Inhibitors of signal peptide peptidase and subtilisin/kexin-isozyme 1 inhibit Ebola virus glycoprotein-driven cell entry by interfering with activity and cellular localization of endosomal cathepsins.

Emerging viruses such as severe fever and thrombocytopenia syndrome virus (SFTSV) and Ebola virus (EBOV) are responsible for significant morbidity and mortality. Host cell proteases that process the glycoproteins of these viruses are potential targets for antiviral intervention. The aspartyl protease signal peptide peptidase (SPP) has recently been shown to be required for processing of the glycoprotein precursor, Gn/Gc, of Bunyamwera virus and for viral infectivity. Here, we investigated whether SPP is also required for infectivity of particles bearing SFTSV-Gn/Gc. Entry driven by the EBOV glycoprotein (GP) and the Lassa virus glycoprotein (LASV-GPC) depends on the cysteine proteases cathepsin B and L (CatB/CatL) and the serine protease subtilisin/kexin-isozyme 1 (SKI-1), respectively, and was examined in parallel for control purposes. We found that inhibition of SPP and SKI-1 did not interfere with SFTSV Gn + Gc-driven entry but, unexpectedly, blocked entry mediated by EBOV-GP. The inhibition occurred at the stage of proteolytic activation and the SPP inhibitor was found to block CatL/CatB activity. In contrast, the SKI-1 inhibitor did not interfere with CatB/CatL activity but disrupted CatB localization in endo/lysosomes, the site of EBOV-GP processing. These results underline the potential of protease inhibitors for antiviral therapy but also show that previously characterized compounds might exert broader specificity than initially appreciated and might block viral entry via diverse mechanisms

Probing Scaffold Size Effects on Multivalent Lectin-Glycan Binding Affinity, Thermodynamics and Antiviral Potency Using Polyvalent Glycan-Gold Nanoparticles

Multivalent lectin-glycan interactions (MLGIs) are pivotal for viral infections and immune regulation. Their structural and biophysical data are thus highly valuable, not only for the understanding of basic mechanisms but also for designing potent glycoconjugate therapeutics against target MLGIs. However, such information for some important MGLIs remain poorly understood, which has greatly limited the research progress in this area. We have recently developed densely glycosylated nanoparticles (e.g., ~4 nm quantum dot (QD) or ~5 nm gold nanoparticle (GNP)) as new mechanistic probes for MLGIs. Using two important tetrameric viral receptors, DC-SIGN and DC-SIGNR as model lectins, we have shown these probes not only can offer sensitive fluorescence readouts for MLGI affinity quantification, but also reveal key structural information (e.g., binding site orientation and binding mode) that are very useful for MLGI targeting. However, the relatively small sizes of scaffolds may not be optimal for maximizing MLGI affinity and targeting specificity. Herein, using -manno-1,2-biose (DiMan) functionalized GNPs (GNP-DiMan) probes, we have systematically studied how GNP scaffold size (e.g., 5, 13, and 27 nm) and glycan density (e.g., 100, 75, 50 and 25%) determine their MLGI affinities, thermodynamics, and antiviral properties. We have developed a new GNP fluorescence quenching assay format for quantifying MLGI affinity to minimize the potential interference from GNP’s strong inner filter effect, revealing that increasing GNP size is highly beneficial to enhance MLGI affinity. We have further determined the MLGI thermodynamics by combining temperature-dependent affinity measurement and Van’t Hoff analysis, revealing that GNP-DiMan-DC-SIGN/R binding is enthalpy driven. Finally, we find that increasing GNP size significantly enhances the antiviral potency. Notably, the DiMan functionalised 27 nm GNP (G27-DiMan) potently and robustly blocks both DC-SIGN and DC-SIGNR mediated pseudo-Ebola virus cellular entry with an EC50 of ~23 and ~49 pM, respectively, placing it the most potent glycoconjugate entry inhibitor against DC-SIGN/R mediated Ebola cellular infections

A polymorphism within the internal fusion loop of the Ebola virus glycoprotein modulates host cell entry.

