Metalloprotease TRABD2A Restriction of HIV-1 Production in Monocyte-Derived Dendritic Cells

Editor: The target cells of HIV-1 are CD4⁺ T lymphocytes and, to a less extent, dendritic cells (DCs) and macrophages. ¹ DCs express the HIV-1 receptor CD4 and are potential target cells for HIV-1. HIV-1 could infect myeloid DCs both in vivo and in vitro, ¹ although this infection is inefficient compared with stimulated CD4⁺ T cells. The HIV-2/simian immunodeficiency virus (SIV) Vpx protein degrades deoxynucleoside triphosphate (dNTP) triphosphohydrolase SAMHD1 to enhance HIV-1 infection in monocyte-derived dendritic cells (MDDCs) in vitro, a cellular enzyme that can deplete intracellular dNTPs to block viral reverse transcription in the early steps of infection. ² In addition to SAMHD1, the other mechanisms responsible for the limited replication of HIV-1 in DCs should also be elucidated.

Quite recently, we have identified cell membrane metalloprotease TRABD2A as a novel host restriction factor inhibiting HIV-1 assembly and production by degrading HIV-1 Gag protein in resting CD4⁺ T cells. ^3,4 Interestingly, we also observed that TRABD2A is expressed in MDDCs but not in primary monocyte-derived macrophages (MDMs), although it is nearly 20–30 times lower in MDDCs than in resting CD4⁺ T cells; therefore, we also wanted to investigate the potential anti-HIV-1 activity of TRABD2A in MDDCs.

At first, we examined the TRABD2A and TRABD2B expression by performing high-throughput RNA-Seq for monocytes and MDDCs from two independent healthy donors. By using Integrative Genomics Viewer (IGV) software, we consistently observed that TRABD2A is expressed in MDDCs at a higher level than those in monocytes (Fig. 1a, b), whereas TRABD2B expression is not detected (data not shown). This suggests the potential anti-HIV-1 activity of TRABD2A in MDDCs. Furthermore, we were curious whether TRABD2A is translocated to the plasma membrane in MDDCs and thus performed immunofluorescence-based analyses for the TRABD2A protein in MDDCs. As a result, we found that the TRABD2A protein could also translocate to the MDDC plasma membrane (Fig. 1c).

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FIG. 1.

TRABD2A restricts HIV-1 production in MDDCs. (a) Monocytes were purified from total PBMCs after Ficoll gradient separation with CD14-positive enrichment. MDDCs were generated by incubating CD14-purified monocytes in an IMDM medium (Gibco) supplemented with 10% FBS, 2 mM l-glutamine, 100 IU/mL penicillin, 100 mg/mL streptomycin, 10 mM HEPES, 1% nonessential amino acids, 1 mM sodium pyruvate, 10 ng/mL GM-CSF, and 50 ng/mL IL-4 (Miltenyi Biotec). On day 4, two-thirds of the culture medium was replaced with a fresh medium containing GM-CSF and IL-4. Immature MDDCs were harvested and used for experiments on day 6. Total RNA was extracted from MDDCs and subjected to high-throughput sequencing, and RNA-Seq data generated for this study were deposited in the SRA under accession code PRJNA533941. IGV software was used to visualize the transcript peaks of TRABD2A, and the relative expression of TRABD2A (normalized by GAPDH) between monocytes and MDDCs was measured according to their transcript reads (b). (c) Immunofluorescence-based analyses of TRABD2A in MDDCs. Alive cells were washed and immunostained with a monoclonal anti-TRABD2A antibody, which was probed with a second antibody conjugated with FITC. Scale bars: 10 μm. (d) MDDCs were transfected with siRNA for TRABD2A or siControl. Twenty-four hours after transfection, MDDCs were aliquoted for Vpx treatment and HIV-1_AD8 infection at low (5 ng) or high (50 ng) doses in the presence or absence of EFV (300 nM) or raltegravir (10 μM). Seventy-two hours after infection, p24 ELISA was used to measure the produced virus in culture supernatants, and total RNA was extracted for RT-qPCR to measure the TRABD2A transcript levels relative to GAPDH expression (e). (f, g) To achieve shRNA-mediated silencing of TRABD2A, shRNA lentiviruses were introduced to MDDCs. The freshly isolated monocytes were treated with Vpx and transduced with shRNA lentivirus particles for TRABD2A or shControl. After puromycin selection, shRNA-transduced MDDCs were treated with Vpx and further infected with HIV-1_AD8 at low (5 ng) or high (50 ng) doses in the presence or absence of EFV (300 nM). Seventy-two hours after infection, p24 ELISA was used to measure the produced virus in culture supernatants. Western blotting was conducted to assess the TRABD2A and GAPDH protein levels (g). (h) The freshly isolated monocytes were treated with Vpx and transduced with shRNA lentivirus particles for TRABD2A or shControl. MDMs were generated through stimulation of monocytes with a 50 ng/mL recombinant human GM-CSF (R&D) for 7 days. After puromycin selection, shRNA-transduced MDMs were treated with Vpx and further infected with HIV-1_AD8 at low (5 ng) or high (50 ng) doses in the presence or absence of EFV (300 nM). After selection with puromycin as in (e), shRNA-transduced MDMs were treated with Vpx and further infected with HIV-1_AD8 at low (5 ng) or high (50 ng) doses. p24 ELISA was used to measure the produced virus in culture supernatants. (i, j) Lentiviral shRNA-transduced MDDCs were infected with HIV-1 at low (5 ng) or high (50 ng) doses for 15 days as indicated. Viral production was measured by p24 ELISA at the indicated time points. *p < .05; **p < .01 (two-tailed unpaired t-test); data are plotted as mean ± standard error of the mean of three independent experiments. All blotting data are representative of three independent experiments. BD, blow the detection limit; EFV, efavirenz; ELISA, enzyme-linked immunosorbent assay; FBS, fetal bovine serum; FITC, fluoresceine isothiocyanate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GM-CSF, granulocyte-macrophage colony-stimulating factor; HEPES, 2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid; IGV: Integrative Genomics Viewer; IL, interleukin; MDDCs, monocyte-derived dendritic cells; MDMs, monocyte-derived macrophages; PBMC, peripheral blood mononuclear cell; R&D, R&D systems; RT-qPCR, real time quantitative PCR; shRNA, short hairpin RNA; siRNA, small interfering RNA; SRA, Sequence Read Archive. Color images are available online.

