Molecular cloning and functional characterization of porcine IFN-beta promoter stimulator 1 (IPS-1).
The IFN-beta promoter stimulator 1 (IPS-1), also known as MAVS/VISA/Cardif, is an adaptor molecule for the retinoic-acid-inducible protein I (RIG-I) or melanoma-differentiation-associated gene 5 (MDA5) that recognizes intracellular double-stranded RNA (dsRNA) and triggers a signal for producing type I IFN. In the present study, porcine IPS-1 cDNA was cloned, using RT-PCR coupled with rapid amplification of cDNA ends (RACE)-PCR, from porcine peripheral blood mononuclear cells. The open reading frame of porcine IPS-1 consists of 1575bp encoding 524 amino acids. The putative porcine IPS-1 protein contains a N-terminal CARD-like domain, a central proline-rich domain, a C-terminal transmembrane domain, and exhibits similarity to mouse, rat, monkey, human and cattle counterparts, ranging from 59% to 79%. Semi-quantitative RT-PCR showed that porcine IPS-1 mRNA was widely expressed in different tissues. Porcine kidney (PK-15) cells transfected with a DNA construct encoding porcine IPS-1 produced type I IFN, and activated IRF3 and NF-kappaB. Deletion mutant analyses further revealed that both the CARD-like domain and transmembrane domain are essential for these functions. In addition, poly(I:C)-induced porcine IFN-beta promoter activation in PK-15 cells was significantly reduced by siRNA targeting IPS-1, indicating that IPS-1 is an important immunoregulator in the porcine innate immune system. The availability of porcine IPS-1 and establishment of its function in the type I IFN signaling pathway provides a useful molecule for defining its role during the course of pig infectious diseases.
Wang
Dang
D
State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, PR China.
Fang
Liurong
L
Li
Tingting
T
Luo
Rui
R
Xie
Lilan
L
Jiang
Yunbo
Y
Chen
Huanchun
H
Xiao
Shaobo
S
D048868:Adaptor Proteins, Signal Transducing / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D006801:Humans; D050835:Interferon Regulatory Factors / Q000378:metabolism; D016899:Interferon-beta / Q000096:biosynthesis; D051379:Mice; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism; D010802:Phylogeny; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D019105:Random Amplified Polymorphic DNA Technique / Q000662:veterinary; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D016415:Sequence Alignment; D013552:Swine / Q000235:genetics / Q000276:immunology*; D014162:Transfection / Q000662:veterinary
D048868:Adaptor Proteins, Signal Transducing; D050835:Interferon Regulatory Factors; D016328:NF-kappa B; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D016899:Interferon-beta