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Intact type I Interferon production and IRF7 function in sooty mangabeys.

Bosinger SE, Johnson ZP, Folkner KA, Patel N, Hashempour T, Jochems SP, Del Rio Estrada PM, Paiardini M, Lin R, Vanderford TH, Hiscott J, Silvestri G - PLoS Pathog. (2013)

Bottom Line: We found that the reported Ser-Gly substitution at position 191 was a sequencing error, and that several of the remaining substitutions represent only minor alleles.Furthermore, in vitro stimulation of SM peripheral blood mononuclear cells with either the TLR7 agonist CL097 or SIV(mac239) induced an 500-800-fold induction of IFN-α and IFN-β mRNA, and levels of IFN-α production by pDCs similar to those of RMs or humans.These data establish that IFN-α and IRF7 signaling in SMs are largely intact, with differences with RMs that are minor and unlikely to play any role in the AIDS resistance of SIV-infected SMs.

View Article: PubMed Central - PubMed

Affiliation: Divison of Microbiology and Immunology, Emory Vaccine Center, Yerkes National Primate Research Center, Atlanta, Georgia, United States of America. sbosing@emory.edu

ABSTRACT
In contrast to pathogenic HIV/SIV infections of humans and rhesus macaques (RMs), natural SIV infection of sooty mangabeys (SMs) is typically non-pathogenic despite high viremia. Several studies suggested that low immune activation and relative resistance of CD4+ central memory T-cells from virus infection are mechanisms that protect SMs from AIDS. In 2008 it was reported that plasmacytoid dendritic cells (pDCs) of SMs exhibit attenuated interferon-alpha (IFN-α) responses to TLR7/9 ligands in vitro, and that species-specific amino acid substitutions in SM Interferon Regulatory Factor-7 (IRF7) are responsible for this observation. Based on these findings, these authors proposed that "muted" IFN-α responses are responsible for the benign nature of SIV infection in SMs. However, other studies indicated that acutely SIV-infected SMs show robust IFN-α responses and marked upregulation of Interferon Stimulated Genes (ISGs). To investigate this apparent disparity, we first examined the role of the reported IRF7 amino acid substitutions in SMs. To this end, we sequenced all IRF7 exons in 16 breeders, and exons displaying variability (exons 2,3,5,6,7,8) in the remainder of the colony (177 animals). We found that the reported Ser-Gly substitution at position 191 was a sequencing error, and that several of the remaining substitutions represent only minor alleles. In addition, functional assays using recombinant SM IRF7 showed no defect in its ability to translocate in the nucleus and drive transcription from an IFN-α promoter. Furthermore, in vitro stimulation of SM peripheral blood mononuclear cells with either the TLR7 agonist CL097 or SIV(mac239) induced an 500-800-fold induction of IFN-α and IFN-β mRNA, and levels of IFN-α production by pDCs similar to those of RMs or humans. These data establish that IFN-α and IRF7 signaling in SMs are largely intact, with differences with RMs that are minor and unlikely to play any role in the AIDS resistance of SIV-infected SMs.

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Transactivation of IFNA4 promoter and nuclear localization by Sooty Mangabey IRF7.(A) HEK293 cells were transfected with the luciferase reporter plasmid containing the human IFNA4 promoter, TBK1 and IRF7 constructs from human, rhesus, and sooty mangabeys, or vehicle, as indicated. Luciferase expressed from the SV40 promoter was transfected as a positive control. Luciferase activity was measured 24 h after transfection. Values represent the average of triplicate wells for Rhesus and Sooty-IRF7 and duplicate wells for the remaining samples. Data are representative of three individual experiments. (B) COS-7 cells were transfected with Sooty-IRF7-GFP and TBK1, or with vehicle for 24 hrs, then stained with DAPI. Magnification is indicated to the right of panels. Data are representative of three experiments.
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ppat-1003597-g001: Transactivation of IFNA4 promoter and nuclear localization by Sooty Mangabey IRF7.(A) HEK293 cells were transfected with the luciferase reporter plasmid containing the human IFNA4 promoter, TBK1 and IRF7 constructs from human, rhesus, and sooty mangabeys, or vehicle, as indicated. Luciferase expressed from the SV40 promoter was transfected as a positive control. Luciferase activity was measured 24 h after transfection. Values represent the average of triplicate wells for Rhesus and Sooty-IRF7 and duplicate wells for the remaining samples. Data are representative of three individual experiments. (B) COS-7 cells were transfected with Sooty-IRF7-GFP and TBK1, or with vehicle for 24 hrs, then stained with DAPI. Magnification is indicated to the right of panels. Data are representative of three experiments.

