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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 further enhances NS1 mediated IFN antagonism in an overexpression system.(A) 293T cells were transfected with IFN-β reporter, renilla reporter, RIG-I N expression plasmid and the indicated combination of expression plasmids. Twenty-four hours post transfection, luciferase and renilla signals were determined by luminometry. In addition, Western blot analyses were applied to determine protein expression levels from cell extracts. (B) Primary human dendritic cells were co-infected with the indicated combination of recombinant NDV viruses at an MOI of 1 per virus. RNA was harvested and subjected to qRT-PCR 14 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; ***, p<0.001. Statistical significance is relative to NS1+pCAGGS and NDV-NS1+NDV-GFP. Data presented in A are representatives of two independent experiments and data in B are representatives of three independent experiments performed in different donors.
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ppat-1002067-g005: PB1-F2 N66S further enhances NS1 mediated IFN antagonism in an overexpression system.(A) 293T cells were transfected with IFN-β reporter, renilla reporter, RIG-I N expression plasmid and the indicated combination of expression plasmids. Twenty-four hours post transfection, luciferase and renilla signals were determined by luminometry. In addition, Western blot analyses were applied to determine protein expression levels from cell extracts. (B) Primary human dendritic cells were co-infected with the indicated combination of recombinant NDV viruses at an MOI of 1 per virus. RNA was harvested and subjected to qRT-PCR 14 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; ***, p<0.001. Statistical significance is relative to NS1+pCAGGS and NDV-NS1+NDV-GFP. Data presented in A are representatives of two independent experiments and data in B are representatives of three independent experiments performed in different donors.

Mentions: To characterize the relationship between NS1 and PB1-F2 regarding IFN antagonism, we overexpressed NS1 with either PB1-F2 66N or 66S in 293T cells and analyzed the induction of the IFN-β reporter activated by RIG-I N. As shown in Figure 5A, PB1-F2 66N did not significantly reduce IFN promoter activation compared to the empty vector control when co-expressed with NS1. In contrast, PB1-F2 66S further decreased IFN induction when co-expressed with NS1 even though PB1-F2 66N is expressed more efficiently as shown in Western blot analyses (Figure 5A). To confirm these findings in the human DC model, we performed a co-infection experiment with the recombinant NDV viruses. Co-infection of human DCs with NDV-NS1 and NDV-PB1-F2 66N induced similar levels of IFN compared to co-infection with NDV-NS1 and NDV-GFP (Figure 5B). In contrast, co-infection of primary human DCs with NDV-NS1 and NDV-PB1-F2 N66S strongly reduced type I IFN and IP-10 mRNA levels (Figure 5B). Collectively, these data indicate that PB1-F2 N66S, but not PB1-F2 WT, enhances the IFN antagonism activity of NS1.


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 further enhances NS1 mediated IFN antagonism in an overexpression system.(A) 293T cells were transfected with IFN-β reporter, renilla reporter, RIG-I N expression plasmid and the indicated combination of expression plasmids. Twenty-four hours post transfection, luciferase and renilla signals were determined by luminometry. In addition, Western blot analyses were applied to determine protein expression levels from cell extracts. (B) Primary human dendritic cells were co-infected with the indicated combination of recombinant NDV viruses at an MOI of 1 per virus. RNA was harvested and subjected to qRT-PCR 14 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; ***, p<0.001. Statistical significance is relative to NS1+pCAGGS and NDV-NS1+NDV-GFP. Data presented in A are representatives of two independent experiments and data in B are representatives of three independent experiments performed in different donors.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3111539&req=5

ppat-1002067-g005: PB1-F2 N66S further enhances NS1 mediated IFN antagonism in an overexpression system.(A) 293T cells were transfected with IFN-β reporter, renilla reporter, RIG-I N expression plasmid and the indicated combination of expression plasmids. Twenty-four hours post transfection, luciferase and renilla signals were determined by luminometry. In addition, Western blot analyses were applied to determine protein expression levels from cell extracts. (B) Primary human dendritic cells were co-infected with the indicated combination of recombinant NDV viruses at an MOI of 1 per virus. RNA was harvested and subjected to qRT-PCR 14 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; ***, p<0.001. Statistical significance is relative to NS1+pCAGGS and NDV-NS1+NDV-GFP. Data presented in A are representatives of two independent experiments and data in B are representatives of three independent experiments performed in different donors.
Mentions: To characterize the relationship between NS1 and PB1-F2 regarding IFN antagonism, we overexpressed NS1 with either PB1-F2 66N or 66S in 293T cells and analyzed the induction of the IFN-β reporter activated by RIG-I N. As shown in Figure 5A, PB1-F2 66N did not significantly reduce IFN promoter activation compared to the empty vector control when co-expressed with NS1. In contrast, PB1-F2 66S further decreased IFN induction when co-expressed with NS1 even though PB1-F2 66N is expressed more efficiently as shown in Western blot analyses (Figure 5A). To confirm these findings in the human DC model, we performed a co-infection experiment with the recombinant NDV viruses. Co-infection of human DCs with NDV-NS1 and NDV-PB1-F2 66N induced similar levels of IFN compared to co-infection with NDV-NS1 and NDV-GFP (Figure 5B). In contrast, co-infection of primary human DCs with NDV-NS1 and NDV-PB1-F2 N66S strongly reduced type I IFN and IP-10 mRNA levels (Figure 5B). Collectively, these data indicate that PB1-F2 N66S, but not PB1-F2 WT, enhances the IFN antagonism activity of NS1.

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