Arenavirus reverse genetics: New approaches for the investigation of arenavirus biology and development of antiviral strategies

dArenaviruses merit significant interest as important human pathogens, since several of them cause severe hemorrhagic fever disease that is associated with high morbidity and significant mortality. Currently, there are no FDA-licensed arenavirus vaccines available, and current antiarenaviral therapy is limited to an off-labeled use of the nucleoside analog ribavirin, which has limited prophylactic efficacy. The pyrimidine biosynthesis inhibitor A3, which was identified in a high-throughput screen for compounds that blocked influenza virus replication, exhibits a broad-spectrum antiviral activity against negative-and positive-sense RNA viruses, retroviruses, and DNA viruses. In this study, we evaluated the antiviral activity of A3 against representative Old World (lymphocytic choriomeningitis virus) and New World (Junin virus) arenaviruses in rodent, monkey, and human cell lines. We show that A3 is significantly more efficient than ribavirin in controlling arenavirus multiplication and that the A3 inhibitory effect is in part due to its ability to interfere with viral RNA replication and transcription. We document an additive antiarenavirus effect of A3 and ribavirin, supporting the potential combination therapy of ribavirin and pyrimidine biosynthesis inhibitors for the treatment of arenavirus infections.

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“…8A) or Candid#1 (Fig. 8B) (36,38,48). At 48 h.p.i., A3 exhibited a dose-dependent inhibitory effect on expression of both GFP (Fig.…”

Section: Resultsmentioning

confidence: 96%

“…At 5 h posttransfection, medium was replaced with medium containing the indicated compounds and concentrations. At 72 h posttransfection, cell lysates were prepared to determine expression levels of CAT protein by using a CAT enzymelinked immunosorbent assay (ELISA) kit (Roche) (38).…”

Section: Methodsmentioning

confidence: 99%

Inhibition of Arenavirus by A3, a Pyrimidine Biosynthesis Inhibitor

Ortiz-Riaño

Ngo

DeVito

et al. 2014

J Virol

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“…JUNV is endemic in regions of Argentina and West Africa and is the cause of severe episodes of hemorrhagic fever with high mortality rates. The search for effective therapeutics to treat arenavirus infections, particularly those caused by Junin and Lassa viruses, has been an active area of research for many years (8,(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42). More recent efforts in the field of antiviral therapeutics for emerging RNA viruses have focused on identifying host factors or virus-host interactions, rather than solely viral proteins, as targets for novel antivirals (6, 8-13, 39, 40, 43, 44).…”

Section: Discussionmentioning

confidence: 99%

A Host-Oriented Inhibitor of Junin Argentine Hemorrhagic Fever Virus Egress

Han

Liu

et al. 2014

J Virol

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IMPORTANCE There are currently no FDA-approved vaccines or therapeutics to prevent or treat Argentine hemorrhagic fever (AHF). The causative agent of AHF is Junin virus (JUNV); a New World arenavirus classified as an NIAID/CDC category A priority pathogen.Here, we describe a prototype therapeutic that blocks budding of JUNV and has the potential to function as a broad-spectrum antiviral drug. J unin virus (JUNV) is a New World arenavirus endemic in Argentina that causes severe Argentine hemorrhagic fever and significant mortality in humans (1). The virus is present in excreta from infected rodents and is typically spread to humans via aerosolization (1). JUNV is important because it is a biosafety level 4 pathogen with potential use as a bioterror agent, for which there are no U.S. Food and Drug Administration (FDA)-approved vaccines or therapeutics. JUNV is an enveloped, bisegmented negative-sense RNA virus that encodes four proteins: surface glycoprotein precursor (GPC), RNA polymerase (L), nucleoprotein (NP), and small RING finger matrix protein (Z). The Z protein of arenaviruses plays a key role in promoting virus egress and spread and is capable of budding independently from mammalian cells in the form of virus-like particles (VLPs) (2-6). Efficient separation of arenavirus Z VLPs from the plasma membrane is promoted by viral L domains within the Z protein (for a review, see reference 7). L domains function by recruiting host proteins that facilitate efficient virus-cell separation ("pinching-off") (7). JUNV Z protein contains a single PTAP type L domain which interacts with host Tsg101, a component of the endosomal sorting complexes required for transport (ESCRT) machinery (5-7). L-domain-mediated recruitment of host proteins by virus represents a potential target for the development of novel antiviral inhibitors to disrupt virus egress from infected cells to reduce virus dissemination and disease progression (3,6,(8)(9)(10)(11)(12)(13).Based upon the known nuclear magnetic resonance structure of the Tsg101-PTAP interaction site, we conducted an in silico screen for competitive binding inhibitors. We identified several compounds, and the most potent (compound 0013) blocks both Z VLP and live JUNV egress at nanomolar concentrations. Moreover, compound 0013 specifically blocks PTAP-Tsg101 interactions. Intriguingly, since L-domain-containing matrix proteins are generally required for efficient virus-cell separation of emerging RNA viruses (filoviruses, arenaviruses, henipaviruses, rhabdoviruses, paramyxoviruses, and retroviruses), we speculate that inhibitors of this virus-host interaction could represent broadspectrum antiviral drugs against a range of enveloped RNA viruses. MATERIALS AND METHODSCell lines, virus, and antibodies. VeroE6 and HEK293T cells were grown in Dulbecco modified Eagle medium supplemented with 10% fetal calf serum. The Candid-1 vaccine strain of JUNV was kindly provided by Robert B. Tesh (University of Texas Medical Branch, Galveston, TX) via

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“…G2 of Z is known to be myristoylated and is critical for cellular membrane attachment and subsequent virion release (Perez et al, 2004;Strecker et al, 2006). The central domain includes the RING domain (zinc-finger motif), which has been shown to regulate genome replication and gene transcription (Cornu & de la Torre, 2001, 2002Cornu et al, 2004;Emonet et al, 2011;Kranzusch & Whelan, 2011;. The C terminus includes L-domains, which are known to regulate the virus budding process.…”

Section: Introductionmentioning

confidence: 99%

Cis- and cell-type-dependent trans-requirements for Lassa virus-like particle production

Urata

Yasuda

2015

Journal of General Virology

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Lassa virus (LASV) small zinc-finger protein (Z), which contains two L-domain motifs, plays a central role in virus budding. Here, we report that co-expression of glycoprotein (GPC) altered the requirements for cholesterol but not the L-domains and host factor, Tsg101, for Z-induced viruslike particle (VLP) production. In particular, the cholesterol requirement for VLP production was cell-type-dependent. In addition, GPC was found to be important for co-localization of Z with CD63, a late endosomal marker. We also found that the N-terminal region (aa 3-10) of Z was critical for its myristoylation and VLP production. These findings will contribute to our understanding of LASV assembly and budding.

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Arenavirus reverse genetics: New approaches for the investigation of arenavirus biology and development of antiviral strategies

Cited by 66 publications

References 121 publications

Inhibition of Arenavirus by A3, a Pyrimidine Biosynthesis Inhibitor

Inhibition of Arenavirus by A3, a Pyrimidine Biosynthesis Inhibitor

A Host-Oriented Inhibitor of Junin Argentine Hemorrhagic Fever Virus Egress

Cis- and cell-type-dependent trans-requirements for Lassa virus-like particle production

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