the family Hantaviridae encode the GPC, which contains two structural glycoproteins, Gn and Gc [33] . HTNV Gn and Gc have their own SPs at the N-termini and the two proteins are co-translationally cleaved at a conserved

The M segment genomes of viruses within the family Hantaviridae encode the GPC, which contains two structural glycoproteins, Gn and Gc [33] . HTNV Gn and Gc have their own SPs at the N-termini and the two proteins are co-translationally cleaved at a conserved 'WAASA' pentapeptide motif by the cellular SPase in the ER lumen [47] . Similar to BUNV described above, SPase cleavage leaves SP Gc connected to the C-terminus of Gn (Pre-Gn). Presumably the SP Gc is processed further by SPP or SPP-like proteases (SPPL) during Gn maturation ( Figure 3B ). The M segment GPC coding pattern of hantaviruses is shared with those of many bunyaviruses within other bunyaviral families, including herbeviruses (Peribunyaviridae), Uukuniemi phlebovirus (UUKV) (Phenuiviridae), Emaraviruses (Fimoviridae) as well as TSWV (Tospoviridae) and RSV tenuivirus (Phenuiviridae) (Figure 2) . Although yet to be experimentally validated, this suggests that the processing of GPCs in these families resembles that observed for HTNV.

The Phenuiviridae family contains 19 genera, which include RVFV and UUKV (genus Uukuvirus), SFTSV (genus Bandavirus) and RSV (genus Tenuivirus) [1, 2] . The GPC composition and coding strategy in this family is varied ( Figure 2 ). This review will focus on the processing of the RVFV GPC.

RVFV, the type species of the Phlebovirus genus, is a human and animal pathogen prevalent in Africa and the Middle East [55, 56] . Its M segment encodes a GPC for the non-structural NSm and P78, and the structural Gn and Gc ( Figure 3C ). The GPC contains three SPs, which are located at the N-termini of P78, Gn, and Gc. Translation initiation takes place at five different AUGs at the N-terminal NSm coding region, producing a nested set of polyprotein precursors, presumably by leaky scanning of the ribosome [37, [57] [58] [59] . The initiation at AUG1 produces a GPC that contains an N-terminal SP (SP p78 ). Cleavage at SP P78 and Gc SP (SP Gc ) by cellular SPase yields the mature Gc and a large P78 glycoprotein, which encompasses NSm and Gn [57, 59] . P78 is dispensable for virus replication in tissue culture [60] but was found to be incorporated into mosquito cell-generated RVFV virions [61] . It has therefore been postulated that RVFV P78 is required for virus dissemination in mosquitoes [62] . Precursors 2 and 3 (P14/NSm-Gn-Gc) are initiated at the 2 nd and 3 rd AUGs, respectively, and are processed into three proteins after cleavage at the SP Gn and SP Gc by the cellular SPase: P14/NSm, pre-Gn, and Gc. Upon SPase cleavage, the SP Gn serves as the C-terminal transmembrane domain of P14/NSm (a type II transmembrane domain that targets the protein to the outer membrane of mitochondria, thereby suppressing apoptosis) [63] . Given the cytosolic location of NSm, it is unsurprising that no carbohydrate was observed at the N-glycosylation sequon (N88) on the protein. However, the same site is glycosylated on P78 [37] . Translation initiation at the 4 th or 5 th AUG produces only Gn and Gc glycoproteins upon SPase cleavage. The precise N-termini of mature Gn and Gc have been determined by amino-acid sequencing of the virion's Gn and Gc, confirming the predicted SP cleavage sites (at residue Glu154 for Gn and Cys691 for Gc) [57, 64] . Presumably, preGns generated from precursors 2/3 (P14/NSm-Gn-Gc) and 4/5 (Gn-Gc) undergo further processing to produce the mature Gn protein by removing the SP Gc from the preGn cytoplasmic tail by SPP or SPPLs, as we described for the maturation of the BUNV Gn protein [46] .

RSV, the type member of the Tenuivirus genus, contains four single strand RNA segments (designated RNA1, 2, 3 and 4) [43] . RNA2 is equivalent to the M segments of other bunyaviruses, has ambisense polarity and encodes a 94 kDa GPC (pc2N-pc2C) in negative sense (cRNA), which is cleaved into two glycoproteins, pc2N and pc2C, corresponding to the Gn and Gc of other bunyaviruses, respectively. A 23 kDa non-structural protein (NSs2) is encoded in the positive sense (gRNA) (Figure 2 ) [30, 65] .

Despite the diversity of nairovirus M segments, which encode a precursor for two to five proteins, the potential furin-like and SKI-1/S1P-like substrate sites separating individual proteins are conserved [66] , suggesting a common processing mechanism, similar to that known for CCHFV. The CCHFV GPC consists of two envelope glycoproteins (Gn and Gc) and three non-structural proteins (MLD, GP38 and NSm) with SPs present at the N-termini of preGn (MLD/GP38/Gn), NSm and Gc ( Figure 3D ) [67, 68] . Co-translational SPase cleavage at these SPs generate PreGn (MLD/GP38/Gn), NSm and preGc. In the Golgi complex, PreGn undergoes cleavage at the motif RRLL by SKI-1/S1P to separate the N-terminal MLD-GP38 from mature Gn [40, 41, 51, 68, 69] . The MLD-GP38 is further cleaved at the RSKR motif by a furin-like proprotein convertase to yield MLD and GP38. MLD and GP38, as well as the uncleaved form (GP85/GP160), are secreted from the CCHFV-infected cells [41] . Using a CCHFV virus-like particle (VLP) approach, it was demonstrated that MLD-GP38 (GP85/GP160) is required for Golgi targeting and maturation of Gc [70] . In the Golgi, Gc matures through the removal of 41 residues from the N-terminus of preGc by an SKI-1/S1P-like protease at the RKPR/RKPL motif [40, 51] . Presumably, an SPP or SPPL removes the SP NSm from the Gn cytoplasmic tail in the ER or Golgi.

The family Arenaviridae was recently placed within the Bunyavirales order [71] . In Hartmanivirus, Mammarenavirus and Reptarenavirus genera the GPCs are encoded in the genomic sense by the S segment of their bi-segmented genomes, whilst in the Antennavirus genus they are encoded by the M segment of their tri-segmented genomes [26] . Unlike other bunyaviruses, the arenavirus glycoprotein precursor contains only a single stable SP (SSP), which is located at the N-terminus ( Figure 3E ). During glycoprotein maturation, the SSP is cleaved from the precursor by a cellular SPase, and GP1 is separated from GP2 by subtilase SK1/S1P [52, [72] [73] [74] [75] . Unique to arenaviruses, the SSP remains stable upon cleavage by cellular SPase in the ER and associates with GP1/GP2 heterodimers to form an SSP/GP1/GP2 complex [76, 77] . The SSP is myristoylated and rearranges to translocate its C-terminus back to the cytosolic side of the membrane, rendering it a double membranespanning protein with both the N-and C-termini residing in the cytosol [78] . The SSP is essential for viral infectivity and plays a crucial role in processing and maturation of the precursor, intracellular trafficking of GP1-GP2 to the Golgi, and pH-dependent membrane fusion activity [79] [80] [81] .

For many enveloped viruses, entry is initiated by the interaction of their envelope glycoproteins with host cell surface receptor(s) [82] [83] [84] [85] . Reflective of the broad range of mammalian, invertebrate, and plant species known to function as virus reservoirs, bunyaviruses utilize a wide variety of cell receptors/co-receptors to achieve this process. While the receptors for the majority of bunyaviruses are still unknown, many cellular receptors and host cofactors have been identified (Table 1) . Some excellent reviews on receptors and co-receptors and other cellular factors for the entry of bunyaviruses were recently published [10, [86] [87] [88] . In Table 1 we list receptors and other cellular factors that have been identified so far to play a role in cell entry of bunyaviruses.

