Identification of H5N1 virus binding protein(s) on neuronal membrane using proteomic-based approaches

Abstract

Viral CNS infection is thought to occur by means of direct neuronal transmission of the virus pathogen. Virus-induced model of post-encephalitic Parkinsonism has been reported following infection with Japanese encephalitis virus (JEV) and a neurotropic influenza virus including H5N1. This study answers several fundamental questions about the susceptibility of the different target brain cells, especially neuron and glia to the well known (JEV) and less known (H5N1) neurotropic virus infection. A parallel study on JEV infection was performed to examine the involvement of microglial cells upon virus infection. It was found that JEV could replicate effectively in microglial cells and during the first 10 h of infection, the infectious progeny is released with high titers resulting in induction of apoptosis but not trigger nitric oxide production. Moreover, microglial cells are able to be persistently infected with JEV for at least 16 week. The persistently JEV-infected microglia also was able to infect neuroblastoma cells. In this part concluded that microglia can serve as a reservoir for JEV infection. Notably, neurotropism of the H5N1 virus has been documented in mammals. In human, H5N1 viruses could disseminate to several organs including brain. In literature, it has been reported that H5N1 virus can be predominantly detected in the dopaminergic neurons of the post-encephalitic parkinsonism model. Moreover, several reports have been proposed that influenza viruses are able to infect the desialylated cells, either directly or in a multistage process. Despite a number of studies, no study has ever been done to identify the H5N1 binding protein(s) on neuronal membrane. Hence, the aim of this study was to determine the human dopaminergic SH-SY5Y cells permissiveness to support the A/Thailand/NK165/05 (H5N1) virus infection and to identify the virus binding protein(s) using 1D-VOPBA, followed by LC-MS/MS applied. In this study showed that NK165 virus antigens could be strongly detected in cytoplasm of the infected cells with progress rapidly in cytopathology of nearly every cell in the monolayers. In a kinetic study demonstrated that the NK165 virus progeny was efficiently produced in SH-SY5Y cells and reached to maximum titers with the entire infected cells were destroyed. The results showed that there was a specific correlation between the degree of cytopathological changes, the increasing of virus antigens and virus production. Mass spectrometry identified the candidate NK165 virus binding proteins to be RACK1 and prohibitin. Although both proteins were unable to inhibit NK165 virus infection on SH-SY5Y cells but a small decrease of virus antigens was able to observe. The co-localization of RACK1 protein and virus antigens was detected in cytoplasm of the infected cells. In contrast, no prohibitin-specific signals can be seen in the infected cells. The results in this study indicated that, SH-SY5Y cells are highly permissive to NK165 virus infection. It is also possible that both RACK1 and prohibitin may be involved in H5N1 virus internalization and infection in human dopaminergic neuronal cells. While the exact mechanism of both proteins to H5N1 virus infection is not clear, the further study should be done to clarify the role of these proteins in mediating H5N1 virus entry on human neurons.