Arbeitsgruppe Prof. Dr. Andrea Maisner
Prof. Dr. Andrea Maisner
Institut für Virologie
Phone: ++49 6421 / 28-65360
Fax: ++49 6421 / 28-68962
Nipah virus infection of polarized epithelial cells
Epithelial cells serve as a primary protective barrier against microorganisms and possess two strictly separated membrane domains. The apical side normally faces the lumen whereas the basolateral side is in contact with underlying tissues and the blood system. Due to polarized protein and lipid sorting in these cells, virus receptors as well as virus-encoded proteins are often expressed in a polarized fashion. As a consequence, virus entry into and virus release from epithelia can be restricted to either the apical or the basolateral plasma membrane. Polarized virus release is assumed to significantly influence viral pathogenesis because viruses which enter at and are released from the apical surface, e.g. influenza viruses, cause a restricted infection of the epithelia without entering the blood stream. In contrast, viruses which are released from the basolateral surface, e.g. vesicular stomatitis virus (VSV), are supposed to easily penetrate the epithelial barrier thereby entering the blood stream and causing a systemic infection. Besides the important role in virus entry, infection of epithelial cells in the respiratory and urinary tract is often important in later stages of infection for virus shedding.
Nipah virus (NiV) is a highly pathogenic paramyxovirus that arised from fruit bats of the genus Pteropus and was first isolated in 1999 after an outbreak of disease in pigs and humans in Malaysia. Because of its zoonotic potential, the high pathogenicity and the lack of therapeutic treatment, NiV was classified as a biosafety level 4 (BSL-4) pathogen. In humans NiV causes a severe acute encephalitis whereas in some animal hosts respiratory symptoms are predominantly observed. Despite the differences in the clinical outcome, microvascular endothelial cell damage predominantly underlies the pathological changes in NiV infections in all susceptible host species. Based on the detection of NiV antigen in the epithelial cells of several species, NiV is assumed to efficiently replicate in the respiratory tract (e.g. oro-nasal, upper and lower respiratory). Since NiV is shedded via aerosols and urine, productive NiV replication in the respiratory and urinary mucosa is important in late infection stages for virus secretion and transmission. By establishing an in vitro system with epithelial cells growing on porous filter membranes, we were able to study the mechanisms of NiV entry into, replication in and release from polarized cell cultures. Using this system we provide evidence that bipolar targeting of the two NiV surface glycoproteins G and F is of biological importance for fusion in polarized epithelia. We could identify basolateral targeting signals in the cytoplasmic domains of the two NiV surface glycoproteins F and G that are required for fusion activity in epithelia (Weise et al., 2010). In late infection stages, NiV particles are released from the apical membranes because of a predominant apical targeting of the viral matrix protein M, the central organizer of NiV assembly and budding (Lamp et al., 2013; Dietzel etal., 2016). Interstingly, virus replication efficiencies and shedding appears to differ in primary airway epithelial cultures from humans and pigs (Sauerhering et al., 2016).
Nipah virus infection of endothelial cells
Histopathological data from infected humans revealed that the systemic NiV replication is confined to endothelial cells, predominantly to those in the CNS. Infection also leads to the overcoming of the blood-brain-barrier and to subsequent infection of brain parenchyma. Even if the endotheliotropism is likely to be controlled by multiple viral and host factors, we have recently shown that the predominant factor is the expression, and the expression levels of the NiV entry receptors ephrin-B2 and ephrin-B3 (Erbar et al., 2008; Thiel et al., 2008). We recently demonstrated that NiV infection of primary brain endothelia not only causes syncytia formation but increases the monolayer permeability thereby impairing the endothelial barrier function (Erbar et al., 2010). Clinical NiV encephalitis is mostly caused inflammation processes, and we could currently demonstrate that early activation of primary brain microvascular endothelial cells can even be triggered in the absence of full NiV replication, just by NiV glycoprotein containing VLPs (Freitag & Maisner, 2016).
Proteolytic activation of the Nipah virus F protein
NiV causes a systemic infection in vivo and is able to replicate in cultured cells of many species and organs. Such pantropic paramyxoviruses generally encode for fusion (F) proteins with multibasic cleavage sites activated by furin or other ubiquitous intracellular host cell proteases. In contrast, NiV has a F protein with a single arginine (R109) at the cleavage site as in the case with paramyxoviruses causing localized infections that are activated by trypsin-like proteases present only in specific cells or tissues. Interestingly, the F protein largely tolerated mutations at the cleavage site and even replacement of the R109 itself did not interfere with F cleavage (Moll et al., 2004; Diederich et al., 2009). This already clearly indicates that NiV F protein is activated by an ubiquitous protease definitely different from the furin-like or trypsin-like proteases known to activate other ortho- and paramyxovirus fusion proteins. Meanwhile, we know that NiV F processing occurs within the endosomal compartment by cathepsin L and B (Diederich, Sauerhering et al., 2012). In contrast to what has been reported for SARS coronavirus and Ebola virus which require endocytosis and cathepsins for successful virus entry, we have shown that endocytosis, acidic pH and cathepsin-mediated cleavage are not necessary for the initiation of NiV infection of new host cells. Proteolytic activation of the NiV F protein thus exclusively occurs after protein synthesis and before incorporation into budding virions (Diederich et al., 2008).To ensure rapid endocytosis and thus cleavage, the NiV F protein contains in its cytoplasmic tail a tyrosine-dependent YSRL motif that perfectly fits into the YxxO-type internalization signals. Disruption of the YSRL motif or substantial deletions in the F cytoplasmic tail interfered with constitutive endocytosis and therefore proteolytic activation by endosomal cathepsins (Vogt et al., 2005; Diederich et al., 2005; Weis & Maisner, 2015).