Mentions: While our extensive sequencing of SM-IRF7 did not reveal any difference with either human or rhesus macaque IRF7 genes that would predict a significant loss of function, these sequencing data do not provide per se any functional information about SM-IRF7. For this reason, we elected to next assess the transactivation potential of SM-IRF7 on an IFNA promoter. We synthesized and cloned into the expression vector pCMV2 a full-length SM-IRF7 gene sequence identical to that of animal FFz, which contained the most representative alleles found in the SMs housed at the Yerkes colony. The ability of SM-IRF7 to initiate IFNA transcription was compared to that of RM-IRF7, human IRF7, and two loss-of-function mutants, using a well-described reporter system in which IRF7, a hu-IFNA4 reporter construct and the activating kinase TBK1 are co-expressed in HEK 293 cells [26]. As shown in Figure 1A, co-expression of human, RM, and SM-IRF7 with the IFNA4 reporter construct alone yielded moderate induction of luciferase activity, consistent with the basal activity that has been reported previously for hu-IRF7 [27]. However, co-transfection of SM-IRF7 together with the IFNA4 reporter and the activating kinase TBK1 resulted in strong induction of transcriptional activity that was >600,000-fold over background (Figure 1A). Importantly, we did not observe any significant difference between the transactivation activity of SM-IRF7 and RM-IRF7. The observed activity of SM-IRF7 was slightly higher than that of human IRF7 (Figure 1A). In addition, we compared SM-IRF7 activity against two loss-of-function IRF7 mutants, IRF7-7A, which contains Ser-to-Ala substitutions in the serine-rich domain, and IRF7-Δ, a dominant-negative mutant missing AA 7-101 of the DNA-binding domain. If the few actual species-specific substitutions in SM-IRF7 impaired its ability to initiate transcription from the IFNA promoter, we would predict that its activity would be closer to the range of the loss-of-function mutants. However, SM-IRF7 transactivation activity was >1000-fold higher than the mutants, and was in general equivalent to, or greater than, human or RM IRF7 constructs (Figure 1A).


Intact type I Interferon production and IRF7 function in sooty mangabeys.

Bosinger SE, Johnson ZP, Folkner KA, Patel N, Hashempour T, Jochems SP, Del Rio Estrada PM, Paiardini M, Lin R, Vanderford TH, Hiscott J, Silvestri G - PLoS Pathog. (2013)

Transactivation of IFNA4 promoter and nuclear localization by Sooty Mangabey IRF7.(A) HEK293 cells were transfected with the luciferase reporter plasmid containing the human IFNA4 promoter, TBK1 and IRF7 constructs from human, rhesus, and sooty mangabeys, or vehicle, as indicated. Luciferase expressed from the SV40 promoter was transfected as a positive control. Luciferase activity was measured 24 h after transfection. Values represent the average of triplicate wells for Rhesus and Sooty-IRF7 and duplicate wells for the remaining samples. Data are representative of three individual experiments. (B) COS-7 cells were transfected with Sooty-IRF7-GFP and TBK1, or with vehicle for 24 hrs, then stained with DAPI. Magnification is indicated to the right of panels. Data are representative of three experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3757038&req=5

