scholarly journals Kinetic expression analysis of the cluster mdv1-mir-M9–M4, genes meq and vIL-8 differs between the lytic and latent phases of Marek’s disease virus infection

2012 ◽  
Vol 93 (7) ◽  
pp. 1519-1529 ◽  
Author(s):  
D. Coupeau ◽  
G. Dambrine ◽  
D. Rasschaert
Keyword(s):  
Disease Virus ◽  
Transcriptional Start Site ◽  
Marek's Disease Virus ◽  
Marek's Disease ◽  
Marek’S Disease Virus ◽  
Marek’S Disease ◽  
First Intron ◽  
Alternatively Spliced ◽  
Infected Cells ◽  
Race Pcr

Marek’s disease virus (GaHV-2) is an alphaherpesvirus that induces T-cell lymphoma in chickens. The infection includes both lytic and latent stages. GaHV-2 encodes three clusters of microRNAs (miRNAs) located in the internal (I)/terminal (T) repeat (R) regions. We characterized transcripts encompassing the mdv1-mir-M9–M4 and mir-M11–M1 clusters located in the IRL/TRL region, upstream and downstream from the meq oncogene, respectively. By 5′- and 3′-RACE-PCR and targeted RT-PCR, we showed that mdv1-mir-M9–M4 could be transcribed from an unspliced transcript or from at least 15 alternatively spliced transcripts covering the IRL/TRL region, encompassing the meq and vIL-8 genes and localizing the mdv1-mir-M9–M4 cluster to the first intron at the 5′-end. However, all these transcripts, whether spliced or unspliced, seemed to start at the same transcriptional start site, their transcription being driven by a single promoter, prmiRM9M4. We demonstrated alternative promoter usage for the meq and vIL-8 genes, depending on the phase of GaHV-2 infection. During the latent phase, the prmiRM9M4 promoter drove transcription of the meq and vIL-8 genes and the mdv1-mir-M9–M4 cluster in the first intron of the corresponding transcripts. By contrast, during the lytic phase, this promoter drove the transcription only of the mdv1-mir-M9–M4 cluster to generate unspliced mRNA, the meq and vIL-8 genes being transcribed principally from their own promoters. Despite the expression of meq and the mdv1-mir-M9–M4 cluster under two different transcriptional processes during the latent and lytic phases, our data provide an explanation for meq expression and mdv1-mir-M4-5P overexpression in miRNA libraries from GaHV-2-infected cells, regardless of the phase of infection.

scholarly journals Glutaminolysis and Glycolysis Are Essential for Optimal Replication of Marek’s Disease Virus

2019 ◽  
Vol 94 (4) ◽  
Author(s):  
Nitish Boodhoo ◽  
Nitin Kamble ◽  
Shayan Sharif ◽  
Shahriar Behboudi
Keyword(s):  
Fatty Acid ◽  
Disease Virus ◽  
Virus Replication ◽  
Tca Cycle ◽  
Marek's Disease Virus ◽  
Marek's Disease ◽  
Host Cells ◽  
Marek’S Disease Virus ◽  
Marek’S Disease ◽  
Infected Cells

ABSTRACT Viruses may hijack glycolysis, glutaminolysis, or fatty acid β-oxidation of host cells to provide the energy and macromolecules required for efficient viral replication. Marek’s disease virus (MDV) causes a deadly lymphoproliferative disease in chickens and modulates metabolism of host cells. Metabolic analysis of MDV-infected chicken embryonic fibroblasts (CEFs) identified elevated levels of metabolites involved in glutamine catabolism, such as glutamic acid, alanine, glycine, pyrimidine, and creatine. In addition, our results demonstrate that glutamine uptake is elevated by MDV-infected cells in vitro. Although glutamine, but not glucose, deprivation significantly reduced cell viability in MDV-infected cells, both glutamine and glucose were required for virus replication and spread. In the presence of minimum glutamine requirements based on optimal cell viability, virus replication was partially rescued by the addition of the tricarboxylic acid (TCA) cycle intermediate, α-ketoglutarate, suggesting that exogenous glutamine is an essential carbon source for the TCA cycle to generate energy and macromolecules required for virus replication. Surprisingly, the inhibition of carnitine palmitoyltransferase 1a (CPT1a), which is elevated in MDV-infected cells, by chemical (etomoxir) or physiological (malonyl-CoA) inhibitors, did not reduce MDV replication, indicating that MDV replication does not require fatty acid β-oxidation. Taken together, our results demonstrate that MDV infection activates anaplerotic substrate from glucose to glutamine to provide energy and macromolecules required for MDV replication, and optimal MDV replication occurs when the cells do not depend on mitochondrial β-oxidation. IMPORTANCE Viruses can manipulate host cellular metabolism to provide energy and essential biosynthetic requirements for efficient replication. Marek’s disease virus (MDV), an avian alphaherpesvirus, causes a deadly lymphoma in chickens and hijacks host cell metabolism. This study provides evidence for the importance of glycolysis and glutaminolysis, but not fatty acid β-oxidation, as an essential energy source for the replication and spread of MDV. Moreover, it suggests that in MDV infection, as in many tumor cells, glutamine is used for generation of energetic and biosynthetic requirements of the MDV infection, while glucose is used biosynthetically.