The large scale of the Ebola virus disease (EVD) outbreak in West Africa in 2013-2016 raised the question whether the host cell interactions of the responsible Ebola virus (EBOV) strain differed from those of other ebolaviruses. We previously reported that the glycoprotein (GP) of the virus circulating in West Africa in 2014 (EBOV2014) exhibited reduced ability to mediate entry into two non-human primate (NHP)-derived cell lines relative to the GP of EBOV1976. Here, we investigated the molecular determinants underlying the differential entry efficiency. We found that EBOV2014-GP-driven entry into diverse NHP-derived cell lines as well as human monocyte-derived macrophages and dendritic cells was reduced as compared to EBOV1976-GP, although entry into most human- and all bat-derived cell lines tested was comparable. Moreover, EBOV2014 replication in NHP but not human cells was diminished relative to EBOV1976, suggesting that reduced cell entry translated into reduced viral spread. Mutagenic analysis of EBOV2014-GP and EBOV1976-GP revealed that an amino acid polymorphism in the receptor-binding domain, A82V, modulated entry efficiency in a cell line-independent manner and did not account for the reduced EBOV2014-GP-driven entry into NHP cells. In contrast, polymorphism T544I, located in the internal fusion loop in the GP2 subunit, was found to be responsible for the entry phenotype. These results suggest that position 544 is an important determinant of EBOV infectivity for NHP- and certain human target cells.IMPORTANCE The Ebola virus disease outbreak in West Africa in 2013 entailed more than 10,000 deaths. The scale of the outbreak and its dramatic impact on human health raised the question whether the responsible virus was particularly adept at infecting human cells. Our study shows that an amino acid exchange, A82V, that the virus acquired during the epidemic and that was not observed in previously circulating viruses, increases viral entry into diverse target cells. In contrast, the epidemic virus showed a reduced ability to enter cells of non-human primates as compared to the virus circulating in 1976 and a single amino acid exchange in the internal fusion loop of the viral glycoprotein was found to account for this phenotype

Hemagglutinin Cleavability, Acid Stability, and Temperature Dependence Optimize Influenza B Virus for Replication in Human Airways

Influenza A virus (IAV) and influenza B virus (IBV) cause yearly epidemics with significant morbidity and mortality. When zoonotic IAVs enter the human population, the viral hemagglutinin (HA) requires adaptation to achieve sustained virus transmission. In contrast, IBV has been circulating in humans, its only host, for a long period of time. Whether this entailed adaptation of IBV HA to the human airways is unknown. To address this question, we compared two seasonal IAVs (A/H1N1 and A/H3N2) and two IBVs (B/Victoria and B/Yamagata lineages) with regard to host-dependent activity of HA as the mediator of membrane fusion during viral entry. We first investigated proteolytic activation of HA by covering all type II transmembrane serine protease (TTSP) and kallikrein enzymes, many of which proved to be present in human respiratory epithelium. The IBV HA0 precursor is cleaved by a broader panel of TTSPs and activated with much higher efficiency than IAV HA0. Accordingly, knockdown of a single protease, TMPRSS2, abrogated spread of IAV but not IBV in human respiratory epithelial cells. Second, the HA fusion pH values proved similar for IBV and human-adapted IAVs (with one exception being the HA of 1918 IAV). Third, IBV HA exhibited higher expression at 33°C, a temperature required for membrane fusion by B/Victoria HA. This indicates pronounced adaptation of IBV HA to the mildly acidic pH and cooler temperature of human upper airways. These distinct and intrinsic features of IBV HA are compatible with extensive host adaptation during prolonged circulation of this respiratory virus in the human population.IMPORTANCE Influenza epidemics are caused by influenza A and influenza B viruses (IAV and IBV, respectively). IBV causes substantial disease; however, it is far less studied than IAV. While IAV originates from animal reservoirs, IBV circulates in humans only. Virus spread requires that the viral hemagglutinin (HA) is active and sufficiently stable in human airways. We resolve here how these mechanisms differ between IBV and IAV. Whereas human IAVs rely on one particular protease for HA activation, this is not the case for IBV. Superior activation of IBV by several proteases should enhance shedding of infectious particles. IBV HA exhibits acid stability and a preference for 33°C, indicating pronounced adaptation to the human upper airways, where the pH is mildly acidic and a cooler temperature exists. These adaptive features are rationalized by the long existence of IBV in humans and may have broader relevance for understanding the biology and evolution of respiratory viruses.status: publishe

