Reduced expression of human metapneumovirus matrix protein impacts virus assembly and ribonucleoprotein localization

Fig 1. PPMO design. (A) Schematic structure of a peptide-conjugated phosphorodiamidate morpholino oligomer. Illustrated are two representative PMO subunits, each containing a morpholine ring and nucleobase, with the subunits connected by phosphorodiamidate linkages. The PMO is conjugated to a single cell-penetrating peptide (CPP) to produce PPMO. For this study, the CPP was (RXR)4XB (sometimes abbreviated “P7”), where R is arginine, X is 6-aminohexanoic acid, and B is beta-alanine, and was conjugated at the 3′ end of each PMO. (B) Table of the main PPMOs used in this study and their respective targets and sequences. The location of the PPMOM target sequence is shown based on the HMPV CAN 97-83 genome (Accession number AY297749.1). (C) The alignment of PPMOM on the target viral mRNA (vmRNA) sequence. Green bases represent the gene start (GS) sequence, and the blue bases show its complement; bolded bases are the initiating codon, and red bases are the PPMO. The M gene ORF sequence is indicated in both the vmRNA and viral genomic RNA (vgRNA).

Fig 2. PPMOM treatment specifically reduces M protein expression and alters N protein localization in infected cells. HMPV-infected or MOCK-infected A549 cells were treated with water or varying concentrations of PPMOM at 1 hpi. (A) At 24 hpi, the cells were lysed and used for immunoblotting with anti-GAPDH, anti-M, and anti-HMPV N antibodies. Data are from three independent experiments and shown as the mean ± SD (n = 3). (B) At 24 hpi, the cells were fixed and stained with an anti-HMPV N (red) antibody and DAPI (blue) and examined by confocal microscopy. Data are from three independent experiments, and a single representative experiment is shown as a single z-slice. The magnified regions are outlined by dashed white boxes, and the white arrows point to the edge of the cell. Scale bars are 10 µm. (C) A549 cells were MOCK-infected or infected with WT HMPV and treated with water, 10 µM PPMONC, or 10 µM PPMOM at 1 hpi. The infection was allowed to progress until 24 hpi, and then the cells were lysed and used for immunoblotting with anti-GAPDH, anti-M, and anti-HMPV N antibodies. Data are from four independent experiments and shown as the mean ± SD (n = 4). **P < 0.01, ***P < 0.001, by…

Fig 3. Viral RNA localization affected by the presence of M protein. A549 cells were infected with WT HMPV and treated with 10 µM PPMONC or PPMOM at 1 hpi. At 6, 12, or 24 hpi, (A) the cells were fixed and stained with anti-HMPV N (green; left) or anti-HMPV F (green; right) antibodies. FISH staining for vRNA (red/white) was then performed, followed by DAPI (blue) staining. Samples were imaged by confocal microscopy and are shown as maximum intensity projections. Data are from three independent experiments, and a single representative experiment is shown. Red arrows show vRNA filaments, and the magnified region is shown by a white box. Scale bar is 10 µm. (B) IB count and volume were quantified (per cell) using vRNA as a marker for IBs. The images were then analyzed to detect individual IBs and their volumes per cell. At least 45 cells from each condition were analyzed across three independent experiments and plotted individually with the mean (black line). (C) IBs were grouped into three sizes: small (<0.2 µm3), medium (between 0.2 and 3 µm3), and large (>3 µm3). The percentage of IBs was calculated by dividing the number of IBs within a group by the total number of IBs in that cell.…

Fig 4. The M protein does not impact viral transcription or replication. A549 cells were MOCK-infected or infected with WT HMPV and subsequently treated with water, 10 µM PPMONC, or 10 µM PPMOM at 1 hpi. At 6, 12, or 24 hpi, the cells were lysed, and RNA was extracted and purified. (A) Total RNA was quantified using one-step RT-qPCR using primers targeting Actinβ, HMPV N, M, and F. (B) Viral mRNA (vmRNA) was quantified by running cDNA synthesis and then qPCR for Actinβ, HMPV N, and HMPV F. (C) Viral genomic RNA (vgRNA) was quantified with cDNA synthesis and then qPCR for HMPV N. Data are from three independent experiments, each in duplicate, and shown as the mean of the experimental replicates ± SD (n = 3). (D) A549 cells were plated and transfected with codon-optimized pCAGGS-HMPV N, pCAGGS-HMPV mCherryP, and varying doses of pCAGGS-HMPV HA-M (69); 24 h post-transfection, the cells were fixed and imaged through confocal microscopy. IB-like structures were thresholded by the mCherry signal, and their volume was measured. Objects below a major axis length of 0.5 µm were excluded from analysis. IB-like structures were grouped into three sizes: small (<0.2 µm3), medium (between 0.2 and 3…