While the enveloped Bunyaviruses enter cells via receptor-mediated endocytosis, many details of their endocytic pathways remain uncharacterized [10] . The wide diversity of the virus species, vectors, hosts and receptors for entry implies that bunyaviruses exploit more than one host endocytic route (Table 2) . Indeed, an increasing number of studies have shown that bunyaviruses are transported into the low pH endosomal lumen via different endocytosis pathways. Fusion of the virion and endosome, which is triggered following exposure of the virion to low pH environment, has been shown to trigger conformational changes to the glycoproteins for many bunyaviruses (see Section 5 structure of bunyavirus envelope glycoproteins for detail). Such a process allows delivery of the viral ribonucleoprotein (RNP) into the cytosol to initiate viral replication. A notable exception is the infection of plant cells by plant-infecting bunyaviruses, where viral RNP is delivered into the cytoplasm by vectors that are able to breach the structural barrier of plant cells [89] . Several comprehensive reviews of bunyaviruses entry were recently published by Albornoz [10] , Leger and Lozach [14] , Chen et al. [89] , Hallam [90] and Mittler et al. [91] . For viruses within the Phenuiviridae, please see an in-depth review of receptors and entry by Koch et al. [88] . 

Clathrin-mediated (CME) OROV [132] , LACV [133] (Orthobunyavirus) SFTSV [134, 135] (Bandavirus) HTNV, SEOV [136] ; ANDV [137] (Orthohantavirus) CCHFV (Orthonairovirus) [138, 139] JUNV [140] , PICV and LASV [141] (Mammarenavirus)

Caveolin-1-mediated (CavME) RVFV (Phlebovirus) [142] ANDV [137] Clathrin and caveolin independent AKAV (Orthobunyavirus) [143] UUK (Phlebovirus) [144] LCMV (Mammarenavirus) [145, 146] Macropinocytosis-like ANDV (Orthohantavirus) [137] 

The bunyavirus glycoproteins Gn and Gc (or SSP, GP1 and GP2 in the case of arenaviruses) form spikes on the lipid bilayer envelope of the virion and facilitate viral invasion of a host cell. High resolution structures of bunyavirus glycoproteins in isolation, and low resolution cryoEM studies of entire virions/VLPs, which are often pleomorphic in shape or deviate from icosahedral symmetry [147] , have been predominantly limited to orthobunyaviruses, phenuiviruses, hantaviruses, tospoviruses and arenaviruses. Although these glycoproteins assemble and give rise to diverse quaternary architectures (Figure 4 ), conserved structural features and folds have been observed within the order (Figures 5 and 6), with the notable exception of arenaviruses (please see Section 5.6 and Figure 7) . In particular and consistent with predictions [148] , the Gc glycoprotein from phleboviruses, bandaviruses, and hantaviruses have been shown to display a class II fusion architecture consisting of three domains, termed I-III, which forms trimers following merger of the virion envelope and target membranes (Figure 8 ; reviewed in [149, 150] ). Although structurally diverse, the cognate Gn glycoprotein from these same virus groups shares similarities with the E2 glycoprotein of alphaviruses [151, 152] . We also describe how these relatively conserved structural features contrast those of the characterized arenaviruses in Section 5.6.

High resolution structural information is currently limited to the N-terminal region of the orthobunyavirus Gc. The crystal structure of the N-terminal half of SBV Gc revealed an elongated multi-domain assembly composed of an α-helical head domain connected to a stalk region composed of two β-sheet subdomains ( Figure 7A ) [153] . Additional crystal structures of the α-helical head domains from BUNV, OROV and LACV Gc demonstrated that this region of the glycoprotein is relatively conserved amongst genetically diverse orthobunyaviruses [153] .

Interestingly, the N-terminal half of the BUNV Gc ectodomain has been shown to be dispensable for the virus replication cycle in vitro, but can serve to modulate the fusogenicity of the protein [157, 158] . The structure of the C-terminal half of the orthobunyaviral Gc ectodomain remains unknown, however, it has been proposed to form a pHdependent class II fusion fold [148, 159] akin to those observed in phleboviruses [160] and hantaviruses [161, 162] . Similarly, the structure of the orthobunyavirus Gn has yet to be reported. to a stalk region composed of two -sheet subdomains ( Figure 7A ) [153] . Additional crystal structures of the -helical head domains from BUNV, OROV and LACV Gc demonstrated that this region of the glycoprotein is relatively conserved amongst genetically diverse orthobunyaviruses [153] . Electron cryo-tomography (cryoET) of purified BUNV, the prototypic orthobunyavirus, has demonstrated that the Gn-Gc spike complex projects approximately 18 nm from the viral membrane ( Figure 4A ,B) [163] . Sub-tomogram averaging of the spike yielded a low resolution (~3-nm) reconstruction and revealed that Gn and Gc assemble to form a pH-sensitive tripod-like arrangement with a locally ordered lattice formed by trimeric surfaces located in both the membrane distal 'head' region and the membrane proximal 'floor' region ( Figure 4A ,B) [163, 164] . Interestingly, the structure of the N-terminal region of BUNV and La Crosse virus (LACV) Gc was observed to form extensive trimeric contacts in the crystal, and this trimer fits well into the membrane distal pyramidal region of the cryoET-derived reconstruction [153] . These combined observations are consistent with the Gn and the C-terminal region of the orthobunyaviral Gc occupying the membrane proximal region of the orthobunyaviral Gn-Gc complex ( Figure 4A,B) . Future studies will shed light on the functional interactions between the head and stalk regions with the fusogenic C-terminal region of the orthobunyaviral Gc, and on the functional role of the smaller Gn. Figure 5 and the C-terminus connects to an α-helical hairpin (grey cartoon), also known as the 'base' domain. (B) The N-terminus of the displayed TSWV Gn structure (PDB: 6Y9L, [167] ) connects to the multi-domain globular region (grey cartoon, also presented in Figure 5 ) and the C-terminus connects to the transmembrane domain. (C) The N-terminus of the displayed CHIKV E2 structure (PDB: 3N43, [170] ) connects to a multi-domain globular region (partially shown by grey cartoon) and the C-terminus connects to the transmembrane domain. The N-and C-termini of the β-sandwich folds are colored blue and red, respectively, and the N-and C-termini of the structures are indicated.

Crystal structures of N-terminal ectodomain regions from RVFV (genus Phlebovirus) and SFTSV (genus Bandavirus) Gn proteins have recently been determined, revealing that both glycoproteins form a three-domain architecture composed of a mixed α-helical/βstranded N-terminal domain, termed herein as 'domain A', a 'β-ribbon domain', and a 'domain B' [151, 166, 171, 172] (Figure 5A ). Although RVFV Gn and SFTSV Gn present the same overall fold, comparison reveals relatively large differences (~3 Å root-mean-square deviation), demonstrating that structural variation exists across the family [166] .

The structure of the phenuivirus Gc ectodomain has been determined for RVFV, SFTSV, and Heartland virus (HRTV) [160, 169, 173, 174] . In contrast to the phleboviral Gn, the Gc is relatively conserved and displays a class-II fusion fold ( Figure 5B ). The three Gc domains, termed I-III, are composed predominantly of β-sheets (reviewed in [149, 150] ). Fusion loops have been identified in domain II, which inserts into the target membrane of Figure 5 and the C-terminus connects to an α-helical hairpin (grey cartoon), also known as the 'base' domain. (B) The N-terminus of the displayed TSWV Gn structure (PDB: 6Y9L, [167] ) connects to the multi-domain globular region (grey cartoon, also presented in Figure 5 ) and the C-terminus connects to the transmembrane domain. (C) The N-terminus of the displayed CHIKV E2 structure (PDB: 3N43, [170] ) connects to a multi-domain globular region (partially shown by grey cartoon) and the C-terminus connects to the transmembrane domain. The N-and C-termini of the β-sandwich folds are colored blue and red, respectively, and the N-and C-termini of the structures are indicated.

