hmpv  (MedChemExpress)

Journal: NAR Molecular Medicine

Article Title: Human pneumovirus induces IFN-dependent expression of the immune-responsive gene 1 and is inhibited by 4-octyl itaconate in human macrophages

doi: 10.1093/narmme/ugag017

Figure Lengend Snippet: Exogenously added 4OI, but not itaconate or citraconate, reduces HMPV levels in human MDMs. (A–C) MDMs were preincubated with 100 or 250 µM 4OI or its vehicle, DMSO, prior to HMPV infection for 24 h. Levels of HMPV N-gene mRNA were quantified by qRT-PCR ( A ; n ≥ 5), HMPV N and GAPDH protein levels were analysed by immunoblotting ( B ; n ≥ 5), while infectious HMPV/HMPV titers in MDM supernatants was determined as TCID 50 /ml ( C ; n = 5). (D, E) MDMs were treated with itaconate (ITA; 0.5, 5, 10, and 20 mM) prior to HMPV infection for 24 h. Levels of HMPV N-gene mRNA were quantified by qRT-PCR ( D ; n ≥ 2), while HMPV N and GAPDH proteins were analysed by immunoblotting ( E ; n ≥ 2). (F, G) MDMs were treated with citraconate (CIT; 1, 10, 20, and 50 mM) prior to HMPV infection for 24 h. Levels of the HMPV N-gene mRNA were quantified by qRT-PCR ( F ; n = 4), while HMPV N and GAPDH protein levels were analysed by immunoblotting ( G ; n = 2). Protein levels were quantified by normalizing band intensities against GAPDH and expressed as fold change compared to untreated (i.e. no itaconate variant) but infected MDMs. Multiple comparisons were analysed by a paired onw-way ANOVA with Tukey post-hoc test. Significance was ranked as * P < .05; ** P < .01; *** P < .001; **** P < .0001, and ns, not significant.

Article Snippet: Inhibition with S-Ruxolitinib (Merck) or GSK 872 (GlaxoSmithKline) was performed 1 h before infection with HMPV, whereas BMS-303141 (MedChemExpress) was preincubated for 2 h. The IFNAR neutralizing antibody (nIFNAR, clone MMHAR-2) was purchased from PBL; 5 or 10 μg/ml were incubated for 30 min before infection with HMPV.

Techniques: Infection, Quantitative RT-PCR, Western Blot, Variant Assay

Journal: NAR Molecular Medicine

Article Title: Human pneumovirus induces IFN-dependent expression of the immune-responsive gene 1 and is inhibited by 4-octyl itaconate in human macrophages

doi: 10.1093/narmme/ugag017

Figure Lengend Snippet: The Nrf2 pathway is induced by 4OI, but not by itaconate or citraconate, and limits HMPV in human macrophages. MDMs were preincubated with ( A ) 100 and 250 µM of 4OI or DMSO, ( B ) 0.5, 5, 10, and 20 mM of itaconate, and ( C ) 1, 10, 20, and 50 mM of citraconate prior to infection with HMPV for 24 h. (A–C) Levels of HO1 and NQO1 mRNA were quantified by qRT-PCR. The expression of HO1 and GAPDH protein was analysed using immunoblotting of whole cell lysates. ( A ) n ≥ 5 for mRNA and protein; ( B ) n ≥ 2 for mRNA and protein; ( C ) n = 4 for mRNA and n = 2 for protein. HO1 and NQO1 mRNA levels were assessed relative to untreated, uninfected MDMs for all experiments. HO1 protein levels were quantified by normalization of band intensities against GAPDH and expressed as fold change compared to untreated, infected MDMs. ( D ) MDMs were transfected with 20 nM NFE2L2 (Nrf2) siRNA or siNTC and infected with HMPV for 24 h. Nrf2, HMPV N protein, and GAPDH were analysed by immunoblotting of whole cell lysates and quantified ( n = 4). Nrf2 protein level is presented as fold change relative to siNTC-treated uninfected MDMs (left panel), while HMPV N-protein is presented as relative to siNTC-treated HMPV-infected MDMs (right panel). In panels (A–C), multiple comparisons were analysed by a paired onw-way ANOVA with Tukey post-hoc test, in panel (D) unpaired Student’s t - test. Significance was ranked as * P < .05; ** P < .01; *** P < .001; **** P < .0001 and ns, not significant.

