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Critical role of the virus-encoded microRNA-155 ortholog in the induction of Marek's disease lymphomas.

Zhao Y, Xu H, Yao Y, Smith LP, Kgosana L, Green J, Petherbridge L, Baigent SJ, Nair V - PLoS Pathog. (2011)

Bottom Line: This loss of oncogenicity appeared to be primarily due to the single miRNA within the cluster, miR-M4, the ortholog of cellular miR-155, since its deletion or a 2-nucleotide mutation within its seed region was sufficient to inhibit the induction of lymphomas.The definitive role of this miR-155 ortholog in oncogenicity was further confirmed by the rescue of oncogenic phenotype by revertant viruses that expressed either the miR-M4 or the cellular homolog gga-miR-155.Furthermore, the use of viruses deleted in miRNAs as effective vaccines against virulent MDV challenge, enables the prospects of generating genetically defined attenuated vaccines.

View Article: PubMed Central - PubMed

Affiliation: The Division of Structural Biology, The Wellcome Trust Centre for Human Genetics, Oxford University, Oxford, United Kingdom.

ABSTRACT
Notwithstanding the well-characterised roles of a number of oncogenes in neoplastic transformation, microRNAs (miRNAs) are increasingly implicated in several human cancers. Discovery of miRNAs in several oncogenic herpesviruses such as KSHV has further highlighted the potential of virus-encoded miRNAs to contribute to their oncogenic capabilities. Nevertheless, despite the identification of several possible cancer-related genes as their targets, the direct in vivo role of virus-encoded miRNAs in neoplastic diseases such as those induced by KSHV is difficult to demonstrate in the absence of suitable models. However, excellent natural disease models of rapid-onset Marek's disease (MD) lymphomas in chickens allow examination of the oncogenic potential of virus-encoded miRNAs. Using viruses modified by reverse genetics of the infectious BAC clone of the oncogenic RB-1B strain of MDV, we show that the deletion of the six-miRNA cluster 1 from the viral genome abolished the oncogenicity of the virus. This loss of oncogenicity appeared to be primarily due to the single miRNA within the cluster, miR-M4, the ortholog of cellular miR-155, since its deletion or a 2-nucleotide mutation within its seed region was sufficient to inhibit the induction of lymphomas. The definitive role of this miR-155 ortholog in oncogenicity was further confirmed by the rescue of oncogenic phenotype by revertant viruses that expressed either the miR-M4 or the cellular homolog gga-miR-155. This is the first demonstration of the direct in vivo role of a virus-encoded miRNA in inducing tumors in a natural infection model. Furthermore, the use of viruses deleted in miRNAs as effective vaccines against virulent MDV challenge, enables the prospects of generating genetically defined attenuated vaccines.

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Expression of miR-M4, miR-M5 and miR-M6 in infected cells.Relative expression of (A) miR-M4, (B) miR-M5 and (C) miR-M6 measured by qRT-PCR in RNA extracted from CEF infected with the wild type RB-1B, the parent pRB-1B5 and each of the mutant viruses. Uninfected CEF, MDV-transformed lymphoblastoid cell line 265L and Meq-deletion mutant RB-1BΔMeq were used as controls. The lowest level of positive samples in each group (miR-00 for miR-M4, miR-4-0-mu2 for miR-M5 and miR-M6) was used as calibrator. Results represent the mean of triplicate assays with error bars showing the standard error of the mean.
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ppat-1001305-g008: Expression of miR-M4, miR-M5 and miR-M6 in infected cells.Relative expression of (A) miR-M4, (B) miR-M5 and (C) miR-M6 measured by qRT-PCR in RNA extracted from CEF infected with the wild type RB-1B, the parent pRB-1B5 and each of the mutant viruses. Uninfected CEF, MDV-transformed lymphoblastoid cell line 265L and Meq-deletion mutant RB-1BΔMeq were used as controls. The lowest level of positive samples in each group (miR-00 for miR-M4, miR-4-0-mu2 for miR-M5 and miR-M6) was used as calibrator. Results represent the mean of triplicate assays with error bars showing the standard error of the mean.

Mentions: We also examined the expression levels of MDV-encoded miRNAs miR-M4, miR-M5 and miR-M6 by qRT-PCR on RNA extracted from CEF infected with wild type RB-1B, the parent pRB-1B5 and the different mutant viruses (Fig. 8A–C). MDV-transformed cell line 265L and RB-1BΔMeq virus were used as controls. The absence of miR-M4 and miR-M5 transcripts in the cells infected by miR-S0 and miR-SS viruses (the low level of miR-M4 but not miR-M5 in miR-00 virus is due to the presence of one copy of miR-M4), confirmed the deletion of these miRNAs in these constructs (Fig. 8A,B). As expected, the expression of miR-M6 located within the LAT region was not affected by the mutations within the cluster 1 miRNA locus (Fig. 8C).


