Virus-Induced Histone Lactylation Promotes Virus Infection in Crustacean

Abstract

As "non-cellular organisms", viruses need to infect living cells to survive themselves. The virus infection must alter host's metabolisms. However, the influence of the metabolites from the altered metabolisms of virus-infected host cells on virus-host interactions remains largely unclear. To address this issue, shrimp, a representative species of crustaceans, is challenged with white spot syndrome virus (WSSV) in this study. The in vivo results presented that the WSSV infection enhanced shrimp glycolysis, leading to the accumulation of lactate. The lactate accumulation in turn promoted the site-specific histone lactylation (H3K18la and H4K12la) in a p300/HDAC1/HDAC3-dependent manner. H3K18la and H4K12la are enriched in the promoters of 75 target genes, of which the H3K18la and H4K12la modification upregulated the expression of ribosomal protein S6 kinases 2 (S6K2) in the virus-infected hosts to promote the virus infection. Further data revealed that the virus-encoded miR-N20 targeted hypoxia inducible factor-1α (HIF-1α) to inhibit the host glycolysis, leading to the suppression of H3K18la and H4K12la. Therefore, the findings contributed novel insights into the effects and the underlying mechanism of the virus-induced histone lactylation on the virus-host interactions, providing new targets for the control of virus infection.

Keywords: glycolysis; histone lactylation; metabolite; miRNA; virus infection.

Figure 1

Promotive impact of virus infection on histone lactylation. A) Influence of virus infection on the histone lactylation. Shrimp were challenged with WSSV. At different time post‐infection, the hemocytes and intestinal tissues of shrimp were subjected to the detection of the histone lactylation using Western blot. the specific antibody against the lactylation sites of H3K9, H3K14, H3K18, H4K8 or H4K12 was used. H3 was used as a control. B) Silencing of lactate dehydrogenase (LDH) in shrimp. Shrimp were simultaneously injected with WSSV and LDH‐siRNA or LDH‐siRNA‐scrambled. At different time after injection, the expression level of LDH in the hemocytes and intestinal tissues of shrimp was examined using quantitative real‐time PCR (*p < 0.05; **p < 0.01). C) Impact of LDH silencing on glycolysis of WSSV‐infected shrimp. The lactate content in the hemocytes and intestinal tissues of shrimp injected with WSSV and LDH‐siRNA or siRNA‐scrambled was evaluated (*p < 0.05; **p < 0.01). As a control, siRNA‐scrambled was included in the injection. D) Influence of LDH silencing on the histone lactylation in the WSSV‐infected shrimp. Shrimp were injected with WSSV and LDH‐siRNA or siRNA‐scrambled. Forty‐eight hours later, the hemocytes and intestinal tissues of shrimp were subjected to Western blot to examine the lactylation of histone H3K18 and H4K12. H3 was used as a control. E) Impact of 2‐deoxy‐D‐glucose on the content of lactate in WSSV‐infected shrimp. The shrimp were injected with WSSV and 2‐deoxy‐D‐glucose or PBS. At different time post‐infection, the contents of lactate in the hemocytes and intestinal tissue of shrimp were examined (**p < 0.01). F) Western blot analysis of lactylation of H3K18 and H4K12 in shrimp. The hemocytes and intestinal tissues of shrimp treated with WSSV, and 2‐deoxy‐D‐glucose or PBS were collected at 48 h post‐infection and then subjected to Western blot to detect the lactylation levels of H3K18 and H4K12. H3 was used as a control. G) Impact of sodium lactate (Nala) on the lactate content of WSSV‐infected shrimp. Shrimp were simultaneously injected with WSSV and Nala or PBS. At different time points post‐infection, the lactate contents in the hemocyte and intestinal tissue of shrimp were examined (**p < 0.01). H) Western blotting analysis of the lactylation of H3K18 and H4K12 in shrimp treated with WSSV and Nala. Shrimp were injected with WSSV and Nala. At 48 h post‐infection, the hemocytes and intestinal tissues were subjected to Western blot to evaluate the levels of H3K18la and H4K12la. H3 was used as a control.

