EFFECT OF DEFENSINS ON HIV INFECTION: MECHANISM(S) OF ACTION
Recent studies indicate that, specific defensins can inhibit or enhance HIV infection. With respect to anti-HIV activities of defensins, these peptides have at least two mechanisms of antiviral activity.
One aspect of antiviral activity involves direct interaction with viral envelopes with possibly disruption of the envelope, similar to their antibacterial activity. This interaction, even in the absence of disruption, could interfere with viral entry. The other antiviral pathway involves indirect effects through interactions with potential target cells. These defensin-cell interactions are complex and at least in part mediated by interacting with cell surface glycoproteins and/or interfering with cell-signaling pathways that are required for viral replication. A recent report demonstrates that HD5 and HD6, induced in cervicovaginal epithelial cells in response to Neisseria gonorrhoeae infection, enhances HIV infectivity [19]. Interestingly, the enhancing effect of HD5 and HD6 is more pronounced with R5 virus compared with X4 virus, which may have clinical relevance relative to the selective transmission of R5 viruses. The enhancing effects, as discussed below may be through similar interactions with viral envelope and cell membranes. The influence of specific defensins on HIV replication is summarized in Table 1.Inhibition of HIV replication by synthetic guinea-pig, rabbit and rat à-defensins was first reported in 1993 [108], when it was shown that these peptides could inhibit HIV-1 infection in vitro following viral entry into transformed CD4+ T cells in the presence of serum [108]. It has subsequently been appreciated that HNPs1-3 block HIV infection through multiple mechanisms [26, 109-111].
Table 1: Effects of defensins on HIV infection and their mechanisms of action.
| Defensin | Effect | Mechanisms of action | Refs |
| NP1 | Inhibitory | Inactivates virion | 34,114 |
| HNPs1-2 | Inhibitory | Induce CC-chemokine production by macrophages | 102 |
| HNPs1-3 | Inhibitory | Bind to gp120 and CD4 | 117 |
| HNP2 | Inhibitory | Binds to gp120 and CD4, blocks fusion Down-regulates CD4 in the absence of serum | 112 |
| HNP1 | Inhibitory | Blocks viral nuclear import and transcription | 114 |
| HNP4 | Inhibitory | Blocks HIV infection in a lectin-independent manner | 115,117 |
| HD5, HD6 | Enhancement | Enhance viral entry | 27 |
| Cryptidin-3 | Enhancement | Not available | 120 |
| HBD1 | None | 81,121 | |
| HBD2 | Inhibitory | Blocks early reverse transcription product formation | 121 |
| HBD2, HBD3 | Inhibitory | Downregulate CXCR4 expression Bind to HIV virions | 81 |
| Retrocyclin | Inhibitory | Blocks viral entry, Binds to gp120 and CD4 | 26,63, 117,122, |
| Retrocyclin1 | Inhibitory | Blocks viral fusion | 124 |
| RTD 1-3 | Inhibitory | Binds to gp120 and CD4 | 117 |
| Guinea-ping, rabbit, rat α-defensins | Inhibitory | Block infection after viral entry | 111 |
HIV, human immunodeficiency virus; HNP, human neutrophil peptide; HBD, human β-defensin; HD5, human defensin 5; RTD, rhesus θ-defensin.
HNPsl -3 all have similar activities against HIV primary isolates [112], in contrast to their differential chemotactic activities on monocytes, where HNP3 has no effect [113]. They can inhibit HIV-1 replication by a direct interaction with the virus as well by affecting multiple steps of the HIV life cycle [26, 107, 109, 111, 114]. In the absence of serum, HNP1 can directly inactivate the virus prior to infection of a cell [107]. The influence of conditions including serum and salt on defensin activity has been well described [17, 73, 107] and clearly influence this direct virus interaction. Some defensins (e.g. HNPs but not HD5 or HD6) at high concentrations are known to cause cytotoxicity in the absence of serum, which is associated with changes in cell membrane permeability, similar to their anti-bacterial activity. This cytotoxicity is abolished by the presence of serum [115, 116]. This type of membrane effect may partially account for the antiviral effect [26] which is also lost in the presence of serum. While most defensins display potent direct antibacterial activities in conditions of low salt [78], neither a low concentration of salt nor the absence of serum are required for the chemotactic effects of defensins [89, 92].
