Identification of a Broad-Spectrum Arenavirus Entry Inhibitor (original) (raw)
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Journal of Virology, 2009
A crucial step in the arenavirus life cycle is the biosynthesis of the viral envelope glycoprotein (GP) responsible for virus attachment and entry. Processing of the GP precursor (GPC) by the cellular proprotein convertase site 1 protease (S1P), also known as subtilisin-kexin-isozyme 1 (SKI-1), is crucial for cell-to-cell propagation of infection and production of infectious virus. Here, we sought to evaluate arenavirus GPC processing by S1P as a target for antiviral therapy using a recently developed peptide-based S1P inhibitor, decanoyl (dec)-RRLL-chloromethylketone (CMK), and the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV). To control for off-target effects of dec-RRLL-CMK, we employed arenavirus reverse genetics to introduce a furin recognition site into the GPC of LCMV. The rescued mutant virus grew to normal titers, and the processing of its GPC critically depended on cellular furin, but not S1P. Treatment with the S1P inhibitor dec-RRLL-CMK resulted in spe...
Inhibition of Arenavirus Infection by a Glycoprotein-Derived Peptide with a Novel Mechanism
Journal of Virology, 2014
The family Arenaviridae includes a number of viruses of public health importance, such as the category A hemorrhagic fever viruses Lassa virus, Junin virus, Machupo virus, Guanarito virus, and Sabia virus. Current chemotherapy for arenavirus infection is limited to the nucleoside analogue ribavirin, which is characterized by considerable toxicity and treatment failure. Using Pichinde virus as a model arenavirus, we attempted to design glycoprotein-derived fusion inhibitors similar to the FDA-approved anti-HIV peptide enfuvirtide. We have identified a GP2-derived peptide, AVP-p, with antiviral activity and no acute cytotoxicity. The 50% inhibitory dose (IC 50) for the peptide is 7 M, with complete inhibition of viral plaque formation at approximately 20 M, and its antiviral activity is largely sequence dependent. AVP-p demonstrates activity against viruses with the Old and New World arenavirus viral glycoprotein complex but not against enveloped viruses of other families. Unexpectedly, fusion assays reveal that the peptide induces virus-liposome fusion at neutral pH and that the process is strictly glycoprotein mediated. As observed in cryo-electron micrographs, AVP-p treatment causes morphological changes consistent with fusion protein activation in virions, including the disappearance of prefusion glycoprotein spikes and increased particle diameters, and fluorescence microscopy shows reduced binding by peptide-treated virus. Steady-state fluorescence anisotropy measurements suggest that glycoproteins are destabilized by peptide-induced alterations in viral membrane order. We conclude that untimely deployment of fusion machinery by the peptide could render virions less able to engage in on-pathway receptor binding or endosomal fusion. AVP-p may represent a potent, highly specific, novel therapeutic strategy for arenavirus infection. IMPORTANCE Because the only drug available to combat infection by Lassa virus, a highly pathogenic arenavirus, is toxic and prone to treatment failure, we identified a peptide, AVP-p, derived from the fusion glycoprotein of a nonpathogenic model arenavirus, which demonstrates antiviral activity and no acute cytotoxicity. AVP-p is unique among self-derived inhibitory peptides in that it shows broad, specific activity against pseudoviruses bearing Old and New World arenavirus glycoproteins but not against viruses from other families. Further, the peptide's mechanism of action is highly novel. Biochemical assays and cryo-electron microscopy indicate that AVP-p induces premature activation of viral fusion proteins through membrane perturbance. Peptide treatment, however, does not increase the infectivity of cell-bound virus. We hypothesize that prematurely activated virions are less fit for receptor binding and membrane fusion and that AVP-p may represent a viable therapeutic strategy for arenavirus infection.
Host Cell Factors as Antiviral Targets in Arenavirus Infection
Viruses, 2012
Among the members of the Arenaviridae family, Lassa virus and Junin virus generate periodic annual outbreaks of severe human hemorrhagic fever (HF) in endemic areas of West Africa and Argentina, respectively. Given the human health threat that arenaviruses represent and the lack of a specific and safe chemotherapy, the search for effective antiviral compounds is a continuous demanding effort. Since diverse host cell pathways and enzymes are used by RNA viruses to fulfill their replicative cycle, the targeting of a host process has turned an attractive antiviral approach in the last years for many unrelated virus types. This strategy has the additional benefit to reduce the serious challenge for therapy of RNA viruses to escape from drug effects through selection of resistant variants triggered by their high mutation rate. This article focuses on novel strategies to identify inhibitors for arenavirus therapy, analyzing the potential for antiviral developments of diverse host factors essential for virus infection.
Receptor use by pathogenic arenaviruses
Virology, 2006
The arenavirus family contains several important human pathogens including Lassa fever virus (LASV), lymphocytic choriomeningitis virus (LCMV) and the New World clade B viruses Junin (JUNV) and Machupo (MACV). Previously, α-dystroglycan (α-DG) was identified as a receptor recognized by LASV and certain strains of LCMV. However, other studies have suggested that α-DG is probably not used by the clade B viruses, and the receptor(s) for these pathogens is currently unknown. Using pseudotyped retroviral vectors displaying arenavirus glycoproteins (GPs), we are able to explore the role played by the GP in viral entry in the absence of other viral proteins. By examining the ability of the vectors to transduce DG knockout murine embryonic stem (ES) cells, we have confirmed that LASV has an absolute requirement for α-DG in these cells. However, the LCMV GP can still direct substantial entry into murine ES cells in the absence of α-DG, even when the GP from the clone 13 variant is used that has previously been reported to be highly dependent on α-DG for entry. We also found that neither LASV or LCMV pseudotyped vectors were able to transduce human or murine lymphocytes, presumably due to the glycosylation state of α-DG in these cells. In contrast, the JUNV and MACV GPs displayed broad tropism on human, murine and avian cell types, including lymphocytes, and showed no requirement for α-DG in murine ES cells. These findings highlight the importance of molecules other than α-DG for arenavirus entry. An alternate receptor is present on murine ES cells that can be used by LCMV but not by LASV, and which is not available on human or murine lymphocytes, while a distinct and widely expressed receptor(s) is used by the clade B viruses.
