HIV-1 Nef Induces Proinflammatory State in Macrophages through Its Acidic Cluster Domain: Involvement of TNF Alpha Receptor Associated Factor 2
Figure 5
Nef-dependent activation of IRF-3 and synthesis of IFNβ requires the integrity of the Acidic Cluster.
(A) MDMs were treated with myr+ wt as well as with ΔN-Term, G2A or myr+ 4EA recNef (100 ng/ml for 30′). Total cell protein extracts (30 µg) were analyzed by Western Blot using anti-IRF3 specific antibodies. IRF-3 activation was visualized as accumulation of the slower migrating form of the protein corresponding to the iperphosphorylated form and the decrease of the faster migrating band. β-tubulin steady-steate expression level was used as an internal loading control. (B) MDMs were treated for 2 h with myr+ wt as well as with ΔN-Term, G2A or myr+ 4EA recNef (100 ng/ml, 2 h). Cell were processed, total RNA was isolated and Real Time PCR was performed as described in Material and Methods section. Results are expressed as fold of induction using the levels of IFNβ mRNA expression in untreated cells as reference. (C) Supernatants collected from MDMs treated for 2 h with twofold dilution of myr+ wt (black line, empty circles), 4EA (gray line, empty squares) or 100 ng/ml of heat-inactivated myr+ wt recNef (black triangle), were tested for the induction of the antiviral state on A549 indicator cells. Results are expressed as International Units (IU) of IFN/ ml. Results were obtained from four independent healthy donors.