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. 2024 Aug 8;25(16):8678.
doi: 10.3390/ijms25168678.

A Combined Transcriptomic and Proteomic Analysis of Monkeypox Virus A23 Protein on HEK293T Cells

Yihao Wang  1   2   3 , Yihan Li  1   2   3 , Mingzhi Li  1   2   3 , Keyi Wang  1   2   3 , Jiaqi Xiong  1   2   3 , Ting Wang  1   2   3 , Yu Wang  1   2   3 , Yunli Guo  1   2 , Lingbao Kong  1   2   3 , Meifeng Li  1   2   3
Affiliations

A Combined Transcriptomic and Proteomic Analysis of Monkeypox Virus A23 Protein on HEK293T Cells

Yihao Wang et al. Int J Mol Sci. .

Abstract

Monkeypox virus (MPXV) is a cross-kingdom pathogen infecting both humans and wildlife, which poses a significant health risk to the public. Although MPXV attracts broad attention, there is a lack of adequate studies to elucidate pathogenic mechanisms associated with viral infections. In this study, a high-throughput RNA sequencing (RNA-seq) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach was used to explore the transcriptional and metabolic responses of MPXV A23 protein to HEK293T cells. The protein-protein interactions and signaling pathways were conducted by GO and KEGG analyses. The localization of A23 protein in HEK293T cells was detected by immunofluorescence. A total of 648 differentially expressed genes (DEGs) were identified in cells by RNA-Seq, including 314 upregulated genes and 334 downregulated genes. Additionally, liquid chromatography-tandem mass spectrometry (LC-MS/MS) detected 115 cellular proteins that interact with the A23 proteins. Transcriptomic sequencing analysis revealed that transfection of MPXV A23 protein modulated genes primarily associated with cellular apoptosis and DNA damage repair. Proteomic analysis indicated that this protein primarily interacted with host ribosomal proteins and histones. Following the identification of the nuclear localization sequence RKKR within the A23 protein, a truncated mutant A23ΔRKKR was constructed to investigate the subcellular localization of A23 protein. The wild-type A23 protein exhibits a significantly higher nuclear-to-cytoplasmic ratio, exceeding 1.5, in contrast to the mutant A23ΔRKKR, which has a ratio of approximately 1. Immunofluorescence assays showed that the A23 protein was mainly localized in the nucleus. The integration of transcriptomics and proteomics analysis provides a comprehensive understanding of the interaction between MPXV A23 protein and the host. Our findings highlight the potential role of this enzyme in suppressing host antiviral immune responses and modulating host gene expression.

