hnRNPM regulates influenza A virus replication through distinct mechanisms in human and avian cells: implications for cross-species transmission

Abstract

The eight-segmented RNA genome of influenza A virus (IAV) is transcribed and spliced into 10 major viral mRNAs in the nucleus of infected cells. Both transcription and splicing are facilitated by the host RNA polymerase II (Pol II) machinery via interactions between the viral ribonucleoprotein (vRNP) complex and various host factors. In this study, we demonstrate that IAV vRNPs recruit species-specific heterogeneous nuclear ribonucleoprotein M (hnRNPM) to support their replication in human and avian cells through distinct mechanisms. In A549 cells, human hnRNPM specifically facilitates the efficient transcription of HA, NA, M, and NS segments of WSN virus in a gene coding sequence-dependent manner. In contrast, in DF-1 cells, chicken hnRNPM restricts excessive splicing of M segment mRNA to ensure proper M2 protein production. Notably, human hnRNPM, with 34 additional amino acids compared with its chicken counterpart, fails to inhibit the M2 expression in DF-1 cells, whereas both human and chicken hnRNPM regulate WSN virus replication similarly in A549 cells. These findings highlight the host-specific roles of M2 levels in IAV replication and reveal how IAV co-opts host factors through virus genome sequence-dependent and host species-specific mechanisms, underscoring its high flexibility and adaptability during cross-species transmission.IMPORTANCEThe transcription and splicing of IAV genome in the nucleus of infected cells are precisely regulated to produce optimal amounts of viral proteins, ensuring efficient virus replication. In this study, we discovered that human hnRNPM regulates the IAV segment-specific differential transcription in a coding sequence-dependent manner in human cells. In contrast, chicken hnRNPM specifically inhibits M2 mRNA splicing to maintain proper M2 protein levels in avian cells. These species-specific regulatory mechanisms highlight the distinct replication strategies employed by IAV in human versus avian cells and underscore the complexity of cross-species transmission.

Keywords: M segment splicing; chicken hnRNPM; human hnRNPM; influenza A virus; segment-specific transcription.

Fig 1

hnRNPM interacts with IAV vRNP as identified by affinity purification. (A) Schematic of the AP-MS screen to investigate influenza virus RNP interactions with host factors. (B) Polymerase activity is not affected by the vRNP with PB2-TAP. Recombinant HA-containing viral vRNP was generated using either PB2 or PB2-TAP. HA protein was assessed using western blot analysis using anti-HA antibodies. (C) Analysis of the purification of the recombinant TAP-tagged RNP complex. Cells were harvested and lysed 24 h post-transfection, followed by purification using IgG-Sepharose. The purified proteins were separated by 8% SDS-PAGE and visualized by silver staining. The positions of PB2-TAP, PB1, PA, NP, and hnRNPM are indicated. (D) Cellular proteins identified by TAP purification of the vRNP complex. The purified proteins were subjected to LC-MS/MS analysis, resulting in the identification of 12 host proteins with high confidence and at least two unique peptide identifications per protein. (E, F) Interaction between hnRNPM and viral RNP confirmed by co-immunoprecipitation. A549 cells were infected with WSN virus at an MOI of 1 for 12 h. Cell lysates were immunoprecipitated with either an anti-hnRNPM antibody (E) or an anti-NP antibody (F), followed by Western blot analysis.