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Next, we examined whether endogenous TRABD2A restricts HIV-1 production in MDDCs by employing two strategies to silence TRABD2A in MDDCs. We also utilized Vpx to antagonize SAMHD1 in MDDCs to promote viral production. We observed that small interfering RNA (siRNA)-mediated depletion of TRABD2A in MDDCs could enhance wild-type CCR5-trophic HIV-1_AD8 production in the absence of efavirenz (EFV) or raltegravir (Fig. 1d, e). HIV-1 production was significantly increased either at low- or high-dose HIV-1 infection, and the expression levels of TRABD2A also inversely responded to HIV-1 production. Moreover, we employed lentiviral short hairpin RNA (shRNA) for TRABD2A to silence its expression into MDDCs and found a consistent upregulation of viral production in the absence of TRABD2A (Fig. 1f, g).

Meanwhile, because primary MDMs do not express TRABD2A, ³ we transduced these shRNAs and did not observe any effect on HIV-1 infection in MDMs (Fig. 1h), thereby indicating their specific usage in MDDCs. Collectively, this indicates that TRABD2A can also restrict HIV-1 production in MDDCs. To this end, we explored HIV-1 spreading infection in MDDCs for 15 days in the presence or absence of TRABD2A. Consequently, silencing of TRABD2A enhanced both CCR5-trophic proviral AD8 and dual-trophic 89.6 infection in MDDCs during viral spreading infection (Fig. 1i, j; knockdown effects are not shown). We found notable differences in virion production by MDDCs in the presence and absence of TRABD2A particularly at low inoculums in which wild-type HIV-1 was allowed to spread to saturation for 15 days.

Overall, TRABD2A is also likely to restrict HIV-1 production in MDDCs in addition to resting CD4⁺ T lymphocytes. Probably, as in resting CD4⁺ T cells, TRABD2A also degrades HIV-1 Gag at plasma membranes to destroy progeny assembly in DCs.

In summary, we have shown here that TRABD2A is expressed to restrict HIV-1 production in MDDCs. The detailed mechanism of TRABD2A combating HIV-1 in MDDCs has not yet been elucidated, including whether TRABD2A degrades the HIV-1 Gag protein at the plasma membrane in MDDCs and whether interferon can utilize TRABD2A to inhibit HIV-1 production.