| Objective:Influenza A virus(IAV)is an enveloped single negative strand RNA virus.The envelope of IAV comes from the phospholipid bilayer of the host cell membrane,which contains two important glycoproteins hemagglutinin(HA)and neuraminidase(NA).What beneath the envelope of IAV is virus matrix protein M1,which interacts with vRNP complex that carris the genome of IAV After the entry of IAV into the host cell,the virus envelope is broken,vRNPs are released and transported to the nucleus.The vRNPs in the nucleus are replicated and transcribed,then the newly synthesized viral RNAs and viral proteins are assembled into new vRNP complexes and gathered to the cell membrane.The progeny viruses are formed by budding,and then new viruses are released to infect other cells.Viral vRNP complex has polymerase and endonuclease activity and plays a key role in the synthesis of cRNA,vRNA and mRNA of IAVCircRNA is a type of circular non-coding RNA without 5’cap structure and 3’poly(A)tail.Because of its unique circular structure,circRNA can not be easily recognized and digested by exonuclease,and is more stable than linear RNA.At present,many studies have shown that circRNA plays an important role in regulating cellular immunity,tumor growth and apoptosis,cell metabolism,and autophagy.The invasion of IAV may interfere the normal physiological state of host cells and affect the expression of some host factors.These differentially expressed host factors can often promote or inhibit IAV replication by regulating innate immunity,viral RNA production,progeny virus release,etc.Until now,many evidences showed that host proteins can participate in the replication process of IAV,but there are few studies on how non-coding RNAs,especially circRNAs,regulate IAV replication.The present study explored the function and regulatory mechanism of circ-IUC1,which was significantly up-regulated after IAV infection in A549 cells,suggesting that circRNA may play a critical role on IAV replication.Methods:In this study,circular RNA circ-IUC1,which was screened out by RNA deepsequencing technology,was up-regulated after IAV infection.The sequencing results were verified by Real-time quantitative PCR.Stimulating cells with SeV,poly(I:C),IFN-β followed by cellular RNA extraction for reverse transcription and Real-time quantitative PCR to detect whether circ-IUC1 can be induced by IFN-β signaling pathway.By PCR and Real-time quantitative PCR,we proved the existence of circIUC1 and verified its biochemical characteristics.We determined the subcellular location of circ-IUC1 through nuclear and cytoplasmic separation assay and FISH.We constructed circ-IUC1-overexpression and-knockdown cell lines to detect the protein levels of M1 and NP protein in cells by Western Blot after infection,and the virus titers in the supernatant of cells after infection were counted by plaque assay to determine whether circ-IUC1 has a regulatory effect on the replication of IAV.In order to explore the specific mechanism by which circ-IUC1 regulates IAV replication,we used RNA pull down technology to enrich the proteins bound by circ-IUC1 during IAV infection,then by mass spectrometry detection,we identifed the proteins which may interact with circ-IUC1.The results showed that NP may interact with circ-IUC 1.The interaction of circ-IUC1 and NP was verified by RIP and RNA pull down assay.In order to determine how circ-IUC1 regulates IAV replication by binding to NP,we explored the effect of circ-IUC1 on the activity of virus polymerase complex by luciferase assay.We detected the effect of overexpression of circ-IUC1 on the expression levels of vRNA,cRNA,and mRNA of IAV by Real-time quantitative PCR.We also explored the effect of circIUC1 on NP oligomerization through co-IP.Finally,we verified the effect of circ-IUC1 on the binding of NP with PB1 and PB2 through co-IP to determine the specific mechanism of circ-IUC1 regulating IAV replication.Results:1.The results of RNA deep sequencing showed that the expression of circIUC1 was significantly increased after IAV infection in A549 cells.Subsequent verification showed that circ-IUC1 was significantly up-regulated in cells infected with IAV strains WSN,PR8,and CA04,and the increase of circ-IUC1 was time-dependent.In addition to IAV,the expression level of circ-IUC1 was significantly up-regulated after SeV infection in 293T cells.A significant increase of circ-IUC1 was also detected in 293T cells transfected with the double-stranded RNA analog poly(I:C).After treatment of 293T cells with IFN-β,the expression of circ-IUC1 increased with the increasing dose of IFN-β.The above results showed that the expression of circ-IUC1 was significantly increased under the induction of various stimuli such as IAV,SeV,poly(I:C)and IFN-β.2.To determine whether circ-IUC1 has