Mechanism of integration of nipah virus into the host genome
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Nipah virus (NiV) is a highly pathogenic member of the genus Henipavirus within the family Paramyxoviridae, originating from fruit bats (1). NiV was first discovered in Malaysia and Singapore in 1999 during an outbreak of severe respiratory disease in pigs and fatal encephalitis in humans (2). Since 2001, smaller outbreaks of NiV infections in India and Bangladesh have been regularly reported (3). Although human infections have been described only in Southeast Asia so far, there is recent evidence for the existence of Henipavirus-related viruses in African fruit bats (4, 5).
Like all paramyxoviruses, NiV is an enveloped virus with a negative-stranded RNA genome (6). Cell infections start with binding of the viral surface glycoprotein G to cellular ephrin-B2 or ephrin-B3 receptors (7–10). After receptor binding, the fusion protein, F, mediates pH-independent fusion of the viral envelope with the host cell membrane to allow virus entry (for a review, see reference 11). While the NiV surface glycoproteins G and F are essential for virus entry processes and later on for cell-to-cell fusion, the third NiV envelope-associated protein, the matrix protein, M, plays an essential role in virus assembly and budding. Similar to many viral matrix proteins, NiV M is a cytoplasmic protein which rapidly associates with cellular membranes. M organizes the assembly of cytoplasmic nucleocapsids and surface glycoproteins at the plasma membrane and is thus needed for efficient release of progeny virus. Conclusive evidence has been provided that late-domain-like motifs and ubiquitin-regulated nuclear-cytoplasmic trafficking are required for NiV M-mediated budding processes (12–14).
The most common route of transmission of NiV is through the oronasopharyngeal route. After initial replication in the airways, NiV disseminates systemically. In the viremic phase of infection, NiV targets endothelial cells in many organs. The pronounced infection of microvascular endothelial cells in the central nervous system (CNS), causing vasculitis, thrombosis, and necrosis, is finally the cause of the multifocal encephalitis generally observed in humans and some animal species (for a review, see reference 15). While CNS pathology was primarily responsible for clinical disease in humans, pigs naturally infected during the first NiV outbreak in Malaysia showed mainly respiratory symptoms, with widespread lesions in the lung epithelium (16). Studies of experimentally infected pigs presented similar results, with the highest virus contents in the upper and lower respiratory tract (15). Studies of NiV infection in a hamster model furthermore showed that the respiratory epithelium is the initial area for NiV replication before it comes to a more systemic spread of infection (17, 18). In agreement with the reported NiV shedding in airway secretions and urine, epithelial cells in the respiratory tract, the kidneys, and the bladder were found to be infected in late stages of natural or experimental NiV infections (reviewed in reference 19). These immunohistological data of in vivo infections clearly demonstrate that NiV efficiently infects epithelial cells in mucosal surfaces.
Epithelial cells differ from most other cell types in their polarized phenotype and their barrier function. The most important feature is their apical and basolateral plasma membrane domains that are strictly separated by tight junctions. Due to specialized protein-sorting machineries in these cells, the two membrane domains differ substantially in their compositions (20, 21). Protein sorting, maintaining the polarity and the specialized functions of epithelial cells, can also influence virus infections. While the polarized distribution of the viral receptor can restrict virus entry to one surface domain, sorting of viral proteins can lead to a vectorial virus release (22–26). Since the handling of NiV is restricted to biosafety level 4 (BSL-4) laboratories, knowledge about the molecular mechanisms underlying the interactions of NiV with epithelial cells based on studies with live virus is extremely limited. We have shown in a previous study that both NiV surface glycoproteins possess tyrosine-dependent sorting signals responsible for the basolateral targeting of the proteins upon single expression in polarized MDCK cells. However, the localization of G and F proteins in infected polarized MDCK cells was found to be bipolar, with most of the glycoproteins concentrating at the apical membrane (27). As it is known for several viruses that the glycoprotein distribution does not necessarily determine the site of virus budding (28–31), the impact of the NiV glycoprotein distribution is not yet known. The aim of this study was thus to elucidate the virus entry and exit pathways in polarized epithelial cells and to clarify the role of vectorial sorting of the NiV envelope proteins in virus spread and release from epithelial cells.