Crystal structures of N-terminal ectodomain regions from RVFV (genus Phlebovirus) and SFTSV (genus Bandavirus) Gn proteins have recently been determined, revealing that both glycoproteins form a three-domain architecture composed of a mixed α-helical/βstranded N-terminal domain, termed herein as 'domain A', a 'β-ribbon domain', and a 'domain B' [151, 166, 171, 172] (Figure 5A ). Although RVFV Gn and SFTSV Gn present the same overall fold, comparison reveals relatively large differences (~3 Å root-mean-square deviation), demonstrating that structural variation exists across the family [166] .

The structure of the phenuivirus Gc ectodomain has been determined for RVFV, SFTSV, and Heartland virus (HRTV) [160, 169, 173, 174] . In contrast to the phleboviral Gn, the Gc is relatively conserved and displays a class-II fusion fold ( Figure 5B ). The three Gc domains, termed I-III, are composed predominantly of β-sheets (reviewed in [149, 150] ). Fusion loops have been identified in domain II, which inserts into the target membrane of the host cell. A conserved cavity nearby the fusion loop of RVFV interacts with glycerophospholipids and contributes to the interaction of Gc with the target membrane [174] . Comparison of putative pre-and post-fusion conformations of RVFV, HRTV, and SFTSV Gc glycoproteins provides a structural basis for understanding the fusogenic rearrangements that these glycoproteins undergo to facilitate merger of the virus and cell membranes following endocytic uptake of the virus. the host cell. A conserved cavity nearby the fusion loop of RVFV interacts with glycerophospholipids and contributes to the interaction of Gc with the target membrane [174] . Comparison of putative pre-and post-fusion conformations of RVFV, HRTV, and SFTSV Gc glycoproteins provides a structural basis for understanding the fusogenic rearrangements that these glycoproteins undergo to facilitate merger of the virus and cell membranes following endocytic uptake of the virus. Initial structural studies of purified RVFV and UUKV by EM revealed that Gn-Gc heterodimers form an icosahedral lattice (T = 12) with 122 distinct capsomers (12 pentons and 110 hexons) ( Figure 4C ,D) [177] [178] [179] [180] . The resolution achieved by these investigations was restricted to approximately 2-nm, a limitation that has been attributed to the flexibility of the Gn-Gc glycoprotein assembly [151] . A more recent set of reconstructions of RVFV by Halldorsson and Li et al. benefited from optimized sample preparation approaches (i.e., virus fixation), improved direct detector technologies and data processing strategies (i.e., a localized reconstruction approach) for EM, and yielded an improved resolution of approximately 1-nm [151] . While the resolution achieved by this study was insufficient to define the specificity of inter-subunit interactions formed by Gn and Gc, it was able to localize the crystallized fragments of the two glycoproteins, where the N-terminal region of the Gn ectodomain is placed at the membrane-distal region of the viral envelope, covering much of the Gc glycoprotein. It has been postulated that placement of the Gn shields the hydrophobic fusion loop of the Gc, preventing premature fusion. Shielding of the Gc is consistent with sera binding data from RVFV infected individuals Initial structural studies of purified RVFV and UUKV by EM revealed that Gn-Gc heterodimers form an icosahedral lattice (T = 12) with 122 distinct capsomers (12 pentons and 110 hexons) ( Figure 4C ,D) [177] [178] [179] [180] . The resolution achieved by these investigations was restricted to approximately 2-nm, a limitation that has been attributed to the flexibility of the Gn-Gc glycoprotein assembly [151] . A more recent set of reconstructions of RVFV by Halldorsson and Li et al. benefited from optimized sample preparation approaches (i.e., virus fixation), improved direct detector technologies and data processing strategies (i.e., a localized reconstruction approach) for EM, and yielded an improved resolution of approximately 1-nm [151] . While the resolution achieved by this study was insufficient to define the specificity of inter-subunit interactions formed by Gn and Gc, it was able to localize the crystallized fragments of the two glycoproteins, where the N-terminal region of the Gn ectodomain is placed at the membrane-distal region of the viral envelope, covering much of the Gc glycoprotein. It has been postulated that placement of the Gn shields the hydrophobic fusion loop of the Gc, preventing premature fusion. Shielding of the Gc is consistent with sera binding data from RVFV infected individuals [181] , which demonstrated that antibodies preferentially target the Gn. Further cryoET imaging and reconstructions of acidified RVFV have given clues to the repositioning of the Gn and are consistent with the requirement for Gc to extend towards the target membrane [151] . Future structural studies will benefit from comparison of the higher-order ultrastructure of genetically diverse phenuiviruses, such as SFTSV and HRTV, and to assess whether such assemblies and structural transitions are conserved across the family.

Crystal structures of fragments from the Gn ectodomains of Maporal virus (MAPV), ANDV, HTNV and PUUV hantaviruses have revealed that the glycoprotein consists of an N-terminal 'head', which forms a fold similar to that exhibited by the Gn of phenuiviruses and the E2 of alphaviruses ( Figure 5A ) [151, 152, 154, 165, 182] . The head connects to a β-sandwich fold ( Figure 6A ) and a C-terminal tetrameric 'base' domain [154] . Crystallographic investigations of the cognate Gc from these viruses have revealed that the glycoprotein comprises a class II fusion fold [154, 161, 162] (Figure 5B ). Different from other class II fusion proteins, hantavirus Gc has an N-terminal extension (Gc N-tail), which has been postulated to stabilize the Gc post-fusion trimer. The structure of the Gn cytoplasmic tail (CT) from ANDV was resolved by nuclear magnetic resonance (NMR) and shown to contain conserved dual CCHC-type classical ββα-fold zinc fingers, suggestive that hantavirus Gn CTs are involved in binding the RNP during virion assembly [183, 184] .

Initial negative-stain EM studies of HTNV revealed a distinct 'grid-like pattern' on the virion surface [185] . Interestingly, (Gn-Gc) 4 , which form the spikes that constitute these grids, has been proposed to exist in equilibrium between two conformational states, termed 'closed' and 'open' [186] . The 'closed' form does not bind membranes at neutral pH, likely due to occlusion of Gc-resident fusion loops, but is capable of undergoing fusogenic rearrangements upon exposure to acidic environments. In the 'open' form, Gc fusion loops have been proposed to be exposed as they can bind target membranes at neutral pH but are unable to fuse the target and viral membranes.

