key: cord-0829835-f3ua8ks3
authors: Zhang, Xing; Zhang, Yunshan; Shi, Xiu; Dai, Kun; Liang, Zi; Zhu, Min; Zhang, Ziyao; Shen, Zenen; Pan, Jun; Wang, Chonglong; Hu, Xiaolong; Gong, Chengliang
title: Characterization of the lipidomic profile of BmN cells in response to Bombyx mori cytoplasmic polyhedrosis virus infection
date: 2020-08-15
journal: Dev Comp Immunol
DOI: 10.1016/j.dci.2020.103822
sha: f53a6a16a02ac5a743e98dc1ddd498104eb2673a
doc_id: 829835
cord_uid: f3ua8ks3

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV)that belongs to the genus Cypovirus in the family of Reoviridae is one of the problematic pathogens in sericulture. In our previous study, we have found that lipid-related constituents in the host cellular membrane are associated with the BmCPV life cycle. It is important to note that the lipids not only affect the cellular biological processes, they also impact the virus life cycle. However, the intracellular lipid homeostasis in BmN cells after BmCPV infection remains unclear. Here, the lipid metabolism in BmCPV-infected BmN cells was studied by lipidomics analysis. Our results revealed that the intracellular lipid homeostasis was disturbed in BmN cells upon BmCPV infection. Major lipids constituents in cellular membrane were found to be significantly induced upon BmCPV infection, which included triglycerides, phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, phospholipids, glucoside ceramide, monoetherphosphatidylcholin, ceramide, ceramide phosphoethanolamineand cardiolipin. Further analysis of the pathways related to these altered lipids (such as PE and PC) showed that glycerophospholipid metabolism was one of the most enriched pathways. These results suggested that BmCPV may manipulate the lipid metabolism of cells for their own interest. The findings may facilitate a better understanding of the roles of lipid metabolic changes during virus infection in future studies.

As one of the members of Cypovirus genus of Reoviridae family, Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is one of the most problematic pathogens in sericulture. The genome of BmCPV comprises ten dsRNA linear segments that are assembled as part of the virion structure [1] . Among different tissue types in silkworms, the midgut is one of the most susceptible organs specifically infected by BmCPV. Silkworms can be infected by BmCPV at any instar stage. BmCPV-infected silkworms show symptoms such as translucent appearance, decreased movement ability and lowered body size. Such symptoms aggravate gradually in line with the infection process. At the event of death, the midgut tissues turn milky in appearance.

The host response of silkworms to BmCPV infection has been comprehensively explored, including the expression pattern analysis of genes [2, 3], proteins [4] and non-coding RNAs [5] . In addition, the function of BmCPV genes [6] , viral encoded non-coding RNAs [7, 8] and viral peptide [9] have also been identified. Our previous studies have found alterations in a large number of genes related to important signaling pathways, including those associated with innate immunity, development and metabolism following BmCPV infection [3, 5] . Although there has been many studies that focus on the pathogenesis of BmCPV, the exact mechanism of BmCPV infection, as well as the interactions between virus and host factors remain unclear. As non-living entities, the life cycles of viruses depend on the host cells that provide essential materials and energy for their replication [10, 11] . Lipid is a vital component of cellular and organelle membranes that plays crucial roles in the regulation of many biological processes including virus-host interaction. An increasing number of studies in recent years have demonstrated changes of lipid metabolism in host cells after virus infection. It has been shown that infection by some single-stranded RNA(ssRNA) viruses can alter the lipid metabolism and other biological processes of the host cells to facilitate the completion of the virus life cycle [11] . Free fatty acids (FFAs), which can be used directly by the body for energy metabolism have been shown to be associated with the life cycle of various RNA viruses, including influenza A virus (IAV) [12] , classical swine fever virus (CSFV) [13] , Middle East respiratorysyndrome J o u r n a l P r e -p r o o f coronavirus (MERS-CoV) [14] , hepatitis C virus (HCV) [15] , zika virus (ZIKV) [16] and ebola virus [17] . Furthermore, some lipids are produced in the cells and others are imported from extracellular environment [12] . Some lipid-related constituents in the cell membrane are indispensable for virus entry and replication. Viral receptor ICAM-5 plays important roles in the replication of Enterovirus D68 [18].

