The Interferon Type I/III Response to Respiratory Syncytial Virus Infection in Airway Epithelial Cells Can Be Attenuated or Amplified by Antiviral Treatment

Abstract

Human respiratory syncytial virus (RSV) is a single-stranded RNA virus that causes acute, and occasionally fatal, lower respiratory illness in young infants, the elderly, and immunocompromised patients. Therapeutic interventions able to cut short viral replication and quickly return the airways to normal function are needed. An understanding of antiviral activities and their effects on host defense mechanisms is important for the design of safe and effective therapy. We targeted functionally and temporally distinct steps within the viral life cycle using small-molecule RSV inhibitors and studied their antiviral activities and their effects on innate interferon responses of airway epithelial cells in vitro. Antivirals acting upstream of RSV polymerase activity (i.e., compounds targeting the fusion protein or the nucleoprotein) reduced viral load immediately postinfection and partially attenuated interferon responses. In contrast, antivirals directed to the RSV polymerase demonstrated activity throughout the viral replication cycle and specifically modulated the RIG-I/mitochondrial antiviral signaling protein (MAVS)/TBK1/IRF3/interferon-stimulated gene (ISG) axis, causing either an upregulation or a downregulation of interferon responses, depending on the mechanism of polymerase inhibition. Notably, polymerase inhibition leading to the accumulation of abortive RNA products correlated with the amplification of interferon-stimulated genes to up to 10 times above normal infection levels. Understanding how antiviral activities and their modulation of innate immunity may affect recovery from RSV infection will help guide the development of safe and effective therapies.

Importance: RSV circulates seasonally, causing acute lower respiratory disease. Therapeutic interventions with efficacy throughout the viral replication cycle, rapid viral clearance, and prevention of potentially harmful inflammatory responses are desirable. Compounds targeting the RSV polymerase inhibited virus replication late in the viral life cycle and, depending on the functional domain targeted, either attenuated or amplified RIG-I and downstream interferon pathways in infected cells. These data will help guide the development of safe and effective therapies by providing new molecular evidence that the mechanism of inhibition by an antiviral compound can directly impact innate antiviral immune responses in the airway epithelium.

FIG 1

Antiviral activities of RSV inhibitors in A549 cells. (A) The EC₅₀ concentration of each compound was determined in a colorimetric RSV F protein cell-based ELISA. The x axis data show the concentrations of compound, and the y axis data represent the measured levels of infection relative to a DMSO control. (B) A 20-fold excess of compound EC₅₀ concentrations was used to treat A549 cells at 24 h post-RSV infection. Cells were harvested at 48 h postinfection and the relative levels of viral RNA determined by sequencing 500 ng of total cellular poly(A) RNA (RSV transcript numbers are indicated above the bars). The data in panel B were verified by RT-qPCR assays (n = 3). (C) Cells were treated and harvested as described for panel B and the levels of viral protein detected in 50 μg total protein/lane using pAb anti-RSV Western blot analysis (lane 1, mock infection; lanes 2, 3, 4, 5, and 6, RSV infected with DMSO, TMC-353121, RSV-604, YM-53403, and BI-D, respectively). The data shown in panels A and C are representative of the results of 3 independent assays.

FIG 2

Helicase sensing of RSV-2 infection and polymerase inhibition in A549 cells. A549 cells infected for 24 h before treatment and harvested at 48 h postinfection were subjected to Western blot analysis of proteins in the innate viral RNA sensing pathway. Total protein was assayed and 50 μg/lane loaded for mock infection (lane 1) and RSV-A2-infected DMSO (lane 2), YM-53403 (lane 3), and BI-D (lane 4). RIG-I and MDA-5 were expressed only upon infection (top row blots, lane 1 versus lanes 2 to 4), whereas MAVS (top row, rightmost blot, lanes 1 to 4), NF-κβ p65, IKKε, TBK1, and IRF3 (middle row blots, lanes 1 to 4) were all constitutively expressed and unaffected by infection. Increased levels of phosphorylation of NF-κβ p65, TBK1, and IRF3 occurred upon infection (bottom blots, lane 1 versus lane 2). Levels of phosphorylation of TBK1 and IRF3 were differentially affected by the polymerase inhibitors, being decreased byYM-53403 (lane 2 versus lane 3) and unchanged by BI-D (lane 2 versus lane 4). These changes were observed in 3 independent experiments.

FIG 3

Compound time-of-addition (ToA) profiles for RSV inhibitors. (A) Single-cycle RSV-A2 infection of A549 (dashed lines) or HEp-2 (solid lines) cells. TMC-353121, RSV-604, YM-53403, or BI-D and ribavirin (80 μM) were added at −4, 0, 2, 4, 6, 8, 12, 16, 20, and 24 h postinfection as indicated on the x axis. All cells were harvested at 36 h postinfection, and the relative amounts of RSV N-gene/GAPDH were determined by RT-qPCR and are expressed as a percentage relative to the averaged data from DMSO-treated infections (n = 6). (B) Multicycle RSV-A2 infections of HAE cells (donor 1) were treated at 0, 24, and 48 h postinfection with compounds as described for panel A. Cells were harvested at 72 h postinfection and analyzed as described for panel A. Data shown represent averages of the results of 3 independent experiments. Error bars corresponding to the single-cycle infections were too large to include.

FIG 4

Effects of postentry inhibitors on RNA synthesis in RNP or L/P biochemical assays. All compounds were tested at a fixed concentration of 50 μM. (A) In the RNP assay, RSV polymerase is extracted from infected HEp-2 cells in heterogeneous, preformed complexes, and the transcription of full-length mRNA products was measured. nt, no transcription. (B) RNA polymerase assay using purified complexes of L and P proteins coexpressed in baculovirus cells and an oligonucleotide template representing the promoter for genome synthesis (with the +3 and +1 start sites calibrated using ladders 1 and 2, respectively). Lanes 3 and 4 show the results of negative-control reactions in which the L/P complex was omitted and of negative-control reactions in which the L/P complex contained a substitution at amino acid N812 of the L protein, respectively. The data are representative of the results of three independent experiments performed with two different wild-type L/P preparations.