The ultrastructure of the putative 'closed' form has been studied by cryoET analysis of Tula virus (TULV) and HTNV, which revealed that (Gn-Gc) 4 form a square-like organization that extends approximately 12 nm from the virion envelope and forms a locally-ordered lattice that interconnects through homodimeric Gc contacts ( Figure 4E,F) [154, 165, [186] [187] [188] . The N-terminal region of the Gn ectodomain was predicted to locate towards the membranedistal region of the lattice [165, 182] . This hypothesis was confirmed following the integration of a cryoET reconstruction of TULV with the crystal structure of a Gn-Gc heterodimer [154] . This study clarified the handedness of previous lower resolution cryoET maps and unambiguously demonstrated that the N-terminal Gn ectodomain obscures the Gc-resident fusion loops through the formation of extensive protein and glycan contacts with Gc domain II [154] . Additionally, the complex of the Gn-Gc heterodimer also revealed a limited structural role of the Gc N-terminal tail in stabilizing the interactions between Gc domains I and III, in the pre-fusion state [154] . The positioning of the Gc and the nature of Gc-mediated cross-linking of adjacent (Gn-Gc) 4 spikes was also further verified by integrated X-ray and cryoET analysis of HTNV VLPs in complex with the Fab fragment from a bank vole-derived neutralizing monoclonal antibody (mAb) specific to the Gc glycoprotein [168] . Future work will likely be focused on elucidating the molecular basis for the interaction of the hantaviral (Gn-Gc) 4 spike complex with host cell surface receptors (Table 1 ) and how this interaction facilitates uptake of the virus into a host cell. [190] ), Rubivirus RUBV (PDB: 4ADJ [191] ); C. elegans (PDB: 4OJC [149] ). The elongated structures of class II fusion proteins are shown as blue shapes (Gc for members of the Bunyavirales, E1 for Togaviridae, E for Flaviviridae and EFF-1 for the cellular C. elegans protein). Putative fusion protein stabilizing entities present on mature viral particles, are shown as purple shapes and have been hypothesized to prevent premature fusion activation (Bunyavirales: Gn, Togaviridae: E2). The E3 protein has been shown to be present in some alphavirus particles [170] but is omitted from this representation for clarity. The level of symmetry of each of the protein assemblies is indicated by symmetry symbols at the bottom right-hand corner. The approximate position of the fusion loop(s) is indicated with an asterisk (*) for each panel. In the case of peribunyaviruses, the exact location of the fusion loop (white asterisk) within the Gc protein is currently not known, but was inferred from the location of the N-terminal extensions within the tripodal EM reconstruction [153, 163] and the C-terminal positioning of Gc transmembrane domains. Note that, although C. elegans EFF-1 (epithelial fusion failure 1) protein presents a class II fusogen architecture, it does not contain a fusion loop. Fusion is believed to be initiated by trimerization of the plasma membrane anchored EFF-1 ectodomains protruding in the extracellular space [149] . The grey region of the column shown for Peribunyaviridae represents the N-terminal extension of the Gc fusion protein, which has not been observed in other bunyavirus glycoproteins. The pre-fusion oligomeric state of EFF-1 has been observed to be monomeric on the plasma membrane [192] . The pre-fusion oligomeric state of rubella virus E1 on the virus membrane is currently unknown and therefore represented as a protomer of an unknown oligomeric assembly. The fusion proteins of alpha-(e.g., Semliki Forest virus (SFV), chikungunya virus (CHIKV)) and flaviviruses (e.g., dengue virus (DENV), zika virus (ZIKV)) are structurally related despite a lack of detectable sequence conservation and are therefore positioned next to each other in the diagram. Similarly, phenuivirus Gc has been shown to be genetically more closely related to the fusion envelope (E) proteins of flaviviruses than to those of other genera in its own order [152] . These proteins are placed next to each other to represent this predicted relationship. The box depicting the cellular EFF-1 protein is colored in yellow as to oppose the boxes in different shades of blue which all contain viral fusion proteins.

Limited structural data currently exists for the nairovirus envelope glycoproteins Gn and Gc. A recent study reported the ultrastructural organization of the Hazara virus (HAZV) envelope glycoproteins to 2.5 nm resolution, revealing that the Gn-Gc assembly displays a putative tetrameric architecture [193] . Due to the low resolution of the reconstruction, it remains unclear which parts represent the Gn and Gc glycoproteins. Given the limited size of the Gn across nairoviral lineages, with respect to the Gc [66] , it has been [190] ), Rubivirus RUBV (PDB: 4ADJ [191] ); C. elegans (PDB: 4OJC [149] ). The elongated structures of class II fusion proteins are shown as blue shapes (Gc for members of the Bunyavirales, E1 for Togaviridae, E for Flaviviridae and EFF-1 for the cellular C. elegans protein). Putative fusion protein stabilizing entities present on mature viral particles, are shown as purple shapes and have been hypothesized to prevent premature fusion activation (Bunyavirales: Gn, Togaviridae: E2). The E3 protein has been shown to be present in some alphavirus particles [170] but is omitted from this representation for clarity. The level of symmetry of each of the protein assemblies is indicated by symmetry symbols at the bottom right-hand corner. The approximate position of the fusion loop(s) is indicated with an asterisk (*) for each panel. In the case of peribunyaviruses, the exact location of the fusion loop (white asterisk) within the Gc protein is currently not known, but was inferred from the location of the N-terminal extensions within the tripodal EM reconstruction [153, 163] and the C-terminal positioning of Gc transmembrane domains. Note that, although C. elegans EFF-1 (epithelial fusion failure 1) protein presents a class II fusogen architecture, it does not contain a fusion loop. Fusion is believed to be initiated by trimerization of the plasma membrane anchored EFF-1 ectodomains protruding in the extracellular space [149] . The grey region of the column shown for Peribunyaviridae represents the N-terminal extension of the Gc fusion protein, which has not been observed in other bunyavirus glycoproteins. The pre-fusion oligomeric state of EFF-1 has been observed to be monomeric on the plasma membrane [192] . The pre-fusion oligomeric state of rubella virus E1 on the virus membrane is currently unknown and therefore represented as a protomer of an unknown oligomeric assembly. The fusion proteins of alpha-(e.g., Semliki Forest virus (SFV), chikungunya virus (CHIKV)) and flaviviruses (e.g., dengue virus (DENV), zika virus (ZIKV)) are structurally related despite a lack of detectable sequence conservation and are therefore positioned next to each other in the diagram. Similarly, phenuivirus Gc has been shown to be genetically more closely related to the fusion envelope (E) proteins of flaviviruses than to those of other genera in its own order [152] . These proteins are placed next to each other to represent this predicted relationship. The box depicting the cellular EFF-1 protein is colored in yellow as to oppose the boxes in different shades of blue which all contain viral fusion proteins.

Limited structural data currently exists for the nairovirus envelope glycoproteins Gn and Gc. A recent study reported the ultrastructural organization of the Hazara virus (HAZV) envelope glycoproteins to 2.5 nm resolution, revealing that the Gn-Gc assembly displays a putative tetrameric architecture [193] . Due to the low resolution of the reconstruction, it remains unclear which parts represent the Gn and Gc glycoproteins. Given the limited size of the Gn across nairoviral lineages, with respect to the Gc [66] , it has been postulated [193] that the membrane distal density may correspond to a portion of the Gc ectodomain, with the Gn being confined to a membrane proximal location.

Of the nairovirus envelope glycoproteins, only a molecular-level structure of the CCHFV Gn tail has been reported to date using NMR spectroscopy [194] . CCHFV Gn CT (~100-residue) contains dual cysteine/histidine rich motifs (C-X-C-X-H-X-C), which are conserved across many bunyavirus families, including orthobunyaviruses, hantaviruses, and tospoviruses. Furthermore, structural analysis revealed a pair of tightly arranged dual ββα zinc fingers similar to those hantavirus Gn CTs [194] .

While little is known about the structure of the Gn ectodomain, the structure of the secreted CCHFV GP38 glycoprotein has been recently elucidated and was shown to consist of a three-helix bundle and a β-sandwich ( Figure 6B ). Interestingly, GP38 has been postulated to share structural features with the ectodomain of the CCHFV Gn glycoprotein due to a gene duplication event [175] . Future high resolution structural studies of the nairovirus Gn and Gc are needed to clarify their mode of assembly and respective functionalities. Additionally, determination of the nairovirus Gc structure will reveal whether the glycoprotein forms the predicted class II fusion glycoprotein architecture observed in hantaviruses, phenuiviruses, alphaviruses and flaviviruses [148] .

