Human neuronal cell protein responses to Nipah virus infection

Nipah virus (NiV) is a recently discovered zoonotic negative-stranded RNA virus of the genus Henipavirus of the Paramyxoviridae family [1, 2]. The virus causes severe to fatal central nervous system (CNS) infection in humans [3, 4]. The virus is acquired from contact with the excretions or secretion of NiV-infected pigs [5‐7] and it has a mortality rate of ~40% in human infection. NiV-infected patients typically present with symptoms of CNS infection with elevated cerebrospinal fluid protein and white cell counts [6]. Severe vasculitis and small lesions with presence of NiV antigen and nucleocapsid inclusion bodies are also detectable in the brain using immunohistochemical staining [8, 9], but no mature viral particles are observed [10, 11]. NiV productively infects several different human cell types and cells of other host origin [12]. In contrast to infections of the fully susceptible human lung fibroblast and pig kidney cells, NiV replicates less efficiently in human neuronal cells. It does not result in immediate cell lysis and releases low number of infectious virus particles. There is evidence to suggest that the infection spreads insidiously through the cell-to-cell spread infection mechanisms and therefore, there is no rapid dissemination of the virus. This is consistent with the observed absence of mature viral particles in the infected human brains [8, 11]. The cytopatologic effects of NiV infection on the neuronal cells and how virus replication is regulated in these cells are still unknown. In the present study, we used two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS) to examine the human neuronal cell protein responses to NiV infection.

NiV infection causes significant cellular morphological changes in the CNS of humans [8]. Infected cells are usually enlarged and giant multinucleated syncytial cells are common [8, 12]. NiV infects cells through ephrin-B2, a common cell surface molecule found especially in neuronal cells [13]. NiV virions are released by budding from the infected cells [11] and high number of extracellular virions is obtained towards the terminal end of the infection [12, 14]. The rate of progression of the cytopathologic effects of NV infection in human neuronal cells, as well as the intracellular and extracellular virus RNA synthesis are relatively low in comparison to the fully susceptible human fibroblast cells or pig kidney cells [12]. Additionally, the production and peak level of NiV release from the neuronal cells are also lower as compared to the other two NiV-infected cell cultures. These suggest that for reasons that are still unknown, NiV replicates less efficiently in neuronal cells despite having high ephrin-B2 on its surface to facilitate NiV entry. One possible mechanism is through specific cellular factors present in the different cell types.

In the present study, we examine the human neuronal cell protein responses to NiV infection and compare it to that of the mock-treated cells. The focus on neuronal cells is to help in understanding the reasons why NiV is not as efficiently replicated in this cell, whilst the infection is perhaps that caused the severe to fatal infection in humans. Total protein comparison is made using cellular proteins separated by the 2D-PAGE. The 2D-PAGE protein profile enabled direct comparison of the differentially expressed proteins between infected and non-infected samples. Moreover, using bioinformatics application, the differences in protein profile can be pin-pointed and the level of significance in expression can be quantitatively estimated. The method for separation of the NiV-infected and mock-infected SK-N-MC human neuronal cell proteins, and the 2D-PAGE protein profiles are described for the first time here. The number of proteins resolved by the 2D-PAGE across the different pI ranges is consistently reproducible and representative of the total number of proteins resolvable using the 2D-PAGE. At least 800 protein spots were used for the comparative analysis and each consensus gel is built from at least triplicate gels. Though sufficient number of proteins are resolved by the 2D-PAGE, there are possibly many other cellular proteins that are missed as these proteins are either inherently difficult to resolve such as the highly basic proteins and some membrane bound proteins, or they are present in very low abundance that is beyond the detection limit of the silver staining used in the 2D-PAGE. In spite of these limitations, invaluable information is still possible from the analysis of the abundantly expressed proteins in the standardized 2D-PAGE gels from the NiV-infected SK-N-MC cells.

