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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.
Antibodies against spike proteins of influenza are used as a tool for characterization of viruses and therapeutic approaches. However, development, production and quality control of antibodies is expensive and time consuming. To circumvent these difficulties, three peptides were derived from complementarity determining regions of an antibody heavy chain against influenza A spike glycoprotein. Their binding properties were studied experimentally, and by molecular dynamics simulations. Two peptide candidates showed binding to influenza A/Aichi/2/68 H3N2. One of them, termed PeB, with the highest affinity prevented binding to and infection of target cells in the micromolar region without any cytotoxic effect. PeB matches best the conserved receptor binding site of hemagglutinin. PeB bound also to other medical relevant influenza strains, such as human-pathogenic A/California/7/2009 H1N1, and avian-pathogenic A/MuteSwan/Rostock/R901/2006 H7N1. Strategies to improve the affinity and to adapt specificity are discussed and exemplified by a double amino acid substituted peptide, obtained by substitutional analysis. The peptides and their derivatives are of great potential for drug development as well as biosensing.