Article Snippet: Inhibition with S-Ruxolitinib (Merck) or GSK 872 (GlaxoSmithKline) was performed 1 h before infection with HMPV, whereas BMS-303141 (MedChemExpress) was preincubated for 2 h. The IFNAR neutralizing antibody (nIFNAR, clone MMHAR-2) was purchased from PBL; 5 or 10 μg/ml were incubated for 30 min before infection with HMPV.

Techniques: Infection, Quantitative RT-PCR, Expressing, Western Blot, Transfection

Journal: NAR Molecular Medicine

Article Title: Human pneumovirus induces IFN-dependent expression of the immune-responsive gene 1 and is inhibited by 4-octyl itaconate in human macrophages

doi: 10.1093/narmme/ugag017

Figure Lengend Snippet: HMPV induction of IRG1 in human macrophages is dependent on type I IFN. ( A ) MDMs were infected with HMPV for the indicated time points. HMPV N gene or IRG1 mRNA levels were determined by qRT-PCR analysis ( n = 3), while protein levels of IRG1, HMPV N, and GAPDH were analysed by immunoblotting ( n = 2). IRG1 protein levels shown are for the same donor sample used in Fig. of , thus panels showing HMPV N and GAPDH levels being identical to the ones shown . ( B ) MDMs were treated with 500 ng/ml LPS for 2 h. IRG1 mRNA levels were assessed by qRT-PCR and normalized to untreated (−) control ( n = 5). ( C ) MDMs were transfected with 10 μg/ml poly IC for 6 or 24 h. IRG1 mRNA levels were assessed by qRT-PCR and normalized to untreated (−) cells ( n ≥ 2). ( D ) MDMs were pre-incubated (30 min) or not with 10 μg/ml of neutralizing IFNAR antibody before infection with HMPV for 24 h. IRG1 mRNA expression was determined using qRT-PCR and normalized to untreated (−) cells ( n = 4). ( E ) MDMs were treated with 100 U/ml recIFN-β for 24 h. IRG1 mRNA expression was determined using qRT-PCR and normalized to untreated (−) cells ( n = 4). ( F ) MDMs were preincubated with the JAK1/JAK2 inhibitor ruxolitinib (5 or 10 μM) prior to infection with HMPV for 18 h or treatment with IFN-β for 3 h. Protein levels in whole cell lysates were analysed for IRG1, STAT1 (Tyr701), STAT1, and GAPDH via immunoblot ( n = 2).

Article Snippet: Inhibition with S-Ruxolitinib (Merck) or GSK 872 (GlaxoSmithKline) was performed 1 h before infection with HMPV, whereas BMS-303141 (MedChemExpress) was preincubated for 2 h. The IFNAR neutralizing antibody (nIFNAR, clone MMHAR-2) was purchased from PBL; 5 or 10 μg/ml were incubated for 30 min before infection with HMPV.

Techniques: Infection, Quantitative RT-PCR, Western Blot, Control, Transfection, Incubation, Expressing

Journal: NAR Molecular Medicine

Article Title: Human pneumovirus induces IFN-dependent expression of the immune-responsive gene 1 and is inhibited by 4-octyl itaconate in human macrophages

