Fluorescent poliovirus for flow cytometric cell surface binding studies
Introduction
Specific cell-surface binding is generally the essential first step for cellular invasion by viruses. Understanding this process is critical to understanding viral pathogenesis, tissue tropism, cellular uptake, interference and other processes. Furthermore, elucidation of virus entry processes aids in researching later steps in viral replication and pathogenesis; for example by permitting generation of advanced reagents (Ren et al., 1990). To understand the receptor binding processes, virological or biochemical methods for determining binding properties of viruses to cells have been developed. An example of the former is the use of cell protection from infection to screen mAbs for anti-receptor activity (Colonno et al., 1986, Nobis et al., 1985).
For biochemical assays, virus must be tagged, either indirectly or directly. Indirect methods are useful because antiviral antibodies are often readily available. Furthermore, potentially minimal disruption to virus structure results from indirect staining methods. However, direct labeling methods, such as those described below, require fewer labeling steps and may be more accurate. Radioactive virus is simple and reliable, but has several disadvantages, including being labor-intensive and producing radioactive waste. Biotinylated adenovirus has been visualized with streptavidin FITC and analyzed by flow cytometry (Zhang et al., 2002). HTLV I has been labeled with rhodamine (Krichbaum-Stenger et al., 1987). Fluorescently labeled virus should have the advantage that flow cytometry permits direct visualization of cells onto which virus is bound.
Understanding tissue tropism of poliovirus in humans is not complete. Both virulent and attenuated poliovirus grow in the gut, facilitating viremia and CNS invasion. However, the specific cell type mediating initial replication, viremia and CNS invasion has not been elucidated. Moreover, the properties determining specific neuronal susceptibility, including appropriate CD155 expression, remain to be elucidated. It has been demonstrated that poliovirus receptor, CD155, is present on primary human monocytes (Freistadt et al., 1993) and CD155-transgenic mouse lymphocytes and monocytes (Freistadt and Eberle, 2000). Poliovirus replicates in these cells. The demonstration of poliovirus binding and replication in blood cells casts doubt on conclusions drawn from virus binding studies that included blood cells (Ren and Racaniello, 1992). Replication in immune cells may have a role in poliovirus pathogenesis, such as in providing sanctuary to virus. However, poliovirus does not replicate in all CD155-expressing monocytes (Eberle et al., 1995). To determine characteristics of the poliovirus-susceptible subset of monocytes and other cells, a fluorescence binding assay for poliovirus was sought.
The production of fluorescent poliovirus (type 1 Mahoney and Sabin), using a commercially available fluoresceination reagent, is described. The fluorescent poliovirus retained biological features of poliovirus, such as specific receptor recognition and cell infectivity. However, some diminution of infectivity, presumably due to destruction of requisite receptor binding sites was noted, necessitating care in titrating fluoresceination conditions. Nevertheless, this procedure should be useful for future studies toward unraveling poliovirus tropism in human tissues and it may be applicable to various approaches with other viruses.
Section snippets
Reagents
Neurovirulent poliovirus type 1 Mahoney stock virus and Hela R19 cells were from E. Wimmer (State University of New York, Stony Brook). Attenuated vaccine strain poliovirus type 1 Sabin was from A. Nomoto (Tokyo Metropolitan Institute of Medical Science, Japan). It was propagated, purified and titrated as described (Freistadt and Eberle, 1996b) on Hela R19 monolayer cultures. U937 cells, a histiocytic lymphoma cell line that is monocyte-like, were from ATCC. The fluoresceination reagent,
Initial studies
To generate fluorescent poliovirus for receptor binding studies, a commercial fluoresceination reagent [N-hydroxy succinimide (NHS)-fluorescein] was used, following the manufacturer's instructions. To assess fluoresceination, published virus-to-cell binding protocols (Ren and Racaniello, 1992) were followed: increasing amounts of fluoresceinated poliovirus was bound to susceptible cells. Initially, partially purified virus (“30 K pellet”) was used. Binding was assessed by flow cytometry. As
Discussion
The production of fluorescent poliovirus (type 1 Mahoney and Sabin) is described, using a commercially available fluoresceination reagent. Flow cytometric detection of virus binding to susceptible cells demonstrated dependence on reagent concentration and input virus dose. Retention of receptor-binding specificity was shown by inhibition of fluoresceinated virus binding to cells with specific anti-receptor antibody. Excess fluoresceination reagent diminished virus function, both in infectivity
Conclusions
- 1.
Poliovirus may be labeled in vitro with N-hydroxy succinimide fluorescein and retain its biological properties.
- 2.
Reduced fluoresceination reagent yields improved the recovery of titer and specificity.
- 3.
Careful titration to balance fluoresceination intensity to functional retention will be critical in future applications of this methodology.
- 4.
These studies will be helpful for studies on viral tissue tropism and virus uptake.
Acknowledgements
We thank Joseph Chaiban for expert flow cytometry performance. Funding was from NIAID, Grant 35104.
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