Fig 5. The M protein is involved in ribonucleoprotein localization. (A) A549 cells were infected with WT HMPV for 3 h, washed, and subsequently transfected with 1 µg GFP-M (green) and 1 µg mCherryP (red) at 3 hpi. At 24 hpi, live cells were imaged. Shown is a representative single z-slice time-lapse acquired every 1 s, and the selected frames are shown. The white arrows show a vRNP complex moving throughout the cell. (B) mCherryP (red) was observed trafficking along eGFP-tagged α-tubulin (Addgene Plasmid #21039) (green) (70). Scale bar is 1 µm. (C) A549 cells were infected with WT HMPV for 3 h, washed, and subsequently transfected with 1 µg GFP-M (green) and 1 µg mCherryP (red) at 3 hpi. At 24 hpi, live cells were imaged. Scale bar is 1 µm. (D) A549 cells were plated and infected with WT HMPV for 3 h, washed, and subsequently transfected with 2 µg of pCAGGs-mCherryP (red) at 3 hpi. At 6 hpi, 10 µM of PPMOs were added. At 24 hpi, the cells were fixed and stained with Alexa Fluor 647-conjugated WGA (pseudo-colored green) and DAPI (blue) and imaged by confocal microscopy. A single z-slice is shown, and the scale bar is 10 µm. (E) At least 10 cells were measured in each replicate, which…

Fig 6. The M protein is not necessary for viral RNP movement. A549 cells were infected with WT HMPV for 3 h, washed, and transfected with mCherryP at 3 hpi. At 6 hpi, the media were changed, and 15 µM PPMOs were added. At 24 hpi, the cells were lysed and analyzed with immunoblots with (A) anti-M, anti-N, and anti-GAPDH antibodies. Before lysis, infected cells were imaged in real time to track mCherryP signal at approximately 500-ms intervals for 1 min. Tracks were manually measured from start to stop. The (B) average and (C) average maximum speeds for at least 150 tracks were analyzed for each treatment condition. Three independent experiments are shown as the mean ± SD (n = 3). (D) The average speed of each track measured is shown in a histogram where the tracks are grouped into bins of 0.2 µm/s. **P < 0.01, by two-way ANOVA.

Fig 7. HMPV M protein levels directly correlate with viral particle production. A549 cells were MOCK-infected or infected with rgHMPV and treated with water, 10 µM PPMONC, or 10 µM PPMOM at 1 hpi. The infection was allowed to progress until 24 hpi, at which point the cultures were treated with 0.3 µg/mL TPCK-trypsin for 1 h. Purified virus was detected by immunoblotting with (A) anti-M and anti-HMPV N antibodies, or (B) particles were titered by immunofluorescence. Blots were quantified by densitometry and normalized to the PPMONC-treated infection. (C) A549 cells were MOCK-infected or infected with WT HMPV and treated with 10 µM of PPMOs at 6, 12, or 18 hpi. After treatment, the infection was allowed to progress until 24 hpi, at which point cell lysates and (D) purified virus were immunoblotted with anti-HMPV N, anti-M, and anti-GAPDH antibodies. Blots were quantified by densitometry and were normalized to the PPMONC-treated infection. (E) A549 cells were infected with WT HMPV and treated with 1, 5, or 10 µM of PPMOs at 1 hpi. At 24 hpi, free virus was purified from the supernatant, and the cells were lysed and used for immunoblotting with anti-HMPV N, anti-M, and anti-GAPDH…

Fig 8. Decreased expression of HMPV M leads to changes in F distribution. A549 cells were infected with WT HMPV or MOCK-infected and then treated with water, 10 µM PPMONC, or 10 µM PPMOM at 1 hpi. At 24 hpi, (A) the cells were fixed and stained with anti-HMPV F (green) and DAPI (blue), followed by confocal microscopy. Magnified images are outlined by a white box. A white arrow points to mature and immature filaments. (B) Cells were stained for flow cytometry with an anti-HMPV F antibody and gated using F as a marker for infection. The mean fluorescence intensity (MFI) of F on the surface was measured by averaging the F signal intensity per infected cell and normalized to vehicle treatment. Data are shown as the mean ± SD (n = 4). (C) An image previously shown was cross-sectioned (shown by the dashed box) to show F protein (green) localization with respect to the nucleus stained with DAPI (blue). (D) Immunofluorescence imaging of A549 cells that were infected with WT HMPV and treated with 10 µM of PPMOs at 6, 12, or 18 hpi. The infected cells were fixed at 24 hpi and stained for cellular nuclei with DAPI (blue), anti-HMPV P (pseudo-colored red), and anti-HMPV F (pseudo-colored green) and…

Fig 9. Proposed mechanism for the M protein in viral ribonucleoprotein localization. Model for the HMPV M protein in vRNP localization. (1) The viral L (purple), N-RNA (black/yellow), P (gray), and M proteins (red) are recruited onto endosomes at the IB periphery. (2) The vRNPs are then trafficked with the movements of endosomes on microtubules (green) until they reach the plasma membrane. (3) Viral RNPs become associated with the plasma membrane through an M interaction with either cortical actin (red), the plasma membrane, or the cytoplasmic tails of the F (yellow) or G (green) proteins. (4) The M-vRNP complex remains associated with the plasma membrane in the absence of endosomes.