Sirt5 Inhibits BmNPV Replication by Promoting a Relish-Mediated Antiviral Pathway in Bombyx mori

Abstract

Silent information regulators (Sirtuins) belong to the family of nicotinamide adenine dinucleotide (NAD⁺)-dependent histone deacetylases (HDACs) that have diverse functions in cells. Mammalian Sirtuins have seven isoforms (Sirt1-7) which have been found to play a role in viral replication. However, Sirtuin members of insects are very different from mammals, and the function of insect Sirtuins in regulating virus replication is unclear. The silkworm, Bombyx mori, as a model species of Lepidoptera, is also an important economical insect. B. mori nucleopolyhedrovirus (BmNPV) is a major pathogen that specifically infects silkworms and causes serious losses in the sericulture industry. Here, we used the infection of the silkworm by BmNPV as a model to explore the effect of Sirtuins on virus replication. We initially knocked down all silkworm Sirtuins, and then infected with BmNPV to analyze its replication. Sirt2 and Sirt5 were found to have potential antiviral functions in the silkworm. We further confirmed the antiviral function of silkworm Sirt5 through its effects on viral titers during both knockdown and overexpression experiments. Additionally, Suramin, a Sirt5 inhibitor, was found to promote BmNPV replication. In terms of molecular mechanism, it was found that silkworm Sirt5 might promote the immune pathway mediated by Relish, thereby enhancing the host antiviral response. This study is the first to explore the role of Sirtuins in insect-virus interactions, providing new insights into the functional role of members of the insect Sirtuin family.

Keywords: BmNPV; Bombyx mori; Relish; Sirt5; sirtuins.

Figure 1

Chromosomal location, subcellular localization and phylogenetic analysis of silkworm members of the Sirtuin Family. (A) Chromosomal location of the silkworm Sirtuin genes. (B) Predicted subcellular localization of silkworm Sirtuin genes. (C) Phylogenetic analysis of Sirtuin family members from the silkworm and other insect, invertebrate and vertebrate species. Bayesian phylogenetic tree was generated based on the Sirtuins nucleotide sequences (The Gene ID of each sequence is shown in Table S2 ). Five branches were color-coded with cyan (Sirt2), purple (Sirt4), yellow (Sirt5), pink (Sirt6) and green (Sirt7). Aa, A; albopictus Hs, H; sapiens; Mm, M; musculus; Dr, D; rerio; Bm, B; mori; Dm, D; melanogaster; Am, A; mellifera; Tc, T; castaneum; Xl, X; laevis, Pt, P; troglodytes, Mam, M; mulatta, Ts, T; scripta elegans, Vl, V; lagopus, Cl, C; lectularius, Sf, S; frugiperda, Ha, H; armigera, De, D; elegans, Si, S; invicta, Vm, V; mandarinia, Pr, P; rapae, Tp, T; palmi, Sl, S; litura.

Figure 2

Expression of Sirtuin family genes in hemocytes, fat body and midgut after BmNPV infection in the silkworm. (A–D) Detection of the expression of BmSirt2, BmSirt4, BmSirt5 and BmSirt6 in hemocytes. (E–H) Transcriptional response of BmSirt2, BmSirt4, BmSirt5 and BmSirt6 to BmNPV infection in fat body. (I–L) Detection of the expression of BmSirt2, BmSirt4, BmSirt5 and BmSirt6 in midgut. Negative control represents uninfected larvae. Each bar represents the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, not significant.

Figure 3

Detection of the expression of the BmNPV capsid gene Vp39 by qRT-PCR after knockdown of Sirtuin genes in BmN cells. (A) The viral gene Vp39 was significantly up-regulated at 72 hpi after knockdown of BmSirt2. (B) The expression of Vp39 did not change significantly after knockdown of BmSirt4. (C) Vp39 expression was significantly up-regulated at 48 and 72 hpi after knockdown of BmSirt5. (D) Vp39 expression did not change significantly after knockdown of BmSirt6. The BmN cells treated with dsRNA-DsRed were used as negative control. Each bar represents the mean ± SD. **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, not significant.

