Fig. 6.

Synthetic peptides corresponding to the Na⁺,K⁺-ATPase α1 C-terminal loops inhibit Tat secretion and HIV-1 infection

(a) Peptides mixture (PMIX) of intracellular loops of the Na⁺,K⁺-ATPase α1 subunit C-terminal domain are internalized by the cells. Different amounts of an equimolar mixture of fluorescein-tagged peptides (PMIX) were added, at the indicated concentrations, to HEK293T cells cultured in OPTIMEM medium. After 4 h, cells were tryspinized, extensively washed, and analyzed by flow cytometry. The overlay plots show the cell mean fluorescence for increasing concentrations of the peptide mix.

(b) Cell treatment with PMIX peptides blocks Tat86-TK release. Cells were transfected with Tat86-TK and the ScVH16 scFv antibody and, after 36 h, washed with heparin and then treated with equimolar amounts of the three peptides (PMIX); presence of Tat86-TK and the scFv antibody in the cell culture supernatant was analyzed by western blotting after a 4 h incubation. The levels of intracellular protein expression were verified on whole cell lysates (WCL).

(c) Scheme of the procedure to assess the effect of PMIX in a single-round HIV-1 infection.

(d) Treatment with the PMIX peptides impairs HIV-1 infection in a single-round cell infection assay. Jurkat cells were infected with HIV-1_NL4.3E-R-Luc pseudotyped with the VSV-G envelope. Before infection, cells were incubated with different concentrations of the PMIX peptides at 37 °C for 1 h. Then, cells were infected with the virus for 4 h, washed and fresh medium with peptides was added; after additional 72 h, cells were harvested and luciferase expression level was measured. *: P < 0.05 and **: P < 0.01 over untreated cells, respectively.

(e) Quantification of viral integration after infection in the presence of PMIX. DNA from Jurkat cells infected as in panel (d) was analyzed for the levels of proviral integration by Alu-PCR. *: P < 0.05 and **: P < 0.01 over untreated cells, respectively.

(f) Scheme of the experiment to assess the effect of PMIX on multiple rounds of infection by wild type HIV-1.

(g) The PMIX peptides inhibit HIV-1 replication. Jurkat cells were infected with wild type HIV-1_BRU virus for 4 h in the presence of the PMIX peptides or of a control peptide (both at 5 μM). After infection, the medium containing the virus was washed and substituted with fresh medium, containing the corresponding peptide preparation. At time = 0 and, subsequently, every 3 days until the 15th day, the supernatants were tested for reverse transcriptase (RT) activity, while the infected cells were diluted 1:2 and fresh peptides were added to the culture media.

(h) Quantification of viral DNA integration after inhibition of viral replication by PMIX. DNA from Jurkat cells treated as in panel (g) was analyzed for the levels of proviral integration by Alu-PCR. *: P < 0.05 and **: P < 0.01 over untreated cells, respectively.