Fig. 4.

cCyAla3,4 inhibits early events of the HIV-1 replication cycle. A) The cyclic peptide cCyAla3,4, but not its linear version, inhibits HIV-1 replication. HeLa-CD4 LTR-LacZ cells were infected with the HIV-1 X4 tropic NL4-3 isolate in presence of the indicated compounds and concentrations. Beta-galactosidase activity was quantified 72 h later. scr: scr cCyAla3,4; NA: not applicable. B) cCyAla3,4 inhibits HIV-1 p24 capsid production. HeLa-CD4 cells were infected with NL4-3 for 5 h. Cells were washed three times and fresh media added with the indicated compounds. Capsid p24 in the supernatant was assessed 72 h later by p24 ELISA. Efav: Efavirenz. Error bars in A) and B) represent standard deviation of three independent experiments done in duplicate (n = 3). C) cCyAla3,4 inhibits viral mRNA expression. Experiment done as in B). Total RNA was extracted 96 h post infection and analyzed by quantitative RT-qPCR. Total HIV mRNA normalized to GAPDH and to 100% DMSO control. Error bars represent standard deviation of two independent experiments done in duplicate (n = 2). D) cCyAla3,4 inhibits acute R5 tropic HIV-1 replication. GHOST cells were infected with the HIV-1 R5 tropic YU2 isolate for 24 h in presence of the indicated compounds and concentrations. Cells were washed and fresh media with compounds added. p24 ELISA was performed 72 h later. Ralt: Raltegravir. Error bars represent standard deviation of four independent experiments (n = 4). E) cCyAla3,4 inhibits an early event hindering proviral integration. HeLa-CD4 cells were infected in presence of the indicated compounds. gDNA was extracted 18 h later and integration events were determined by Alu-PCR followed by nested quantitative qPCR. HI: Heat inactivated. Error bars represent standard deviation of at least three independent experiments done in duplicate (n = 4 except cCyAla3,4 scr n = 3). F) cCyAla3,4 does not inhibit Rev-RRE/CRM1 dependent viral mRNA export. HeLa-CD4 cells were infected for 48 h. Cells were washed and compounds added for 24 h. After this period of time, cytoplasmic and nuclear RNA were extracted and analyzed by quantitative RT-qPCR using primers recognizing total HIV mRNA, multiply spliced (Tat, Rev, Nef), singly spliced (Env) or unspliced mRNA (GagPol). All mRNA normalized to GAPDH. The ratio nuc/cyt RNA represents the fold enrichment (accumulation) of viral mRNA in the nucleus with the DMSO control set to 1. KPT-330 is a selective CRM1-mediated nuclear mRNA export inhibitor. Error bars represent standard deviation of two independent experiments done in duplicate (n = 2).