The recent structural elucidation of a Gn ectodomain region from TSWV provides the first detailed insights [167] into the glycoprotein architecture of this important group of viruses. TSWV Gn presents structural features similar to the Gn of hanta-and phenuiviruses and the E2 of alphaviruses, consisting of a pincer domain (analogous to the β-ribbon domain), an N-terminal domain A, and a domain B that is inserted between two β-strands of the β-ribbon/pincer domain ( Figure 5) . Interestingly, the TSWV Gn crystal structure presents a C-terminally positioned domain of a topology similar to that observed in the C-terminal ectodomain regions of hantavirus Gn, alphavirus E2 and flavivirus prmE ( Figure 6 ) [154, 170, 195] . Distinctively, TSWV Gn contains a reduced version of domain B, which forms a β-hairpin that faces the opposing domain A. Studies of the glycoprotein assemblies found on tospovirus particles indicate the existence of both Gn homodimers and Gn/Gc heterodimers [196] While partial disulfide bond-mediated dimerization of Gn was observed in solution, crystal contacts of the reported TSWV Gn structure may reflect a second dimerization mode through its pincer domain (equivalent of the β-ribbon domain) [167] .

Similar to other bunyaviruses, the Gc protein of tospoviruses has been predicted to be a class II fusion protein [197] and, based on sequence alignments, most closely resembles that of orthobunyaviruses [162] . High resolution structural characterization of the orthobunyavirus and tospovirus Gc glycoproteins will be necessary to validate this hypothesis. Given the structural distinctiveness of the tospovirus Gn with respect to other bunyavirus Gns [167, 196] , it seems possible that the tospovirus envelope Gn/Gc assembly may represent another variation on the assembly modes of bunyaviruses. The strong conservation of Gc fusogens, combined with greater structural diversity of the Gn glycoprotein ( Figure 5 ), suggests an instrumental role for the latter protein for dictating the distinct ultrastructural glycoprotein organizations on the viral membrane.

Although some cryoET analysis has been performed for the University of Helsinki virus (UHV) (genus Reptarenavirus) [198] , which demonstrated a trimeric glycoprotein assembly reminiscent to the GP of the Ebola virus [155, 199] , structural information of the envelope glycoproteins within the Arenaviridae family is mostly limited to the GP from the Mammarenavirus genus. Each protomer of the highly N-linked glycosylated trimeric mammarenavirus GP is composed of a non-covalently associated heterotrimer consisting of an SSP, GP1, and a transmembrane GP2 [155, 200] . The non-covalently associated GP1 is responsible for receptor recognition and has been hypothesized to be shed from the GP following exposure to acidic pH during internalization of the virus into a host cell [155, 201, 202] .

Crystal structures of the GP1 have been solved for both New World (NW) and Old World (OW) arenaviruses and shown to be composed of a compact α/β fold ( Figure 7C ) [203] . Studies of the OW GP1 from LASV, lymphocytic choriomeningitis virus (LCMV), Morogoro virus (MORV), and Loei River virus (LORV) have revealed contrasting conformations when produced alone or in complex with the cognate GP2 [204] [205] [206] [207] [208] . While a NW GP1-GP2 structure has yet to be reported, the NW GP1 from Machupo virus (MACV) [209, 210] , Junín virus (JUNV), and Whitewater Arroyo virus (WWAV), solved alone, and in complex with the TfR1 receptor and Fab fragments of neutralizing mAbs, have revealed only a single conformation, which may resemble the GP2-bound pre-fusion state [207, [209] [210] [211] [212] [213] [214] . The observation that the OW arenavirus GP1 undergoes conformational changes provides a structure-based hypothesis for how it may constitute an immunological decoy following release from the pre-fusion GP complex during infection [202, [215] [216] [217] and is consistent with a study showing that recombinantly-derived NW GP1 is more effective at raising a neutralizing antibody response than OW GP1 [217] . Interestingly, crystallographic analysis of receptor-bound Lujo virus (LUJV) GP1 revealed a structure that contrasts known OW and NW GP1 structures [218] , an observation that likely reflects its unique usage of the NRP2 host cell molecule (Table 1 ) [103] .

Unlike the other bunyaviruses reviewed above, detailed structural data has been acquired for several arenavirus GP-receptor interactions. X-ray crystallography of a MACV GP1-hTfR1 complex provides a structural basis for recognition of human TfR1 by the GP1 of certain zoonotic clade B and D NW arenaviruses [210, 219] . Interestingly, this binding site on TfR1 is distal from that used by the physiological ligands transferrin and hereditary hemochromatosis associated protein [210] . The crystal structure of a LUJV GP1-NRP2 complex revealed a metal ion-dependent mechanism of recognition that may involve the full trimeric spike during native binding [218] . Finally, although limited information exists for how most OW arenaviruses and clade C NW arenaviruses interact with the C-type lectin, DC-SIGN, or the O-mannose glycans presented on α-DG [92, 93, 101, 220] , functional studies have identified residues on LASV GP1 that are important in modulating the pH-dependent recognition of LAMP1 [205, 206] and a cryoET-derived reconstruction of acidified LASV VLPs in the presence of recombinantly-derived LAMP1 supports this interaction taking place at a membrane distal region of the molecule [155] .

CryoET ultrastructure analysis of mammarenavirus GPs, as presented on the virion membrane, has been limited to LASV and has revealed that the GP forms a tripodal organization that extends approximately 9 nm from the virus surface and contrasts the single stem assembly of the UHV reptarenavirus [198] . The low resolution (~1.4 nm) of the reconstruction of LASV GP was largely in agreement with the crystal structure of a trimeric LASV GP1-GP2 complex bound to the Fab fragment of a neutralizing mAb, termed 37.7H [156] . Interestingly, several mAbs (including mAb 37.7H) have been shown to target a site, termed 'GPC-B', and to specifically bind a quaternary epitope thereby cross-linking the GP1-GP2 protomers [156, 201, 221] . These structural data provide first molecular-level insights into the trimeric GP1-GP2 architecture ( Figure 4G,H) .

Upon endocytic uptake and detachment of GP1, GP2 is responsible for catalyzing the merger of viral and target membranes in a pH-dependent process. In contrast to other structurally characterized bunyavirus fusion glycoproteins reviewed above, the mammarenavirus GP2 has been shown to be a class I fusion protein [222] . Other class I fusion proteins have been observed in paramyxoviruses, coronaviruses, HIV, and influenza viruses [203, 223] . Crystal structures of the GP2 when bound to the GP1 glycoprotein have revealed a GP1-stabilized pre-fusion conformation ( Figure 7C ), while post-fusion structures of GP2 fragments have shown that the glycoprotein forms a trimeric coiled-coil ( Figure 7D ) [156, 176, 201, 204, 221, [223] [224] [225] . The NMR structure of the GP2 cytoplasmic tail has also been determined and shown to comprise a zinc binding domain, which interacts with the SSP [226] .

Bunyaviruses include several important human, animal and plant pathogens. Elucidation of the biological structure and function of bunyaviral glycoproteins is essential for the rational development of vaccines, drugs and other preventive strategies. Our improving understanding of bunyavirus glycoproteins has enhanced our appreciation of the pathobiological diversity within this important group of pathogens.

However, there remain many questions and challenges concerning the function and structure of bunyavirus glycoproteins. Indeed, much remains to be elucidated on the process of bunyavirus glycoprotein folding and biosynthesis, their diverse assemblies and host-interactions, and how they interact and recruit the N protein during virus assembly. Furthermore, it remains poorly understood how virus pathogenesis differs between human and animal pathogens. We anticipate that clarification of these fundamental elements of bunyavirus biology will further rely on the utilization of cutting-edge technologies and approaches. For example, haploid screening approaches have been essential for the identification of several bunyavirus host cell receptors [94, 103, 111, 123] , and may continue to serve as key method for the identification of novel bunyavirus receptors [227] . Similarly, while integrated cryoEM/cryoET and X-ray studies have provided numerous insights into the glycoprotein structure of phenuiviruses, hantaviruses, nairoviruses, and orthobunyaviruses, the low level of glycoprotein sequence conservation across the order suggests that there is still much to learn about bunyavirus glycoprotein assembly and functionality. Indeed, this paucity of structural knowledge both hinders our understanding of how bunyaviruses utilize their glycoproteins to interact with specific host cell types and limits our ability to rationally target these important pathogens.

