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. 2018 Jul 17;9(4):e01115-18.
doi: 10.1128/mBio.01115-18.

Liver X Receptors Suppress Activity of Cholesterol and Fatty Acid Synthesis Pathways To Oppose Gammaherpesvirus Replication

P T Lange  1 , C Schorl  2 , D Sahoo  3   4 , V L Tarakanova  5   6
Affiliations

Liver X Receptors Suppress Activity of Cholesterol and Fatty Acid Synthesis Pathways To Oppose Gammaherpesvirus Replication

P T Lange et al. mBio. .

Abstract

Gammaherpesviruses are oncogenic pathogens that persist in ~95% of the adult population. Cellular metabolic pathways have emerged as important regulators of many viral infections, including infections by gammaherpesviruses that require several lipid synthetic pathways for optimal replication. Liver X receptors (LXRs) are transcription factors that are critical regulators of cellular fatty acid and cholesterol synthesis pathways. Not surprisingly, LXRs are attractive therapeutic targets in cardiovascular disease. Here we describe an antiviral role for LXRs in the context of gammaherpesvirus infection of primary macrophages. We show that type I interferon increased LXR expression following infection. Surprisingly, there was not a corresponding induction of LXR target genes. Rather, LXRs suppressed the expression of target genes, leading to decreased fatty acid and cholesterol synthesis, two metabolic pathways that support gammaherpesvirus replication. This report defines LXR-mediated restriction of cholesterol and lipid synthesis as an intrinsic metabolic mechanism to restrict viral replication in innate immune cells.IMPORTANCE Fatty acid and cholesterol synthesis pathways of the host play important roles in diverse biological systems. Importantly, these two metabolic pathways are also usurped by a number of viruses to facilitate viral replication. In this report, we show that suppression of these pathways by liver X receptors in primary macrophages creates an intrinsic antiviral state that attenuates gammaherpesvirus replication by limiting viral access to the two metabolic pathways.

Keywords: cholesterol synthesis; fatty acid synthesis; gammaherpesvirus; liver X receptors; macrophages.

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Figures

FIG 1
FIG 1
LXRs attenuate MHV68 replication in macrophages, which show increased LXR expression in a type I IFN-dependent manner. Bone marrow-derived macrophages (BMDM) of indicated genotypes were infected at an MOI of 0.01 (A and C), 5 (B, E, F, G, J, and K), or 1 (D) for the indicated time or for 24 h. Data represent total viral titers (combined cell associated and extracellular) (A and B), relative viral DNA levels (cell associated) (C and D), protein levels of LXRα and β-actin in triplicate (E), and mRNA levels of LXRα and LXRβ (F to I). The BL6 macrophages represented in panels H and I were mock treated or treated with10 U/ml of IFN-β for 24 h. (J and K) BL6 macrophages were infected with MHV68 or UV-inactivated MHV68 or were mock infected for 24 h. Expression of LXRα and LXRβ was assessed by qRT-PCR. Data shown are representative results from 2 to 4 independent experiments. Error bars represent standard errors of the means here and in the other figures. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant (here and in the other figures). Veh, vehicle.
FIG 2
FIG 2
25-Hydroxycholesterol (25HC) is not required for LXR expression and suppresses MHV68 replication in an LXR-independent manner. (A) Primary macrophages derived from BL6 and CH25H−/− mice were infected at an MOI of 5 for 24 h. LXRα and LXRβ expression was measured by qRT-PCR. (B) Primary macrophages derived from BL6 and LXR−/− mice were infected at an MOI of 0.01 and then immediately treated with 2 µM 25HC or vehicle control for 72 h. Data shown are representative results from at least two independent experiments.
FIG 3
FIG 3
Increased expression of LXRs in MHV68-infected macrophages does not produce a corresponding increase in the expression of LXR target genes. BL6 BMDM were mock treated or infected with MHV68 at an MOI of 5 for 24 h. mRNA levels of the indicated genes were measured by qRT-PCR. Data were pooled from at least two independent experiments.
FIG 4
FIG 4
LXR deficiency results in increased expression of classical LXR target genes in primary macrophages. (A) Three independent batches of BL6 and LXR−/− BMDM were infected at an MOI of 10 for 24 h. Total RNA was isolated, and relative gene expression levels were compared via Affymetrix microarray. Differentially expressed genes were analyzed by the use of Ingenuity Pathway Analysis software. Gray bars represent the percentages of genes in each statistically significant category for which the expression was lower in BL6 macrophages than in LXR−/− macrophages. Diamonds indicate corresponding –Log data (P value) for each category. (B) Volcano plot representation of differentially expressed genes. (C to F) BMDM from BL6 and LXR−/− mice were infected at an MOI of 5 for 24 h or were mock infected, with subsequent measurement of relative gene expression levels by qRT-PCR. Data are representative of results from two or more experiments.
FIG 5
FIG 5
LXRs do not regulate MHV68 replication in mouse embryonic fibroblasts (MEFs). MEFs were prepared from BL6 embryos or embryos genetically deficient in both LXR isoforms (LXR−/−) and were mock treated, infected with MHV68 (MOI = 1), or treated with exogenous IFN-β. (A and B) MRNA levels of indicated genes were measured at 24 h postinfection/treatment by qRT-PCR for SCD2 (A) or FADS2 (B). (C) Viral titers were determined at the indicated times postinfection. Data are representative of results from two or more experiments.
FIG 6
FIG 6
Treatment with artificial LXR agonists promotes expression of LXR target genes but does not increase MHV68 replication. (A to C) BL6 BMDM were infected at an MOI of 5 or mock infected. At 20 h postinfection, macrophages were treated with 5 µM GW3965 (GW) or vehicle control (Veh) for 4 h, followed by isolation of total RNA. qRT-PCR was performed to determine the relative expression levels of the indicated genes. (D) Experimental parameters were identical to those described for panels A to C with both BL6 and LXR−/− macrophages used. (E) BL6 BMDM were infected at an MOI of 0.01 and treated with 5 µM GW3965 or vehicle control. Viral titers were determined at the indicated times. Data shown are representative results from at least two independent experiments.
FIG 7
FIG 7
Activity of fatty acid and cholesterol synthesis pathways is increased and antiviral effects of fatty acid synthesis inhibition are attenuated in LXR−/− macrophages. (A to C) BMDM of the indicated genotypes were infected at an MOI of 5 or mock infected. Macrophages were exposed to 5 µCi [3H]acetic acid for 8 h (16 to 24 h postinfection). At 24 h postinfection, macrophages were harvested; cholesterol (A), fatty acids (B), and triglycerides (C) data were resolved by TLC; and incorporation of 3H label was quantified by scintillation counting. DPM, disintegration per minute. (D and E) BMDM of indicated genotypes were infected at an MOI of 0.01 and treated with 10 µg/ml TOFA or vehicle control. Viral titers were measured at the indicated times postinfection. Data are representative of results from at least two independent experiments.
FIG 8
FIG 8
Working model. Macrophages respond to type I IFN produced during viral infection by increasing expression of LXRs. The LXRs interact with nuclear corepressors to limit the expression of select LXR target genes. This repression of gene expression limits the activity of the proviral cholesterol and fatty acid synthesis pathways and restricts MHV68 replication.
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