Journal of Virology, 2015
Several arenaviruses cause hemorrhagic fever disease in humans and represent important public health problems in the regions where these viruses are endemic. In addition, evidence indicates that the worldwide-distributed prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is an important neglected human pathogen. There are no licensed arenavirus vaccines and current antiarenavirus therapy is limited to the use of ribavirin that is only partially effective. Therefore, there is an unmet need for novel antiarenaviral therapeutics. Here, we report the generation of a novel recombinant LCM virus and its use to develop a cell-based high-throughput screen to rapidly identify inhibitors of LCMV multiplication. We used this novel assay to screen a library of 30,400 small molecules and identified compound F3406 (chemical name: N -[3,5-bis(fluoranyl)phenyl]-2-[5,7-bis(oxidanylidene)-6-propyl-2-pyrrolidin-1-yl-[1,3]thiazolo[4,5-d]pyrimidin-4-yl]ethanamide), which exhibited strong an...
Research efforts to control highly pathogenic arenaviruses: A summary of the progress and gaps
Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology, 2015
Significant progress has been made in the past 10 years in unraveling the molecular biology of highly pathogenic arenaviruses that are endemic in several West African countries (Lassa fever virus) and in some regions of South America (Argentine and Bolivian hemorrhagic fever viruses). While this has resulted in proof-of-concept studies of novel vaccine candidates in non-human primates and in the discovery of several novel antiviral small molecule drug candidates, none of them has been tested in the clinic to date. The recent Ebola outbreak in West Africa has demonstrated very clearly that there is an urgent need to develop the prophylactic and therapeutic armamentarium against viral hemorrhagic fever viruses as part of a global preparedness for future epidemics. As it pertains to this goal, the present article summarizes the current knowledge of highly pathogenic arenaviruses and identifies opportunities for translational research.
A broad-spectrum antiviral targeting entry of enveloped viruses
Proceedings of the National Academy of Sciences, 2010
We describe an antiviral small molecule, LJ001, effective against numerous enveloped viruses including Influenza A, filoviruses, poxviruses, arenaviruses, bunyaviruses, paramyxoviruses, flaviviruses, and HIV-1. In sharp contrast, the compound had no effect on the infection of nonenveloped viruses. In vitro and in vivo assays showed no overt toxicity. LJ001 specifically intercalated into viral membranes, irreversibly inactivated virions while leaving functionally intact envelope proteins, and inhibited viral entry at a step after virus binding but before virus–cell fusion. LJ001 pretreatment also prevented virus-induced mortality from Ebola and Rift Valley fever viruses. Structure–activity relationship analyses of LJ001, a rhodanine derivative, implicated both the polar and nonpolar ends of LJ001 in its antiviral activity. LJ001 specifically inhibited virus–cell but not cell–cell fusion, and further studies with lipid biosynthesis inhibitors indicated that LJ001 exploits the therapeu...
Different Mechanisms of Cell Entry by Human-Pathogenic Old World and New World Arenaviruses
Journal of Virology, 2008
The Old World arenavirus Lassa virus (LASV) is the causative agent of severe viral hemorrhagic fever (VHF) in humans and is the most prevalent human pathogen among arenaviruses. The present study investigated the largely unknown mechanisms of cell entry of LASV, a process know to be mediated solely by the virus envelope glycoprotein (GP). To circumvent biosafety restrictions associated with the use of live LASV, we used reverse genetics to generate a recombinant variant of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) expressing the LASV GP (rLCMV-LASVGP). The rescued rLCMV-LASVGP grew to titers comparable to that of LCMV and showed the receptor binding characteristics of LASV. We used rLCMV-LASVGP to characterize the cellular mechanisms of LASV entry in the context of a productive arenavirus infection. The kinetics of pH-dependent membrane fusion of rLCMV-LASVGP resembled those of the human-pathogenic New World arenavirus Junin virus (JUNV) and other enveloped...
2001
Background: Recent studies of viral entry proteins from influenza, measles, human immunodeficiency virus, type 1 (HIV-1), and Ebola virus have shown, first with molecular modeling, and then X-ray crystallographic or other biophysical studies, that these disparate viruses share a coiled-coil type of entry protein. Results: Structural models of the transmembrane glycoproteins (GP-2) of the Arenaviruses, lymphochoriomeningitis virus (LCMV) and Lassa fever virus, are presented, based on consistent structural propensities despite variation in the amino acid sequence. The principal features of the model, a hydrophobic amino terminus, and two antiparallel helices separated by a glycosylated, antigenic apex, are common to a number of otherwise disparate families of enveloped RNA viruses. Within the first amphipathic helix, demonstrable by circular dichroism of a peptide fragment, there is a highly conserved heptad repeat pattern proposed to mediate multimerization by coiled-coil interactions. The amino terminal 18 amino acids are 28% identical and 50% highly similar to the corresponding region of Ebola, a member of the Filovirus family. Within the second, charged helix just prior to membrane insertion there is also high similarity over the central 18 amino acids in corresponding regions of Lassa and Ebola, which may be further related to the similar region of HIV-1 defining a potent antiviral peptide analogue. Conclusions: These findings indicate a common pattern of structure and function among viral transmembrane fusion proteins from a number of virus families. Such a pattern may define a viral transmembrane superfamily that evolved from a common precursor eons ago.