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1.
Vet Res ; 52(1): 20, 2021 Feb 12.
Article in English | MEDLINE | ID: mdl-33579382

ABSTRACT

Processing and packaging of herpesvirus genomic DNA is regulated by a packaging-associated terminase complex comprising of viral proteins pUL15, pUL28 and pUL33. Marek's disease virus (MDV) homologs UL28 and UL33 showed conserved functional features with high sequence identity with the corresponding Herpes simplex virus 1 (HSV-1) homologs. As part of the investigations into the role of the UL28 and UL33 homologs of oncogenic MDV for DNA packaging and replication in cultured cells, we generated MDV mutant clones deficient in UL28 or UL33 of full-length MDV genomes. Transfection of UL28- or UL33-deleted BAC DNA into chicken embryo fibroblast (CEF) did not result either in the production of visible virus plaques, or detectable single cell infection after passaging onto fresh CEF cells. However, typical MDV plaques were detectable in CEF transfected with the DNA of revertant mutants where the deleted genes were precisely reinserted. Moreover, the replication defect of the UL28-deficient mutant was completely restored when fragment encoding the full UL28 gene was co-transfected into CEF cells. Viruses recovered from the revertant construct, as well as by the UL28 co-transfection, showed replication ability comparable with parental virus. Furthermore, the transmission electron microscopy study indicated that immature capsids were assembled without the UL28 expression, but with the loss of infectivity. Importantly, predicted three-dimensional structures of UL28 between MDV and HSV-1 suggests conserved function in virus replication. For the first time, these results revealed that both UL28 and UL33 are essential for MDV replication through regulating DNA cleavage and packaging.


Subject(s)
DNA, Viral/chemistry , Endodeoxyribonucleases/genetics , Mardivirus/physiology , Receptors, Chemokine/genetics , Viral Proteins/genetics , Virus Replication , Amino Acid Sequence , Animals , Chick Embryo , Endodeoxyribonucleases/chemistry , Endodeoxyribonucleases/metabolism , Mardivirus/enzymology , Mardivirus/genetics , RNA Cleavage , Receptors, Chemokine/chemistry , Receptors, Chemokine/metabolism , Sequence Alignment , Specific Pathogen-Free Organisms , Viral Proteins/chemistry , Viral Proteins/metabolism
2.
J Exp Med ; 203(5): 1143-5, 2006 May 15.
Article in English | MEDLINE | ID: mdl-16682501

ABSTRACT

Telomerase, the enzyme that elongates our telomeres, is crucial for cancer development based on extensive analyses of human cells, human cancers, and mouse models. New data now suggest that a viral telomerase RNA gene encoded by Marek's disease virus (MDV), an oncogenic herpesvirus of chickens, promotes tumor formation. These findings highlight the importance of telomerase in cancer and raise new questions regarding the mechanisms by which the telomerase RNA component supports tumorigenesis.


Subject(s)
Cell Transformation, Viral , Lymphoma, T-Cell/enzymology , Mardivirus/enzymology , Telomerase/metabolism , Viral Proteins/metabolism , Animals , Cell Line , Chickens , Fibroblasts/metabolism , Fibroblasts/pathology , Fibroblasts/virology , Gene Expression Regulation, Leukemic/physiology , Gene Expression Regulation, Viral/physiology , Genome , Humans , Integrin alpha Chains/biosynthesis , Integrin beta Chains/biosynthesis , Lymphoma, T-Cell/genetics , Lymphoma, T-Cell/pathology , Lymphoma, T-Cell/virology , Mardivirus/genetics , Marek Disease/enzymology , Marek Disease/genetics , Marek Disease/pathology , Marek Disease/virology , Mice , Mutation , Sequence Homology, Amino Acid , Telomerase/genetics , Viral Proteins/genetics
3.
J Exp Med ; 203(5): 1307-17, 2006 May 15.
Article in English | MEDLINE | ID: mdl-16651385

ABSTRACT

Telomerase is a ribonucleoprotein complex consisting of two essential core components: a reverse transcriptase and an RNA subunit (telomerase RNA [TR]). Dysregulation of telomerase has been associated with cell immortalization and oncogenesis. Marek's disease herpesvirus (MDV) induces a malignant T cell lymphoma in chickens and harbors in its genome two identical copies of a viral TR (vTR) with 88% sequence identity to chicken TR. MDV mutants lacking both copies of vTR were significantly impaired in their ability to induce T cell lymphomas, although lytic replication in vivo was unaffected. Tumor incidences were reduced by >60% in chickens infected with vTR- viruses compared with animals inoculated with MDV harboring at least one intact copy of vTR. Lymphomas in animals infected with the vTR- viruses were also significantly smaller in size and less disseminated. Constitutive expression of vTR in the chicken fibroblast cell line DF-1 resulted in a phenotype consistent with transformation as indicated by morphological alteration, enhanced anchorage-independent cell growth, cell growth beyond saturation density, and increased expression levels of integrin alpha v. We concluded that vTR plays a critical role in MDV-induced T cell lymphomagenesis. Furthermore, our results provide the first description of tumor-promoting effects of TR in a natural virus-host infection model.


