Key Determinants of Human α-Defensin 5 and 6 for Enhancement of HIV Infectivity

Abstract

Defensins are antimicrobial peptides important for mucosal innate immunity. They exhibit a broad spectrum of activity against bacteria, viruses, and fungi. Levels of α-defensins are elevated at the genital mucosa of individuals with sexually transmitted infections (STIs). Somewhat paradoxically, human α-defensin 5 and 6 (HD5 and HD6) promote human immunodeficiency virus (HIV) infectivity, and contribute to STI-mediated enhancement of HIV infection in vitro. Specific amino acid residues of HD5 and HD6 that are crucial for antimicrobial activities have been characterized previously; however, the key determinants of defensins responsible for enhancement of HIV infectivity are not known. Here, we have identified residues of HD5 and HD6 that are required for enhancement of HIV attachment and infection. Most of these residues are involved in hydrophobicity and self-association of defensins. Specifically, we found that mutant defensins L16A-HD5, E21me-HD5, L26A-HD5, Y27A-HD5, F2A-HD6, H27W-HD6, and F29A-HD6 significantly lost their ability to promote HIV attachment and infection. L29A mutation also reduced HIV infection-enhancing activity of HD5. Additionally, a number of mutations in charged residues variably affected the profile of HIV attachment and infectivity. One HD5 charged mutation, R28A, notably resulted in a 34–48% loss of enhanced HIV infectivity and attachment. These results indicate that defensin determinants that maintain high-ordered amphipathic structure are crucial for HIV enhancing activity. In a comparative analysis of the mutant defensins, we found that for some defensin mutants enhancement of HIV infectivity was associated with the reverse transcription step, suggesting a novel, HIV attachment-independent, mechanism of defensin-mediated HIV enhancement.

Defensins: A Double-Edged Sword in Host Immunity

“…HNP1-and HD5-promoted viral infection has also been observed with certain serotypes of HAdV as reported by 41), ( 5) inhibition of nuclear import of viral RNA (30,39), and ( 6) suppression of HIV transcription (30,39,167). Infection-promoting mechanisms include: (7) enhancing viral adhesion/attachment (25,56,57,59), and (8, 9) disrupting tight junction to promote trans-epithelial transmission of HIV (60).…”

“…More recent studies, mostly by the same research groups who demonstrated the beneficial role of defensins in controlling viral infection, unveil infection-promoting effects of defensins in HIV-1 and certain serotypes of HAdV infections (25,51,(56)(57)(58)(59)(60). Chang and colleagues reported that HD5 and HD6, induced by Neisseria gonorrhoeae infection in a cervicovaginal tissue culture system, increase HIV infectivity in a CD4-and HIV coreceptor-independent manner (25).…”

“…For human α-defensins, their hydrophobicity and selective cationicity segregated on a dimeric structure stabilized by intramolecular disulfides are critical for antimicrobial activity (11). Several mutational studies have identified the functional determinants of α-defensins in promoting viral and bacterial infections (25,56,57,59,60,(90)(91)(92). Briefly, disulfide bonding in defensins is absolutely required for their ability to enhance HIV-1 infection (25,60) and to promote Shigella adhesion and invasion (91,92); hydrophobic residues in α-defensins, i.e., Trp26 and Phe28 in HNP1, Leu16, Leu26, Tyr27 and Leu29 in HD5, and Phe2 and Phe29 in HD6, play a pivotal functional role (59,90,91); dimerization and/or oligomerization of α-defensins are functionally indispensable (59,83,91,101,102); selective cationicity, as exemplified by Arg28 in HD5, can be critical for promoting HIV and Shigella infection (59,91,92).…”

“…Several mutational studies have identified the functional determinants of α-defensins in promoting viral and bacterial infections (25,56,57,59,60,(90)(91)(92). Briefly, disulfide bonding in defensins is absolutely required for their ability to enhance HIV-1 infection (25,60) and to promote Shigella adhesion and invasion (91,92); hydrophobic residues in α-defensins, i.e., Trp26 and Phe28 in HNP1, Leu16, Leu26, Tyr27 and Leu29 in HD5, and Phe2 and Phe29 in HD6, play a pivotal functional role (59,90,91); dimerization and/or oligomerization of α-defensins are functionally indispensable (59,83,91,101,102); selective cationicity, as exemplified by Arg28 in HD5, can be critical for promoting HIV and Shigella infection (59,91,92). Obviously, although α-defensins are highly variable in amino acid sequence, their functional determinants are rather conserved, irrespective of their pathogenic and protective roles in host immunity.…”

“…Infection-inhibiting mechanisms include: (1) direct inactivation of the virus (39, 40), (2) blockade of gp120-CD4 interactions (164, 165), (3) coreceptor downregulation (41, 166), (4) inhibition of gp41-and Env-mediated viral fusion (41), (5) inhibition of nuclear import of viral RNA(30,39), and (6) suppression of HIV transcription(30,39,167). Infection-promoting mechanisms include:(7) enhancing viral adhesion/attachment(25,56,57,59), and (8, 9) disrupting tight junction to promote trans-epithelial transmission of HIV(60).…”