GABAA receptor binding is transiently increased in rat whisker barrels during the second postnatal week, at a time when neurons in the developing rat cortex are vulnerable to excitotoxic effects. To test whether these GABAA receptors might serve to protect neurons from excessive excitatory input, polymer implants containing the GABAA receptor antagonist bicuculline were placed over barrel cortex for a 4-day period in young (postnatal days 8 - 12) and adult rats. In the cortex of young, but not adult rats, the chronic blockade of GABAA receptors resulted in substantial tissue loss and neuron loss. The greater loss of neurons in young rats supports the hypothesis that a high density of GABAA receptors protects neurons from excessive excitatory input during a sensitive period in development.

Traditional bacterial disease therapies utilize compounds that ultimately kill the target bacteria but it exerts a strong selective pressure on the bacteria to develop multi-drug resistance mutants. The increasing occurrence of resistance in common pathogens has highlighted the need to identify new anti-microbials that target the control of bacterial pathogenicity in a non-extermination manner to reduce the incidence of bacteria resistance. One new strategy exploits the discrete signaling molecules that regulate the various bacterial signaling pathways, which are responsible for the expression of pathogenicity traits. Halogenated furanones (fimbrolides) from the marine red alga, Delisea pulchra have been shown to interfere with the key signaling pathway present in Gram-negative bacteria by competitively displacing the cognate signaling molecule from the transcription protein. This project focused on the design and synthesis of 1,5-dihydropyrrol-2-ones, a new class of fimbrolide derivatives capable of displaying strong antagonistic properties of the fimbrolides. Primary synthetic methodologies examined include the halolactamization of allenamides and the direct lactone-lactam transformation. No doubt, both methodologies yielded the lactam ring, the former failed to introduce the crucial C-5 bromomethylene group essential for bioactivity. A facile high yielding two-step lactone-lactam transformation method was developed and using this method, a wide range of substituted 5-bromomethyl- and 5-dibromomethylene-1,5-dihydropyrrol-2-ones were synthesized. Furthermore, a new class of tricyclic crown-ether type compounds with no literature precedent were discovered. To vary the diversity of the compounds, a related class of compounds, 5,6-dihydroindol-2-ones, were examined. A general versatile method for the synthesis of 7-substituted 5,6-dihydroindol-2-ones was developed. The synthetic strategy proceeds via the established Suzuki-Miyaura cross-coupling reaction of halogenated dihydroindol-2-ones with arylboronic acids/esters. The Suzuki methodology was found to be reliable in furnishing a wide range of 7-substituted products in high yields. A preliminary molecular modeling approach was used to assist in the design of new anti-microbials via the ligand-docking analyses of the TraR and LasR protein. A positive correlation was observed between the docking scores and biological activity and the methodology was further developed into an initial screening tool to filter potential active and non-active compounds. The newly synthesized compounds were analysed for their efficacy in reducing the expression of the Green Fluorescent Protein (GFP) in the presence of natural AHL signaling molecules in an AHL-monitor strain, indicative of the inhibition of bacterial phenotype expression. The dihydropyrrol-2-one class of compounds showed significant biological activity and this highlighted their potential for further development.

The G-protein coupled receptors (GPCRs) comprise the largest family of receptors in humans and other vertebrates. They are embedded in the cell membrane and are activated by many different signaling molecules. Activation modulates cellular signal transduction pathways and influences many physiological processes. Therefore the GPCRs are important as targets for numerous drugs. The receptors for NPY (neuropeptide Y) belong to GPCRs of Class A (rhodopsin-like). NPY and its related peptides PYY and PP are involved in the regulation of appetite, blood pressure and many other processes. They share a common structure and interact with the receptors Y1, Y2, Y4 and Y5 in mammals, and, in addition, Y7 and Y8 in amphibians and bony fishes. This thesis is focused on the human Y2 receptor, known to reduce appetite, by investigating the importance of thirteen amino acid residues for ligand binding. Mutagenesis followed by functional expression and receptor binding was conducted. During the course of this work several new GPCR crystal structures have been resolved, thereby improving the receptor modeling in papers I-III. The major finding is that even though the Y1 and Y2 receptors have evolved from a common ancestor, their points of ligand interaction differ and have thus changed during evolution. In general, the positions investigated resulted in milder changes in the ligands’ affinities for Y2 compared to Y1. These findings were incorporated in the design of new Y1 and Y2 receptor models, leading to improved understanding of how such divergent receptors, sharing only 30 percent sequence identity, can still interact with the same ligands. Notably, several of the mutations introduced in Y2 resulted in increased affinity. A novel NPY receptor gene named Y2-2 was identified in the genomes of zebrafish and medaka. This brings the number of zebrafish NPY receptors to seven. The binding characteristics of zebrafish Y2-2 differed from zebrafish Y2 mainly in the interaction with NPY13-36 and the antagonist BIIE0246. In conclusion, these results increase our understanding of ligand interactions with GPCRs and will be useful for refinement of ligand-receptor models for future development of receptor subtype-selective drugs.

