Previous studies have shown that human defensins inhibit infection of various viruses, including HIV (reviewed in Ref. 31
). In this study we provide evidence that HD5 and HD6 can promote HIV infection in various experimental settings. HD5 and HD6 enhanced HIV infection at the step of viral entry, and these peptides promoted R5 virus, the predominant strain transmitted, to a greater extent than X4 virus. In light of the report demonstrating elevation of HD5 proteins by 10- to 30-fold in urethral fluid from men with STIs (29
), we hypothesized that STIs may induce expression of HD5 and HD6 and, in turn, could enhance vaginal transmission of HIV. Indeed, we found that GC infection of cervicovaginal epithelial cells induced HD5 and HD6 expression (). Importantly, conditioned medium from GC-exposed epithelial cells contained HD5 and promoted HIV infection (). Furthermore, introduction of siRNAs targeting HD5 or HD6 blocked GC-mediated enhanced HIV infectivity. Because HD5 is normally present at a concentration of 1 μ
g/ml in vaginal fluid (52
), an increase of HD5 and HD6 levels to 10 μ
g/ml, which is sufficient to enhance HIV infection in vitro, could promote HIV transmission in women with STIs.
Our findings are in contrast to those reported by Tanabe et al. (54
), who demonstrated that recombinant HD5 had no effect on HIV replication in the transformed T cell line MT-2. This discrepancy is most likely due to the choice of target cells, as has been demonstrated with HNP1-mediated inhibition of HIV (34
). Importantly, we observed the enhancement of HIV in the primary target cells, CD4+
T cells. Additionally, we incubated defensins and virus in PBS rather than water, which may also account for the discrepancy, as the effect of HBDs on the HIV virion only occurs under a low salt conditions (10 mM phosphate buffer) (32
). Interestingly, Tanabe et al. did found that cryptdin3, a mouse enteric defensin ortholog, enhanced HIV infection (54
). The effects of defensins on virus replication, particularly HIV, are quite complex and defensin specific.
Our results suggest that interactions with HIV glycoproteins may play a role in defensin-mediated enhancement of HIV infectivity. The maximal HIV-enhancing effect of HD5 and HD6 was achieved when HIV was preincubated with defensins. In addition, the promoting effect of HD5 and HD6 was more pronounced on R5 virus compared with X4 virus. It is possible that the relatively modest enhancing effect of defensins on X4 viruses was due to the high positive charge of X4 gp120 proteins, which may inhibit the interactions with the positively charged defensins. As HD5 and HD6 appeared to enhance HIV entry, we are currently dissecting specific steps of the early HIV life cycle, including attachment and fusion modulated by these peptides, as well as determining the specific region(s) of HIV envelopes that interact with HD5 and HD6 and the influence of HIV gp120 charge to these interactions and to the HIV-enhancing effect.
Polycationic polymers, such as Polybrene, have been used to enhance the infection of HIV and other retroviruses (56
). It is possible that defensins act like polycationic polymers to promote HIV infection as they are positively charged. The viral enhancement of Polybrene is thought to be mediated through increased viral adsorption as a consequence of decreased repulsive forces between virions and cells, both of which contain negatively charged lipid membranes. Davis et al. have shown that enhancement of retrovirus transduction by polybrene is receptor and envelope independent (59
). In addition to a neutralizing membrane charge, positive charge polymers >15 kDa in size can promote infection through virus aggregation (58
). We demonstrated that HD5 and HD6 increased HIV infection in a glycoprotein-dependent and HIV receptor-independent manner. Furthermore, HD5 and HD6 linear analogues with the same charge as their structured counterparts failed to promote HIV infection (). These results suggest that neutralizing negative charges on viral envelopes or target cell membranes cannot fully account for defensin-mediated HIV enhancement, although charge may be important in the context of proper conformation. It remains to be determined whether proper formation of hydrophobic domains facilitates membrane fusion upon viral entry.
HD5 and HD6 have been shown to exhibit antiviral activity against other sexually transmitted viruses such as human papillomavirus (a nonenveloped virus) and HSV-2 (64
), although their antiviral mechanisms are distinct. HD5 does not affect binding and endocytosis of human papillomavirus but prevents virion release from the endosome (64
). HD5 and HD6 appear to block HSV-2 infection via different mechanisms (65
). Although HD6 inhibits HSV-2 attachment and penetration, HD5 blocks later stages of the HSV-2 life cycle. HD6 binds to heparan sulfate, required for HSV-2 attachment on the target cells, whereas HD5 binds to HSV-2 gB, which is essential for viral penetration but does not bind to heparan sulfate. These interactions are quite specific and distinct for each defensin.
