Figure 1.

TLR4 Activation Induces G0 Arrest, Dephosphorylates SAMHD1, and Blocks HIV-1 Infection in an Interferon-Independent Manner

(A) MDMs were treated with TAK242, BX795, and RUXO 6 h before addition of LPS. Cells were infected by vesicular stomatitis virus G protein (VSV-G)-pseudotyped HIV-1 18 h later. The percentage of infected cells was determined 48 h post-infection (n = 5, mean ± SEM). Cells from a representative donor were used for immunoblotting.

(B) A simplified diagram of TLR4 signaling in response to LPS. LPS activates both MyD88-dependent and -independent signaling pathways. BX795, an inhibitor of TBK1; RUXO (ruxolitinib), an inhibitor of JAK1/2 kinase that suppresses IFN signaling; TAK242, an inhibitor of TLR4 signaling.

(C) IRF3/NF-κB nuclear translocation assay. Cells were exposed to LPS in the absence or presence of TAK242, BX795, and RUXO, and 2 h later stained for IRF3/NF-κB. The percentage of cells with nuclear staining was determined (n = 3, mean ± SEM). Scale bars, 20 μm.

(D) Expression data of TRIF and TBK1 in MDMs, displayed as cycle threshold (Ct) values (n = 3, mean ± SEM).

(E–H) MDMs were transfected with control or TRIF, TBK1 siRNA. mRNA expression is shown as fold change relative to control (n = 3, mean ± SEM) (E). Cells from a representative donor were used for immunoblotting (F). Cells were exposed to LPS in control or KD cells, and 2 h later stained for IRF3/NF-κB. % of cells with nuclear staining was determined (n = 3, mean ± SEM) (G). MDMs transfected with control or TRIF, TBK1 siRNA were treated with LPS. Cells were infected by VSV-G-pseudotyped HIV-1 18 h later. The percentage of infected cells was determined 48 h post-infection (n = 3 donors, mean ± SEM). Cells from a representative donor were used for immunoblotting (H).

^∗∗∗p ≤ 0.001; ^∗∗p ≤ 0.01; ^∗p ≤ 0.1; ^nsp, non-significant, paired t test.