The Effect And Potential Mechanism Of LncRNA#61,#45 On Viral Replication Of H5N1 Subtype Of Avian Influenza Virus

Wild waterfowl is the primary natural host reservoir for the H5N1 avian influenza virus(AIVs).However,these viruses can occasionally infect other host species,including wild birds,terrestrial poultry and various mammals.The H5N1AIVs also pose a serious threat to human public health,mainly damage the host's respiratory system.Some severe cases also shown as typical pneumonia and subsequent acute respiratory distress syndromes(ARDS)and multiple organ dysfunction syndrome(MODS),which ultimately lead to death.Although the pathogenesis of the H5N1 subtype AIVs has been extensively studied,the molecular mechanisms responsible for the disease remain unclear.Therefore,it is particularly important to understand the pathogenesis through analyzing the intimate interactions between the virus and host.Current researches of virus-host interactions mainly focused on the protein level.However,the majority of the mammalian genomes are transcribed into non-coding RNAs(ncRNAs),including long non-coding RNAs(lncRNAs).LncRNA is a type of ncRNA with a length more than 200bp,and were previously thought to be"transcriptional noise" in the genome that did not have a biological function.However,with the development of RNA-Seq technology,more and more lncRNAs have been identified and many of its function also have been progressively revealed.It was found that the expression pattern of lncRNAs is both time-specific and species-specific.The primary sequence of lncRNAs is poorly conserved,and lncRNAs can regulate gene expression through multiple levels,including epigenetic,transcriptional and post-transcriptional modifications.Recently,lncRNAs also have been found to play an important role in viral replication and antiviral immunity,and can participate in the host innate immune response by regulating inflammatory cell differentiation,migration,activation and production of inflammatory mediators.Therefore,systematically exploring the potential role of lncRNAs in the pathogenesis of influenza viruses can lay the foundation for the development of effective antiviral strategies and provide potential targets for the prevention and control of the highly pathogenic H5N1 subtype AIVs.1.Effect of LncRNA#61 and LncRNA#45 on influenza virus replicationTo assess the potential role of lncRNAs in the pathogenesis of H5N1 subtype AIVs,using RNA-Seq,our laboratory previously compared the differential expression profiles of lung lncRNAs of a pair of model viruses(CK10 and GS10)which have similar genetic background but differ greatly in pathogenicity in mice.Two differentially expressed potentially functional lncRINAs,NONMMUT011061(LncRNA#61)and NONMMUT061245(LncRNA#45),were successfully screened out.In this study,we further systematically explored the specific role of LncRNA#61 and LncRNA#45 on the replication of H5N1 subtype AIVs.Through qRT-PCR,we first demonstrated that the expression of LncRNA#61 and LncRNA#45 were highly up-regulated by H5N1 subtype of AIVs.In an attempt to define the expression pattern of LncRNA#61 and LncRNA#45,we found that LncRNA#61 and LncRNA#45 could be significantly induced by various subtypes of IAVs,including H1N1 subtype IAVs(PR8)and H7N9 subtype AIVs(S8).The subcellular localization of LncRNA#61 and LncRNA#45 were detected by RNA-FISH,and the result showed that LncRNA#61 and LncRNA#45 were translocated from the nucleus to the cytoplasm after CK10 virus infection.Altogether,these results showed that LncRNA#61 and LncRNA#45 can be highly induced by various subtypes of IAVs and can translocate from the nucleus to the cytoplasm after H5N1 virus infection,suggesting LncRNA#61 and LncRNA#45 may play a role during influenza virus infection.To achieve an optimal infection,IAVs need to hijack the replication system in the host cell nucleus.In addition,entry of the viral ribonucleoprotein complex(RNP)into the nucleus is necessary for virus replication.In order to determine the effect of