Error bars represent mean +/- standard deviation. cells, but several post-transcriptional blocks in mammalian cells inhibit late events in the virus life-cycle, limiting infection to a single round in these cells [2,3]. ASV-GFP infection of mammalian cells, however, recapitulates key early events of the retroviral life-cycle, including entry, uncoating, reverse-transcription and integration. As diminished GFP expression is a faithful readout of Daxx-dependent silencing, we have previously employed ASV-GFP to identify post-integration silencing of retroviral gene-expression as a Daxx-sensitive step [2,3]. After treating HeLa cells with either IFN- or IFN- for 18?h, we infected these cells with ASV-GFP in the presence of DEAE-Dextran (20?g/mL), as described previously [6], and quantified viral gene expression by measuring GFP fluorescence 48?h post-infection. As the IFN-induced antiviral state is rarely maintained for more than 30?h post-treatment [7], cells were supplemented with IFN 6?h and 24?h post infection. Vesicular stomatitis virus encoding GFP (VSV-GFP) [8] was used as a positive control for IFN activity, as VSV is a well-established IFN-sensitive virus [9,10]. We found that treatment of HeLa cells with either IFN- or IFN- efficiently diminished GFP positivity (by ~70% and ~85%, respectively) following ASV infection, demonstrating that type I IFNs are capable of blocking ASV gene expression (Figure?1A,B). As expected, IFN- and IFN- inhibited VSV-GFP replication almost completely (from 75% GFP-positive cells in untreated controls to 1% GFP-positive cells after IFN-/ treatment; Figure?1C,D). Open in a separate window Figure 1 Type I IFNs inhibit ASV replication. (A) Fluorescence-activated Cell Sorter (FACS) analysis of ASV-GFP replication (indicated by % GFP-positive cells) in untreated, human IFN- (1000 U/ml)- or human IFN- (1000 U/ml)-treated HeLa cells 48?h post-infection from a representative experiment. GFP fluorescence data were collected on an LSR II flow cytometer (Becton Dickinson), and analyzed using FlowJo software. FSC?=?Forward scatter. (B) Quantification of GFP-positive cells from four independent replicates of the experiment described in panel A. Error bars represent mean +/- standard deviation. * 0.05. (C) VSV-GFP replication (indicated by % GFP-positive cells) in untreated, IFN- (1000 U/ml)- or IFN- (1000 U/ml)-treated HeLa cells 24?h post-infection from a representative experiment. (D) Quantification of GFP-positive cells from four independent replicates of the experiment described in panel C. Error bars represent mean +/- standard deviation. *** 0.001. Type I IFNs Inhibit ASV replication in avian cells To extend this investigation to cells of natural ASV hosts, we performed similar experiments in DF-1 chicken cells. We limited ASV replication to a single round in these cells by using a self-inactivating ASV-based alpharetroviral GFP-transducing (+)-Piresil-4-O-beta-D-glucopyraside vector with diminished LTR transcriptional activity [11]. After treating DF-1 cells with chicken IFN- for 18?h, we infected these with 5?L of self-inactivating ASV-GFP in the presence of Polybrene (10?g/mL) at 37C for 1?h. To ensure continued maintenance of the antiviral state, we supplemented cells with IFN- 6?h and 24?h p.i. When we examined these cells by GFP-based flow cytometry 48?h p.i., we observed that treatment with chicken type I IFN diminished proviral reporter gene expression by a significant amount (by ~70%, Figure?2), as observed in mammalian cells (Figure?1A-D). Collectively, these results demonstrate that type I IFNs exert antiviral activity against ASV, and set the stage for experiments designed to determine if Daxx is an essential component of the IFN anti-ASV program. Open in a separate window Figure 2 Chicken IFN- inhibits ASV replication in DF-1 cells. (A) FACS analysis of ASV-GFP replication (indicated by % GFP-positive cells) in untreated or chicken IFN- (1000 U/ml)-treated DF-1 cells 48?h post-infection from a representative experiment. FSC?=?Forward scatter. (B) Quantification of GFP-positive cells from three independent replicates of the experiment described in panel A. Error bars represent mean +/- standard deviation. * 0.05. Daxx is induced by type I IFNs in mammalian.