doi: 10.1093/narmme/ugag017

Figure Lengend Snippet: TBK1 and NF-κB positively regulate IRG1 levels in HMPV-infected human macrophages. MDMs were incubated with 5 or 10 μM of the TBK1 inhibitor BX795 for 30 min before infection with HMPV for 24 h. ( A ) IRG1 and IFN-β mRNA levels ( n ≥ 3) were analysed by qRT-PCR (left panels), while in panel ( B ) IRG1 and GAPDH protein levels were determined by immunoblotting, quantified, and presented with SD relative to uninfected cells treated with DMSO ( n = 2; right panel). (C–K) MDMs were transfected with siRNAs targeting the NF-κB subunit RELA (C–E), IRF1 (F–H), RIPK3 (I–K), or control siRNA (siNTC) before infection with HMPV for 6, 9, or 24 h and analysis of IRG1 mRNA levels by qRT-PCR or IRG1, RelA/p65, IRF1, RIPK3, or GAPDH protein levels by immunoblotting. IRG1 mRNA levels were assessed relative to siNTC-transfected, uninfected MDMs for all experiments. ( C, E ) IRG1 mRNA after 6 h ( n = 3) or 24 h HMPV ( n = 3). ( D ) Protein levels of IRG1, RelA/p65, and GAPDH ( n = 3) after 9 h HMPV. ( F, H ) IRG1 mRNA after 6 h ( n = 4) or 24 h of infection ( n = 3). ( G ) Protein levels of IRG1, IRF1, and GAPDH ( n = 3) after 9 h HMPV. ( I, K ) IRG1 mRNA after 6 h ( n = 3) or 24 h of infection ( n = 3). ( J ) Protein levels of IRG1, IRF1, and GAPDH ( n = 3) after 9 h HMPV. ( L ) MDMs were incubated with 100 nM of the RIPK3 inhibitor GSK 872 for 1 h before infection with HMPV for 24 h. IRG1 mRNA levels were normalized relative to uninfected cells treated with DMSO ( n = 2). Single comparison between control (NTC) siRNA and target-siRNA conditions were calculated using paired t -test with Tukey post-hoc test. * P < .05, ** P < .01, *** P < .001, **** P < .0001; ns = non-significant. See also

Article Snippet: Inhibition with S-Ruxolitinib (Merck) or GSK 872 (GlaxoSmithKline) was performed 1 h before infection with HMPV, whereas BMS-303141 (MedChemExpress) was preincubated for 2 h. The IFNAR neutralizing antibody (nIFNAR, clone MMHAR-2) was purchased from PBL; 5 or 10 μg/ml were incubated for 30 min before infection with HMPV.

Techniques: Infection, Incubation, Quantitative RT-PCR, Western Blot, Transfection, Control, Comparison

Journal: NAR Molecular Medicine

Article Title: Human pneumovirus induces IFN-dependent expression of the immune-responsive gene 1 and is inhibited by 4-octyl itaconate in human macrophages

doi: 10.1093/narmme/ugag017

Figure Lengend Snippet: 4OI, itaconate, and citraconate differ in regulating IRG1 and the IFN-β response upon HMPV infection. MDMs were preincubated with ( A ) 100 and 250 µM of 4OI or DMSO, ( B ) 0.5, 5, 10, and 20 mM of itaconate, and ( C ) 1, 10, 20, and 50 mM of citraconate prior to infection with HMPV for 24 h. Levels of IRG1 and IFNB mRNA (left panels) were quantified by qRT-PCR. The expression of STAT1 (Tyr701), STAT1, IRG1, and GAPDH protein (right panels) was analysed using immunoblotting of whole-cell lysates and quantification STAT1 (Tyr701) expression. ( A ) n ≥ 5 for mRNA and protein; ( B ) n ≥ 2 for mRNA and protein; and ( C ) n = 4 for mRNA and n = 2 for protein. IFNB and IRG1 mRNA levels were assessed relative to untreated, uninfected MDMs for all experiments. Protein levels were quantified by normalization of band intensities against GAPDH and was expressed as fold change compared to untreated, uninfected MDMs. Multiple comparisons were analysed by a paired onw-way ANOVA with Tukey post-hoc test. Significance was ranked as * P < .05; ** P < .01; *** P < .001; **** P < .0001, and ns, not significant.

Article Snippet: Inhibition with S-Ruxolitinib (Merck) or GSK 872 (GlaxoSmithKline) was performed 1 h before infection with HMPV, whereas BMS-303141 (MedChemExpress) was preincubated for 2 h. The IFNAR neutralizing antibody (nIFNAR, clone MMHAR-2) was purchased from PBL; 5 or 10 μg/ml were incubated for 30 min before infection with HMPV.

