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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 inhibits NDV induced IFN in primary human dendritic cells.(A) Primary human dendritic cells were infected with NDV-GFP, NDV-PB1-F2 WT or NDV-PB1-F2 N66S at an MOI of 2 and NDV HN was determined via qRT-PCR at 6,14,18 and 22 hpi. (B and C) At 14 hpi, qRT-PCR analyses were performed for IFN-alpha and IP-10 mRNA levels. In addition, supernatants were collected for ELISA analyses to quantify IFN-α and IP-10 protein secretion. 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; ***, p<0.001. Statistical significance is relative to NDV-GFP. The data presented in B and C are representatives of five independent experiments with different donors.
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ppat-1002067-g003: PB1-F2 N66S inhibits NDV induced IFN in primary human dendritic cells.(A) Primary human dendritic cells were infected with NDV-GFP, NDV-PB1-F2 WT or NDV-PB1-F2 N66S at an MOI of 2 and NDV HN was determined via qRT-PCR at 6,14,18 and 22 hpi. (B and C) At 14 hpi, qRT-PCR analyses were performed for IFN-alpha and IP-10 mRNA levels. In addition, supernatants were collected for ELISA analyses to quantify IFN-α and IP-10 protein secretion. 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; ***, p<0.001. Statistical significance is relative to NDV-GFP. The data presented in B and C are representatives of five independent experiments with different donors.

Mentions: Newcastle disease virus (NDV) has been shown to be a potent IFN inducer by activating RIG-I [42]. Therefore, we asked the question whether PB1-F2 could block NDV induced IFN activation in primary human dendritic cells. Dendritic cells are important immune effector cells that are involved in clearing viral infections by secreting pro-inflammatory cytokines as well as type I IFN and bridging the innate and adaptive immune responses. It has been demonstrated that primary human dendritic cells are a suitable ex vivo model to study human influenza A virus host responses [43]. We generated recombinant NDV viruses that express GFP or PB1-F2 (WT and N66S) and infected primary human dendritic cells to investigate the induction of type I IFN in this model. As shown in Figure 3A, NDV-PB1-F2 N66S replicated more efficiently than the NDV-PB1-F2 WT and NDV-GFP viruses at 18 hpi and 22 hpi. To test whether this growth advantage may be due to suppressed IFN induction by PB1-F2 N66S expressed from the NDV vector, we analyzed IFN induction at 14 hpi by the three recombinant NDV viruses when replication was still found to be similar. NDV-GFP induced high levels of IFN as well as IP-10 as measured by qRT-PCR and ELISA (Figure 3B and 3C). Expression of PB1-F2 WT did not suppress IFN and IP-10 levels compared to GFP, whereas PB1-F2 N66S efficiently inhibited IFN induction in NDV infected dendritic cells (Figure 3B and 3C). These results indicate that PB1-F2 N66S is also able to suppress IFN production in primary human dendritic cells confirming that this inhibitory effect is not strictly cell type-specific.


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 inhibits NDV induced IFN in primary human dendritic cells.(A) Primary human dendritic cells were infected with NDV-GFP, NDV-PB1-F2 WT or NDV-PB1-F2 N66S at an MOI of 2 and NDV HN was determined via qRT-PCR at 6,14,18 and 22 hpi. (B and C) At 14 hpi, qRT-PCR analyses were performed for IFN-alpha and IP-10 mRNA levels. In addition, supernatants were collected for ELISA analyses to quantify IFN-α and IP-10 protein secretion. 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; ***, p<0.001. Statistical significance is relative to NDV-GFP. The data presented in B and C are representatives of five independent experiments with different donors.
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getmorefigures.php?uid=PMC3111539&req=5

ppat-1002067-g003: PB1-F2 N66S inhibits NDV induced IFN in primary human dendritic cells.(A) Primary human dendritic cells were infected with NDV-GFP, NDV-PB1-F2 WT or NDV-PB1-F2 N66S at an MOI of 2 and NDV HN was determined via qRT-PCR at 6,14,18 and 22 hpi. (B and C) At 14 hpi, qRT-PCR analyses were performed for IFN-alpha and IP-10 mRNA levels. In addition, supernatants were collected for ELISA analyses to quantify IFN-α and IP-10 protein secretion. 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; ***, p<0.001. Statistical significance is relative to NDV-GFP. The data presented in B and C are representatives of five independent experiments with different donors.
Mentions: Newcastle disease virus (NDV) has been shown to be a potent IFN inducer by activating RIG-I [42]. Therefore, we asked the question whether PB1-F2 could block NDV induced IFN activation in primary human dendritic cells. Dendritic cells are important immune effector cells that are involved in clearing viral infections by secreting pro-inflammatory cytokines as well as type I IFN and bridging the innate and adaptive immune responses. It has been demonstrated that primary human dendritic cells are a suitable ex vivo model to study human influenza A virus host responses [43]. We generated recombinant NDV viruses that express GFP or PB1-F2 (WT and N66S) and infected primary human dendritic cells to investigate the induction of type I IFN in this model. As shown in Figure 3A, NDV-PB1-F2 N66S replicated more efficiently than the NDV-PB1-F2 WT and NDV-GFP viruses at 18 hpi and 22 hpi. To test whether this growth advantage may be due to suppressed IFN induction by PB1-F2 N66S expressed from the NDV vector, we analyzed IFN induction at 14 hpi by the three recombinant NDV viruses when replication was still found to be similar. NDV-GFP induced high levels of IFN as well as IP-10 as measured by qRT-PCR and ELISA (Figure 3B and 3C). Expression of PB1-F2 WT did not suppress IFN and IP-10 levels compared to GFP, whereas PB1-F2 N66S efficiently inhibited IFN induction in NDV infected dendritic cells (Figure 3B and 3C). These results indicate that PB1-F2 N66S is also able to suppress IFN production in primary human dendritic cells confirming that this inhibitory effect is not strictly cell type-specific.

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