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      Kissing-loop interaction between 5′ and 3′ ends of tick-borne Langat virus genome ‘bridges the gap’ between mosquito- and tick-borne flaviviruses in mechanisms of viral RNA cyclization: applications for virus attenuation and vaccine development

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          Abstract

          Insertion of microRNA target sequences into the flavivirus genome results in selective tissue-specific attenuation and host-range restriction of live attenuated vaccine viruses. However, previous strategies for miRNA-targeting did not incorporate a mechanism to prevent target elimination under miRNA-mediated selective pressure, restricting their use in vaccine development. To overcome this limitation, we developed a new approach for miRNA-targeting of tick-borne flavivirus (Langat virus, LGTV) in the duplicated capsid gene region (DCGR). Genetic stability of viruses with DCGR was ensured by the presence of multiple cis-acting elements within the N-terminal capsid coding region, including the stem-loop structure (5′SL6) at the 3′ end of the promoter. We found that the 5′SL6 functions as a structural scaffold for the conserved hexanucleotide motif at its tip and engages in a complementary interaction with the region present in the 3′ NCR to enhance viral RNA replication. The resulting kissing-loop interaction, common in tick-borne flaviviruses, supports a single pair of cyclization elements (CYC) and functions as a homolog of the second pair of CYC that is present in the majority of mosquito-borne flaviviruses. Placing miRNA targets into the DCGR results in superior attenuation of LGTV in the CNS and does not interfere with development of protective immunity in immunized mice.

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          Most cited references42

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          Molecular Cloning : A Laboratory Manual

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            R: A Language and environmental for statistical computing

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              The MicroRNA miR-124 promotes neuronal differentiation by triggering brain-specific alternative pre-mRNA splicing.

              Both microRNAs and alternative pre-mRNA splicing have been implicated in the development of the nervous system (NS), but functional interactions between these two pathways are poorly understood. We demonstrate that the neuron-specific microRNA miR-124 directly targets PTBP1 (PTB/hnRNP I) mRNA, which encodes a global repressor of alternative pre-mRNA splicing in nonneuronal cells. Among the targets of PTBP1 is a critical cassette exon in the pre-mRNA of PTBP2 (nPTB/brPTB/PTBLP), an NS-enriched PTBP1 homolog. When this exon is skipped, PTBP2 mRNA is subject to nonsense-mediated decay (NMD). During neuronal differentiation, miR-124 reduces PTBP1 levels, leading to the accumulation of correctly spliced PTBP2 mRNA and a dramatic increase in PTBP2 protein. These events culminate in the transition from non-NS to NS-specific alternative splicing patterns. We also present evidence that miR-124 plays a key role in the differentiation of progenitor cells to mature neurons. Thus, miR-124 promotes NS development, at least in part by regulating an intricate network of NS-specific alternative splicing.
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                Author and article information

                Journal
                Nucleic Acids Res
                Nucleic Acids Res
                nar
                nar
                Nucleic Acids Research
                Oxford University Press
                0305-1048
                1362-4962
                20 April 2016
                04 February 2016
                04 February 2016
                : 44
                : 7
                : 3330-3350
                Affiliations
                [1 ]Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD, 20892-3203 USA
                [2 ]Bioinformatics and Computational Biosciences Branch, NIAID, NIH, Bethesda, MD, 20892, USA
                Author notes
                [* ]To whom correspondence should be addressed. Tel: +1 301 402 7754; Fax: +1 301 480 0501; Email: apletnev@ 123456niaid.nih.gov
                Article
                10.1093/nar/gkw061
                4838367
                26850640
                4f1c72d4-dc31-4235-9b40-ed84f5a27754
                Published by Oxford University Press on behalf of Nucleic Acids Research 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.
                History
                : 24 January 2016
                : 20 January 2016
                : 10 September 2015
                Page count
                Pages: 21
                Categories
                RNA
                Custom metadata
                20 April 2016

                Genetics
                Genetics

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