Distinct Patterns of IFITM-Mediated Restriction of Filoviruses, SARS Coronavirus, and Influenza A Virus

Interferon-inducible transmembrane proteins 1, 2, and 3 (IFITM1, 2, and 3) are recently identified viral restriction factors that inhibit infection mediated by the influenza A virus (IAV) hemagglutinin (HA) protein. Here we show that IFITM proteins restricted infection mediated by the entry glycoproteins (GP1,2) of Marburg and Ebola filoviruses (MARV, EBOV). Consistent with these observations, interferon-β specifically restricted filovirus and IAV entry processes. IFITM proteins also inhibited replication of infectious MARV and EBOV. We observed distinct patterns of IFITM-mediated restriction: compared with IAV, the entry processes of MARV and EBOV were less restricted by IFITM3, but more restricted by IFITM1. Moreover, murine Ifitm5 and 6 did not restrict IAV, but efficiently inhibited filovirus entry. We further demonstrate that replication of infectious SARS coronavirus (SARS-CoV) and entry mediated by the SARS-CoV spike (S) protein are restricted by IFITM proteins. The profile of IFITM-mediated restriction of SARS-CoV was more similar to that of filoviruses than to IAV. Trypsin treatment of receptor-associated SARS-CoV pseudovirions, which bypasses their dependence on lysosomal cathepsin L, also bypassed IFITM-mediated restriction. However, IFITM proteins did not reduce cellular cathepsin activity or limit access of virions to acidic intracellular compartments. Our data indicate that IFITM-mediated restriction is localized to a late stage in the endocytic pathway. They further show that IFITM proteins differentially restrict the entry of a broad range of enveloped viruses, and modulate cellular tropism independently of viral receptor expression

Pfn-1 knockdown abolishes anti-migratory activity of tyrphostin A9 but not purvalanol. A. Trajectories of individual cells

<p>treated with vehicle (DMSO), 10 µM purvalanol, or 10 µM tyrphostin A9. <b>B. Box and whiskers plot</b> documenting significant reduction in cell motility by both agents and reversal of anti-migratory activity by Pfn-1 siRNA of tyrphostin A9 but not purvalanol. Data are pooled from two independent experiments comprised of 28 cells each. *, p<0.0001. <b>C. Western blot</b> analysis of Pfn-1 expression in the presence or absence of Pfn-1 siRNA. Data are from a single experiment that has been repeated twice with identical results.</p

Confirmation of positives from the high-content cell migration screen. A. Multiparameter concentration-response confirmation in the Oris™ Pro assay.

<p>MDA-MB-231 cells were treated for 48 h with ten point, two-fold concentration gradients of test agents. Cells were stained with Hoechst 33342 and analyzed for cell migration, cell density, and condensed nuclei as described in Materials and Methods. Data are the averages ± SE from quadruplicate determinations and are from a single experiment that has been repeated twice. B. and C. Single-cell motility assay. Agents that showed selective, concentration-dependent inhibition of cell migration in the primary assay format were tested in a single-cell motility assay. Two compounds significantly reduced cell migration velocity compared with vehicle control. Box, 25th and 75th percentiles, whiskers, 10th and 90^th percentiles; line, median. Data are the combined values from two independent experiments, each comprising 28 individual cells. Rose plots in C. illustrate motility patterns of individual cells. Each line represents the trajectory of a single cell over a period of 48 h.</p

A high-throughput cell motility assay that enables multiplexed image-based analysis of cell migration and associated pharmacodynamic markers.

<p>A. Schematic of Oris™ Pro 384 Cell Migration Assay. Cells are seeded and allowed to adhere in an annular monolayer surrounding a Biocompatible Gel (BCG). The BCG dissolves to reveal a cell-free Detection Zone into which cells migrate. B. Wells are imaged via microscopy or High Content Imagers, and images analyzed for cell migration as well as phenotypic changes. C. Quantitation of readouts enables assembly of compound activity profiles including multiparameter toxicity (<i>left panel</i>), cell morphology, and pharmacodynamic markers of compound activity (<i>right panel</i>), exemplified by the actin-depolymerizing agent, cytochalasin D. All readouts are correlated with the primary functional phenotypic readout (<i>cell migration</i>).</p

HTS assay development.

<p>MDA-MB-231 cells were plated in Oris™ Pro 384 plates and allowed to attach for 2 h. Plates were stained with Hoechst 33342 immediately thereafter (<i>pre-migration</i>) or after 2 days in culture (<i>2-day migration</i>), and imaged on the ArrayScan II. <b>A. Seeding density.</b> Optimal gap closure with minimal background was obtained at 15,000 cells/well. <b>B. DMSO tolerance.</b> 16 wells each of minimum (pre-migration) and maximum (two-day migration) controls were treated with a ten-point, two-fold gradient of vehicle (DMSO) and numbers of cells that had migrated into the exclusion zone were enumerated. Assay performance decreased at concentrations above 0.6% DMSO due to toxicity. <b>C. Three-day variability.</b> Two full microplates of minimum and maximum controls were treated with vehicle (0.1% DMSO) on three consecutive days using equipment to be used in HTS. Intra-plate and inter-plate variability parameters were calculated (<i>Table</i>). SD, standard deviation; CV, coefficient of variance; PL to PL, plate to plate comparison; S:B ratio, signal-to-background ratio. Scatter plots illustrate day to day performance; the lower Z-factor on day 3 was a result of a partially obstructed dispense manifold. <b>D. Control inhibitor studies.</b> Using optimized assay conditions, identical IC₅₀ curves for cytochalasin D were obtained in three independent runs (<i>left panel</i>). Multiparametric profiling of cell migration, toxicity (cell density), and nuclear morphology (brightness and area) document selective inhibition of cell migration in the absence of overt cytotoxicity (<i>right panel</i>).</p

LOPAC library screening for inhibitors of cell migration.

<p>Cells were treated in duplicate Oris™ Pro 384 plates with vehicle (<i>green</i>), 1 µM cytochalasin D (<i>red</i>), 0.1 µM cytochalasin D (<i>pink</i>), <i>or</i> 10 µM of compounds (<i>blue</i>) for 2 days. Cells that migrated into the exclusion zone were enumerated by high-content analysis on the ArrayScan II. <b>A. Histograms</b> show that positive and negative controls were well separated and data largely followed a normal distribution. <b>B. Trellis plots.</b> Z-scores were calculated for each data point based on plate average and plotted against well number. Data are from one replicate run; the y-axis shows z-scores of migrated cells.</p

Confirmation of anti-migratory activity by Pfn-1 inducing agents.

<p>Cells were treated with compounds for 48-1 coated cell culture dishes, replated and imaged by time-lapse videomicroscopy. <b>A. Trajectories of individual MDA-MB-231 cells</b> of different experimental groups in time-lapse motility assay. Data are from a single experiment that has been repeated once with identical results. <b>B. Box and whisker plots</b> representing the average speed of migration of MDA-MB-231 cells treated with DMSO (control) vs. 10 µM of either purvalanol or tyrphostin A9. Box, 25th and 75th percentiles; whiskers, 10th and 90th percentiles; line, median. Data are pooled cell data from two independent experiments (n = 20 cells per group; *p<0.001).</p

Prioritized cell migration inhibitors from the LOPAC screen.

a<p>according to SIGMA-Aldrich LOPAC description.</p