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. 2019 Jul:533:137-144.
doi: 10.1016/j.virol.2019.05.007. Epub 2019 May 23.

Immune function of an angiotensin-converting enzyme against Rice stripe virus infection in a vector insect

Xue Wang  1 , Wei Wang  2 , Wenzhong Zhang  3 , Jing Li  2 , Feng Cui  4 , Luqin Qiao  5
Affiliations

Immune function of an angiotensin-converting enzyme against Rice stripe virus infection in a vector insect

Xue Wang et al. Virology. 2019 Jul.

Abstract

Angiotensin-converting enzyme (ACE) plays diverse roles in the animal kingdom. However, whether ACE plays an immune function against viral infection in vector insects is unclear. In this study, an ACE gene (LsACE) from the small brown planthopper was found to respond to Rice stripe virus (RSV) infection. The enzymatic activities of LsACE were characterized at different pH and temperature. Twenty planthopper proteins were found to interact with LsACE. RSV infection significantly upregulated LsACE expression in the testicle and fat body. When the expression of LsACE in viruliferous planthoppers was inhibited, the RNA level of the RSV SP gene was upregulated 2-fold in planthoppers, and all RSV genes showed higher RNA levels in the rice plants consumed by these planthoppers, leading to a higher viral infection rate and disease rating index. These results indicate that LsACE plays a role in the immune response against RSV transmission by planthoppers.

Keywords: Angiotensin-converting enzyme; Enzyme activity; Immune function; Rice stripe virus; Small brown planthopper.

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Figures

Image 1
Graphical abstract
Fig. 1
Fig. 1
Sequence characteristics of LsACE. (A) The amino acid sequence of LsACE. The secretory signal peptide is underlined. N-glycosylation sites are marked with triangles. Active site sequences are boxed. The transmembrane region at the C-terminal is underlined with two lines. (B) The phylogenetic tree of LsACE and six homologs of Drosophila melanogaster constructed with the neighbor-joining method using pairwise deletion and the p-distance model. Bootstrap analysis with 1000 replicates was performed. The accession numbers for D. melanogaster ACE homologs are NP_477195.1 for DmACER, NP_477046.1 for DmAnCE, NP_788042.1 for DmAnCE2, NP_001033904.1 for DmAnCE3, NP_610442.2 for DmAnCE4, and NP_573392.2 for DmAnCE5.
Fig. 2
Fig. 2
Enzymatic activity of LsACE. (A) SDS-PAGE of the recombinantly expressed and purified LsACE fragment from amino acid residues 50 to 716. The arrow indicates the 79.5 kDa His-tagged recombinant protein that was eluted from Ni Sepharose using different concentrations of imidazole. (B) Standard curves of N-[3-(2-furyl)acryloyl]-L-phenylalanyl-glycylglycine (FAPGG) and a mixture of equal molar N-(3-[2-furyl]acryloyl)-L-phenylalanine (FAP) and glycylglycine (GG) measured at 340 nm. Points represent the means ± SE of three replicates. The linear regression equation and coefficient of determination (R2) for each curve are presented. (C) Enzymatic activity of LsACE at different pH values. (D) Enzymatic activity of LsACE at different temperatures. Points represent means ± SE of five replicates. Differences were statistically evaluated using one-way ANOVA followed by Tukey's test for multiple comparisons. Significant differences are indicated by different lowercase letters.
Fig. 3
Fig. 3
Yeast two-hybrid assay to screen planthopper proteins that putatively interact with LsACE. (A) Numbers and positions of the 38 clones that were further verified with the yeast two-hybrid system after screening the cDNA library of a small brown planthopper using LsACE as bait. The clones with red numbers are deemed to interact with LsACE in Fig. 3B a, pGBKT7-53 + pGADT7-T, positive control; b, pGBKT7-Lam + pGADT7-T, negative control; c, pGBKT7-LsACE + pGADT7, self-activation. (B) Verification of the interaction between LsACE and the 38 clones on triple dropout medium (SD/-Leu/-Trp/-His) and quadruple dropout medium (SD/-Leu/-Trp/-His/-Ade) in the yeast two-hybrid system. Only 29 clones were determined to interact with LsACE on the quadruple dropout medium and encoded 20 different proteins (Table 1).
Fig. 4
Fig. 4
Temporal and spatial expression of LsACE in viruliferous and nonviruliferous planthoppers. (A) Relative transcript levels of LsACE in the brain, salivary glands, gut, fat body, testis and ovary. (B) Relative transcript levels of LsACE in different developmental stages. The transcript level of LsACE was normalized to that of the elongation factor 2 transcript and was represented as the mean ± SE. Differences were statistically evaluated by Student's t-test to compare two means. *, P < 0.05.
Fig. 5
Fig. 5
Effect of LsACE on RSV infection in planthoppers and rice plants. (A) Relative transcript level of LsACE to that of elongation factor 2 5 d after dsLsACE-RNA or dsGFP-RNA injection in planthoppers. (B) Relative RNA levels of seven viral genes to that of elongation factor 2 5 d after dsLsACE-RNA or dsGFP-RNA injection in viruliferous planthoppers. (C) Western blot to show the protein levels of SP in viruliferous planthoppers after dsLsACE-RNA or dsGFP-RNA injection using monoclonal anti-SP antibodies. Beta-tubulin of the small brown planthoppers was applied as the internal control against the monoclonal anti-beta-tubulin antibody. (D) Relative RNA levels of seven viral genes to that of ubiquitin 5 in rice plants 4 d after consumed by dsLsACE-RNA or dsGFP-RNA-injected viruliferous planthoppers. (E) Western blot to show the protein levels of NS3, CP, SP, and NSvc4 in rice plants 4 d after consumed by dsLsACE-RNA or dsGFP-RNA-injected viruliferous planthoppers using monoclonal anti-CP, anti-SP, anti-NS3 antibodies and polyclonal anti-NSvc4 antibody. Beta-actin of the rice was applied as the internal control against the monoclonal anti-beta-actin antibody. (F) Infection rates of the rice plants consumed by dsLsACE-RNA or dsGFP-RNA-injected viruliferous planthoppers at 10, 13, and 16 d after viral inoculation (DAI). (G) Disease rating index of the rice plants consumed by dsLsACE-RNA or dsGFP-RNA-injected viruliferous planthoppers at 10, 13, and 16 DAI. (H) Disease symptoms of the rice leaves consumed by dsLsACE-RNA or dsGFP-RNA-injected viruliferous planthoppers at 16 DAI. Values represent means ± SE. Differences were statistically evaluated by Student's t-test to compare two means. *, P < 0.05; **, P < 0.01.
Fig. S1
Fig. S1
Densitometry analysis for the viral protein image bands from Fig. 5C and E. (A) The relative densities of SP in planthoppers from Fig. 5C were normalized with those of beta-tubulin and presented as mean ± SE. (B) The relative densities of NS3, CP, SP, and NSvc4 in rice plants from Fig. 5E were normalized with those of beta-actin and presented as mean ± SE. ***, P < 0.001.
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References

