Die Nipahvirus Glykoproteine - Ihre Verteilung in infizierten und transfizierten polarisierten Epithelzellen und die Identifizierung basolateraler Transportsignale

The highly pathogenic Nipah virus (NiV) is aerially transmitted and replicates initially in polarized epithelial cells of the respiratory tract. Subsequently NiV spreads systemically, with extensive endothelial involvement leading to vasculitis, which is mostly responsible for the clinical disease. In pigs and in about half of human infections symptomatic respiratory illness is also observed.(27, 87, 100, 119) It is assumed that the patients with symptomatic respiratory tract infections were responsible for human-to-human transmission.(87) Thus, NiV infection of the airway mucosa is relevant not only for primary NiV infection, serving as a portal of virus entry, but also for virus shedding and transmission to other hosts. Beside respiratory epithelia, epithelial cells in the kidney and bladder have been shown to be infected in vivo and are suggested to be important sites of release of progeny virions into the urine.(26, 91, 98, 109, 162) The aim of this study was to elucidate the molecular mechanisms of NiV spread within epithelial cells, focusing on the roles of the two surface glycoproteins G and F. Preliminary analysis showed the both glycoproteins are expressed at the basolateral surface of polarized MDCK cells.(150) It is known that basolateral expression of viral gylcoproteins in polarized cells can mediate cell-to-cell fusion and therefore disruption of the epithelial barrier in vivo. In this study it could be shown that NiV infection of polarized MDCK cells leads to the formation of viral foci. The finding that both NiV glycoproteins are not only apically expressed but also at lateral membranes in infected cells adjacent to noninfected cells suggests that the infection spreads by cell-to-cell fusion. Supporting this idea basolateral targeting signals were identified in the cytoplasmic domains of both NiV glycoproteins. Tyrosine 525 in the F protein is part of an endocytosis signal(151) and is also responsible for basolateral sorting. Surprisingly, a dityrosine motif at position 28/29 in the G protein was identified to mediate polarized targeting. A dileucine motif predicted to function as sorting signal is not involved. Mutation of the targeting signal in one of the NiV glycoproteins prevented the fusion of polarized cells, suggesting that basolateral or bipolar F and G expression facilitates the spread of NiV within epithelial cell monolayers, thereby contributing to efficient virus spread in mucosal surfaces in early and late phases of infection. To elucidate the molecular mechanisms of the basolateral transport colocalization and Yeast Two-Hybrid studies with adaptor protein (AP) complexes were performed. The results indicate that AP-1B seems to be involved in basolateral sorting of NiV F and AP-4 may play a role for polarized targeting of NiV G.