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Human immunodeficiency virus type 1 (HIV-1) is the leading cause of acquired immunodeficiency syndrome worldwide. Since the AIDS epidemics in the early 1980s, nearly 70 million people have been infected with the virus, resulting in the deaths of about 30 million people [1]. Combined antiretroviral therapy (cART) has increased the life expectancy in people infected with HIV [2]. Despite the efforts, no effective vaccine is available yet. HIV encodes three primary genes, including gag, pol and env, and five accessory genes including vpr, vpu, vif, nef, rev and tat [3]. The Nef is a 27-34 kDa cell membrane-associated protein [4]. HIV infection progression in humans and animals has shown to be related to this protein [5]. Nef has a role in T cell signaling pathway activation [6]. On the other hand, cell-penetrating peptides (CPPs) are short peptides which can transfer their cargoes into the cells [7]. CPPs contain a large number of positive amino acids, especially lysine and arginine, which increase cell penetration [8]. Size and polarity are two essential factors that determine the mechanism for entering peptides and other molecules into the cells. Small non-polar molecules usually penetrate the cell membrane through passive diffusion [9]. Larger molecules enter the cell using two mechanisms of direct translocation and endocytosis [10]. MPG is one of the cellpenetrating peptides used to deliver DNA cargoes into the cells. Peptides such as MPG, penetratin, and CADY containing both polar and nonpolar domains, are defined as amphipathic peptides. Some of the primary amphipathic CPPs are chimeric peptides which bind to the nuclear localization sequences (NLS) via covalent bonds for effective penetration through the cell membranes. MPG is based on the NLS sequence of SV40 and HIV gp41 protein. In MPG, the hydrophobic region is separated from NLS with a linker [11]. Recently, a new cysteine-rich cationic CPP, called CyLoP-1, has been developed for the delivery of peptide and protein cargoes. CyLoP-1 was derived from the nuclear localization sequence of a snake toxin, called crotamine [12]. The presence of cysteine and tryptophan amino acids is necessary to maintain its function. Besides, the cysteine oxidation status plays an essential role in the uptake efficiency of CyLoP-1, and the disulfide-containing form plays a more active role in the CyLoP-1 uptake efficiency [13]. Introduction: The human immunodeficiency virus type 1 (HIV-1) Nef regulatory protein is known as a candidate for the design of therapeutic HIV DNA and protein vaccines. One of the limitations of these vaccines is the inability of DNA and protein to pass through the cell membrane. Various delivery systems have been developed to transfer DNA and protein into cells. Cell penetrating systems such as MPG and CyloP-1 are among delivery systems, which can deliver DNA and protein cargoes into the cells, respectively. Methods: In this study, we produced the recombinant Nef protein in Escherichia coli expression system. Then, the formation of CPP/DNA and CPP/protein nanoparticles was confirmed by agarose gel retardation, scanning electron microscope (SEM), Zetasizer and SDS-PAGE, and their stability was evaluated against nucleases and proteases. Finally, the delivery of the nanoparticles into HEK-293T cells was assessed by fluorescent microscopy, flow cytometry, and western blotting. Results: Our data confirmed the formation of stable nanoparticles through non-covalent bonds with a diameter of less than 200 nm. Moreover, the results of fluorescence microscopy, flow cytometry, and western blotting demonstrated that these CPPs could successfully deliver the Nef protein and DNA into HEK-293T cells. Conclusion: Our results indicated that the MPG and CyLoP-1 CPPs are suitable candidates for the delivery of DNA and protein cargoes into mammalian cells, respectively. J Med Microbiol Infec Dis, 2019, 7 (1-2): 37-43.

Enhancing HIV-1 Nef Penetration into Mammalian Cells as an Antigen Candidate