iTRAQ protein profile analysis of neuroblastoma (NA) cells infected with the rabies viruses rHep-Flury and Hep-dG

Abstract

The rabies virus (RABV) glycoprotein (G) is the principal contributor to the pathogenicity and protective immunity of RABV. In a previous work, we reported that recombinant rabies virus Hep-dG, which was generated by reverse genetics to carry two copies of the G-gene, showed lower virulence than the parental virus rHep-Flury in suckling mice with a better immune protection effect. To better understand the mechanisms underlying rabies virus attenuation and the role of glycoprotein G, isobaric tags for relative and absolute quantitation (iTRAQ) was performed to identify and quantify distinct proteins. 10 and 111 differentially expressed proteins were obtained in rHep-Flury and Hep-dG infection groups, respectively. Selected data were validated by western blot and qRT-PCR. Bioinformatics analysis of the distinct protein suggested that glycoprotein over-expression in the attenuated RABV strain can induce activation of the interferon signaling. Furthermore, it may promote the antiviral response, MHC-I mediated antigen-specific T cell immune response, apoptosis and autophagy in an IFN-dependent manner. These findings might not only improve the understanding of the dynamics of RABV and host interaction, but also help understand the mechanisms underlying innate and adaptive immunity during RABV infection.

Rhabdovirus Infection Is Dependent on Serine/Threonine Kinase AP2-Associated Kinase 1

“…Therefore, AAK1 is involved in the entry of viruses via its function of regulating clathrin-mediated endocytosis. In our previous study, we showed that RABV infection increases AP-2-associated protein kinase 1 (AAK1) expression in mouse neuroblastoma (N2a) cells through proteomics analysis based on isobaric tags for relative and absolute quantitation (iTRAQ) [27]. In this study, we further confirmed the role of AAK1 in RABV infection.…”

“…Our previous study showed that rabies viruses (HEP-Flury strain and rHEP-dG strain) proliferation increased AAK1 expression in N2a cells by iTRAQ protein profile analysis [27]. To confirm these results, N2a cells were infected with RABV rHEP-GFP, which carries a green fluorescent protein gene between the N and P genes [29], to investigate the expression of AAK1.…”

Phenotypic Consequences In vivo and In vitro of Rearranging the P Gene of RABV HEP-Flury

“…Cell apoptosis is a particular factor attenuating the pathogenic potential of RABV ( Préhaud et al, 2003 ; Thoulouze et al, 2003 ; Kassis et al, 2004 ). Previous work has shown that, at a MOI of 0.01, rHEP-Flury does not cause toxicity in NA cells, and only induced 2.9% NA cell apoptosis at 48 h post-infection ( Yang et al, 2015b ; Peng et al, 2016 ). In this study, at a MOI of 3, the percentage of early stage apoptotic cells as well as late stage apoptotic or even necrotic cells was about 11.8%.…”

“…Meanwhile, we found that the RABV P4 which G mRNA ratio increased, its pathogenicity was significant weaker than others. As HEP-Flury G protein could induce cell apoptosis which was contribute to attenuate the pathogenicity of RABV ( Yang et al, 2015b ; Peng et al, 2016 ). We speculated that the pathogenicity of RABV was correlated with the ratio of viral gene.…”

“…G protein was a key element for viral spread in CNS and a G gene deletion-mutant RABV cannot spread beyond initially infected cells ( Takayama-Ito et al, 2006 ; Wickersham et al, 2007 ; Beier et al, 2013 ). But as for HEP-Flury, G protein could induce the apoptosis of NA cells ( Liu et al, 2014 ; Yang et al, 2015b ), consequently limiting the viral spread in CNS ( Lay et al, 2003 ; Sarmento et al, 2005 , 2006 ). Here the results indicated that the viral replication mainly affected efficiency of cell-to-cell spread but not apoptosis-inducing ability.…”