关键词: 酵母双杂交, 流感病毒, PA蛋白, PCBP1蛋白, 蛋白互作

Abstract: 【Background】The PA protein is an integral component of the influenza virus RNA polymerase complex and plays an important role in the transcription and replication of the viral genome. The yeast two-hybrid system was used in our laboratory to screen for host proteins that interact with PA protein, and one of them was identified to be poly(rC)-binding protein 1 (PCBP1). 【Objective】This study investigated the interaction between PCBP1 protein and influenza virus PA protein and the role of PCBP1 on the replication of influenza virus, which would provide scientific data for the in-depth understanding of the replication regulation mechanism of influenza virus in the host. 【Method】In order to verify the interaction between PA protein and PCBP1 protein in yeast system, the bait plasmid pGBKT7-PA and the selected prey plasmid pGADT7-PCBP1 as well as the negative control and positive control were cotransformed into yeast competent cells, respectively, according to the LiAc transformation procedure. The transformed yeasts were then spread on three kinds of auxotrophic medium and incubated at 30℃ for 5-7 days to observe the growth and color of colonies. According to the sequences of PA protein and human PCBP1 protein registered in the GenBank, specific amplification primers were designed to construct eukaryotic recombinant expression plasmids pCAGGS-Flag-PA and pCAGGS-Myc-PCBP1. These two eukaryotic expression plasmids were individually transfected or co-transfected into HEK293T cells. The transfected cells were lysed 48 hours after transfection to harvest the supernatant. A small portion of the supernatant was used as a control, and the rest of supernatant was successively added with the FLAG monoclonal antibody and Protein G agarose beads for the preparation of immunoprecipitates. Protein samples were analyzed by SDS-PAGE and Western blot to detect the interaction between PA protein and PCBP1 protein in mammalian cells. To establish a PCBP1-overexpressing cell line, the pseudovirus was packaged in HEK293T cells by using the lentivirus packaging system pLVX-IRES-ZsGreen1, followed by transduction of A549 cells and ultra-fast flow cytometry sorting. After verification of PCBP1 overexpression by Western blot, the overexpressing cell line was infected with WSN virus at an MOI of 0.01. Supernatants were collected at 24 h and 48 h post infection and virus titers were determined by means of plaque assay on MDCK cells. To downregulate the expression of PCBP1 protein, the specific siRNA targeting PCBP1 was synthesized and transfected into A549 cells. At 48 h post treatment with siRNA, the downregulation of PCBP1 expression was confirmed by Western blot, and the siRNA-treated cells were infected with WSN virus at an MOI of 0.01. Supernatants were harvested at 24 h and 48 h after infection and titrated by plaque assay.【Result】Yeast competent cells co-transformed with bait plasmid pGBKT7-PA and recombinant plasmid pGADT7-PCBP1 could grow on SD/-2, SD/-4, and SD/-4/X/A auxotrophic medium plates and produced blue colonies on SD/-4/X/A plates by breaking down X-α-Gal, which was consistent with the positive control group, indicating that the PA protein interacted with PCBP1 protein in the yeast system. Co-immunoprecipitation experiments showed that PA protein bound PCBP1 protein, indicating that they interacted with each other in mammalian cells. The influenza virus replication titer was decreased in the PCBP1-overexpressing cell line. In contrast, the virus titer was increased in cells transfected with siRNA targeting PCBP1. Together, these results indicated that PCBP1 protein negatively regulated influenza virus replication.【Conclusion】In this study, we demonstrated that influenza virus PA protein can interact with PCBP1 protein in yeast and mammalian cells, and found that PCBP1 protein negatively regulated influenza virus replication.

Key words: yeast two-hybrid, influenza virus, PA protein, PCBP1 protein, protein interaction