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Heterocellular induction of interferon by negative-sense RNA viruses.

Chen S, Short JA, Young DF, Killip MJ, Schneider M, Goodbourn S, Randall RE - Virology (2010)

Bottom Line: The infection of cells by RNA viruses is associated with the recognition of virus PAMPs (pathogen-associated molecular patterns) and the production of type I interferon (IFN).Here we present data on the dynamics of IFN production and response during developing infections by paramyxoviruses, influenza A virus and bunyamwera virus.We show that only a limited number of infected cells are responsible for the production of IFN, and that this heterocellular production is a feature of the infecting virus as opposed to an intrinsic property of the cells.

View Article: PubMed Central - PubMed

Affiliation: School of Biology, Centre for Biomolecular Sciences, BMS Building, North Haugh, University of St. Andrews, St. Andrews, Fife, KY16 9ST, UK.

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Characterisation of the A549/pr(IFN-β).GFP reporter cells. (A). Monolayers of A549 cells were either mock-infected or infected with 5 pfu per cell of a stock of MuV (ori) known to be a good inducer of IFN; at 4, 6 and 8 h p.i. the cells were trypsinised to a single cell suspension and the percentage of GFP-positive cells estimated by FACS analysis. (B). Confluent monolayers of A549/pr(IFN-β).GFP cells were grown in 60 mm dishes that contained coverslips and infected at various multiplicities of infection of MuV(ori). At 12 h p.i. the coverslips were fixed and those cells expressing GFP visualised using a Nikon Microphot-FXA fluorescence microscopy. In addition, the amount of IFN secreted into the culture medium was determined (right hand column). (C). A549/pr(IFN-β).GFP, A549/pr(IFN-β).GFP/BVBV.Npro or A549/pr(IFN-β).GFP/HCV.NS34a cells were either mock-infected of infected with MuV(ori) at 5 pfu/cell. At 12 h p.i. the cells were fixed and immunostained with an anti-NP mAb. GFP-positive and virus-infected cells were visualised by fluorescence microscopy. (D). A549/pr(IFN-β).GFP were, or were not (control), treated with aphidicolin (1 μg/ml) for 16 h, infected with MuV (ori) at 5 pfu/cell in the presence or absence of aphidicolin as appropriate, fixed at 16 h p.i. and GFP-positive cells visualised by fluorescence microscopy.
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f0010: Characterisation of the A549/pr(IFN-β).GFP reporter cells. (A). Monolayers of A549 cells were either mock-infected or infected with 5 pfu per cell of a stock of MuV (ori) known to be a good inducer of IFN; at 4, 6 and 8 h p.i. the cells were trypsinised to a single cell suspension and the percentage of GFP-positive cells estimated by FACS analysis. (B). Confluent monolayers of A549/pr(IFN-β).GFP cells were grown in 60 mm dishes that contained coverslips and infected at various multiplicities of infection of MuV(ori). At 12 h p.i. the coverslips were fixed and those cells expressing GFP visualised using a Nikon Microphot-FXA fluorescence microscopy. In addition, the amount of IFN secreted into the culture medium was determined (right hand column). (C). A549/pr(IFN-β).GFP, A549/pr(IFN-β).GFP/BVBV.Npro or A549/pr(IFN-β).GFP/HCV.NS34a cells were either mock-infected of infected with MuV(ori) at 5 pfu/cell. At 12 h p.i. the cells were fixed and immunostained with an anti-NP mAb. GFP-positive and virus-infected cells were visualised by fluorescence microscopy. (D). A549/pr(IFN-β).GFP were, or were not (control), treated with aphidicolin (1 μg/ml) for 16 h, infected with MuV (ori) at 5 pfu/cell in the presence or absence of aphidicolin as appropriate, fixed at 16 h p.i. and GFP-positive cells visualised by fluorescence microscopy.

