The Envelope Proteins of the Bunyavirales

Abstract

The Bunyavirales Order encompasses nine families of enveloped viruses containing a single-stranded negative-sense RNA genome divided into three segments. The small (S) and large (L) segments encode proteins participating in genome replication in the infected cell cytoplasm. The middle (M) segment encodes the viral glycoproteins Gn and Gc, which are derived from a precursor polyprotein by host cell proteases. Entry studies are available only for a few viruses in the Order, and in each case they were shown to enter cells via receptor-mediated endocytosis. The acidic endosomal pH triggers the fusion of the viral envelope with the membrane of an endosome. Structural studies on two members of this Order, the phleboviruses and the hantaviruses, have shown that the membrane fusion protein Gc displays a class II fusion protein fold and is homologous to its counterparts in flaviviruses and alphaviruses, which are positive-sense, single-stranded RNA viruses. We analyze here recent data on the structure and function of the structure of the phlebovirus Gc and hantavirus Gn and Gc glycoproteins, and extrapolate common features identified in the amino acid sequences to understand also the structure and function of their counterparts in other families of the Bunyavirales Order. Our analysis also identified clear structural homology between the hantavirus Gn and alphavirus E2 glycoproteins, which make a heterodimer with the corresponding fusion proteins Gc and E1, respectively, revealing that not only the fusion protein has been conserved across viral families.

Introduction

The Bunyavirales Order is one of the largest group of RNA viruses, including more than 350 members distributed among nine families: Feraviridae, Fimoviridae, Hantaviridae, Jonviridae, Nairoviridae, Peribunyaviridae, Phasmaviridae, Phenuiviridae and Tospoviridae (https://talk.ictvonline.org/taxonomy/). Four of the nine families contain viruses that cause disease in humans, including, among others: the highly pathogenic Crimean-Congo hemorrhagic fever virus (CCHFV) in the Nairoviridae family; the Rift Valley fever virus (RVFV), the severe fever with thrombocytopenia syndrome virus (SFTSV) and Toscana virus in the Phenuiviridae family (genus Phlebovirus); Lacrosse virus and Oropouche virus in the Peribunyaviridae family (genus orthobunyavirus); and several hantaviruses such as Hantaan virus, Sin Nombre virus, Andes virus (Hantaviridae family). Hantaviruses, which cause persistent infections in rodents or in insectivores and often spill over to humans where they can cause severe disease, are the only bunyaviruses that are not arboviruses (short for arthropod-borne viruses). All the others, including the plant viruses in the family Tospoviridae, are transmitted by insect vectors (mosquitoes, sand flies, thrips) or by tick vectors. Bunyaviruses thus infect a broad range of hosts: vertebrates (in particular, mammals), invertebrates, and plants. Among the newly discovered bunyaviruses are: Gouleako virus (family Phenuiviridae, genus Goukovirus), which is similar to the phleboviruses but infects only mosquitoes (Marklewitz et al., 2011) and is not transmitted to vertebrates or plants; the Jonchet (family Jonviridae), Ferak (family Feraviridae), and Herbert virus (family Peribunyaviridae, genus Herbevirus) groups, which have been isolated from mosquitoes in Côte d’Ivoire, Ghana, and Uganda and replicate efficiently in insect cells but not in mammalian cells (Marklewitz et al., 2013, Marklewitz et al., 2015); and phasmaviruses (Phasmaviridae family), which were discovered in arctic phantom midges (Ballinger et al., 2014). Many other bunyaviruses have been detected and sequenced using metagenomics approaches on arthropods (Li et al., 2015) and other invertebrate species (Shi et al., 2016a), but have not yet been isolated and characterized.

Bunyavirus particles are in general spherical, with a diameter of about 80–120 nm, and their morphology has been studied by a number of biochemical, biophysical, and electron microscopy studies (Battisti et al., 2011, Bowden et al., 2013, Freiberg et al., 2008, Hepojoki et al., 2010, Huiskonen et al., 2009, Huiskonen et al., 2010, Martin et al., 1985, Overby et al., 2008, Rusu et al., 2012, Salanueva et al., 2003, Sherman et al., 2009). Viruses in some of the families (like the hantaviruses) also exhibit elongated particles. The virion contains a negative- or ambisense single-stranded RNA genome divided into three segments, called S (small), M (medium), and L (large) (Fig. 1) (Elliott and Schmaljohn, 2013). The S and the L segments code for the proteins required for genome replication within a cell: the nucleocapsid protein (N), which encapsidates the genomic RNA into ribonucleoprotein complexes is encoded in the S segment, and the large RNA-dependent RNA-polymerase (or “L” protein), responsible for transcription and replication of the genomic RNA, in the L segment. Some genera code for a nonstructural protein (NSs) in the S segment. The L and S segments are very similar to the two segments of the Arenaviridae family of bisegmented ambisense RNA viruses, and very likely have the same origin, except that in arenaviruses the S gene also codes for the envelope protein (Buchmeier et al., 2013).

The M segment of the bunyaviruses codes for proteins required in particle budding and for entry into target cells, in the form of a polyprotein precursor of the envelope glycoproteins Gn and Gc. As discussed later, the M gene appears to be related to the structural genes of positive strand viruses of the Togaviridae (Kuhn, 2013) and Flaviviridae (Lindenbach et al., 2013) families, which appear to have evolved from a distant ancestor in common with the bunyavirus M gene. This finding is a further illustration of the mosaic nature of viral genomes, which are a patchwork of genes having different origins. Some bunyavirus families also encode one or several nonstructural proteins (NSm) in the M segment (Fig. 1). The individual glycoproteins Gn and Gc—which are the focus of this review—are generated by signalase cleavage in the ER, followed by additional processing depending on the family. Gn/Gc heterodimers associate in the ER and travel to the site of particle morphogenesis (Petterson, 1996). Budding was shown to occur in the Golgi apparatus, although some hantaviruses were reported to bud at the plasma membrane (Goldsmith et al., 1995). Gn/Gc heterodimers associate into larger complexes that interact laterally to drive budding of newly formed virions by inducing membrane curvature, resulting in closed particles with projections 5–10 nm long at the viral surface. There is no proteolytic maturation processing of the envelope proteins upon assembly in the Golgi, and the “priming” process necessary for activation by the endosomal acidic pH upon entering a new cell is not currently understood. This is an important difference with flaviviruses (Flaviviridae family) and alphaviruses (Togaviridae family), which have a similar envelope protein organization, but require cleavage of a companion, chaperone protein occupying the place of Gn in the precursor polyprotein of those viruses. In this respect, the bunyaviruses are more similar to Rubella virus (Togaviridae family), which also has a similar envelope protein organization with no maturation cleavage (Kuhn, 2013).