Identification and Characterization of a Novel Broad-Spectrum Virus Entry Inhibitor

Abstract

Virus entry into cells is a multistep process that often requires the subversion of subcellular machineries. A more complete understanding of these steps is necessary to develop new antiviral strategies. While studying the potential role of the actin network and one of its master regulators, the small GTPase Cdc42, during Junin virus (JUNV) entry, we serendipitously uncovered the small molecule ZCL278, reported to inhibit Cdc42 function as an entry inhibitor for JUNV and for vesicular stomatitis virus, lymphocytic choriomeningitis virus, and dengue virus but not for the nonenveloped poliovirus. Although ZCL278 did not interfere with JUNV attachment to the cell surface or virus particle internalization into host cells, it prevented the release of JUNV ribonucleoprotein cores into the cytosol and decreased pH-mediated viral fusion with host membranes. We also identified SVG-A astroglial cell-derived cells to be highly permissive for JUNV infection and generated new cell lines expressing fluorescently tagged Rab5c or Rab7a or lacking Cdc42 using clustered regularly interspaced short palindromic repeat (CRISPR)-caspase 9 (Cas9) gene-editing strategies. Aided by these tools, we uncovered that perturbations in the actin cytoskeleton or Cdc42 activity minimally affect JUNV entry, suggesting that the inhibitory effect of ZCL278 is not mediated by ZCL278 interfering with the activity of Cdc42. Instead, ZCL278 appears to redistribute viral particles from endosomal to lysosomal compartments. ZCL278 also inhibited JUNV replication in a mouse model, and no toxicity was detected. Together, our data suggest the unexpected antiviral activity of ZCL278 and highlight its potential for use in the development of valuable new tools to study the intracellular trafficking of pathogens. IMPORTANCEThe Junin virus is responsible for outbreaks of Argentine hemorrhagic fever in South America, where 5 million people are at risk. Limited options are currently available to treat infections by Junin virus or other viruses of the Arenaviridae, making the identification of additional tools, including small-molecule inhibitors, of great importance. How Junin virus enters cells is not yet fully understood. Here we describe new cell culture models in which the cells are susceptible to Junin virus infection and to which we applied CRISPR-Cas9 genome engineering strategies to help characterize early steps during virus entry. We also uncovered ZCL278 to be a new antiviral small molecule that potently inhibits the cellular entry of the Junin virus and other enveloped viruses. Moreover, we show that ZCL278 also functions in vivo, thereby preventing Junin virus replication in a mouse model, opening the possibility for the discovery of ZCL278 derivatives of therapeutic potential. T he Junin virus (JUNV) is an important member of the NewWorld arenavirus family (clade B) and is responsible for outbreaks of Argentine hemorrhagic fever in South America, where 5 million people are considered to be at risk (1). The reservoir of JUNV is the rodent Calomy...

Dynamics of phosphoinositide conversion in clathrin-mediated endocytic traffic

“…9b, d), but the TIRF geometry did not allow us to follow most of the uncoated vesicles as they moved into the cell. We obtained similar results in gene-edited human SVGA cells27 (Extended Data Fig. 9e) and in SUM159 cells transiently expressing EGFP-Rab5a (Extended Data Fig.…”

Transcription and replication mechanisms of Bunyaviridae and Arenaviridae L proteins

“…Crystallographic and CryoEM structures show that the Nterminus of the LACV L protein, corresponding to the EN responsible for cap-snatching, is flexibly hanging from the central core. This peripheral extension of the L proteins has also been observed for the MACV L protein, and described in detail by high resolution structures for the polymerases of influenza and VSV, and were associated in all the structures to the transcriptional processes of cap-snatching for sNSV and capping for nsNSV (Chou et al, 2016;Kranzusch et al, 2010;Pflug et al, 2014;Reguera et al, 2016a). These peripheral extensions are in all these polymerases located at the opposite side of the template RNA entry and exit channels showing a protein compartmentalization where the RNA reading occurs in one side and the product RNA in the other.…”

Membrane dynamics of dividing cells imaged by lattice light-sheet microscopy

“…The single PCR product obtained in the genome-edited cell line was larger than the corresponding product obtained in the parental SUM159 cells (WT) using the same set of PCR probes. (C) Immunoprecipitation with the monoclonal antibody specific for β1/β2 of AP-2 (Clairmont et al , 1997) from cell-free lysates obtained from SUM159 cells (WT), stably expressing σ2-eGFP (σ2-eGFP overexp ), or genome-edited (σ2-eGFP edited+/+ ), followed by Western blot analysis with an antibody specific for σ2. The data show presence of σ2-eGFP and undetectable amounts of nontagged σ2 in SUM-AP-2.1 cells.…”