Abstract
Hantavirus assembly and budding are governed by the surface glycoproteins Gn and Gc. In this study, we investigated the glycoproteins of Puumala, the most abundant Hantavirus species in Europe, using fluorescently labeled wild-type constructs and cytoplasmic tail (CT) mutants. We analyzed their intracellular distribution, co-localization and oligomerization, applying comprehensive live, single-cell fluorescence techniques, including confocal microscopy, imaging flow cytometry, anisotropy imaging and Number&Brightness analysis. We demonstrate that Gc is significantly enriched in the Golgi apparatus in absence of other viral components, while Gn is mainly restricted to the endoplasmic reticulum (ER). Importantly, upon co-expression both glycoproteins were found in the Golgi apparatus. Furthermore, we show that an intact CT of Gc is necessary for efficient Golgi localization, while the CT of Gn influences protein stability. Finally, we found that Gn assembles into higher-order homo-oligomers, mainly dimers and tetramers, in the ER while Gc was present as mixture of monomers and dimers within the Golgi apparatus. Our findings suggest that PUUV Gc is the driving factor of the targeting of Gc and Gn to the Golgi region, while Gn possesses a significantly stronger self-association potential.
Highlights
Hantaviruses (HVs) pose a global health threat, infecting ca. 30000 individuals each year worldwide[1]
By assessing the oligomerization of the chimeric proteins via fluorescence anisotropy imaging microscopy (FAIM) and Number&Brightness (N&B) analysis, we demonstrate that Gn possesses a significantly higher tendency to oligomerize than Gc, supporting previous data that suggests that Gn may form the tetrameric spikes on the hantavirus surface[16,36]
The fluorescent tag was attached to the N-termini of the Puumala virus (PUUV) glycoproteins (Fig. 1b) to minimize any interference with putative interactions of cytoplasmic factors that could be required for glycoprotein trafficking and localization, since several important interaction sites and functional motifs have been identified within the cytoplasmic tail (CT) of Bunyavirus glycoproteins[13,16,27,37,38]
Summary
Hantaviruses (HVs) pose a global health threat, infecting ca. 30000 individuals each year worldwide[1]. The medium size vRNA segment (M-vRNA) encodes for two glycoproteins, Gn and Gc, which together form the spike complexes of the viral envelope[9]. Through its interaction with the viral nucleocapsid protein and the vRNA segments, Gn mediates the assembly and budding of infectious virus particles[12,13,14,15,16,17]. Where this process takes place is still under debate[11], but the Golgi complex is generally believed to be the major budding site of HVs18–20. The genesis of the spike complex has been only insufficiently characterized and novel studies of other members of the Bunyaviridae family revealed a surprisingly high diversity in the organization of the spike complexes[31,32,33,34,35]
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