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The influenza virus protein PB1-F2 inhibits the induction of type I interferon at the level of the MAVS adaptor protein.

Varga ZT, Ramos I, Hai R, Schmolke M, García-Sastre A, Fernandez-Sesma A, Palese P - PLoS Pathog. (2011)

Bottom Line: Of note, the PB1-F2 N66S protein showed enhanced IFN antagonism activity compared to PB1-F2 wildtype.By inducing the IFN-β reporter at different levels in the signaling cascade, we found that PB1-F2 inhibited IFN production at the level of the mitochondrial antiviral signaling protein (MAVS).Furthermore, immunofluorescence studies revealed that PB1-F2 co-localizes with MAVS.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Mount Sinai School of Medicine, New York City, New York, United States of America.

ABSTRACT
PB1-F2 is a 90 amino acid protein that is expressed from the +1 open reading frame in the PB1 gene of some influenza A viruses and has been shown to contribute to viral pathogenicity. Notably, a serine at position 66 (66S) in PB1-F2 is known to increase virulence compared to an isogenic virus with an asparagine (66N) at this position. Recently, we found that an influenza virus expressing PB1-F2 N66S suppresses interferon (IFN)-stimulated genes in mice. To characterize this phenomenon, we employed several in vitro assays. Overexpression of the A/Puerto Rico/8/1934 (PR8) PB1-F2 protein in 293T cells decreased RIG-I mediated activation of an IFN-β reporter and secretion of IFN as determined by bioassay. Of note, the PB1-F2 N66S protein showed enhanced IFN antagonism activity compared to PB1-F2 wildtype. Similar observations were found in the context of viral infection with a PR8 PB1-F2 N66S virus. To understand the relationship between NS1, a previously described influenza virus protein involved in suppression of IFN synthesis, and PB1-F2, we investigated the induction of IFN when NS1 and PB1-F2 were co-expressed in an in vitro transfection system. In this assay we found that PB1-F2 N66S further reduced IFN induction in the presence of NS1. By inducing the IFN-β reporter at different levels in the signaling cascade, we found that PB1-F2 inhibited IFN production at the level of the mitochondrial antiviral signaling protein (MAVS). Furthermore, immunofluorescence studies revealed that PB1-F2 co-localizes with MAVS. In summary, we have characterized the anti-interferon function of PB1-F2 and we suggest that this activity contributes to the enhanced pathogenicity seen with PB1-F2 N66S- expressing influenza viruses.

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PB1-F2 N66S induces less IFN compared to PB1-F2 WT in the context of viral infection.(A) Growth curves of PR8 WT and PR8 N66S viruses in A549 cells and in ovo. A549 cells were infected with virus at an MOI of 0.01 and aliquots of the supernatants were taken at 4, 24, 48 and 72 hpi. To determine viral titers, supernatants were plaqued on MDCK cells. Ten-day old embryonated chicken eggs were inoculated with 100 PFU of virus and placed at 4°C at 12, 24, 48 and 72 hpi. The following days, allantoic fluids were harvested, spun down and titered via plaque assays on MDCK cells. (B) An MDCK cell line expressing an IFN-β promoter driven luciferase reporter (MDCK-IFN-beta luc) was infected with PR8 WT and PR8 N66S viruses at an MOI of 2. Cells were lysed at 4, 8 and 12 hpi and luciferase activity was measured. In addition, cell lysates were analyzed for protein expression via Western blots. (C and D) Primary murine dendritic cells (C) or murine lung epithelial cells (D) were infected with the indicated PR8 viruses at an MOI of 2. RNA was isolated 8 hpi and subjected to qRT-PCR. All data represent means ± standard deviations of one representative experiment (n = 3). Statistical significance was determined using Student's t test. *, p<0.05; **, p<0.01. Statistical significance is relative to PR8 WT. All data shown are representatives of at least two independent experiments.
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ppat-1002067-g002: PB1-F2 N66S induces less IFN compared to PB1-F2 WT in the context of viral infection.(A) Growth curves of PR8 WT and PR8 N66S viruses in A549 cells and in ovo. A549 cells were infected with virus at an MOI of 0.01 and aliquots of the supernatants were taken at 4, 24, 48 and 72 hpi. To determine viral titers, supernatants were plaqued on MDCK cells. Ten-day old embryonated chicken eggs were inoculated with 100 PFU of virus and placed at 4°C at 12, 24, 48 and 72 hpi. The following days, allantoic fluids were harvested, spun down and titered via plaque assays on MDCK cells. (B) An MDCK cell line expressing an IFN-β promoter driven luciferase reporter (MDCK-IFN-beta luc) was infected with PR8 WT and PR8 N66S viruses at an MOI of 2. Cells were lysed at 4, 8 and 12 hpi and luciferase activity was measured. In addition, cell lysates were analyzed for protein expression via Western blots. (C and D) Primary murine dendritic cells (C) or murine lung epithelial cells (D) were infected with the indicated PR8 viruses at an MOI of 2. RNA was isolated 8 hpi and subjected to qRT-PCR. All data represent means ± standard deviations of one representative experiment (n = 3). Statistical significance was determined using Student's t test. *, p<0.05; **, p<0.01. Statistical significance is relative to PR8 WT. All data shown are representatives of at least two independent experiments.