Techniques: Infection, Quantitative RT-PCR, Expressing, Western Blot

Journal: NAR Molecular Medicine

Article Title: Human pneumovirus induces IFN-dependent expression of the immune-responsive gene 1 and is inhibited by 4-octyl itaconate in human macrophages

doi: 10.1093/narmme/ugag017

Figure Lengend Snippet: 4OI reduces expression of ATP-dependent citrate lyase required for HMPV replication. ( A ) 4OI reduces ATP-dependent citrate lyase ( ACLY) expression. MDMs were treated with 4OI (250 µM) or DMSO prior to infection with HMPV for 24 h. Expression levels of ACLY, FASN , and SCD1 mRNA were quantified relative to untreated, uninfected MDMs via qRT-PCR ( n ≥ 4). ( B ) ACLY inhibition reduces HMPV levels. MDMs were pretreated with 10 or 20 µM of the ACLY inhibitor BMS-303141 or DMSO prior to infection with HMPV for 24 h. HMPV N-gene mRNA was determined by qRT-PCR ( n = 4). Protein expression of HMPV N and GAPDH was analysed via immunoblotting of whole cell lysates ( n = 2). Protein levels were quantified by normalizing of band intensities against GAPDH and expressed as fold change compared to siNTC-transfected, infected MDMs. Multiple comparisons were analysed by a paired one-way ANOVA with Tukey post-hoc test. Significance was ranked as * P < .05; ** P < .01; *** P < .001; **** P < .0001 and ns, not significant. ( C ) HMPV stimulates increased level of neutral lipids in infected MDMs. MDMs were left uninfected (−) or infected with a MOI 1 of GFP-expressing recombinant HMPV for 24 h. HCS LipidTOX™ Deep Red neutral lipid stain was used to monitor lipids by confocal microscopy. Left panels: Representative images for each treatment showing HCS LipidTOX™ Deep Red (magenta) and HMPV (green). Right panel: Quantification of the LipidTOX™ signal per area. Signal was quantified from z-stacks of six fields of view per condition using the 20× numerical aperture yielding to ~2000 cells per condition and normalized to the area. Scalebar is adjusted to 100 µm ( n = 1). The data were assessed for normality and then compared with a non-parametric, two-tailed Mann–Whitney test. Significance was ranked as * P < .05; ** P < .01; *** P < .001; **** P < .0001, and ns, not significant.

Article Snippet: Inhibition with S-Ruxolitinib (Merck) or GSK 872 (GlaxoSmithKline) was performed 1 h before infection with HMPV, whereas BMS-303141 (MedChemExpress) was preincubated for 2 h. The IFNAR neutralizing antibody (nIFNAR, clone MMHAR-2) was purchased from PBL; 5 or 10 μg/ml were incubated for 30 min before infection with HMPV.

Techniques: Expressing, Infection, Quantitative RT-PCR, Inhibition, Western Blot, Transfection, Recombinant, Staining, Confocal Microscopy, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Development of a potent monoclonal antibody for treatment of human metapneumovirus infections

doi: 10.1038/s41467-026-69328-w

Figure Lengend Snippet: a Bait-and-switch approach using an antigen from one HMPV strain to identify B cells that cross-neutralize another strain of the virus. HMPV B2-binding B cells from human peripheral blood and spleen were labeled with APC-conjugated tetramers of HMPV B2 preF and sorted. b Flow cytometry plot of HMPV B2 preF-binding B cells after gating for live, CD3 − CD14 − CD16 − CD19 + CD20 + (B cells), IgD − (isotype-switched), and APC/Dylight755 − His tag − (to exclude cells binding to the His tag, APC, or streptavidin). PE-conjugated tetramers of RSV B preF and PE/DyLight650-conjugated control tetramers of the His tag were also included for comparison. The bound fraction contains cells magnetically enriched using APC- and PE-specific microbeads. The flowthrough fraction contains cells that did not bind the magnet and were thus depleted of antigen-specific cells. Numbers in plots are percentages of total cells in the gate. c Vero cells were infected with HMPV A2 in the presence of serial dilutions of the indicated mAbs. Data points are the average ± SD from two independent experiments. Neutralization potency was determined by a 50% plaque reduction neutralization test (PRNT 50 ), which is also indicated by the dotted line. d Vero cells were infected with HMPV subtypes A1, A2, B1, or B2 in the presence of serial dilutions of 4F11. WT-GFP indicates the same wild-type HMPV-A2 expressing a GFP reporter used in the neutralization assay shown in ( c ). The recombinant virus was generated based on the wild-type sequence of CAN97-83. The dotted midline represents the PRNT 50. Data points are the average ±SD of two replicates. a was created in BioRender. Boonyaratanakornkit, J. (2026) https://BioRender.com/rum3jsc .

