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. 2022 Apr 24;13(5):750.
doi: 10.3390/genes13050750.

Impact of Group II Baculovirus IAPs on Virus-Induced Apoptosis in Insect Cells

Hao Zheng  1 , Yong Pan  1 , Mian Muhammad Awais  1 , Weibin Tian  1 , Jingyang Li  1 , Jingchen Sun  1
Affiliations

Impact of Group II Baculovirus IAPs on Virus-Induced Apoptosis in Insect Cells

Hao Zheng et al. Genes (Basel). .

Abstract

Apoptosis plays an important role in virus-host interactions and is a major element of the insect immune response. Exploring the regulatory mechanisms of virus-induced apoptosis through the expression of apoptotic genes holds important research and application value. Functional research on the reported inhibitor of apoptosis proteins (IAPs) mainly focuses on the group I baculovirus, while the functions of the group II baculovirus IAPs remains unclear. To explore its role in the regulation of the apoptosis of insect cells, we constructed the transient expression vector (pIE1 vectors) and the recombinant baculovirus expressing Bsiap genes (from the Buzura suppressaria nucleopolyhedrovirus) of the group II baculovirus. Apoptosis gene expression results and the virus-induced apoptosis rate show that the overexpression of BsIAP1 could promote apoptosis in insect cells. However, the overexpression of BsIAP2 and BsIAP3 decreases the expression of apoptotic genes, revealing an inhibitory effect. Results on the impact of baculovirus-induced apoptosis also confirm that BsIAP1 reduces viral nucleocapsid expression and the baculovirus titer, while BsIAP2 and BsIAP3 increase them significantly. Furthermore, compared with single expression, the co-expression of BsIAP2 and BsIAP3 significantly reduces the rate of virus-induced apoptosis and improves the expression of nucleocapsids and the titer of offspring virus, indicating the synergistic effect on BsIAP2 and BsIAP3. In addition, combined expression of all three BsIAPs significantly reduced levels of intracellular apoptosis-related genes (including apoptosis and anti-apoptosis genes), as well as apoptosis rate and progeny virus titer, indicating that life activities in insect cells are also inhibited. These findings reveal the relationship between apoptosis and group II baculovirus IAP, which provide an experimental and theoretical basis for further exploration of the molecular mechanism between group II baculoviruses and insect cells.

Keywords: apoptosis; group II baculovirus; inhibitor of apoptosis protein; insect cell.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Figure 1
Figure 1
Construction of transient expression vector. Maps of the pIE1-His-Bsiap1 (A), pIE1-V5-Bsiap2 (B), and pIE1-Flag-Bsiap3 plasmids (C).
Figure 2
Figure 2
Construction of recombinant plasmids carrying Bsiaps and fluorescent genes. Maps of the pFBDM-HisBsiap1-egfp (A), pUCDM-V5Bsiap2-mCherry (B), pFBDM-FlagBsiap3-egfp (C), and pFBDM-HisBsiap1-FlagBsiap3-egfp plasmids (D).
Figure 3
Figure 3
Schematic illustrating the construction of recombinant BV-Bsiaps baculovirus through multi-gene co-expression and liposome-free transfection technology. Bsiap genes carried by the recombinant plasmids, pFBDM and pUCDM, were introduced into an E. coli Sw106 bacmid using Tn7 and Cre-loxP sites, respectively. Due to the lack of diaminopimelic acid in Sf9 cells, E. coli Sw106, which invade Sf9 cells via invasin activity, cannot synthesize cell walls, and bacmids are released and BV-Bsiaps proliferation completed.
Figure 4
Figure 4
Transfection of insect Sf9 cells and identification of BsIAPs by western blot. The cells of the control group were cultured for the same time after transfection with pIE vector. After the transient expression plasmids were transfected into Sf9 cells (48 h, Bar = 25 μm), the intracellular BsIAPs were identified by western blot with three anti-tag antibodies (His, V5, and Flag), respectively. Western blot identification showed that different BsIAPs (and their combinations) were highly expressed in insect Sf9 cells after co-transfection (see the Figure S1 for details).
Figure 5
Figure 5
Impact of transient overexpression of BsIAPs on apoptotic gene expression. The expression of apoptosis genes, including the control group, were relative to those of non-transfected insect Sf9 cells. (A) Heatmap illustrating the effect of transient overexpressing BsIAPs on apoptotic gene expression. (B–I) Detailed results of the effects of transient overexpressing BsIAPs on the expression of caspase−1 (B), caspase−2 (C), caspase−9 (D), dredd (E), dronc (F), Sf9−iap (G), and p53 (H). * p < 0.05, ** p < 0.01.
Figure 5
Figure 5
Impact of transient overexpression of BsIAPs on apoptotic gene expression. The expression of apoptosis genes, including the control group, were relative to those of non-transfected insect Sf9 cells. (A) Heatmap illustrating the effect of transient overexpressing BsIAPs on apoptotic gene expression. (B–I) Detailed results of the effects of transient overexpressing BsIAPs on the expression of caspase−1 (B), caspase−2 (C), caspase−9 (D), dredd (E), dronc (F), Sf9−iap (G), and p53 (H). * p < 0.05, ** p < 0.01.
Figure 6
Figure 6
Construction of recombinant baculovirus and identification of BsIAPs. (A) Expression of eGFP and mCherry were observed by fluorescence microscopy, indicating that the seven recombinant baculoviruses successfully infected Sf9 cells (Bar, 25 μm). Control cells were infected with BV-gfp under the same operation. (B) Western blot analysis using the three anti-tag antibodies (His, V5, and Flag) showed that BsIAPs were highly expressed after 72 h of Sf9 cell infection with the seven recombinant baculoviruses. The abscissa shows the cell samples infected by the seven groups of baculoviruses (BV) respectively. The ordinate indicates that different samples were identified by western blot using three tag antibodies. The result shows that different BsIAP proteins (or their combinations) are successfully and efficiently expressed in cells (B).
Figure 7
Figure 7
Effects of overexpression of BsIAPs on Sf9 cell apoptosis rate. * p < 0.05, ** p < 0.01. After Sf9 cells were infected with recombinant baculovirus stably expressing BsIAPs for 48 h, the apoptosis rate was determined by flow cytometry. Control cells were infected with BV-gfp under the same operation.
Figure 8
Figure 8
Effects of BsIAP overexpression on VP39 capsid protein levels. * p < 0.05, ** p < 0.01. After 48 h of infection with recombinant baculovirus stably expressing BsIAPs, total RNA was extracted, and the expression of vp39 gene was determined by reverse transcription PCR. The VP39 expression is relative to the control group (baculovirus AcBV-gfp expressing fluorescent protein only).
Figure 9
Figure 9
Effects of overexpression of BsIAPs on progeny virus titers after different infection time periods (* p < 0.05, ** p < 0.01). (A) Overexpression of BsIAPs for 36 h. (B) Overexpression of BsIAPs for 48 h. (C) Overexpression of BsIAPs for 60 h. (D) Overexpression of BsIAPs for 72 h. Control cells were infected with BV-gfp under the same operation.
Figure 10
Figure 10
Analysis of BsIAPs amino acid domains and active sites. (A) BsIAP1. (B) BsIAP2. (C) BsIAP3.
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