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© Christine Schmitt, Anubis Vega Rua, Jean-Marc Panaud
Tête de moustique femelle Aedes albopictus, vecteur du virus de la dengue et du chikungunya. Microphotographie électronique à balayage, image colorisée.
Publication : Virology

Growth-restricted dengue virus mutants containing deletions in the 5′ noncoding region of the RNA genome

Scientific Fields
Diseases
Organisms
Applications
Technique

Published in Virology - 20 Feb 1995

Cahour A, Pletnev A, Vazielle-Falcoz M, Rosen L, Lai CJ

Link to Pubmed [PMID] – 7871753

Virology 1995 Feb;207(1):68-76

The dengue type 4 virus (DEN4) RNA genome contains a 101-nt 5′ noncoding (NC) sequence which is predicted to form a stable secondary structure. DEN4 cDNA from which infectious RNA can be transcribed was used to engineer deletions in the 5′ NC region for functional analysis of RNA structure and for isolation of DEN4 mutants that could be evaluated as candidates for use in a live attenuated vaccine. Eleven distinct deletions in the region of the DEN4 genome between nts 18 and 98 were constructed; each mutation was predicted to alter or disrupt the local base-parings in the 5′ NC RNA structure. An infectious virus was not recovered from the RNA transcripts of five of these deletion mutants. Significantly, four of the five apparently lethal deletions were located in a 5- to 6-nt base-paired region of a predicted long stem or adjacent to it. In contrast, with one exception, mutants which yielded infectious virus had deletions which were located in a loop or short stem region. The effect of the deletions on the efficiency of translation of viral RNA transcripts was examined in vitro. The RNA transcripts of deletion constructs which did not yield viable virus were translated at an efficiency ranging from 40 to 160% that of wild-type virus transcripts. The translation efficiency of infectious RNA transcripts also varied. Deletion mutants recovered from RNA transcripts that exhibited low to moderate efficiency of translation had a small plaque morphology and exhibited reduced growth in simian LLC-MK2 and mosquito C6/36 cells compared to the wild-type virus. Among the 11 mutant constructs, deletion of nts 82-87 caused the greatest reduction in translation efficiency. Nevertheless, an infectious virus was recovered from LLC-MK2 cells transfected with the RNA transcripts of mutant d(82-87). The progeny of this mutant produced small plaques on LLC-MK2 cells and grew to low titer in these cells. Unlike wild-type DEN4 or other DEN4 deletion mutants tested, mutant d(82-87) failed to produce plaques on C6/36 cells and was also replication-defective in Aedes aegypti and Aedes albopictus following intrathoracic inoculation.

http://www.ncbi.nlm.nih.gov/pubmed/7871753