Direct visualization of HIV-enhancing endogenous amyloid fibrils in human semen - PubMed (original) (raw)

Onofrio Zirafi 1, Janis A Müller 1, Nathallie L Sandi-Monroy 2, Jay K Yadav 3, Christoph Meier 4, Tanja Weil 4, Nadia R Roan 5, Warner C Greene 6, Paul Walther 7, K Peter R Nilsson 8, Per Hammarström 8, Ronald Wetzel 9, Christopher D Pilcher 10, Friedrich Gagsteiger 11, Marcus Fändrich 3, Frank Kirchhoff 1, Jan Münch 1

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Direct visualization of HIV-enhancing endogenous amyloid fibrils in human semen

Shariq M Usmani et al. Nat Commun. 2014.

Abstract

Naturally occurring fragments of the abundant semen proteins prostatic acid phosphatase (PAP) and semenogelins form amyloid fibrils in vitro. These fibrils boost HIV infection and may play a key role in the spread of the AIDS pandemic. However, the presence of amyloid fibrils in semen remained to be demonstrated. Here, we use state of the art confocal and electron microscopy techniques for direct imaging of amyloid fibrils in human ejaculates. We detect amyloid aggregates in all semen samples and find that they partially consist of PAP fragments, interact with HIV particles and increase viral infectivity. Our results establish semen as a body fluid that naturally contains amyloid fibrils that are exploited by HIV to promote its sexual transmission.

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Figures

Figure 1

Figure 1. Detection of endogenous amyloid in human semen with fluorescent dyes and fibril-specific antibodies

(a) Number of Proteostat-stained structures in semen from individual donors. Liquefied ejaculates were stained with the amyloid-specific Proteostat dye at room temperature for 15- 20 min and imaged using a LSM710 confocal microscope. (b) Size distribution of endogenous amyloid in semen, and in vitro generated SEVI fibrils in buffer. (c) Confocal microscopy images of in vitro generated SEVI fibrils and of semen samples (SE-A to SE-C) stained with amyloid-specific probes Proteostat, pFTAA, and ThT. Scale bar = 5 µm. PBS, negative control. (d) Detection of amyloid fibrils in semen using amyloid-specific conformational antibodies. Semen was treated with WO1 and WO2 antibodies, and then amyloid/antibody complexes were pelleted, washed, and detected using Alexa488-coupled appropriate secondary antibody (green), and counterstained with Proteostat dye (red). Scale bar = 5 µm. (e) Effect of semen samples A-C on HIV infection. R5-tropic HIV-1 was exposed to indicated concentrations of semen (%), and used to infect TZM-bl cells. After 2 h, semen-virion mixtures were replaced with fresh medium. HIV-1 infection rates were measured three days later by quantifying β-galactosidase activities in cell lysates. Values shown are average values derived from triplicate infection ± standard deviation. The numbers above the columns give the n-fold enhancement of infection observed after treatment with 10 % semen relative to infection with mock-treated virus.

Figure 2

Figure 2. Ultrastructural analyses of endogenous amyloid in semen

(a) Transmission electron micrographs of semen. In vitro generated SEVI fibrils or semen samples SE-B and SE-C were dropped onto EM grids which were then washed with PBS after 5 min, and then negatively stained with 2 % uranyl acetate. All samples were immediately analyzed. Scale bars indicate 500 nm for top panel images and 200 nm for bottom panel images. (b) Atomic force micrographs of synthetic SEVI fibrils and endogenous fibrils in semen samples SE-B and Se-C, which were placed on silicon substrates, washed with H2O, and imaged in air. Scale bar = 200 nm. The height profiles represent the averages of multiple cross-sections along the fibrils

Figure 3

Figure 3. Endogenous amyloids partially consists of SEVI

(a) Semen was treated with pre-immune (top) or anti-SEVI antiserum (α-SEVI, bottom). The amyloid/antibody complexes were pelleted, washed, and incubated with an Alexa488-coupled secondary antibody (green), and counterstained with Proteostat dye (red). Scale bar = 5 µm. (b) Immunogold-labeling of endogenous SEVI fibrils in semen. Transmission electron micrographs of semen treated with a pre-immune serum or an anti-SEVI antiserum as primary antibodies, and gold conjugated anti-rabbit secondary antibody. Scale bar = 100 nm. White arrows indicate gold particles bound to amyloid fibrils.

Figure 4

Figure 4. Seminal amyloid interacts with retroviral particles

EYFP-labeled virions (green) were mixed with PBS or semen that was stained with Proteostat dye (red). Images were acquired 15 min later on a laser scanning confocal microscope. Single plane images from the centre of the Z-stack are shown. Treatment of semen with heparin (100 µg/ml) abrogates the ability of seminal amyloid to bind viral particles. Scale bar = 5 µm. Also see Supplementary Movie 1.

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References

    1. Lederman MM, Offord RE, Hartley O. Microbicides and other topical strategies to prevent vaginal transmission of HIV. Nat Rev Immunol. 2006;6:371–382. - PubMed
    1. Shaw GM, Hunter E. HIV transmission. Cold Spring Harb Perspect Med. 2012;2(11) - PMC - PubMed
    1. Gupta K, Klasse PJ. How do viral and host factors modulate the sexual transmission of HIV? Can transmission be blocked? PLoS Med. 2006;3(2):e79. - PMC - PubMed
    1. Haase AT. Early events in sexual transmission of HIV and SIV and opportunities for interventions. Annu Rev Med. 2011;62:127–139. - PubMed
    1. Allen RD, Roberts TK. The relationship between the immunosuppressive and cytotoxic effects of human seminal plasma. Am J Reprod Immunol Microbiol. 1987;11:59–64. - PubMed

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