Figure 4

Knockdown of BmSirt5 significantly promoted BmNPV replication in BmN cells. (A) Green fluorescence images of BmNPV-eGFP infected cells observed by an inverted microscope (100×) at 24, 48 and 72 hpi. (B) Determination of viral titers in dsRNA-BmSirt5-transfected BmN cells at 24, 48 and 72 hpi. DsRNA of DsRed was used as a negative control. Each bar represents the mean ± SD. ***p < 0.001. ns, not significant.

Figure 5

Over-expression of BmSirt5 inhibited the replication of BmNPV. (A) The expression level of exogenous BmSirt5 in BmN cells was determined by Western blotting with flag antibody at 24, 48, 72 and 96 h post transfection. BmN cells transfected with pIEX-eGFP were used as a negative control. (B) Quantification of BmNPV gene Vp39 expression by qRT-PCR in BmN cells transfected with pIEX-BmSirt5 or pIEX-eGFP at 24, 48 and 72 h after BmNPV infection. (C) Viral titer determination using the TCID₅₀ assay in supernatants of BmN cells transfected with pIEX-BmSirt5 or pIEX-eGFP at 24, 48 and 72 hpi. Each bar represents the mean ± SD. *p < 0.05, **p < 0.01. ns, not significant.

Figure 6

The Sirt5 inhibitor, Suramin, promoted BmNPV replication. (A) Schematic diagram of treatment of silkworms with Suramin. (B) Quantification of the expression of the BmNPV capsid gene Vp39 by qRT-PCR after treatment with Suramin in the silkworm fat body at 24, 48 and 72 hpi. (C) Cytotoxicity assay of different concentrations of Suramin in BmN cells. (D) Quantification of Vp39 expression by qRT-PCR after treatment with Suramin in BmN cells at 24, 48 and 72 hpi. (E) Determination of viral titers using the TCID₅₀ assay. Each bar represents the mean ± SD. **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, not significant.

Figure 7

Induction of BmRelish expression after BmNPV infection. (A–C) Quantification of the expression of BmRelish, CecA and CecB by qRT-PCR in the fat body of virus-infected silkworm larvae. (D–F) Quantification of the expression of BmRelish, CecA and CecB in BmN cells by qRT-PCR at 24, 48 and 72 h after BmNPV infection. Negative control corresponds to uninfected samples. Each bar represents the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, not significant.

Figure 8

Enhancement of BmRelish-mediated immunity by BmSirt5 during BmNPV infection. (A–C) Quantification of the expression of BmRelish, CecA and CecB by qRT-PCR after knockdown of BmSirt5 in BmN cells at 24, 48 and 72 h after BmNPV infection. BmN cells treated with dsRNA-DsRed were used as negative control. (D–F) Quantification of the expression of BmRelish, CecA and CecB by qRT-PCR after over-expression of BmSirt5 in BmN cells at 24, 48 and 72 hpi. BmN cells treated with pIEX-eGFP were used as negative control. (G–I) Quantification of the expression of BmRelish, CecA and CecB by qRT-PCR after treatment with Suramin in BmN cells. BmN cells treated with ddH₂O were used as negative control. Each bar represents the mean ± SD. *p < 0.05, **p < 0.01, ****p < 0.0001. ns, not significant.

Figure 9

Schematic diagram of the putative antiviral mechanism by which BmSirt5 inhibited BmNPV proliferation in the silkworm. (A) BmSirt5 expression was significantly induced by BmNPV infection in the fat body or midgut, together with the activation of other host antiviral pathways such as STING. (B) The innate immune pathway mediated by BmRelish can induce the production of downstream antimicrobial peptide (AMP) genes and other antiviral genes to resist BmNPV infection. (C) Nuclear translocation of Relish may be stimulated by de-succinylation or de-acetylation activities of BmSirt5. (D) Inhibition of the NAD⁺-dependent de-acetylase activity of BmSirt5 by Suramin could interfere with the activation of BmRelish, for instance by inhibition of its transfer to the nucleus.