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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 reduces IFN induction by a dsRNA and TRIM25 binding mutant NS1 influenza virus.(A) An MDCK cell line expressing an IFN-β promoter driven luciferase reporter (MDCK-IFN-beta luc) was infected with PR8 viruses expressing the dsRNA/TRIM25 binding mutant form of NS1 (R38A/K41A) as well as PB1-F2 WT or PB1-F2 N66S at an MOI of 0.5. 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 blot analyses. (B and C) Primary human dendritic cells were infected with the viruses described in (A) at an MOI of 0.5 and RNA was harvested for qRT-PCR analyses at 6 and 8 hpi (B). In addition, supernatants were analyzed for IFN-alpha secretion via ELISA (C). (D) A549 cells were infected with the viruses described in (A) at an MOI of 0.5 and RNA was analyzed via qRT-PCR at 6 hpi. 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 PB1-F2 WT. Data shown are representatives of two independent experiments.
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ppat-1002067-g004: PB1-F2 N66S reduces IFN induction by a dsRNA and TRIM25 binding mutant NS1 influenza virus.(A) An MDCK cell line expressing an IFN-β promoter driven luciferase reporter (MDCK-IFN-beta luc) was infected with PR8 viruses expressing the dsRNA/TRIM25 binding mutant form of NS1 (R38A/K41A) as well as PB1-F2 WT or PB1-F2 N66S at an MOI of 0.5. 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 blot analyses. (B and C) Primary human dendritic cells were infected with the viruses described in (A) at an MOI of 0.5 and RNA was harvested for qRT-PCR analyses at 6 and 8 hpi (B). In addition, supernatants were analyzed for IFN-alpha secretion via ELISA (C). (D) A549 cells were infected with the viruses described in (A) at an MOI of 0.5 and RNA was analyzed via qRT-PCR at 6 hpi. 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 PB1-F2 WT. Data shown are representatives of two independent experiments.

Mentions: The NS1 protein is the major IFN antagonist of influenza viruses as the knockout of NS1 strongly attenuates the virus in IFN competent cells [44]. NS1 employs multiple strategies to counteract the initiation and establishment of an antiviral state in virus infected cells [45]. One of these strategies is the masking of viral RNA species from recognition by RIG-I [16], [17]. Within the N-terminal domain of NS1, the basic residues R38 and K41 were found to mediate binding to dsRNA species [16] and mutating these residues to alanine was shown to induce a robust IFN response in influenza virus infected cells [15]. In addition, these mutations also result in loss of NS1 interaction with TRIM25, an interaction that is required for inhibition of RIG-I activation mediated by this E3 ligase [20]. We thus aimed to examine the IFN induction by viruses that express a dsRNA/TRIM25 binding mutant form of NS1 and PB1-F2 66N or 66S. For this purpose we rescued PR8 viruses with the described point mutations in NS1 (R38A/K41A) and those that express the WT or N66S form of PB1-F2. Infection of the MDCK-IFN-β reporter cell line with these two mutant viruses resulted in approximately 3 log higher induction of the IFN-β reporter compared to the PR8 viruses with WT NS1 at 8 hpi (Figure 2B and 4A). At 4 hpi, no remarkable difference in IFN induction was seen between the PB1-F2 66N or 66S expressing viruses. At 8 hpi, however, the NS1 dsRNA/TRIM25 binding mutant virus expressing PB1-F2 66S induced significantly less IFN than the isogenic virus expressing PB1-F2 66N (Figure 4A). At 12 hpi, the difference in IFN induction became minimal (Figure 4A) and a substantial detachment of cells was observed at this time point (data not shown). At 4 hpi and 8 hpi, the NP and NS1 protein levels were comparable in cells infected with both viruses. Of note, the NP protein levels in cells infected with the NS1 mutant viruses do not increase as dramatically between 4 and 8 hpi compared to those in cells infected with PR8 viruses expressing wildtype NS1 (Figure 2B). This finding indicates that the NS1 dsRNA binding mutant viruses are attenuated compared to the wildtype NS1 expressing viruses. Next, we infected primary human dendritic cells with the NS1 dsRNA/TRIM25 binding mutant viruses and analyzed the induction of type I IFN via qRT-PCR and ELISA. At 6 hpi and 8 hpi, IFN-β mRNA as well as IFN-α protein levels were decreased by the PB1-F2 N66S expressing virus compared to the isogenic virus (Figure 4B and 4C) which confirms the data obtained in the MDCK IFN-β reporter cell line (Figure 4A). We also performed qRT-PCR analyses to quantify IFN-α mRNA levels and found a similar trend to IFN-β mRNA levels (data not shown). To test this phenomenon in epithelial cells, we also infected A549 cells with the dsRNA/TRIM25 binding mutant NS1 viruses. As shown in Figure 4D, the PB1-F2 N66S expressing virus induced less IFN and IP-10 compared to the PB1-F2 WT virus at 6 hpi and 8 hpi (data not shown). In summary, these data demonstrate that even in the presence of an NS1 protein that lacks the dsRNA and TRIM25 binding function, PB1-F2 66S is able to reduce the induction of IFN compared to PB1-F2 66N.


