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Lactate facilitates classical swine fever virus replication by enhancing cholesterol biosynthesis

Summary An emerging topic in virology is that viral replication is closely linked with the metabolic reprogramming of host cells. Understanding the effects of reprogramming host cell metabolism due to classical swine fever virus (CSFV) infection and the underling mechanisms would facilitate controlling the spread of classical swine fever (CSF). In the current study, we found that CSFV infection enhanced aerobic glycolysis in PK-15 cells. Blocking glycolysis with 2-deoxy-d-glycose or disrupting the enzymes PFKL and LDHA decreased CSFV replication. Lactate was identified as an important molecule in CSFV replication, independent of the pentose phosphate pathway and tricarboxylic acid cycle. Further analysis demonstrated that the accumulated lactate in cells promoted cholesterol biosynthesis, which facilitated CSFV replication and disrupted the type I interferon response during CSFV replication, and the disruption of cholesterol synthesis abolished the lactate effects on CSFV replication. The results provided more insights into the complex pathological mechanisms of CSFV.


INTRODUCTION
The replication and propagation of viruses in cells are dependent on host cell metabolism (Varanasi and Rouse, 2018). In recent years, we have made significant advances with respect to how viruses interact with host cell metabolism (Mayer et al., 2019). Viruses reshape host cell metabolism during infections by targeting essential metabolic regulatory proteins and generating unique metabolic states that facilitate their replication (Sumbria et al., 2020). By modulating complex signaling pathways, some viruses increase available energy and promote their reproduction by manipulating glucose metabolism (Thaker et al., 2019). It has been discovered that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hijacks the mitochondria of cells and manipulates the metabolic pathways of the host cell for replication (Ajaz et al., 2021). The Epstein-Barr virus (EBV) and the Zika virus enhance glucose uptake due to a reduction in AMPK phosphorylation (Lyu et al., 2018;Singh et al., 2020). The PI3K/AKT pathway is activated by the norovirus, rhinovirus, and the human immunodeficiency virus (HIV) to increase glucose transporter type 1 (GLUT1) phosphorylation and increase glycolysis flux (Gualdoni et al., 2018;Passalacqua et al., 2019;Sorbara et al., 1996).
Meanwhile, numerous reports have emphasized the effects of viral infections and replication on the important roles of host cell lipid metabolism. To gain a selective advantage and to facilitate its replication, a virus actively alters host cell lipid metabolism. For example, human cytomegalovirus and Middle East respiratory syndrome coronavirus increase SREBP1 translocation to the nuclei, and thus, increase fatty acid synthesis (Spencer et al., 2011;Yuan et al., 2019). The EBV, coxsackievirus B3, respiratory syncytial virus, and hepatitis C virus (HCV) increase the expression of fatty acid synthase and induce lipogenic gene expression (Hulse et al., 2021;Kapadia and Chisari, 2005;Ohol et al., 2015;Wilsky et al., 2012). In addition, SARS-CoV-2, HCV, Zika virus, dengue virus, West Nile virus, rotavirus, and influenza virus can alter lipid droplet (LD) compartments, trafficking, formation, or recruitment (Cheung et al., 2010;Dias et al., 2020;Episcopio et al., 2019;Hou et al., 2017;Martins et al., 2019;Samsa et al., 2009;Vogt et al., 2013).
Previous reports of proteomic analyses have indicated that CSFV infection in PK-15 cells improved the glycolytic process by increasing phosphoglycerate mutase 1 (PGAM1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and triosephosphate isomerase (Gou et al., 2017). However, later research on the metabolic profiles in CSFV-infected PK-15 cells have shown that the glycolytic intermediates were decreased, but the lactate level was upregulated (fold change [FC] = 0.09) (Sun et al., 2008). Thus, a consensus has not been reached on the role of aerobic glycolysis in CSFV infection and replication.
Herein, we investigated the relationship between CSFV infection and aerobic glycolysis in PK-15 cells and the underlying mechanisms. The results showed that CSFV infection enhanced aerobic glycolysis and that lactate was an essential molecule in CSFV replication. In addition, lactate exerted functions by promoting cholesterol biosynthesis. The data provided more insights into the complex pathological mechanisms of CSFV.