common biochemical properties as circRNAs,we examined the ability of resisting RNaseR digestion and stability of circ-IUC1.The results showed that the content of circ-IUC1 did not change significantly after being digested by RNaseR.After 72 hours of treating A549 cells with actinomycin D,the expression level of circ-IUC1 was not significantly changed,proving that circ-IUC1 can effectively resist the digestion of RNaseR,and has strong stability.The localization of circ-IUC1 in uninfected A549 and infected A549 cells were detected by nuclear and cytoplasmic separation assay and FISH technology,and the results showed that most of circ-IUC1 existed in cytoplasm.3.In order to explore whether circ-IUC1 can regulate IAV replication,the A549 circIUC1 overexpression stable cell line was constructed through the lentiviral expression system.Circ-IUC1 was up-regulated by 12 times in the overexpression cell line.The A549 overexpression stable cell line and the control cell line were infected with IAV,and the expression levels of M1 and NP in the cells were measured and the supernatant virus titers were detected.The results showed that overexpression of circ-IUC1 can significantly inhibit the expression levels of IAV protein M1,NP and the virus titers in supernatants.Meanwhile,the A549 circ-IUC1 knockdown stable cell lines were also constructed through the lentiviral expression system.The expression of circ-IUC1 in the knockdown cell lines was significantly reduced,while VAMP3 mRNA was not affected.The A549 knockdown stable cell lines and the control cell line were infected to detect the expression levels of M1 and NP of IAV and virus titers in the supernatant.The results showed that knocking down circ-IUC1 can significantly promote the expression levels of M1 and NP and the virus titers in supernatant.The above results show that circ-IUC1 can significantly inhibit the replication of IAV4.In order to explore the specific mechanism of circ-IUC1 regulating IAV replication,we enriched the proteins bound by circ-IUC1 in infected cells by RNA pull down technology and performed mass spectrometry detection.It was found that circ-IUC1 may interact with the IAV protein NP post infection.The interaction was then verified by RIP and RNA pull down.In order to determine whether the binding of circ-IUC1 to NP needs to be mediated by other IAV proteins,we transfected FLAG-NP in 293T cells to detect the interaction of circ-IUC1 to NP,and the results showed that in the absence of other IAV proteins,circ-IUC1 can still interact with NP significantly.The above results demonstrate that circ-IUC1 can interact with NP in infected cells,and the interaction between the circ-IUC1 and NP is not mediated by other IAV proteins.5.In view of the key role of NP protein in vRNP complex,the effect of circ-IUC1 on the activity of vRNP complex was detected in 293T cells and 293T-IAV-luc cell line.It was found that overexpression of circ-IUC1 had a significant inhibitory effect on the relative luciferase activity of vRNP complex.In addition,we found that the vRNA,cRNA,and mRNA levels of M1 and NP were significantly reduced after overexpression of circ-IUC1 in 293 T cell lines,indicating that circ-IUC1 can interfere with the transcription and replication of IAV by inhibiting the activity of vRNP complex.6.In order to explore the mechanism of circ-IUC1 regulating the activity of vRNP complex,we first detected the effect of circ-IUC1 on NP oligomerization.The oligomerization level of NP was detected by co-IP in 293T cells and it was found that after overexpression of circ-IUC1,the binding of FLAG-NP and myc-NP in cells was not affected.We further confirmed the effect of circ-IUC1 on the interaction between NP and RdRP complex proteins PB1 and PB2.The results showed that the interaction between NP and PB1 and PB2 were both significantly weakened after overexpression of circ-IUC1 in 293T cell lines,which proved that circ-IUC1 inhibited the activity of vRNP complex by interfering with the interaction between NP and PB1 as well as PB2.Conclusion:1.The expression level of circ-IUC1 increased significantly when A549 cells were infected with WSN,CA04 and PR8.Circ-IUC1 can be induced when the IFN-β signaling pathway is activated.2.Circ-IUC1 can resist RNaseR digestion and shows relatively strong stability.CircIUC1 was mainly located in the cytoplasm no matter whether A549 cells were infected by IAV.3.Circ-IUC1 can significantly inhibit IAV replication.4.During IAV infection,circ-IUC1 has a significant interaction with NP.From the perspective of mechanism,circ-IUC1 does not affect NP oligomerization,but interferes with the interaction of NP with PB1 and PB2 by binding to NP,thereby inhibiting the activity of vRNP complex and the production of cRNA,vRNA and mRNA,then inhibits IAV replication. |