Fig 2

Human hnRNPM positively regulates IAV replication in human cells. (A, B) SiRNA knockdown of hnRNPM in A549 cells. A549 cells were transfected with two individual siRNAs targeted to hu-hnRNPM (sihu-hM-1# and sihu-hM-2#) or a scrambled siRNA as a negative control (siNC) for 48 h. The mRNA (A) and protein (B) levels of hnRNPM were quantified by RT-qPCR and western blotting, respectively. ****P < 0.0001. (C) Cell viability of sihu-hnRNPM-treated A549 cells was assessed using a Cell Counting Kit-8 (CCK-8) assay. ns, not significant. (D–F) Growth curves of WSN, PR8, and H9N2 viruses in A549 cells. A549 cells were treated with the indicated siRNAs for 36 h and then infected with WSN (MOI = 0.001) (D), A/Puerto Rico/8/34 (PR8, H1N1) (MOI = 0.1) (E), or H9N2 virus (MOI = 0.01) (F). Supernatants were collected at the indicated time points and subjected to plaque assays on MDCK cells. **P < 0.01, ***P < 0.001, ****P < 0.0001. (G, H) Overexpression of hu-hnRNPM restores the replication of WSN virus in sihu-hnRNPM-treated 293T cells. Sihu-hnRNPM- or siNC-treated 293T cells were transfected with the plasmids encoding either an empty vector or hu-hnRNPM (hu-hM). At 24 h post-transfection, the cells were infected with WSN virus at an MOI of 0.001 for 30 h post-infection. Virus titers in the supernatants were determined by performing plaque assays (G), and whole-cell lysates were analyzed by Western blotting (H). Data are representative of at least three independent experiments. Means ± SD are shown in (A, C–G) (n = 3).

Fig 3

Human hnRNPM regulates IAV replication independently of STAT1-mediated innate immune pathways. (A–D) Effect of hu-hnRNPM knockdown on IRF3 (A) and ISGs (B–D) transcripts in A549 cells. A549 cells were transfected with the indicated siRNAs for 36 h, then uninfected (UI) or infected with WSN virus (MOI = 1) or VSV virus (MOI = 3). Cellular mRNAs were analyzed using qPCR at the indicated time points. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant. (E) Hu-hnRNPM knockdown affects IRF3 and its phosphorylated protein levels. A549 cells were treated with the indicated siRNA, infected with WSN virus, and subsequently subjected to western blot analysis using specific antibodies. (F) Analysis of STAT1-knockout and hnRNPM knockdown in A549 cells using immunoblotting. (G) Knockdown of hnRNPM reduces WSN virus replication in STAT1-knockout A549 cells. A549 cells and STAT1-knockout A549 cells, with or without hu-hnRNPM knockdown, were infected with WSN virus (MOI = 0.001) for 48 h post-infection. Virus titers in the supernatants were quantified using the plaque assay. ***P < 0.001, ****P < 0.0001. Data are representative of at least three independent experiments. Means ± SD are shown in (A–D, G) (n = 3).

Fig 4

Human hnRNPM differentially regulates the transcription efficiency of the IAV genome in a segment-specific manner. (A-C) Hu-hnRNPM knockdown results in the differential expression of IAV genes in A549 cells. A549 cells were transfected with the indicated siRNAs for 36 h and infected with the WSN virus at an MOI of 10. Cells were harvested at specific time points. Proteins were analyzed by western blotting (A). Quantification of viral proteins is shown in the right panel. RNAs were analyzed using primer extension analysis to detect the vRNA, mRNA, and cRNA of PB2 (B) and NA (C) segments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (D, E) Quantification of IAV eight-gene mRNA (E) and vRNA (F) by qPCR analysis at 6 h post-infection. ****P  <  0.0001. (F) Hu-hnRNPM does not regulate IAV mRNA degradation. 293T cells were transfected with the indicated siRNA and subsequently transfected with expression plasmids (pcDNA) encoding PB2, NP, NA, and M. After 24 h, total RNAs were harvested at 0, 3, and 6 h post-actinomycin D treatment, and the mRNA levels of PB2, NP, NA, and M were assessed by qPCR analysis. ns, not significant. (G) Overexpression of hu-hnRNPM restores the differential expression of the WSN virus protein in sihu-hnRNPM-treated A549 cells. Sihu-hnRNPM- or siNC-treated A549 cells were transfected with the plasmids encoding either an empty vector (EV) or hu-hnRNPM (hu-hM). At 24 h post-transfection, the cells were infected with WSN virus at an MOI of 10. Cells were harvested at 7 h post-infection. Proteins were analyzed by western blotting. Quantification of viral proteins is shown in the right panel. **P < 0.01, ***P < 0.001, ****P < 0.0001. Data are representative of at least three independent experiments. Means ± SD are shown in (A, D-G) (n = 3).

