CV-A10-associated HFMD typically manifests mild and self-limiting symptoms, but occasionally exhibits severe CNS manifestations and even death in a few cases[4]. In recent years, the increasing incidence of CV-A10 infection has been an alarming trend, and even have exceeded EV-A71 and CV-A16 infections in several countries and regions, which causes a widely concern for public health[29]. However, there is still no specific medicine or vaccine registered against CV-A10[4]. Moreover, the detailed molecular mechanism of CV-A10 has not been thoroughly studied, thereby there is an urgent need to develop effective antiviral drugs and vaccines, and meanwhile further reveal the molecular determinants of CV-A10-induced HFMD[4, 23]. Previous studies have clearly indicated that after the virus enters the infected cell, it generally begins to replicate and produce many host-pathogen interactions[15, 36]. Complex interactions between host and virus have been shown to cause changes in protein levels which further lead to specific host cell processes, thus the alterations of cellular proteins are crucial for identifying the pathogenesis of viral infections[36]. During the past decade, proteomic methods have become important tools with which to study host cellular responses to viral infection and provide specific insights into the cellular mechanisms involved in viral pathogenesis[9, 37]. Thus, in this work, we adopted the TMT labeling-based quantitative proteomics and found a large number of differentially expressed proteins that may inform function under physiologically relevant, unbiased conditions, and provide new therapeutic targets for CV-A10-induced HFMD.
Here, we were able to quantitate 6615 proteins and identify 293 proteins as stably and significantly differentially regulated. And it was observed that 104 proteins were remarkably up-regulated and 189 proteins were markedly down-regulated. Thereafter, a bioinformatic analysis revealed that these differentially expressed cellular proteins were assignable to several biological processes and signaling pathways. It was found that dysregulated proteins were mainly focused on metabolic-related processes, such as small molecule metabolic process, organic substance metabolic process, protein metabolic process, organonitrogen compound metabolic process, glutamine metabolic process, and so on. However, metabolism consists of a series of reactions that occur within cells of living organisms to sustain life[20]. The process of metabolism involves many interconnected cellular pathways to ultimately provide cells with the energy required to carry out their function[5, 20]. Thus, the perturbation of metabolic process in host cells was considered to be directly affect the process of CV-A10 infection. In the enrichment results of KEGG pathway, it was seen that “Proteasome” is the top 1 pathway in up-regulated proteins, and “Ribosome” is the top 1 in down-regulated proteins. “Proteasome”, a class of molecules in eukaryotes, cleans up misfolded proteins in cells, prevents them from producing cytotoxicity, and plays a crucial role in cell signal transduction and cell function regulation[3]. However, “Ribosome”, a staggeringly complex molecular machine, is crucial for the interpretation of genetic information by translating the encoded messenger RNA (mRNA) into functional proteins in all cell types[44] Thence, “Proteasome” and “Ribosome” actually dominate the process of protein degradation and synthesis in the cell. Currently, it was revealed that “Proteasome” and “Ribosome” are closely involved in the virus-host interactions. For example, viruses could utilize the ubiquitin-proteasome system for their own survival which includes various stages of the viral life cycle and interferes with the host’s antiviral response in a variety of ways, such as immune evasion by down-regulating cell-surface immune molecules, regulation of viral gene transcription, inhibition of cell apoptosis, and promotion of viral budding and release[8]. Meanwhile, viruses completely rely on the protein synthesis machinery of host cells to propagate and have evolved various mechanisms to redirect the host’s ribosomes toward their viral mRNAs[16]. Thus, the opposite expression pattern of “Proteasome” and “Ribosome” just showed that CV-A10 uses host proteins for its survival and proliferation. Additionally, many nervous-related pathways, such as Neurotrophin signaling pathway, GABAergic synapse, Glutamatergic synapse, were enriched. Actually, previous studies have confirmed that neurotrophins are a family of closely related proteins that were identified initially as survival factors for sensory and sympathetic neurons, and have since been shown to control many aspects of survival, development and function of neurons in both the peripheral and the central nervous systems[18, 33]. GABAergic synaptic plays a crucial role in neuronal firing, excitability, and synaptic plasticity to regulate neuronal circuits[24, 30]. Astrocytes are widely acknowledged as inseparable element of glutamatergic synapses and the aberrant changes in glutamatergic astroglial signaling are found to be highly linked to both neuroprotective and pathogenic in neurological and neurodegenerative diseases[14, 27]. However, it has been demonstrated that CV-A10-associated HFMD commonly shows mild and self-limiting symptoms; nonetheless, a few cases present with various severe nervous system manifestations, such as encephalitis, acute flaccid paralysis, neurorespiratory syndrome, and even death[4]. Hence, the changes of these nervous-related pathways might be closely associated with the neuropathogenesis of CV-A10 infection. Meanwhile, MAPK signaling pathway was also observed to be up-regulated, and mounting evidence indicated that MAPK signaling pathway is a classical inflammatory pathway. However, substantial inflammatory pathological damages were observed in multi-organs, including the lung, heart, liver, and kidney of CV-A10-infected rhesus macaques model, which was consistent in severe and critically ill patients with CV-A10 infection[12].
