A medium-throughput screen for inhibitors of human metapneumovirus

Abstract

Human metapneumovirus, a paramyxovirus discovered in 2001, is a major cause of lower respiratory infection in adults and children worldwide. There are no licensed vaccines or drugs for human metapneumovirus. We developed a fluorescent, cell-based medium-throughput screening assay for human metapneumovirus that captures inhibitors of all stages of the viral lifecycle except budding of progeny virus particles from the cell membrane. We optimized and validated the assay and performed a successful medium-throughput screening. A number of hits were identified, several of which were confirmed to inhibit viral replication in secondary assays. This assay offers potential to discover new antivirals for human metapneumovirus and related respiratory viruses. Compounds discovered using the medium-throughput screening may also provide useful probes of viral biology.

Keywords: Human metapneumovirus; high-throughput screen; paramyxovirus; statins; topoisomerase inhibitors.

Figure 1.

Plate layout for MTS showing image of experimental plate with merged red (cell) and green (virus) fluorescent signals.

Figure 2.

Example plate of raw data used to calculate Z′ for MTS assay. Virus-positive wells are infected with HMPV at MOI 5 PFU/cell; negative wells are uninfected cells.

Figure 3.

Compound test plate from screen. Layout of plate is identical to Figure 1.

Figure 4.

In vitro inhibitory activity of mevastatin (MEV) and simvastatin (SIM). Cell monolayers were treated with the compounds or DMSO control at the indicated concentrations, infected with HMPV, and infectivity scored by fluorescence at 48 h (left y axis, bar indicates mean ± SD). MEV and SIM were tested for cytotoxicity using the CellTiterGlo assay (Promega) and the result plotted on the right y axis (dots, mean ± SD). HMPV: human metapneumovirus.

Figure 5.

In vitro inhibitory activity of irinotecan (IRI), etoposide (ETO), ciclopirox (CIC), isoproterenol (ISO), and acacetin (ACA). Cell monolayers were treated with the compounds or DMSO control at the indicated concentrations, infected with HMPV, and infectivity scored by fluorescence read at 800 nm at 48 h. Cell viability was measured by cell signal read at 700 nm. HMPV: human metapneumovirus.