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Cloning, sequencing, and functional analysis of a Marek's disease virus origin of DNA replication.

ABSTRACT: Previously, we isolated a replicon from a defective Marek's disease virus (MDV), analogous to defective herpes simplex viruses (amplicons). Defective viruses contain cis-acting elements required for DNA synthesis and virus propagation such as an origin of DNA replication and a packaging-cleavage signal site. In this report, the MDV replicon was utilized to locate an origin of MDV DNA replication. A comparison of MDV replicon sequences with other herpesvirus replication origin sequences revealed a 90-bp sequence containing 72% identity to the lytic origin (oris) of herpes simplex virus type 1. This 90-bp sequence displayed no similarity to betaherpesvirus or gammaherpesvirus replication origins. The 90-bp sequence is arranged as an imperfect palindrome centered around an A+T-rich region. This sequence also contains a 9-bp motif (5'CGTTCGCAC3') highly conserved in alphaherpesvirus replication origins. To test functionality of the 90-bp putative MDV replication origin, we conducted DpnI replication assays with subclones generated from the 4-kbp MDV replicon. A 700-bp MDV replicon subfragment containing the 90-bp putative MDV replication origin sequence is capable of replicating in chicken embryo fibroblast cells cotransfected with helper virus DNA. In conclusion, we identified a functional origin of DNA replication in MDV. Similarity of MDV origin sequences to those of alphaherpesviruses supports the current contention that MDV is more closely related to alphaherpesviruses than to gammaherpesviruses.

SUBMITTER: Camp HS

PROVIDER: S-EPMC250344 | biostudies-other | 1991 Nov

REPOSITORIES: biostudies-other

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Cloning, sequencing, and functional analysis of a Marek's disease virus origin of DNA replication.

Camp H S HS Coussens P M PM Silva R F RF

Journal of virology 19911101 11

Previously, we isolated a replicon from a defective Marek's disease virus (MDV), analogous to defective herpes simplex viruses (amplicons). Defective viruses contain cis-acting elements required for DNA synthesis and virus propagation such as an origin of DNA replication and a packaging-cleavage signal site. In this report, the MDV replicon was utilized to locate an origin of MDV DNA replication. A comparison of MDV replicon sequences with other herpesvirus replication origin sequences revealed ...[more]

PMID: 1656099

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Project description:Marek's disease virus (MDV) is a highly contagious alphaherpesvirus that infects chickens and causes a deadly neoplastic disease. We previously demonstrated that MDV infection arrests cells in S phase and that the tegument protein VP22 plays a major role in this process. In addition, expression of VP22 induces double-strand breaks (DSBs) in the cellular DNA, suggesting that DNA damage and the associated cellular response might be favorable for the MDV life cycle. Here, we addressed the role of DNA damage in MDV replication and pathogenesis. We demonstrated that MDV induces DSBs during lytic infection in vitro and in the peripheral blood mononuclear cells of infected animals. Intriguingly, we did not observe DNA damage in latently infected MDV-induced lymphoblastoid cells, while MDV reactivation resulted in the onset of DNA lesions, suggesting that DNA damage and/or the resulting DNA damage response might be required for efficient MDV replication and reactivation. In addition, reactivation was significantly enhanced by the induction of DNA damage using a number of chemicals. Finally, we used recombinant viruses to show that VP22 is required for the induction of DNA damage in vivo and that this likely contributes to viral oncogenesis.IMPORTANCE Marek's disease virus is an oncogenic alphaherpesvirus that causes fatal T-cell lymphomas in chickens. MDV causes substantial losses in the poultry industry and is also used in small-animal models for virus-induced tumor formation. DNA damage not only is implicated in tumor development but also aids in the life cycle of several viruses; however, its role in MDV replication, latency, and reactivation remains elusive. Here, we demonstrate that MDV induces DNA lesions during lytic replication in vitro and in vivo DNA damage was not observed in latently infected cells; however, it was reinitiated during reactivation. Reactivation was significantly enhanced by the induction of DNA damage. Recombinant viruses that lacked the ability to induce DNA damage were defective in their ability to induce tumors, suggesting that DNA damage might also contribute to cellular transformation processes leading to MDV lymphomagenesis.

Project description: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.

Project description:Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus of chickens that causes lymphomas in various organs. Most MDV genes are conserved among herpesviruses, while others are unique to MDV and may contribute to pathogenesis and/or tumor formation. High transcript levels of the MDV-specific genes MDV082, RLORF11, and SORF6 were recently detected in lytically infected cells; however, it remained elusive if the respective proteins are expressed and if they play a role in MDV pathogenesis. In this study, we first addressed if these proteins are expressed by inserting FLAG tags at their N or C termini. We could demonstrate that among the three genes tested, MDV082 is the only gene that encodes a protein and is expressed very late in MDV plaques in vitro. To investigate the role of this novel MDV082 protein in MDV pathogenesis, we generated a recombinant virus that lacks expression of the MDV082 protein. Our data revealed that the MDV082 protein contributes to the rapid onset of Marek's disease but is not essential for virus replication, spread, and tumor formation. Taken together, this study sheds light on the expression of MDV-specific genes and unravels the role of the late protein MDV082 in MDV pathogenesis. IMPORTANCE MDV is a highly oncogenic alphaherpesvirus that causes Marek's disease in chickens. The virus causes immense economic losses in the poultry industry due to the high morbidity and mortality, but also the cost of the vaccination. MDV encodes over 100 genes that are involved in various processes of the viral life cycle. Functional characterization of MDV genes is an essential step toward understanding the complex virus life cycle and MDV pathogenesis. Here, we have identified a novel protein encoded by MDV082 and two potential noncoding RNAs (RLORF11 and SORF6). The novel MDV082 protein is not needed for efficient MDV replication and tumor formation. However, our data demonstrate that the MDV082 protein is involved in the rapid onset of Marek's disease.

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Cloning, sequencing, and functional analysis of a Marek's disease virus origin of DNA replication.

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Cloning, sequencing, and functional analysis of a Marek's disease virus origin of DNA replication.

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