Annexin A2 antibodies but not inhibitors of the annexin A2 heterotetramer impair productive HIV-1 infection of macrophages in vitro.

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Fig. 1

A2 from macrophage lysates is captured on HIV-1 gp120-coated SiMPull slides. Lysis buffer (a) or macrophage cell lysates (b) were flowed onto SiMPull slides coated with increasing amounts of biotinylated gp120, and the number of captured complexes (c) were detected following staining with a rabbit anti-A2 antibody and an anti-rabbit 568-conjugated secondary antibody using TIRF microscopy, where each white dot represents one protein-protein complex (scale bar = 5 μm). Controls included no gp120 and no lysate. Data are presented as the means ± SD of five fields of view of a representative example of an experiment performed three times. *p < 0.05 **p < 0.01 as determined by a one-way ANOVA followed by a Kruskal-Wallis multiple comparisons test against the no gp120 control group. d In a separate experiment, lysates were flowed onto SiMPull slides coated with an anti-A2 antibody, and captured complexes were detected with mouse anti-S100A10 or anti-SLPI primary antibodies and an anti-mouse 568-conjugated secondary antibody. ***p < 0.001 as determined by an unpaired two-tailed Student’s T-test against the no capture control group

Generally, HIV-1 infects macrophages through the canonical CD4 receptor CCR5 coreceptor pathway [2, 9], though numerous cofactors can affect the efficiency of this process and the rate of infection [5, 6]. Entry inhibitors, such as the CCR5 antagonist maraviroc [10], often lead to the emergence of resistant HIV-1 strains that can use alternative pathways [9]. Moreover, alternative pathways of HIV-1 infection are likely to differ in macrophages and CD4⁺ T cells as they express different membrane components such as PS and A2, which are found on the macrophage cell membrane but not on viable T cells [4, 7]. A2 can be found on the cell surface as a heterotetramer (A2t) consisting of two A2 monomers and an S100A10 dimer [11], which are co-expressed by macrophages [7]. Additionally, data from the HIV-1 Human Interaction Database from the National Center for Biotechnology Information (NCBI) suggests that there may be interactions between HIV-1 gp120 and host A2 [12], though direct evidence is lacking. Recently, our collaborators developed triazole-based small molecule inhibitors of A2t (A2ti) that specifically disrupt the interaction between A2 and S100A10 [13], and we showed that these small molecules block infection of the A2t-utilizing human papillomavirus type 16 (HPV16) [14], but have yet to be explored in the context of HIV. While A2 has already been implicated in HIV-1 infection of macrophages [6, 15], it is not understood if A2t acts as a cofactor for infection. Therefore, the goals of the current study were to investigate potential protein-protein interactions between A2 and the HIV-1 envelope protein gp120, and the ability of A2ti to inhibit HIV-1 infection of macrophages in vitro.

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Methods

HEK293T cells maintained in in DMEM with L-glutamine (Lonza, Walkersville, MD, USA), 10% FBS, and 1% sodium pyruvate were transfected at 50–75% confluence with the HIV-1_JR-CSF plasmid using the Calcium Phosphate Transfection Kit (Invitrogen, Life technologies) according to manufacturer instructions. Supernatants containing HIV-1 (viral inoculum) were harvested after 72 h, filtered through 0.45 μM filters, and titered using the GHOST-indicator cell line as previously described [16]. Monocytes were isolated from total peripheral blood mononuclear cells (PBMC) via the EasySep negative selection Human Monocyte Enrichment kit (STEMCELL technologies) according to manufacturer instructions. Isolated monocytes were then used to derive MDMs via 7-day incubation with 1000 U/mL granulocyte-macrophage colony-stimulating factor (GM-CSF) (Leukine, Sanofi-Aventis, Bridgewater, NJ, USA) as previously described [17], and were confirmed as CD11c⁺CD14⁻CD4^lowCCR5⁺A2⁺S100A10⁺ (data not shown). Recombinant A2 and S100A10 proteins were gifts from Ralf Langen [18], and recombinant purified HIV-1 gp120 biotin was purchased from Immune Technology (New York, NY, USA). A2ti were synthesized as described [13].

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Results

Through a series of enzyme-linked immunosorbent assays (ELISA) and co-immunoprecipitation (co-IP) assays, we investigated whether A2 directly interacts with the primary HIV-1 envelope protein gp120. Following published procedures that we utilized for the evaluation of A2 interactions with HPV16 [18], we found that recombinant gp120 was not able to bind to purified A2 or S100A10 when measured via ELISA, and that gp120 incubated with macrophages was unable to co-IP with A2 (data not shown). However, in addition to traditional co-IP assays, we used a highly-sensitive single molecule pulldown (SiMPull) technique that combines classical pulldown methodology with single molecule fluorescence imaging [19]. We found that when biotinylated gp120 (1 μg/mL) was captured onto SiMPull slides as a bait protein via NeutrAvidin [19], and macrophage lysates (protein concentration 0.5 μg/mL) were then added, A2-gp120 complexes were detected with a rabbit anti-A2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) followed by an anti-rabbit 568-conjugated secondary antibody (Abcam, Cambridge, MA, USA) using total internal reflection fluorescence (TIRF) microscopy. Max gp120 binding to the slides (100%) was determined as the amount of gp120 added at which no more could be detected directly with an anti-gp120 antibody (Immune Technology). The slides were washed after each step to remove unbound gp120, proteins in the lysate, and detection antibodies. The maximum number of A2-gp120 complexes detected was approximately 50% of those detected for A2-S100A10 or A2-SLPI complexes detected in control experiments with an anti-A2 capture antibody (BD Bioscience) and S100A10 (BD Bioscience) and SLPI (R&D Systems) detection antibodies, but was 3-fold higher than background levels (Fig. 1c and d). These results indicate that A2 from macrophage lysates interacts with gp120 in the absence of other viral proteins, though it is possible that this interaction is indirect and is mediated by other binding partners present in macrophage lysate (Fig. 1).

Next, we validated the effects of an antibody against the N-terminus of A2 on productive HIV-1 infection of macrophages [6]. Macrophages were pre-treated with the anti-A2 antibody (Santa Cruz), or maraviroc (NIH AIDS Research and Reference Reagent Program) for 4 h prior to the addition of HIV-1_JR-CSF (MOI = 1), and infection was measured via HIV-1 p24 ELISA (ABL, Kensington, MD, USA) following published procedures [6]. Maraviroc efficiently impaired HIV-1 particle production as demonstrated by a three-fold reduction in p24 detection, which was expected as maraviroc is a CCR5 antagonist known to block HIV-1 infection [10]. The antibody against the N-terminus of A2 (Santa Cruz) also effectively inhibited HIV-1 particle production by macrophages as efficiently as maraviroc at the same concentration (Fig. 2b ), indicating that A2 is involved in productive HIV infection of macrophages, which is in agreement with previous results [6]. To assess whether A2 is functional as the A2t heterotetramer or as the A2 monomer in HIV infection, we next determined the effects of three different A2ti that we previously developed, two of which had half maximal inhibitory concentration (IC₅₀) values in the low μM range, and one that was chemically analogous, but had a much higher IC₅₀ value as a control (Fig. 2a) [13]. We found that viral load as measured by p24 levels did not decrease with any of the A2ti, even at concentrations well above the reported IC₅₀ values for the two more potent compounds, compared to that of the untreated group (Fig. 2b). However, in agreement with our previous results and following our published procedures [14], A2ti were able to effectively block A2t-utlizing HPV16 infection of HaCaT epithelial cells, serving as a positive control for A2ti activity (Fig. 2c , with data for other A2ti available in [14]).