Function Of Host Cell Factors HCF-1 And HCF-2 In HIV-1 Transcription Elongation

In eukaryotic cells,the gene transcription consists of different stages,including initiation,elongation and termination.During most genes transcription,RNA polymerase Ⅱ pauses near the transcription start site(TSS).Positive transcription elongation factor b(P-TEFb)antagonizes the pausing through phosphorylating the CTD of RNA polymerase Ⅱ and negative transcription factors DSIF and NELF.HIV-1 virus employs the transcriptional system in host cells to regulate viral DNA transcription.HIV-1 viral protein Tat protein binds with different transcription factors and co-factors to form super elongation complexes(SEC).There are two important transcription factors in SEC complex:P-TEFb and elongation factor ELL2.Host cell factorl(HCF-1)binds and recruits different transcription initiation factors to herpes simplex virus(HSV)Immediate Early(IE)gene promoter in order to regulate HSV gene transcription initiation.Recently,through endogenous HCF-1 IP and MS,Alfonso-Dunn R group found that HCF-1 can also regulate HSV IE genes transcription elongation by binding with several elongation complexes including SECs.But the mechanism is unclear,and whether HCF-1 can regulate HIV-1 transcription elongation is still unknown.This project found that both two host cell factors(HCFs,including HCF-1 and HCF-2)activate HIV-1 transcription through stabilizing ELL2.The main research contents are summarized as below:(1)Over-expression and knockdown HCF-1/HCF-2 in different HIV-1 transcription cell models suggest that HCF-1 and HCF-2 activate Tat dependent HIV-1 transcription elongation.(2)The protein levels of three important transcription elongation factors(ELL1/ELL2 and CDK9)were checked in HCF-1 KD and HCF-2 KD cells.The ELL2 protein level decreased dramatically after HCF-1/2 KD.Besides,the half life of ELL2 was prolonged by HCF-1 or HCF-2.So HCF-1 and HCF-2 increase the stability of ELL2.(3)The whole cell lysates after Ub-His/HCF-1/HCF-2 and ELL2 or Siahl over-expression were objected to in vivo ubiquitination assay,the results showed HCF-1 and HCF-2 inhibit Siahl E3 ligase activity.(4)According to the structures of HCF-1 and HCF-2,the N-and C-terminal of HCF-1 and HCF-2 share high identity.So the N-terminal/C-terminal and middle region of HCF-1/2 were co-expressed with Siah1.Co-IP results suggest that the N-teminal of HCF-1 or HCF-2 binds with the SBD(substrate binding domain)of Siahl,thus stabilizing ELL2 and other substrates of Siah1.(5)AFF4 was knockout in HCF-1/2 KD cells using CRISPR-Cas9 system.After AFF4 KO in HCF-1 KD or HCF-2 KD cells,the ELL2 protein level decreased further.This indicates that HCF-1 and HCF-2 regulate HIV-1 transcription in AFF4 independent manner.Collectively,this project demonstrates the mechanism of HCFs in HIV-1 transcription regulation.This provides an significant molecular function of HCFs in regulating HIV-1 and some other genes transcription elongation.HIV-1 virus employs the transcription system in host cells for its viral DNA transcription elongation,so the mechanism of HCFs can be a potential target for HIV-1 therapy.Especially,the function of Siah1 were identified and studied in cancer cells,and some Siahl inhibitors such as Menadione were used for cancer therapy and clinical study.So the discovery and mechanism that HCFs regulate Siah1-dependent protein ubiquitination and degradation provide a new direction for novel drugs design to cure Siah1 related cancers.