Figure 2

Mechanism of site‐specific lactylation in vivo. A) Silencing of p300, HDAC1 and HDAC3 in shrimp. Shrimp were co‐injected with WSSV and siRNA (p300‐siRNA, HDAC1‐siRNA or HDAC3‐siRNA). At different time after the injection (12, 24, and 36 h), siRNA alone was injected into the same shrimp. As controls, p300‐siRNA‐scramble, HDAC1‐siRNA‐scramble and HDAC3‐siRNA‐ scramble were included in the injections. At 48 h after the first injection, the expression levels of p300, HDAC1 and HDAC3 in the hemocytes and intestinal tissues of shrimp were examined (**p < 0.01). B) Roles of p300, HDAC1 and HDAC3 in the lactylation of H3K18 and H4K12 in shrimp. The expression of p300, HDAC1 or HDAC3 was silenced in the WSSV‐infected shrimp using sequence‐specific siRNA. At 48 h after the first injection of siRNA, the shrimp hemocytes and intestinal tissues were subjected to Western blot analysis to detect the levels of H3K18la and H4K12la. H3 was used as a control. C) Influence of inhibitor of p300 or HDAC1 and HDAC3 on the lactylation of H3K18 and H4K12 in shrimp. The WSSV‐infected shrimp were treated with Vorinostat (SAHA), an inhibitor of histone deacetylase or C646, an inhibitor of p300, at different concentrations. At different time after treatment, the H3K18la and H4K12la levels in shrimp hemocytes and intestinal tissues were examined using Western blot. H3 was used as a control.

Figure 3

Target genes directly regulated by H3K18la and H4K12la. A) Genome‐wide enrichment analysis of DNAs bound to H3K18la and H4K12la. Shrimp were infected with WSSV. Forty‐eight hours later, the hemocytes of WSSV‐infected shrimp were subjected to the CUT&Tag analysis. The heatmap presented the counts of genes bound to H3K18la (left) or H4K12la (right). The binding density of genes with transcription start site (TSS) and transcription end site (TES) was visualized using deepTools. B) Genome‐wide distribution of the DNAs bound to H3K18la or H4K12la. C) Number of target genes whose promotors were bound to H3K18la and/or H4K12la. D) Kyoto encyclopedia of genes and genomes (KEGG) analysis of the target genes regulated by H3K18la and H4K12la. E) Number of the genes targeted by H3K18la and H4K12la. Shrimp were challenged by WSSV. At 48 h after infection, the shrimp hemocytes were subjected to transcriptome analysis. A total of 75 upregulated genes were overlapped in the CUT&Tag and RNA‐seq data. F) Effects of WSSV infection on the expressions of target genes of H3K18la and H4K12la. Shrimp were injected with WSSV or PBS. PBS was included in the injection as a control. At 48 h after injection, the expression profiles of shrimp ribosomal protein S6 kinases 2 (S6K2), phospholipase D1 (PLD1), TGF‐beta receptor type‐1 (TGFBR1), atypical protein kinase C (aPKC) and ATP‐dependent translocase (ABCB1) in the shrimp hemocytes were examined using quantitative real‐time PCR (**p < 0.01). G) Genome browser tracks of CUT&Tag signals at the S6K2 gene loci. The blue arrows indicated the peak regions of H3K18la and H4K12la around the transcription start sites (TSS) and the enrichment at the target gene's promoters. The number showed the peak height. H) H3K18la and H4K12la levels in the S6K2 promoter of WSSV‐infected shrimp. Shrimp were injected with WSSV or PBS. PBS was used as a control. At 48 h after injection, ChIP‐qPCR assay was performed to detect the enrichment of H3K18la and H4K12la in the S6K2 promoter (**p < 0.01).

