Fig 1.
hPIV1 and hPIV3 persist in human airway cells without cytopathic effects.
A549 or HPBE cells were mock-, hPIV1-, hPIV3-, or SeV-infected at an MOI of 5 and cultured for up to 15 days. (A) Cell viability was determined by cellular dehydrogenase assay (n = 3). *P≤0.05 ***P≤0.001. (B) Cell surface expression of viral HN was determined by immuno-fluorescence assay (left) or by flow cytometry analysis in A549 cells (right).
Fig 2.
hPIV1, but not hPIV3 establishes quiescent infection in human airway cells.
A549 or HPBE cells were mock-, hPIV1-, or hPIV3-infected at an MOI of 5 and cultured for up to 15 days. (A) Infectious progeny virions released into the medium for 24 h at various time points were titrated (n = 3). *P≤0.05. Accumulated NP proteins in cell lysates at each time point were determined by Western blotting. (B) De novo protein synthesis in A549 cells was determined by radio-immunoprecipitation after 3 h labeling with 35S-Met/Cys at 2 and 15 dpi. Radiolabeled viral HN and NP (or HN and N) were immunoprecipitated with specific mAbs or polyclonal serum. (C) Released and 35S-Met/Cys radio-labeled virus from A549 cells was purified by sucrose gradient centrifugation and analyzed by a Personal Molecular Imager after SDS-PAGE. The same samples were also tested for the presence of NP protein by Western blotting.
Fig 3.
Impaired raft association and formation of large vRNP aggregates in hPIV1 infected cells at late time points.
A549 cells were infected with hPIV1 at an MOI of 5 for 2 or 8 dpi. (A) IF analysis of hPIV1 M and NP at early and late time points. (B and C) A549 cells constitutively expressing Rab11- or Rab8-mRFP were infected with hPIV1 and analyzed at 2 and 8 dpi by IF. (D) Membrane floatation assay showing NP association with lipid raft at 2 and 8 dpi (left). Caveolin-1 association with lipid rafts was analyzed in mock and hPIV1 infected cells at 8 dpi (right).
Fig 4.
Expression of viral and host genes at 2 and 10 dpi.
(A-C) A549 cells were mock-, hPIV1-, or hPIV3-infected at an MOI of 3 and total RNA was collected at 2 and 10 dpi for RNAseq analysis (n = 3). (A) Counts of viral mRNA reads. (B) Counts of IFN genes. (C) Fold induction of ISGs in hPIV1 or hPIV3 infected cells relative to mock. (D-H) A549 cells were mock-, hPIV1-, or hPIV3-infected at an MOI of 3. (D) Release of IFN-λ1 into culture medium was quantitated by ELISA (n = 3). *P≤0.05, **P≤0.01. (E) Expression of MX1, IFIT2, IFN-λ1 and IFN-β1 mRNAs in infected cells was determined by qRT-PCR (n = 3). Fold increase relative to mock after normalization to GAPDH is shown. *P≤0.05, **P≤0.01. (F) Expression of MX1, MX2 and β-actin in infected cell lysates was analyzed by immunoblotting. (G) Co-localization of MX proteins with vRNPs in hPIV1 infected cells at 5 dpi was determined by IF using anti MX1, MX2 or NP Abs. (H) Interaction of MX proteins with NP at 3 dpi was determined by immunoprecipitation of NP and immunoblotting to detect MX1, MX2 and viral NP.
Fig 5.
Downregulation of genes involved in cholesterol biogenesis in hPIV1 infected cells.
(A) Top 10 GO terms of DEGs downregulated in hPIV1 infected cells compared to hPIV3. (B) Fold change in expression of genes involved in cholesterol biosynthesis in hPIV1 or hPIV3 infected cells compared to mock infected cells. (C) Total cellular cholesterol in A549 cells infected with hPIV1 or hPIV3 at an MOI of 5. (n = 3) *P≤0.05, **P≤0.01. (D) Total cellular cholesterol in A549 cells infected with various doses of hPIV1. (n = 3) *P≤0.05, **P≤0.01 ***P≤0.001.
Fig 6.
Reduced HMGCR expression in hPIV1-infected cells.
(A) A549 cells were either mock- or hPIV1-infected at an MOI of 5 and expression levels of HMGCR, SQLE, viral NP and β-actin were assessed by immunoblotting. (B) HPBE cells were either mock- or hPIV1-infected at an MOI of 5 and expression levels of HMGCR, SQLE, viral NP and β-actin were assessed by immunoblotting. (C) A549 cells were either mock-, hPIV1-, or hPIV3-infected at an MOI of 5 and HMGCR and β-actin expression was assessed at late time points by immunoblotting. (D) A549 cells were infected with hPIV1 at an MOI of 5 and HMGCR transcripts were determined by qRT-PCR. HMGCR mRNA levels were normalized to GAPDH mRNA and relative mRNA levels are shown (n = 3). (E) A549 cells infected with hPIV1 at an MOI of 5 were labeled with 35S-Met/Cys for 3 h at 8 dpi and de novo HMGCR synthesis was analyzed by radio-immunoprecipitation.
Fig 7.
Ubiquitination and proteasomal degradation of HMGCR in hPIV1 infected cells.
(A) A549 cells were infected with hPIV1 or hPIV3 at an MOI of 5 for 2 days. Cells were then labeled with 35S-Met/Cys for 30 min and chased for either 2 or 4 h. HMGCR stability was analyzed by radio-immunoprecipitation. (B) A549 cells infected with hPIV1 at an MOI of 5 were treated with either MG132 (10 μM) and/or NH4Cl (20 mM) at 8 hpi for 8 h. Expression of HMGCR, SQLE, viral NP and β-actin were analyzed by immunoblotting. (C) A549 or HPBE cells were mock- or hPIV1-, hPIV3- infected at an MOI of 3 for 18 h. MG132 (10 μM) or DMSO was added and cells were cultured for an additional 8 h. HMGCR in cell lysates was immunoprecipitated using an HMGCR specific Ab and immunoblotting was performed to detect ubiquitin or HMGCR.