A novel high throughput quantum dot-based fluorescence assay for quantitation of virus binding and attachment

Abstract

Quantum dots (QDots) are fluorescent semiconductor nanocrystals with a narrow emission spectrum, high quantum yield, and excellent photostability. These unique properties of QDots have been utilized to develop a fluorescent binding assay using biotinylated human T cell leukemia virus type 1 (biot-HTLV-1) conjugated with streptavidin-coated Qdots that enabled both qualitative and quantitative analyses of viral binding. The specificity and linearity of the assay was demonstrated utilizing T cells, the primary HTLV-1-susceptible cell population. Furthermore, differential binding of HTLV-1 was analyzed in additional cell types of clinical relevance including primary CD4 + and CD8 + T cells, dendritic cells (DCs), monocytes, bone marrow progenitor cells, and epithelial cells. DCs exhibited maximum binding affinity when compared to other examined cell types except the Jurkat and SUP-T1 T cell lines. Finally, blocking antibodies directed against a putative HTLV-1 receptor on DCs; DC-SIGN (dendritic cell-specific ICAM-3-grabbing non-integrin), were utilized to study the inhibition of HTLV-1 binding to target cells. Overall, these results demonstrated that this novel high throughput assay can be utilized to study the binding of a biotinylated virus and has implications for screening of viral binding inhibitors as well as host membrane proteins that may serve as receptors for viral entry. KeywordsHTLV-1; quantum dot; viral binding assay 1.IntroductionViral binding and attachment to a host cell membrane, while seemingly simplistic, is a complex area of research for a wide range of viruses. It is often viewed as the first step in infection, whereby a virion is able to attach to a target cell, fuse to the cell membrane, and deliver the contents of the capsid to the cytoplasm of the newly infected cell. The exact mechanism of binding to a host cell varies between viruses and is usually determined by the composition of attachment proteins located within the viral and cellular membranes. The population of cells † A part of this work was presented during 7th International Symposium on NeuroVirology (ISNV), Philadelphia, PA, USA, May 30 - Street, Philadelphia, PA 19102, USA, Tel.: 215-762-8586; Fax: 215-762-1955, E-mail: pjain@drexelmed.edu, Web site: http://www.drexelmed.edu/ Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. (Dhawan et al., 1991;Inghirami et al., 1988). Occasionally, radioactive labels are also utilized for the quantitative estimation of binding (Hubbard, 2003). However, organic fluorophores conventionally used for labeling nucleotides and proteins have poor pho...

DC-SIGN Mediates Cell-Free Infection and Transmission of Human T-Cell Lymphotropic Virus Type 1 by Dendritic Cells

“…For DC-SIGN, MAb clone 507 has been used in several previous studies as a blocking Ab and did inhibit HTLV-1 binding to DC-SIGN-expressing cells in our previous studies. Conversely, L-SIGN-specific MAb 604 (specificity control) did not exhibit any effect in similar studies (20), giving us confidence with respect to the use of this clone in the studies reported here. Similarly, with respect to GLUT-1 inhibition, an extensive body of literature on glucose transport indicates that Cyto B directly binds to GLUT-1 (21,24).…”

“…Both RNA interference (RNAi) (3,32) and carbohydrate-binding agents (4) have been shown as potential means to inhibit the DC-SIGN-mediated transmission of human immunodeficiency virus type 1 (HIV-1). We have previously demonstrated that monoclonal antibodies (MAbs) to DC-SIGN inhibit the binding of HTLV-1 to DCs (20). In this study, we have shown that the silencing of DC-SIGN inhibits the HTLV-1 infection of DCs.…”

“…5A). These observations were expected, because all of these inhibitors are known to operate by reducing the accessibility of the receptor rather than by directly affecting their expression (12,18,20,21,24,30,35,48); therefore, the detection of their respective receptors by flow cytometry depends on the binding site for the corresponding Ab versus the site occupied by the inhibitor. The functionality of inhibitors was evident with respect to their impact on HTLV-1 binding to MDDCs, however.…”

“…HTLV-1 binding to B-THP-1 cells was examined by using cell-free infectious virus to circumvent the problem of improper conformation associated with isolated envelope components as used in previous studies (30). The quantitative estimation of binding was determined by the QDot-based viral binding assay in which Biot-HTLV-1 was used in conjunction with streptavidin-QDots (Strep-QDots) (20). To confirm that the labeled virus was structurally intact and functional, the immunoreactivity of the viral envelope was tested with an anti-HTLV-1 Env Ab (data not shown).…”

“…Dendritic cells (DCs) are of particular significance in HTLV-1 pathogenesis, because progression to HAM/TSP is associated with the rapid maturation of DCs (25), whereas ATL is associated with defects in their maturation (29). We previously demonstrated that DCs exhibited a greater degree of binding to HTLV-1 virions than to other cell types examined, including T cells, the primary target cell population during the course of disease (20). DCs from HAM/TSP patients were found to be infected with HTLV-1 (25) and were shown to be infected in vitro by both cell-free (19,27) and cellassociated (5) virus.…”