Article Snippet: Clinical strains of HMPV were obtained from ZeptoMetrix, including A1 (strain IA10-2003, lot#331204, cat#0810161CF), A2 (strain IA27-2004, lot#331149, cat#0810164CF), B1 (strain Peru3-2003, lot#328937, cat#0810158CF), and B2 (strain IA18-2003, lot#329066, cat#0810162CF).

Techniques: Virus, Binding Assay, Labeling, Flow Cytometry, Control, Comparison, Infection, Neutralization, Plaque Reduction Neutralization Test, Expressing, Recombinant, Generated, Sequencing

Journal: Nature Communications

Article Title: Development of a potent monoclonal antibody for treatment of human metapneumovirus infections

doi: 10.1038/s41467-026-69328-w

Figure Lengend Snippet: a Biolayer interferometry (BLI) measurements of the ability of the mAb listed on the left side of the chart to block binding of the mAb listed on the top. Competition is expressed as the percent drop in maximum signal compared to the maximum signal in the absence of competing mAb (red, 80–100%; yellow, 40–80%; white, 0–40%). b Cryo-EM map of 4F11 (heavy chain, purple; light chain, light purple) and MxR (heavy chain, brown; light chain, light brown) Fabs in complex with DS-CavEs2 P185A monomer (gray). Only the VH/VL domains are shown. c Cryo-EM map of 4F11 (purple) Fab in complex with MPV-2c trimer (shades of gray) (left). Top view surface representation (right). The footprint of 4F11 VH is shown in dark purple and VL in light purple in the top view. d Plot showing buried surface area (BSA) contribution of protomer A and adjacent protomer B residues of the MPV-2c trimer when interacting with 4F11. Interactions with 4F11 VH are shown in dark purple (protomer A), and interactions with 4F11 VL are shown in light purple (protomer A) or green (protomer B). The sequences of MPV-2c, DS-CavEs2, v3B, and RSV are aligned with the HMPV subtypes in parentheses. e Kinetics of 4F11 Fabs binding to HMPV A2 monomer and MPV-2c trimer by BLI. f Zoomed in view of the cartoon representation of MPV-2c F trimer and 4F11 Fab with site Ø shown in shades of blue and site Ø glycans in green (left). BSA of the N172 glycan with N-acetylglucosamine (NAG) represented as blue squares and mannose represented as a green circle (right). g Binding measured by ELISA of 4F11 and MxR IgG to HMPV trimer with and without the N172 glycan. Data points are the average ± SD from three independent experiments.

Article Snippet: Clinical strains of HMPV were obtained from ZeptoMetrix, including A1 (strain IA10-2003, lot#331204, cat#0810161CF), A2 (strain IA27-2004, lot#331149, cat#0810164CF), B1 (strain Peru3-2003, lot#328937, cat#0810158CF), and B2 (strain IA18-2003, lot#329066, cat#0810162CF).

