伪狂犬病毒早期蛋白UL9通过抑制宿主应激颗粒形成促进自身复制OA

To identify key viral proteins responsible for inhibiting host stress granules(SGs)formation during the early stage of pseudorabies virus(PRV)infection and promoting viral replication and immune evasion,dithiothreitol(DTT)was used to induce cellular stress,and the effect of early PRV infection on SGs formation was evaluated by laser scanning confocal immunofluorescence assay.Eukaryotic expression plasmids of PRV immediate-early/early proteins were constructed and screened to identify the inhibitory effect of the unique long region 9(UL9)protein on SGs formation.Truncated UL9 eukaryotic expression plasmids were constructed and transfected into cells to determine the functional domain responsible for SGs inhibition.Small interfering RNA(siRNA)-mediated knockdown of UL9 was performed,followed by viral growth curve analysis to evaluate the effect of UL9 on PRV replication.A PRV HB1201-ΔUL9 strain was constructed using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9(CRISPR/Cas9)gene-editing system,and its replication ability and effect on SGs formation were further evaluated.The results showed that PRV infection at the early stage antagonized host SGs formation and thereby promoted viral replication.Overexpression of UL9 significantly inhibited SGs formation(P<0.000 1),and this inhibitory effect depended on the structural integrity of UL9.Truncated of UL9 significantly reduced PRV replication(P<0.01 or P<0.000 1).The PRV HB1201-ΔUL9 strain was successfully constructed.Compared with the parental PRV HB1201 strain,the UL9-deleted virus exhibited significantly reduced replication ability and impaired inhibition of SGs formation(P<0.01,P<0.000 1,or P<0.000 1).These results indicate that the PRV early protein UL9 is a key viral factor involved in inhibiting SGs formation.This study expands the understanding of PRV-mediated immune evasion mechanisms and provides a potential target for the development of attenuated vaccines.