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

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.

Human Alpha-Defensin HNP1 Increases HIV Traversal of the Epithelial Barrier: A Potential Role in STI-Mediated Enhancement of HIV Transmission.

Alpha-defensins, including human neutrophil peptides 1-3 (HNP1-3) and human defensin 5 (HD5), are elevated at the genital mucosa in individuals with sexually transmitted infections (STIs). The presence of STIs is associated with an increased risk of human immunodeficiency virus (HIV) transmission, suggesting there may be a role for defensins in early events of HIV transmission. HD5 has been demonstrated to contribute to STI-mediated increased HIV infectivity in vitro. HNPs exhibit anti-HIV activity in vitro. However, increased levels of HNPs have been associated with enhanced HIV acquisition and higher viral load in breast milk. This study found that HNP1, but not HD5, significantly disrupted epithelial integrity and promoted HIV traversal of epithelial barriers. Linear HNP1 with the same charges did not affect epithelial permeability, indicating that the observed effect of HNP1 on the epithelial barrier was structure dependent. These results suggest a role for HNP1 in STI-mediated enhancement of HIV transmission.

Anti-HIV activity of human defensin 5 in primary CD4+ T cells under serum-deprived conditions is a consequence of defensin-mediated cytotoxicity.

BACKGROUND: We have previously shown that human defensin 5 (HD5) promotes HIV infectivity in both primary CD4+ T cells and HeLa cells expressing CD4 and CCR5. HD5 is induced in response to sexually transmitted infections (STIs) such as Chlamydia trachomatis and Neisseria gonorrhoeae, suggesting it plays a role in STI-mediated enhancement of HIV transmission. In contrast to our findings, a recent study reports that HD5 has an anti-HIV effect in primary CD4+ T cells under serum-deprived conditions. To resolve these apparently contradictory observations, we investigated experimental parameters that might contribute to contrasting effects of HD5. RESULTS: Serum-deprived culture conditions were associated with anti-HIV activity. In contrast to the dependence of the HIV enhancing effect on HD5 structure, the anti-HIV activity in serum-deprived primary CD4+ T cells was independent of HD5 structure as the linear peptide [Abu] HD5 exhibited similar anti-HIV activity. Under serum deprived conditions, HD5 blocked CD4-receptor-independent HIV-1vsv infection before or after viral entry. We found that HD5 and its linear form induced significant cell death in primary CD4+ T cells under serum-deprived culture conditions. HD5-mediated apoptosis was observed as early as 2 h after addition of defensins to serum-deprived primary CD4+ T cells. In contrast to primary CD4+ T cells, HD5 did not induce cytotoxicity and promote HIV infectivity of HeLa-CD4-CCR5 cells under serum-deprived conditions. CONCLUSIONS: These results indicate that under serum-deprived culture conditions HD5 is toxic for primary CD4+ T cells, warranting caution in data interpretation.