    1. Aguilar R., Jedlicka A.E., Mintz M., Mahairaki V., Scott A.L., Dimopoulos G. Global gene expression analysis of Anopheles gambiae responses to microbial challenge. Insect Biochem. Mol. Biol. 2005;35:709–719. - PubMed
    1. Asp L., Johansson A.-S., Mann A., Owe-Larsson B., Urbanska E.M., Kocki T., Kegel M., Engberg G., Lundkvist G.B., Karlsson H. Effects of pro-inflammatory cytokines on expression of kynurenine pathway enzymes in human dermal fibroblasts. J. Inflamm. 2011;8:25. - PMC - PubMed
    1. Baumann R., Casaulta C., Simon D., Conus S., Yousefi S., Simon H.-U. Macrophage migration inhibitory factor delays apoptosis in neutrophils by inhibiting the mitochondria-dependent death pathway. FASEB J. 2003;17:2221–2230. - PubMed
    1. Bernstein K.E., Ong F.S., Blackwell W.L.B., Shah K.H., Giani J.F., Gonzalez-Villalobos R.A., Shen X.Z., Fuchs S. A modern understanding of the traditional and nontraditional biological functions of angiotensin-converting enzyme. Pharmacol. Rev. 2013;65:1–46. - PMC - PubMed
    1. Cheung H.S., Wang F.L., Ondetti M.A., Sabo E.F., Cushman D.W. Binding of peptide substrates and inhibitors of angiotensin-converting enzyme. Importance of the COOH-terminal dipeptide sequence. J. Biol. Chem. 1980;255:401–407. - PubMed

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