Evidence against the Human Metapneumovirus G, SH, and M2-2 Proteins as Bona Fide Interferon Antagonists

FIG 1. Schematic overview of HMPV NL/1/00 genomes lacking G, SH, or M2-2 protein expression. Protein expression was ablated by either deletion (dashed line) of the ORF (e.g., HMPVΔG-ORF), deletion of the ORF and gene-start and gene-end signals (e.g., HMPVΔG), or introduction of several mutations leading to stop codons in the ORF (e.g., HMPVStopG) (red stars). The figure was generated using BioRender (https://biorender.com).

FIG 2. Replication kinetics of HMPV lacking SH (a and b), G (c and d), or M2-2 (e and f) protein expression in Vero-118 cells (a, c, and e) and A549 cells (b, d, and f). Samples were collected at the indicated time points. Data shown are representative of three individual experiments. Error bars indicate standard deviations (n = 2).

FIG 3. Fold increases in IFN production of A549 cells inoculated with HMPV mutants. (a) IFN expression levels were indirectly quantified by a luciferase-based bioassay and plotted as fold induction versus mock inoculated cells. (b) Infection percentages were determined by FACS analysis. Data shown are representative of three individual experiments. Error bars indicate standard deviations (n = 3).

FIG 4. IFN induction in A549 WT, A549ΔRIG-I, A549ΔPKR, and A549ΔMAVS cells upon inoculation with HMPV mutants. (a to c) Western blot analysis of clonal A549 cells with a knockout of MAVS (a), PKR (b), or RIG-I (c). M, marker; C, control (A549 WT cells). All clones analyzed for MAVS or PKR contained a knockout, and two of four clones analyzed for RIG-I had a knockout. (d to f) IFN levels produced by cells inoculated with HMPV lacking M2-2 (d), G (e), or SH (f) protein expression in (knockout) A549 cells. Each virus was used to inoculate three replicates that were used to quantify IFN production in the supernatant and three replicates that were used to determine the percentage of infected cells by FACS analysis. IFN production was quantified by a luciferase-based bioassay at 18 h postinfection in (knockout) A549 cells inoculated with HMPV mutants at an MOI of 3. Data shown are representative of three individual experiments. Error bars indicate standard deviations (n = 3). ****, P < 0.0001, as calculated by an unpaired t test.

FIG 5. Mutations in HMPVWT and mutant genomes detected by Illumina next-generation sequencing. The percentages of reads with specific mutations are shown. A schematic presentation of the genome drawn to scale is shown at the top. A variant analysis with a minimum coverage of 100 reads per nucleotide and a 2% cutoff value was performed using CLC Genomics Workbench software.

FIG 6. ADAR1 editing of M2-2 mutant virus genomes. (a) Percentages of ADAR1 knockdown (KD) by CRISPR interference in Vero-118 cells quantified by Western blotting. Two different gRNAs were used for knockdown of ADAR1 (gRNA1 and gRNA2). Different exposure times were used to visualize the ADAR-p110 and ADAR-p150 bands. (b to e) Mutation maps of viral genomes generated in Vero-118 cells expressing gRNA 2, using passage 3 virus stocks. (f and g) Magnification of nucleotide positions 12500 to 13378 from the HMPVStopM2-2 virus genomes, to highlight mutation hot spots. Control cells used in these experiments (b, d, and f) were transduced cells that were not treated with doxycycline. A variant analysis with a minimum coverage of 100 reads per nucleotide and a 2% cutoff value was performed using CLC Genomics Workbench software.

FIG 7. Northern blot analysis of RNA isolated from virus stocks of HMPV mutants. An RNA equivalent of 107 TCID50 was loaded as input in each gel lane. The probe was complementary to a 180-nucleotide stretch of the trailer region.