Keywords: A23R; innate immune signaling pathway; mpox virus; proteomic; transcriptome.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Construction and expression of recombinant A23R in HEK293T cells. (A) Model of a cloned fragment. (B) Double digestion of the pCAGGS−HA−A23R plasmid. Lane 1–4: A23R recombinants were digested by EcoR I and Xho I. (C) Expression of recombinant A23R in HEK293T cells, followed by immunoblot analysis using HA−tag antibodies or anti β−actin antibodies. Lane 1: Transfected with pCAGGS−HA. Lane 2: Transfected with pCAGGS−HA−A23R. (D) Western blot analysis of the Co−IP sample. Flow Through: the WCL after anti−HA incubate.
Figure 1
Figure 1
Construction and expression of recombinant A23R in HEK293T cells. (A) Model of a cloned fragment. (B) Double digestion of the pCAGGS−HA−A23R plasmid. Lane 1–4: A23R recombinants were digested by EcoR I and Xho I. (C) Expression of recombinant A23R in HEK293T cells, followed by immunoblot analysis using HA−tag antibodies or anti β−actin antibodies. Lane 1: Transfected with pCAGGS−HA. Lane 2: Transfected with pCAGGS−HA−A23R. (D) Western blot analysis of the Co−IP sample. Flow Through: the WCL after anti−HA incubate.
Figure 2
Figure 2
Identification of differentially expressed genes (DEGs). (A) The volcano diagram of DEGs. The horizontal axis represents the fold change in gene expression between the experimental and control groups (log2FoldChange). The vertical axis represents the significance of the DEGs between the experimental and control groups (−log10padj or −log10pvalue). Up−regulated genes are shown as red dots. Down−regulated genes are shown as green dots. Blue dots indicate no statistically significant genes (NO 25029). Threshold lines for DEGs screening criteria are indicated by blue dashed lines. (B) Heatmap of DEGs with length, type, and chr. The horizontal coordinate represents the sample name. The vertical coordinates on the left represent the cluster analysis. The vertical coordinates on the right represent length/type/chr. The heatmap specifies the length of each gene (length), categorizes its functions (type), and determines its position in the chromosome (chr). The red color in the middle of the heatmap represents high expression, and the green color represents low expression.
Figure 2
Figure 2
Identification of differentially expressed genes (DEGs). (A) The volcano diagram of DEGs. The horizontal axis represents the fold change in gene expression between the experimental and control groups (log2FoldChange). The vertical axis represents the significance of the DEGs between the experimental and control groups (−log10padj or −log10pvalue). Up−regulated genes are shown as red dots. Down−regulated genes are shown as green dots. Blue dots indicate no statistically significant genes (NO 25029). Threshold lines for DEGs screening criteria are indicated by blue dashed lines. (B) Heatmap of DEGs with length, type, and chr. The horizontal coordinate represents the sample name. The vertical coordinates on the left represent the cluster analysis. The vertical coordinates on the right represent length/type/chr. The heatmap specifies the length of each gene (length), categorizes its functions (type), and determines its position in the chromosome (chr). The red color in the middle of the heatmap represents high expression, and the green color represents low expression.
Figure 3
Figure 3
RT−qPCR verified the expression of seven genes. We examined the gene expression levels of IL9R, PLA2G4C, MAFA, CYP2E1, H3C1, H2BC17, HLA−DPB1 LIG4, BIRC3, SMAC, RAD9, and SGO2 by RT−qPCR in HEK293T cells after the expression of A23 protein. RNA expression levels in each system were normalized to β−actin. The error bars indicate the SD of repeated RT−qPCR. All experiments were conducted in−dependently, at least three times. Statistical significance is indicated by ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 4
Figure 4
Functional enrichment analysis of DEGs. The scatter plot of GO (A) and KEGG (B) analysis. The vertical axis represents the top thirty terms with the most significance. The horizontal axis represents the gene ratio. Count: the number of DEGs. Gene ratio: the ratio of DEG number to background gene number. p-value: indicators of the significance of the term; the smaller the p-value, the more significant the term.
Figure 5
Figure 5
Functional enrichment analysis of the interacting proteins. The scatter plot of GO (A) and KEGG (B) analysis. The vertical axis represents the top thirty terms with the most significance. The horizontal axis represents the ratio. Count: the number of proteins. Gene ratio: the ratio of protein number to background protein number. p-value: indicators of the significance of the term, the smaller the p-value, the more significant the term.
Figure 5
Figure 5
Functional enrichment analysis of the interacting proteins. The scatter plot of GO (A) and KEGG (B) analysis. The vertical axis represents the top thirty terms with the most significance. The horizontal axis represents the ratio. Count: the number of proteins. Gene ratio: the ratio of protein number to background protein number. p-value: indicators of the significance of the term, the smaller the p-value, the more significant the term.
Figure 6
Figure 6
Mutation of the RKKR impairs nuclear import of A23 protein. The nuclear location signal in A23 protein was identified through NLStradamus (A), NucPred (B), and cNLS Mapper (C). HEK293T cells were transiently transfected with plasmic coding for HA-A23 and the A23△RKKR. (D) Transiently transfected cells were fixed, stained with DAPI, and analyzed by fluorescent Inverted microscope. (E) Nuclear localization of A23 protein and mutants was assessed in transiently transfected cells as a ratio of nuclear to cytoplasmic fluorescence using the Image J software version 1.54j. Data are from n = 4 fluorescent cells analyzed. ** p < 0.05 difference from WT-A23 transfected cells. Scale bar = 50 µm.
Figure 6
Figure 6
Mutation of the RKKR impairs nuclear import of A23 protein. The nuclear location signal in A23 protein was identified through NLStradamus (A), NucPred (B), and cNLS Mapper (C). HEK293T cells were transiently transfected with plasmic coding for HA-A23 and the A23△RKKR. (D) Transiently transfected cells were fixed, stained with DAPI, and analyzed by fluorescent Inverted microscope. (E) Nuclear localization of A23 protein and mutants was assessed in transiently transfected cells as a ratio of nuclear to cytoplasmic fluorescence using the Image J software version 1.54j. Data are from n = 4 fluorescent cells analyzed. ** p < 0.05 difference from WT-A23 transfected cells. Scale bar = 50 µm.
Figure 7
Figure 7
A combined transcriptomic and proteomic analysis of monkeypox virus A23 protein on HEK293T cells.
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