Critical Role of the Virus-Encoded MicroRNA-155 Ortholog in the Induction of Marek's Disease Lymphomas

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

Homodimerization of the Meq Viral Oncoprotein Is Necessary for Induction of T-Cell Lymphoma by Marek's Disease Virus ▿

Marek's disease virus (MDV) is a lymphotropic alphaherpesvirus that induces fatal rapid-onset T-cell lymphomas in chickens, its natural host. The MDV-encoded nuclear oncoprotein Meq is essential for lymphomagenesis and acts as a regulator of transcription. Meq has structural features, including a basic domain adjacent to a leucine zipper motif (B-ZIP), that suggest it is related to the Jun/Fos family of transcription factors. Via the leucine zipper, Meq can form homodimers or heterodimerize with c-Jun. Meq/Meq homodimers are associated with transrepression, and Meq/Jun heterodimers can transactivate target genes carrying an AP-1-like binding site. In order to determine the role of the leucine zipper and of Meq dimerization in T lymphomagenesis, specific point mutations were engineered into the highly oncogenic RB-1B strain of MDV to produce virus completely lacking a functional Meq leucine zipper (RB-1B MeqBZIP/BZIP) or virus encoding Meq that cannot homodimerize but can still bind to c-Jun and an AP-1-like site on DNA (RB-1B MeqHom/Hom). Both of these mutant viruses were capable of replication in cultured chicken embryo fibroblasts. However both mutations resulted in a complete loss of oncogenicity, since no lymphomas were produced up to 90 days postinfection in experimentally infected chicks. We conclude that the leucine zipper is necessary for the oncogenic activity of Meq and/or the efficient establishment of long-term MDV latency in T cells. Moreover, it appears that the ability to form homodimers is an absolute requirement and the ability to bind c-Jun alone is insufficient for the T-cell lymphomagenesis associated with virulent MDV

A Functional MicroRNA-155 Ortholog Encoded by the Oncogenic Marek's Disease Virus▿ †

Kaposi's sarcoma-associated herpesvirus-encoded microRNA (miRNA) MiR-K12-11 was recently shown to be a functional ortholog of miR-155, a miRNA that plays a major role in lymphoid malignancies and the modulation of immune responses. Here we show that miR-M4, encoded by the highly oncogenic Marek's disease virus of chickens, shares common targets with miR-155 and thus is also a functional ortholog of miR-155, the first one identified in an alphaherpesvirus. The observation that two distinct oncogenic herpesviruses associated with distinct types of lymphomas in different species encode functional miR-155 orthologs suggested the importance of this miRNA in regulatory pathways and the biology of lymphomagenesis

Poly(A) Binding Protein 1 Enhances Cap-Independent Translation Initiation of Neurovirulence Factor from Avian Herpesvirus

<div><p>Poly(A) binding protein 1 (PABP1) plays a central role in mRNA translation and stability and is a target by many viruses in diverse manners. We report a novel viral translational control strategy involving the recruitment of PABP1 to the 5' leader internal ribosome entry site (5L IRES) of an immediate-early (IE) bicistronic mRNA that encodes the neurovirulence protein (pp14) from the avian herpesvirus Marek’s disease virus serotype 1 (MDV1). We provide evidence for the interaction between an internal poly(A) sequence within the 5L IRES and PABP1 which may occur concomitantly with the recruitment of PABP1 to the poly(A) tail. RNA interference and reverse genetic mutagenesis results show that a subset of virally encoded-microRNAs (miRNAs) targets the inhibitor of PABP1, known as paip2, and therefore plays an indirect role in PABP1 recruitment strategy by increasing the available pool of active PABP1. We propose a model that may offer a mechanistic explanation for the cap-independent enhancement of the activity of the 5L IRES by recruitment of a <i>bona fide</i> initiation protein to the 5' end of the message and that is, from the affinity binding data, still compatible with the formation of ‘closed loop’ structure of mRNA.</p></div

Experiment 3

Data and R code for Fig3_and_S

Reverse genetic mutation analysis shows that MDV1 miRNAs from Lat-cluster are responsible for paip2 repression.

<p>(<b>A</b>) Schematic representation of the bicistronic transcripts that we and others have cloned as cDNA and that encode for pp14a and pp14b isoforms, modified from Tahiri-Alaoui et al, J. Virol. Dec. 2009, Vol.83, No. 24, p12769-12778. (<b>B</b>) & (<b>C</b>) Chicken embryo fibroblasts (CEF) were transfected with BAC clone pRB1B5 Lat-miR-Revertant or pRB1B5 Lat-miR- deletion, respectively. RNA and proteins were simultaneously extracted using Trizol at the indicated time points. Viral and host proteins were detected by immunoblotting with the indicated antibodies. (<b>D</b>) Quantitative RT-PCR of host (paip2) and of viral transcripts (pp14a and pp14b isoforms) at the indicated time points. GAPDH is used as the endogenous control and time zero is used as the calibrator. All experiments were repeated three times and the error bars indicate the SEM.</p

Effect of MDV1 infection on paip2 expression, PAPB1 level and localization.

<p>(<b>A</b>) Chicken embryo fibroblasts (CEF) were transfected with oncogenic BAC clone pRB1B5 of MDV1 or mock-transfected for 72 h. Total proteins were harvested and analysed by immunoblotting with the indicated antibodies. Quantification of the immunoblots from panel <b><i>A</i></b> using ImageQuant software is shown to the right. The results are from two independent experiments each in duplicate. (<b>B</b>) Total proteins were extracted from control samples or from samples taken from chicken infected with the oncogenic BAC clone pRB1B5 derived from archive samples. Proteins were analysed by immunoblotting as in panel <b><i>A</i></b>. Quantification of the immunoblots from panel <b><i>B</i></b> using ImageQuant software is shown to the right. The results are repeats from two different archive samples derived from the same chicken challenge experiment. (<b>C</b>) Indirect immunofluorescence of pRB1B5-infected CEF 72 h posttransfection. A series of optical sections were taken sequentially for each channel along the z-axis using a step size of 0.290 µm. The resulting 3D confocal image was reconstructed using IMARIS software. DAPI-staining shows the nucleus in blue, PABP1 in red and pp14 in green, the scale bar: 10 µm.</p