Triggering TLR8 and/or 7/8 blocks HIV replication after HIV entry but before integration.

<p>(A) Unspecific inhibitory effects of the compounds triggering TLR7 and/or 8 on HIV entry was examined with a cell-cell-based fusion assay, in which one cell line expresses the HIV-gp120 and an LTR-driven <i>lacZ</i> gene (HeLa SX CCR5) and the other cell line expresses the HIV receptor complex (CD4 and CCR5) and Tat (HeLa 243). Fusion of these cell lines will result in Tat driven LTR <i>lacZ</i> gene expression which we quantified histochemically by staining for β-galactosidase activity (n = 2). (B and C) Percentage of C4+ T-cells expressing CCR5 and CXCR4 was quantified by flow-cytometry two days after triggering TLR7 and/or 8 of PBMC Dashed lines indicate the matched samples. (D) Replication incompetent lentiviral viruses pseudotyped with VSV envelope which encodes a luciferase reporter gene were used to assess whether TLR7 and/or 8 triggering affects the early step in the replication cycle of HIV. Notably VSV uses a totally different entry mechanism than HIV which is highly unlikely to be affected by the TLR7 and/or 8 agonists. (E) Assessment whether TLR agonists have any effects on HIV replication at the transcriptional level or later in the HIV replication cycle. For this purpose, CD4+ T-cells were infected with HIV and spreading infection was blocked by adding the fusion inhibitor enfurvitide. These CD4+ T-cells with integrated HIV were co-cultured with either autologous PBMC devoid of CD4+ T-cells previously stimulated by TLR7 and/or 8 agonists vs unstimulated autologous PBMC devoid of CD4+ T-cells. Thus, any change in HIV output would be due to the effects on either transcription or later events in the HIV replication cycle due to TLR7 and/or 8 triggering (analysis of variance: n.s.; n = 6).