Since the recovery of BUNV by reverse genetics from cDNA clones of viral genomes [228] , numerous viruses from different families in the order Bunyavirales have successfully been rescued, including LCMV, JUNV, LASV, LUJV, Pichinde virus (PICV) (Arenaviridae) [229] [230] [231] [232] [233] [234] ; BUNV, LACV and AKAV, SBV, OROV, Cache Valley fever virus (CVV) and Kari virus (KRIV) (Peribunyaviridae) [228, [235] [236] [237] [238] [239] ; RVFV, UUKV and SFSTV (Phenuiviridae) [240] [241] [242] ; CCHFV and Hazara virus (HAZV) (Nairoviridae) [243, 244] , and TSWV (Tospoviridae) [245] . The broader availability of reverse genetics techniques will empower studies of bunyavirus pathobiology and aid the development of preventive strategies (e.g., vaccine and antiviral development) that may be used to protect against infection.

In summary, bunyaviruses constitute a growing order of biomedically and economically impactful pathogens. Continuation of the already successful efforts to characterize these viruses will not only enhance our appreciation of the seemingly limitless genomic, structural, and functional diversity within the order, but also enhance our preparedness for their future emergence. 

Taxonomy of the order Bunyavirales: Update

taxonomic update for phylum Negarnaviricota (Riboviria: Orthornavirae), including the large orders Bunyavirales and Mononegavirales

The WHO R&D blueprint: 2018 review of emerging infectious diseases requiring urgent research and development efforts

Virus Taxonomy

Lassa fever in West African sub-region: An overview

Emerging problems of Tospoviruses (Bunyaviridae) and their management in the Indian subcontinent

Exotic emerging viral diseases: Progress and challenges

The genus Tospovirus: Emerging Bunyaviruses that threaten food security

Early Bunyavirus-host cell interactions

Orthobunyaviruses: Recent genetic and structural insights

The molecular biology of nairoviruses, an emerging group of tick-borne arboviruses

Thrips transmission of Tospoviruses

Bunyaviruses: From transmission by arthropods to virus entry into the mammalian host first-target cells

Bunyavirus-vector interactions

Biology and molecular biology of viruses in the genus Tenuivirus

Nosocomial spread of viral disease

Person-to-person transmission of Andes virus

Super-Spreaders" and person-to-person transmission of Andes virus in Argentina

MAFFT multiple sequence alignment software version 7: Improvements in performance and usability

trimAl: A tool for automated alignment trimming in large-scale phylogenetic analyses

New algorithms and methods to estimate maximum-likelihood phylogenies: Assessing the performance of PhyML 3.0

Smart model selection in PhyML

Interactive Tree Of Life (iTOL) v4: Recent updates and new developments

Characterization of viruses in a tapeworm: Phylogenetic position, vertical transmission, and transmission to the parasitized host

Negative-strand RNA viruses: The plant-infecting counterparts

Current insights into research on rice stripe virus

The nucleotide sequence of the M RNA segment of tomato spotted wilt virus, a bunyavirus with two ambisense RNA segments

Nucleotide sequence and possible ambisense coding strategy of rice stripe virus RNA segment 2

Arenavirus genetic diversity and its biological implications

Hantaan virus MRNA: Coding strategy, nucleotide sequence, and gene order

Discovery of a unique novel clade of mosquito-associated bunyaviruses

Complete nucleotide sequence of the M RNA segment of Uukuniemi virus encoding the membrane glycoproteins G1 and G2

ICTV virus taxonomy profile: Fimoviridae

Rift Valley fever virus M segment: Phlebovirus expression strategy and protein glycosylation

Discovery and evolution of bunyavirids in arctic phantom midges and ancient bunyavirid-like sequences in insect genomes

Evolutionary and phenotypic analysis of live virus isolates suggests arthropod origin of a pathogenic RNA virus family

Crimean-Congo hemorrhagic fever virus glycoprotein proteolytic processing by subtilase SKI-1

Crimean-Congo hemorrhagic fever virus glycoprotein precursor is cleaved by furin-like and SKI-1 proteases to generate a novel 38-kilodalton glycoprotein

Genomic characterization of the genus Nairovirus (Family Bunyaviridae). Viruses

Molecular biology of tenuiviruses, a remarkable group of plant viruses

The evolutionary history of vertebrate RNA viruses

Processing and membrane topology of the spike proteins G1 and G2 of Uukuniemi virus

Bunyamwera orthobunyavirus glycoprotein precursor is processed by cellular signal peptidase and signal peptide peptidase

The Hantaan virus glycoprotein precursor is cleaved at the conserved pentapeptide WAASA

Signal peptidase I: Cleaving the way to mature proteins

Signal peptidase can cleave inside a polytopic membrane protein

Identification of signal peptide peptidase, a presenilin-type aspartic protease

Characterization of the glycoproteins of Crimean-Congo hemorrhagic fever virus

The Lassa virus glycoprotein precursor GP-C is proteolytically processed by subtilase SKI-1/S1P

ICTV virus taxonomy profile: Peribunyaviridae

Requirement of the N-terminal region of the orthobunyavirus non-structural protein NSm for virus assembly and morphogenesis

Understanding Rift Valley fever: Contributions of animal models to disease characterization and control

Rift Valley fever: Biology and epidemiology

Synthesis, proteolytic processing and complex formation of N-terminally nested precursor proteins of the Rift Valley fever virus glycoproteins

Molecular biology and genetic diversity of Rift Valley fever virus

Expression strategy of a phlebovirus: Biogenesis of proteins from the Rift Valley fever virus M segment

NSm and 78-kilodalton proteins of Rift Valley fever virus are nonessential for viral replication in cell culture

Rift Valley fever virus incorporates the 78 kDa glycoprotein into virions matured in mosquito C6/36 cells

The Rift Valley fever accessory proteins NSm and P78/NSm-GN are distinct determinants of virus propagation in vertebrate and invertebrate hosts

The C-terminal region of Rift Valley fever virus NSm protein targets the protein to the mitochondrial outer membrane and exerts antiapoptotic function

Complete nucleotide sequence of the M RNA segment of rift valley fever virus

Processing and intracellular localization of rice stripe virus Pc2 protein in insect cells

A global genomic characterization of Nairoviruses identifies nine discrete genogroups with distinctive structural characteristics and host-vector associations

Recent advances in research on Crimean-Congo hemorrhagic fever

Molecular insights into Crimean-Congo hemorrhagic fever virus

Identification of a novel C-terminal cleavage of Crimean-Congo hemorrhagic fever virus PreGN that leads to generation of an NSM protein

The interplays between Crimean-Congo hemorrhagic fever virus (CCHFV) M segment-encoded accessory proteins and structural proteins promote virus assembly and infectivity

Envelope glycoprotein of Arenaviruses

Cleavage of the glycoprotein of Arenaviruses

Site 1 protease is required for proteolytic processing of the glycoproteins of the South American hemorrhagic fever viruses Junin, Machupo, and Guanarito

Endoproteolytic processing of the lymphocytic choriomeningitis virus glycoprotein by the subtilase SKI-1/S1P

Identification of Lassa virus glycoprotein signal peptide as a trans-acting maturation factor

The signal peptide of the Junín Arenavirus envelope glycoprotein is myristoylated and forms an essential subunit of the mature G1-G2 complex

