Self-association and subcellular localization of Puumala hantavirus envelope proteins

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.

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

“…These observations are consistent with the small Gc:Gc contact patch, which appears too weak to maintain Gc dimers in solution. Similarly, membrane-anchored Gc expression in the absence of Gn was predominantly monomeric in live cells, although some Gc dimers were detected (Sperber et al, 2019). Therefore, it is likely that Gc anchoring in the context of lateral inter-spike constrains may be required for efficient Gc:Gc association.…”

“…A higher resolution 15.6 Å cryo-ET map allowed the docking of the Gn ectodomain into the central lobes on the tetrameric spike, at the membrane distal side, and masking the Gc fusion loops, suggesting that the 2-fold related spike-spike interactions are made by the Gc moiety (Li et al, 2016). Moreover, a central role of Gn in self-association to form spikes has recently been confirmed by number and brightness analysis in single live cells showing that separate Gn expression allows detection of Gn oligomers while separate Gc expression predominantly leads to Gc monomers and some Gc dimers (Sperber et al, 2019).…”

Detection of Envelope Glycoprotein Assembly from Old World Hantaviruses in the Golgi Apparatus of Living Cells

“…Unless otherwise specified, glycoprotein gene sequences refer to the PUUV strain. Original plasmids of signal peptide (SP)-mEYFP-Gn and SP-mEYFP-Gc were previously described (16). Plasmids encoding Gn and Gc preceded by three HA tags (HAtag-Gn, HAtag-Gc) were a gift from A. Herrmann (Humboldt Universität zu Berlin).…”

“…Early studies that aimed to localize singularly expressed Gn and Gc showed variations for different members of the Bunyavirales order (10,11). However, co-expression of both HV GPs results, in general, in their translocation to the Golgi apparatus (GA) (12)(13)(14)(15)(16). This holds true also for recombinant proteins that are not derived from a common precursor (17).…”

“…This approach was based on the bulk analysis of several cells, in a restricted protein concentration range, and required some assumptions on the actual presence of specific multimeric states (e.g. Gn tetramers) (16).…”