Sean M. Amberg

Characterization of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike glycoprotein-mediated viral entry

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is a rapidly emerging pathogen with potentially serious consequences for public health. Here we describe conditions that result not only in the efficient expression of the SARS-CoV spike (S) protein on the surface of cells, but in its incorporation into lentiviral particles that can be used to transduce cells in an S glycoprotein-dependent manner. We found that although some primate cell lines, including Vero E6, 293T and Huh-7 cells, could be efficiently transduced by SARS-CoV S glycoprotein pseudoviruses, other cells lines were either resistant or very poorly permissive to virus entry. Infection by pseudovirions could be inhibited by several lysosomotropic agents, suggesting a requirement for acidification of endosomes for efficient S-mediated viral entry. In addition, we were able to develop a cell–cell fusion assay that could be used to monitor S glycoprotein-dependent membrane fusion. Although proteolysis did not enhance the infectivity of cell-free pseudovirions, trypsin activation is required for cell–cell fusion. Additionally, there was no apparent pH requirement for S glycoprotein-mediated cell–cell fusion. Together, these studies describe important tools that can be used to study SARS-CoV S glycoprotein structure and function, including approaches that can be used to identify inhibitors of the entry of SARS-CoV into target cells.

Identification of a Broad-Spectrum Arenavirus Entry Inhibitor

ABSTRACT Several arenaviruses, including Lassa virus (LASV), are causative agents of hemorrhagic fever, for which effective therapeutic options are lacking. The LASV envelope glycoprotein (GP) gene was used to generate lentiviral pseudotypes to identify small-molecule inhibitors of viral entry. A benzimidazole derivative with potent antiviral activity was identified from a high-throughput screen utilizing this strategy. Subsequent lead optimization for antiviral activity identified a modified structure, ST-193, with a 50% inhibitory concentration (IC50) of 1.6 nM against LASV pseudotypes. ST-193 inhibited pseudotypes generated with other arenavirus envelopes as well, including the remaining four commonly associated with hemorrhagic fever (IC50s for Junín, Machupo, Guanarito, and Sabiá were in the 0.2 to 12 nM range) but exhibited no antiviral activity against pseudotypes incorporating either the GP from the LASV-related arenavirus lymphocytic choriomeningitis virus (LCMV) or the unrelated G protein from vesicular stomatitis virus, at concentrations of up to 10 μM. Determinants of ST-193 sensitivity were mapped through a combination of LASV-LCMV domain-swapping experiments, genetic selection of viral variants, and site-directed mutagenesis. Taken together, these studies demonstrate that sensitivity to ST-193 is dictated by a segment of about 30 amino acids within the GP2 subunit. This region includes the carboxy-terminal region of the ectodomain and the predicted transmembrane domain of the envelope protein, revealing a novel antiviral target within the arenavirus envelope GP.

pH-Induced Activation of Arenavirus Membrane Fusion Is Antagonized by Small-Molecule Inhibitors

ABSTRACT The arenavirus envelope glycoprotein (GPC) mediates viral entry through pH-induced membrane fusion in the endosome. This crucial process in the viral life cycle can be specifically inhibited in the New World arenaviruses by the small-molecule compound ST-294. Here, we show that ST-294 interferes with GPC-mediated membrane fusion by targeting the interaction of the G2 fusion subunit with the stable signal peptide (SSP). We demonstrate that amino acid substitutions at lysine-33 of the Junín virus SSP confer resistance to ST-294 and engender de novo sensitivity to ST-161, a chemically distinct inhibitor of the Old World Lassa fever virus. These compounds, as well as a broadly active inhibitor, ST-193, likely share a molecular target at the SSP-G2 interface. We also show that both ST-294 and ST-193 inhibit pH-induced dissociation of the G1 receptor-binding subunit from GPC, a process concomitant with fusion activation. Interestingly, the inhibitory activity of these molecules can in some cases be overcome by further lowering the pH used for activation. Our results suggest that these small molecules act to stabilize the prefusion GPC complex against acidic pH. The pH-sensitive interaction between SSP and G2 in GPC represents a robust molecular target for the development of antiviral compounds for the treatment of arenavirus hemorrhagic fevers.