ppat-1003597-g001: Transactivation of IFNA4 promoter and nuclear localization by Sooty Mangabey IRF7.(A) HEK293 cells were transfected with the luciferase reporter plasmid containing the human IFNA4 promoter, TBK1 and IRF7 constructs from human, rhesus, and sooty mangabeys, or vehicle, as indicated. Luciferase expressed from the SV40 promoter was transfected as a positive control. Luciferase activity was measured 24 h after transfection. Values represent the average of triplicate wells for Rhesus and Sooty-IRF7 and duplicate wells for the remaining samples. Data are representative of three individual experiments. (B) COS-7 cells were transfected with Sooty-IRF7-GFP and TBK1, or with vehicle for 24 hrs, then stained with DAPI. Magnification is indicated to the right of panels. Data are representative of three experiments.
Mentions: While our extensive sequencing of SM-IRF7 did not reveal any difference with either human or rhesus macaque IRF7 genes that would predict a significant loss of function, these sequencing data do not provide per se any functional information about SM-IRF7. For this reason, we elected to next assess the transactivation potential of SM-IRF7 on an IFNA promoter. We synthesized and cloned into the expression vector pCMV2 a full-length SM-IRF7 gene sequence identical to that of animal FFz, which contained the most representative alleles found in the SMs housed at the Yerkes colony. The ability of SM-IRF7 to initiate IFNA transcription was compared to that of RM-IRF7, human IRF7, and two loss-of-function mutants, using a well-described reporter system in which IRF7, a hu-IFNA4 reporter construct and the activating kinase TBK1 are co-expressed in HEK 293 cells [26]. As shown in Figure 1A, co-expression of human, RM, and SM-IRF7 with the IFNA4 reporter construct alone yielded moderate induction of luciferase activity, consistent with the basal activity that has been reported previously for hu-IRF7 [27]. However, co-transfection of SM-IRF7 together with the IFNA4 reporter and the activating kinase TBK1 resulted in strong induction of transcriptional activity that was >600,000-fold over background (Figure 1A). Importantly, we did not observe any significant difference between the transactivation activity of SM-IRF7 and RM-IRF7. The observed activity of SM-IRF7 was slightly higher than that of human IRF7 (Figure 1A). In addition, we compared SM-IRF7 activity against two loss-of-function IRF7 mutants, IRF7-7A, which contains Ser-to-Ala substitutions in the serine-rich domain, and IRF7-Δ, a dominant-negative mutant missing AA 7-101 of the DNA-binding domain. If the few actual species-specific substitutions in SM-IRF7 impaired its ability to initiate transcription from the IFNA promoter, we would predict that its activity would be closer to the range of the loss-of-function mutants. However, SM-IRF7 transactivation activity was >1000-fold higher than the mutants, and was in general equivalent to, or greater than, human or RM IRF7 constructs (Figure 1A).

Bottom Line: We found that the reported Ser-Gly substitution at position 191 was a sequencing error, and that several of the remaining substitutions represent only minor alleles.Furthermore, in vitro stimulation of SM peripheral blood mononuclear cells with either the TLR7 agonist CL097 or SIV(mac239) induced an 500-800-fold induction of IFN-α and IFN-β mRNA, and levels of IFN-α production by pDCs similar to those of RMs or humans.These data establish that IFN-α and IRF7 signaling in SMs are largely intact, with differences with RMs that are minor and unlikely to play any role in the AIDS resistance of SIV-infected SMs.

View Article: PubMed Central - PubMed

Affiliation: Divison of Microbiology and Immunology, Emory Vaccine Center, Yerkes National Primate Research Center, Atlanta, Georgia, United States of America. sbosing@emory.edu

ABSTRACT
In contrast to pathogenic HIV/SIV infections of humans and rhesus macaques (RMs), natural SIV infection of sooty mangabeys (SMs) is typically non-pathogenic despite high viremia. Several studies suggested that low immune activation and relative resistance of CD4+ central memory T-cells from virus infection are mechanisms that protect SMs from AIDS. In 2008 it was reported that plasmacytoid dendritic cells (pDCs) of SMs exhibit attenuated interferon-alpha (IFN-α) responses to TLR7/9 ligands in vitro, and that species-specific amino acid substitutions in SM Interferon Regulatory Factor-7 (IRF7) are responsible for this observation. Based on these findings, these authors proposed that "muted" IFN-α responses are responsible for the benign nature of SIV infection in SMs. However, other studies indicated that acutely SIV-infected SMs show robust IFN-α responses and marked upregulation of Interferon Stimulated Genes (ISGs). To investigate this apparent disparity, we first examined the role of the reported IRF7 amino acid substitutions in SMs. To this end, we sequenced all IRF7 exons in 16 breeders, and exons displaying variability (exons 2,3,5,6,7,8) in the remainder of the colony (177 animals). We found that the reported Ser-Gly substitution at position 191 was a sequencing error, and that several of the remaining substitutions represent only minor alleles. In addition, functional assays using recombinant SM IRF7 showed no defect in its ability to translocate in the nucleus and drive transcription from an IFN-α promoter. Furthermore, in vitro stimulation of SM peripheral blood mononuclear cells with either the TLR7 agonist CL097 or SIV(mac239) induced an 500-800-fold induction of IFN-α and IFN-β mRNA, and levels of IFN-α production by pDCs similar to those of RMs or humans. These data establish that IFN-α and IRF7 signaling in SMs are largely intact, with differences with RMs that are minor and unlikely to play any role in the AIDS resistance of SIV-infected SMs.

Show MeSH
Related in: MedlinePlus