To date, many researchers have applied proteomics to study whole-cell proteomics during infection with influenza viruses such as H5N1, H3N2, and H1N1. However, we believe that compared with whole-cell proteomics, subcellular proteomics is more capable of identifying early diagnostic markers of influenza virus infection and is more conducive to the analysis of disease-related proteins and observation of the dynamic process of host cell infection with the virus. Influenza A virus can induce apoptosis [12], and the apoptotic pathway occurs in mitochondria [13]. To study the effect of infection with two different subtypes of influenza virus on the mitochondrial proteome, thus, there is no need to set a blank control. We performed subcellular mitochondrial proteomic analysis of A549 cells infected with H5 and H9 subtype avian influenza viruses was conducted. Compared with Control, H5N1 and H9N2 basically appeared different proteins after 24 hours of infection [14–15]. In order to better study the process of virus infecting the host, we chose the 24 hours time point to study the mitochondrial protein difference between two viruses with different pathogenicity.
2-D electrophoresis allows differential distribution of many protein isotypes. After data redundancy removal, we found that 16 proteins were upregulated and 16 were downregulated in the H5N1-infected group compared with the H9N2-infected group. However, further validation of the subcellular localization of some proteins is needed. Among the identified mitochondrial proteins, 6 mitochondrial proteins were downregulated and 2 mitochondrial proteins were upregulated in the H5N1-infected group compared with the H9N2-infected group.
After GO analysis, most differentially expressed proteins were binding proteins. A variety of binding proteins have been discovered that affect the virulence of influenza viruses, such as poly (rC)-binding protein 2 and nuclear export protein 1 [16–17]. We found that these differentially expressed binding proteins may be related to the mechanism of influenza virus infection. Therefore, our findings are helpful for further analysis of the mechanism that binds proteins to influenza viruses.
Among the upregulated mitochondrial proteins was the molecular chaperone HSPA1L, a member of the 70-kDa heat shock protein (HSP70) family that is localized to the mitochondrial matrix and whose coding gene is located on chromosome 6p21 in the HLA class III region [18]. Other studies, showed that HSP70 appears to be upregulated in HPAI compared to LPAI IAVs. This chaperone is involved in a variety of cellular processes, including folding and transport of newly synthesized polypeptides, proteolytic activation of misfolded proteins, and formation and dissociation of protein complexes [19]. The ECHS1 protein is found in mitochondria, peroxisomes, and smooth endoplasmic reticulum. The upregulated protein enoyl-CoA hydratase, encoded by ECHS1 on chromosome 10, is a 160-kDa hexamer enzyme consisting of 290 amino acids and is located in the mitochondrial matrix. ECHS1 is associated with mitochondrial short-chain and medium-chain fatty acid β-oxidation and branched-chain amino acid catabolic pathways, as well as other catabolic pathways[20]. In the absence of hepatitis B virus infection, the ECHS1 gene was subjected to RNA interference, and the proapoptotic genes Bid and Bax were found to be upregulated after transfection into HepG2 cells. However, in Xiao et al.’s study in hepatitis B virus-infected HepG2 cells, ECHS1, a binding protein of hepatitis B virus surface antigen, promoted HepG2 cell apoptosis. The coexistence of ECHS1 and hepatitis B virus surface antigen changed the expression of Bcl-2 family proteins, 12 proapoptotic proteins were upregulated, and 8 antiapoptotic proteins were downregulated [21]. The results of this study are consistent with those obtained after RNA interference in the absence of hepatitis B virus infection, indicating that not all viruses can use ECHS1 as a binding protein for viral surface antigens, thereby promoting apoptosis. Related studies, have confirmed that influenza virus can induce apoptosis. In our study, the difference in BAX expression detected by western blotting showed that the level of endogenous apoptosis induced by the highly pathogenic H5N1 virus was higher than that induced by the low pathogenic H9N2 virus. Endogenous apoptosis leads to mitochondrial swelling, disappearance of internal cristae and permeabilization, a possible reason for the difference in the virulence of these two viruses. In addition, downregulation of ECHS1 protein expression affects its fatty acid β oxidation pathway and reduces the replication ability of RNA viruses such as measles virus, vesicular stomatitis virus, and Semliki Forest virus [22]. In our results, the expression of ECHS1 protein was upregulated in the H5N1 virus-infected group compared with the H9N2 virus-infected group, which may explain why the H5N1 virus is more pathogenic than the H9N2 virus.
Among these downregulated mitochondrial proteins, the heat shock 70-kDa protein 1-like, malate dehydrogenase, mitochondrial membrane ATP synthase, and stomatin-like 2 proteins are located in the mitochondrial inner membrane, while the peroxiredoxin 5 and 60-kDa heat shock proteins are located in the mitochondrial matrix. HSPA1L indirectly affects body metabolism and biological function by regulating iron-sulfur protein maturation [23]; malate dehydrogenase is associated with the TCA cycle [24]; ATP synthase is involved in energy production and permeability transition pores (PTP, key players in cell death) ; stomatin-like protein 2 is involved in cell T cell activation, calcium homeostasis, and the stress response [25]; peroxiredoxin-5, which plays an anti-oxidative stress role in cell protection [26–27]; and 60-kDa heat shock protein is involved in controlling protein folding, the stress response, and the delivery of endogenous peptides to antigen presenting cells [28].
These eight differentially expressed mitochondrial proteins, especially ECHS1, may be used as new antiviral targets, but the results need to be further verified by a series of methods, such as RNA interference.
In IAV proteomic studies by other groups, 60-kDa heat shock protein, 70-kDa heat shock protein and ATP synthase subunits often appear as differentially expressed proteins. Is differential expression of these proteins shared by different IAVs? To date, a relatively small amount of proteomic data has been obtained for different IAVs; thus, the proteomic profiles of additional IAVs must be compared to augment this research to provide a basis for this possibility.
We hypothesized that H5N1 is highly pathogenic compared with H9N2, probably because of the upregulation and downregulation of the above eight mitochondrial proteins, which in turn inhibit T cell activation, antigen presentation, stress responses, and other processes. The increased mortality from H5N1 may also be due to metabolic abnormalities. A total of 42.3% of these differentially expressed proteins were involved in the apoptotic process, and we speculate that the altered levels of mitochondrial protein expression during IAV pathogenesis are due mainly to the difference in the endogenous apoptotic process. Our analysis also identified many other influencing factors, indicating that infection of the host cell is a complex process. Mechanistic analysis of these specific processes needs to be continuously augmented through numerous experimental studies.