Traditional bacterial disease therapies utilize compounds that ultimately kill the target bacteria but it exerts a strong selective pressure on the bacteria to develop multi-drug resistance mutants. The increasing occurrence of resistance in common pathogens has highlighted the need to identify new anti-microbials that target the control of bacterial pathogenicity in a non-extermination manner to reduce the incidence of bacteria resistance. One new strategy exploits the discrete signaling molecules that regulate the various bacterial signaling pathways, which are responsible for the expression of pathogenicity traits. Halogenated furanones (fimbrolides) from the marine red alga, Delisea pulchra have been shown to interfere with the key signaling pathway present in Gram-negative bacteria by competitively displacing the cognate signaling molecule from the transcription protein. This project focused on the design and synthesis of 1,5-dihydropyrrol-2-ones, a new class of fimbrolide derivatives capable of displaying strong antagonistic properties of the fimbrolides. Primary synthetic methodologies examined include the halolactamization of allenamides and the direct lactone-lactam transformation. No doubt, both methodologies yielded the lactam ring, the former failed to introduce the crucial C-5 bromomethylene group essential for bioactivity. A facile high yielding two-step lactone-lactam transformation method was developed and using this method, a wide range of substituted 5-bromomethyl- and 5-dibromomethylene-1,5-dihydropyrrol-2-ones were synthesized. Furthermore, a new class of tricyclic crown-ether type compounds with no literature precedent were discovered. To vary the diversity of the compounds, a related class of compounds, 5,6-dihydroindol-2-ones, were examined. A general versatile method for the synthesis of 7-substituted 5,6-dihydroindol-2-ones was developed. The synthetic strategy proceeds via the established Suzuki-Miyaura cross-coupling reaction of halogenated dihydroindol-2-ones with arylboronic acids/esters. The Suzuki methodology was found to be reliable in furnishing a wide range of 7-substituted products in high yields. A preliminary molecular modeling approach was used to assist in the design of new anti-microbials via the ligand-docking analyses of the TraR and LasR protein. A positive correlation was observed between the docking scores and biological activity and the methodology was further developed into an initial screening tool to filter potential active and non-active compounds. The newly synthesized compounds were analysed for their efficacy in reducing the expression of the Green Fluorescent Protein (GFP) in the presence of natural AHL signaling molecules in an AHL-monitor strain, indicative of the inhibition of bacterial phenotype expression. The dihydropyrrol-2-one class of compounds showed significant biological activity and this highlighted their potential for further development.

Influenza A viruses (IAVs) cause occasional pandemics and seasonal epidemics, thus presenting continuous challenges to public health. Vaccination is the primary strategy for the prevention and control of influenza outbreaks. The antigenicity matched high-yield seed strain is critical for the success of influenza vaccine. Currently, there are several limitations for the influenza vaccine manufacture: 1) the conventional methods for generating such strains are time consuming; 2) egg-based vaccines, the predominant production platform, have several disadvantages including the emergence of viral antigenic variants that can be induced during egg passage; 3) vaccine seed viruses often do not grow efficiently in mammalian cell lines. Previous studies suggested that mutations in the receptor binding site (RBS) that locates at the globular head of the HA1 can change IAVs’ binding specificity, antigenicity, and yield and thus RBS would be an potential target for engineering vaccine seed strain. However, systematic analysis of the mutations on RBS affecting those viral phenotypes is lacking. Specifically, this dissertation has following aims: Firstly, we developed a novel method to rapidly generate high-yield candidate vaccine strains by integrating error-prone PCR, site-directed mutagenesis strategies, and reverse genetics. The error-prone PCR- based reverse genetic system could also be applied to gain-ofunction studies for influenza virus and other pathogens; Secondly, in this dissertation, we identified an Y161F mutation in the hemagglutinin (HA) that enhanced the infectivity and thermostability of virus without changing its original antigenic properties which would prompted the development of cell-based vaccines; Thirdly, the molecular mechanisms underlying host adaption of equine-origin influenza A(H3N8) virus from horses to dogs are unknown. This dissertation identified that a substitution of W222L in the HA of the equine-origin A(H3N8) virus facilitated its host adaption to dogs. This mutation increased binding avidity of the virus specifically to sialyl Lewis X motifs, which were found abundantly in the submucosal glands of dog trachea but not in equine trachea. To summary, this dissertation investigated the role of RBS in IAVs biology and expanded the current knowledge toward IAV vaccine strain engineering, IAV host adaption and evolution.

