The effects of cellular hnRNP M interaction with viral IRES on Senecavirus A replication
CAO Zhi-yuan;WEI Dan-dan;WANG Hai-wei;SUN Chao;YU Li;State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences;
To investigate whether hnRNP M is involved in Senecavirus A(SVA) replication and infection, the recombinant plasmid PCAGGS-HA-hnRNP M was constructed in this study. After transfection into BHK-21 cells, the recombinant plasmid was detected by western blot, and the results showed that hnRNP M protein could be correctly expressed in cells. Subsequently, hnRNP M overexpression or knockdown of BHK-21 cells was infected with SVA, and the effect of hnRNP M on SVA replication was evaluated by Western blot and TCID50. The results showed that overexpression of hnRNP M significantly inhibited SVA replication in BHK-21 cells(P0.01), while down-regulation of hnRNP M protein significantly promoted the replication of SVA in BHK-21 cells(P0.01). Further, the immunoprecipitation complex was obtained from SVA-infected BHK-21 cells by RNA immuno-precipitation assay, and the RNA was extracted, reverse-transcribed and detected by PCR using SVA genome-specific primers. The RNA immunoprecipitation results showed an interaction between hnRNP M protein and SVA RNA in the case of SVA infection. Confocal laser microscopy detected the localization of the SVA genome and hnRNP M. The results showed that hnRNP M was transferred from the nucleus to the cytoplasm after SVA infection and co-localized with SVA RNA in the cytoplasm. In addition, hnRNP M overexpression of hnRNP M or knockdown of BHK-21 cells were detected by the dual-luciferase reporter assay to determine the SVA IRES activity. The dual-luciferase reporter assay showed that overexpression of hnRNP M protein inhibited IRES-mediated translation activity. In contrast, down-regulation of hnRNP M protein promoted IRES activity. This study demon-strated for the first time that host protein hnRNP M could interact with the SVA genome to affect the activity of SVA IRES and inhibit viral replication,laying a foundation to further understanding the molecular regulation mechanism of SVA replication in the host.