In the presence of serum and at non-cytotoxic concentrations (low dose), HNP1 acts on primary CD4+ T cells and blocks HIV-1 infection at the steps of nuclear import and transcription by interfering with PKC signaling [107]. The post-entry inhibitory effect of HIV infection occurs in primary CD4+ T cells and macrophages but not in several transformed T-cell lines [107, 111]. In the presence of serum, HNP1 does not affect expression of cell-surface CD4 and HIV-co-receptors on primary CD4+ T cells [107], whereas HNP2 down-regulates CD4 expression in the absence of serum [109]. HNPs block HIV-mediated cell-cell fusion and the early steps of HIV infection by interacting with HIVgp120 and CD4 through their lectin-like properties [109].
In macrophages, HNPs 1 and 2 upregulate the expression of CC-chemokines, which could contribute to inhibition of HIV through competition for receptors [99]. CC-chemokines can also induce the release of HNPs from neutrophils by degranulation [61]. Both effects could play a role in vivo in an innate immune response to HIV; at the mucosal surface, HNPs might work to directly inactivate the virions in the absence of serum; however, in the presence of serum, their inhibitory effect would largely be on the infected cell.HNPs are positively charged, so direct binding to HIV virions through charge interactions may account for some of their direct inhibition of HIV virions as well as account for sensitivity to serum through competing interactions with serum proteins. Acting as lectins, HNPs1-3 bind to HIV envelope glycoprotein gp120 and to its receptor, CD4 with high affinity [114]. Binding to gp120 is strongly attenuated by serum. Interestingly, in contrast to HNPs 1-3, HNP4 acts in a lectin-independent manner and does not bind to CD4 or HIV gp120 [112, 114]. However, HNP4 inhibits HIV replication more effectively than HNPs-13 [112].
Other α-defensins, including HD5 and HD6, mouse Paneth cell cryptdin-3 and cryptdin-4, and rhesus macaque myeloid α-defensin-3 (RMAD3) and RMAD4 have been tested for their ability to block HIV infection [19, 117]. While HD5 does not exhibit any effect on X4 HIV-1lai infection of transformed CD4+ T cell lines [117], HD5 and HD6 significantly enhances infectivity of HIV-1R5 strains [19]. At high concentrations associated with cytotoxicity, RMAD4 blocks HIV replication, whereas similar to human Paneth cell defensins, cryptdin-3 enhances viral replication.
Similar to HNP1 [107], HBD2 and HBD3 have dual anti-HIV activities through direct interactions with the virus and by altering the target cell. The binding of defensins to cellular membranes and HIV virions has been demonstrated by electron microscopy, although membrane disruption is not apparent [73].
HBD2 does not affect viral fusion but inhibits the formation of early reverse transcribed HIV DNA products [118]. There are conflicting reports on the downregulation of expression of HIV co-receptors by β-defensins. Sun et al. [118] reported that HBD1 and HBD2 did not modulate cell-surface HIV co-receptor expression by primary CD4+ T cells, whereas Quinones-Mateu et al. [73] showed HBD2- and HBD3-mediated downregulation of surface CXCR4 but not CCR5 expression on peripheral blood mononuclear cells (PBMCs) at high salt conditions and in the absence of serum. Interestingly, HBD2 is constitutively expressed in the healthy adult oral mucosa but the level seems to be diminished in HIV-infected individuals [118].Retrocyclins, and RTD1, -2 and -3 act as lectins and can inhibit entry of X4 and R5 viruses, including primary isolates [18, 55, 114, 119]. Unlike α- and β-defensins, retrocyclin does not appear to directly inactivate the HIV virion [55]. Retrocyclin does however bind to HIV gp120 as well as CD4 with high affinity, which is consistent with inhibition of viral entry [55, 119]. This high-binding affinity to glycosylated gp120 and CD4 is mediated through interactions with their ^-linked and A-linked sugars [120] and is strongly reduced in the presence of serum [114]. RTD1 binds directly to the C-terminal heptad repeat of HIV envelope gp41, blocking formation of the six helix bundle required for fusion [121]. Studies on retrocyclin-1 analogues indicate that modification of this peptide can enhance its potency against HIV in vitro [122], suggesting a therapeutic potential.