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P.M. Scanlan, V. Tiwari, S. Bommireddy, D. Shukla; Spinoculation of Heparinase Treated CHO Cells Show gD Receptor Type Dependent Levels of Viral Infectivity That Can Be Used to Identify Relevant Receptors in Primary Ocular Cell Types . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1020.
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Purpose: The goals of this study were to determine the role of HS in viral entry, and produce a method to identify which viral entry receptors are involved in infection of primary cell types. Methods: Viral spinoculation (the use of low speed centrifugation) at 1,200 x g was used to infect both GAG deficient and GAG expressing CHO cells transiently transfected with HVEM, nectin–1, or 3–OST–3. Beta–galactosidase expressing KOS gL86 virus was used as a reporter virus and results were read using ONPG or X–gal substrates. Fluorescent GFP tagged virus was spinoculated with CHO cells to determine the surface binding of virus in 96 well plates. Standard cell fusion assays using luciferase activity were used to determine the effect of centrifugation on viral fusion. Results: Spinoculation of GAG deficient cells expressing nectin–1 or HVEM at 1,200 x g increased viral entry compared to unspinoculated cells at 1 x g. Additionally, HVEM expressing spinoculated 745 CHO cells showed restoration of HSV–1 entry to near normal levels when compared to unspinoculated GAG+ HVEM expressing CHO cells. In contrast, nectin–1 expressing spinoculated 745 CHO cells showed less entry than unspinoculated HS+ nectin–1 expressing CHO cells. Similar results were seen in GAG+ cells treated with or without heparinase using the spinoculation technique. Additionally, cell to cell fusion was not affected by centrifugation; nor could any of the four essential glycoproteins be replaced by centrifugation. Conclusions: All three known gD receptors HVEM, nectin–1, and 3–OS–HS show different patterns of viral entry when GAG+ CHO cells are treated with or without heparinase and with or without spinoculation. By using the spinoculation techniques on ocular primary cell types the relevant receptors leading to viral entry can be identified.
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