Extracellular Vesicles (EVs) are small membranous particles secreted by the cells. They play an important role in intercellular communication and can transport a variety of biomolecules, including proteins, lipids, and nucleic acids, to target cells. The scientific community recently considered EVs attractive candidates for developing targeted drug delivery systems (DDSs), given their biocompatibility, low immunogenicity, stability in biofluids, and capability to cross biological barriers. Most studies have shown the feasibility of incorporating desired moieties at the EV surface through the genetic modification of EV-producing cells, exploiting the fusion with proteins enriched at the EV membrane. Tetraspanins are transmembrane proteins enriched in EVs, already exploited for EV isolation or tracking upon fusion with fluorescent reporters. CD81 is a well-characterized tetraspanin with ubiquitous protein expression, overexpression tolerance and a limited number of encoded protein isoforms with respect to other EV-associated tetraspanins. Here, I have explored a CD81-based approach for EV targeting. CD81, in full-length or truncated form, was used to guide the expression into EVs of an anti-HER2 moiety, namely the light chains of trastuzumab, within three different constructs, including turbo-GFP (tGFP) as a reporter: CD81-tGFP as master control, CD81-antiHER2-tGFP and CD81delta-antiHER2-tGFP. The first part of the project was dedicated to the characterization of chimeric proteins at cellular and vesicular levels. CD81-based constructs were successfully expressed in HEK239T cells with a preferential enrichment in organelle fractions, underlying the expected involvement in the intracellular vesicular trafficking. Next, chimeric proteins were also found in the derived EVs, with a similar expression trend, corroborated by imaging flow cytometry. Nanoparticle tracking analysis and cryogenic electron microscopy acquisitions confirmed that CD81-fusion proteins boosted EV release without altering the size distribution. Subsequently, I tested the binding capacity of the chimeric proteins to HER2 receptor through orthogonal techniques, such as AlphaLISA and immunoprecipitation. Confocal imaging, also on live cells, confirmed EV internalization into breast cancer cells, depending on the recipient cell type and the presence of HER2 receptor. Moreover, chimeric EVs loaded with doxorubicin were able to mediate a concentration-dependent cytotoxic effect on recipient breast cancer cells. Of note, messenger RNA provided a valuable readout of the in vivo delivery capability of the CD81-engineered EVs, since detected by digital droplet PCR in breast cancer tumour xenografts from mice treated with chimeric EVs. The results presented in this thesis highlighted the feasibility of using CD81 fusion proteins for cell targeting and cargo delivery, ultimately opening new perspectives for the development of EV-based therapeutics.

Wild birds are the natural reservoirs of Influenza A viruses (IAVs) which cause occasional pandemics and seasonal epidemics. Avian IAVs can be transmitted from wild birds to domestic poultry, low mammals, and humans. It is well accepted that avian IAVs prefer to sialic acids (Sia) α2,3-linked galactose (SAα2,3-Gal), whereas human IAVs to α2,6-linked galactose (SAα2,6-Gal). However, SAα2,3-Gal in wild bird tissues is widely distributed with little variation while some subtypes of avian IAVs have species preference. The different isolation rates among wild bird species cannot be explained by avian IAVs binding to SAα2,3-Gal alone. Specifically, this dissertation had the following aims: Firstly, to determine distribution of glycan receptors across respiratory and gastrointestinal tissues of wild birds and domestic poultry; Secondly, to determine the viral-receptor binding specificity of avian IAVs; Thirdly, to understand the role of glycan motifs in shaping virus evolution during the natural history of IAVs, especially from wild bird to poultry transmission. We found that avian H7 and H10 viruses acquired the binding ability to SAα2,6-Gal without adaptation, furthermore, we evaluated one of these H10 virus that possess the ability of binding to SAα2,6-Gal in ferret model and found it could cause aerosol and contact transmissions. On the other hand, H7 viruses have strong binding avidity to SLex which are present widely in epithelial cells of chicken trachea, which could facilitate the transmission of avian H7 viruses from waterfowl to poultry. Lastly, we found that H7 viruses from waterfowl bound both Neu5Ac and Neu5Gc while chicken isolates from China only prefer to Neu5Ac. Of interest, we found Neu5Gc was found in mallards but not in chickens, which indicated that viruses reduce or lost binding ability to Neu5Gc by adapting in chicken. In summary, this dissertation focused on certain subtypes of avian IAVs, which have caused threats to domestic poultry and public health, and primary avian species for influenza risk. The glycan substructures may play an important role in avian IAVs transmission and adaptation. The knowledge derived from this dissertation will help identify species for influenza surveillance in wild birds and facilitate risk assessment of avian IAVs.

