Influenza A virus proteins NS1 and hemagglutinin along with M2 are involved in stimulation of autophagy in infected cells - PubMed (original) (raw)
Influenza A virus proteins NS1 and hemagglutinin along with M2 are involved in stimulation of autophagy in infected cells
O P Zhirnov et al. J Virol. 2013 Dec.
Abstract
The NS1 protein of influenza A virus is known to downregulate apoptosis early in infection in order to support virus replication (O. P. Zhirnov, T. E. Konakova, T. Wolff, and H. D. Klenk, J. Virol. 76:1617-1625, 2002). In the present study, we analyzed the development of autophagy, another mechanism to protect cells from degradation that depends on NS1 expression. To this end, we compared autophagy in cells infected with wild-type (WT) influenza virus and virus lacking the NS1 gene (delNS1 virus). The results show that in WT-infected cells but not in delNS1 virus-infected cells, synthesis of the autophagy marker LC3-II, the lipidated form of microtubule light chain-associated protein LC3, is stimulated and that LC3-II accumulates in a perinuclear zone enriched with double-layered membrane vesicles characteristic of autophagosomes. Transfection experiments revealed that NS1 expressed alone was unable to upregulate autophagy, whereas hemagglutinin (HA) and M2 were. Proteolytic cleavage of HA increased autophagy. Taken together, these observations indicate that NS1 stimulates autophagy indirectly by upregulating the synthesis of HA and M2. Thus, it appears that NS1, besides downregulating apoptosis, is involved in upregulation of autophagy and that it supports the survival of infected cells by both mechanisms.
Figures
Fig 1
LC3-I and LC3-II profiles in CV-1 cells infected with WT and delNS1 viruses. (A) CV-1 cells were infected with either WT or delNS1 influenza A/PR/8/34 (H1N1) viruses at an MOI of 1. At 4, 6.5, 9.5, and 15 h p.i., equivalent amounts of cell homogenates were subjected to PAGE and WB analysis using antibodies specific for beclin-1, LC3, actin, and secondary species-specific HRP conjugates. Protein bands were visualized by exposure to Agfa film using the enhanced chemiluminescent West Dura reagent. Mo, mock-infected cells. (B) The amount of LC3-II relative to that of LC3-I (100%) was calculated by scanning analysis using the TINA program.
Fig 2
Subcellular distribution of LC3-I and LC3-II in cells infected with WT and delNS1 viruses. MDCK cells were infected with either WT or delNS1 influenza A/PR/8/34 (H1N1) viruses at an MOI of 1. At 7 and 11 h p.i., equal amounts of cells were disrupted by short sonication and cytoplasmic homogenates were prepared. Supernatant (S40) and membrane pellet (P40) fractions were obtained by centrifugation at 40,000 rpm (∼110,000 × g) for 1 h. Polypeptides were analyzed by PAGE-WB using LC3- and viral HA-specific antibodies and the ECL procedure. Equivalent aliquots of each subcellular fraction in relation to the volume of the initial cell sample were loaded into the wells.
Fig 3
LC3 localization in CV-1 cells infected with WT and delNS1 viruses. CV-1 cells were infected with either WT or delNS1 influenza A/PR/8/34 (H1N1) virus at an MOI of 1. (A) Uninfected and infected cells were fixed at 9.5 h p.i. with 4% paraformaldehyde, permeabilized with 0.1% NP-40, and stained with rabbit monoclonal antibody specific to LC3 (Cell Signaling) and secondary affinity-purified anti-rabbit–TRITC conjugate (Jackson) (red). Nuclei were stained with DAPI (blue). Stained cells were analyzed under a confocal microscope. Magnifications, ×250. (B) Fluorescence-labeled LC3 was quantified by image processing analysis as described in Materials and Methods. The fluorescence of 10 separate 20-by-20 pixel squares in the perinuclear zones of 3 separate cells was measured and normalized by measuring nuclear fluorescence. Arbitrary units were calculated as the ratio of perinuclear red channel pixels/intranuclear blue channel pixels. *, significant difference (P < 0.01) between the values for delNS1 and WT viruses. (C) Uninfected and infected cells were pelleted at 1,000 × g for 5 min and processed for electron microscopy, as described in Materials and Methods. NC, nucleus. Bar, 0.5 μm.