Next, we performed an analysis of protein domains and sub-cellular localization. Protein domains are independent, functional, and stable structural units of proteins[28]. Accurate protein domain boundary prediction plays an important role in understanding protein structure and evolution, as well as for protein structure prediction[26, 28]. It was observed that the most significant changed protein domain was “Cold-shock protein, DNA-binding”. It was previously reported that cold-shock proteins are small, acidic proteins which are primarily responsible for nucleic acid binding and perform mRNA translation acting as “RNA chaperones”[34]. Hence, the perturbance of “Cold-shock protein, DNA-binding” might be involved in the process of mRNA translation during CV-A10 infection. However, the subcellular localization of a protein directly determines its physiological function[11]. Our results presented that the dysregulated proteins were localized on 14 subcellular components and it was also visualized that the dysregulated proteins were mostly distributed in nuclei. It was well-known that the eukaryotic nucleus shows organized structures of chromosomes, transcriptional components and their associated proteins, but the proteins located in nuclei are often not fixed, and under different physiological and pathological environment, some protein located in the nucleus might change its position[11, 39]. For instance, in its inactive form, NF-κB is sequestered in the cytoplasm, while in its active form, the exposure of the nuclear localization signals on NF-κB subunits and the subsequent translocation of the molecule to the nucleus which further activates its downstream factors[25]. Thence, the alterations of subcellular location of these differentially expressed proteins in response to CV-A10 infection might be cause extensive intracellular activities. Finally, to understand the impact of CV-A10 on the interactions in host cells, global protein network of differentially expressed protein was constructed.
Finally, among these differentially expressed proteins, it was found that HMGB1 was significantly decreased during CV-A10 infection in this work. Actually, HMGB1, a highly abundant, multi-functional, DNA-binding nuclear protein, predominantly regulates gene transcription, DNA repair, as well as chromatin stability in quiescent cells[7]. However, in the recent years, it was reported that potentially participates in the regulation of viral replication cycles with complicated mechanisms[10]. For example, Japanese encephalitis virus restricts HMGB1 expression to maintain MAPK pathway activation for viral replication[40]. HMGB1 promotes hepatitis C virus replication by interaction with stem-loop 4 in the viral 5’-untranslated region[42]. However, whether HMGB1 plays a role in viral replication of CV-A10 during infection is still unknown. Thus, in this work, we adopted the knockdown and overexpression vectors of HMGB1 to examine the role of HMGB1 in viral replication. Treatment with shRNA of HMGB1 dramatically restricted the expression of VP1 and CV-A10 titer, but treatment with HMGB1-overexpressed vector notably promoted the expression of VP1 and CV-A10 titer. Therefore, these data indicated that HMGB1 might be required for the enhancement of CV-A10 replication.
Collectively, proteomic analysis is primarily a tool to generate hypothesis on the mechanisms and protein involvement in viral infectious disease. And in this work, we reported a large data set of changes in cellular proteomes of 16HBE cells infected with CV-A10, and it was mainly highlighted specific cellular proteome changes related to specific biological processes, signaling pathways, protein domains, subcellular localizations, as well as PPI network. To our knowledge, this study is the first attempt to present the proteomic analysis of 16HBE cells with CV-A10 infection, and then after a set of bioinformatics analysis, our findings advance our understanding of the pathogenesis of CV-A10 infection and may facilitate the search for potential protein targets for further studies and antiviral development. In addition, according to our proteomic results, we also screened HMGB1 to further explore. Interestingly, it was identified a key function of HMGB1 on CV-A10 replication, suggesting that the use of HMGB1 as possible targets for CV-A10 inhibition in the future.