Calcium Regulation of Hemorrhagic Fever Virus Budding: Mechanistic Implications for Host-Oriented Therapeutic Intervention

<div><p>Hemorrhagic fever viruses, including the filoviruses (Ebola and Marburg) and arenaviruses (Lassa and Junín viruses), are serious human pathogens for which there are currently no FDA approved therapeutics or vaccines. Importantly, transmission of these viruses, and specifically late steps of budding, critically depend upon host cell machinery. Consequently, strategies which target these mechanisms represent potential targets for broad spectrum host oriented therapeutics. An important cellular signal implicated previously in EBOV budding is calcium. Indeed, host cell calcium signals are increasingly being recognized to play a role in steps of entry, replication, and transmission for a range of viruses, but if and how filoviruses and arenaviruses mobilize calcium and the precise stage of virus transmission regulated by calcium have not been defined. Here we demonstrate that expression of matrix proteins from both filoviruses and arenaviruses triggers an increase in host cytoplasmic Ca²⁺ concentration by a mechanism that requires host Orai1 channels. Furthermore, we demonstrate that Orai1 regulates both VLP and infectious filovirus and arenavirus production and spread. Notably, suppression of the protein that triggers Orai activation (Stromal Interaction Molecule 1, STIM1) and genetic inactivation or pharmacological blockade of Orai1 channels inhibits VLP and infectious virus egress. These findings are highly significant as they expand our understanding of host mechanisms that may broadly control enveloped RNA virus budding, and they establish Orai and STIM1 as novel targets for broad-spectrum host-oriented therapeutics to combat these emerging BSL-4 pathogens and potentially other enveloped RNA viruses that bud via similar mechanisms.</p></div

Synta66 inhibits egress of authentic filoviruses and arenaviruses.

<p><b>A</b>. HeLa cells were infected with LASV (MOI 0.01), JUNV (MOI 0.1), MARV (MOI 0.1), or EBOV (MOI 0.1) and treated with Synta66 at indicated concentrations. Cellular virus levels were detected by immunofluorescence staining of fixed cells at 72 (LASV, JUNV) or 96 (MARV, EBOV) hours post infection with virus specific antibodies. The percent of cells infected (relative to total cells) was determined using Harmony High Content Imaging and Analysis software (PerkinElmer). Data is expressed relative to vehicle (DMSO) control treated cells for each virus. The percent of infected cells for vehicle control treatment was as follows (LASV = 12% ± 2.69%, JUNV = 9% ± 1.11%, MARV = 20% ± 1.92%, EBOV = 15% ± 1.55%). Error bars indicate standard error of mean (SEM) and statistical significance was determined by two way ANOVA with Bonferroni multiple comparisons (** p < 0.01, **** p < 0.0001). <b>B</b>. Representative images from a single live virus experiment demonstrate Synta66 dose (0, 5, 10, 30, 50μM) dependent inhibition of virus spread. For each condition, respective viruses were detected with virus specific antibodies (green). The value in the upper left hand corner of each image is the percentage of total cells infected. For each condition, the total number of cells was determined by staining nuclear DNA with Hoechst DNA dye.</p

Viral matrix protein expression mobilizes cytoplasmic Ca²⁺.

<p>Viral matrix proteins or vector controls and the genetically encoded Ca²⁺ indicator R-GECO-1 were co-expressed in WT HEK293T cells, HEK293T cells stably expressing the dominant negative permeation defective Orai (E106A) mutant, or WT HEK293T cells treated with the Orai inhibitor Synta66 (50 μM, lower right). Cytoplasmic R-GECO-1 fluorescence emission (580nM) was monitored between 6 and 24 hours post transfection from cells cultured in an environmentally controlled chamber on the microscope stage. Each trace represents the average +/- SEM of at least two experiments of at least 20 cells.</p

Synta66 and 2-APB inhibit egress of live JUNV (Candid-1 strain) from VeroE6 cells.