Critical role of the virus-encoded microRNA-155 ortholog in the induction of Marek's disease lymphomas.

Zhao Y, Xu H, Yao Y, Smith LP, Kgosana L, Green J, Petherbridge L, Baigent SJ, Nair V - PLoS Pathog. (2011)

Expression of miR-M4, miR-M5 and miR-M6 in infected cells.Relative expression of (A) miR-M4, (B) miR-M5 and (C) miR-M6 measured by qRT-PCR in RNA extracted from CEF infected with the wild type RB-1B, the parent pRB-1B5 and each of the mutant viruses. Uninfected CEF, MDV-transformed lymphoblastoid cell line 265L and Meq-deletion mutant RB-1BΔMeq were used as controls. The lowest level of positive samples in each group (miR-00 for miR-M4, miR-4-0-mu2 for miR-M5 and miR-M6) was used as calibrator. Results represent the mean of triplicate assays with error bars showing the standard error of the mean.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3044692&req=5

ppat-1001305-g008: Expression of miR-M4, miR-M5 and miR-M6 in infected cells.Relative expression of (A) miR-M4, (B) miR-M5 and (C) miR-M6 measured by qRT-PCR in RNA extracted from CEF infected with the wild type RB-1B, the parent pRB-1B5 and each of the mutant viruses. Uninfected CEF, MDV-transformed lymphoblastoid cell line 265L and Meq-deletion mutant RB-1BΔMeq were used as controls. The lowest level of positive samples in each group (miR-00 for miR-M4, miR-4-0-mu2 for miR-M5 and miR-M6) was used as calibrator. Results represent the mean of triplicate assays with error bars showing the standard error of the mean.
Mentions: We also examined the expression levels of MDV-encoded miRNAs miR-M4, miR-M5 and miR-M6 by qRT-PCR on RNA extracted from CEF infected with wild type RB-1B, the parent pRB-1B5 and the different mutant viruses (Fig. 8A–C). MDV-transformed cell line 265L and RB-1BΔMeq virus were used as controls. The absence of miR-M4 and miR-M5 transcripts in the cells infected by miR-S0 and miR-SS viruses (the low level of miR-M4 but not miR-M5 in miR-00 virus is due to the presence of one copy of miR-M4), confirmed the deletion of these miRNAs in these constructs (Fig. 8A,B). As expected, the expression of miR-M6 located within the LAT region was not affected by the mutations within the cluster 1 miRNA locus (Fig. 8C).

Bottom Line: This loss of oncogenicity appeared to be primarily due to the single miRNA within the cluster, miR-M4, the ortholog of cellular miR-155, since its deletion or a 2-nucleotide mutation within its seed region was sufficient to inhibit the induction of lymphomas.The definitive role of this miR-155 ortholog in oncogenicity was further confirmed by the rescue of oncogenic phenotype by revertant viruses that expressed either the miR-M4 or the cellular homolog gga-miR-155.Furthermore, the use of viruses deleted in miRNAs as effective vaccines against virulent MDV challenge, enables the prospects of generating genetically defined attenuated vaccines.

View Article: PubMed Central - PubMed

Affiliation: The Division of Structural Biology, The Wellcome Trust Centre for Human Genetics, Oxford University, Oxford, United Kingdom.

ABSTRACT
Notwithstanding the well-characterised roles of a number of oncogenes in neoplastic transformation, microRNAs (miRNAs) are increasingly implicated in several human cancers. Discovery of miRNAs in several oncogenic herpesviruses such as KSHV has further highlighted the potential of virus-encoded miRNAs to contribute to their oncogenic capabilities. Nevertheless, despite the identification of several possible cancer-related genes as their targets, the direct in vivo role of virus-encoded miRNAs in neoplastic diseases such as those induced by KSHV is difficult to demonstrate in the absence of suitable models. However, excellent natural disease models of rapid-onset Marek's disease (MD) lymphomas in chickens allow examination of the oncogenic potential of virus-encoded miRNAs. Using viruses modified by reverse genetics of the infectious BAC clone of the oncogenic RB-1B strain of MDV, we show that the deletion of the six-miRNA cluster 1 from the viral genome abolished the oncogenicity of the virus. This loss of oncogenicity appeared to be primarily due to the single miRNA within the cluster, miR-M4, the ortholog of cellular miR-155, since its deletion or a 2-nucleotide mutation within its seed region was sufficient to inhibit the induction of lymphomas. The definitive role of this miR-155 ortholog in oncogenicity was further confirmed by the rescue of oncogenic phenotype by revertant viruses that expressed either the miR-M4 or the cellular homolog gga-miR-155. This is the first demonstration of the direct in vivo role of a virus-encoded miRNA in inducing tumors in a natural infection model. Furthermore, the use of viruses deleted in miRNAs as effective vaccines against virulent MDV challenge, enables the prospects of generating genetically defined attenuated vaccines.

Show MeSH
Related in: MedlinePlus