INTRODUCTION

Influenza A viruses are among the most common causes of human respiratory diseases which cause infections with high mortality, especially in the elderly, infants, and people with chronic diseases and weakened immune systems1. These viruses have an RNA genome consisting of eight single-stranded segments with negative polarity 2,3. Replication and transcription of the viral genome are catalyzed by the RNA-dependent RNA polymerase (RdRP) enzyme, which is a complex composed of PB2, PB1, and PA subunits4-6. The RdRP enzyme resides in the virus particle attached to the vRNP complexes consisting of the NP proteins and RNA molecules. The PA subunit of the RdRP enzyme has endonuclease activity, binds to the 5’-cap of the host pre-mRNAs along with the PB2 subunit, and cleaves. It has a role in binding to the genomic RNA promoter along with the PB1 subunit 7. It is known that the PA protein, which is effective in viral pathogenesis, is associated with several cellular protein factors. Some of the PA protein-related proteins have a negative regulatory effect on influenza virus replication including HAX1 8 and SNX29; however, some of the PA-associated cellular proteins stimulate viral replication. It has been reported that the pyruvate kinase M2 (PKM2) binds the c-terminal region of the PA subunit and is essential for virus replication10. Kawaguchi & Nagata11 suggest that the minichromosome maintenance protein complex (MCM), a DNA helicase, interacts with the influenza A virus PA protein and stimulates virus replication. The humanChromosome 14 Open Reading Frame 166 (C14orf166), which is the subject of this study, is a stimulatory factor for the influenza A virus12. This protein is encoded by a gene located on chromosome 14 (14q22.1) and has an average weight of 28 kDa. The C14orf166 is also known as RTRAF, CLE, CLE7, hCLE, CGI99, RLLM1, hCLE1, CGI-99, and LCRP369. In recent reports, it was shown that C14orf166 is a member of nucleo-cytosolic shuttle protein complexes involving DDX1, HSPC117, and FAM98B proteins and has a role in the transport of the molecules involved in RNA metabolism between nucleus and cytoplasm13. This protein is associated with several factors required for RNA synthesis and processing, including transcription factor 4, heterogeneous nuclear ribonucleoprotein R, poly A binding protein 1, and the nuclear pore complex Nup15314-16. C14orf166 is not only involved in the regulation of RNA polymerase activity but is also upregulated in some tumors. Overexpression of the C14orf166 has been found to contribute to oncogenesis and invasive behaviors in various tumors. C14orf166 is a JAK2-related protein that activates STAT3 signaling which may cause cervical and esophageal cancer17,18. It has also been known that expression of the C14orf166 is upregulated in breast cancer cell and tissues compared with normal breast tissues. The overexpression of C14orf166 inhibits the expression of cell cycle inhibitors p21 and p27 and increases the phosphorylation level of the Rb protein19. From these results, it is suggested that C14orf166 contributes to cell proliferation by regulating the G1/S transition of the cells, but its mechanism has not yet been fully elucidated. Furthermore, the C14orf166 interacts with RNA polymerase II and directly regulates RNA transcription, suggesting that C14orf166 has a vital role in cell growth and organ development 20. It has been reported that the C14orf166 protein is also associated with some virus replication/transcription processes. Huarte et al. reported the interaction of the C14orf166 protein with influenza A virus PA21. Silencing of the C14orf166 gene expression results in decreased vRNA transcription/replication, and virus production12. The C14orf166 protein involves in acute/chronic hepatitis C virus (HCV) infection by interacting with HCVc174, a core protein of HCV. It is thought that the C14orf166/HCVc174 complex may cause abnormal mitosis of infected hepatocytes, resulting in hepatic carcinoma22. These reports show that the c14orf166 protein is a cellular factor involved in the replication of some viruses as well as some cancer types in humans. In this study, full-size and truncated C14orf166 proteins interacting with the influenza A virus PA protein that is used as bait in the yeast two-hybrid (Y2H) screening were defined, and the interaction patterns of these protein with PA were analyzed by in silico tools. It was concluded that some amino acid residues located at the carboxyl terminal end of C14orf166 are significant in the PA protein interaction.