Fig 5

Human hnRNPM regulates the segment-specific transcription efficiency in a coding sequence-specific manner. (A) Hu-hnRNPM specifically inhibits M1 and M2 expression in an RNP reconstitution system. 293T cells were treated with the indicated siRNAs, then transfected with bidirectional expression plasmids pHW2000 encoding PB2, PB1, PA, and NP, along with Pol I-driven RNA expression plasmids encoding M vRNA. Cellular proteins were analyzed by immunoblotting. (B) Hu-hnRNPM regulates viral gene expression independently of the viral gene’s NCR. 293T cells were transfected with the indicated siRNAs and RNP reconstitution plasmids. Pol I-driven RNA expression plasmids encode chimeric PB2(GFP) or NA(GFP) RNAs. Proteins were analyzed by immunoblotting. (C–F) Hu-hnRNPM regulates viral gene expression based on the ORF sequence. siRNA-treated 293T cells were transfected with RNP reconstitution plasmids and Pol I-driven recombinant plasmids. These Pol I-driven plasmids retained the NCRs of PB2 or NA and replaced their ORFs with GFP, NA, M, or M1. Specifically, the Pol I-driven plasmids encoded the following chimeric RNAs: NA(GFP) and PB2(NA-Flag) in (C), PB2(GFP) and PB2(M) in (D), PB2(NA-Flag) and PB2(M) in (E), PB2(M1) and PB2(M1^mut-Flag) in (F). Cellular proteins were analyzed by immunoblotting in (C, D, and F). mRNAs were identified by qPCR analysis in (E). Each protein band was quantified by ImageJ. ****P < 0.0001; ns, not significant. Data are representative of at least three independent experiments. Means ± SD are shown in (E, F) (n = 3).

Fig 6

Chicken hnRNPM inhibits the splicing of IAV M2 mRNA to support IAV replication in DF-1 cells. (A) Identify the predominantly expressed chicken hnRNPM isoforms in DF-1 cells. The protein sequences of human hnRNPM and three predicted isoforms of chicken hnRNPM (ch-hM772, ch-hM738, and ch-hM709, named according to their amino acid lengths) were aligned to identify conserved domains and species-specific domains among the orthologs. Based on the alignment results, plasmids expressing the three predicted isoforms of chicken hnRNPM were transfected into DF-1 cells, and the predominantly expressed isoform in DF-1 cells was determined using hnRNPM-specific antibodies. (B) SiRNA knockdown of ch-hnRNPM (ch-hM) in DF-1 cells. DF-1 cells were transfected with control siRNA (siNC) or ch-hnRNPM-specific siRNA (sich-hM) for 48 h. The mRNA and protein levels of ch-hnRNPM were quantified by RT-qPCR and western blotting. ****P < 0.0001. (C) Cell viability of sich-hM-treated DF-1 cells was assessed using a CCK-8 assay. ns, not significant. (D–G) Growth curves of WSN, H9N2, PR8, and H5N1W viruses in DF-1 cells. DF-1 cells treated with the indicated siRNAs were infected with 0.001 MOI of WSN (D), 0.001 MOI of H9N2 (E), 0.1 MOI of PR8 (F), and 0.001 MOI of H5N1W (G). Supernatants were collected at the indicated time points post-infection, and virus titers were determined by plaque assay using MDCK cells. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant. (H, I) Knockdown of ch-hnRNPM specifically increases M2 expression in DF-1 cells. DF-1 cells were transfected with the indicated siRNAs and infected with WSN (H) or H9N2 (I) viruses at an MOI of 10. Cells were harvested at specific time points, and proteins were analyzed by Western blotting. Quantification of WSN viral proteins is shown in the right panel. ****P  <  0.0001; ns, not significant. (J) Detection of IAV eight-gene mRNA by qPCR analysis at 6 h post-infection. mRNAs from (H) cells were quantified using RT-qPCR. ****P  <  0.0001; ns, not significant. (K, L) The splicing ratios of M and NS mRNA in DF-1 (K) and A549 cells (L). DF-1 and A549 cells were treated with control or targeting host-specific hnRNPM siRNAs, followed by infection with WSN virus at an MOI of 10. Total RNA was harvested at 6 h post-infection, and the splicing ratios of the M and NS segments were determined using RT-qPCR. ****P  <  0.0001; ns, not significant. (M) Overexpression of ch-hnRNPM re-inhibits the expression of M2 protein in sich-hnRNPM-treated DF-1 cells. DF-1 cells treated with either sich-hnRNPM or siNC were transfected with the indicated plasmids. At 24 h post-transfection, the cells were infected with WSN virus at an MOI of 10. Cells were harvested at 8 h post-infection, and viral proteins were analyzed by Western blotting. Quantification of viral proteins is shown in the right panel. **P < 0.01, ***P < 0.001, ****P < 0.0001. Data are representative of at least three independent experiments. Means ± SD are shown in (B–H, J–M) (n ≥ 3).