A Polyvalent Nano-Lectin Potently Neutralizes SARS-CoV-2 by Targeting Glycans on the Viral Spike Protein

Mutations in spike (S) protein epitopes allow SARS-CoV-2 variants to evade antibody responses induced by infection and/or vaccination. In contrast, glycosylation sites in the S protein are conserved across SARS-CoV-2 variants, making glycans a potential robust target for developing antivirals. However, this target has not been adequately exploited for SARS-CoV-2, mostly due to intrinsically weak monovalent protein-glycan inter-actions. We hypothesize that polyvalent nano-lectins with flexibly linked carbohydrate-recognition-domains (CRDs) can adjust their relative positions and bind multivalently to S protein glycans, potentially exerting potent antiviral activity. Herein, we displayed the CRDs of DC-SIGN, a dendritic cell lectin known to bind to diverse viruses, polyvalently onto 13 nm gold nanoparticles (named as G13-CRD). G13-CRD bound strongly and specifically to target glycan-coated quantum dots with sub-nM Kd. Moreover, G13-CRD neutralized particles pseudo-typed with the S proteins of Wuhan Hu-1, B1, Delta variant and Omicron subvariant BA.1 with low nM EC50. In contrast, natural tetrameric DC-SIGN and its G13 conjugate were ineffective. Further, G13-CRD potently and completely inhibited authentic SARS-CoV-2 Wuhan Hu-1 and BA.1, with <10 pM and <10 nM EC50, respectively. These results identify G13-CRD as a polyvalent nano-lectin with broad activity against SARS-CoV-2 variants that merits further exploration as a novel approach to antiviral therapy

The glycoprotein of vesicular stomatitis virus promotes release of virus-like particles from tetherin-positive cells

<div><p>Vesicular stomatitis virus (VSV) release from infected cells is inhibited by the interferon (IFN)-inducible antiviral host cell factor tetherin (BST-2, CD317). However, several viruses encode tetherin antagonists and it is at present unknown whether residual VSV spread in tetherin-positive cells is also promoted by a virus-encoded tetherin antagonist. Here, we show that the viral glycoprotein (VSV-G) antagonizes tetherin in transfected cells, although with reduced efficiency as compared to the HIV-1 Vpu protein. Tetherin antagonism did not involve alteration of tetherin expression and was partially dependent on a GXXXG motif in the transmembrane domain of VSV-G. However, mutation of the GXXXG motif did not modulate tetherin sensitivity of infectious VSV. These results identify VSV-G as a tetherin antagonist in transfected cells but fail to provide evidence for a contribution of tetherin antagonism to viral spread.</p></div

The GXXXG motif is dispensable for viral spread in tetherin-positive cells.

<p>(A) HeLa cells were transfected with the indicated siRNAs and subsequently infected with VSV wt or LXXXL mutant at an MOI of 0.005 for 1 h. Virus titers in culture supernatants were determined at the indicated time points post infection. The results of a single representative experiment carried out with triplicate samples are shown and were confirmed in two separate experiments. (B) The experiment was carried out as described for panel (A) but relative titers measured at 12 h post infection are shown. The results represent the average of three independent experiments performed with triplicate samples. Titers measured for untransfected control cells were set to 1. Error bars indicate standard error of the mean (SEM). (C) Vero cells stably expressing human tetherin (Vero-Tetherin) or stably containing empty vector (Vero) were infected with VSV wt or mutant LXXXL. Viral titers in culture supernatants were determined at the indicated time points post infection. The results of a single representative experiment carried out with triplicate samples are shown and were confirmed in two separate experiments. (D) The experiment was carried out as described for panel (C) but relative titers measured at 12 h post infection are shown. The results represent the average of three independent experiments performed with triplicate samples. Titers obtained from the respective control Vero cells were set to 1. Error bars indicate SEM. One-way ANOVA with Bonferroni post-test analyses were performed (B and D) to test statistical significance between selected groups (***, p ≤ 0.001).</p

Mutagenesis of the GXXXG motif in the transmembrane domain of VSV-G.