(A) Fluorescence-activated Cell Sorter (FACS) analysis of ASV-GFP replication (indicated by % GFP-positive cells) in untreated, human IFN- (1000 U/ml)- or human IFN- (1000 U/ml)-treated HeLa cells 48?h post-infection from a representative experiment. reporter virus is pseudotyped to express the murine leukemia virus (MuLV) amphotropic envelope protein, and is therefore capable of entry into mammalian cells. ASV-GFP contains an intact complement of replicative genes, and is fully-capable of productive infection in its natural avian host cells, but several post-transcriptional blocks in mammalian cells inhibit late events in the virus life-cycle, limiting infection to a single round in these cells [2,3]. ASV-GFP infection of mammalian cells, however, recapitulates key early events of the retroviral life-cycle, including entry, uncoating, reverse-transcription and integration. As diminished GFP expression is a faithful readout of Daxx-dependent silencing, we have previously employed ASV-GFP to identify post-integration silencing of retroviral gene-expression as a Daxx-sensitive step [2,3]. After treating Rabbit Polyclonal to PHCA HeLa cells with either IFN- or IFN- for 18?h, we infected these cells with ASV-GFP in the presence of DEAE-Dextran (20?g/mL), as described previously [6], and quantified (+)-Piresil-4-O-beta-D-glucopyraside viral gene expression by measuring GFP fluorescence 48?h post-infection. As the IFN-induced antiviral state is rarely maintained for more than 30?h post-treatment [7], cells were supplemented with IFN 6?h and 24?h post infection. Vesicular stomatitis virus encoding GFP (VSV-GFP) [8] was used as a positive control for IFN activity, as VSV is a well-established IFN-sensitive virus [9,10]. We found that treatment of HeLa cells with either IFN- or IFN- efficiently diminished GFP positivity (by ~70% and ~85%, respectively) following ASV infection, demonstrating that type I IFNs are capable of blocking ASV gene expression (Figure?1A,B). As expected, IFN- and IFN- inhibited VSV-GFP replication almost completely (from 75% GFP-positive cells in untreated controls to 1% GFP-positive cells after IFN-/ treatment; Figure?1C,D). Open in a separate window Figure 1 Type I IFNs inhibit ASV replication. (A) Fluorescence-activated Cell Sorter (FACS) analysis of ASV-GFP replication (indicated by % GFP-positive cells) (+)-Piresil-4-O-beta-D-glucopyraside in untreated, human IFN- (1000 U/ml)- or human IFN- (1000 U/ml)-treated HeLa cells 48?h post-infection from a representative experiment. GFP fluorescence data were collected on an LSR II flow cytometer (Becton Dickinson), and analyzed using FlowJo software. FSC?=?Forward scatter. (B) Quantification of GFP-positive cells from four independent replicates (+)-Piresil-4-O-beta-D-glucopyraside of the experiment described in panel A. Error bars represent mean +/- standard deviation. * 0.05. (C) VSV-GFP replication (indicated by % GFP-positive cells) in untreated, IFN- (1000 U/ml)- or IFN- (1000 U/ml)-treated HeLa cells 24?h post-infection from a representative experiment. (D) Quantification of GFP-positive cells from four independent replicates of the experiment described in panel C. Error bars represent mean +/- standard deviation. *** 0.001. Type I IFNs Inhibit ASV replication in avian cells To extend this investigation to cells of natural ASV hosts, we performed similar experiments in DF-1 chicken cells. We limited ASV replication to a single round in these cells by using a self-inactivating ASV-based alpharetroviral GFP-transducing vector with diminished LTR transcriptional activity [11]. After treating DF-1 cells with chicken IFN- for 18?h, we infected these with 5?L of self-inactivating ASV-GFP in the presence of Polybrene (10?g/mL) at 37C for 1?h. To ensure continued maintenance of the antiviral state, we supplemented cells with IFN- 6?h and 24?h p.i. When we examined these cells by GFP-based flow cytometry 48?h p.i., we observed that treatment with chicken type I IFN diminished proviral reporter gene expression by a significant amount (by ~70%, Figure?2), as observed in mammalian cells (Figure?1A-D). Collectively, these results demonstrate that type I IFNs exert antiviral activity against ASV, and set the stage for experiments designed to determine if Daxx is an essential component of the IFN anti-ASV program. Open in a separate window Figure 2 Chicken IFN- inhibits ASV replication in DF-1 cells. (A) FACS analysis of ASV-GFP replication (indicated by % GFP-positive cells) in untreated or chicken IFN- (1000 U/ml)-treated DF-1 cells 48?h post-infection from a representative experiment. FSC?=?Forward scatter. (B) Quantification of GFP-positive cells from three independent replicates of the experiment described in panel A. Error bars symbolize mean +/- standard deviation. * 0.05. Daxx is definitely induced by type I IFNs in mammalian and avian cells We previously shown that treatment with IFN- results in induction of mRNA in HeLa cells [3]. To evaluate Daxx protein levels following IFN treatment, we treated HeLa or DF-1 cells with either human being or chicken IFN-, respectively, and examined whole-cell lysates prepared from these cells at numerous occasions post-treatment by immunoblotting. As demonstrated in Number?3A, IFN treatment increased Daxx protein levels ~3-fold by 24?h in HeLa cells. In DF-1 cells, IFN- induction of Daxx was confirmed to occur in the mRNA level (~2.5-fold, Figure?3B). A protein band of the approximate size of the putative avian Daxx ortholog.