<p> This study addresses the issue of how to convert peptides into drug-like non-peptides while retaining the biological activity at peptide receptors. Angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe, Ang II) was used as a model peptide.</p><p> Small bioactive peptides are in most cases conformationally flexible molecules. Rigidified peptide analogues or peptidomimetic scaffolds can be introduced into the peptide, to enforce a particular backbone conformation, and thereby locate the side-chains at defined positions in space. The conformationally constrained analogues are of considerable value in determining biologically active conformation(s) of the studied peptide. The strategy applied in this thesis includes identification of non-pharmacophoric amino acid residues, rigidification, conformational analysis and incorporation of turn mimicking scaffolds in </p><p>Ang II. Several side-chain cyclized (disulfide and methylendithioether) Ang II analogues have been synthesized. The binding studies of the rigidified analogues demonstrated that the compounds designed for the AT₁-receptor had affinity for both receptor subtypes, while the compounds designed for the AT₂-receptor displayed high selectivity only for this receptor subtype. Conformational evaluation revealed that several of the cyclized Ang II analogues most probably adopt a <i>γ</i>-turn like conformation around Tyr-4 while interacting with the </p><p>Ang II receptor. Based on this hypothesis, three different <i>γ</i>-turn mimetics replacing amino acid residues 3-5 were designed, synthesized and incorporated into Ang II. One of the synthesized pseudopeptides, incorporating an azepine-containing <i>γ</i>-turn mimetic, exerted high binding affinity and agonistic activity. These results strongly support the theory that Ang II adopts a <i>γ</i>-turn like conformation when activating the AT₁ receptor. The other Ang II analogues, incorporating bicyclic and aromatic <i>γ</i>-turn mimetics, did not display any binding to the AT₁ receptor.</p>

This study addresses the issue of how to convert peptides into drug-like non-peptides while retaining the biological activity at peptide receptors. Angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe, Ang II) was used as a model peptide. Small bioactive peptides are in most cases conformationally flexible molecules. Rigidified peptide analogues or peptidomimetic scaffolds can be introduced into the peptide, to enforce a particular backbone conformation, and thereby locate the side-chains at defined positions in space. The conformationally constrained analogues are of considerable value in determining biologically active conformation(s) of the studied peptide. The strategy applied in this thesis includes identification of non-pharmacophoric amino acid residues, rigidification, conformational analysis and incorporation of turn mimicking scaffolds in Ang II. Several side-chain cyclized (disulfide and methylendithioether) Ang II analogues have been synthesized. The binding studies of the rigidified analogues demonstrated that the compounds designed for the AT1-receptor had affinity for both receptor subtypes, while the compounds designed for the AT2-receptor displayed high selectivity only for this receptor subtype. Conformational evaluation revealed that several of the cyclized Ang II analogues most probably adopt a γ-turn like conformation around Tyr-4 while interacting with the Ang II receptor. Based on this hypothesis, three different γ-turn mimetics replacing amino acid residues 3-5 were designed, synthesized and incorporated into Ang II. One of the synthesized pseudopeptides, incorporating an azepine-containing γ-turn mimetic, exerted high binding affinity and agonistic activity. These results strongly support the theory that Ang II adopts a γ-turn like conformation when activating the AT1 receptor. The other Ang II analogues, incorporating bicyclic and aromatic γ-turn mimetics, did not display any binding to the AT1 receptor.