Figure 4

Role of S6K2 in virus infection. A) Differential expression of S6K2 in the WSSV‐challenged and healthy shrimp. Shrimp were injected with WSSV or PBS. At different time after injection, the expression of S6K2 in the hemocytes and intestinal tissues of shrimp was examined by quantitative real‐time PCR (**p < 0.01) (left) and Western blot (right). β‐actin was used as a control. B) Knockdown of S6K2 in shrimp. The WSSV‐infected shrimp was injected with S6K2‐specific siRNA (S6K2‐siRNA) or S6K2‐siRNA‐scrambled as a control. At 48 h after injection, the expression level of S6K2 in the hemocytes and intestinal tissues of shrimp was examined using quantitative real‐time PCR (**p < 0.01) and Western blot. C) Impact of S6K2 silencing on virus infection in shrimp. The WSSV‐infected shrimp were injected with S6K2‐siRNA and S6K2‐siRNA‐scrambled. At different time after injection, the virus copy in the hemocytes and intestinal tissues of shrimp was quantified by quantitative real‐time PCR (**p < 0.01). D) Effects of S6K2 silencing on the mortality of WSSV‐infected shrimp. Shrimp were co‐injected with WSSV and S6K2‐siRNA or S6K2‐siRNA‐scrambled. The numbers on the horizontal axis indicated the days post‐infection (*p < 0.05; **p < 0.01). E) Influence of 2‐DG on the expression of S6K2 in shrimp. WSSV‐infected shrimp was injected with 2‐DG. PBS was included in the injection as a control. At 48 h after injection, the expression level of S6K2 in the hemocytes and intestinal tissues of shrimp was examined using quantitative real‐time PCR (**p < 0.01) and Western blot. F) Impact of 2‐DG on virus infection in shrimp. The WSSV‐infected shrimp were injected with 2‐DG. At different time after injection, the virus copies in the hemocytes and intestinal tissues of shrimp were examined by quantitative real‐time PCR (**p < 0.01). G) Effects of 2‐DG on the mortality of WSSV‐infected shrimp. The number on the horizontal axis indicated the days post‐infection (*p < 0.05; **p < 0.01). H) Impact of LDH silencing on the S6K2 expression in shrimp. The WSSV‐infected shrimp was injected with LDH‐siRNA or LDH‐siRNA‐scramble. At 48 h after injection, the expression level of S6K2 in the hemocytes and intestinal tissue of shrimp was examined (**p < 0.01). I) Examination of virus copy in the LDH‐silenced shrimp. The virus copy was quantified using quantitative real‐time PCR (**p < 0.01). (J) Mortality of the virus‐infected shrimp. The WSSV‐infected shrimp were injected with LDH‐siRNA or siRNA‐scrambled. At different time post‐infection, the shrimp mortality was determined (*p < 0.05; **p < 0.01).

Figure 5

Regulation of H3K18la and H4K12la mediated by viral miRNAs. A) Prediction of WSSV miRNAs targeting hypoxia inducible factor‐1α (HIF‐1α). Based on the prediction, WSSV‐miR‐N20 could target shrimp HIF‐1α. The underline showed the seed sequence of miR‐N20. B) Direct interaction between miR‐N20 and HIF‐1α. The insect High Five cells were co‐transfected with miR‐N20 or miR‐N20‐scrambled and plasmid EGFP‐HIF‐1α 3′UTR or EGFP‐HIF‐1α 3′UTR mutant. At 36 h after tansfection, the fluorescence of insect cells was observed under a fluorescence microscope. The relative fluorescence intensity was calculated with Image J (**p < 0.01). Scale bar, 100 µm. C) Effects of miR‐N20 overexpression on the expression of HIF‐1α in the hemocytes and intestine of shrimp. WSSV‐infected shrimp were injected with miR‐N20. Forty‐eight hours later, the mRNA or protein level of HIF‐1 α was detected using quantitative real‐time quantitative PCR (*p < 0.05; **p < 0.01) (left) or Western blot. D) Impact of miR‐N20 overexpression on the LDH expression in shrimp. The WSSV‐infected shrimp were injected with miR‐N20. Forty‐eight hours later, the mRNA or protein level of LDH was detected using quantitative real‐time quantitative PCR (*p < 0.05; **p < 0.01) (left) or Western blot (right). E) Western blot analysis of H3K18la and H4K12la in shrimp overexpressing miR‐N20. The WSSV‐infected shrimp were injected with miR‐N20. At 48 h after injection, the levels of H3K18la and H4K12la in the hemocytes and intestines of shrimp were detected using Western blot. H3 was used as a control.

Figure 6

Influence of miR‐N20‐mediated H3K18la and H4K12la on S6K2 and virus infection. A) Effects of miR‐N20 overexpression on the expression of S6K2 in the hemocytes and intestine of shrimp. WSSV‐infected shrimp were injected with miR‐N20. Forty‐eight hours later, the mRNA or protein level of S6K2 was detected using quantitative real‐time PCR (*p < 0.05; **p < 0.01) (left) or Western blot (right). B) Effects of miR‐N20 overexpression on virus infection in shrimp. The WSSV‐infected shrimp were injected with miR‐N20. At different time after injection, the virus copy in the hemocytes and intestinal tissue of shrimp was quantified by real‐time PCR (*p < 0.05; **p < 0.01). C) Influence of miR‐N20 overexpression on the mortality of WSSV‐infected shrimp. The numbers on the horizontal axis indicated the post‐infection days (**p < 0.01). D) Model for the role of glycolysis‐mediated histone lactylation in virus infection.