CSFV enhances aerobic glycolysis in PK-15 cells
We investigated the glucose levels in a culture medium of PK-15 cells during CSFV replication. As shown in Figure 1A, a significant decrease in glucose levels was detected in the PK-15 cells during CSFV replication. Meanwhile, extracellular lactate was increased significantly when compared with that of the control group ( Figure 1B). Furthermore, the ATP levels in CSFV-infected cells were elevated significantly ( Figure 1C). iScience Article According to the one-step growth curve of CSFV ( Figure S1A), the copy numbers of viral genomic RNAs peaked at 48 h post infection (hpi). Therefore, CSFV-infected PK-15 cells were prepared at 48 hpi for transcriptome analysis. There were 824 differentially expressed genes (DEGs) in the CSFV-infected PK-15 cells, with 460 upregulated genes and 364 downregulated genes ( Figure 1D). The ''glycolytic process'' was discovered to be a highly enriched biological pathway ( Figure 1E). In the RNA-sequencing data, transcriptional and translational expression analyses confirmed significant changes in the genes involved in glycolysis, such as the HK2, PFKL, and LDHA genes ( Figures 1F-1H). Taken together, CSFV replication enhanced aerobic glycolysis in PK-15 cells.

Aerobic glycolysis direct involvement in CSFV replication
To investigate the effects of glycolysis on CSFV replication, the glycolytic pathway was manipulated (Figure 2B and Table 1). First, we discovered that the glucose analog 2-deoxy-d-glycose inhibited CSFV replication significantly, whereas UK-5099, a selective inhibitor of mitochondrial pyruvate carrier, enhanced the glycolysis flux and increased CSFV replication ( Figure 2A). Then, to further investigate the effect of glycolysis on CSFV replication, the PFKL gene (PO) and the LDHA gene (LO) in the PK-15 cells were separately iScience Article disrupted by the CRISPR-Cas9 system. As shown in Figure 2C, CSFV replication was effectively decreased in the PO and LO cells. The disruption of the LDHA gene, which plays a role in the downstream of glycolysis, decreased CSFV replication more than that of the PFKL gene ( Figure S2A). Moreover, the levels of intracellular and extracellular lactate were significantly lower in the PO and LO cells when compared with the control group. However, the addition of lactate into the culture media reversed the inhibition of CSFV replication in the LO cells ( Figures 2D, 2E, and S2B-S2D). Taken together, these findings suggested that glycolysis plays an important role in CSFV replication.

Intracellular lactate maintains CSFV replication as the effector molecule of glycolysis
Previous studies have shown that many tumor and cancer cells undergo glycolysis under aerobic conditions to produce large amounts of lactate for proliferation and metastasis (Becker, 2020), and that lactate appears to be the primary cause of decreased extracellular pH in tumor tissues (Kato et al., 2013). Thus, we examined the role of extracellular lactate in CSFV replication. As shown in Figure 3A, the levels of extracellular lactate were gradually increased and extracellular pH levels were continuously decreased along with the time of CSFV replication. To identify the pH effects caused by lactate on CSFV replication, we prepared culture mediums with various pH values by using the addition of lactate. When compared with the control group, different pH values did not affect CSFV replication in the PK-15 cells ( Figure 3C). Along with the CSFV replication, the level of intracellular lactate reached a peak at 48 hpi when compared with the control group ( Figure 3D). To investigate the effects of intracellular lactate, we disrupted lactate monocarboxylate transporter 1 (MCT1), which is responsible for the transport of exogenous lactate into PK-15 cells (MO) (Figures S3A and S3B and, Table 1, S1, and S2). As shown in Figures 3E and 3F, the deletion of the MCT1 gene itself had no effect on CSFV replication or intracellular lactate levels. However, exogenous lactate could not restore CSFV replication or intracellular lactate levels by knocking out PFKL (PMO) and LDHA (LMO) on the basis of MCT1 and knockout (Figures 3G-3I). Collectively, intracellular, but not extracellular, lactate promotes CSFV replication.

