Study Of The Regulation Mechanism Of LINE-1 Retrotransposition By Viral And Cellular Factors

LINE-1 or L1,is the only autonomous transposable element in the human genome.L1 participates in genome evolution,shapes the morphology and function of genes,and is associated with aging and immunity.L1 also causes genome instability,induces genetic mutations,and is associated with tumor development and the autoimmune disease.Therefore,it is important to clarify the regulation network of L1.L1 is an endogenous activator of the innate antiviral immune response.Excessive L1 activity can lead to AGS disease,which further supports the activation of innate antiviral immunity by L1.However,the reverse effects and anti-L1 regulation by viruses have not been fully elucidated.Cells have evolved a series of restriction factors that regulate the negative effects of transposons on genomic stability via different mechanisms.However,the extensive cellular regulation of L1 requires further exploration.By investigating the influence of enterovirus,SARS-CoV-2encoded proteins,and the Neddylation pathway on L1 retrotransposition,the present study provides a comprehensive understanding of the human L1 regulation network by viral and cellular factors.Systematic research on L1 regulators revealed the following:1.Enterovirus infection restricts L1 retrotranspositionBoth EV-D68 and EV-A71 infections suppressed L1 retransposition activity.We screened the effects of different viral non-structural proteins on the activity of L1.L1 transposition activity was inhibited by 2A,3A,3C,and EV-ORF2 p.Viral protein 2A inhibited L1 activity by inhibiting L1 protein expression.Viral proteins 3A and 3C restricted ORF2p-mediated L1 reverse transcription in isolated L1 ribonucleoproteins.The newly discovered viral protein ORF2 p inhibited intracellular L1 ORF1 p accumulation.The collective findings elucidated the strict modulation of L1 during enterovirus replication and revealed the mechanisms of the regulation of L1 mobility by 2A,3A,3C,and EV-ORF2 p.The findings also presented the experimental basis for the regulation network of retrotransposon L1 by enterovirus,and implicated viral proteins as L1 inhibitors.2.SARS-CoV-2-encoded inhibitors of human L1 retrotranspositionWe screened the effects of individual SARS-CoV-2-encoded proteins on L1 mobility.Using a well-established reporter system,we found that several viral proteins restricted L1 transposition activity at different levels.In particular,Nsp1,Nsp3,Nsp5,and Nsp14 showed much higher inhibitory activity on L1.The accumulation of L1 ORF1 p proteins was strongly downregulated by Nsp1 and Nsp14 compared with that in the presence of Nsp3 and Nsp5.However,Nsp3,Nsp5,and Nsp14 specifically suppressed L1 ORF2 p reverse transcriptase activity.The data indicate that SARS-CoV-2 generates a complex network that modulates L1 mobility.3.Neddylation promotes human L1 retrotranspositonWe revealed that the post-translational modification,Neddylation,promoted L1 activity,and that the Neddylation inhibitor,Pevonedistat,was a potent suppressor of L1 activity.Neddylation E1(NAE)and E2(UBE2M)are important components of the Neddylation pathway.Inhibition of NAE and UBE2 M markedly downregulated L1 retrotransposition activity.Further molecular mechanistic studies revealed that blocking Neddylation NAE and UBE2 M did not affect L1 ORF1 p expression levels,but significantly suppressed ORF2p-mediated L1 reverse transcription in isolated L1 ribonucleoproteins.Moreover,the dominant negative Cullin family members(Cul1-DN,Cul3-DN,and Cul4A-DN)restricted L1 mobility by targeting the reverse transcription step.The data demonstrate that Neddylation is a novel cellular regulator of the L1 regulation network.Neddylation signaling may be a breakthrough for discovering more L1 regulators.The Neddylation signaling pathway could be a potential therapeutic target against L1 dysregulation.