Apolipoprotein A-I binding protein is an intrinsic factor that suppresses human immunodeficiency virus (HIV) replication [19] . Zebrafish C-reactive protein-like protein inhibits spring viraemia of carp rhabdovirus replication by causing alterations of cholesterol ratios in the host cellular membranes [20] . Lipids have also been shown to play important roles in the replication of coxsackievirus B3 [21] . An increased level fatty acid biosynthesis coupled with accumulation of free fatty acids that are associated with virus replication has been observed in host cells following CSFV infection [13] . Phosphorylated 5'-adenosine monophosphate-activated protein kinase induced by the infection of porcine reproductive and respiratory syndrome virus can inactivate the fatty acid biosynthesis pathway, playing an antagonistic role in the virus replication [22] . Upon enterovirus A71 and coxsackievirus A16 infection, disturbed lipid homeostasis in the infected cells has been shown to be related to virus replication [23] . Fatty acid synthase and stearoyl-CoA desaturase required for fatty acid metabolism have been demonstrated to be required for chikungunya virus infection [24] . Cholesterol can also affect hemagglutinin fusion activity and hence the virus assembly of influenza virus [25] . ZIKV-triggered lipid metabolism that has been found in patient serum samples may also be associated with virus replication [16] .

Host cell lipid response has been observed to be significantly altered upon human coronavirus 229E infection [14] . The increased levels of polyunsaturated fatty acids that have been found upon HCV infection are essential for viral progeny production [15] . Our previous study has shown that ganglioside GM2 and cholesterol in the cellular membrane are required for BmCPV attachment and entry [3]. Analysis of differential expressed circRNAs has revealed changes in the metabolism of fatty acids upon BmCPV infection [5] . All these reports have demonstrated that lipid-related metabolisms play vital roles in the life cycles of RNA viruses. It is worth noting that J o u r n a l P r e -p r o o f the virus-induced changes of cellular metabolic activity generally facilitate the progress of virus infection [26, 27] . To date, the lipid metabolism in cells infected by double-stranded RNA (dsRNA) viruses, such as, BmCPV remains unknown.

In this study, silkworm ovary-derived BmN cells, which are normally unsusceptible of but infectable by BmCPV were used as the model to investigate the lipid metabolomics of BmCPV-infected cells. We found that the expression of multiple lipid classes was changed in BmN cells upon BmCPV infection. An understanding of the post-infection cellular metabolism changes may provide some insights on the mechanism of viral pathogenesis in order to contribute to the development of novel therapeutic strategies for viral diseases.

BmN cells were cultured at 26 in complete TC-100 medium supplemented with 10% fetal bovine serum. The virion solution of BmCPV-SZ strain used for infecting BmN cells was stored in our lab.

BmN cells (1×10 7 ) were seeded in T25 flasks and cultured in complete medium 

To study the effects of lipid metabolism in vitro after BmCPV infection,

BmCPV-infected BmN cells were collected and analyzed by western blotting and real-time PCR to evaluate the virus multiplication. The results showed that BmCPV structural protein VP7 was identified in BmCPV-infected BmN cells at 48 hours post infection (hpi). Furthermore, the expression level of vp1 was also detected in the same cell pools ( Figure 1A) . Thus, BmN cells were successfully infected by BmCPV.

Lipidomic analysis using UHPLC-QTOF-MS were performed with lipids isolated from CASE and CON at 48 hpi ( Figure 1B) . To validate the reliability of UHPLC-QTOF-MS in BmN cells, both the positive (+) or negative ion mode (-) were featured in the base peak ion chromatograms (matching similarity >80%) to display the distribution of metabolites. The results revealed that metabolite retention in samples within the same group were not significantly changed and that the signal representing each substance was detected with disparity ( Figure 2) . Therefore, we concluded that the data from UHPLC-QTOF-MS analysis are suitable and reliable, J o u r n a l P r e -p r o o f which can be used in the following study.