Bitopic membrane topology of the stable signal peptide in the tripartite Junín virus GP-C envelope glycoprotein complex

The curious case of Arenavirus entry, and its inhibition

Mapping the landscape of the lymphocytic choriomeningitis virus stable signal peptide reveals novel functional domains

Signal peptide requirements for lymphocytic choriomeningitis virus glycoprotein C maturation and virus infectivity

Viral entry

Virus entry: Open sesame

The cell biology of receptor-mediated virus entry

Dynamics of virus-receptor interactions in virus binding, signaling, and endocytosis

Lassa virus cell entry reveals new aspects of virus-host cell interaction

Novel insights into cell entry of emerging human pathogenic Arenaviruses

Entry of phenuiviruses into mammalian host cells

Entry of bunyaviruses into plants and vectors

Hantavirus entry: Perspectives and recent advances

Identification of α-dystroglycan as a receptor for lymphocytic choriomeningitis virus and Lassa fever virus

New World Arenavirus clade C, but not clade A and B viruses, utilizes α-dystroglycan as its major receptor

Lassa virus entry requires a trigger-induced receptor switch

Role of LAMP1 binding and pH sensing by the spike complex of Lassa virus

Transferrin receptor 1 is a cellular receptor for New World haemorrhagic fever Arenaviruses

Structural basis for receptor selectivity by the whitewater arroyo Mammarenavirus

New World clade B Arenaviruses can use transferrin receptor 1 (TfR1)-dependent and -independent entry pathways, and glycoproteins from human pathogenic strains are associated with the use of TfR1

A novel circulating tamiami mammarenavirus shows potential for zoonotic spillover

Cell surface molecules involved in infection mediated by lymphocytic choriomeningitis virus glycoprotein

Identification of cell surface molecules involved in dystroglycan-independent Lassa virus cell entry

Axl can serve as entry factor for Lassa virus depending on the functional glycosylation of dystroglycan

NRP2 and CD63 are host factors for Lujo virus cell entry

siRNA screen for genes that affect Junín virus entry uncovers voltage-gated calcium channels as a therapeutic target

Heparan sulfate proteoglycan is an important attachment factor for cell entry of Akabane and Schmallenberg viruses

DC-SIGN as a receptor for phleboviruses

Severe fever with Thrombocytopenia virus glycoproteins are targeted by neutralizing antibodies and can use DC-SIGN as a receptor for pH-dependent entry into human and animal cell lines

Cellular entry of hantaviruses which cause hemorrhagic fever with renal syndrome is mediated by β 3 integrins

β 3 Integrins mediate the cellular entry of hantaviruses that cause respiratory failure

Andes virus recognition of human and syrian hamster β 3 integrins is determined by an L33P substitution in the PSI domain

Protocadherin-1 is essential for cell entry by New World hantaviruses

Schönrich, G. β2 integrin mediates hantavirus-induced release of neutrophil extracellular traps

Hantavirus causing hemorrhagic fever with renal syndrome enters from the apical surface and requires decay-accelerating factor (DAF/CD55)

Equilibrium and kinetics of sin nombre hantavirus binding at DAF/CD55 functionalized bead surfaces

A hantavirus causing hemorrhagic fever with renal syndrome requires gC1qR/p32 for efficient cell binding and infection

Cellular entry of Hantaan virus A9 strain: Specific interactions with β 3 integrins and a novel 70 kDa protein

Analysis of the entry mechanism of Crimean-Congo hemorrhagic fever virus, using a vesicular stomatitis virus pseudotyping system

Identification of a putative Crimean-Congo hemorrhagic fever virus entry factor

Differential use of the C-Type lectins L-SIGN and DC-SIGN for phlebovirus endocytosis

Characterization of glycoprotein-mediated entry of severe fever with thrombocytopenia syndrome virus

Heparan sulfate facilitates Rift Valley fever virus entry into the cell

Bovine lactoferrin inhibits Toscana virus infection by binding to heparan sulphate

A haploid genetic screen identifies heparan sulfate proteoglycans supporting Rift Valley fever virus infection

RNASEK is required for internalization of diverse acid-dependent viruses

Nonmuscle myosin heavy chain IIA is a critical factor contributing to the efficiency of early infection of severe fever with thrombocytopenia syndrome virus

Efficient functional screening of a cellular cDNA library to identify severe fever with thrombocytopenia syndrome virus entry factors

The α-tubulin of Laodelphax striatellus mediates the passage of rice stripe virus (RSV) and enhances horizontal transmission

Immunoprecipitation of a 50-kDa protein: A candidate receptor component for tomato spotted wilt Tospovirus (Bunyaviridae) in its main vector, Frankliniella occidentalis

Interaction of Tomato Spotted Wilt Tospovirus (TSWV) glycoproteins with a thrips midgut protein, a potential cellular receptor for TSWV

Binding of tomato spotted wilt virus to a 94-kDa thrips protein

Discovery of novel thrips vector proteins that bind to the viral attachment protein of the plant bunyavirus tomato spotted wilt virus

Oropouche virus entry into HeLa cells involves clathrin and requires endosomal acidification

Orthobunyavirus entry into neurons and other mammalian cells occurs via clathrin-mediated endocytosis and requires trafficking into early endosomes

Single-particle tracking reveals the sequential entry process of the bunyavirus severe fever with thrombocytopenia syndrome virus

Entry of severe fever with thrombocytopenia syndrome virus

Hantaan virus enters cells by clathrin-dependent receptor-mediated endocytosis

Endocytic pathways used by Andes virus to enter primary human lung endothelial cells

Crimean-Congo hemorrhagic fever virus entry and replication is clathrin-, pH-and cholesterol-dependent

Crimean-Congo hemorrhagic fever virus utilizes a clathrin-and early endosome-dependent entry pathway

Characterization of Junin arenavirus cell entry

Arenavirus entry occurs through a cholesterol-dependent, non-caveolar, clathrin-mediated endocytic mechanism

Rift Valley fever virus strain MP-12 enters mammalian host cells via caveola-mediated endocytosis

Akabane virus utilizes alternative endocytic pathways to entry into mammalian cell lines

Entry of bunyaviruses into mammalian cells

Different mechanisms of cell entry by human-pathogenic Old World and New World arenaviruses

Cellular entry of lymphocytic choriomeningitis virus

Structures of enveloped virions determined by cryogenic electron microscopy and tomography

Proteomics computational analyses suggest that the carboxyl terminal glycoproteins of bunyaviruses are class II viral fusion protein (beta-penetrenes)

Structural basis of eukaryotic cell-cell fusion

Class II enveloped viruses

Shielding and activation of a viral membrane fusion protein

Chapter three-The envelope proteins of the Bunyavirales

Orthobunyavirus spike architecture and recognition by neutralizing antibodies

The hantavirus surface glycoprotein lattice and its fusion control mechanism

Acidic pH-induced conformations and LAMP1 binding of the Lassa virus glycoprotein spike

Structural basis for antibody-mediated neutralization of Lassa virus

Functional analysis of the Bunyamwera orthobunyavirus Gc glycoprotein

Visualizing the replication cycle of Bunyamwera orthobunyavirus expressing fluorescent protein-tagged gc glycoprotein

Mutagenesis of the La Crosse virus glycoprotein supports a role for Gc (1066-1087) as the fusion peptide

Crystal structure of glycoprotein C from Rift Valley fever virus

Crystal structure of glycoprotein C from a hantavirus in the post-fusion conformation

Mechanistic insight into bunyavirus-induced membrane fusion from structure-function analyses of the hantavirus envelope glycoprotein Gc

Orthobunyavirus ultrastructure and the curious tripodal glycoprotein spike

Averaging of viral envelope glycoprotein spikes from electron cryotomography reconstructions using Jsubtomo

