Identification of a Broad-Spectrum Arenavirus Entry Inhibitor

Larson, Ryan A.; Dai, Dongcheng; Hosack, Virginia T.; Tan, Ying; Bolken, Tove’ C.; Hruby, Dennis E.; Amberg, Sean M.

doi:10.1128/jvi.00941-08

Cited by 128 publications

(162 citation statements)

References 55 publications

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“…ST-193 had originally been identified as an inhibitor of LASV GPC and was subsequently found to be active against New World arenaviruses (33). Although this molecule shows nanomolar potency against both Old World and New World GPC in pseudotyped virions (33), the IC 50 against JUNV GPC in the cell-cell fusion assay is markedly poorer than that with LASV GPC (0.9 M versus Ͻ Ͻ50 nM, respectively) (Fig. 5, right panel).…”

Section: Resultsmentioning

confidence: 99%

“…These factors may have been differentially selected for during screening and lead optimization. It is worth noting that ST-294 was identified through its inhibition of New World virus replication (3), whereas ST-193 and ST-161 were developed as inhibitors of Old World GPC (33). Furthermore, the pH requirement for viral entry in the endosome is itself undefined, and thus, in vitro measures of inhibitor potency at pH 5.0 may differ from those determined by virus infection.…”

Section: Resultsmentioning

confidence: 99%

“…SIGA Technologies has independently identified two distinct chemical classes of Old World arenavirus fusion inhibitors (Fig. 2) through high-throughput screening for molecules that prevent cell entry by pseudotyped retroviruses bearing the LASV GPC (33). We characterized the inhibitory activity of these new compounds, ST-161 and ST-193, by using JUNV and LASV GPCs in cell-cell fusion assays.…”

Section: Resultsmentioning

confidence: 99%

“…These two components associate in trans and reconstitute the native GPC complex (18). The GPC open reading frame of LASV-Josiah (33) and that of the vesicular stomatitis virus (VSV) G glycoprotein (kindly provided by John K. Rose, Yale University) were also expressed in pcDNA3.1 plasmids. Transient expression utilized a recombinant vaccinia virus expressing T7 RNA polymerase (vTF7-3) (23).…”

Section: Molecular and Chemical Reagents And Monoclonal Antibodies (Mmentioning

confidence: 99%

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

York

Dai

Amberg

et al. 2008

J Virol

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159

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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 GPCmediated 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.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Molecular and Chemical Reagents And Monoclonal Antibodies (Mmentioning

confidence: 99%

See 2 more Smart Citations

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

York

Dai

Amberg

et al. 2008

J Virol

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159

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“…The key role of GPC in virus entry underscores its potential as a target for antiviral intervention (9,33,34,72). GPC is synthesized as a precursor polypeptide that undergoes two proteolytic cleavage events to form the mature envelope glycoprotein complex (13).…”

mentioning

confidence: 99%

Assembly of Arenavirus Envelope Glycoprotein GPC in Detergent-Soluble Membrane Microdomains

Agnihothram

Dancho²,

Grant

et al. 2009

J Virol

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The family Arenaviridae includes a number of highly pathogenic viruses that are responsible for acute hemorrhagic fevers in humans. Genetic diversity among arenavirus species in their respective rodent hosts supports the continued emergence of new pathogens. In the absence of available vaccines or therapeutic agents, the hemorrhagic fever arenaviruses remain a serious public health and biodefense concern. Arenaviruses are enveloped virions that assemble and bud from the plasma membrane. In this study, we have characterized the microdomain organization of the virus envelope glycoprotein (GPC) on the cell surface by using immunogold electron microscopy. We find that Junín virus (JUNV) GPC clusters into discrete microdomains of 120 to 160 nm in diameter and that this property of GPC is independent of its myristoylation and of coexpression with the virus matrix protein Z. In cells infected with the Candid#1 strain of JUNV, and in purified Candid#1 virions, these GPC microdomains are soluble in cold Triton X-100 detergent and are thus distinct from conventional lipid rafts, which are utilized by numerous other viruses for assembly. Virion morphogenesis ultimately requires colocalization of viral components, yet our dual-label immunogold staining studies failed to reveal a spatial association of Z with GPC microdomains. This observation may reflect either rapid Z-dependent budding of virus-like particles upon coassociation or a requirement for additional viral components in the assembly process. Together, these results provide new insight into the molecular basis for arenavirus morphogenesis.

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High‐Throughput Screening of Viral Entry Inhibitors Using Pseudotyped Virus

2010

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Virus entry into a host cell is an attractive target for therapy because propagation of virus can be blocked at an early stage, minimizing chances for the virus to acquire drug resistance. Anti-infective drug discovery for BSL-4 viruses like Ebola or Lassa hemorrhagic fever virus presents challenges due to the requirement for a BSL-4 laboratory containment facility. Pseudotyped viruses provide a surrogate model in which the native envelope glycoprotein of a BSL-2 level virus (e.g., vesicular stomatitis virus) is replaced with envelope glycoprotein of a foreign BSL-4 virus (e.g., Ebola virus). Because the envelope glycoprotein determines interaction of virus with its cellular receptors, pseudotyped viruses can mimic the viral entry process of the original virus. Moreover, they are competent for only a single cycle of infection, and therefore can be used in BSL-2 facilities. Pseudotyped viruses have been used in high-throughput screening of entry inhibitors for a number of BSL-4 level viruses. This unit includes protocols for preparing pseudotyped viruses using lentiviral vectors and use of pseudotyped viruses for high-throughput screening of viral entry inhibitors.

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Identification of a Broad-Spectrum Arenavirus Entry Inhibitor

Cited by 128 publications

References 55 publications

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

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

Assembly of Arenavirus Envelope Glycoprotein GPC in Detergent-Soluble Membrane Microdomains

High‐Throughput Screening of Viral Entry Inhibitors Using Pseudotyped Virus

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