The six significant differentially expressed proteins confidently identified using MS and MS/MS are important cellular proteins associated with various cell functions. The hnRNPs in particular are involved in the regulation of RNA synthesis of both cells and virus RNAs, and influence mRNA processing, trafficking, and stability [15, 16]. The hnRNPs H and H2 found suppressed in NiV-infected cells bind to a guanine-rich sequence in pre-mRNAs, downstream of the polyadenine [poly(A)] addition site, and activate or influence the efficiency of pre-mRNA processing [17]. The binding of H and H2 is affected by hnRNP F, found in abundance in NiV-infected SK-N-MC cells. The hnRNP F binds to the same sequence region as the hnRNPs H and H2 but it blocks the binding of the cleavage stimulatory factor 74 kDa subunit that results in the inhibition of the cleavage-polyadenylation reaction [18, 19]. The abundance of hnRNP F perhaps results in inhibition of polyadenylation of NiV mRNAs in neuronal cells infection [20, 21] and this may have affected the efficiency of NiV replication resulting in the low number of NiV released from infection of the human neuronal cells [12]. As the expression levels of hnRNP F and hnRNPs H and H2 is differentially regulated in pairs [18, 22], the findings from the present study could reflect the importance of the hnRNP F/hnRNP H and H2 ratio in the regulation of neuronal cell responses to NiV infection and replication. We also found that the G protein and the mitochondria associated proteins, VDAC2 and cytochrome bc1 are significantly increased in the NiV-infected human neuronal cells. The specific roles of these proteins in NiV infection are presently unknown. The G protein, however, is usually peripherally associated with the plasma membrane and plays important role in the signal transduction mechanism. One possible association between the increase in G protein and NiV infection is perhaps related to binding of NiV to ephrin-B2, a protein highly expressed in the neurons [13] that acts as receptor for NiV [23, 24] and activation of the G protein signaling pathways [25]. It is possible that increased expression of the G protein is to compensate for the lost of the G protein function following binding of NiV to ephrin-B2. Alternatively, the abundance of this protein in NiV infection could be important in controlling the infection, perhaps by modulating cellular responses to the infection through the Src-kinase and mitogen-activated protein kinase mediated pathways [26, 27]. The mitochondrial proteins VDAC2 and cytochrome bc1 found in abundance in NiV-infected human neuronal cells, on the other hand, are two proteins that could be associated with the induction of apoptosis and cellular pathologic response to the infection. Increase in VDAC2, a mitochondrial porin family [28] may contribute to the increase in the permeability and subsequently, causes the swelling of the mitochondrial matrix observed previously in NiV infected cells [12]. This can lead to the rupture of the mitochondrial outer membrane and release of the mitochondrial proapoptotic factors [29]. These factors then induce apoptosis to the neuronal cell cultures seen in the present study. Increased abundance of cytochrome bc1, a component of the ubiquinol-cytochrome c reductase complex (cytochrome bc1 complex) in NiV infection, on the other hand, is perhaps to help sustain the cytochrome bc1 complex/mitochondrial-associated activities as a consequent to the dysfunction of the mitochondrial respiratory chain or electron transport, or in providing extra energy required to support enhanced protein synthesis, particularly the proteins for virus replication and virus production [30]. While these are all possible, further investigation is required as the cytochrome bc1 complex is also associated with other cell functions including signal transduction and cytokine induction of intercellular adhesion molecule 1 (ICAM-1) expression [31, 32].

Our findings in this study identify the human neuronal cell proteins that are differentially expressed following NiV infection. This represents the first study using proteomic technologies that determine and identify cellular protein modifications in the course of NiV infection. The proteins identified are associated with various cellular functions and their abundance reflects the potential significance in the cytopathologic responses to the infection and the regulation of NiV replication. Whether these proteins differentiate human neuronal cells against the cellular responses of other highly susceptible cells to NiV infection remain to be investigated. Thus, future studies shall focus on the specific roles of each protein, in particular the role of hnRNPs and their relevance in the development of antiviral strategies against NiV and other henipaviruses.