Dual role of α-defensin-1 in anti–HIV-1 innate immunity (original) (raw)
Effect of α-defensin-1 on HIV-1 virions. Our previous results suggest that direct inactivation of virions is not required for HIV-1 inhibition by α-defensin-1. We examined whether α-defensin-1 at low, noncytotoxic but physiologic concentrations can directly inactivate HIV-1 virions. The direct effect of α-defensin-1 on HIV-1 virions was examined by incubation of replication-competent HIV-1IIIB at an MOI of 0.01 or 0.1 with α-defensin-1 at initial concentrations of 0, 1, 5, and 20 μg/ml at 37°C for 1 hour. Samples were then diluted 100-fold in complete media to a final MOI of 0.0001 or 0.001 before infection of primary CD4+ T cells. These final concentrations of α-defensin-1 had no postentry effect on HIV. HIV-1 virus particles released into media were measured by HIV p24 assay. Because serum can block the direct effect of α-defensin-1 on virions (16, 36), we also examined whether the presence of serum affected the direct inactivation of HIV-1 virions. α-Defensin-1 at a concentration as low as 1 μg/ml displayed a direct inhibitory effect on the viruses at a low MOI in the absence of serum, whereas the inhibitory effect was abolished in the presence of serum (Figure 1A). Moreover, the inhibitory effect on HIV virions was abolished by a 10-fold increase in virus particles to an MOI of 0.1 in the absence of serum (Figure 1B). Increasing concentrations of α-defensin-1 up to 20 μg/ml did not restore its inhibitory effect. We further demonstrated that α-defensin-1 had a direct effect on the R5 HIV-1 virion using a single-cycle infection assay. Replication-defective recombinant HIV-1JR-FL–pseudotyped virus containing a luciferase reporter gene was produced in media without serum. Viruses were incubated with α-defensin-1 at concentrations of 0, 1, 10, and 20 μg/ml in the absence or presence of serum at 37°C for 1 hour and then diluted 100-fold with complete media before infection of HeLa-CD4 cells expressing CCR5 coreceptors. In the absence of serum, α-defensin-1 had a direct effect on HIV-1JR-FL–pseudotyped virus, and the inhibitory effect was abolished by serum (Figure 1C). No inhibition was observed when α-defensin-1 at 0.01, 0.1, and 0.2 μg/ml, the final concentrations present in samples after 100-fold dilution, was added during viral inoculation for 2 hours followed by wash out (data not shown). Therefore, the presence of residual low concentrations of α-defensin-1 during viral inoculation did not account for the inhibitory effect.
Effect of α-defensin-1 on HIV-1 virions. (A and B) HIV-1IIIB virions at MOI 0.01 (A) or 0.1 (B) were incubated with α-defensin-1 at concentrations of 0, 1, 5, and 20 μg/ml in the absence or presence of serum at 37–C for 1 hour. The mixtures were then diluted 100-fold before addition to primary CD4+ T cells (5 × 105 per sample). The levels of HIV-1 p24 at day 10 after infection are shown. P < 0.05, α-defensin-1–treated samples at MOI 0.01 vs. nontreated controls, α-defensin-1–treated samples at MOI 0.1 vs. nontreated controls, calculated by Student’s t test. (C) HIV-1JR-FL–pseudotyped replication-defective luciferase virus was incubated with α-defensin-1 at concentrations of 0, 1, 10, and 20 μg/ml in the absence or presence of serum at 37–C for 1 hour. The samples were then diluted 100-fold before infection of HeLa-CD4 cells expressing CCR5 coreceptors. After 2 hours of incubation, cells were washed with PBS and incubated in the complete media for 48 hours before luciferase activity was measured (in RLUs). P < 0.05, α-defensin-1–treated virus vs. nontreated controls. Data are mean ± SD of triplicate samples and represent 3 independent experiments.
α_-Defensin-1 inhibits X4 and R5 primary isolates following viral entry_. We have previously shown that α-denfesin-1 inhibits HIV infection after viral entry. This was shown using replication-defective, HIVHxB2- or HIVVSV-pseudotyped luciferase-expressing viruses as well as replication-component HIVBaL (15). To determine whether α-defensin-1 can inhibit HIV primary isolates at a postentry level, primary CD4+ T cells were infected with HIV X4, R5, or X4R5 primary isolates at 37°C for 2 hours. Infected cells were then treated with α-defensin-1 at different concentrations, and virus production was measured by quantitation of HIV p24 antigens. As a control, cells were also infected with the laboratory-adapted strain HIVIIIB. As expected, HIVIIIB replication was inhibited when cells were treated with α-defensin-1 after viral infection (Figure 2D). More importantly, α-defensin-1 inhibited different subtypes of X4, R5, and dual-tropic HIV primary isolates following viral entry (Figure 2, A–C).