Lactate promotes cholesterol biosynthesis to maintain the replication of CSFV
When compared with the control group, along with lactate reduction, cholesterol was decreased in the LO cells, and the exogenous addition of cholesterol, similar to lactate addition, reversed the inhibition of CSFV replication in the LO cells. Moreover, the increased total SREBP2 protein in CSFV-infected cells was reduced when LDHA was deleted. iScience Article response in a STING-dependent manner (Fritsch and Weichhart, 2016;York et al., 2015). Thus, we investigated the roles of cholesterol on the regulation of glycolysis via IFNs during CSFV replication. As shown in Figures 5A-5H and S5A-S5H, the levels of phosphorylated TBK1 (p-TBK1) and interferon-stimulated genes (ISGs) such as CCL2, CXCL10, and MX1 in CSFV-infected PK-15 cells were significantly suppressed at 48 hpi. Disruption of glycolysis or the cholesterol biosynthesis pathway both facilitated the expression of p-TBK1 and ISGs. Exogenous lactate and cholesterol could both restore p-TBK1 and ISG levels in the LO cells. However, only the addition of cholesterol, rather than lactate, was able to reverse p-TBK1 and ISG expression in the SO cells. Collectively, these data suggest that CSFV infection enhances aerobic glycolysis to produce a large amount of lactate that activates cholesterol biosynthesis to attenuate type I IFN signaling.

DISCUSSION
In this study, we showed that CSFV infection enhanced aerobic glycolysis in PK-15 cells and the end product of glycolysis, i.e., lactate, was the essential molecule that maintained CSFV replication. Meanwhile, lactate accumulation promoted the synthesis of cholesterol, which disrupted the type I IFN response and further facilitated CSFV replication. iScience Article Accumulating evidence has suggested that a virus and its encoded genes or proteins alter host cell metabolism by modifying the expression and activity of glucose transporters and glycolytic enzymes and modulating the function of key signaling molecules involved in host cell metabolism for viral replication (Yu et al., 2018). The adenoviral gene product E4ORF1 enhances MYC transcriptional activity to upregulate glycolytic target genes such as HK2, LDHA, and PFK1 for optimal adenovirus replication (Thai et al., 2014). DENV nonstructural protein 1 (NS1) enhances glycolysis flux and energy production by suppressing the function of GAPDH (Allonso et al., 2015;Silva et al., 2019). EBV-encoded LMP1 activates the FGFR1 signaling pathway to increase the cellular uptake of glucose and to enhance LDHA activity and regulates HoxC8 expression via PolII stalling to stabilize the expression of glycolytic genes such as GLUT1 and HK2 (Jiang et al., 2015;Lo et al., 2015). In addition, C-terminal-activating regions of LMP1 can promote GLUT1 transcription via the mTORC1/NF-kB signaling pathway . The hepatitis B virus core protein (HBc) and x protein (HBx) can increase key glycolytic enzymes and related genes NAMPT and NRF2 to enhance glycolysis (Guo et al., 2021;Xie et al., 2017). Moreover, HCV severely impairs mitochondrial oxidative phosphorylation to promote HIF-1a expression and transcriptional activation, and its encoded proteins (NS5A and NS5B) can promote glycolytic enzymes' activity to enhance glycolysis for viral replication (Ramiè re et al., 2014;Ripoli et al., 2010;Wu et al., 2008). Previous studies have used 2D polyacrylamide gel electrophoresis followed by MALDI-TOF tandem mass spectromentry identification to perform proteomic analyses in CSFV-infected PK-15 cells and have found that CSFV replication affects glycolytic enzyme expression including upregulated (PGAM1), triosephosphate isomerase, and downregulated GAPDH in PK-15 cells (Gou et al., 2017). However, in CSFV-infected PK-15 cells, a metabolic analysis revealed that CSFV replication decreased glycolytic intermediates such as glucose 6-phosphate (FC = À1.94) and glyceraldehyde-3phosphate (FC = À1.83) (Sun et al., 2008). In our study, increased glucose consumption, lactate production,  iScience Article and the expression of glucose transporter GLUT1 and the glycolytic enzyme LDHA were also found in CSFV-infected PK-15 cells. The transcriptome results further confirmed that the glycolytic flux was significantly enhanced in CSFV-infected cells when compared with the control group. Inhibition of the tricarboxylic acid (TCA) cycle or addition of lactate in PFKL or LDHA knockout (KO) cells both increased CSFV replication. The data confirmed the relationship between glycolysis flux or lactate and CSFV replication.
