Interaction between HIV-1 Rev and Integrase Proteins

Amnon Hizi,Joseph Rosenbluh,Zvi Hayouka,Moshe Kotler,Aviad Levin,Shoshana Loya,Abraham Loyter,Elena Britan,Ayelet Armon-Omer,Assaf Friedler

doi:10.1074/jbc.m609864200

Amnon Hizi, Joseph Rosenbluh + Show 8 more

Open Access

https://doi.org/10.1074/jbc.m609864200

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Abstract

Human immunodeficiency virus 1 (HIV-1) Rev and integrase (IN) proteins are required within the nuclei of infected cells in the late and early phases of the viral replication cycle, respectively. Here we show using various biochemical methods, that these two proteins interact with each other in vitro and in vivo. Peptide mapping and fluorescence anisotropy showed that IN binds residues 1-30 and 49-74 of Rev. Following this observation, we identified two short Rev-derived peptides that inhibit the 3'-end processing and strand-transfer enzymatic activities of IN in vitro. The peptides bound IN in vitro, penetrated into cultured cells, and significantly inhibited HIV-1 in multinuclear activation of a galactosidase indicator (MAGI) and lymphoid cultured cells. Real time PCR analysis revealed that the inhibition of HIV-1 multiplication is due to inhibition of the catalytic activity of the viral IN. The present work describes novel anti-HIV-1 lead peptides that inhibit viral replication in cultured cells by blocking DNA integration in vivo.

Highlights

EXPERIMENTAL PROCEDURESCells—Monolayer adherent HeLa and HEK293T cells were grown in Dulbecco’s modified Eagle’s medium
A Peptide numbers are as given in the NIH AIDS Research and Reference Reagent Program. b The peptide sequences are from the HIV-1 Rev consensus B sequence
IN5 was practically unable to block IN enzymatic activity (Fig. 5c) or HIV-1 replication (Fig. 8, a–c), despite its ability to penetrate cultured cells. These results support the view that a peptide, which does not inhibit IN activity in vitro, cannot block HIV replication in vivo

Summary

EXPERIMENTAL PROCEDURES

Cells—Monolayer adherent HeLa and HEK293T cells were grown in Dulbecco’s modified Eagle’s medium. Following a 3-h incubation with protein G-agarose beads (Santa Cruz) at 4 °C, the samples were washed three times with PBS containing 1% Nonidet P-40. Following a 1-h incubation at 37 °C, 60 ␮l of a buffer containing 20 mM Tris-HCl (pH 8), 400 mM NaCl, 10 mM EDTA and salmon sperm DNA was added. This overall integrase reaction was followed by an immunosorbent assay on avidin-coated plates as described (22). Quantization of Integrated HIV-1 DNA in the Cellular Genome—Following incubation of Sup T1 cells with the indicated peptides for 2 h, the cells were infected with a HIV-1 ⌬env/VSV-G virus at a m.o.i. of 2 (as described above) for 24 h. The Me2SO-solubilized cells were transferred to a 96-well ELISA plate, and OD values were monitored at a wavelength of 570 nm (25)

RESULTS

Amino acid sequence

Hill coefficient

Peptide numbera

Inhibition of IN strand transfer

DISCUSSION