Effect Of LncRNA TCONS₀0179042 On The Replication Of PRRSV In Marc-145 Cells

The porcine reproductive and respiratory syndrome?PRRS?has been found for more than 30 years.However,it has not been controlled effectively throughout the world.Since 2006,highly pathogenic PRRSV?HP-PRRSV?has emerged and become a dominant strain in China,which has caused great economic losses to China's pig industry.Recently,functions of long non-coding RNA?lncRNAs?have been increasingly reported,which expressed in a wide range of mammalian cells and play important roles in the regulation of gene function at transcription and post-transcriptional levels in different organisms.LncRNA can also exert antiviral innate immune functions through the formation of immune complexes.In our previous research,we found that the expression of lncRNA TCONS₀0179042 is significantly decreased in PRRSV infected PAM cells than in mock infected PAM cells by high-throughput sequencing.To verify the function of this lncRNA during PRRSV infection,the sequence of lncRNA TCONS₀0179042 was cloned by RT-PCR at first.The secondary structure of this lncRNA was predicted with bioinformatics methods.And then,this lncRNA was overexpressed in Marc-145 cells,its effect on the replication of GSWW15 strain of PRRSV was analyzed by qPCR,Western blotting and TCID₅₀.The distribution of lncRNA TCONS₀0179042 in PAM cells was analyzed by nuclear fraction isolation experiment.Further,the targeted genes of lncRNA TCONS₀0179042 were preliminarily screened by RNA-seq and bioinformatics prediction methods.The results showed that the lncRNA TCONS₀0179042 transcript was approximately 430 nt and had a relatively complex secondary structure.qPCR results demonstrated that the relative expression level of lncRNA TCONS₀0179042 in the pcDNA3.1-TCONS₀0179042 transfected cells was about 3×10⁵ folds of the control group,indicating that lncRNA TCONS₀0179042 coule be overexpressed in Marc-145 cells.After infection of Marc-145 cells expressing lncRNA TCONS₀0179042 and PRRSV GSWW15,qPCR results showed that the PRRSV RNA copies number in the lncRNA overexpressing group was about 60%of the control group.And the expression of PRRSV N protein in lncRNA overexpression group was about 25%of the control group.We also found that the PRRSV titer was significantly lower in lncRNA overexpression group than in the control group by TCID₅₀.These results suggested that lncRNA TCONS₀0179042 could inhibit the replication of PRRSV GSWW15 in Marc-145 cells.The nuclear separation experiment showed that the lncRNA TCONS₀0179042 was existed both in the cytoplasm and the nucleus,but the expression level in the nucleus was higher than that in the cytoplasm.The targeted gene preliminary screening results showed that there were 8up-regulated genes related to the host antiviral immune responses,which could be founctional targets of lncRNA TCONS₀0179042,laying a good foundation for further study on the molecular mechanism of lncRNA regulating viral replication.In summary,this study demonstrates that lncRNA TCONS₀0179042 has significant anti-PRRSV functions in mammalian cells.It is necessary to study the molecular mechanisms of this lncRNA in regulating the antiviral immune responses.This study also provides some clues for the development of new anti-PRRSV strategies.