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. 2022 Sep 14:13:990978.
doi: 10.3389/fmicb.2022.990978. eCollection 2022.

Isobaric tags for relative and absolute quantification-based proteomic analysis of host-pathogen protein interactions in the midgut of Aedes albopictus during dengue virus infection

Jiatian Wang  1 , Peiyang Fan  1 , Yong Wei  1 , Jiaqi Wang  1 , Weihao Zou  1 , Guofa Zhou  2 , Daibin Zhong  2 , Xueli Zheng  1
Affiliations

Isobaric tags for relative and absolute quantification-based proteomic analysis of host-pathogen protein interactions in the midgut of Aedes albopictus during dengue virus infection

Jiatian Wang et al. Front Microbiol. .

Abstract

Aedes albopictus (Ae. albopictus), an important vector of dengue virus (DENV), is distributed worldwide. Identifying host proteins involved in flavivirus replication in Ae. albopictus and determining their natural antiviral mechanisms are critical to control virus transmission. Revealing the key proteins related to virus replication and exploring the host-pathogen interaction are of great significance in finding new pathways of the natural immune response in Ae. albopictus. Isobaric tags for relative and absolute quantification (iTRAQ) was used to perform a comparative proteomic analysis between the midgut of Ae. albopictus infected with DENV and the control. 3,419 proteins were detected, of which 162 were ≥ 1.2-fold differentially upregulated or ≤ 0.8-fold differentially downregulated (p < 0.05) during DENV infections. Differentially expressed proteins (DEPs) were mainly enriched in ubiquitin ligase complex, structural constituent of cuticle, carbohydrate metabolism, and lipid metabolism pathways. We found that one of the DEPs, a putative pupal cuticle (PC) protein could inhibit the replication of DENV and interact with the DENV-E protein. In addition, the result of immunofluorescence (IF) test showed that there was co-localization between ubiquitin carboxyl-terminal hydrolase (UCH) protein and the DENV-E protein, and virus infection reduced the level of this protein. iTRAQ-based proteomic analysis of the Ae. albopictus midgut identified dengue infection-induced upregulated and downregulated proteins. The interaction between the PC and UCH proteins in the midgut of Ae. albopictus might exert a natural antiviral mechanism in mosquito.

Keywords: Ae. albopictus; DENV; PC protein; UCH protein; iTRAQ.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Quantitative proteomics of the Ae. albopictus midgut during DENV infection. (A) Schematic diagram of the experimental procedure. Ae. albopictus mosquitoes were infected with DENV. Quantitative proteomics analysis was carried out at 14 days post infection. (B) The levels of 3,419 proteins between the infected and uninfected Ae. albopictus groups were determined by three repeated experiments. (C) Heat map of differential expressed proteins with a ratio of more than 1.2 times. Each column in the diagram represents a sample and each row represents a protein, and red represents a high levels and blue represents a low level. MP means mosquito-positive group, MN means mosquito-negative group.
FIGURE 2
FIGURE 2
KEGG functional classification of the DEPS in the midgut of Ae. albopictus identified by iTRAQ analysis. (A) KEGG functional classification of upregulated proteins; (B) KEGG functional classification of downregulated proteins. DEP, differential expressed protein; KEGG, Kyoto Encyclopedia of Genes and genomes.
FIGURE 3
FIGURE 3
GO enrichment analysis of DEPs. The abscissa is the (A) GO-biological process enrichment; (B) GO-cellular component enrichment; and (C) GO-molecular function enrichment. The abscissa is the p-value of log10 conversion. The larger the value, the higher the significance of the enrichment in this category. The ordinate represents the name of the enrichment category, and the larger the circle, the more the number of differential proteins in this category. The color of the circle represents the fold enrichment of the protein, and the redder the color, the greater the enrichment.
FIGURE 4
FIGURE 4
qPCR validation of transcript levels between infection and infection groups. Midguts were dissected from mosquitoes and RNA was extracted on day 14th after the mosquito infected with DENV, qPCR was used to verify the expression levels of DEPs between the infected group and the uninfected group. (A) The down-regulation proteins of infection group. (B) The up-regulation proteins of infection group (**P < 0.01, ***P < 0.001).
FIGURE 5
FIGURE 5
PC protein inhibits the expression of DENV-E protein. (A) Empty vector were transfected into C6/36 cells, and cells were inoculated with DENV (MOI = 1) 48 h after transfection, lysates of C6/36 cells at 24 hpi were detected by SDS-PAGE and WB using the antibodies as indicated. (B,C) 0, 1, 2 μgpAC5.1b-myc-YFP-Cuticle-HA was transfected into C6/36 cells, respectively, and DENV (MOI = 1) was infected 48 h after transfection. The cells’ RNA and protein were extracted from the cells at 24 hpi and detected by qRT-PCR and WB as indicated, respectively. (D) BHK-21 cells were transfected with pcDNA3.1(+)-YFP-Cuticle-HA plasmids and infected with DENV (MOI = 1) at 24 h after transfection. Cells were fixed at 24 hpi, and incubated with a DENV-E protein monoclonal antibody and the corresponding fluorescent secondary antibody. A YFP-PC-HA fusion protein was expressed to detect PC protein (green), localization of DENV-E was detected with antibodies to fluorescent secondary antibody (red), nuclei were stained with DAPI (blue). The upper and lower images in each column are of two fields of view in the same slide. (A,E) COS7 cells were transfected with pcDNA3.1 (+)-YFP-EEV6-HA plasmid and infected with DENV (MOI = 1) at 24 h after transfection, and proteins were extracted at 24 hpi for WB and Co-ip analysis. All experiments were performed at least three times, *denotes a P-value < 0.005, **denotes a P-value < 0.001.
FIGURE 6
FIGURE 6
DENV infection results in degradation of UCH. (A) pAC5.1b-myc-UCH-HA plasmids were transfected into C6/36 cells with a gradient of 0, 1, and 2 μg, and DENV (MOI = 1) was infected 48 h after transfection. Cellular proteins were extracted at 24 hpi. (B) 1.5 μg of pcDNA3.1 (+)-UCH-HA plasmids were transfected into BHK-21 cells, and 24 h after transfection, dengue virus (MOI = 1) was infected. The cells were fixed at 24 hpi for immunofluorescence assay. Localization of DENV-E and HA-UCH were detected by co-immunostaining with antibodies to fluorescent secondary antibody (green) and HA tag (red). Nuclei were stained with DAPI (blue). The upper and lower images in each column are of two fields of view in the same slide.
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