LncRNA#61 and LncRNA#45 on the replication of H5N1 subtype AIVs,LncRNA#61 and LncRNA#45 were over expressed and were further evaluated their effect on viral replication and polymerase activity.And the results showed that overexpression of LncRNA#61 and LncRNA#45 significantly inhibited viral NP protein expression,polymerase activity,viral mRNA,cRNA and vRNA synthesis and viral titers.In contrast,abolish of LncRNA#61 and LncRNA#45 expression by RNA interference favored replication of these viruses,including H1N1 subtype IAVs(PR8)and H7N9 subtype AIVs(S8),highlighting the potential broad antiviral activity of LncRNA#61 and LncRNA#45 to IAVs.In addition,we also found that LncRNA#61 and LncRNA#45 significantly inhibited virus-induced apoptosis but did not affect virus-induced cell necrosis.In summary,here we conclude that LncRNA#61 and LncRNA#45 may exert broad-spectrum anti-influenza viral effects.2.Molecular mechanisms of LncRNA#61 and LncRNA#45 against influenza viral replicationThe above results have confirmed that LncRNA#61 and LncRNA#45 have an inhibitory effect on the replication of influenza virus.To further investigate the effect of LncRNA#61 and LncRNA#45 on viral single-cycle replication,NP gene expression levels at single-cycle replication time points were detected by qRT-PCR,and the results showed that LncRNA#61 and LncRNA#45 significantly inhibited viral entry,early and mid-late viral replication stages.In order to further investigate the molecular mechanism for the antiviral effect of the LncRNA#61 and LncRNA#45 against AIVs,we then further evaluate the effect of LncRNA#61 and LncRNA#45 on RNP component protein nuclear accumulation.And the result of IFA confirmed that LncRNA#61 and LncRNA#45 significantly inhibited the nuclear accumulation ability of NP and PA proteins of CK10 virus.Furthermore,transmission electron microscopy results revealed that LncRNA#61 and LncRNA#45 had no effect on the morphological changes of the viral particles upon release.Therefore,LncRNA#61 and LncRNA#45 may inhibit viral replication by suppressing nuclear accumulation of NP and PA protein.As more and more biological functions of lncRNAs are discovered,the secondary and tertiary structures are receiving increasing attention.To further explore the key functional regions for the antiviral activity of LncRNA#61 and LncRNA#45,we then constructed a series of truncation mutants of LncRNA#61 and LncRNA#45.And the result revealed that except for LncRNA#45-MutF(580bp-730bp)and LncRNA#45-MutI(1200bp-1310bp),other regions all contribute to the antiviral activity of the LncRNA#45.As for LncRNA#61,LncRNA#61-MutB,LncRNA#61-MutC,LncRNA#61-MutE and LncRNA#61-MutI all inhibited the mRNA level of viral NP gene and polymerase activity,suggesting their role in regulating the antiviral ability of LncRNA#61.Subsequently,the antiviral mechanism of LncRNA#61 was further explored,and we found that LncRNA#61-MutA,LncRNA#61-MutB,LncRNA#61-MutC,LncRNA#61-MutD,LncRNA#61-MutE and LncRNA#61-MutG inhibited NP and PA protein nuclear accumulation.Therefore,the above findings suggest that except for LncRNA#45-MutF and LncRNA#45-MutI,all secondary structural domains contribute to the antiviral activity of LncRNA#45.Moreover,the secondary structural domains of the LncRNA#61-MutB,LncRNA#61-MutC,LncRNA#61-MutE and LncRNA#61-MutI are the key functional domains contribute to the antiviral activity of LncRNA#61.In conclusion,in this study,we found that LncRNA#61 and LncRNA#45 were highly induced by different subtypes of IAVs and exerted broad-spectrum antiviral functions.More importantly,we also demonstrated that LncRNA#61 and LncRNA#45 function as a broad-spectrum antiviral factor to inhibit influenza virus replication probably through inhibiting polymerase activity and NP and PA nuclear accumulation via multiple secondary structural domains.Further exploration of the antiviral mechanism of LncRNA#61 and LncRNA#45 will contribute to the pathogenesis of IAVs and also provides new ideas to accelerate the development of antiviral drugs.