scholarly journals Infection of macrophages by a lymphotropic herpesvirus: a new tropism for Marek's disease virus

2003 ◽  
Vol 84 (10) ◽  
pp. 2635-2645 ◽  
Author(s):  
Alexander D. Barrow ◽  
Shane C. Burgess ◽  
Susan J. Baigent ◽  
Ken Howes ◽  
Venugopal K. Nair
Keyword(s):  
Disease Virus ◽  
Marek's Disease Virus ◽  
Marek's Disease ◽  
Cell Protein ◽  
Marek’S Disease Virus ◽  
Marek’S Disease ◽  
Macrophage Infection ◽  
T Helper Lymphocytes ◽  
Infected Cells

Marek's disease virus (MDV) is classified as an oncogenic lymphotropic herpesvirus of chickens. MDV productively and cytolytically infects B, αβT and γδT lymphocytes and latently infects T-helper lymphocytes. The aims of this study were to identify whether MDV infects macrophages in vivo and, if so, whether quantitative differences in macrophage infection are associated with MDV strain virulence. Chickens were infected with either virulent MDV (HPRS-16) or ‘hypervirulent’ MDV (C12/130). Flow cytometry with monoclonal antibodies recognizing MDV pp38 antigen and leukocyte antigens was used to identify MDV lytically infected cells. Macrophages from HPRS-16- and C12/130-infected chickens were pp38+. It is demonstrated that macrophages are pp38+ because they are infected and not because they have phagocytosed MDV antigens, as assessed by confocal microscopy using antibodies recognizing MDV antigens of the three herpesvirus kinetic classes: infected cell protein 4 (ICP4, immediate early), pp38 (early) and glycoprotein B (gB, late). Spleen macrophages from MDV-infected chickens were ICP4+, pp38+ and gB+, and ICP4 had nuclear localization denoting infection. Finally, MDV pp38+ macrophages had high inherent death rates, confirming cytolytic MDV infection, although production of virus particles has not been detected yet. These results have two fundamental implications for understanding MDV pathogenesis: (i) MDV evolved to perturb innate, in addition to acquired, immunity and (ii) macrophages are excellent candidates for transporting MDV to primary lymphoid organs during the earliest stages of pathogenesis.

scholarly journals Involvement of the Oncoprotein c-Myc in Viral Telomerase RNA Gene Regulation during Marek's Disease Virus-Induced Lymphomagenesis

2007 ◽  
Vol 81 (9) ◽  
pp. 4848-4857 ◽  
Author(s):  
Marina Shkreli ◽  
Ginette Dambrine ◽  
Denis Soubieux ◽  
Emmanuel Kut ◽  
Denis Rasschaert
Keyword(s):  
Transcriptional Regulation ◽  
Disease Virus ◽  
Transcriptional Start Site ◽  
Marek's Disease Virus ◽  
Marek's Disease ◽  
Luciferase Reporter ◽  
Telomerase Rna ◽  
Marek’S Disease Virus ◽  
Marek’S Disease ◽  
Protein Subunit

ABSTRACT Marek's disease virus (MDV) is an alphaherpesvirus that induces a highly malignant T-lymphoma in chickens. The viral genome encodes two identical copies of a viral telomerase RNA subunit (vTR) that exhibits 88% sequence identity to its chicken ortholog chTR. The minimal telomerase ribonucleoprotein complex consists of a protein subunit with reverse transcriptase activity (TERT) and an RNA subunit (TR). The active complex compensates for the progressive telomere shortening that occurs during mitosis and is involved in the cell immortalization process. We show here that the upregulation of telomerase activity is associated with an increase in vTR gene expression in chickens infected with the highly oncogenic MDV strain RB-1B. A comparative functional analysis of the viral and chicken TR promoters, based on luciferase reporter assays, revealed that the vTR promoter was up to threefold more efficient than the chTR promoter in avian cells. We demonstrated, by directed mutagenesis of the vTR promoter region, that the stronger transcriptional activity of the vTR promoter resulted largely from an E-box located two nucleotides downstream from the transcriptional start site of the vTR gene. Furthermore, transactivation assays and chromatin immunoprecipitation assays demonstrated the involvement of the c-Myc oncoprotein in the transcriptional regulation of vTR. Finally, an Ets binding site was specifically implicated in the transcriptional regulation of vTR in the MDV-transformed lymphoblastoid cell line MSB-1.

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