<p>Schematic representation of the VSV* genome in which the authentic viral open reading frames (ORFs, VSV-N, -P, -M, -G and -L; light grey) are surrounded by non-translated regulatory elements (white). The additional ORF for eGFP (dark grey) was inserted between the ORFs for VSV-G and -L (restriction sites used for cloning and further modification of the genome are highlighted). The transmembrane domain (underlined amino acids) of VSV-G wt contains a GXXXG motif (amino acid residues 473–477) that has been mutated to LXXXL.</p

Probing Multivalent Lectin-Carbohydrate Binding via Multifunctional Glycan-Gold Nanoparticles: Implications for Blocking Virus Infection

Multivalent lectin-glycan interactions are widespread in biology and are often exploited by pathogens to bind and infect host cells. Glycoconjugates can block such interactions and thereby prevent infection. The inhibition potency strongly depends on matching the spatial arrangement between the multivalent binding partners. However, the structural details of some key lectins remain unknown and different lectins may exhibit overlapping glycan specificity. This makes it difficult to design a glycoconjugate that can potently and specifically target a particular multimeric lectin for therapeutic interventions, especially under the challenging in vivo conditions. Conventional techniques such as surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) can provide quantitative binding thermodynamics and kinetics. However, they cannot reveal key structural information, e.g. lectin’s binding site orientation, binding mode, and inter-binding site spacing, which are critical to design specific multivalent inhibitors. Herein we report that gold nanoparticles (GNPs) displaying a dense layer of simple glycans are powerful mechanistic probes for multivalent lectin-glycan interactions. They can not only quantify the GNP-glycan-lectin binding affinities via a new fluorescence quenching method, but also reveal drastically different affinity enhancing mechanisms between two closely-related tetrameric lectins, DC-SIGN (simultaneous binding to one GNP) and DC-SIGNR (inter-crosslinking with multiple GNPs), via a combined hydrodynamic size and electron microscopy analysis. Moreover, a new term, potential of assembly formation (PAF) has been proposed to successfully predict the assembly outcomes based on the binding mode between GNP-glycans and lectins. Finally, the GNP-glycans can potently and completely inhibit DC-SIGN-mediated augmentation of Ebola virus glycoprotein-driven cell entry (with IC50 values down to 95 pM), but only partially block DC-SIGNR-mediated virus infection. Our results suggest that the ability of a glycoconjugate to simultaneously block all binding sites of a target lectin is key to robust inhibition of viral infection

The GXXXG motif in the transmembrane domain of VSV-G is required for tetherin antagonism in transfected cells.

<p>(A) 293T cells were transfected with plasmids encoding the indicated glycoproteins or mock-transfected. Expression was determined by Western blot analysis using anti-VSV-G antibody (concentrated supernatants from hybridoma CL-2700). Detection of β-actin expression served as loading control. (B) The average of four independent experiments conducted as described for panel (A) and quantified via the ImageJ program is presented. Expression of VSV-G wt was set to 100%. (C) Incorporation of VSV-G wt and mutant LXXXL into VSV pseudotypes was investigated by Western blot analysis using an anti-VSV-G antibody (concentrated supernatants from hybridoma CL-2700). To ensure that similar amounts of pseudotypes were analyzed, levels of particle-associated M proteins were determined using an anti-VSV-M antibody. Pseudotypes harboring no glycoprotein (Mock) served as negative control. The results of a single immunoblot are shown from which irrelevant lanes were cut out. Similar results were obtained in two separate experiments. (D) 293T cells were transduced with equal volumes of the VSV pseudotypes described in panel (C). At 24 h post transduction, luciferase activity in cell lysates was measured. Transduction driven by VSV-G wt was set as 100%. The average of three independent experiments is shown. Error bars indicate SEM. (E) 293T cells were cotransfected with plasmids encoding Gag, the indicated glycoproteins and tetherin. Expression of Gag in supernatants and cell lysates was determined by Western blot. Detection of β-actin expression served as loading control. (F) The average of three independent experiments conducted as described for panel (E) and quantified via the ImageJ program is presented. Error bars indicate standard error of the mean (SEM). Release of Gag from cells coexpressing the highest amount of VSV-G and tetherin was set to 100%. For all graphs (B, D, F), paired two-tailed Students’ t-tests were performed to assess whether differences between VSV-G wt and LXXXL mutant were of statistical significance.</p