Fig 4
LC3-I/LC3-II profiles in 293T cells transfected with plasmids expressing individual virus proteins. (A and B) 293T cells transfected with pCAGGS vectors expressing protein PB1, PA, PB2, NP, NS1, NEP, M1, M2, HA, or NA of A/WSN/33 virus (A) or protein HA of wild-type viruses A/North Carolina/1918 (H1-wt), A/FPV/Rostock/34 (H7N1) (H7-wt), A/quail/Shantou/782/00 (H9N2) (H9-wt), and A/Aichi/68 (H3N2) (H3-wt) (B). At 40 h after transfection, intracellular LC3-I and LC3-II were detected by PAGE-WB analysis. Mock-transfected cells were also analyzed. Equal amounts of each cellular sample were loaded in each well of the gels. (C) H7 and H5 HAs containing polybasic cleavage sites of strains A/FPV/Rostock/34 (H7N1) (H7-wt), A/Kurgan/05/2001 (H5N1) (H5-wt), and HA mutants of strain A/FPV/Rostock/34 (H7N1) with monobasic cleavage sites (H7-del and H7-aaa) were expressed in 293T cells. After 40 h, equal aliquots of each sample of transfected cells were loaded in each well of the gels and analyzed by PAGE-WB for the expression of uncleaved HA0 and cleaved HA1, actin, LC3-I, and LC3-II. The amounts of LC3-II relative to those of LC3-I plus LC3-II (100%) were also determined by scanning of bands in the WB membrane image with the TINA program. (D) 293T cells were transfected with pCAGGS expressing HA, A/Aichi/68 (H3-wt), and the A/FPV/Rostock/34 mutant (H7-del) and incubated in DMEM containing 0.9% FCS. After 25 h, the medium was replaced with DMEM alone containing acetylated trypsin (T6763; Sigma) at a concentration of 3.5 μg/ml and cells were incubated for an additional 27 h. Cells were harvested, and equal amounts were loaded in each well of the gel and analyzed by PAGE-WB.
Fig 5
Early and late proteins in cells infected with WT and delNS1 influenza viruses. CV-1 cells were infected with either WT or delNS1 influenza A/PR/8/34 (H1N1) viruses at an MOI of 1. (A) At 6.5 h p.i., cells were incubated in medium containing a mixture of [35S]methionine and cysteine for 1.5 h and cellular polypeptides were analyzed by PAGE and autoradiography. Mar, (C-14 molecular size markers: 14, 20, 30, 45, 66, 97, and 220 kDa; Amersham). (B) Cell homogenates prepared at 6.5, 10, and 15 h p.i. were processed by PAGE-WB. Polypeptides were detected with antibodies specific for HA, NP, M1, and M2 and for HRP conjugates using ECL, and positive bands were calculated by scanning with the TINA program.
Fig 6
Apoptotic and autophagic markers in cells infected with WT and delNS1 influenza A/PR/8/34 viruses. (A) MDCK cells were infected with WT and delNS1 viruses at an MOI of 1. At 6.5, 9.5, and 14 h p.i., cellular polypeptides were analyzed by PAGE-WB using antibodies specific for AKTpho, 32K and 18K units of activated caspase 3 (Casp3a 32K, Casp3a 18K), actin, and viral NP and for secondary host species-specific HRP conjugates. (B) S6k/S6k-pho profiles were identified by PAGE-WB using rabbit anti-S6k and -SK6-pho antibodies and anti-rabbit antibody–HRP conjugate. Positive bands were detected by the ECL procedure using the West Dura luminescent reagent.
Fig 7
NS1-dependent regulatory links between autophagy and apoptosis in influenza virus-infected cells. NS1, synthesized as early as 1 h p.i., regulates autophagy and apoptosis by different mechanisms. First, NS1 initiates host protein kinase AKT, also known as protein kinase B (PKB), activation through binding to the p85beta subunit of PI3K class I (11, 12), resulting in the downregulation of apoptosis (9, 10). Second, NS1 amplifies synthesis of the late virus proteins HA, NA, M1, M2, and NEP. Amplification of M2 and HA stimulates formation of autophagosomes and suppresses their fusion with lysosomes (6). Thus, autophagy develops in influenza virus-infected cells after inhibition of apoptosis (19). The AKT-mTOR loop, a negative regulator of autophagosome generation (25), is not stimulated in cells infected with influenza H1N1 viruses and might be downregulated only in certain cell types infected with H5N1 virus (27). Late in virus replication (>13 h p.i.), AKT activation ceases and apoptosis develops when virus production has already declined (9). The exact mechanisms of apoptosis in influenza virus-infected cells remain to be discovered. Many influenza virus proteins, including NS1, NA, PB1-F2, and M1, possess proapoptotic abilities, and their overaccumulation late in infection can lead to apoptosis. An approximate time scale as times (hours) postinfection is shown on the left. Gray and dashed arrows and gray and dashed borders and hammer-like lines show up- and downregulatory actions, respectively.
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