Subject(s)
Cell Transformation, Viral , Lymphoma, T-Cell/enzymology , Mardivirus/enzymology , Telomerase/metabolism , Viral Proteins/metabolism , Animals , Cell Line , Chickens , Fibroblasts/metabolism , Fibroblasts/pathology , Fibroblasts/virology , Gene Expression Regulation, Leukemic/physiology , Gene Expression Regulation, Viral/physiology , Genome , Integrin alpha Chains/biosynthesis , Integrin beta Chains/biosynthesis , Lymphoma, T-Cell/genetics , Lymphoma, T-Cell/pathology , Lymphoma, T-Cell/virology , Mardivirus/genetics , Marek Disease/enzymology , Marek Disease/genetics , Marek Disease/pathology , Marek Disease/virology , Mutation , Sequence Homology, Amino Acid , Telomerase/genetics , Viral Proteins/genetics
4.
J Virol ; 79(11): 6984-96, 2005 Jun.
Article in English | MEDLINE | ID: mdl-15890938

ABSTRACT

The genome of Marek's disease virus (MDV) has been predicted to encode a secreted glycoprotein, vLIP, which bears significant homology to the alpha/beta hydrolase fold of pancreatic lipases. Here it is demonstrated that MDV vLIP mRNA is produced via splicing and that vLIP is a late gene, due to its sensitivity to inhibition of DNA replication. While vLIP was found to conserve several residues essential to hydrolase activity, an unfavorable asparagine substitution is present at the lipase catalytic triad acid position. Consistent with structural predictions, purified recombinant vLIP did not show detectable activity on traditional phospholipid or triacylglyceride substrates. Two different vLIP mutant viruses, one bearing a 173-amino-acid deletion in the lipase homologous domain, the other having an alanine point mutant at the serine nucleophile position, caused a significantly lower incidence of Marek's disease in chickens and resulted in enhanced survival relative to two independently produced vLIP revertants or parental virus. These data provide the first evidence that vLIP enhances the replication and pathogenic potential of MDV. Furthermore, while vLIP may not serve as a traditional lipase enzyme, the data indicate that the serine nucleophile position is nonetheless essential in vivo for the viral functions of vLIP. Therefore, it is suggested that this particular example of lipase homology may represent the repurposing of an alpha/beta hydrolase fold toward a nonenzymatic role, possibly in lipid bonding.


Subject(s)
Lipase/physiology , Mardivirus/enzymology , Mardivirus/pathogenicity , Amino Acid Sequence , Amino Acid Substitution , Animals , Base Sequence , Cell Line , Chickens , Chromosome Mapping , DNA, Viral/genetics , Genes, Viral , Genome, Viral , Glycosylation , Lipase/chemistry , Lipase/genetics , Mardivirus/genetics , Marek Disease/etiology , Marek Disease/virology , Molecular Sequence Data , Mutagenesis, Insertional , RNA Splicing , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Virulence/genetics , Virulence/physiology
5.
J Biol Chem ; 280(25): 23502-15, 2005 Jun 24.
Article in English | MEDLINE | ID: mdl-15811851

ABSTRACT

Telomerase activity is present in most malignant tumors and provides a mechanism for the unlimited potential for division of neoplastic cells. We previously characterized the first identified viral telomerase RNA (vTR) encoded by the Marek's disease virus (MDV) (Fragnet, L., Blasco, M. A., Klapper, W., and Rasschaert, D. (2003) J. Virol. 77, 5985-5996). This avian herpesvirus induces T-lymphomas. We demonstrated that the vTR subunit of the oncogenic MDV-RB1B strain is functional and would be more efficient than its chicken counterpart, cTR, which is 88% homologous. We take advantage of the functionality of those natural mutant TRs to investigate the involvement of the mutations of vTR on its efficiency in a heterologous murine cell system and in a homologous in vitro system using the recombinant chicken telomerase reverse transcriptase. The P2 helix of the pseudoknot seems to be more stable in vTR than in cTR, and this may account for the higher activity of vTR than cTR. Moreover, the five adenines just upstream from the P3 helix of vTR may also play an important role in its efficiency. We also established that the substitution of a single nucleotide at the 3'-extremity of the H-box of the vaccine MDV-Rispens strain vTR resulted in a lack of its accumulation within the cell, especially in the nucleus, correlated with a decrease in telomerase activity.


Subject(s)
Mardivirus/enzymology , Mutation , RNA, Viral/physiology , Telomerase/genetics , Animals , Base Sequence , Cell Line , Chickens , DNA Primers , Mice , Molecular Sequence Data , Nucleic Acid Conformation , RNA, Viral/chemistry , Sequence Homology, Nucleic Acid
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