The 3-O-(3',3'-dimethylsuccinyl) derivative of betulinic acid (DSB) inhibits the assembly of virus-like particles in HIV-1 Gag precursor-expressing cells

BACKGROUND: The 3-O-(3',3'-dimethylsuccinyl) derivative of betulinic acid (DSB) blocks HIV-1 maturation by interfering with viral protease (PR) at the capsid (CA)-SP1 cleavage site, a crucial region in HIV-1 morphogenesis. METHODS: We analysed the effect of DSB on the assembly of HIV-1 Gag precursor (Pr55Gag(HIV)) into membrane-enveloped virus-like particles (VLP) in baculovirus-infected cells expressing Pr55Gag(HIV), in a cellular context devoid of viral PR. RESULTS: DSB showed a dose-dependent negative effect on VLP assembly, with an IC50 approximately 10 microM. The DSB inhibitory effect was p6-independent and was also observed for intracellular assembly of non-N-myristoylated Gag core-like particles. HIV-1 VLP assembled in the presence of DSB exhibited a lower stability of their inner cores upon membrane delipidation compared with control VLP, suggesting weaker Gag-Gag interactions. DSB also inhibited the assembly of simian immunodeficiency virus SLVmac251 VLP, although with a twofold lower efficacy (IC50 approximately 20 microM). No detectable inhibitory activity was observed for murine leukaemia virus (MLV) VLP; however, fusion of the SP1-NC-p6 domains from HIV-1 to the matrix (MA)-CA domains from MLV conferred DSB sensitivity to the chimaeric Gag precursor Pr72Gag(MLV-HIV) (IC50 = 30 microM). This observation suggested that the main DSB target on Pr55Gag was the SP1 domain, but the higher degree of DSB resistance for Pr72Gag(MLV-HIV) compared with Pr55Gag(HIV) implied that other upstream Gag region(s) might contribute to DSB reactivity. CONCLUSIONS: Sequence alignment and three-dimensional modelling by homology of the CA-SP1-NC junction in HIV-1, SLVmac251 and Pr72Gag(MLV-HIV) suggested that a higher hydrophilic character of the CA region immediately upstream to the HIV-1 CA-SP1 junction, as occurred in Pr72Gag(MLV-HIV), correlated with a lower DSB sensitivity.