Previous research has reported that the accumulation of lactate contributed to the acidification of a tumor microenvironment, and that the acidosis favored metastasis, angiogenesis, and immunosuppression (de la Cruz-Lopez et al., 2019). In the present study, we employed various pH values using the addition of lactate to observe the effect on CSFV replication. The data demonstrated that lactate, and not the acidic environment, maintained CSFV replication. When MCT1 was deleted, which blocked the transportation of exogenous materials into cells, especially lactate, which is the predominant carbon source of TCA, the CSFV replication was not affected. Thus, endogenous lactate is sufficient to support the replication of CSFV. It is well known that the glycolytic intermediate, glucose-6-phosphate, can promote the pentose phosphate pathway (PPP), and the enhanced PPP produces pentoses, which is used to synthesize DNA, RNA, and cofactors in enzymes. In cancer, aerobic glycolysis provides cellular energy, and also increases PPP to process macromolecular biosynthesis, conferring a selective advantage under diminished nutrition (Patra and Hay, 2014). However, in the present study, PFKL KO, which forced PPP enhancement, blocked lactate production and the TCA cycle, and still decreased CSFV replication significantly, similar to that of LDHA KO, which blocked lactate production and enhanced the TCA cycle. The data demonstrated that neither PPP nor the TCA cycle was the determinant factor, and lactate played an essential role in CSFV replication.
How does lactate maintain CSFV replication? Numerous reports have indicated that lipid metabolism plays an important role during the viral replication cycle (Monson et al., 2021). HIV, via its accessory protein Nef, downregulates ATP-binding cassette transporter A1 (ABCA1) expression and alters intracellular distribution of ABCA1, thus consequently impairing cholesterol efflux of infected macrophages and increasing infectivity of HIV virions (Mujawar et al., 2006). DENV infection induces autophagy, causing the depletion of LD triglyceride stores and the release of free fatty acids, and facilitates robust viral RNA replication (Heaton and Randall, 2010). HBx induces the expression of liver x receptor (LXR) and its lipogenic target genes such as sterol regulatory element-binding protein-1c (SREBP-1c), and this is accompanied by the accumulation of LDs (Na et al., 2009). HCV and its viral proteins such as core, NS2, and NS4B modulate lipid homeostasis by increasing lipogenesis via SREBP activation and reducing oxidation and lipid export (Moriishi et al., 2007;Negro and Sanyal, 2009;Oem et al., 2008;Park et al., 2009). Regarding CSFV, many studies have demonstrated that interference with cholesterol biosynthesis and trafficking affected its life cycle (Liang et al., 2019;Yu et al., 2019;Zou et al., 2022). In the present study, we observed that exogenous lactate could increase either CSFV replication or intracellular cholesterol level in LDHA KO cells. Moreover, LDHA KOinduced CSFV replication could be elevated by cholesterol addition. Furthermore, lactate could not enhance CSFV replication when the synthesis of cholesterol was disrupted. Collectively, CSFV-induced lactate production enhances cholesterol biosynthesis, which facilitates the replication of itself.
Current mechanistic studies have revealed that limiting the cholesterol biosynthetic pathway spontaneously engaged a type I IFN response in a STING-dependent manner (York et al., 2015). Meanwhile, studies have showed that lactate inhibited retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) signaling, resulting in decreased type I interferon production and viral clearance . Our data also showed that CSFV infection induced lactate accumulation, which disturbed the type I IFN response by altering the cholesterol level in PK-15 cells.
In conclusion, our findings show that CSFV infection induces reprogramming of host cell metabolism, which facilitates its replication. Mechanistically, the aerobic glycolysis induced by CSFV infection enhances the accumulation of lactate in cells, and the lactate promotes cholesterol biosynthesis, which facilitates the replication of CSFV and disrupts the type I IFN response.

