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

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

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Product# 1039 HIV-1 YU2 (M Tropic)Envelope Glycoprotein gp41

Product# 1081 HIV-1 gp120 (ADA)

Product# 1011 HIV-1 gp120 (subtype C)

Product# 1031 HIV-1 gp120 (YU2)

Abstract

During sexual transmission of human immunodeficiency virus (HIV), macrophages are initial targets for HIV infection. Secretory leukocyte protease inhibitor (SLPI) has been shown to protect against HIV infection of macrophages through interactions with annexin A2 (A2), which is found on the macrophage cell surface as a heterotetramer (A2t) consisting of A2 and S100A10. Therefore, we investigated potential protein-protein interactions between A2 and HIV-1 gp120 through a series of co-immunoprecipitation assays and a single molecule pulldown (SiMPull) technique. Additionally, inhibitors of A2t (A2ti) that target the interaction between A2 and S100A10 were tested for their ability to impair productive HIV-1 infection of macrophages. Our data suggest that interactions between HIV-1 gp120 and A2 exist, though this interaction may be indirect. Furthermore, an anti-A2 antibody impaired HIV-1 particle production in macrophages in vitro, whereas A2ti did not indicating that annexin A2 may promote HIV-1 infection of macrophages in its monomeric rather than tetrameric form.

Keywords: Annexin A2, Annexin A2 heterotetramer, HIV-1, Inhibitor, Macrophage, Receptor

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Introduction

During sexual transmission of human immunodeficiency virus (HIV), macrophages of the cervical, anal, and foreskin epithelium are among the first immune cells to encounter the virus, which makes them initial targets for HIV infection [12]. It is well established that secretory leukocyte protease inhibitor (SLPI), a protein found in high concentrations in mucosal fluids, protects against HIV-1 infection of macrophages independent of its anti-protease activity [34]. Moreover, when the host-cell membrane constituent phospholipid phosphatidylserine (PS) is incorporated into the viral envelope during the budding process, it acts as a cofactor for HIV-1 infection of macrophages [5]. The ability of host-derived PS to influence HIV-1 infection led to the prediction that an unknown factor on target-cell membranes facilitated viral binding and/or fusion through PS. It was later revealed that SLPI directly interacted with annexin A2 (A2), a PS-binding moiety, and that SLPI could disrupt the interaction between A2 and PS on the HIV-1 envelope to prevent infection in vitro [6] (also see Fig. 1d). Additionally, antibodies against A2 or RNA silencing of A2 significantly inhibited HIV-1 infection similar to that of SLPI. It was also shown that A2 is involved in HIV-1 replication in monocyte-derived macrophages (MDMs) [7], and that HIV-1 produced from MDMs that had been treated with A2 siRNA exhibited decreased infectivity [8].

 

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Fig. 2

in vitro HIV-1 infection of macrophages following treatment with A2ti, anti-A2 antibody, or maraviroc. a Structures and reported IC50 values of the three triazole-based A2ti tested [13]. b Macrophages were treated with different A2ti (25 or 50 μM), an anti-A2 antibody (25 μg/mL), or maraviroc (25 μg/mL) prior to exposure to HIV-1JR-CSF (MOI = 1). After 24 h, the supernatants were collected and the relative amount of the HIV-1 capsid protein p24 (pg/mL) was measured via ELISA. Controls included uninfected, HIV only, heat-inactivated virus (H.I. HIV), and DMSO at a concentration matched to that of 50 μM A2ti. Data are presented as the means ± SD of three independent experiments. **p < 0.01 and ***p < 0.001 as determined by unpaired two-tailed Student’s t-tests compared to the HIV only group. c HaCaT cells were left untreated or treated with 50 μM A2ti-1. The following day cells were infected with GFP-plasmid-containing HPV16 pseudovirions (PsV). GFP-positive cells were measured after 48 h by flow cytometry. The mean percentage ± SD of infected cells normalized to the untreated group from a representative experiment performed in triplicate is presented. **p < 0.01 as determined by unpaired two-tailed Student’s t-test compared to the untreated group

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Discussion

In summary, an anti-A2 antibody targeting the N-terminus of A2 successfully blocked productive HIV-1 infection of macrophages in vitro, but A2t-targeting drugs did not, indicating that A2 may promote HIV-1 infection of macrophages in its monomeric form. This mechanism is different from HPV infection, which involves the heterotetrameric form of A2 [18]. Additionally, the highly-sensitive SiMPull assay demonstrated that immobilized HIV-1 gp120 captured A2 from macrophage lysates. However, this interaction was not robust enough to detect via standard co-IP techniques, and may be indirectly mediated by unidentified cellular factors. Thus, it is possible that other factors interact with gp120 and A2, and these interactions may be blocked by anti-A2 antibodies. As HIV-1 entry inhibitors function to prevent initial or ongoing spread of infection while the current standards of care largely target viral enzymes to which HIV-1 resistance continues to emerge [20], novel entry inhibitors are of great interest, though future research is required to determine if monomeric A2 is a viable target for such approaches.

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Acknowledgements

The authors would like to thank Heike Brand for excellent technical assistance.

Funding

This research was supported by Public Health Service grant R01 CA074397 from the National Cancer Institute (to WMK) and P30 CA014089 (to the Norris Comprehensive Cancer Center). LVD was supported by grants from Cancer Research UK (grant references C21559/A11597 and C21559/A7252). AWW is an Arnold O. Beckman Postdoctoral Fellow and received additional support from NIH grant F32 CA206330. Contributions from the Netherlands-American Foundation, Sammie’s Circle, Christine Ofiesh, Yvonne Bogdanovich, and Johannes van Tilburg are also gratefully acknowledged. WMK holds the Walter A. Richter Cancer Research Chair. Additional support for JRT from the ARCS Foundation John and Edith Leonis Award is greatly appreciated. JGS was supported by the Keck School of Medicine/USC Graduate School PhD Fellowship. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Availability of data and materials

The datasets analyzed for the current study are available from the corresponding author upon reasonable request.

Authors’ contributions

AWW, AMS, and JRT performed experiments. AWW and AMS analyzed the data. AWW and JGS prepared the figures. AWW, AMS, JRT, JGS, DMD, LVD, and WMK wrote/edited the manuscript. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not applicable.

Ethics approval and consent to participate

Not applicable.

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Abbreviations

A2 Annexin A2
A2t Annexin A2 heterotetramer
A2ti A2t small molecule inhibitor
Co-IP Co-immunoprecipitation
ELISA Enzyme-linked immunosorbent assay
GM-CSF Granulocyte-macrophage colony-stimulating factor
HIV Human immunodeficiency virus
HPV16 Human papillomavirus type 16
IC50 Half maximal inhibitory concentration
MDM Monocyte-derived macrophage
PBMC Peripheral blood mononuclear cell
PS Phosphatidylserine
SiMPull Single molecule pulldown
SLPI Secretory leukocyte protease inhibitor
TIRF Total internal reflection fluorescence

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Contributor Information

Andrew W. Woodham, Email: ude.tim.iw@mahdoow.

Adriana M. Sanna, Email: es.ul.dem@annas.anairda.

Julia R. Taylor, Email: ude.csu@yatailuj.

Joseph G. Skeate, Email: ude.csu.dem@etaekS.hpesoJ.

Diane M. Da Silva, Email: ude.csu.dem@avlisad.enaid.

Lodewijk V. Dekker, Email: ku.ca.mahgnitton@rekkeD.kjiwedoL.

  1. Martin Kast, Email: ude.csu.dem@tsaK.nitraM.

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