CTCF modulates HIV infection CTCF is a CCCTC DNA binding factor with 11 zinc finger mostly known as an insulator protein. Recent studies suggest that it has nuclear organizing activity with a number of important functions in higher order chromatin structure, imprinting, RNA binding and alternative splicing, DNA virus latency and reactivation. CTCF knockdown results in global drop in the binding of silenced chromatin marker H3K27me3, indicating a role in organization of silenced chromatin. HIV is subject to transcriptional silencing by cellular repressive chromatin when it intergrates into the host genome. To determine if CTCF play a role in HIV infection, we used HIV-1ⅢB infected human leukemia T cell C8166, and examined the effect of CTCF knock down (KD) on HIV infection. We found that in CTCF KD cells, HIV CPE increased significantly compared to controls. In contrast, in overexpression treatment, HIV induction reduced significantly than CTCF empty treatment. To determine whether CTCF may control the expression of host defense genes, we examined the effect of CTCF knockdown on host antiviral genes and found that IFNβ, ISG15, IRF7, OASL, Mx2 were down regulated in KD than the control in HIV-1ⅢBinfection. But TRIM5α, CXCR4 were upregulaed in CTCF KD cells. But the expression of IFNβ, ISG15, IRF7, OASL, TRIM5α had no significant change in KD HeLa cells infected with GFP HIV. Next, we tested if CTCF acts as a classically defined restriction factor to HIV. We found that when FACS were used to measure HIV pseudovirus vector drived GFP, the induction of HIV increased two-fold than controls. But there is no significant changein viral genome copy number as measure by qRT-PCR. This result suggests that CTCF is not a restrictive factor for HIV. We then tested the hypothesis that CTCF acts to help silence HIV genome. In TZM-bl cells dived by LTR promoter, we found transcriptional luciferase increased 10% when knocked down. Andit reduced 50% when overexpressed.We found CTCF indeed binds the HIV promoter (LTR) by ChIP, and the enrichment reduced 55% in KD than the control treatment. We also found that CTCF acts at transcription level to limit RNA Pol II recruitment. When CTCF knocked down, RNA Pol II ser2P stalled the +1-+39 increased 7.49 folds than the control. In summary, we found that CTCF plays an important role in limiting HIV infection first by regulating antiviral genes in T cell and by silencing HIV LTR promoter limit RNA Pol II recruitment.