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 reduces IFN induction by a dsRNA and TRIM25 binding mutant NS1 influenza virus.(A) An MDCK cell line expressing an IFN-β promoter driven luciferase reporter (MDCK-IFN-beta luc) was infected with PR8 viruses expressing the dsRNA/TRIM25 binding mutant form of NS1 (R38A/K41A) as well as PB1-F2 WT or PB1-F2 N66S at an MOI of 0.5. 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 blot analyses. (B and C) Primary human dendritic cells were infected with the viruses described in (A) at an MOI of 0.5 and RNA was harvested for qRT-PCR analyses at 6 and 8 hpi (B). In addition, supernatants were analyzed for IFN-alpha secretion via ELISA (C). (D) A549 cells were infected with the viruses described in (A) at an MOI of 0.5 and RNA was analyzed via qRT-PCR at 6 hpi. 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 PB1-F2 WT. Data shown are representatives of two independent experiments.
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ppat-1002067-g004: PB1-F2 N66S reduces IFN induction by a dsRNA and TRIM25 binding mutant NS1 influenza virus.(A) An MDCK cell line expressing an IFN-β promoter driven luciferase reporter (MDCK-IFN-beta luc) was infected with PR8 viruses expressing the dsRNA/TRIM25 binding mutant form of NS1 (R38A/K41A) as well as PB1-F2 WT or PB1-F2 N66S at an MOI of 0.5. 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 blot analyses. (B and C) Primary human dendritic cells were infected with the viruses described in (A) at an MOI of 0.5 and RNA was harvested for qRT-PCR analyses at 6 and 8 hpi (B). In addition, supernatants were analyzed for IFN-alpha secretion via ELISA (C). (D) A549 cells were infected with the viruses described in (A) at an MOI of 0.5 and RNA was analyzed via qRT-PCR at 6 hpi. 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 PB1-F2 WT. Data shown are representatives of two independent experiments.
Mentions: The NS1 protein is the major IFN antagonist of influenza viruses as the knockout of NS1 strongly attenuates the virus in IFN competent cells [44]. NS1 employs multiple strategies to counteract the initiation and establishment of an antiviral state in virus infected cells [45]. One of these strategies is the masking of viral RNA species from recognition by RIG-I [16], [17]. Within the N-terminal domain of NS1, the basic residues R38 and K41 were found to mediate binding to dsRNA species [16] and mutating these residues to alanine was shown to induce a robust IFN response in influenza virus infected cells [15]. In addition, these mutations also result in loss of NS1 interaction with TRIM25, an interaction that is required for inhibition of RIG-I activation mediated by this E3 ligase [20]. We thus aimed to examine the IFN induction by viruses that express a dsRNA/TRIM25 binding mutant form of NS1 and PB1-F2 66N or 66S. For this purpose we rescued PR8 viruses with the described point mutations in NS1 (R38A/K41A) and those that express the WT or N66S form of PB1-F2. Infection of the MDCK-IFN-β reporter cell line with these two mutant viruses resulted in approximately 3 log higher induction of the IFN-β reporter compared to the PR8 viruses with WT NS1 at 8 hpi (Figure 2B and 4A). At 4 hpi, no remarkable difference in IFN induction was seen between the PB1-F2 66N or 66S expressing viruses. At 8 hpi, however, the NS1 dsRNA/TRIM25 binding mutant virus expressing PB1-F2 66S induced significantly less IFN than the isogenic virus expressing PB1-F2 66N (Figure 4A). At 12 hpi, the difference in IFN induction became minimal (Figure 4A) and a substantial detachment of cells was observed at this time point (data not shown). At 4 hpi and 8 hpi, the NP and NS1 protein levels were comparable in cells infected with both viruses. Of note, the NP protein levels in cells infected with the NS1 mutant viruses do not increase as dramatically between 4 and 8 hpi compared to those in cells infected with PR8 viruses expressing wildtype NS1 (Figure 2B). This finding indicates that the NS1 dsRNA binding mutant viruses are attenuated compared to the wildtype NS1 expressing viruses. Next, we infected primary human dendritic cells with the NS1 dsRNA/TRIM25 binding mutant viruses and analyzed the induction of type I IFN via qRT-PCR and ELISA. At 6 hpi and 8 hpi, IFN-β mRNA as well as IFN-α protein levels were decreased by the PB1-F2 N66S expressing virus compared to the isogenic virus (Figure 4B and 4C) which confirms the data obtained in the MDCK IFN-β reporter cell line (Figure 4A). We also performed qRT-PCR analyses to quantify IFN-α mRNA levels and found a similar trend to IFN-β mRNA levels (data not shown). To test this phenomenon in epithelial cells, we also infected A549 cells with the dsRNA/TRIM25 binding mutant NS1 viruses. As shown in Figure 4D, the PB1-F2 N66S expressing virus induced less IFN and IP-10 compared to the PB1-F2 WT virus at 6 hpi and 8 hpi (data not shown). In summary, these data demonstrate that even in the presence of an NS1 protein that lacks the dsRNA and TRIM25 binding function, PB1-F2 66S is able to reduce the induction of IFN compared to PB1-F2 66N.

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