Interactions and Oligomerization of Hantavirus Glycoproteins

In this report the basis for the structural architecture of the envelope of hantaviruses, family Bunyaviridae, is systematically studied by the interactions of two glycoproteins N and C (Gn and Gc, respectively) and their respective disulfide bridge-mediated homo- and heteromeric oligomerizations. In virion extracts Gn and Gc associated in both homo- and hetero-oligomers which were, at least partially, thiol bridge mediated. Due to strong homo-oligomerization, the hetero-oligomers of Gn and Gc are likely to be mediated by homo-oligomeric subunits. A reversible pH-induced disappearance of a neutralizing epitope in Gc and dissociation of the Gn-Gc complex at pH values below 6.2 provide proteochemical evidence for the fusogenicity of Gc. Incomplete inactivation of virions at acidic pH indicates that additional factors are required for hantavirus fusion, as in the case of pestiviruses of the Flaviviridae. Based on similarities to class II fusion proteins, a structure model was created of hantavirus Gc using the Semliki Forest virus E1 protein as a template. In total, 10 binding regions for Gn were found by peptide scanning, of which five represent homotypic (Gn_I to Gn_V) and five represent heterotypic (Gc_I to Gc_V) interaction sites that we assign as intra- and interspike connections, respectively. In conclusion, the glycoprotein associations were compiled to a model wherein the surface of hantaviruses is formed of homotetrameric Gn complexes interconnected with Gc homodimers. This organization would create the grid-like surface pattern described earlier for hantaviruses in negatively stained electron microscopy specimens.Hantaviruses, a genus in the family Bunyaviridae, are rodent- and insectivore-borne zoonotic viruses that are seemingly apathogenic to the carrier rodents (39, 57). A number of hantaviruses are human pathogens that in areas of endemicity are responsible for two diseases: hemorrhagic fever with renal syndrome in Eurasia and hantavirus cardiopulmonary syndrome in the Americas (49, 57, 61). Hantaviruses are enveloped viruses and have a trisegmented, single-stranded, negative-sense RNA genome that encodes an RNA-dependent RNA polymerase, two glycoproteins, and a nucleocapsid protein (22, 34, 49, 60). During assembly, the four proteins and the RNA genome are packed into a round or a pleomorphic particle enveloped with a lipid bilayer. The interactions among the structural components of hantavirus have not been described in sufficient detail to construct the basic architecture of the virus particle or to understand the mechanisms of its assembly and entry.The envelope glycoproteins are expressed as a precursor polypeptide, which is cotranslationally cleaved after a conserved pentapeptide WAASA into an N- and C-terminal portion prior to maturation of the envelope glycoproteins proteins N and C (Gn and Gc, respectively) (27). In the family Bunyaviridae the transport of newly synthesized glycoproteins from endoplasmic reticulum to the Golgi apparatus requires the presence of both Gn and Gc (36, 37, 50, 53). Recombinant coexpression of the hantavirus glycoproteins is sufficient to achieve proper folding and the expected cellular localization at the Golgi even when the glycoproteins are not expressed from a common precursor (6, 36, 50). This suggests that the expression of the precursor is not a prerequisite for interactions between Gn and Gc during maturation in which the formation of a Gn-Gc complex results in exposure of a conformational Golgi apparatus-targeting signal, present only in the heterodimeric Gn-Gc complex (6, 50).Entry of enveloped viruses via recognition of the cell surface receptors and subsequent fusion of the virus and cell membranes are accomplished by viral glycoproteins which often appear in homo- and/or heteromeric complexes. For example, the E1 and E2 of Semliki Forest virus (SFV) form a trimer of heterodimers (45), and the E protein of tick-borne encephalitis virus (TBEV) forms a homodimer (41) while the hemagglutinin of influenza A virus (67) and the S protein of severe acute respiratory syndrome coronavirus associate in homotrimers (4, 5). The mature glycoproteins extracted from virions of Uukuniemi phlebovirus exist as homodimers (44), whereas glycoprotein complex formations of many other members of the Bunyaviridae have not been defined. The viral fusion proteins can be classified into class I, class II, and class III (25). Between classes I and II, a distinguishing property is the orientation of a fusion protein in the metastable state. The class I proteins are oriented perpendicular to the viral membrane, and the class II protein is parallel to the viral membrane (7). The class II viral fusion proteins assemble in virions as metastable homo- or heterodimeric complexes which, upon exposure to low pH, fuse the viral and target cellular membranes (7). This process begins with a conformational change in the fusion protein, leading to the revelation of its fusion loop, which binds to the cellular target membrane (7). Additionally, the formation of a homotrimeric fusion protein complex and structural changes that drive the fusion into completion occur (7).Understanding the multimeric status, protein-protein interactions, and pH-dependent conformational changes of glycoproteins is paramount to our understanding of selectivity in cell receptor binding and mechanisms of virus entry. It is unknown whether higher-order oligomeric complexes are found in hantavirus particles. Many neutralizing monoclonal antibodies (MAbs) have been isolated and by MAb escape mutants shown to recognize epitopes in both Gn and Gc, typically localized at discontinuous sites (15). Different neutralization mechanisms for hantavirus MAbs have been elucidated. These range from inhibiting receptor binding to inhibition of virus fusion (2, 23, 28, 30, 65). It is known that hantaviral glycoproteins possess fusogenic activity. Glycoproteins of hantaviruses that cause hemorrhagic fever with renal syndrome can induce syncytia when subjected to low pH (32, 35), and infection by Hantaan virus was shown to use low-pH-dependent clathrin-mediated endocytosis (19). Hantavirus Gc is suggested to be a class II fusion protein (13, 55), and the N-linked glycosylation of Gc is essential for cell fusion activity (70); but no clear understanding exists of the fusion mechanism or conformational changes that mediate uncoating of virions after entry.Our study supports the hypothesis that the Gc of hantaviruses is a class II fusion protein. We show the interaction between Gn and Gc to be pH sensitive and dissociation to start at a pH below 6.4. The low-pH-induced Gc dissociation from Gn was reversible, suggesting that the conformational changes in Gc are also reversible. Both glycoproteins were found to form homodimeric and hetero-oligomeric complexes in virion extracts through thiol bridging. Interaction studies further suggested that the protruding part of the spike complex in the hantavirus virion consists of four Gn subunits and that the spike complexes interconnect with homodimeric Gc subunits. Finally, we mapped and compiled the interaction sites of Gn and Gc proteins in a class II fusion protein three-dimensional (3D) model of Gc. The identified Gn-Gn, Gn-Gc, and Gc-Gc interaction sites may play an important role in glycoprotein folding and maturation, spike assembly, virus fusion, and neutralization of infection.