To truly display the alterations of intracellular lipid homeostasis between CASE and CON, SIMCA software package was applied to integrate the positive and negative ion data for principle component analysis (PCA). A significant trend of lipid distribution was found between CASE and CON in the PCA score chart ( Figure 3A and 3B) . To

show the altered metabolics between CASE and CON, OPLS-DA model was applied. accompanied by data comparison from the two groups was considered satisfactory. To assess the quality of the current model, R2Y and Q2 were used as the parameters. As shown in Figure 3D and 3E, our analysis (R2Y> 0.5 and Q2> 0.5) indicated that our established model was suitable and reliable. These results demonstrated that the established model is reliable, and can be used to identify differential lipids between CASE and CON.

To screen the significant differential lipids between CASE and CON, univariate and multivariate analyses were performed based on the lipid mass spectrum. In univariate analysis, a total of 345 differential lipids were detected between CASE and CON. A total of 194 and 151 differential lipids were respectively identified in the positive and negative mode (Supplementary Table 1 ).

To further investigate the significant differential lipids from the data obtained from univariate analysis, parameters, where VIP >1 and p value< 0.05 were applied in the screening process by combining multidimensional and one-dimensional analysis. A total of 34 significant differential lipids were detected between CASE and CON, where, 24 and 10 lipids were respectively identified in the positive and negative J o u r n a l P r e -p r o o f model (Table 1 ). These results indicated that BmCPV infection can markedly alter the intracellular lipid homeostasis in BmN cells.

To further characterize the differential lipids, volcano plots were applied to visualize the di erential metabolites ( Figure 4A) . Meanwhile, the correlation between identified lipids was shown using Hierarchical Clustering Analysis (HCA), which indicated that the differential lipids identified from CASE were markedly distinguished from those identified from CON ( Figure 4B ).

A total of 34 significantly differential lipids were identified between CASE and CON, 

To understand the potential regulatory mechanisms of significant differential lipids associated with the metabolic pathway upon BmCPV infection, several pathway maps in the KEGG database were constructed based on the pathway information. As shown in Figure 6 , upon BmCPV infection, pathways related to glycerophospholipid metabolism, glycosylphosphatidylinositol (GPI)-anchor biosynthesis, autophagy, linoleic acid metabolism, alpha-linolenic acid metabolism and arachidonic acid metabolism were found to be most significantly affected. These results suggested that these metabolic pathways may be applied for intracellular signaling upon virus J o u r n a l P r e -p r o o f infection.

In our previous study, multiple components in membrane lipid rafts, including In our previous study, cellular membrane constituents, such as ganglioside GM2 and cholesterol have been found to be required for BmCPV attachment and entry [29] . In addition, fatty acid metabolism has been observed to be significantly changed by the differentially expressed circRNAs in the midgut of silkworms infected by BmCPV [5] .

These KEGG enrichment analysis of differential proteins from the midgut of in some viruses' replication [35] [36] [37] . The phospholipids metabolism was also found to be related to the lipid regulation, lipoprotein, whole-body energy metabolism and metabolic disorders [38] . Therefore, we speculated that the disorder of phospholipids metabolism may be associated with the BmCPV entry. Most of the glycerophospholipids were identified with unsaturated fatty acids, and it is noted that the condensation of phospholipid films was negative correlated with unsaturated fatty acids [39] . Therefore, we speculated that the increased level of glycerophospholipids with unsaturated fatty acids under BmCPV infection may be related to the geometry of phospholipid monolayers in cellular membrane. PE derived from PS was validated to contribute to the formation of neurite in cells [40] . 

Identification and characterization of circular RNAs in the silkworm midgut following Bombyx mori cytoplasmic polyhedrosis virus infection

Identification and characterization of vp7 gene in Bombyx mori cytoplasmic polyhedrosis virus

Functional analysis of a miRNA-like small RNA derived from Bombyx mori cytoplasmic polyhedrosis virus

Viral Small-RNA Analysis of Bombyx mori

Larval Midgut during Persistent and Pathogenic Cytoplasmic Polyhedrosis Virus Infection

Bombyx mori cypovirus encoded small peptide inhibits viral multiplication

Metabolic Instruction of Immunity

Viral activation of cellular metabolism

Influence of cellular lipid content on influenza A virus replication

Serum Lipidomics Analysis of Classical Swine Fever Virus Infection in Piglets and Emerging Role of Free Fatty Acids in Virus Replication in vitro