MATERIAL AND METHODS
Yeast cells
The yeast strain Saccharomyces cerevisiae PJ69-4A was used to detect the proteins interacting with the influenza A virus PA protein by the yeast dual-hybrid method (Genotype: MATa trp1-901 leu2-3, 112 ura3-52 his3-200 gal4(deletion) gal80(deletion) LYS2::GAL1-HIS3 GAL2-ADE2 met2::GAL7-lacZ). The cells were grown in YPAD (yeast extract- peptone-dextrose plus adenine) rich medium at 30°C at 175 rpm shaking and stored at -80°C in a YPD (yeast extract peptone dextrose) medium containing 20% glycerin.
cDNA library
The cDNA library constructed by cloning human embryonic kidney cells (HEK293) cDNA after the sequence encoding the yeast GAL4 activation domain (GAL4-AD) in pAKT2 Y2H plasmid (pAKT2-cDNAn) was purchased as transformed in E. coli cells (BNN132) (Clontech #638826)). The cells were grown on LB agar (+amp) plates according to the manufacturer’s instructions, and the plasmid DNA was isolated.
Plasmids
Construction of the pGBD-PA plasmid vector encoding the viral PA as a bait protein has been previously described9. Briefly, the pGBD-C1 plasmid DNA was digested with EcoRI restriction enzyme just after the sequence coding the yeast GAL4 DNA binding domain (GAL4-BD), was blunted with the Klenow enzyme (New England Biolabs, UK), and then was dephosphorylated with shrimp alkaline phosphatase (Thermo Fisher Scientific, USA). The influenza A/duck/Pennsylvania/10,218/84/H5N2 (DkPen) virus PA gene open reading frame (ORF) was generated from the pCAGGS-PA (DkPen) plasmid23 with PCR by using phosphorylated oligonucleotide primers 5’-CGGAGGATCTGGAATG GAAGACTTTGTGCGACAATG-3’ and 5’- CTATTTCAGTGCATGTGCGAG-3’. The PCR was carried out with high-fidelity KOD DNA polymerase. The PCR amplified DNA was purified with a gel extraction kit (Invitrogen # K210012, USA) and ligated with linear pGBD-C1 using a T4 DNA ligase kit (Ligation High v.II, TaKaRa, Japan). To construct a mammalian expression vector coding the human C14orf166 protein, the ORF of the gene was cloned into the EcoRV site of pCHA plasmid DNA24. C14orf166 ORF was generated with PCR using the Y2H cDNA library as a template and phosphorylated primers having 5’-ATCATG TTCCGACGCAAGTTG-3’ and 5’-ATCTA TCTTCCAACTTTTCCCAG-3’ sequences. The PCR amplified DNA was purified by a gel extraction kit and ligated with the pCHA plasmid which was digested with EcoRV restriction enzyme (New England Biolabs, UK) and dephosphorylated with SAP. The resulting plasmid was named pCHA-C14orf166. The plasmid constructs were verified by colony PCR, restriction digestion, and finally checked by Sanger sequencing.
Yeast two-hybrid screening
A small-scale yeast culture (5 ml) was prepared in the YPAD medium and transformed with the pGBD-PA plasmid DNA coding the PA bait protein by the lithium acetate/polyethylene glycol (LiAc/PEG) method. Transformants were selected on a yeast synthetic drop-out (SD) agar medium (without Trp). One of the colonies harboring the bait plasmid was grown in the YPAD medium, transformed with the Y2H M Matchmaker cDNA library and seeded on SD agar plates (without Her, Leu, Ade, and Trp) for selection of positive colonies.