Mentions: To investigate whether PB1-F2 66S is a stronger IFN antagonist than PB1-F2 66N in the context of influenza virus infection, we employed reverse genetics to generate a PR8 influenza virus expressing PB1-F2 66S or 66N (WT). We then examined the growth kinetics of these two viruses in A549 cells, a human lung epithelial cell line which supports efficient viral replication, and in 10-day old embryonated chicken eggs. Both viruses displayed similar replication kinetics in both A549 cells and in ovo (Figure 2A). We next examined IFN induction by these two viruses at early timepoints of infection. For this purpose we infected an MDCK IFN-β reporter cell line with the PR8 viruses either expressing PB1-F2 66N or 66S. At 4 hours post infection (hpi), PR8 WT virus induced the IFN-β reporter approximately 5-fold over mock infected cells. This weak induction is due to the strong IFN antagonist activity of the NS1 protein. Yet, PR8 N66S suppressed the IFN-β reporter by 2-fold compared to the PR8 WT virus (Figure 2B). At 8 and 12 hpi similar results were observed. Of note, this phenomenon was not due to increased PB1-F2 protein expression by the PR8 N66S virus (data not shown). NS1 and NP proteins from both viruses were expressed at equal levels with increasing protein expression over time, as determined by Western blot analysis, indicating that the enhanced IFN suppression activity by the PR8 N66S virus was not due to increased replication in these cells or higher NS1 protein expression (Figure 2B). In Western blots with higher exposure, NP protein levels are visible at 4 hpi showing similar levels in PR8 WT and PR8 N66S virus infected cells (Figure 2B).


The influenza virus protein PB1-F2 inhibits the induction of type I interferon at the level of the MAVS adaptor protein.

Varga ZT, Ramos I, Hai R, Schmolke M, García-Sastre A, Fernandez-Sesma A, Palese P - PLoS Pathog. (2011)

PB1-F2 N66S induces less IFN compared to PB1-F2 WT in the context of viral infection.(A) Growth curves of PR8 WT and PR8 N66S viruses in A549 cells and in ovo. A549 cells were infected with virus at an MOI of 0.01 and aliquots of the supernatants were taken at 4, 24, 48 and 72 hpi. To determine viral titers, supernatants were plaqued on MDCK cells. Ten-day old embryonated chicken eggs were inoculated with 100 PFU of virus and placed at 4°C at 12, 24, 48 and 72 hpi. The following days, allantoic fluids were harvested, spun down and titered via plaque assays on MDCK cells. (B) An MDCK cell line expressing an IFN-β promoter driven luciferase reporter (MDCK-IFN-beta luc) was infected with PR8 WT and PR8 N66S viruses at an MOI of 2. Cells were lysed at 4, 8 and 12 hpi and luciferase activity was measured. In addition, cell lysates were analyzed for protein expression via Western blots. (C and D) Primary murine dendritic cells (C) or murine lung epithelial cells (D) were infected with the indicated PR8 viruses at an MOI of 2. RNA was isolated 8 hpi and subjected to qRT-PCR. All data represent means ± standard deviations of one representative experiment (n = 3). Statistical significance was determined using Student's t test. *, p<0.05; **, p<0.01. Statistical significance is relative to PR8 WT. All data shown are representatives of at least two independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3111539&req=5