Limitations of the study
Although our study highlights that lactate promotes cholesterol biosynthesis to facilitate CSFV replication, several limitations of our study remain to be explored that will further shed light on detailed mechanism of action, for example, experiments probing how lactate regulates cholesterol biosynthesis and its mechanism. We believe these are important future research avenues that will elucidate the exact mechanism of action of lactate in CSFV replication.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

ACKNOWLEDGMENTS
This work was financially supported by the National Natural Science Foundation of China (no. 31972710 and 31772747). We thank our colleagues from the College of Animal Sciences, Jilin University, for their discussion and feedback.

AUTHOR CONTRIBUTIONS
X.Z., H.O., D.P., and X.T. conceived the study and wrote the manuscript. X.Z., Y.Y., F.L., J.C., H.Z., and L.L. performed the experiments, analyzed the data, and drafted the manuscript. L.R. reviewed and revised the manuscript.

DECLARATION OF INTERESTS
The authors declare that they have no conflicts of interest.

Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Xiaochun Tang at xiaochuntang@jlu.edu.cn.

Materials availability
This study did not generate new unique reagents. All unique reagents generated in this study are available from the lead contact with a completed Material Transfer Agreement.
Data and code availability d All data reported in this paper will be shared by the lead contact upon request.
d This study did not generate original code.
d Any additional information required to reanalyze the data reported in this paper is available form the lead contact upon request.

Cell lines
Porcine kidney cell line (PK-15) cells were purchased from ATCC. PK-15 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum and penicillin-streptomycin. All cells were maintained at 37 C and 5% CO 2 . Most culture media were from Gibco unless otherwise noted. Heatinactivated FBS was purchased from Gibco.

Virus strain
CSFV (Shimen strain) was kindly provided by Dr. Changchun Tu (Academy of Military Medical Sciences, Changchun, China).

Detection of lactate concentrations
The lactate concentrations were measured using the lactate assay kit (Nanjing Jiancheng Bioengineering Institute, China, A019-2-1), as directed by the manufacturer. An Infinite 2000 PRO Microplate Reader was used to determine the OD (Tecan, Switzerland). The absorbance of the samples was monitored at 37 C and measured in triplicate. The total protein concentration was used to normalize the data.

Detection of the content of adenosine triphosphate (ATP)
According to the manufacturer's instructions, the content of ATP was detected using an Enhanced ATP Assay Kit (Beyotime, China, S0027 TRNzol-A + Reagent (Tiangen, China, DP424) was used to extract total RNA from PK-15 cells, and the FastKing RT Kit (with gDNase) (Tiangen, China, KR116) was used to reverse transcribe the RNA into cDNA. A BIO-RAD iCycler Thermal Cycler with iQ5 Optical Module for RT-PCR was used to measure fluorescence intensity and amplification plots (Bio-Rad, ABI 7500, iQ5). The primers used in qPCR are listed in Table S1.

Virus one-step growth curve
Infection of PK-15 cells with 200 TCID50 of CSFV (Shimen strain) in a 6-well plate with 80 percent confluence was done in three replicates for each group. The inoculums were replaced with fresh medium one hour after inoculation. At 12,24,36,48,60,72,84,200 mL of viral supernatant was harvested, extract viral RNA and reverse transcribe the RNA into cDNA. The viral-genomic copy numbers were detected by qPCR. The virus one-step growth curve was created according to viral-genomic copy numbers.

Transcriptome analysis
PK-15 cells were seeded at a density of 2 3 10 5 cells per well in 6-well plates to obtain 80-90% confluence before CSFV infection. CSFV (Shimen strain) was infected into PK-15 cells at 200 TCID 50 and harvested 48 h after infection. TRNzol-A+ reagent was used to isolate total RNA, which was then subjected to RNA deep sequencing and analysis as directed by the manufacturer. The Metascape platform (http://metascape.org/) was used to perform GO and KEGG enrichment pathways .

Western blotting (WB)
Western blotting was performed according to the manufacturer's instructions. Equal amounts of proteins were separated on the separating gel using SDS-PAGE, and the protein bands were electrophoretically transferred to PVDF membranes. The ECL-Plus western blotting reagent was used to detect the protein bands. The primary and secondary antibodies involved in the process were shown in Table S2.

Cell transfection and genotyping of cell clones
Approximately, 3 3 10 6 PK-15 cells were electrotransfected with 30 mg plasmids in 200 mL of Opti-MEM (GIBCO) using 2-mm gap cuvettes and a BTX ECM 2001 electroporator. The parameters for electrotransfection were as follows: 300 V, 1 ms, 3 pulses for 1 repeat. At 36 h after electrotransfection, cells were plated into five 10-cm dishes at 1.5 3 10 3 cells per dish. In 48-well plates, single-cell colonies were chosen and cultured. The cell clones were subcultured after reaching 80% confluence, and 20% of each clone was lysed for 1 h at 56 C and 10 min at 95 C in 20 mL of NP40 lysis buffer (0.45 percent NP-40 plus 0.6 percent Proteinase K).
The lysate was used as a PCR template and was subjected to 1% agarose gel electrophoresis. The primers for genotyping are shown in the following table.

Off-target analysis
Cas-OFFinder was used to predict all possible off-target sites (OTSs) for each sgRNA. To identify off-target effects, OTS were analyzed using PCR and DNA sequencing. The primer sequences used for analyzing the off-target activities are listed in Tables S3 and S4.

Detection of extracellular pH (pHe)
The extracellular pH of samples was measured using a high-precision pH meter according to manufacturer's protocol. Samples were monitored at 37 C and measured in triplicate.