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Identification of amino acid residues critical for the B cell growth-promoting activity of HIV-1 matrix protein p17 variants

He, Wangxiao, Mazzuca, Pietro, Yuan, Weirong, Varney, Kristen, Bugatti, Antonella, Cagnotto, Alfredo, Giagulli, Cinzia, Rusnati, Marco, Marsico, Stefania, Diomede, Luisa, Salmona, Mario, Caruso, Arnaldo, Lu, Wuyuan, Caccuri, Francesca
Biochimica et biophysica acta 2019 v.1863 no.1 pp. 13-24
B-lymphocytes, HIV infections, Human immunodeficiency virus 1, Western blotting, agar, amino acids, cell proliferation, circular dichroism spectroscopy, denaturation, disulfide bonds, epitopes, non-Hodgkin lymphoma, nuclear magnetic resonance spectroscopy, patients, protein conformation, receptors, signal transduction, surface plasmon resonance, synthetic peptides
HIV-1 matrix protein p17 variants (vp17s) detected in HIV-1-infected patients with non-Hodgkin's lymphoma (HIV-NHL) display, differently from the wild-type protein (refp17), B cell growth-promoting activity. Biophysical analysis revealed that vp17s are destabilized as compared to refp17, motivating us to explore structure-function relationships.We used: biophysical techniques (circular dichroism (CD), nuclear magnetic resonance (NMR) and thermal/GuHCL denaturation) to study protein conformation and stability; Surface plasmon resonance (SPR) to study interactions; Western blot to investigate signaling pathways; and Colony Formation and Soft Agar assays to study B cell proliferation and clonogenicity.By forcing the formation of a disulfide bridge between Cys residues at positions 57 and 87 we obtained a destabilized p17 capable of promoting B cell proliferation. This finding prompted us to dissect refp17 to identify the functional epitope. A synthetic peptide (F1) spanning from amino acid (aa) 2 to 21 was found to activate Akt and promote B cell proliferation and clonogenicity. Three positively charged aa (Arg15, Lys18 and Arg20) proved critical for sustaining the proliferative activity of both F1 and HIV-NHL-derived vp17s. Lack of any interaction of F1 with the known refp17 receptors suggests an alternate one involved in cell proliferation.The molecular reasons for the proliferative activity of vp17s, compared to refp17, relies on the exposure of a functional epitope capable of activating Akt.Our findings pave the way for identifying the receptor(s) responsible for B cell proliferation and offer new opportunities to identify novel treatment strategies in combating HIV-related NHL.