α-Defensin-1 inhibited HIV-1 primary isolates following viral entry. (A–C) Primary CD4+ T cells were infected with HIV-1 primary isolates using R5, X4, or X4R5 coreceptors. The names and tropisms of virus isolates are indicated, and viral genotypes are shown in parentheses. (D) As a control, cells were also infected with a laboratory-adapted strain, HIVIIIB. After a 2-hour viral inoculation, cells were washed and treated with α-defensin-1 at different concentrations. HIV-1 production was measured, and the levels of HIV-1 p24 at day 10 after infection are shown. P < 0.05, α-defensin-1–treated infected cells vs. nontreated controls. Data are mean ± SD of triplicate samples and represent 2 independent experiments.
HIV-1 inhibition in α-defensin-1–pretreated CD4+ cells is not due to downregulation of CD4, CCR5, or CXCR4 surface expression. We have previously observed that pretreatment of HeLa-CD4 cells with α-defensin-1 blocks HIV-1 infection. To analyze whether pretreatment of CD4+ T cells with α-defensin-1 would also lead to HIV-1 inhibition during a single life cycle, cells were treated with α-defensin-1 at 5 μg/ml for 16 hours, washed, and cultured in media without or with the inhibitor during HIV-1 infection. In agreement with our previous finding, HIV-1 infection was inhibited by 70% in cells pretreated with α-defensin-1 even though the inhibitor was washed out before infection (Figure 3A). This result indicated that in the presence of serum α-defensin-1 had an antiviral effect on the target cell and that this effect persisted after wash-out of α-defensin-1.
HIV-1 inhibition in α-defensin-1–pretreated primary CD4+ T cells and the effect of α-defensin-1 on CD4+ T cell proliferation and CD4, CCR5, and CXCR4 surface expression. (A) CD4+ T cells were pretreated with α-defensin-1 at 5 μg/ml for 16 hours. Cells were washed and infected with HIV-1VSV–pseudotyped replication-defective luciferase virus. Infected cells were then placed in complete media with (Add back) or without (Wash out) α-defensin-1 during viral infection. P < 0.05, α-defensin-1–treated cells vs. nontreated controls. Luciferase activity was measured at 48 hours after infection. Data are mean ± SD of triplicate samples and represent 2 independent experiments. (**B**) Cell viability of activated CD4+ T cells incubated with α-defensin-1 at different concentrations for 48 hours was measured by CellTiter 96 aqueous 1-solution cell proliferation assay (Promega Corp.). No significant difference was observed in cells in the absence or presence of α-defensin-1 (_P_ > 0.05). (C) Activated CD4+ T cells were treated without (white bars) or with (black bars) α-defensin-1 at 5 μg/ml for 16 hours. Surface expression of CD4, CXCR4, and CCR5 in activated CD4 T cells was determined by flow cytometry. No significant difference was observed between α-defensin-1–treated cells and controls (P > 0.05). Results are mean ± SD of 2 independent experiments.
To ensure that the effect of α-defensin-1 on cells was not due to cytotoxicity, cell proliferation assay was performed. Activated CD4+ T cells were treated with α-defensin-1 at different concentrations for 48 hours, and cell proliferation was determined. α-Defensin-1 had no effect on CD4+ T cell proliferation up to 10 μg/ml for 48 hours (Figure 3B).
CD4, CXCR4, and CCR5 receptors are required for productive HIV infection of primary T cells (reviewed in ref. 37). A recent report shows that human β-defensin-2 downregulates cell surface CXCR4 but not CCR5 in unstimulated PBMCs in the absence of serum (24). We examined whether pretreatment of cells with α-defensin-1 altered the expression of these receptors, subsequently leading to HIV-1 inhibition. The effect of α-defensin-1 on expression of CD4, CXCR4, and CCR5 was analyzed by FACS analysis. Activated primary CD4+ T cells were incubated without or with α-defensin-1 at 5 μg/ml for 24 hours in complete media containing 10% FBS, and the expression of receptors on cell surfaces was determined. An isotype antibody was included as a control. α-Defensin-1 had no effect on expression of CD4, CXCR4, and CCR5 receptors (Figure 3C). Examination of the effect of α-defensin-1 on CD4 and CXCR4 expression in CD4+ T cells was also performed in the absence of serum. Activated primary CD4+ T cells were incubated in a serum-free medium, AIM-V, overnight before treatment with α-defensin-1. Cell surface CD4 and CXCR4 were analyzed by FACS analysis. Although expression of CXCR4 was downregulated in the serum-free condition, no significant change was observed in both CD4 and CXCR4 expression in cells treated with α-defensin-1 (data not shown). These results show that HIV-1 inhibition by α-defensin-1 pretreatment of CD4+ T cells was not due to downregulation of CD4, CXCR4, or CCR5 on cell surfaces.