<p>JUNV foci were visualized and the number of all detectable foci was plotted for each Synta66 <b>(A)</b> or 2-APB concentration <b>(B)</b>. Statistical significance was analyzed by one way ANOVA (*<i>p</i> < 0.01 for Synta66) (*<i>p</i> < 0.05, **<i>p</i> < 0.01 for 2-APB). MTT based cell viability measurements demonstrate no toxicity of Synta66 <b>(A)</b> or 2-APB <b>(B)</b> under identical conditions as those used for JUNV infection of VeroE6 cells. Each value is normalized to cells treated with DMSO alone. Comparable levels of JUNV GP were detected by immunoblot analysis in JUNV infected VeroE6 cells in the absence (DMSO alone) or presence of Synta66 <b>(A)</b> or 2-APB <b>(B)</b>.</p

STIM1 suppression inhibits egress of eVP40 VLPs.

<p><b>A</b>. VP40 was expressed in control and STIM1 suppressed HEK293T cells as indicated and eVP40 VLP production quantified by immunoblot analysis demonstrates significant inhibition of eVP40 production in STIM1 suppressed cells (n = 4 independent experiments). <b>B</b>. Immunoblot analysis of cell extracts and VLPs from HEK293T cells expressing eVP40 in cells co-transfected with indicated amounts of control vector (top panel), STIM1 suppression plasmid (middle panel), or shSTIM1-STIM1 suppression-rescue plasmid (bottom panel). <b>C</b>. Cellular VP40 and STIM1 levels were measured in mock-transfected HEK293T cells, or cells transfected with the indicated plasmids and siRNAs. Results are representative of three independent experiments.</p

Genetic inactivation of Orai Ca²⁺ permeation inhibits egress of authentic filoviruses and arenaviruses.

<p>Wild type HEK293T cells and a Ca²⁺ permeation defective HEK293T line that stably overexpresses the dominant negative Orai E106A mutant were infected with LASV (MOI 0.05), JUNV (MOI 0.1), MARV (MOI 0.1), or EBOV (MOI 0.5). Cellular virus levels were detected by immunofluorescence staining of fixed cells at 72 (LASV) or 96 (JUNV, MARV, EBOV) hours post infection. The percent of infected cells relative to total viable cells per condition is plotted and error bars represent standard error of mean (SEM). Statistical significance was determined by student t test, two-tailed (*** p < 0.0001, **** p < 0.0001 as indicated)</p

Budding of filovirus and arenavirus VLPs from WT and E106A cells.

<p>WT or Orai1 E106A mutant HEK293T cells were transfected as indicated with eVP40 <b>(A)</b>, mVP40 <b>(B)</b>, JUNV-Z <b>(C)</b>, or LASV-Z <b>(D)</b> for 24 hours and VP40 or Z protein levels in cells and VLPs were quantified by immunoblot analysis. Expression of cellular actin served as a loading control. Results are representative of three independent experiments.</p

Pharmacological effect of Synta66 and 2-APB on egress of filovirus VLPs.

<p>HEK293T expressing eVP40 (<b>A</b> and <b>B</b>) or mVP40 (<b>C</b> and <b>D</b>) were treated with indicated concentrations of 2-APB or Synta66. eVP40 and mVP40 levels in cell extracts and VLPs were detected by immunoblot analysis and quantified in VLPs (bar graphs). 2-APB <b>(E)</b> and Synta66 <b>(F)</b> cytotoxicity was assessed with an MTT viability assay under conditions that mimicked those used for the VLP experiments. In all experiments, cellular actin served as a loading control. Relative protein bands densities were normalized to untreated sample densities (0 μM). Plotted values represent the average (+/- S.E.M) of 3–6 independent experiments and were analyzed with either Welch's t-test or Wilcoxon rank-sum test (*p = 0.01, **p = 0.05).</p

Synta66 decreases the number of viral particles in supernatants.

<p>A virus plaque assay used to quantify infectious virions in supernatants of virus infected cells demonstrates that Synta66 (30 μM) significantly inhibits the budding and spread of live LASV, JUNV, MARV, and EBOV. Error bars indicate standard error of mean (SEM) and statistical significance was determined by two way ANOVA with Bonferroni multiple comparisons (**p<0.01, ***p < 0.001, ****p < 0.0001).</p