Protocols to Assess Coagulation Following In Vitro Infection with Hemorrhagic Fever Viruses.

During the course of infection with a hemorrhagic fever virus (HFV), the checks and balances associated with normal coagulation are perturbed resulting in hemorrhage in severe cases and, in some patients, disseminated intravascular coagulopathy (DIC). While many HFVs have animal models that permit the analyses of systemic coagulopathy, animal infection models do not exist for all HFVs and moreover do not always recapitulate the pathology observed in human tissues. Furthermore, molecular analyses of how coagulation is affected are not always straightforward or practical when using ex-vivo animal-derived samples, thus reinforcing the importance of cell culture studies. This chapter highlights procedures utilizing human umbilical vein endothelial cells (HUVECs) as a model system to evaluate components of the intrinsic (prekallikrein (PK), factor XII (FXII), kininogen, and bradykinin (BK)) and extrinsic (Tissue Factor (TF)) systems. Specifically, protocols are included for the generation of a coculture blood vessel model, plating and infection of HUVEC monolayers and assays designed to measure activation of PK and FXII, cleavage of kininogen, and to measure the expression of TF mRNA and protein.

Epstein-Barr virus nuclear antigen 3A promotes cellular proliferation by repression of the cyclin-dependent kinase inhibitor p21WAF1/CIP1.

Latent infection by Epstein-Barr virus (EBV) is highly associated with the endemic form of Burkitt lymphoma (eBL), which typically limits expression of EBV proteins to EBNA-1 (Latency I). Interestingly, a subset of eBLs maintain a variant program of EBV latency - Wp-restricted latency (Wp-R) - that includes expression of the EBNA-3 proteins (3A, 3B and 3C), in addition to EBNA-1. In xenograft assays, Wp-R BL cell lines were notably more tumorigenic than their counterparts that maintain Latency I, suggesting that the additional latency-associated proteins expressed in Wp-R influence cell proliferation and/or survival. Here, we evaluated the contribution of EBNA-3A. Consistent with the enhanced tumorigenic potential of Wp-R BLs, knockdown of EBNA-3A expression resulted in abrupt cell-cycle arrest in G0/G1 that was concomitant with conversion of retinoblastoma protein (Rb) to its hypophosphorylated state, followed by a loss of Rb protein. Comparable results were seen in EBV-immortalized B lymphoblastoid cell lines (LCLs), consistent with the previous observation that EBNA-3A is essential for sustained growth of these cells. In agreement with the known ability of EBNA-3A and EBNA-3C to cooperatively repress p14(ARF) and p16(INK4a) expression, knockdown of EBNA-3A in LCLs resulted in rapid elevation of p14(ARF) and p16I(NK4a). By contrast, p16(INK4a) was not detectably expressed in Wp-R BL and the low-level expression of p14(ARF) was unchanged by EBNA-3A knockdown. Amongst other G1/S regulatory proteins, only p21(WAF1/CIP1), a potent inducer of G1 arrest, was upregulated following knockdown of EBNA-3A in Wp-R BL Sal cells and LCLs, coincident with hypophosphorylation and destabilization of Rb and growth arrest. Furthermore, knockdown of p21(WAF1/CIP1) expression in Wp-R BL correlated with an increase in cellular proliferation. This novel function of EBNA-3A is distinct from the functions previously described that are shared with EBNA-3C, and likely contributes to the proliferation of Wp-R BL cells and LCLs.