Abstract

Rodent-borne hantaviruses are human pathogens that can produce fatality rates up to 50%. While their internal structural proteins are conserved with other segmented negative-strand viruses, their Gn and Gc envelope glycoproteins are different. These glycoproteins are derived from a precursor protein and upon assembly onto viral particles, they form spikes with apparent 4-fould symmetry. These surface glycoproteins mediate viral cell entry by receptor binding and membrane fusion. While Gn remains largely uncharacterized and displays a new fold, Gc is known to mediate membrane fusion after viral uptake into endosomes. Recent structural studies of Gc confirm that it is folded as a class II viral fusion protein, such as those of phleboviruses (which form another genus in the Bunyaviridae family) and of flaviviruses and alphaviruses, which belong to different families of positive-sense, single-stranded RNA viruses. Here we aimed to characterize Gc in functional terms, including aspects of fusion activation and the relevance of certain novel structural features within the class II proteins, and challenged its structural classification mechanistically. Our results confirm that acidification is the trigger of membrane fusion, upon which Gc inserts into target membranes and undergoes homotrimerization into a stable post-fusion conformation. We further identified residues that drive membrane insertion and pore opening as well as residues promoting the stable, trimeric post-fusion structure. Finally, by using inhibitory strategies used against class II fusion proteins, the Gc fusion activity was arrested at a stage preceding hemifusion. Together, these data suggest that hantavirus Gc shares not only structural, but also mechanistic similarity with class II viral fusion proteins, while also presenting specific, yet undescribed features.