Recognition of decay accelerating factor and αvβ3 by inactivated hantaviruses: Toward the development of high-throughput screening flow cytometry assays

Abstract

Hantaviruses cause two severe diseases in humans: hemorrhagic fever with renal syndrome (HFRS) or hantavirus cardio-pulmonary syndrome (HCPS). The lack of vaccines or specific drugs to prevent or treat HFRS and HCPS, and the requirement for conducting experiments in a biosafety level 3 laboratory (BSL-3) limit the ability to probe the mechanism of infection and disease pathogenesis. In this study we have developed a generalizable spectroscopic assay to quantify saturable fluorophore sites solubilized in envelope membranes of Sin Nombre virus (SNV) particles. We then use flow cytometry and live cell confocal fluorescence microscopy imaging to show that UV-killed SNV bind to the cognate receptors of live virions, namely, decay accelerating factor (CD55/DAF) expressed on Tanoue B cells and α v β 3 integrins expressed on Vero E6 cells. SNV binding to DAF is multivalent and of high affinity (K d ≈ 26pM). Selfexchange competition binding assays between fluorescently labeled SNV and unlabeled SNV are used to evaluate an infectious unit-to-particle ratio of ∼1:14000. We have configured the assay for measuring the binding of fluorescently labeled SNV to Tanoue B suspension cells using a high throughput flow cytometer. In this way, we establish a proof of principle high throughput screening assay for binding inhibition. This is a first step towards the development of HTS format assays for small molecule inhibitors of viral-cell interactions, as well as dissecting the mechanism of infection in a BSL-2 environment.

Pathogenic Old World Hantaviruses Infect Renal Glomerular and Tubular Cells and Induce Disassembling of Cell-to-Cell Contacts

“…The infection of the glomerular endothelium may allow the entry of the hantavirus into the kidney with subsequent infection of podocytes and tubular cells. The expression of the hantaviral coreceptor CD55 on different renal cell types may also play a role in the susceptibility (8,40). An analysis of the impact of infection on the integrity of the cell-to-cell contacts revealed structural alterations in tubular and glomerular cells.…”

Maporal Virus as a Surrogate for Pathogenic New World Hantaviruses and its Inhibition by Favipiravir

“…Notably, complete inhibition could not be achieved, suggesting that other mechanisms of virus entry are likely. Recent studies have shown that in addition to the β integrins, hantaviruses also exploit decay-accelerating factor (DAF) to successfully infect cells [47, 48]. This may explain why, after incubation with ECM proteins or antibodies to the β integrins, viruses still were able to infect cells with ‘blocked’ receptors resulting in less than complete inhibition.…”

Rapid parallel flow cytometry assays of active GTPases using effector beads

“…For preparation of UV-inactivated SNV, we placed 100 μL of virus stock (typically 1.5–2×10 6 focus forming units/ml) in each well of a 96-well plate and subjected the virus to UV irradiation at 254 nm for various time intervals (~5 mW/cm 2 ) as described elsewhere [12]. We verified efficiency of virus inactivation by focus assay before removing from the BSL-3 facility.…”