LGP2 expression is enhanced by interferon regulatory factor 3 in olive flounder, Paralichthys olivaceus.

In innate immunity, LGP2 (laboratory of genetics and physiology 2) plays a very important role in the production of type I interferon (IFN) through recognition of cytosolic viral RNA. Although viral infection or stimulation with double-strand RNA dramatically induces expression of the LGP2 gene, the underlying transcriptional mechanism has never been studied. Here, we cloned and characterized the 5'-upstream region (-1,337 bp) of the LGP2 gene in olive flounder (Paralichthys olivaceus). Numerous canonical motifs for IFN-regulatory factors (IRFs) were found in this region, and reporter assays identified a poly I:C-responsive promoter region (-506 to -398) that regulated LGP2 transcription. Transcriptional activity of the LGP2 promoter was strongly enhanced by IRF3, which bound to IRF3 motif #3 (-480). The LGP2 promoter was also responsive to viral infection in vitro. These results suggest that LGP2 transcriptional control is crucially involved to regulated by IRF3 function after viral infection or stimulation with poly I:C

An IRF3 canonical motif in the olive flounder LGP2 promoter region possessing strong transcriptional activity in HINAE cells.

<p>(<b>A</b>) Schematic structure of the reporter constructs, pGL3-LP(−506) and pGL3-LP(−453). The schematic diagrams indicate the canonical motifs, and numbers on the diagrams indicate the types of IRFs. (<b>B</b>) Luciferase activity in IRF3-cotransfected HINAE cells was measured 48 hours after transfection. Activity is shown relative to pGL3-LP(−506) activity in IRF3-cotransfected cells, expressed as a percentage. Activity values represent means ± SEMs of three individual experiments (<i>*p</i><0.01, pGL3-empty vs. pcDNA4-transfected cells; Student’s <i>t</i>-test).</p

Expression of olive flounder LGP2, MDA5, Mx, and ISG15 mRNAs in HINAE cells stimulated with poly I:C for 6, 12, 24 or 48 hours.

<p>Expression levels of LGP2 (<b>A</b>), MDA5 (B), Mx (<b>C</b>), and ISG15 (<b>D</b>) were calculated relative to β-actin mRNA levels. The expression values represent the means ± SEMs of three individual experiments (*<i>p</i><0.05, **<i>p</i><0.01, mock control vs. poly I:C-transfected HINAE cell at each time point; Student’s <i>t</i>-test).</p

Transcriptional activity of the 5′-upstream region of the olive flounder LGP2 gene in HINAE cells.

<p>(<b>A</b>) Schematic structure of the olive flounder LGP2 reporter constructs, pGL3-LP(−1271), pGL3-LP(−1096), pGL3-LP(−796), pGL3-LP(−507), pGL3-LP(−400), and pGL3-LP(−51). The schematic diagrams indicate transcription factor canonical motifs, and numbers on the diagrams indicate the types of IRFs. Luciferase activity of reporter constructs transfected into HINAE cells stimulated by poly I:C-transfection (<b>B</b>) or poly I:C-extracellular addition to the medium (<b>C</b>) was measured 48 hours post-transfection. Luciferase activity is expressed relative to the activity in empty vector-transfected (mock) cells. The activity values represent the means ± SEMs of three individual transfections (<i>*p</i><0.05, mock vs. poly I:C-transfected cells; Student’s <i>t</i>-test).</p

Changes in the transcriptional activity of the LGP2 promoter in LGP2- or MDA5-overexpressing cells stimulated with poly I:C.

<p>(<b>A</b>) Different amounts of LGP2 or MDA5 constructs (0, 2, 20 and 100 ng) were cotransfected into HINAE cells with 50 ng of poly I:C (transfected). (<b>B</b>) Different amounts of poly I:C constructs (0, 16, 80 and 400 ng) were cotransfected into LGP2- or MDA5-overexpressing HINAE cells. Activity is shown relative to pGL3-LP(−506) activity in empty vector cotransfected cells. Activity values represent means ± SEMs of six individual experiments (<i>*p</i><0.05, pGL3-empty mock control vs. poly I:C- and pcDNA4-transfected cells; Student’s <i>t</i>-test). Abbreviation: TF, transfection.</p

Potential transcription factor binding motifs in the 5′-upstream region of the olive flounder LGP2 gene.

<p>(<b>A</b>) Nucleotide sequence of the 5′-upstream region is shown with canonical motifs underlined. The transcription initiation site is indicated by an arrow (±0). The TATA box motif is italicized and underlined, and the coding region is in bold. (<b>B</b>) Comparison of the 5′-upstream region of the <i>LGP2</i> gene in olive flounder (−1,337 to −1) with those of green puffer (Chr. 3∶13152455-13154190), Japanese medaka (Chr. 8∶6135706-6138037, complement), and humans (Chr. 17∶40264652-40265128, complement). The 5′-upstream region between LGP2 and KAT2A genes in humans, green puffer, and Japanese medaka was analyzed using MatInspector software (Genomatix). The schematic diagrams indicate transcription factor canonical motifs, and numbers on the diagrams indicate the types of IRFs.</p

Transcription factor binding sites in the olive flounder LGP2 promoter.

*<p>Core similarity: the element denotes the match of an element to the canonical motif as calculated by MatInspector (TRANSFAC-based analysis).</p>**<p>Capital letters indicate the core region of the motif sequence.</p

Transcriptional activity of the LGP2 promoter.

<p>(<b>A</b>) LP(−506) was fused with the open reading frame of the GFP gene and cotransfected with poly I:C (300 ng) or pcDNA4-IRF (300 ng) into HINAE cells. After 24 and 48 hours, the expression of GFP protein was detected by fluorescence microscopy. (<b>B</b>) The LP(−506)-GFP vector was transfected into HINAE cells. After 24 hours, cells were infected by VHSV (MOI = 1). After 18 hours, the expression of GFP protein was detected.</p

Enhancement of olive flounder LGP2 promoter transcriptional activity by IRF3 in HINAE cells.

<p>(<b>A</b>) Schematic structure of the reporter constructs, pGL3-LP(−506) and pGL3-LP(−396). The schematic diagrams indicate the canonical motifs, and numbers on the diagrams indicate the types of IRFs. (<b>B</b>) Luciferase activity in IRF-cotransfected HINAE cells was measured 48 hours post-transfection. Activity is expressed relative to that of cells transfected with pGL3 and pcDNA4 empty vectors. (<b>C</b>) Expression of olive flounder IRF1, IRF3, and IRF7 mRNAs in HINAE cells stimulated with poly I:C for 6, 12, 24 or 48 hours. IRF1, IRF3, and IRF7 expression levels were calculated as fold increase in mRNA levels, normalized to β-actin values, between poly I:C-transfected HINAE cells and mock controls at each time point (6, 12, 24, and 48 hours). Luciferase activity and expression values represent means ± SEMs of three individual experiments (<i>*p</i><0.01, pGL3-empty vs. pcDNA4-transfected cells [in B], and mock control vs. poly I:C-transfected HINAE cell at each time point [in C]; Student’s <i>t</i>-test).</p

Oligonucleotide sequences.

*<p>Amplification efficiency.</p