Protein Scaffold and Expression Level Determine Antiviral Activity of Membrane-Anchored Antiviral Peptides

Abstract

Cell membrane-anchored (ma) antiviral peptides derived from the C-terminal heptad repeat of the HIV-1 transmembrane glycoprotein gp41 (C-peptides) and expressed from retroviral vectors were shown to efficiently inhibit HIV-1 entry into target cells. Here, we analyzed the influence of the vector backbone, the scaffold modules that anchor the peptide to the membrane and the length of the C-peptide on expression level and antiviral activity. In general, antiviral activity was determined primarily by the density of the C-peptide on the cell surface. By influencing expression levels, the scaffold elements indirectly also determined antiviral activity. Additional direct effects of the scaffold on antiviral activity were minor. At comparable expression levels, the elongated C-peptide (maC46) was found to be more potent than the shorter maC36. On the basis of these findings, a dose-response assay was established that quantifies antiviral activity relative to the expression level of the antiviral gene product. Taken together, these data demonstrate the importance of analyzing the efficacy of therapeutic genes relative to the dose of the gene product.

Mutations in gp120 Contribute to the Resistance of Human Immunodeficiency Virus Type 1 to Membrane-Anchored C-Peptide maC46

“…Mutations in gp120 and gp41 were found to contribute to maC46 resistance. C46c (original name, M87/omc) (15), and M87o (10) have been described previously. For a schematic overview of all vectors, see Fig.…”

“…The gamma-retroviral vectors C46a, C46b, C46c, C46b1, and M87o-dPRE express identical transgenes (C-peptide maC46) but differ in their expression levels. The diverse expression is achieved by differences in the vector backbone, codon optimization of the transgene, and the presence of cis elements (15). The viruses NL4-3, BaL, and BaL_V_K (original name, BaL/sel [19]) were propagated on PM-1 cells.…”

“…Determination of cell surface expression level of membrane-anchored peptides by a quantitative flow cytometry assay. Quantification of the antibodybinding sites (ABS) of the maC46-expressing cell lines was performed as described previously by Lee et al (17) with minor modifications (15,33). In short, the mean fluorescence intensity was converted into ABS using a Quantum Simply Cellular Microbead Kit (Bangs Laboratory, Fishers, IN).…”

“…The titer was normalized relative to the control cell line. For the dose-response assay, the reciprocal of the titer on the C-peptide-expressing cell line relative to the titer on the control cell line was used as a measure for the inhibitory capacity of each construct, as described previously (15,33).…”

Gene Therapy for HIV-1 Infection

“…Our group has previously developed a membraneanchored gp41-derived HIV-1 fusion inhibitor, maC46. This protein is expressed on the surface of T cells after transduction with retroviral or lentiviral vectors (Egelhofer et al, 2004;Hermann et al, 2009b;Perez et al, 2005). The protein binds to the HIV-1 gp41 heptad repeat 1 region thereby interfering with six-helix bundle formation during the viral and cellular membrane fusion process.…”