Background:  a-defensins are abundant antimicrobial peptides in polymorphonuclear leukocytes and play an important role in innate immunity. Human a-denfesin-1 is an effective inhibitor of HIV-1; however, the mechanism of its anti-HIV-1 activity is not clear. Here we examined the molecular mechanism of a-defensin-1-mediated HIV-1 inhibition.

Methods:  The effect of a-defensin-1 on HIV virions was determined by incubating HIV-1 at different multiplicity of infection (moi) with a-defensin-1 in the presence or absence of serum. The anti-HIV activity of a-defensin-1 against X4 and R5 primary isolates after viral entry was examined. We then studied the kinetics of HIV life cycle in primary CD4 T cells in the presence of a-defensin-1 in comparison with inhibitors such as zidovudine and L731 988, which block reverse transcription and integration, respectively. The effect of a-defensin-1 on PKC activity was determined by analyzing protein kinase C (PKC) phosphoylation. A PKC activator, bryostatin-1, was applied to reverse the anti-HIV activity of a-defensin-1. The effect of a PKC inhibitor Go6976 on HIV-1 replication was also studied. HIV products in cells treated with a-defensin-1 or Go6976 were analyzed.

Results:  a-defensin-1 had a direct effect on HIV-1 virions at a low moi in the absence of serum, but that the effect was abolished by serum or an increase in virus particles. In the presence of serum, a-denfensin-1 inhibited both X4 and R5 primary isolates. a-defensin-1 inhibited PKC activity in CD4 T cells. Pretreatment of infected CD4 T cells with a PKC activator partially reversed a-defensin-1-mediated HIV inhibition. A PKC isoform-selective inhibitor Go6976 blocked HIV-1 infection in a dose-dependent manner, suggesting that PKCa and b are important for HIV-1 infection in primary CD4 T cells. Furthermore, kinetic studies and analysis of HIV-1 products indicated that a-defensin-1 and Go6976 blocked the steps of nuclear import and transcription in the virus life cycle.

Conclusions:  Our studies demonstrate that in the absence of serum, a-defensin-1 may act directly on the virus but in the presence of serum, its effects are on the cell where it inhibits HIV-1 replication at the steps of nuclear import and transcription. At least one of the cellular effects associated with HIV inhibition is interference with PKC signaling in CD4 T cells. Studying the complex function of a-defensin-1 in innate immunity against HIV has implications for prevention as well as therapeutics.

Keywords: alpha-defensin; anti-HIV innate immunity; cell signaling