Mentions: The heterogeneity of the MxA induction seen in the above experiment could result from either heterogeneity or temporal lags in the production or response of IFN. To distinguish between these possibilities we generated an A549 reporter cell line in which IFN production was monitored by placing the eGFP gene under the control of the IFN-β promoter. To generate such a cell line, the first 282 bp of the IFN-β promoter were cloned into a bicistronic lentivirus vector, generating a plasmid (pdl′pIFNβ′GFP) such that following activation of the IFN-β promoter both eGFP and pac (which confers puromycin resistance) are expressed. Following transduction of A549 cells with the vector, cells were first subjected to puromycin selection for 2 days following stimulation with dsRNA for 8 h. Subsequently surviving cells were transfected with dsRNA for 8 h and GFP-positive single cells separated by FACS into individual wells of a 96-well microtitre plate such that a colony appeared in approximately 10–20% of wells. The resulting colonies were screened for their ability to express GFP in response to infection with a stock of MuV that we knew to be an extremely good inducer of IFN as it contained large amounts of defective interfering particles (DIs) that are powerful activators of the IFN-induction cascade (this stock will be referred to hereafter as MuV[ori]). The best colony of responding cells (termed A549/pr(IFN-β).GFP) in terms of numbers of positive cells and intensity of GFP expression was then further characterised. Fig. 2A shows that by 4 h p.i. approximately 35% of cells were positive for GFP expression, by 6 h p.i. 80% were positive and by 8 h p.i. 90% of the cells were strongly positive upon infection with MuV(ori). FACS analysis of the MuV(ori) infected cells showed a discrete population of GFP-positive cells rather than a gradient of GFP-positive cells, suggesting that the IFN-β promoter is either ‘on’ or ‘off’ in infected cells. Next, A549/pr(IFN-β).GFP cells that had been grown on coverslips in 24-well Linbro plates were infected with differing multiplicities of MuV(ori). At 24 h p.i. the number of cells expressing GFP was visualised by fluorescence and the amount of IFN in the culture medium estimated using a CPE-reduction bio-assay (Fig. 2B), showing that the number of cells expressing GFP correlated with the amount of IFN produced.


Heterocellular induction of interferon by negative-sense RNA viruses.

Chen S, Short JA, Young DF, Killip MJ, Schneider M, Goodbourn S, Randall RE - Virology (2010)

Characterisation of the A549/pr(IFN-β).GFP reporter cells. (A). Monolayers of A549 cells were either mock-infected or infected with 5 pfu per cell of a stock of MuV (ori) known to be a good inducer of IFN; at 4, 6 and 8 h p.i. the cells were trypsinised to a single cell suspension and the percentage of GFP-positive cells estimated by FACS analysis. (B). Confluent monolayers of A549/pr(IFN-β).GFP cells were grown in 60 mm dishes that contained coverslips and infected at various multiplicities of infection of MuV(ori). At 12 h p.i. the coverslips were fixed and those cells expressing GFP visualised using a Nikon Microphot-FXA fluorescence microscopy. In addition, the amount of IFN secreted into the culture medium was determined (right hand column). (C). A549/pr(IFN-β).GFP, A549/pr(IFN-β).GFP/BVBV.Npro or A549/pr(IFN-β).GFP/HCV.NS34a cells were either mock-infected of infected with MuV(ori) at 5 pfu/cell. At 12 h p.i. the cells were fixed and immunostained with an anti-NP mAb. GFP-positive and virus-infected cells were visualised by fluorescence microscopy. (D). A549/pr(IFN-β).GFP were, or were not (control), treated with aphidicolin (1 μg/ml) for 16 h, infected with MuV (ori) at 5 pfu/cell in the presence or absence of aphidicolin as appropriate, fixed at 16 h p.i. and GFP-positive cells visualised by fluorescence microscopy.
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Related In: Results  -  Collection

