OBJECTIVES: A dynamic G-quadruplex region has been previously shown to form in the long terminal repeat (LTR) promoter of the HIV-1 integrated DNA genome. Inhibition of promoter activity and antiviral effects have been observed when this region was stabilized by BRACO-19, a trisubstituted acridine derivative that binds G-quadruplexes. Here, we aimed at characterizing the antiviral mechanism of action of BRACO-19 by analysing its activity towards a broad range of HIV-1 strains, host cells and infection modes. METHODS: The antiviral activity of BRACO-19 in cell lines and primary cells infected or persistently infected by HIV-1 strains was evaluated at different times post-infection. Virucidal, viral binding, time-dependent and drug-dependent assays were performed to identify the viral target step. Circular dichroism, UV spectroscopy and a reverse transcriptase (RT) stop assay were used to assess RNA G-quadruplex folding and inhibition of RT processing. RESULTS: Thorough virological assays demonstrated that BRACO-19 acts both at the reverse transcription and the post-integration level during the virus life cycle. This behaviour was rationalized by the observation that a G-quadruplex-forming sequence identical to that of the LTR DNA is present at the 3'-end of the virus RNA genome. Biophysics and biomolecular testing showed that this region has the ability to fold into very stable G-quadruplex structures that are even more stabilized by BRACO-19, therefore inhibiting the reverse transcription process at the template level. CONCLUSIONS: Our findings strongly support the activity of BRACO-19 at the viral G-quadruplex level and therefore strengthen the use of viral G-quadruplexes as new anti-HIV-1 targets.