Abstract
Marek's disease virus (MDV) transforms CD4+ T cells and causes a deadly neoplastic disease that is associated with metabolic dysregulation leading to atherosclerosis in chickens. While MDV-infected chickens have normal serum concentrations of cholesterol, their aortic tissues were found to have elevated concentrations of free and esterified cholesterol. Here, we demonstrate that infection of chicken embryonated fibroblasts (CEFs) with highly pathogenic MDV-RB1B increases the cellular cholesterol content and upregulates the genes involved in cholesterol synthesis and cellular cholesterol homeostasis using comprehensive two-dimensional gas chromatography-mass spectrometry and real-time PCR (RT-PCR), respectively. Using small pharmacological inhibitors and gene silencing, we established an association between MDV-RB1B replication and mevalonic acid, sterol, and cholesterol biosynthesis and trafficking/redistribution. We propose that MDV trafficking is mediated by lysosome-associated membrane protein 1 (LAMP-1)-positive vesicles based on short hairpin RNA (shRNA) gene silencing and the colocalization of LAMP-1, glycoprotein B (gB) of MDV, and cholesterol (filipin III) fluorescence signal intensity peaks. In conclusion, our results demonstrate that MDV hijacks cellular cholesterol biosynthesis and cholesterol trafficking to facilitate cell-to-cell spread in a LAMP-1-dependent mechanism.IMPORTANCE MDV disrupts lipid metabolism and causes atherosclerosis in MDV-infected chickens; however, the role of cholesterol metabolism in the replication and spread of MDV is unknown. MDV-infected cells do not produce infectious cell-free virus in vitro, raising the question about the mechanism involved in the cell-to-cell spread of MDV. In this report, we provide evidence that MDV replication depends on de novo cholesterol biosynthesis and uptake. Interruption of cholesterol trafficking within multivesicular bodies (MVBs) by chemical inhibitors or gene silencing reduced MDV titers and cell-to-cell spread. Finally, we demonstrated that MDV gB colocalizes with cholesterol and LAMP-1, suggesting that viral protein trafficking is mediated by LAMP-1-positive vesicles in association with cholesterol. These results provide new insights into the cholesterol dependence of MDV replication.
Highlights
Marek’s disease virus (MDV) transforms CD4ϩ T cells and causes a deadly neoplastic disease that is associated with metabolic dysregulation leading to atherosclerosis in chickens
The relative levels of three metabolites involved in cholesterol biosynthesis were determined in cell lysates with a total of 6 technical replicates per biological replicate obtained from mock- and MDV-infected primary chicken embryonated fibroblasts (CEFs) at 48 and 72 h postinfection using comprehensive two-dimensional gas chromatography-mass spectrometry (GC/GC-MS)
The salient findings of this study are that (i) MDV infection increases the cholesterol content of infected cells and activates de novo cholesterol synthesis; (ii) exogenous and de novo cholesterol biosynthesis are required for efficient MDV replication and spread; (iii) MDV induces the upregulation of lysosome-associated membrane protein 1 (LAMP-1), involved in cholesterol trafficking, within multivesicular bodies (MVBs); and (iv) glycoprotein B of MDV, an essential viral protein for replication and egress, is associated with cholesterol and LAMP-1 within MVBs
Summary
Marek’s disease virus (MDV) transforms CD4ϩ T cells and causes a deadly neoplastic disease that is associated with metabolic dysregulation leading to atherosclerosis in chickens. Using small pharmacological inhibitors and gene silencing, we established an association between MDV-RB1B replication and mevalonic acid, sterol, and cholesterol biosynthesis and trafficking/redistribution. We demonstrated that MDV gB colocalizes with cholesterol and LAMP-1, suggesting that viral protein trafficking is mediated by LAMP-1-positive vesicles in association with cholesterol. These results provide new insights into the cholesterol dependence of MDV replication. The final stage involves the conversion of lanosterol to cholesterol, which is distributed via lysosome-associated membrane protein 1 (LAMP-1)-positive vesicles between intracellular compartments, including cell membranes [11,12,13]. The final envelopment of varicella-zoster virus, a highly cell-associated alphaherpesvirus similar to MDV, in the cytoplasm occurs at TGN-derived vesicles [17, 18]
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