Our previous work demonstrated that the KSHV-encoded K8.1A virion glycoprotein is essential for infection of B cells (MC116 human B cell line, human tonsillar B cells). This requirement stands in marked contrast to other non-B cell types, for which our results confirm published findings that K8.1A is dispensable for infection of other cell types (the 293 human embryonic kidney epithelium cell line, the African green monkey Vero cell line, human microvascular endothelial cells. In FY2016 we extended this work by demonstrating that another anti-K8.1A monoclonal antibody inhibits KSHV infection selectively for B cells. Thus we have identified a specific virus-encoded tropism determinant for the major cell type harboring KSHV in infected persons, and associated with two KSHV-associated lymphoproliferative diseases. We have extended studies on the effects of producer cell type on KSHV tropism, by expanding the number and types of cell lines constitutively infected with the KSHV.219 reporter virus (GFP linked to a constitutive cellular promoter; RFP linked to the KSHV lytic phase PAN promoter). In these inducible cells, the KSHV lytic phase can be induced by the lytic switch protein RTA (replication and transcription activator), linked to the tet-operator and thereby inducible with doxycycline. We now have inducible variants of the unusually KSHV-susceptible human B cell line MC116, the telomerase-immortalized human microvascular endothelium cell line TIME, the 293 cell line and the Vero cell line. We have characterized in detail the effects of specific activation with doxycycline versus nonspecific activation with BUdR, by measuring both RFP (fluorescence microscopy, flow cytometry) and the production of infectious KSHV (titration on fresh Vero cells). These inducible cell lines will be most valuable in our study of the impact of producer cell type and mode of activation on the target cell tropism of KSHV virions. We have also found a second mode for KSHV infection B cells, namely antibody-dependent entry. This mechanism is K8.1A independent, and in fact was first observed when the anti-K8.1A mAbs were found to greatly stimulate infection of Raji cells, which are otherwise refractory to KSHV. Preliminary data suggest that the antibody-dependent infection pathway is mediated by Fc receptors on the B cell surface. In collaboration with Dr. Robert Yarchoan, NCI, we are analyzing the sera from infected people with different KSHV-mediated syndromes. Preliminary data suggest that sera from MCD and PEL subjects have antibodies that greatly stimulate KSHV infection of Raji cells, whereas sera from KS subjects have lower levels of KSHV-stimulating antibodies. As yet, we do not know the relationships between the stimulating activities and the total levels of anti-KSHV antibodies or antibodies against any specific KSHV-encoded proteins.
| Triyatni, Miriam; Berger, Edward A; Saunier, Bertrand (2016) Assembly and release of infectious hepatitis C virus involving unusual organization of the secretory pathway. World J Hepatol 8:796-814 |
| Berger, Edward A (2015) Finding Fusin/CXCR4, the First ""2nd Receptor"" for HIV Entry. Front Immunol 6:283 |
| Dollery, Stephen J; Santiago-Crespo, Rey J; Kardava, Lela et al. (2014) Efficient infection of a human B cell line with cell-free Kaposi's sarcoma-associated herpesvirus. J Virol 88:1748-57 |
| Triyatni, Miriam; Berger, Edward A; Saunier, Bertrand (2011) A new model to produce infectious hepatitis C virus without the replication requirement. PLoS Pathog 7:e1001333 |
