E2 Ubiquitin-Conjugating Enzymes Regulates Dengue Virus-2 Replication in Aedes albopictus

Abstract

Aedes albopictus, a major vector of dengue virus (DENV), has a global distribution. Identifying the key components of the ubiquitin system of A. albopictus essential for the replication of viruses could help identify targets for developing broad-spectrum antiviral strategies. This study explores the interaction between E2 ubiquitin-conjugating enzymes (Ubc9) and DENV-2 proteins (NS1, NS5, and E) using cell culture and mosquito models. The replication of DENV-2 and the knockdown efficiency of the Ubc9 gene were assessed through reverse transcription-quantitative polymerase chain reaction. The DENV-2-related protein expression was evaluated via Western blot analysis. The interaction between Ubc9 and DENV E and NS5 proteins was investigated through confocal immunofluorescence and co-immunoprecipitation. RNA interference technology was employed to silence Ubc9 expression in C6/36 cells and in A. albopictus mosquitoes. The expression level of Ubc9 in the DENV-2-infected group was 3.5-fold higher than that in the control group. The Ubc9 gene expression in the midgut tissue of the mosquito was significantly upregulated. Transfection of C6/36 and BHK-21 cells with the pAc5.1b-EGFP-Ubc9-HA vector led to the overexpression of Ubc9, which decreased the transcription levels of DENV E and NS1, NS5 proteins. The difference was statistically significant (F = 24.27, p < 0.01). The expression levels of DENV NS5 and E proteins significantly decreased after infection with DENV-2, suggesting that the depletion of Ubc9 may limit the replication of DENV-2. Ubc9 regulates DENV-2 replication through SUMOylation in the cells and A. albopictus, potentially affecting vector competence and DENV transmission. This is the first study to demonstrate that the Ubc9 of A. albopictus plays a significant role in regulating the replication of DENV in both mosquito cells and the mosquito itself. The study results may prove useful in designing appropriate therapeutic approaches for dengue and associated complications.

Keywords: Aedes albopictus; E2 ubiquitin-conjugating enzymes; SUMOylation; dengue virus-2; regulates.

Figure 1

qPCR validation of different expression levels of Ubc9 between the virus-infected group and the control group. A t-test was used for statistical analysis, and the results showed that the expression level of Ubc9 in DENV-2-infected A. albopictus was significantly upregulated (t = 12.83, p < 0.001).

Figure 2

High expression of Ubc9 protein inhibits the expression of DENV NS1 and E in C6/36 cells. (A) Expression levels of DENV E and NS1 proteins after the gradient transfection of an empty vector into C6/36 cells. (B) Expression levels of DENV E and NS1 proteins after the gradient transfection of Ubc9. When the concentration of pAc5.1b-EGFP-Ubc9-HA in C6/36 cells was increased, the expression of DENV-2 E and NS1 proteins began to decrease. (C) The copy number of the DENV transcription level after the gradient transfection of Ubc9 exhibited a downward trend compared with that in control group (p < 0.05), which was determined by using ANOVA statistical analysis.

Figure 3

High expression of Ubc9 protein inhibits the expression of DENV NS5 in BHK-21 cells. (A) Expression level of DENV NS5 after empty vector transfection into BHK-21 cells. (B) Expression levels of DENV NS5 after gradient transfection of Ubc9. ANOVA statistical analysis was conducted, which showed that the transcription level of the DENV E protein exhibited a downward trend compared with that in control group (p < 0.05).

Figure 4

Interaction between Ubc9 and DENV E, as confirmed by CO-IP assay. Lane 1: blank control group; lane 2: 3 μg of pcDNA3.1(+)-EGFP-Ubc9-HA plasmid group; lane 3: DENV infection group; lane 4: 3 μg of pcDNA3.1(+)-EGFP-Ubc9-HA plasmid+DENV infection group. Anti-HA-specific rabbit monoclonal antibody and protein A agarose bead were added in the Co-IP experimental group (IP group). The cell lysate was detected by Western blot with anti-HA antibody. Positive control (Input): Ubc9, the target protein, and the DENV E protein, the protein with which it was predicted to interact, were confirmed to be present in the protein sample. Western blot analysis was performed directly on the cell lysate using anti-HA antibody and anti-DENV E protein-specific antibody to verify the presence of Ubc9 and DENV E proteins in the cell lysate. Output group: Equal amounts of normal rabbit IgG and protein A agar agar beads were added to conduct the Western blot assay of Ubc9 and DENV E proteins, respectively. The experimental results prove that Ubc9 and DENV E interact with each other.

Figure 5

Colocalization of Ubc9 with DENV-2 E protein, as depicted through IF.

Figure 6

Silencing of Ubc9 inhibited the expression of DENV E and NS5 proteins. (A) ANOVA statistical analysis showed significant difference in the expression level of Ubc9 compared with that in the blank control group and the no-load treatment group (p < 0.5), in which the C6/36 cells were transfected with an empty lentiviral plasmid (pLKO.1-CMV-copGFP-PURO) and a lentiviral vector comprising a Ubc9-specific shRNA. After transfection for 24–48 h, the C6/36 cells transfected with different plasmids were infected with DENV-2 (MOI = 1). The results show that the expression levels of DENV-2 NS5 and E proteins was lower in experiment group (silencing of Ubc9 group). (B) Expression of DENV decreased after silencing the Ubc9 gene. The viral replication level was analyzed by RT-qPCR. Compared with the blank control and no-load treatment groups, the expression level of Ubc9 in the experimental group decreased, and the difference was statistically significant (F = 11.05, p < 0.05). (C) Expression of DENV E and NS5 proteins decreased after silencing the Ubc9 gene. Western blot results show that after transfecting C6/36 cells with a lentiviral vector containing Ubc9-specific shRNA, the expression levels of DENV NS5 and E protein significantly decreased after infection with DENV-2, suggesting that the depletion of Ubc9 may limit the replication of DENV-2.

Figure 7

Silencing of the Ubc9 gene in Aedes albopictus decreased the replication of DENV proteins. (A) RT-qPCR test confirmed the silencing of the Ubc9 gene in A. albopictus; a: blank control group, b: PBS injection group, c: dsRNA injection group. Female mosquitoes were injected with approximately 300 nL of dsRNA. The control group was injected with 300 nL of PBS. The total RNA of the mosquitoes was collected after 3 days of injection. The silencing efficiency of Ubc9 was measured by RT-qPCR on day 3. The results show that female A. albopictus mosquitoes were injected with dsRNA to silence the Ubc9 gene, the expression level of Ubc9 in the dsRNA experimental group was lower, and the difference was statistically significant (F = 18.78, p < 0.05), based on using ANOVA analysis. (B) RT-qPCR was used to detect the viral transcription levels of silenced Ubc9 mosquitoes on day 7 of infection with DENV. The DENV transcription level decreased in the experimental group (DENV-infected mosquitoes in the dsRNA-injection). ANOVA statistical analysis showed a significant difference in the copy number level of DENV transcription level compared with that in the blank control group, showing a decrease (F = 21.5, p < 0.05). (C) Expression levels of DENV NS5 and E proteins in mosquitoes with silenced Ubc9 on the 7th day of DENV infection, as detected by Western blotting.