Characterization of the Lipidomic Profile of Human Coronavirus-Infected Cells: Implications for Lipid Metabolism Remodeling upon Coronavirus Replication. Viruses

Methyl-beta-cyclodextrin inhibits EV-D68 virus entry by perturbing the accumulation of virus particles and ICAM-5 in lipid rafts

Inhibition of HIV Replication by Apolipoprotein A-I Binding Protein Targeting the Lipid Rafts

Zebrafish C-reactive protein isoforms inhibit SVCV replication by blocking autophagy through interactions with cell membrane cholesterol

Baicalin Inhibits Coxsackievirus B3 Replication by Reducing Cellular Lipid Synthesis

Fatty Acids Regulate Porcine Reproductive and Respiratory Syndrome Virus Infection via the AMPK-ACC1

Signaling Pathway. Viruses

Lipidomic Profiling Reveals Significant Perturbations of Intracellular Lipid Homeostasis in Enterovirus-Infected Cells

Fatty acid synthase and stearoyl-CoA desaturase-1 are conserved druggable cofactors of Old World Alphavirus genome replication

Cholesterol Binding to the Transmembrane Region of a Group 2 Hemagglutinin (HA) of Influenza Virus Is Essential for Virus Replication, Affecting both Virus Assembly and HA Fusion Activity

Zika virus infection modulates the metabolomic profile of microglial cells

Vitro and In Vivo Metabolomic Profiling after Infection with Virulent Newcastle Disease Virus. Viruses

Viruses: hostages to the cell

Host Cell Plasma Membrane Phosphatidylserine Regulates the Assembly and Budding of Ebola Virus

Ebola virus requires a host scramblase for externalization of phosphatidylserine on the surface of viral particles

The Ebola virus matrix protein VP40 selectively induces vesiculation from phosphatidylserine-enriched membranes

The critical role of phosphatidylcholine and phosphatidylethanolamine metabolism in health and disease

Effect of saturation degree on the interactions between fatty acids and phosphatidylcholines in binary and ternary Langmuir monolayers

Differential Utilization of the Ethanolamine Moiety of Phosphatidylethanolamine Derived from Serine and Ethanolamine during NGF-Induced Neuritogenesis of PC12 Cells

Fatty acid composition of adipose tissue and blood in humans and its use as a biomarker of dietary intake

Perturbations in the antioxidant metabolism during Newcastle disease virus (NDV) infection in chicken : protective role of vitamin E

Vitamin E Supplementation Ameliorates Newcastle Disease Virus-Induced Oxidative Stress and Alleviates Tissue Damage in the Brains of Chickens

PS(35:1) PS(35:1)-H PS (35:1) 1.13005 6

MePC(33:1) MePC(33:1)+Na MePC (33:1) 1.5893 2

PC(34:4) PC(34:4)+H PC

CL(78:1) CL

PS(39:1) PS(39:1)-H PS

) MePC(38:2)+NH4 MePC (38:2) 1.29956 1

SM(d34:1) SM(d34:1)+Na SM

Hex1Cer(d14:1/22:0) Hex1Cer(d36:1)+H Hex1Cer

PC(16:1/16:1) PC(32:2)+HCOO PC

TG

:1)+HCOO PC

PC

TG

PE

SM(d38:1) SM(d38:1)+H SM

SM(d38:4) SM(d38:4)+H SM (d38:4) 1.19202 9

CerPE(d36:2) CerPE(d36:2)+H CerPE

PC(33:1) PC(33:1)+H PC

PC(38:5) PC(38:5)+H PC (38:5) 1.28944 5.9E-05

PS(38:3) PS(38:3)-H PS

CerPE(d34:1) CerPE(d34:1)+H CerPE

CerPE(d36:1) CerPE(d36:1)+H CerPE (d36:1) 2.28767 6

PC(38:1) PC(38:1)+H PC

) PC(38:2)+H PC (38:2) 1.27056 6E-05

PE(36:1) PE(36:1)+Na PE

SM(d36:1) SM(d36:1)+H SM

The authors declare that they have no conflict of interest.