Mutagenesis of the Signal Sequence of Yellow Fever Virus prM Protein: Enhancement of Signalase Cleavage In Vitro Is Lethal for Virus Production

ABSTRACT Proteolytic processing at the C-prM junction in the flavivirus polyprotein involves coordinated cleavages at the cytoplasmic and luminal sides of an internal signal sequence. We have introduced at the COOH terminus of the yellow fever virus (YFV) prM signal sequence amino acid substitutions (VPQAQA mutation) which uncoupled efficient signal peptidase cleavage of the prM protein from its dependence on prior cleavage in the cytoplasm of the C protein mediated by the viral NS2B-3 protease. Infectivity assays with full-length YFV RNA transcripts showed that the VPQAQA mutation, which enhanced signal peptidase cleavage in vitro, was lethal for infectious virus production. Revertants or second-site mutants were recovered from cells transfected with VPQAQA RNA. Analysis of these viruses revealed that single amino acid substitutions in different domains of the prM signal sequence could restore viability. These variants had growth properties in vertebrate cells which differed only slightly from those of the parent virus, despite efficient signal peptidase cleavage of prM in cell-free expression assays. However, the neurovirulence in mice of the VPQAQA variants was significantly attenuated. This study demonstrates that substitutions in the prM signal sequence which disrupt coordinated cleavages at the C-prM junction can impinge on the biological properties of the mutant viruses. Factors other than the rate of production of prM are vitally controlled by regulated cleavages at this site.

Heptad repeat 2-based peptides inhibit avian sarcoma and leukosis virus subgroup a infection and identify a fusion intermediate.

ABSTRACT Fusion proteins of enveloped viruses categorized as class I are typified by two distinct heptad repeat domains within the transmembrane subunit. These repeats are important structural elements that assemble into the six-helix bundles characteristic of the fusion-activated envelope trimer. Peptides derived from these domains can be potent and specific inhibitors of membrane fusion and virus infection. To facilitate our understanding of retroviral entry, peptides corresponding to the two heptad repeat domains of the avian sarcoma and leukosis virus subgroup A (ASLV-A) TM subunit of the envelope protein were characterized. Two peptides corresponding to the C-terminal heptad repeat (HR2), offset from one another by three residues, were effective inhibitors of infection, while two overlapping peptides derived from the N-terminal heptad repeat (HR1) were not. Analysis of envelope mutants containing substitutions within the HR1 domain revealed that a single amino acid change, L62A, significantly reduced sensitivity to peptide inhibition. Virus bound to cells at 4°C became sensitive to peptide within the first 5 min of elevating the temperature to 37°C and lost sensitivity to peptide after 15 to 30 min, consistent with a transient intermediate in which the peptide binding site is exposed. In cell-cell fusion experiments, peptide inhibitor sensitivity occurred prior to a fusion-enhancing low-pH pulse. Soluble receptor for ASLV-A induces a lipophilic character in the envelope which can be measured by stable liposome binding, and this activation was found to be unaffected by inhibitory HR2 peptide. Finally, receptor-triggered conformational changes in the TM subunit were also found to be unaffected by inhibitory peptide. These changes are marked by a dramatic shift in mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, from a subunit of 37 kDa to a complex of about 80 kDa. Biotinylated HR2 peptide bound specifically to the 80-kDa complex, demonstrating a surprisingly stable envelope conformation in which the HR2 binding site is exposed. These experiments support a model in which receptor interaction promotes formation of an envelope conformation in which the TM subunit is stably associated with its target membrane and is able to bind a C-terminal peptide.

Evaluation of Lassa antiviral compound ST-193 in a guinea pig model.

Abstract Lassa virus (LASV), a member of the Arenaviridae family, causes a viral hemorrhagic fever endemic to West Africa, where as many as 300,000 infections occur per year. Presently, there are no FDA-approved LASV-specific vaccines or antiviral agents, although the antiviral drug ribavirin has shown some efficacy. A recently identified small-molecule inhibitor of arenavirus entry, ST-193, exhibits submicromolar antiviral activity in vitro. To determine the antiviral utility of ST-193 in vivo, we tested the efficacy of this compound in the LASV guinea pig model. Four groups of strain 13 guinea pigs were administered 25 or 80mg/kg ST-193, 25mg/kg of ribavirin, or the vehicle by the intraperitoneal (i.p.) route before infection with a lethal dose of LASV, strain Josiah, and continuing once daily for 14 days. Control animals exhibited severe disease, becoming moribund between days 10 and 15 postinfection. ST-193-treated animals exhibited fewer signs of disease and enhanced survival when compared to the ribavirin or vehicle groups. Body temperatures in all groups were elevated by day 9, but returned to normal by day 19 postinfection in the majority of ST-193-treated animals. ST-193 treatment mediated a 2–3-log reduction in viremia relative to vehicle-treated controls. The overall survival rate for the ST-193-treated guinea pigs was 62.5% (10/16) compared with 0% in the ribavirin (0/8) and vehicle (0/7) groups. These data suggest that ST-193 may serve as an improved candidate for the treatment of Lassa fever.