α-Defensin-1 inhibits HIV-1 infection following reverse transcription and integration. To dissect the stages of HIV-1 infection inhibited by α-defensin-1 after viral entry, we studied the kinetics of the HIV-1 life cycle in primary CD4+ T cells in the presence of α-defensin-1 using a single-cycle viral infection assay. Activated CD4+ T cells were infected with replication-defective recombinant HIV-1HxB2–pseudotyped virus containing a luciferase reporter gene. Infected cells were treated with α-defensin-1 at 2, 6, 9, and 16 hours after infection as indicated in Figure 4A. Samples treated with α-defensin-1 were compared with those treated with the reverse transcriptase inhibitor azidothymidine (AZT) at 5 μM. AZT or α-defensin-1 inhibited HIV-1 infection by 99% or 80%, respectively, when inhibitors were added at 2 hours after infection. AZT lost its inhibitory effect at 9 hours after infection, indicating that reverse transcription was complete, whereas the inhibitory effect of α-defensin-1 was sustained at this time point. The difference between levels of HIV infection in α-defensin-1–treated and AZT-treated cells at 9 and 16 hours was significant (P < 0.05, calculated by Student’s t test). This result suggests that the block in the HIV-1 life cycle by α-defensin-1 occurred after reverse transcription in primary CD4+ T cells.
α-Defensin-1 inhibited HIV-1 infection following reverse transcription and integration. Activated CD4+ T cells infected with HIV-1HxB2–pseudotyped replication-defective luciferase virus were treated with α-defensin-1 at 5 μg/ml, or with AZT at 5 μM (A) or L-731,988 at 10 μM (B), at different time points after infection. Infected cells were collected at 48 hours after infection, and luciferase activity was measured. P < 0.05, α-defensin-1–treated cells vs. nontreated controls at different time points, α-defensin-1–treated vs. AZT-treated cells at 9 and 16 hours after infection, α-defensin-1–treated vs. L-731,988–treated cells at 24 hours after infection. Data are mean ± SD of triplicate samples and represent 3 independent experiments.
The anti-HIV activity of α-defensin-1 gradually decreased in the kinetic study in Figure 4A, suggesting that α-defensin-1 may affect more than 1 step in the HIV-1 life cycle. Therefore, we compared the kinetics of the HIV-1 life cycle in the presence of α-defensin-1 or an integrase inhibitor, L-731,988 (38). L-731,988 and α-defensin-1 inhibited HIV-1 infection by 75–80%, when inhibitors were added at 0 or 2 hours after infection (Figure 4B). At 24 hours after infection, L-731,988 lost its inhibitory effect, consistent with completion of integration, whereas α-defensin-1 blocked HIV-1 replication by 50%. Taken together, these results suggest that α-defensin-1 affects more than 1 step of the HIV-1 life cycle following reverse transcription, including a postintegration effect.
PKC signaling pathway(s) is involved in α-defensin-1–mediated HIV-1 inhibition in primary CD4+ T cells. α-Defensin-1 can inhibit PKC in vitro (39). In addition, it is internalized and interacts with PKCα and β in smooth muscle cells (40). Because PKC plays an important role in HIV-1 infection (41–43), we investigated whether HIV-1 inhibition by α-defensin-1 is mediated through its effects on PKC activity. The activity of PKC is under the control of distinct serine/threonine phosphorylation (44). Therefore, we analyzed phosphorylated PKC proteins in whole-cell extracts from primary CD4+ T cells treated with α-defensin-1 using phospho-PKC–specific antibody. The blot was then stripped and reprobed with antibodies against PKCα, β, and γ as a control for equal loading. PKC phosphorylation was detected in activated CD4+ T cells without treatment (Figure 5A, lane 1). The level of PKC phosphorylation was decreased by 30% and 60% in cells treated with α-defensin-1 for 5 and 15 minutes, respectively (Figure 5A, lanes 2 and 3), indicating that α-defensin-1 inhibited PKC activity in primary CD4+ T cells.