The yeast colonies grown on the selective medium were tested for reporter β-galactosidase activity, which is encoded from the second reporter gene. The plasmids carrying cDNA were isolated from the colonies having high β-galactosidase activity with a yeast plasmid DNA extraction kit. The cDNAs carried on the plasmids were amplified with PCR using the oligonucleotide primers 5’-AATACCACTACAATGGATGATGT-3’ and 5’-CCAAGATTGAAACTTAGAGGAGT-3’. The PCR products were purified with a gel extraction kit and the sequence of the cDNAs was defined with Sanger sequencing. DNA sequence results were evaluated with the Basic Local Alignment Search Tool (BLAST), and the genes were identified.
β-galactosidase assay
The yeast cells in a 250 µl of saturated yeast cultures were precipitated with centrifugation at 3500 rpm for 5 minutes, and the precipitates were suspended in 300 µl of Z buffer/β-ME (100 mM Phosphate Buffer, pH. 7, 10 mM KCl, 1mM MgSO4, 50 mM 2-β-Mercaptoethanol). The samples were subjected to 10 cycles of freezing (in liquid nitrogen) and thawing (at 37°C). Then, 60 µl of an ONPG substrate solution (4 mg/ml) was added to the samples and incubated at 30°C for 60 minutes. After incubation, the reaction was terminated by adding 300 µl of 0.5 M Na2CO3 to the samples. The cell debris was precipitated with centrifugation at 15000 rpm for 5 minutes, and OD420 of the supernatants was determined.
Minireplicon assay
The effect of C14orf166 overexpression on the human type influenza A/WSN/33/H1N1 (WSN) and the avian type DkPen virus RNA polymerase enzyme was investigated with a minireplicon model in HEK293 cells. The assays were performed using minireplicon plasmid DNAs, as previously described23,25.
In silico predictions
The shortest C14orf166 peptide (consisting of 69 amino acid residues at the carboxy-terminal end) having a positive interaction with the viral PA protein in yeast cells was used as a basic structure for the in silico prediction of three-dimensional (3D) structures and protein interactions. The 3D models of the peptide/proteins were generated with the online I-TASSER algorithm (https://zhanggroup.org/I-TASSER/)26. The best-predicted models were determined by using the c-score [-5 - +2], TM-score (>0.5), and root-mean-square deviation (RMSD) that were used to predict the quality of the models. A reference 3D model of C14orf166 was received in PDB format from the AlphaFold Protein Structure Database(https://alphafold.ebi.ac.uk/entry/Q9Y224). A homology model of the influenza A DkPen PA protein predicted with the I-TASSER algorithm was used as a reference model in all protein-protein docking analyses. The docking analyses were carried out using the ClusPro protein docking algorithm (https://www.cluspro.org)27. The most likely C14orf166/PA interaction models were defined by considering the experimental data of the Y2H screening, the reports of the structure of the influenza A virus RdRP enzyme complex consisting of PB2-PB1 and PA proteins and 3D models of this complex28 (PDB # 6QPF), binding energies and/or clustering rates revealed by docking algorithms, and amino acid residues with possible binding potential in the models. PyMOL software and an online PDBsum server (http://www.ebi.ac.uk) were used in the analysis of selected models for PA and C14orf166 interaction29.
RESULTS
Identification of human C14orf166 proteins interacting with influenza a virus PA protein in yeast cells
Several host proteins that interact with influenza virus PA were identified using the Y2H screening assay carried out with transformation of the HEK293 Matchmaker cDNA library into the yeast cells harboring the pGBD-PA bait plasmid. Among the cDNAs isolated from the positive yeast colonies, the most often encountered cDNA belonged to theC14orf166 gene. It was also noteworthy that the bait proteins with different sizes encoded from differently truncated C14orf166 cDNAs gave positive reactions to the viral PA protein. From the sequencing results of cDNAs isolated from the yeast cells, seven different sizes of C14orf166 cDNA were defined (four samples are given in Figure 1).