Role Of Transcription Factor JunD In Regulating HIV-1 Proviral DNA Transcription

Combined antiretroviral therapy has no scavenging effect on latently infected HIV-1 viruses in resting CD4+ T cells,macrophages,and other cells,since the latent HIV-1 virus can maintain transcriptional silencing for a long period of time.The HIV-1 latent state is closely related to the activity of transcription factors such as NF-?B and AP-1.Loss of key cellular transcription factor activity is a major reason in the inhibition of viral transcription initiation in resting CD4+ T cells.JunD,one of the members of the Activating protein-1(AP-1)family,regulates transcription of target genes by binding to the promoters of the genes.Previous studies have demonstrated that the members of AP-1family,c-Jun and JunB can bind to the AP-1 binding sites on the LTR region of different HIV-1subtypes,and play a role of activating the transcription of the latent HIV-1 proviruses.However,whether JunD can also regulate the transcription of HIV-1 proviral DNA by binding to AP-1 binding sites on LTR and further contribute to the formation of latent infection of HIV-1 as what c-Jun or JunB did remains unclear.To answer this question,in this study,a JunD knock out(KO)Jurkat cell line was constructed using CRISPR / Cas9 technique and a JunD overexpression Jurkat cell line was established based on a lentiviral vector-based deliver system.By utilizing the above cell lines,whether JunD is involved in regulating transcriptional activation of HIV-1 proviral DNA was investigated.First,HIV-1 Luc reporter pseudovirus was rescued and infected to JunD KO,overexpression and control cell lines separately.The transcription level of HIV-1 proviral DNA was tested by luciferase assay and real-time quantitive PCR(qPCR).The results showed that JunD KO significantly inhibited HIV-1 provirus DNA transcription compared with control by approximately 57.05 ± 7.80%;whereas JunD overexpression promoted HIV-1 proviral DNA transcription by 3.50 ± 0.88 folds.At the same time,the results of qPCR showed that the relative HIV-1 mRNA level in JunD KO cell lines was significantly lower than that in control cells,and was down-regulated by approximately 85.15 ± 4.90%;but the relative HIV-1 mRNA levels in Jun D overexpressing cell lines were significantly increased to 1.64 ± 0.12 times compared to the control groups.In addition,an AP-1 specific inhibitor T5224 treatment on Jun D KO cells show the contribution of JunD to the transcriptional activation of HIV-1 proviral DNA accounted for approximately 40.40 ± 8.45% of the total contribution of all AP-1 family members.Second,by using a luciferase reporter system that was construted by inserting predicted JunD binding motifs on HIV-1 LTR into the reporter vector plasmid pGL3-Luc,it was confirmed that JunD can bind to the LTR by the specific binding site TGACATC and show a promotion effect on transcription of HIV-1 provirus DNA.Consistantly,the infection ability of HIV-1 pseudovirus on Jurkat cells was sharply declined if the JunD binding motif TGACATC on LTR was mutated to AAAAAA.Third,when using SAHA to activate the JNK signaling pathway in Jurkat cells,the amount of phosphorylated JNK and phosphorylated Jun D were significantly up-regulated in a dose-dependent manner,as well as the transcription level of HIV-1 proviral DNA.However,this promoting effect ofSAHA treatment on HIV-1 provirus DNA transcription was partialy inhibited if JunD expression was downregulated in Jurkat cells.Collectively,in this study,the role of JunD in regulating HIV-1 provirus DNA transcription was evaluated.It is first time to prove that Jun D can bind to the LTR of HIV-1 via specific binding site and work as an activator in regulating the transcription of HIV-1 provirus DNA.In addition,the phosphorylation status of JunD is positively correlated with the transcriptional activation of HIV-1provirus DNA.The role of JunD in regulating HIV-1 provirus DNA transcription we found here strongly suggests that JunD may be a new potential target for intervention of latent HIV-1 transcription.It must be helpful to understand the mechanism that underlies the latent infection of HIV-1 and also for the exploration of more effective strategies for the activation and eradication of latent HIV-1 infection in vivo.