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Mechanism and immunological consequences of human cytomegalovirus entry into porcine cells

Anne-Laure Millard¹, Aline M. Taveira¹, Nicolas Ponroy¹, Jörg D. Seebach², Nicolas J. Mueller¹

¹Division of Infectious Diseases and Hospital Epidemiology, University Hospital Zürich, Zürich; ²Department of Immunology and Allergology, University Hospital and Medical Faculty, Geneva; Switzerland

Reactivation of human cytomegalovirus (HCMV) is a potential risk of pig-to-human xenotransplantation. We previously demonstrated in vitro that HCMV is able to enter and replicate in porcine cells. Using this model, we herein compared the mechanisms used by HCMV to enter into human and porcine endothelial cells (hEC and pEC, respectively) and evaluated the consequences of pEC infection on human NK cell activation.

First, EC were analyzed for HCMV entry receptor expression. While EGFR was undetectable, membrane expression of ß1 integrins and PDGFRa was observed on both human and porcine EC. Moreover, siRNA experiments revealed that PDGFRa was not involved in HCMV entry into pEC. The endotheliotropic HCMV strain TB40/E was able to similarly infect both human and porcine EC, whereas the fibrotropic strain TB40/F, which failed to replicate in hEC, rapidly translocated to the nucleus and efficiently replicated in different types of pEC. We further investigated the entry pathways (endocytosis versus macropinocytosis) utilized by TB40/E and TB40/F, by treating the cells with various specific inhibitors. TB40/E entry into both human and porcine EC was significantly blocked by both clathrin-mediated and caveolar endocytosis inhibitors as well as by macropinocytosis inhibitors. On the contrary, TB40/F entry into pEC was not affected by endocytosis inhibitors suggesting that TB40/F enters into pEC via phagocytosis and/or macropinocytosis. We finally evaluated the consequences of HCMV entry into pEC on human NK cell activation. We demonstrated that IFNg secretion and CD107a degranulation by human NK cells were increased upon coculture with infected pEC and correlated to increased susceptibility of HCMV-infected pEC to NK cell-mediated lysis.

In conclusion, HCMV entry differs between human and porcine cells and considerably increases their susceptibility to human NK cell cytotoxicity.