Fig 7

Species-specific regulation of M2 expression by hnRNPM influences IAV replication in different host cells. (A) Overexpression of chicken or human hnRNPM differentially regulates M2 protein expression in sich-hnRNPM-treated DF-1 cells. DF-1 cells treated with either sich-hnRNPM or siNC were transfected with the indicated plasmids. At 24 h post-transfection, the cells were infected with WSN virus at an MOI of 10. Cells were harvested 8 h post-infection and analyzed by western blotting. Quantification of viral proteins is shown in the right panel. **P < 0.01, ***P < 0.001, ****P < 0.0001. (B) Overexpression of human or chicken hnRNPM restores the differential expression of WSN virus protein in sihu-hnRNPM-treated A549 cells. A549 cells treated with either sihu-hnRNPM or siNC were transfected with the indicated plasmids. At 36 h post-transfection, the cells were infected with WSN virus at an MOI of 10. Cells were harvested at 7 h post-infection and analyzed by western blotting. Quantification of viral proteins is shown in the right panel. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant. (C) Knockdown of host-specific hnRNPM differentially regulates M segment expression in human and avian cells. A549 and DF-1 cells were treated with control or targeting host-specific hnRNPM siRNAs and then infected with WSN virus for 8 h. Viral proteins were analyzed by immunoblotting. (D–G) Overexpression of WSN M2 differentially regulates IAV replication in human and avian cells. A549 and DF-1 cells were transfected with WSN M2 for 24 h and then infected with WSN or H5N1W at 1 MOI for 12 h (D, F) or at 0.001 MOI for 36 h (E, G). Virus titers in the supernatants were determined by plaque assays (E, G), and whole-cell lysates were analyzed by western blotting (D, F). *P < 0.05, **P < 0.01. Data are representative of at least three independent experiments. Means ± SD are shown in (A, B, E, G) (n ≥ 3).

Fig 8

Proposed model for species-specific hnRNPM regulates influenza A virus replication in human and avian cells. During IAV infection, viral vRNPs are transported to the nucleus, where they drive viral RNA transcription and replication. Concurrently, the M and NS mRNAs undergo critical splicing events to generate M2 and NS2 mRNA, respectively. In human cells, hnRNPM regulates the transcription of the HA, NA, M, and NS segments, thereby enhancing their protein expression and promoting IAV virion production. In contrast, in avian cells, ch-hnRNPM specifically inhibits M segment mRNA splicing, thereby reducing M2 mRNA and protein levels, which in turn promotes IAV replication.