Construction of chicken lung cDNA library Confirmation of interaction Chicken serine/arginine-rich splicing factor interacts with Nucleoprotein of Highly Pathogenic Avian Influenza Virus Vinod RMT Balasubramaniam1, Iekhsan Othman1, Abdul R Omar2 and Sharifah S Hassan1 1.Virus-Host Interaction Group, Infectious Disease Laboratory (MR3), School of Medicine and Health Sciences, Monash University, Sunway Campus, 46150 Sunway, Malaysia 2. Institute of Bioscience, University Putra Malaysia, UPM Serdang, Selangor, 43400, Malaysia MONASH UNIVERSITY INTRODUCTION Screening for potential host cellular proteins that interacts with HPAI H5N1 Nucleoprotein (NP) was conducted using yeast 2-hybrid system . A chicken (Gallus gallus) lung cDNA library was constructed and fused inside yeast acting domain vector (pGADT7). This was mated with the yeast binding domain vector (pGBKT7) carrying the HPAI H5N1 NP. A novel chicken serine/arginine-rich splicing factor (SRSF) protein was shown to have interacted with the NP viral protein. This protein was hypothesized as to play a role in viral mRNA transport and export into the cytoplasm for translation. The SRSF was shown to localize in the nucleus and co-localization with the NP viral protein also in the nucleus of H5N1-infected human and canine kidney cell lines was observed. Docking using 3D models and pull down co-immunoprecipitation assays confirmed these interaction. The SRSF protein, therefore, plays an important structural and functional role and might represent an attractive drug target. METHODOLOGY & RESULTS Construction of chicken lung cDNA library Interaction of Avian H5N1 NP [A/chicken/Malaysia/5858/2004(H5N1)] with chicken lung cDNA library Confirmation of interaction M 1 2 Fig.5: showing the confirmation of interaction between the Avian H5N1 NP and Avian SRSF. A549 cells were transfected with pcDNA3.1-NP and pcDNA3.1-SRSF alone or in combination. Cells were harvested 48hrs post transfection and immunoprecipitation pull-down assays using anti-NP antibody and anti-SRSF antibody, followed by western blotting were conducted. These results showed that NP co-precipitated with SRSF and vice-versa. Total RNA extraction First strand cDNA synthesis Double stranded cDNA synthesis Column purification of ds cDNA Fig.1: Electrophoresis of the constructed cDNA library. Lane 1: abundance of genes from 100bp to 10kb. Lane 2: Purified cDNA library Fig.3: Mating process between the yeast containing cDNA library and bait protein H5N1 NP. The blue arrow indicates blue colonies (activation of all reporter genes) confirming authentic interaction Fig.2: Transformation of the cDNA library into yeast strain Y187. 2 million clones in total, at least 70 cfu per plate with 1/100 dilution Fig.4: Yeast colony PCR from the extracted plasmids of the blue positive colonies. These bands are sequenced to identify the interacting partners of H5N1 NP M CONCLUSION Co-localization of Avian H5N1 NP and SRSF in the nucleus 3D Modeling of the interaction We hypothesize that identification of this SRSF protein could help us to better understand the molecular mechanisms regulating the NP-dependent stages of the virus life cycle. This protein might play a role in facilitating viral mRNA transport from nucleus to the cytoplasm. A B A B ACKNOWLEDGEMENTS C D C D We would like to thank Prof. Dato Dr. Anuar Zaini for his support in this project. We would also like to thank Dr.Satoshi Ogawa from BRIMS for his assistance in fluorescence microscopy and Tham Hong Wai for his assistance in the construction of the cDNA library. Fig.8: NP residues that interact with SRSF (blue color): Arg65, Arg74, Tyr78, Gly94, Arg106, Leu108, Arg152, Arg156, Ser170, Thr171, Met191, Arg195, Arg199, Ala366, Asn368, and Asn370. NP residues that interact with RNA (red color): Arg65, Leu68, Ser69, Asp72, Glu73, Tyr78, Lys87, Asp88, and Lys113. NP residues that interact with both RNA and SRSF (magenta color): Tyr78 and Arg65. REFERENCE Fig 7: Avian H5N1-infected MDCK cells with at MOI of 5. A. Cells were fixed and labelled with anti-NP antibody conjugated to Alexa Fluor 594 (red). B. Cells were fixed and labelled with anti-SRSF antibody conjugated to Alexa Fluor 488 (green). C. DAPI nuclear stain. D. Merged picture showing the localization of NP and SRSF are in the nucleus of the cell Fig 6: A. A549 cells transfected with pcDNA3.-NP and labelled with Alexa Fluor 594 (red). B. A549 cells transfected with pcDNA3.1-SRSF and labelled with Alexa Fluor 488 (green). C. DAPI nuclear stain. D. Merged picture showing the localization of NP and SRSF are in the nucleus of the cell Sharma K, Tripathi S, Ranjan P, Kumar P, Garten R, et al. (2011) Influenza A Virus Nucleoprotein Exploits Hsp40 to Inhibit PKR Activation. PLoS ONE 6(6): e20215. doi:10.1371/journal.pone.0020215