title: 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.
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.
authors: Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD, 20892-3203 USA.
authors: Konstantin A
authors: KA
authors: Tsetsarkin
authors: Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD, 20892-3203 USA.
authors: Guangping
authors: G
authors: Liu
authors: Bioinformatics and Computational Biosciences Branch, NIAID, NIH, Bethesda, MD, 20892, USA.
authors: Kui
authors: K
authors: Shen
authors: Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD, 20892-3203 USA apletnev@niaid.nih.gov.
authors: Alexander G
authors: AG
authors: Pletnev
mesh_terms: D000818:Animals; D001921:Brain / Q000821:virology; D036022:Capsid Proteins / Q000235:genetics*; D002522:Chlorocebus aethiops; D004669:Encephalitis Viruses, Tick-Borne / Q000235:genetics* / Q000254:growth & development / Q000502:physiology; D004675:Encephalitis, Tick-Borne / Q000276:immunology / Q000821:virology; D020131:Genes, Duplicate; D016679:Genome, Viral; D051379:Mice; D035683:MicroRNAs / Q000235:genetics*; D009154:Mutation; D009711:Nucleotides / Q000737:chemistry; D016366:Open Reading Frames; D009928:Organ Specificity; D011401:Promoter Regions, Genetic; D012367:RNA, Viral / Q000737:chemistry*; D038621:Regulatory Sequences, Ribonucleic Acid; D014613:Vaccines, Attenuated; D014709:Vero Cells; D014765:Viral Vaccines; D014779:Virus Replication
chemical_list: D036022:Capsid Proteins; D035683:MicroRNAs; D009711:Nucleotides; D012367:RNA, Viral; D038621:Regulatory Sequences, Ribonucleic Acid; D014613:Vaccines, Attenuated; D014765:Viral Vaccines