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f0010: Characterisation of the A549/pr(IFN-β).GFP reporter cells. (A). Monolayers of A549 cells were either mock-infected or infected with 5 pfu per cell of a stock of MuV (ori) known to be a good inducer of IFN; at 4, 6 and 8 h p.i. the cells were trypsinised to a single cell suspension and the percentage of GFP-positive cells estimated by FACS analysis. (B). Confluent monolayers of A549/pr(IFN-β).GFP cells were grown in 60 mm dishes that contained coverslips and infected at various multiplicities of infection of MuV(ori). At 12 h p.i. the coverslips were fixed and those cells expressing GFP visualised using a Nikon Microphot-FXA fluorescence microscopy. In addition, the amount of IFN secreted into the culture medium was determined (right hand column). (C). A549/pr(IFN-β).GFP, A549/pr(IFN-β).GFP/BVBV.Npro or A549/pr(IFN-β).GFP/HCV.NS34a cells were either mock-infected of infected with MuV(ori) at 5 pfu/cell. At 12 h p.i. the cells were fixed and immunostained with an anti-NP mAb. GFP-positive and virus-infected cells were visualised by fluorescence microscopy. (D). A549/pr(IFN-β).GFP were, or were not (control), treated with aphidicolin (1 μg/ml) for 16 h, infected with MuV (ori) at 5 pfu/cell in the presence or absence of aphidicolin as appropriate, fixed at 16 h p.i. and GFP-positive cells visualised by fluorescence microscopy.
Mentions: The heterogeneity of the MxA induction seen in the above experiment could result from either heterogeneity or temporal lags in the production or response of IFN. To distinguish between these possibilities we generated an A549 reporter cell line in which IFN production was monitored by placing the eGFP gene under the control of the IFN-β promoter. To generate such a cell line, the first 282 bp of the IFN-β promoter were cloned into a bicistronic lentivirus vector, generating a plasmid (pdl′pIFNβ′GFP) such that following activation of the IFN-β promoter both eGFP and pac (which confers puromycin resistance) are expressed. Following transduction of A549 cells with the vector, cells were first subjected to puromycin selection for 2 days following stimulation with dsRNA for 8 h. Subsequently surviving cells were transfected with dsRNA for 8 h and GFP-positive single cells separated by FACS into individual wells of a 96-well microtitre plate such that a colony appeared in approximately 10–20% of wells. The resulting colonies were screened for their ability to express GFP in response to infection with a stock of MuV that we knew to be an extremely good inducer of IFN as it contained large amounts of defective interfering particles (DIs) that are powerful activators of the IFN-induction cascade (this stock will be referred to hereafter as MuV[ori]). The best colony of responding cells (termed A549/pr(IFN-β).GFP) in terms of numbers of positive cells and intensity of GFP expression was then further characterised. Fig. 2A shows that by 4 h p.i. approximately 35% of cells were positive for GFP expression, by 6 h p.i. 80% were positive and by 8 h p.i. 90% of the cells were strongly positive upon infection with MuV(ori). FACS analysis of the MuV(ori) infected cells showed a discrete population of GFP-positive cells rather than a gradient of GFP-positive cells, suggesting that the IFN-β promoter is either ‘on’ or ‘off’ in infected cells. Next, A549/pr(IFN-β).GFP cells that had been grown on coverslips in 24-well Linbro plates were infected with differing multiplicities of MuV(ori). At 24 h p.i. the number of cells expressing GFP was visualised by fluorescence and the amount of IFN in the culture medium estimated using a CPE-reduction bio-assay (Fig. 2B), showing that the number of cells expressing GFP correlated with the amount of IFN produced.

Bottom Line: The infection of cells by RNA viruses is associated with the recognition of virus PAMPs (pathogen-associated molecular patterns) and the production of type I interferon (IFN).Here we present data on the dynamics of IFN production and response during developing infections by paramyxoviruses, influenza A virus and bunyamwera virus.We show that only a limited number of infected cells are responsible for the production of IFN, and that this heterocellular production is a feature of the infecting virus as opposed to an intrinsic property of the cells.

View Article: PubMed Central - PubMed

Affiliation: School of Biology, Centre for Biomolecular Sciences, BMS Building, North Haugh, University of St. Andrews, St. Andrews, Fife, KY16 9ST, UK.

Show MeSH
Related in: MedlinePlus