ppat-1002067-g002: PB1-F2 N66S induces less IFN compared to PB1-F2 WT in the context of viral infection.(A) Growth curves of PR8 WT and PR8 N66S viruses in A549 cells and in ovo. A549 cells were infected with virus at an MOI of 0.01 and aliquots of the supernatants were taken at 4, 24, 48 and 72 hpi. To determine viral titers, supernatants were plaqued on MDCK cells. Ten-day old embryonated chicken eggs were inoculated with 100 PFU of virus and placed at 4°C at 12, 24, 48 and 72 hpi. The following days, allantoic fluids were harvested, spun down and titered via plaque assays on MDCK cells. (B) An MDCK cell line expressing an IFN-β promoter driven luciferase reporter (MDCK-IFN-beta luc) was infected with PR8 WT and PR8 N66S viruses at an MOI of 2. Cells were lysed at 4, 8 and 12 hpi and luciferase activity was measured. In addition, cell lysates were analyzed for protein expression via Western blots. (C and D) Primary murine dendritic cells (C) or murine lung epithelial cells (D) were infected with the indicated PR8 viruses at an MOI of 2. RNA was isolated 8 hpi and subjected to qRT-PCR. All data represent means ± standard deviations of one representative experiment (n = 3). Statistical significance was determined using Student's t test. *, p<0.05; **, p<0.01. Statistical significance is relative to PR8 WT. All data shown are representatives of at least two independent experiments.
Mentions: To investigate whether PB1-F2 66S is a stronger IFN antagonist than PB1-F2 66N in the context of influenza virus infection, we employed reverse genetics to generate a PR8 influenza virus expressing PB1-F2 66S or 66N (WT). We then examined the growth kinetics of these two viruses in A549 cells, a human lung epithelial cell line which supports efficient viral replication, and in 10-day old embryonated chicken eggs. Both viruses displayed similar replication kinetics in both A549 cells and in ovo (Figure 2A). We next examined IFN induction by these two viruses at early timepoints of infection. For this purpose we infected an MDCK IFN-β reporter cell line with the PR8 viruses either expressing PB1-F2 66N or 66S. At 4 hours post infection (hpi), PR8 WT virus induced the IFN-β reporter approximately 5-fold over mock infected cells. This weak induction is due to the strong IFN antagonist activity of the NS1 protein. Yet, PR8 N66S suppressed the IFN-β reporter by 2-fold compared to the PR8 WT virus (Figure 2B). At 8 and 12 hpi similar results were observed. Of note, this phenomenon was not due to increased PB1-F2 protein expression by the PR8 N66S virus (data not shown). NS1 and NP proteins from both viruses were expressed at equal levels with increasing protein expression over time, as determined by Western blot analysis, indicating that the enhanced IFN suppression activity by the PR8 N66S virus was not due to increased replication in these cells or higher NS1 protein expression (Figure 2B). In Western blots with higher exposure, NP protein levels are visible at 4 hpi showing similar levels in PR8 WT and PR8 N66S virus infected cells (Figure 2B).

Bottom Line: Of note, the PB1-F2 N66S protein showed enhanced IFN antagonism activity compared to PB1-F2 wildtype.By inducing the IFN-β reporter at different levels in the signaling cascade, we found that PB1-F2 inhibited IFN production at the level of the mitochondrial antiviral signaling protein (MAVS).Furthermore, immunofluorescence studies revealed that PB1-F2 co-localizes with MAVS.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Mount Sinai School of Medicine, New York City, New York, United States of America.

ABSTRACT
PB1-F2 is a 90 amino acid protein that is expressed from the +1 open reading frame in the PB1 gene of some influenza A viruses and has been shown to contribute to viral pathogenicity. Notably, a serine at position 66 (66S) in PB1-F2 is known to increase virulence compared to an isogenic virus with an asparagine (66N) at this position. Recently, we found that an influenza virus expressing PB1-F2 N66S suppresses interferon (IFN)-stimulated genes in mice. To characterize this phenomenon, we employed several in vitro assays. Overexpression of the A/Puerto Rico/8/1934 (PR8) PB1-F2 protein in 293T cells decreased RIG-I mediated activation of an IFN-β reporter and secretion of IFN as determined by bioassay. Of note, the PB1-F2 N66S protein showed enhanced IFN antagonism activity compared to PB1-F2 wildtype. Similar observations were found in the context of viral infection with a PR8 PB1-F2 N66S virus. To understand the relationship between NS1, a previously described influenza virus protein involved in suppression of IFN synthesis, and PB1-F2, we investigated the induction of IFN when NS1 and PB1-F2 were co-expressed in an in vitro transfection system. In this assay we found that PB1-F2 N66S further reduced IFN induction in the presence of NS1. By inducing the IFN-β reporter at different levels in the signaling cascade, we found that PB1-F2 inhibited IFN production at the level of the mitochondrial antiviral signaling protein (MAVS). Furthermore, immunofluorescence studies revealed that PB1-F2 co-localizes with MAVS. In summary, we have characterized the anti-interferon function of PB1-F2 and we suggest that this activity contributes to the enhanced pathogenicity seen with PB1-F2 N66S- expressing influenza viruses.

Show MeSH
Related in: MedlinePlus