[The transfection of lentiviral siRNA vectors targeting ezrin, radixin and moesin facilitates influenza virus replication in primary pulmonary microvascular endothelial cells].

OBJECTIVE

To construct lentivirus siRNA vectors targeting ezrin (E), radixin (R) and moesin (M), transfect rat primary pulmonary microvascular endothelial cells (PMVECs) with the vectors and observe the effects of the transfection on E, R, M gene expressions and influenza virus replication.

METHODS

According to the principles of siRNA synthesis, siRNA sequences targeting E, R and M genes were synthesized and cloned into lentiviral plasmid GV248 to construct lentiviral siRNA vectors Lenti-E-siRNA, Lenti-R-siRNA and Lenti-M-siRNA, respectively. Random Lenti-control-siRNA was also constructed targeting nonsense sequences. Rat PMVECs were cultured by peripheral lung tissue-sticking method. Rat PMVECs were transfected with Lenti-E-siRNA, Lenti-R-siRNA and Lenti-M-siRNA, respectively. The levels of ezrin, radixin and moesin mRNA and proteins were detected by real-time quantitative PCR and Western blotting, the distributions of ERM proteins were detected using confocal laser scanning microscope; After lentivirus-transfected PMVECs were infected with influenza virus, the effects of lentiviral vectors on influenza virus replication were measured by real-time quantitative PCR.

RESULTS

Lenti-E-siRNA, Lenti-R-siRNA, Lenti-M-siRNA and Lenti-control-siRNA were successfully constructed according to sequencing identification of recombinant plasmids. The infection efficacy of lentiviral siRNA vectors in rat PMVECs was above 80% at a multiplicity of infection (MOI) of 10. The transcription and expressions of ezrin, radixin and moesin in rat PMVECs were significantly inhibited by Lenti-E-siRNA, Lenti-R-siRNA, Lenti-M-siRNA respectively. Lentivirus control (containing no target genes) and Lenti-control-siRNA inhibited influenza virus replication in influenza virus-infected PMVECs, while Lenti-E-siRNA, Lenti-R-siRNA and Lenti-M-siRNA increased the levels of influenza virus mRNA in the infected PMVECs.

CONCLUSION

Lentiviral siRNA vectors targeting E, R and M genes were constructed successfully and could inhibit E, R and M gene expressions specifically and effectively. Lentiviral vectors could inhibit influenza virus replication whereas lentiviral siRNA vectors targeting E, R and M facilitated influenza virus replication.