As the membrane protein of ASFV, the function of p22 was rarely known. In attempt to get acquired of the p22 function, p22 interacted protein of the host were identified by a high throughput method and analyzed by Go terms and KEGG pathways, numerous cellular proteins in 293-T that interacted with p22 proteins were identified. This study provides a large database and useful tool. It will help to figure out the function of the p22.
In this study, Go terms mainly covered three parts: biological process, molecular function and cellular component. The top two enriched GO terms of biological process were cellular process and metabolic process, implying that p22 might utilize the host proteins directly or indirectly to affect the cell growth, function and stability. Main enriched GO terms of molecular function were binding, catalytic activity. GO analyses revealed that the most significant ontology categories of molecular function is binding, as the protein at inner envelop, we wish to dig the possible role of p22, like, the entry process of the virus, the high number of proteins that interacted with p22 protein were related to binding process, suggesting a role of p22 in virus binding and entry into the cell. The interesting result would inspire us to dig out the real function of p22 in virus entry.
The GO terms analysis of the interacted proteins in cell component mainly included cell part, organelle, protein-containing complex, membrane-enclose lumen, membrane, the results further verified the conclusion that p22 was located in the membrane of the viron and might participate in virus structure maintence and contact with the host membrane via the binding and endocytosis process. Of course the suspect needs further to be proven.
For KEGG pathways analysis, a large number of KEGG pathways were screened out as high as 165, the KEGG pathways that related proteins participated in mainly were Ribosomes, splicesome. Ribosomes are nanomachines essential for protein production and protein synthesis. The initial steps of ribosome biogenesis take place in the cell compartment. Spliceosome executes eukaryotic precursor messenger RNA (pre-mRNA) splicing to remove noncoding introns. It depends on the interaction of RNA-RNA, RNA-protein and protein-protein. It is composed of several nucleoproteins and has the function of recognizing 5 'splicing site, 3' splicing site and branching point of mRNA precursor. It indicated that p22 interacted proteins mainly participated in the process of gene expression in the host cells, gave us a hint that p22 affected the gene and protein expression of cell host, directly or indirectly affected the function of the biological process.
Morever, KEGG pathways that less number of p22 interacted proteins were involved in included pathogenic Escherichia coli infection, tight junction, necroptosis, ribosome biogenesis in eukaryotes, RNA transport, regulation of actin cytoskeleton, cardiac muscle contraction, adrenergic signaling in cardiomyocytes, Alzheimer disease, Huntington disease, etc. The widely affected pathways reflected the wide range of the functions that p22 or its related proteins.
It is noteworthy that KEGG pathways analysis showed that 7 p22-interacted proteins participated in endocytosis. As the results in GO analysis, a large number of p22 interacted proteins participated in binding. Above all, p22 was predicted to be involved in the entry process at the envelop of the virus.
At last, it was possible that other pathways had an important influence on the progression of ASFV entry via some biological process, such as cGMP-PKG signaling pathway, cAMP signaling pathway and AMPK signaling pathway which were screened out by KEGG analysis. cGMP is the intracellular second messenger that mediates the action of nitric oxide (NO) and natriuretic peptides (NPs), affecting a wide range of physiologic processes . cAMP is also one of the most common and universal second messengers, cAMP regulates pivotal physiologic processes including metabolism, secretion, calcium homeostasis, muscle contraction, cell fate, and gene transcription . AMPK is a central regulator of cellular energy homeostasis, regulating growth and reprogramming metabolism as well as in cellular processes including autophagy and cell polarity .
In those hub proteins connected in PPI network, the ADP-ribosylation factor (Arf) protein family is part of the large Ras superfamily which encompasses small GTPases . Among this family, ARF6 stimulates actin polymerization, drives phagocytosis through multiple mechanisms, and also assists autophagy as well . Other than Arf6, RAB10 also influenced the GTPase activity . Rab10 localizes on both Golgi and early endosomal/recycling compartments, plays an important roles in lysosome exocytosis and plasma membrane repair . Alpha actinins belong to the spectrin gene superfamily which represents a diverse group of cytoskeletal proteins. Alpha actinin is an actin-binding protein. In nonmuscle cells, it is involved in binding actin to the membrane. In skeletal, cardiac, and smooth muscle isoforms, it localized to the Z-disc and analogous dense bodies, participates in anchoring the myofibrillar actin filaments. ACTN4 encodes a nonmuscle, alpha actinin isoform which is concentrated in the cytoplasm and involved in metastatic processes . MYH9 is involved in several important functions, including cytokinesis, cell motility and maintenance of cell shape . ARPC2, Actin-related protein 2/3 complex subunit 2, containing 7 subunits, of which Arp2 and Arp3 belong to actin-related proteins . The activation of Arp2/3 complex could promote the synthesis of F-actin in the suitable condition . The Arp2/3 complex is involved in the rearrangement of the macrophage cytoskeleton and affected the phagocytosis of macrophages . Knockout of the Arp2/3 complex APC2 gene in mouse macrophages results in decreased F-actin polymerization, followed by decreased phagocytic ability . In summary, the key proteins mentioned above and other hub proteins in PPI network were closely related to actin filament organization and movement, resulting in affecting the process of phagocytosis and endocytosis. Additional studies on the role of p22 in the process of endocytosis should be conducted.