Enveloped Virus Glycoprotein/receptor Interactions

National Institute Of Allergy And Infectious Diseases

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Abstract

We have extended analyses of our finding that the KSHV virion glycoprotein K8.1A is a major determinant for KSHV infection of B cells (human MC116 B cell line; primary B cells from human tonsils), but not for commonly used non-B cell target cells (293 human embryonic kidney epithelial cells, African green monkey Vero cells). The K8.1 A requirement for B cell infection, initially based on the infection-blocking activities of anti-K8.1A monoclonal antibodies, has now been extended using siRNA to down-regulate K8.1A expression in virus producer cells. We have improved immunostaining assays to quantitate infected cells by measurement of KSHV gene products, to complement reporter gene expression assays. Recombinant soluble K8.1A proteins have been produced as probes to identify the putative cell surface receptor mediating KSHV infection of B cells. In our studies of variables influencing KSHV tropism, we have been analyzing infection by virus induced from constitutively infected cell lines of diverse lineage, including MC116 B cells, telomerase-immortalized human microvascular endothelial TIME cells, as well as 293 and Vero cells. Results indicate that virus tropism is influenced not only by the producer cell type, but also by the induction protocol, i.e sodium butyrate versus the KSHV lytic switch protein RTA (under the Tet operator). Once conditions are defined for obtaining virions with distinct tropisms, studies will be performed to assess the steps in the virus life cycle as well as associated variations in virion biochemical composition. We previously noted an alternate K8.1A-independent pathway by which KSHV can infect B cells, i.e. antibody-mediated entry. Studies with blocking antibodies clearly indicate the Fc receptor involvement in this infection route. We plan to assess the role of Fc receptors in the antibody enhancement of B cell infection previously noted with sera from KSHV infected subjects (samples provided by Dr. Robert Yarchoan, NCI).