Research On Human Rhinovirus 16 2B Protein Induced ER-stress Through PERK-eIF2α Pathway

The majority of cold and flu-like illnesses are caused by human rhinovirus (HRV). Improved detection of HRV has shown that HRV is also associated with more serious lower respiratory illness, such as asthma, wheezing illnesses in children, and fatal pneumonia in immune-compromised patients. Besides, HRV can co-infect with other viruses and bacteria to exacerbate asthma and chronic obstructive pulmonary disease (COPD). According to the gene sequence homology analysis, HRV can be divided into three groups, which are HRV-A,-B and HRV-C. The major group of HRVs, more than 90% of HRV-A and-B,bind to intercellular adhesion molecule (ICAM)-1,whereas only 10 serotype bind to low density lipoprotein (LDL) receptor. Upon entering a host cell, the HRV replicate in local epithelial cells and lymphoid tissues, causing inflammation and cell injury.However, the mechanism for HRV causing respiratory disease is not clear.HRV are members of the positive-sense single-strand RNA Picornaviridae family, for which the best studied prototype is poliovirus (PV) and which includes other important pathogens, such as foot-and-mouth disease virus (FMDV), hepatitis A virus(HAV), and coxsackie virus (CV). After infecting sensitive cells, HRVs disrupt epithelial barrier functions, which are mainly regulated by intercellular junctions, referred to as tight junctions (TJs) and adherens junctions (AJs), causing mucus overproduction and vascular permeability, especially destroyed the TJs. HRV does disrupt epithelial cell barrier function by decreasing both mRNA levels and protein levels of ZO-1, occludin, claudin-1, and E-cadherin after infecting cultured human nasal epithelial cells. In addition to increasing permeability, this disruption of the epithelial barrier will facilitate translocation of pathogens (including non-RV pathogens) and their soluble products, and expose basolateral epithelial receptors, where TLR and other PRRs are prominently located, inducing innate immune response. After infecting susceptible cells with Picornavirus, the structure and function of the host cells significantly change. These changes include increased intracellular vesicle formation, increased membrane permeability, disrupted Ca2+ balance in cells, which then induced cell apoptosis and autophagy.The endoplasmic reticulum (ER) widely exists in eukaryotic cells, it is primarily recognized as the main site for protein synthesis and modification. Also as the intracellular Ca2+ store place, it plays an important role in regulating the Ca2+ level, cellular stress response. Normally, there are 3 major receptors associated with ER stress exist on cell ER membrane:inositol-requiring enzyme 1 (Irel), RNA activated protein kinase(PKR)-like ER kinese(PERK) and activating transcription factor 6 (ATF6). In resting cells, all three ER stress receptors are maintained in an inactive state through binding with the ER chaperone, GRP78. On accumulation of unfolded proteins, GRP78 dissociates from the three receptors, which leads to their activation and triggers the unfolded proteins response(UPR). As a pro-survival response, the UPR reduces the accumulation of unfolded proteins and restore normal ER function. However, if persistent protein aggregation and stress cannot be resolved in time, signal of cellular pro-survival will switch into pro-apoptotic.Some researchs showed that above functional and morphological changes can also be observed in cells expressing the Enterovirus 2B protein only, inclu ding increased membrane permeability, disrupted Ca2+ balance in cells and so o n. Rhinoviruses non-structural protein 2B is comprised of only 95 amio acid. Using this amino acid sequence, we analyzed the topology of the integral mem brane protein and found the transmembrane region using available algorithms. The first region (39 VKWMLRIISAMⅧRNSS 57) is predicted to span the m embrane with a partial amphipathic cationic helix. The second predicted region (61 TIIATLTLIGCNGSPWRFL 79) is thought to be a transmembrane domain (TMD). HRV16 2B protein may anchor, span, and rearrange ER membranes, w hich leads to permeability changes by altering intracellular calcium concentratio ns and triggering ER stress.In order to understand the impact of HRV non-structural protein 2B on cells, we constructed eukaryotic expression vector of HRV16-2B (p2B-GFP), transfecting BHK-21 cells, and we found that nonstructural protein 2B of HRV 16 localized to ER and induced an ER stress response in BHK-21 cells. HRV 162B induced significantly high levels of ER chaperone glucose-regulated protein 78(GRP78), and activated activating transcription factor (ATF) 4 and ATF6 in a time-dependent manner. HRV 16 2B protein also significantly activated the important PERK-eIF2a pathway with marked increased in expression of p-PERK, p-eIF2a, and C/EBP homologous protein (CHOP). Additionally, HRV16 2B decreased the expression of phosphorylated inositol requiring protein-1(p-IRE1). Also,we don’t find the increase of ATF6 active form. Results suggest that HRV 16 protein 2B mediates ERS by activating PERK-eIF2 alpha signaling pathways, rather than the IRE1 and ATF6 pathways.In this study, we also found that HRV 16 protein 2B induces nuclear condensation and degradation of apoptosis marker proteins PARP, suggesting HRV16 protein2B induced cell apoptosis. Our study show that HRV 16 non-structural protein 2B induces ERS and cells apoptosis, which lays a certain foundation to illustrate the mechanism for cell damage caused by HRV protein 2B, also provides a new potential target for antiviral drugs.