Abstract
Marek's Disease (MD) is a widespread lymphoid neoplastic disease with tumours caused by infection with the Marek's Disease Virus (MDV). MD is one of the major poultry health and welfare problems throughout the world, with losses estimated to be up to US$ 2,000 million despite more than 20 billion doses of the MD vaccines used annually. MDV has proven to be a valuable model for understanding some of the principles of oncogenesis. It provides a well defined small-animal model of general tumorigenesis, and virus-induced lymphomagenesis in particular. The reproducible kinetics of disease induction and progression, in a natural virus-host system, make MDV unique allowing studies that are impossible to perform in other non-natural models of herpesvirus-induced oncogenesis. Studies of virus-infected cells have been a prominent source of information on the mechanism of translational control. As obligate intracellular parasites, viruses depend on cells for their replication. Nowhere is this dependency seen more clearly than in the translation system, as viruses lack a translational apparatus. Viruses have evolved ways to gain a translational advantage for their mRNAs. This is particularly crucial during the very early stages of viral infection where immediate-early transcripts are produced and need to be translated. Internal ribosome entry site (IRES)-mediated translation initiation is one of the strategies that viruses use during time of cellular stress and when cap-dependent translation might be inhibited. We have discovered that an immediate-early transcript from MDV has two IRESes. The first IRES is contained within the 5' leader sequence and controls the translation of the downstream open reading frame that encode for the pp14 lytic protein. The 5' leader IRES appears to be regulated by base-pairing with sequences within the 18S rRNA and by a ribosomal protein within the 40S ribosomal subunit. The second IRES is contained within the intercistronic region (ICR) and controls the translation of the second protein known as RLORF9. The ICR IRES activity appears to be controlled by a set of viral small non-coding RNA known as micro-RNAs. This transcript is known to be associated with oncogenicity and the maintenance of latency of MDV transformed cells. This transcript is within a locus that undergoes specific sequence amplification during viral attenuation. This sequence amplification is the only validated marker that always correlates with viral attenuation. What benefits this research proposal will bring to the field of MDV oncogenesis in particular and to translational control in general? Preliminary work has allowed us to put forward novel concepts in translational control with direct impact on the understanding of MDV oncogenesis. The availability of an infectious BAC clone of the highly oncogenic RB-1B strain will allow us to use recombination-mediated genetic engineering to investigate the biological function of the 1.8-kb transcript in cell culture and in chicken. This highly reproducible disease model in the natural host will be used to obtain insights into the mechanisms of herpesvirus-induced lymphomagenesis for designing better vaccines. Detailed analysis of the structure and the function of the two IRESes within the 1.8-kb transcript will allow us to dissect fundamental mechanisms in translation such as miRNA-IRES mediated translational control, ribosomal filtering and IRES allostery. The results will also be exploited in designing novel multicistronic vectors to allow the expression of a gene of interest inside the target cells with the aid of a reporter, a drug selection marker, or a suicidal gene, all expressed from the same transcript. The emerging application of viruses in cancer, gene therapy and delivery mandate an understanding of the interplay between viruses and the host mechanisms. The study of translational control impinges on several fields including biotechnology, cell biology, Medicine and Agriculture.
Technical Summary
The basis for the current investigation emerged from an important discovery that we have made while investigating the targets of MDV-encoded miRNAs. Identification of miRNA targets is extremely difficult, and the current methods rely heavily on bioinformatics based on seed homology and sequence conservation. Such predictions are less useful in the case of viral or host miRNAs. As part of the effort to identify the targets of MDV-encoded miRNAs, we developed a novel approach to isolate transcripts that are bound to the biotinylated mature viral miRNAs. This method of identification of miRNA targets was very successful and has helped to identify several transcripts as potential targets of MDV-encoded miRNAs. One of the targets identified by this approach is an MDV-encoded mRNA that is the product of an immediate-early gene and that belongs to the 1.8-kb family of transcripts suspected to be associated with oncogenicity and maintenance of latency of MDV transformed cells. We will examine the importance of this immediate-early transcript, and the benefits that this study will bring to the field of MDV oncogenesis, in particular, to the field of translational regulation. Availability of the infectious BAC clone of the highly oncogenic RB-1B strain of MDV will allow us to use recombination-mediated genetic engineering to investigate the biological function of the 1.8-kb transcript in cell culture and in chicken. Furthermore, detailed analysis of the structure and the function of the two IRESes within the 1.8-kb transcript will allow us to dissect fundamental mechanisms in translation such as miRNA-IRES mediated translational control, ribosomal filtering and IRES allostery. The results will be exploited in the design of novel multicistronic vectors to allow the expression of a gene of interest inside the target cells with the aid of a reporter, a drug selection marker, or a suicidal gene, all expressed from the same transcript.
Planned Impact
Who will benefit from this research? The proposed research will benefit all scientists working in the field of translational control of eukaryotic genes and that in both academic and commercial private sector. We have discovered a naturally occurring model system (bicistronic viral transcript that is capped and polyadenylated) that offers the possibility of studying novel concepts in translational regulation of eukaryotic genes. The novel concepts that we are proposing to investigate includes, miRNA-IRES interactions, IRES allostery and ribosomal filtering, are for the first time put in an integrated approach that will lead to major breakthrough. The impact of our research is highlighted by the increasing use of bicistronic vectors as a research tool and in gene delivery. Our discovery that an IRES from a DNA virus contains a translational enhancer element that functions through base-pairing with ribosomal RNA provides a means to increase protein productivity using existing manufacturing methods in traditional cell lines. Therefore, it offers exceptional and broad technological applications. The proposed research will benefit the poultry industry as an improved understanding of the molecular mechanisms of MDV pathology could lead to new strategies of designing new vaccine. Our research proposal offers a key advantage as it investigates the translational regulation of a transcript known to be in a locus that undergoes specific sequence amplification during viral attenuation as a result of multiple in vitro cell passages. This is a standard procedure of producing large amounts of cell-associated live MDV vaccine. Judging from the interest in the lay press, there is a considerable interest among the public for the scientific understanding of how some diseases produce damage to animal health. This proposal could unravel some of the intricate pathways of viral-induced tumours, and hence will have significant impact on animal welfare through all poultry production systems. How they will benefit from this research? Our work expands the repertoire of mRNA base-pairing interactions in eukaryotes to include rRNA in addition to microRNAs, small nucleolar RNA and antisene RNAs. It uncovers a novel function for viral miRNAs. These translational control strategies will benefit protein production technologies. The groundbreaking technology will exploits naturally occurring regulatory mechanisms of mammalian and avian cells, allowing the platform to be used to: Boost protein production capacity, foster DNA vaccine development and drive breakthroughs in gene therapy. What will be done to ensure that they have the opportunity to benefit from this research? To ensuring that our research in the field of translational control will have the competitive edge at the international level we have initiated collaboration with the Dr. Vincent P. Mauro and the Nobel Laureate Dr. Gerald M. Edelman from the Scripps research institute, La Jolla, Ca. Our initial collaboration has led to the filing of joint patent that covers our discovery of the MDV IRES that harbours a translation enhancer element.