We observed differential HIV enhancing effects of HD5 and HD6 between HeLa-CD4-CCR5 and primary CD4+
T cells, implicating factors on the target cell that influence the effect. The levels of glycosaminoglycans and ICAM vary between adherent cells (HeLa cells) and suspension cells (CD4+
T cells) and can modulate HIV attachment (reviewed in Ref. 66
). It is possible that the defensin-mediated effect alters HIV envelope interactions with glycosaminoglycans, including heparan sulfate and chondroitin sulfate on the cell surface, as glycosaminoglycans influence HIV attachment in an envelope-dependent and coreceptor-independent manner (56
In response to an invasion of pathogens, HBDs are known to be induced through TLR2 and TLR4 activation as well as via proinflammatory cytokines such as IL-1 (23
). Activation of TLR2 or TLR4 induces HBD2 in immortalized vaginal epithelial cells via the NF-κ
B pathway (68
). Although it has been suggested that TNF-α
and LPS can induce HD5 (52
), the HD5 gene contains an IFN response element in the promoter region (29
) and can be induced in response to IFN-γ in HeLa cells (E. Porter, unpublished data). With respect to induction of HD5 and HD6 by GC infection, it remains to be determined whether TLR activation via bacterial LOS or porins directly modulates expression of HD5 and HD6 or via the production of cytokines such as IFNs and TNF-α
Gonococcal pili and colony Opa proteins play a role in GC adherence to and invasion of epithelial cells (69
), although GC can invade epithelial cells via a LOS-dependent and Opa-independent manner (72
). Fichorova et al. have demonstrated that both piliated and nonpiliated gonococci can induce proinflammatory host cytokine responses despite their different abilities to invade epithelial cells (19
). It remains to be determined whether the presence of pili or Opa affects GC-mediated induction of defensin gene expression and enhanced HIV infectivity as we did not characterize the expression of pili and Opa by the gonococcal strain used in these studies.
HD5 is primarily secreted as precursor molecules in the lumen of the normal small intestine and female vaginal tract and the inflamed male urethra (29
). In contrast to HNPs that are processed intracellularly in neutrophils (23
), HD5 is cleaved upon secretion by trypsin in the small intestine and neutrophil protease in inflamed penile urethra (29
). We observed that cervicovaginal epithelial cells produced HD5 precursor molecules in response to GC infection and that pro-HD5 enhanced HIV infection at similar molar levels as HD5. The concentration of HD5 precursor molecules in the conditioned medium from GC-exposed vaginal epithelial cells that enhanced HIV infection was ~1 μ
g/ml by Western blot analysis using synthetic HD5 as a standard, ~10-fold less than that required to enhance HIV infection in vitro. However, considering the much larger medium volume covering cultured epithelial cells compared with the much smaller volume of mucus covering vaginocervical cells in vivo, it is likely that HD5 concentrations affecting HIV infectivity are reached in vivo. Furthermore, native HD5 appears to be in part glycosylated (53
), affecting its antibacterial activity, and it needs to be determined whether vaginocervical cells release glycosylated HD5 with stronger HIV-inducing activity. Venkataraman et al. demonstrated that the anti-HIV activity of cation-depleted vaginal fluid can be fully restored by adding back the cationic polypeptide fraction, but the activity is only restored partially by using a mixture of different recombinant cationic polypeptides (76
). It is also likely that native HD5 proteins at a lower concentration could promote HIV infectivity by interacting with other proteins in conditioned medium from GC-exposed epithelial cells. In support of these possibilities, introduction of siRNAs targeting HD5 and HD6 blocked the HIV-enhancing effect of the conditioned medium from GC-exposed cells, demonstrating that HD5 and HD6 play a direct role in GC-mediated enhanced HIV infectivity.
Induction of HNPs and HD5 has been reported in the male urethra during C. trachomatis
and N. gonorrhoeae
). Although neutrophil infiltration accompanied by elevation of HNPs is the major vaginal immune response to most STIs (27
), induction of other defensins such as HBDs, HD5, and HD6 in women with C. trachomatis
and N. gonorrhoeae
infection has not been reported. N. gonorrhoeae
is resistant to HNPs but not to HD5 and its precursor (29
). Pro-HD5, processed by neutrophil proteases in the urogenital mucosa in men with STIs, also exerts antibacterial activity against N. gonorrhoeae
). It remains to be determined whether HD5 and HD6 are elevated in women with C. trachomatis
and N. gonorrhoeae
infection and whether elevation of HD5 and HD6 by GC results in controlling bacterial growth but at the same time enhancing HIV infection in vivo. Furthermore, as GC can induce anti-HIV factors such as HBDs, RANTES, MIP-1α
, and MIP-1β
, which may moderate the HIV enhancing effect of HD5, the interaction between these anti-HIV factors and HD5/HD6 and their net effect on HIV infection may be complex and dynamic. Thus, further studies are warranted to determine how different defensins inhibit or enhance HIV entry as well as affect the overall outcome of HIV infection in the presence of various peptides at physiologic concentrations.
In summary, we report a novel mechanism by which STIs, specifically GC, could enhance HIV transmission through up-regulation of the defensins HD5 and HD6. In addition to promoting HIV entry, HD5 can induce cytokines such as IL-8 (50
) that may increase HIV transmission within the cervicovaginal mucosa (82
). Understanding the mechanism of HD5- and HD6-mediated HIV enhancement and the complex contribution of these host factors to transmission is critically important for the development of new strategies for HIV prevention, particularly those that target or alter the vaginal mucosa.