A molecular-level account of the antigenic hantaviral surface

Structures of phlebovirus glycoprotein Gn and identification of a neutralizing antibody epitope

Crystal structure of tomato spotted wilt virus GN reveals a dimer complex formation and evolutionary link to animal-infecting viruses

Molecular rationale for antibody-mediated targeting of the hantavirus fusion glycoprotein

Structure of a phleboviral envelope glycoprotein reveals a consolidated model of membrane fusion

Glycoprotein organization of Chikungunya virus particles revealed by X-Ray crystallography

A protective monoclonal antibody targets a site of vulnerability on the surface of Rift Valley fever virus

Neutralization mechanism of human monoclonal antibodies against Rift Valley fever virus

The postfusion structure of the heartland virus Gc glycoprotein supports taxonomic separation of the bunyaviral families Phenuiviridae and Hantaviridae

A glycerophospholipid-specific pocket in the RVFV class II fusion protein drives target membrane insertion

Structure and characterization of Crimean-Congo hemorrhagic fever virus GP38

Variations in core packing of GP2 from old world mammarenaviruses in their post-fusion conformations affect membrane-fusion efficiencies

Insights into bunyavirus architecture from electron cryotomography of Uukuniemi virus

Three-dimensional organization of Rift Valley fever virus Revealed by cryoelectron tomography

Single-particle cryo-electron microscopy of Rift Valley fever virus

Electron cryo-microscopy and single-particle averaging of Rift Valley fever virus: Evidence for GN-GC glycoprotein heterodimers

Structural transitions of the conserved and metastable hantaviral glycoprotein envelope

The hantavirus glycoprotein G1 tail contains a dual CCHC-type classical zinc fingers

The structure of the hantavirus zinc finger domain is conserved and represents the only natively folded region of the Gn cytoplasmic tail

Distinction between Bunyaviridae genera by surface structure and comparison with Hantaan virus using negative stain electron microscopy

Molecular organization and dynamics of the fusion protein Gc at the hantavirus surface

Electron cryotomography of Tula hantavirus suggests a unique assembly paradigm for enveloped viruses

Structural studies of Hantaan virus

Recognition determinants of broadly neutralizing human antibodies against dengue viruses

Cryo-EM structures of Eastern equine encephalitis virus reveal mechanisms of virus disassembly and antibody neutralization

Functional and evolutionary insight from the crystal structure of rubella virus protein E1

The full-length cell-cell fusogen EFF-1 is monomeric and upright on the membrane

Potassium is a trigger for conformational change in the fusion spike of an enveloped RNA virus

Structural characterization of the Crimean-Congo hemorrhagic fever virus gn tail provides insight into virus assembly

The flavivirus precursor membrane-envelope protein complex: Structure and maturation

Expression and characterization of a soluble form of tomato spotted wilt virus glycoprotein GN

Genetic organisation of Iris yellow spot virus M RNA: Indications for functional homology between the G(C) glycoproteins of tospoviruses and animal-infecting bunyaviruses

Isolation, identification, and characterization of novel arenaviruses, the etiological agents of boid inclusion body disease

Spatial localization of the Ebola virus glycoprotein mucin-like domain determined by cryo-electron tomography

Characterization of Lassa virus glycoprotein oligomerization and influence of cholesterol on virus replication

Most neutralizing human monoclonal antibodies target novel epitopes requiring both Lassa virus glycoprotein subunits

Shedding of soluble glycoprotein 1 detected during acute Lassa virus infection in human subjects

Lassa virus glycoprotein: Stopping a moving target

Crystal structure of the prefusion surface glycoprotein of the prototypic arenavirus LCMV

Molecular mechanism for LAMP1 recognition by Lassa virus

Mapping of the Lassa virus LAMP1 binding site reveals unique determinants not shared by other old world arenaviruses

Structure-based classification defines the discrete conformational classes adopted by the arenaviral GP1

Unraveling virus relationships by structure-based phylogenetic classification

Unusual molecular architecture of the machupo virus attachment glycoprotein

Structural basis for receptor recognition by New World hemorrhagic fever arenaviruses

Convergent immunological solutions to Argentine hemorrhagic fever virus neutralization

Vaccine-elicited receptor-binding site antibodies neutralize two New World hemorrhagic fever arenaviruses

Molecular basis for antibodymediated neutralization of New World hemorrhagic fever mammarenaviruses

Native functionality and therapeutic targeting of arenaviral glycoproteins

Effective vaccine for Lassa fever

Safety, immunogenicity, and efficacy of the ML29 reassortant vaccine for Lassa fever in small non-human primates

Differential antibody-based immune response against isolated GP1 receptor-binding domains from Lassa and Junin viruses

Structural basis for receptor recognition by Lujo virus

Rational design of universal immunotherapy for TfR1-tropic arenaviruses

Role of DC-SIGN in Lassa virus entry into human dendritic cells

Convergent structures illuminate features for germline antibody binding and pan-Lassa virus neutralization

Identification of an N-terminal trimeric coiled-coil core within arenavirus glycoprotein 2 permits assignment to class I viral fusion proteins

X-Ray structure of the arenavirus glycoprotein GP2 in its postfusion hairpin conformation

Crystal structure of Venezuelan hemorrhagic fever virus fusion glycoprotein reveals a class 1 postfusion architecture with extensive glycosylation

Crystal structure of refolding fusion core of Lassa virus GP2 and design of Lassa virus fusion inhibitors

Structure of a zinc-binding domain in the Junin virus envelope glycoprotein

Hunting viral receptors using haploid cells

Rescue of a segmented negative-strand RNA virus entirely from cloned complementary DNAs

Recovery of an arenavirus entirely from RNA polymerase I/II-driven cDNA

Rescue of the prototypic arenavirus LCMV entirely from plasmid

Efficient reverse genetics generation of infectious Junin viruses differing in glycoprotein processing

Efficient rescue of recombinant Lassa virus reveals the influence of S segment noncoding regions on virus replication and virulence

Reverse genetics recovery of Lujo virus and role of virus RNA secondary structures in efficient virus growth

Development of infectious clones for virulent and avirulent pichinde viruses: A model virus to study arenavirus-induced hemorrhagic fevers

Efficient cDNA-based rescue of La crosse bunyaviruses expressing or lacking the nonstructural protein NSs

Rescue of Akabane virus (family Bunyaviridae) entirely from cloned cDNAs by using RNA polymerase I

Establishment of a reverse genetics system for Schmallenberg virus, a newly emerged orthobunyavirus in Europe

Generation of recombinant oropouche viruses lacking the nonstructural protein NSm or NSs

Development of reverse genetics systems and investigation of host response antagonism and reassortment potential for Cache Valley and Kairi viruses, two emerging orthobunyaviruses of the Americas

Rescue of infectious rift valley fever virus entirely from cDNA, analysis of virus lacking the NSs gene, and expression of a foreign gene

Generation of mutant Uukuniemi viruses lacking the nonstructural protein NSs by reverse genetics indicates that NSs is a weak interferon antagonist

Reverse genetics system for severe fever with thrombocytopenia syndrome virus

Recovery of recombinant Crimean Congo hemorrhagic fever virus reveals a function for non-structural glycoproteins cleavage by furin

Rescue of infectious recombinant hazara nairovirus from cDNA reveals the nucleocapsid protein DQVD caspase cleavage motif performs an essential role other than cleavage

Rescue of tomato spotted wilt virus entirely from complementary DNA clones

Acknowledgments: This paper is dedicated to the memory of our late colleague Richard M. Elliott who passed away in 2015. We thank Jayna Raghwani for helpful comments and advice.

The authors declare no conflict of interest.Viruses 2021, 13, 353