Involvement of PKC signaling pathway(s) in α-defensin-1–mediated HIV-1 inhibition. (A) Whole-cell extracts were prepared from cells treated without or with α-defensin-1 at 10 μg/ml for 0, 5, and 15 minutes. PKC phosphorylation was analyzed using a phospho-PKC antibody. The blot was then stripped and reprobed with an antibody against PKC as a control. (B) Activated CD4+ T cells were infected with HIV-1HxB2–pseudotyped replication-defective luciferase virus. Infected cells were then treated with bryostatin 1 at 10 nM for 30 minutes, washed, and incubated without or with α-defensin-1 for 48 hours before measurement of luciferase activity. Data are mean ± SD of triplicate samples and represent 2 independent experiments. (C) Primary activated CD4+ T cells were pretreated without (lanes 1 and 2) or with (lanes 3 and 4) bryostatin 1 for 30 minutes and then not treated (lanes 1 and 3) or treated (lanes 2 and 4) with α-defensin-1 for 15 minutes. Whole-cell extracts were prepared and PKC phosphorylation was analyzed as described above.
We then examined whether enhancement of PKC activity before treatment with α-defensin-1 would affect α-defensin-1–mediated HIV-1 inhibition during a single-cycle infection. HIV-1HxB2–infected primary CD4+ T cells were incubated with a PKC activator, bryostatin 1, at 10 nM for 30 minutes before treatment with α-defensin-1 for 48 hours. Pretreatment of infected cells with bryostatin 1 reduced the HIV-inhibitory effect of α-defensin-1 from 80% to 20% (Figure 5B). In addition, α-defensin-1 had no effect on PKC phosphorylation in bryostatin 1–treated cells (Figure 5C). These results suggest that PKC signaling pathways are involved in α-defensin-1–mediated HIV-1 inhibition in primary CD4+ T cells.
α-Defensin-1 and a PKC inhibitor, Go6976, block the HIV-1 life cycle at a similar stage. To examine the role of specific PKC isoforms in HIV-1 infection in primary CD4+ T cells, we studied HIV-1 infection in the presence of a PKC isoform–selective inhibitor, Go6976, which blocks activities of PKC isoforms α and β. Primary CD4+ T cells were infected with HIV-1HxB2–pseudotyped luciferase reporter virus, treated with Go6976 at 2 hours after infection, and incubated for 48 hours before measurement of luciferase activity. Go6976 inhibited HIV-1 infection in a dose-dependent manner (Figure 6A), indicating that PKCα and β were important for HIV-1 infection in primary CD4+ T cells.
α-Defensin-1 and a PKC isoform–selective inhibitor, Go6976, blocked the HIV-1 life cycle at similar stages. (A) The effect of the PKCα and β inhibitor Go6976 on HIV-1 infection in primary CD4+ T cells was determined by a single-cycle infection assay. Cells infected with HIV-1HxB2–pseudotyped luciferase viruses were treated with Go6976 at different concentrations at 2 hours after infection. (B) The kinetics of the HIV-1 life cycle in primary CD4+ T cells in the presence of α-defensin-1 or Go6976 were studied as described in Figure 4. P < 0.05, inhibitor-treated cells vs. nontreated controls at different time points. Data are mean ± SD of triplicate samples and represent 3 independent experiments.
We then determined whether the PKC inhibitor Go6976 and α-defensin-1 blocked at a similar stage of HIV-1 infection by studying the kinetics of the HIV-1 life cycle in primary CD4+ T cells in the presence of these inhibitors. Infected cells were treated with α-defensin-1 at 5 μg/ml or Go6976 at 500 nM at 2, 6, and 16 hours after infection, and luciferase activity was measured at 48 hours after infection. Similar kinetics of HIV-1 inhibition were observed in cells treated with α-defensin-1 or Go6976, which suggests that the block in the HIV-1 life cycle may occur at the same stage(s) (Figure 6B).
To analyze whether α-defensin-1 or Go6976 also exhibited the anti–HIV-1 activity in transformed T cell lines, several transformed cell lines were infected with HIV-1HxB2–pseudotyped luciferase reporter virus and treated with α-defensin-1 or Go6976 at 2 hours after infection. Infected cells were incubated for 48 hours before measurement of luciferase activity. In contrast to the results found in primary CD4+ T cells, no effect of α-defensin-1 on HIV-1 infection was observed in transformed T cell lines including H9, CEM, and Jurkat cells. In these transformed T cells, Go6976 was found to actually enhance HIV-1 infection (data not shown). The results with Go6976, in parallel with those with α-defensin-1, suggest that signaling pathways involved with HIV infection in primary CD4+ T cells are not the same as those in transformed T cells.