Spumaretroviren, oder Foamyviren (FV), unterscheiden sich von Orthoretroviren durch mehrere Besonderheiten in ihrer Replikationsstrategie. Das Partikel-assoziierte Hüllglykoprotein (Env-Protein) des „Prototype Foamy Virus“ (PFV) ist im Vergleich zu anderen retroviralen Hüllglykoproteinen einzigartig. Die Koexpression des PFV Env-Proteins für die PFV-Partikelfreisetzung ist essenziell und die spezifische Funktion kann nicht von heterologen viralen Env-Proteinen übernommen werden. Das Env-Protein des PFV durchläuft eine für ein Membranglykoprotein ungewöhnliche Biosynthese. Das Env-Vorläuferprotein besitzt zu Beginn eine Typ-III-Membrantopologie, bei der der N- und der C-Terminus im Zytoplasma lokalisiert sind. Während des Transports zur Zelloberfläche wird es posttranslational durch bisher unbekannte zelluläre Proteasen in mindestens drei Untereinheiten gespalten. Das N-terminale Signalpeptid bzw. Leader-Peptid (LP) hat eine Typ-II-Membrantopologie, mit dem N-Terminus im Zytoplasma und dem C-Terminus im Lumen, wohingegen die Transmembran (TM)-Untereinheit eine Typ-IMembrantopologie besitzt, bei der der N-Terminus im Lumen und der C-Terminus im Zytoplasma lokalisiert sind. Die interne Oberflächen (SU)-Untereinheit assoziiert vermutlich im Lumen mit der extrazellulären Domäne der TM-Untereinheit. Im Rahmen dieser Arbeit wurde der Beweis erbracht, dass Furin oder Furin-ähnliche Proteasen und nicht der Signalpeptidase-Komplex für beide proteolytischen Spaltungen verantwortlich sind. Durch die N-terminale Sequenzierung der SU- und der TM-Untereinheit eines aufgereinigten PFV Env-Immunoadhäsionsproteins wurden N-terminal von beiden Spaltstellen Furin- Konsensussequenzen identifiziert. Mutationsanalysen von zwei sich in diesem Bereich überlappenden minimalen Furin-Konsensussequenzen an der PFV LP/SU-Spaltstelle im wildtypischen PFV Env-Protein bestätigten die Ergebnisse der N-terminalen Sequenzierung und bewiesen, dass nur die erste Spaltstelle genutzt wird. Obwohl diese Mutanten aufgrund geringerer Partikelfreisetzung einen signifikanten Verlust der Infektiosität zeigten, wurde keine Korrelation zur Inhibierung der Spaltung beobachtet, da andere Mutanten mit normaler LP/SU-Spaltung einen ähnlichen Defekt besaßen. Virale Env-Proteine initiieren den Eintritt membranumhüllter Viren in die Wirtszelle durch die Bindung an zelluläre Rezeptoren. Dabei führen Konformationsänderungen in den Env- Proteinen zum Verschmelzen der Virusmembran mit der Zellmembran und weiterhin zur Aufnahme des Kapsids in das Zytoplasma der Wirtszelle. Die foamyviralen Env-Proteine sind in dieser Hinsicht keine Ausnahme und vermitteln die Anheftung an die Wirtszelle durch die Bindung an den bisher unbekannten zellulären Rezeptor. Der zelluläre foamyvirale Rezeptor ist vermutlich ein ubiquitäres Molekül, denn bisher konnte keine Zelllinie identifiziert werden, die gegen FV-Infektionen resistent ist. Bislang existieren nur sehr wenig strukturelle und funktionelle Informationen der extrazellulären Domänen des PFV Env-Proteins. Deshalb wurde im Hauptteil dieser Arbeit die PFV Env-Rezeptorbindungsdomäne (RBD) charakterisiert. Hierfür wurden rekombinante PFV Env-Immunoadhäsionsproteine verwendet und deren Bindungskapazitäten an Zielzellen in der durchflusszytometrischen Analyse bestimmt. Untersuchungen zeigten, dass sowohl die extrazelluläre Domäne der C-terminalen TM-Untereinheit als auch der Transport der Immunoadhäsionsproteine durch das spezifische PFV Env LP zum sekretorischen Weg für die Bindung an Zielzellen entbehrlich sind und ließen vermuten, dass die PFV Env-RBD innerhalb der SU-Untereinheit lokalisiert ist. N- und C-terminale Deletionsanalysen der PFV Env SU-Untereinheit enthüllten eine minimale kontinuierliche RBD von AS 225 bis 555. Interne Deletionen im PFV Env-Protein von AS 397 bis 483 wurden im Gegensatz zu deletierten Regionen von AS 262 bis 300 und AS 342 bis 396 ohne signifikanten Einfluss auf die Wirtszellbindung in Immunoadhäsionsproteinen toleriert. Die Analyse der Immunoadhäsionsproteine mit einzelnen substituierten Cysteinen in der PFV Env SU-Untereinheit zeigten, dass nur die Immunoadhäsionsproteine, die in der nicht essenziellen Region von AS 397 bis 483 lokalisierte Cysteine ersetzt hatten, eine Restbindungskapazität