Fig 2: TLR4 Activation by Whole E. coli Induces Interferon-Independent G0 Arrest(A) pHrodo-labeled E. coli were added to MDMs for 1 h. MDMs were washed and fixed. 104 cells were recorded and analyzed. Percentage of E. coli-positive cells was determined using automated cell imaging system Hermes WiScan and ImageJ.(B) IRF3/NF-κB translocation assay. Cells were exposed to pHrodo E. coli in the presence or absence of inhibitors and 2 h later stained for IRF3/NF-κB. The percentage of cells with nuclear staining was determined (n = 3, mean ± SEM). No IRF3 or NF-κB translocation was detected in un-treated cells.(C and D) MDMs were treated with (C) TAK242 or RUXO 6 h or (D) BX795 2 h before addition of pHrodo E. coli. 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.(E and F) Relative expression levels (fold changes) of ISGs (E) and cell-cycle-associated transcripts (F). MDMs were treated with RUXO 6 h before addition of pHrodo E. coli. Cells were collected 24 h later (n = 3 donors, mean ± SEM).(G) Diagram of G0 arrest following exposure to E. coli and TLR4/TRIF activation resulting in block to HIV-1 infection.∗∗∗p ≤ 0.001; ∗∗p ≤ 0.01; ∗p ≤ 0.1, paired t test.

Fig 3: 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.