A specific interaction of small-molecule entry inhibitors with the envelope glycoprotein complex of the Junin hemorrhagic fever arenavirus

Arenaviruses are responsible for acute hemorrhagic fevers worldwide and are recognized to pose significant threats to public health and biodefense. Small molecule compounds have recently been discovered that inhibit arenavirus entry and protect against lethal infection in animal models. These chemically distinct inhibitors act on the tripartite envelope glycoprotein (GPC) through its unusual stable signal peptide subunit to stabilize the complex against pH-induced activation of membrane fusion in the endosome. Here, we report the production and characterization of the intact transmembrane GPC complex of Junín arenavirus and its interaction with these inhibitors. The solubilized GPC is antigenically indistinguishable from the native protein and forms a homogeneous trimer in solution. When reconstituted into a lipid bilayer, the purified complex interacts specifically with its cell-surface receptor transferrin receptor-1. We show that small molecule entry inhibitors specific to New World or Old World arenaviruses bind to the membrane-associated GPC complex in accordance with their respective species selectivities and with dissociation constants comparable with concentrations that inhibit GPC-mediated membrane fusion. Furthermore, competitive binding studies reveal that these chemically distinct inhibitors share a common binding pocket on GPC. In conjunction with previous genetic studies, these findings identify the pH-sensing interface of GPC as a highly vulnerable target for antiviral intervention. This work expands our mechanistic understanding of arenavirus entry and provides a foundation to guide the development of small molecule compounds for the treatment of arenavirus hemorrhagic fevers.

Discovery and optimization of potent broad-spectrum arenavirus inhibitors derived from benzimidazole

A chemically diverse library of about 400,000 small molecules was screened for antiviral activity against lentiviral pseudotypes with the Lassa virus envelope glycoprotein (LASV GP) gene incorporated. High-throughput screening resulted in discovery of a hit compound (ST-37) possessing a benzimidazole core which led to a potent compound series. Herein, we report SAR studies which involved structural modifications to the phenyl rings and methylamino linker portion attached to the benzimidazole core. Many analogs in this study possessed single digit nanomolar potency against LASV pseudotypes. Compounds in this benzimidazole series also exhibited nanomolar antiviral activity against pseudotypes generated from other arenavirus envelopes indicating the potential for development of a broad-spectrum inhibitor. Ultimately, lead compound ST-193 was identified and later found to be efficacious in a lethal LASV guinea pig model showing superior protection compared to ribavirin treatment.

SAR analysis of a series of acylthiourea derivatives possessing broad-spectrum antiviral activity.

A series of acylthiourea derivatives were designed, synthesized, and evaluated for broad-spectrum antiviral activity with selected viruses from Poxviridae (vaccinia virus) and two different genera of the family Bunyaviridae (Rift Valley fever and La Crosse viruses). A compound selected from a library screen, compound 1, displayed submicromolar antiviral activity against both vaccinia virus (EC(50)=0.25 μM) and La Crosse virus (EC(50)=0.27 μM) in cytopathic effect (CPE) assays. SAR analysis was performed to further improve antiviral potency and to optimize drug-like properties of the initial hits. During our analysis, we identified 26, which was found to be nearly fourfold more potent than 1 against both vaccinia and La Crosse viruses. Selected compounds were further tested to more fully characterize the spectrum of antiviral activity. Many of these possessed single digit micromolar and sub-micromolar antiviral activity against a diverse array of targets, including influenza virus (Orthomyxoviridae), Tacaribe virus (Arenaviridae), and dengue virus (Flaviviridae).

Discovery and optimization of potent broad-spectrum arenavirus inhibitors derived from benzimidazole and related heterocycles

A series of potent arenavirus inhibitors sharing a benzimidazole core were previously reported by our group. SAR studies were expanded beyond the previous analysis, which involved the attached phenyl rings and methylamino linker portion, to include modifications focused on the benzimidazole core. These changes included the introduction of various substituents to the bicyclic benzimidazole ring system along with alternate core heterocycles. Many of the analogs containing alternate nitrogen-based bicyclic ring systems were found to retain antiviral potency compared to the benzimidazole series from which we derived our lead compound, ST-193. In fact, 21 h, built on an imidazopyridine core, possessed a near tenfold increase in potency against Lassa virus pseudotypes compared to ST-193. As found with the benzimidazole series, broad-spectrum arenavirus activity was also observed for a number of the analogs discovered during this study.