behielten. Interessanterweise zeigte die Analyse von verschiedenen N-Glykosylierungsmutanten eine bedeutende Rolle der Kohlenhydratkette an Position N391 im PFV Env-Protein entweder hinsichtlich der direkten Interaktion mit dem zellulären Rezeptor oder für die korrekte Faltung der PFV Env-RBD. Diese Ergebnisse weisen darauf hin, dass ein diskontinuierliches Sequenzmotiv von AS 225 bis 396 und AS 484 bis 555 für die Bildung der PFV Env-RBD essenziell ist und die darin lokalisierte potenzielle achte N-Glykosylierungsstelle eine entscheidende Rolle bei der Wirtszellbindung spielt. / Spumaretroviruses or foamy viruses (FVs) use a replication pathway with features distinctive from orthoretroviruses. The particle-associated envelope (Env) glycoprotein of prototype foamy virus (PFV) is unique compared to other retroviral envelope proteins since its coexpression is strictly required for the FV particle release process and its function cannot be replaced by heterologous viral glycoproteins. The PFV Env glycoprotein shows a highly unusual biosynthesis. Its precursor protein has a type III membrane topology with both the N-and C-terminus located in the cytoplasm. During its transport to the cell surface, it is posttranslationally processed by yet-unidentified cellular proteases into at least three subunits. The N-terminal signal or leader peptide (LP) has a type II membrane topology, whereas the C-terminal transmembrane (TM) subunit has a type I membrane topology. The internal surface (SU) subunit presumably associates with extracellular domains of TM on the luminal side. Here we provide strong evidence that furin itself or furin-like proteases and not the signal peptidase complex are responsible for both processing events. N-terminal protein sequencing of the SU and TM subunits of purified PFV Env-immunoglobulin immunoadhesin identified furin consensus sequences upstream of both cleavage sites. Mutagenesis analysis of two overlapping minimal furin consensus sequences at the PFV LP/SU cleavage site in the wild-type protein confirmed the sequencing data and demonstrated utilization of only the first site. Although these mutants displayed a significant loss in infectivity as a result of reduced particle release, no correlation to processing inhibition was observed, since another mutant having normal LP/SU processing had a similar defect. Viral Env proteins initiate entry of membrane enveloped viruses into cells by binding to cell surface receptors followed by conformational changes leading to membrane fusion and delivery of the genome containing viral capsid to the cytoplasm. The Env glycoproteins of FVs are no exception and mediate attachment to host cells through binding to an yet unknown ubiquitous cellular receptor molecule because no cell type is currently known that is resistant to FV entry. Little structural and functional information on the extracellular domains of PFV Env is available. In this study we characterized the PFV Env receptor-binding-domain (RBD) by flow-cytometric analysis of recombinant PFV Env immunoadhesin binding to target cells. Analysis showed that the extracellular domains of the C-terminal TM subunit as well as targeting of the recombinant immunoadhesins by the cognate LP to the secretory pathway were dispensable for target cell binding suggesting that the PFV Env RBD is contained within the SU subunit. N- and C- terminal deletion analysis of the SU domain revealed an minimal continuous RBD spanning aa 225-555, however internal deletions covering the region from aa 397-483, but not aa 262-300 or aa 342-396, were tolerated without significant influence on host cell binding. Analysis of individual cysteine point mutants in PFV Env SU revealed that only most of those located in the non-essential region from aa 397-483 retained residual binding activity. Interestingly, analysis of various N-glycosylation site mutants suggests an important role of the carbohydrate chain attached to N391 either for direct interaction with the cellular receptor or for correct folding of the PFV Env RBD. Taken together these results suggest that a bipartite sequence motif spanning aa 225-396 and aa 484-555 is essential for formation of the PFV Env RBD, with N-glycosylation site 8 playing a crucial role for host cell binding.