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Immune responses, subcellular localization, and antiviral activity of interferon-induced protein 35 (IFP35) in rock bream (Oplegnathus fasciatus)

원문정보

W.S.P. Madhuranga, M.D. Neranjan Tharuka, J.C. Harasgama, Hyukjae Kwon, Qiang Wan, Jehee Lee

제주대학교 해양과학연구소 해양과학연구소 연구논문집 제45권 2021.12 pp.225-234
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초록

영어

Interferon-induced protein 35 kDa (IFP35) has been demonstrated to play important roles in antiviral defense, inflammatory response and cancer progression. However, its precise function in teleost fish remains to be elucidated. Herein, we functionally characterized the rock bream (Oplegnathus fasciatus) IFP35 (OfIFP35) to understand its expression pattern, subcellular localization, antiviral activity, and regulation of downstream genes. OfIFP35 consists of an 1107 bp open reading frame encoding 368 amino acids, including two N-mycinteractor (Nmi)/IFP35 domains (NIDs). The predicted molecular weight of OfIFP35 was 42 kDa, with a theoretical isoelectric point (pI) of 5.10. Evolutionary conservation of IFP35 was analyzed using multiple, pairwise alignments and phylogenetic tree analysis. OfIFP35 in rock bream was found to be highest expressed in the gills. Immune challenges with iridovirus, polyinosinic:polycytidylic acid, lipopolysaccharide, and live bacteria (Streptococcus iniae and Edwardsiella tarda) significantly upregulated its mRNA expression in gill and liver tissues of the rock bream. GFP-tagged OfIFP35 was localized in the cytoplasm of FHM cells, and its overexpression significantly suppressed VHSV transcription in vitro. Moreover, the analysis of downstream gene expression revealed that OfIFP35 could activate the type I interferon pathway. Collectively, these findings indicate that OfIFP35 is important for the immune system of rock bream as it promotes defense responses during viral infections.

목차

ABSTRACT
1. Introduction
2. Materials and methods
2.1. Experimental fish and tissue collection
2.2. Identification of OfIFP35 cDNA sequence and bioinformatics analysis
2.3. Immune challenge experiment
2.4. Total RNA extraction and cDNA synthesis
2.5. Expression analysis of OfIFP35
2.6. Cloning and plasmid construction
2.7. Cell culture, transfection, and optimization of viral load
2.8. OfIFP35 subcellular localization
2.9. Antiviral activity assay of OfIFP35
2.10. Regulation of downstream interferon-stimulated genes (ISGs)
2.11. Statistical analysis
3. Results
3.1. O. fasciatus IFP35 sequence and in silico characterization
3.2. Tissue distribution of OfIFP35 transcripts
3.3. Temporal expression of OfIFP35 against pathogens and immune stimulants
3.4. Subcellular localization of OfIFP35 and antiviral effect in FHM cells
3.5. Involvement of OfIFP35 in the type I IFN pathway
4. Discussion
5. Conclusion
References

키워드

저자정보

  • W.S.P. Madhuranga Department of Marine Life Sciences & Fish Vaccine Research Center, Jeju National University, Jeju Self-Governing Province, 63243; Marine Science Institute, Jeju National University, Jeju Self-Governing Province, 63333, Republic of Korea
  • M.D. Neranjan Tharuka Department of Marine Life Sciences & Fish Vaccine Research Center, Jeju National University, Jeju Self-Governing Province, 63243; Marine Science Institute, Jeju National University, Jeju Self-Governing Province, 63333, Republic of Korea
  • J.C. Harasgama Department of Marine Life Sciences & Fish Vaccine Research Center, Jeju National University, Jeju Self-Governing Province, 63243; Marine Science Institute, Jeju National University, Jeju Self-Governing Province, 63333, Republic of Korea
  • Hyukjae Kwon Department of Marine Life Sciences & Fish Vaccine Research Center, Jeju National University, Jeju Self-Governing Province, 63243; Marine Science Institute, Jeju National University, Jeju Self-Governing Province, 63333, Republic of Korea
  • Qiang Wan Department of Marine Life Sciences & Fish Vaccine Research Center, Jeju National University, Jeju Self-Governing Province, 63243; Marine Science Institute, Jeju National University, Jeju Self-Governing Province, 63333, Republic of Korea
  • Jehee Lee Department of Marine Life Sciences & Fish Vaccine Research Center, Jeju National University, Jeju Self-Governing Province, 63243; Marine Science Institute, Jeju National University, Jeju Self-Governing Province, 63333, Republic of Korea

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