Abstract
Semen contains amyloid fibrils that enhance HIV-1 infection (Münch et al., 2007; Kim et al., 2010; Roan et al., 2011; Arnold et al., 2012; Usmani et al., 2014; Roan et al., 2014). Positively charged semen amyloids capture negatively charged viral particles and increase their attachment rates to the cell surface resulting in enhanced fusion and infection (Roan et al., 2009). Since semen is highly cytotoxic, we developed an assay that allows quantification of the infection enhancing activity of semen while minimizing its cell damaging activity. Here, we describe two protocols that allow the quantification of the infectivity enhancing activity of semen using a reporter cell line (TZM-bl cells) or peripheral blood mononuclear cells (PBMCs).
Keywords: HIV, Semen, Amyloid, SEVI, Sexual transmission
Materials and Reagents
Equipment
Software
Procedure
Representative data
Figure 1. Typical layout of a microtiter plate used in experiments to study the effect of semen on HIV-1 infection. Cells seeded in the inner 60 wells are inoculated with HIV-1 of indicated MOIs pretreated with 0, 0.4, 2, 10 and 50 % semen, or are left uninfected. grey: PBS; white: uninfected; colored: different MOIs of HIV-1. Figure 2. Light microscopy analysis of infected TZM-bl cells in presence of semen. TZM-bl cells were inoculated with PBS (uninfected) or HIV-1 that has been preincubated with indicated concentrations of semen. Final semen concentrations on cells are given in brackets. Minor (0% semen), little (2% semen) and strong (10% semen) CPE. Scale bars are 100 µm. Figure 3. Results and evaluation of a representative reporter gene assay. Different MOIs (red: 0.1, blue: 0.01, green: 0.001) of HIV-1 NL4-3 92Th014-2 (R5-tropic) were preincubated with indicated concentrations of semen before infection of TZM-bl cells. A. Raw data representing relative light units/second of β-galactosidase activity measured in each well. B. Average background (uninfected cells) is calculated and subtracted. C. Average and standard deviations of triplicate infections are calculated. D. Fold enhancement is calculated by setting 0% semen sample = 1. Figure 4. Graphical presentation of data described above. A. Shown are average β-galactosidase activities (n = 3) measured 3 days after virus exposure. RLU/s: relative light units per sec. The numbers above the bars give n-fold enhancement of HIV infection by semen relative to that measured for the corresponding PBS control. B. n-fold enhancement values represented as bar graphs. Values represent average values obtained from triplicate infection ± standard deviation. Note that 50% semen during virion treatment (corresponding to final cell culture concentrations of semen of 3.3 %) are cytotoxic resulting in reduced infection rates.
Notes
The following issues need to be carefully considered:
If these points are considered, the experimental set up can be adapted and modified to allow measurement of infection of different target cells with any HIV strain and reporter virus (i.e., GFP followed by a flow cytometry readout), as well as other viruses such as HCMV and HSV (Tang et al., 2013; Torres et al., 2015). Infection enhancement by in vitro generated amyloid fibrils can be examined using the same protocol, except that the toxicity avoiding steps, i.e., addition of gentamicin, high volumes of medium, removal of toxic semen after 2-3 h can be omitted.
Recipes
Acknowledgments
This assay was first published in (Münch et al., 2007) and the protocol described in detail in (Kim et al., 2010). Thanks to Annika Röcker, Edina Lump, and Onofrio Zirafi for carefully reading and revising the protocol. Janis A. Müller is part of the International Graduate School in Molecular Medicine Ulm.
References
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