Antibody mimetic peptides have evolved as versatile tools for biomedical applications, based on their ability to interfere with protein-protein interactions. We had previously designed a functional mimic of the broadly neutralizing HIV-1 antibody b12 that recognizes the CD4 binding site of the HIV-1 envelope glycoprotein gp120. The molecular details of the interaction of a linear variant of this peptide (H1H3s) with gp120 have now been characterized through cross-linking mass spectrometry, confirming the proposed involvement of the CD4 binding site of gp120 in the interaction. In addition, a variant of the b12 mimetic peptide composed mostly of D-amino acids was shown to be stable towards proteolytic degradation, while the binding and HIV-1 neutralizing properties were largely preserved. Furthermore, a peptide variant in which aspartate residues were replaced with lysine was shown to strongly enhance infection of cells with HIV-1 and GALV glycoprotein pseudotyped viral vectors, respectively, introducing this peptide as a tool to facilitate retroviral gene transfer. Collectively, the presented results highlight the versatile potential therapeutic and gene transfer applications of H1H3s and its variants in particular, as well as antibody mimetic peptides in general.