Both α-defensin-1 and Go6976 inhibit HIV-1 infection at the steps of nuclear import and transcription. To confirm that Go6976 and α-defensin-1 inhibited HIV in primary CD4+ T cells following reverse transcription, real-time PCR analysis was performed to amplify HIV-1 strong-stop (R/U5) and full-length (R/gag) reverse-transcribed products. These represent early and late reverse-transcribed DNA, respectively (45). Activated primary CD4+ T cells were infected with HIV-1HxB2–pseudotyped luciferase reporter virus and then treated with α-defensin-1 at 5 μg/ml or Go6976 at 500 nM at 2 hours after infection. Genomic DNA was extracted at 48 hours after infection, and HIV reverse-transcribed DNA products were examined. There was no reduction of early and late HIV reverse-transcribed PCR products in primary CD4+ T cells in the presence of α-defensin-1 or Go6976 (Figure 7A), which suggests that the block occurred after reverse transcription.
α-Defensin-1 and Go6976 inhibited HIV-1 infection at the steps of nuclear import and transcription. (A) CD4+ T cells were infected with HIV-1HxB2–pseudotyped viruses and then treated with α-defensin-1 or Go6976 for 48 hours. Quantitation of HIV-1 early strong-stop (R/U5) and late full-length (R/gag) reverse-transcribed products was performed. Data are mean ± SD of 3 independent experiments. No significant difference was observed between control (HIV-1–infected, no treatment) and α-defensin-1–treated cells, or between control and Go6976-treated cells, calculated by Student’s t test (P > 0.05). (B) To assess whether α-defensin-1 or Go6976 suppressed HIV-1 nuclear import, real-time PCR analysis was performed to measure c2-LTR circles in CD4+ T cells infected with replication-defective HIV-1VSV–pseudotyped virus (left panel) or replication-competent HIVIIIB at MOI 0.1 (right panel) upon treatment with inhibitors at 2 hours after infection. Samples were prepared at 24 h after infection, and c2-LTR circles were measured. *P < 0.05, control (HIV-1–infected, no treatment) vs. α-defensin-1–treated, control vs. Go6976-treated, calculated by Student’s t test. Data represent 2 independent experiments. (C) To determine whether α-defensin-1 or Go6976 inhibited HIV transcription, primary CD4+ T cells were infected with HIVIIIB at MOI 0.05 for 2 hours. Cells were washed and treated with Nelfinavir at 20 μM for 48 hours before exposure to inhibitors for 3 additional days. Total RNA was analyzed by Northern blot analysis using 32P-labeled HIV-nef DNA fragment. Longer exposure of the blot is shown in the right panel. The blot was stripped and then probed with GAPDH as a control.
To determine whether the inhibitory effect on HIV-1 infection occurred at nuclear import, real-time PCR analysis of closed 2–long-terminal repeat (c2-LTR) circles was performed. Extrachromosomal closed circular forms of HIV DNA (E-DNA), which form only after nuclear import of fully reverse-transcribed linear DNA and contain either a single or a tandem double copy of the LTR (c1-LTR or c2-LTR, respectively), are considered a marker of nuclear import (46). Primary CD4+ T cells were infected with replication-defective recombinant HIV-1VSV–pseudotyped luciferase-expressing virus and then treated with α-defensin-1 or Go6976 at 2 hours after infection. Genomic DNA was prepared at 24 hours after infection, and c2-LTR circles were analyzed. α-Defensin-1 and Go6796 inhibited nuclear import by 55% and 37%, respectively (Figure 7B, left panel). Similar inhibition of c2-LTR circle formation was observed when replication-component HIVIIIB was used to infect primary CD4+ T cells in the presence of the inhibitors (Figure 7B, right panel).
The kinetic study in Figure 4B indicated that α-defensin-1 had persistent inhibition of HIV-1 even after integration was complete. To examine whether the inhibitors affected HIV transcription in primary CD4+ T cells, cells were infected with replication-competent HIVIIIB and then treated with a protease inhibitor, Nelfinavir, to prevent new rounds of viral replication. Infected cells were incubated for 48 hours to allow completion of viral integration before treatment with α-defensin-1 or Go6976. Total RNA was prepared at 5 days after infection and analyzed by Northern blot analysis using a probe from the HIV-1 nef region. The level of all major species of viral RNAs was decreased in the presence of α-defensin-1 or Go6976 (Figure 7C). Longer exposure of the blot revealed that both inhibitors suppressed the full-length unspliced 9.2-kb mRNA by 43% (Figure 7C, right panel). These results and the kinetic studies demonstrated that α-defensin-1 and Go6976, a PKCα and β inhibitor, blocked HIV-1 infection at the steps of nuclear import and transcription.