Techniques: Blocking Assay, Binding Assay, Cryo-EM Sample Prep, Glycoproteomics, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Development of a potent monoclonal antibody for treatment of human metapneumovirus infections

doi: 10.1038/s41467-026-69328-w

Figure Lengend Snippet: a Passaging results after HMPV A2, expressing a GFP reporter, were mixed with serial dilutions of MxR, 4F11, or no antibody, as a control. An “X” indicates the sample from which virus was sequenced by metagenomics. Percent infection was determined by assessing GFP expression with fluorescence microscopy. ND, not done. Each passaging experiment was performed once. The entire well was visually scanned, and a representative fluorescence microscopy image is shown for the sample with MxR at 15 µg/mL at passage 3 (top) and for the sample with 4F11 at 0.24 µg/mL at passage 3 (bottom). The scale bar represents 500 µm. b Metagenomic sequencing of viruses. Mutations listed are those with at least 10% allele frequency and that were absent in the no antibody control samples. Recombinant HMPV F with the listed mutations was produced by transient transfection in 293 cells, and the yield of mutant F was compared to wild-type HMPV F. c Binding of MxR IgG (top) and 4F11 IgG (bottom) to recombinant HMPV preF protein was measured by biolayer interferometry. Measurements are normalized against an isotype control antibody. Asterisks indicate mutations that led to loss of binding by mAb. d Biolayer interferometry measurements of binding of 4F11 IgG to stabilized HMPV preF containing a N466K substitution in the presence or absence of a K179E substitution. e Vero cells were infected with HMPV-WT (wild-type), HMPV-K179E, or HMPV-K179E + N466K in the presence of serial dilutions of 4F11 antibody. The dotted midline represents the PRNT 50 . Data points are the average ± SD of two replicates. f Viral growth kinetics of HMPV-WT, HMPV-K179E, and HMPV-K179E + N466K. Vero cells were infected at an MOI of 0.1. Cell culture supernatant was collected at each timepoint and quantified using a viral titer assay. Data points are the average ± SD of three replicates. g Electrostatics of 4F11 and HMPV F around amino acid position 179. The 4F11 paratope (left) is colored by electrostatic values. The binding site for K179 is circled in black. 180° rotation showing the MPV-2c trimer surface colored by electrostatic charge with K179 (middle) and E179 mutation (right).

Article Snippet: Clinical strains of HMPV were obtained from ZeptoMetrix, including A1 (strain IA10-2003, lot#331204, cat#0810161CF), A2 (strain IA27-2004, lot#331149, cat#0810164CF), B1 (strain Peru3-2003, lot#328937, cat#0810158CF), and B2 (strain IA18-2003, lot#329066, cat#0810162CF).

Techniques: Passaging, Expressing, Control, Virus, Metagenomics, Infection, Fluorescence, Microscopy, Sequencing, Recombinant, Produced, Transfection, Mutagenesis, Binding Assay, Cell Culture, Titer Assay

Journal: Nature Communications

Article Title: Development of a potent monoclonal antibody for treatment of human metapneumovirus infections

doi: 10.1038/s41467-026-69328-w

Figure Lengend Snippet: a Schematic of experiments in which hamsters were injected intramuscularly with mAb, 24 h after intranasal challenge with 10 5 pfu of HMPV A2. b Dose–response of 4F11 on HMPV replication ( n = 5 experimental animals/dose, n = 9 control animals/dose). c Comparison of HMPV replication in hamsters treated with 2.5 mg/kg of 4F11 or MxR mAb ( n = 5 animals per group). Viral titers were measured by plaque assay in nasal homogenates (left) and lungs (right) from individual hamsters at 4 days post-infection. Dashed lines indicate the limit of detection. Bars represent the mean, error bars represent standard deviations, and p values are calculated by two-sided Mann–Whitney test. Control hamsters were injected with 1× DPBS. a was created in BioRender. Boonyaratanakornkit, J. (2026) https://BioRender.com/zbugyzr .

Article Snippet: Clinical strains of HMPV were obtained from ZeptoMetrix, including A1 (strain IA10-2003, lot#331204, cat#0810161CF), A2 (strain IA27-2004, lot#331149, cat#0810164CF), B1 (strain Peru3-2003, lot#328937, cat#0810158CF), and B2 (strain IA18-2003, lot#329066, cat#0810162CF).

Techniques: Injection, Control, Comparison, Plaque Assay, Infection, MANN-WHITNEY