GROWTH-RESTRICTED DENGUE VIRUS MUTANTS CONTAINING DELETIONS IN THE 5'NONCODING REGION OF THE RNA GENOME

The dengue type 4 virus (DEN4) RNA genome contains a 101-nt 5' noncodi ng (NC) sequence which is predicted to form a stable secondary structu re. 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 a s candidates for use in a live attenuated vaccine. Eleven distinct del etions in the region of the DEN4 genome between nts 18 and 98 were con structed; each mutation was predicted to alter or disrupt the local ba se-parings in the 5' NC RNA structure. An infectious virus was not rec overed from the RNA transcripts of five of these deletion mutants. Sig nificantly, four of the five apparently lethal deletions were located in a 5- to 6-nt base-paired region of a predicted long stem or adjacen t to it. In contrast, with one exception, mutants which yielded infect ious virus had deletions which were located in a loop or short stem re gion. The effect of the deletions on the efficiency of translation of viral RNA transcripts was examined in vitro. The RNA transcripts of de letion constructs which did not yield viable virus were translated at an efficiency ranging from 40 to 160% that of wild-type virus transcri pts. The translation efficiency of infectious RNA transcripts also var ied. Deletion mutants recovered from RNA transcripts that exhibited lo w to moderate efficiency of translation had a small plaque morphology and exhibited reduced growth in simian LLC-MK(2) and mosquito C6/36 ce lls compared to the wild-type virus. Among the 11 mutant constructs, d eletion of nts 82-87 caused the greatest reduction in translation effi ciency. Nevertheless, an infectious virus was recovered from LLC-MK(2) cells transfected with the RNA transcripts of mutant d(82-87). The pr ogeny of this mutant produced small plaques on LLC-MK(2) cells and gre w to low titer in these cells. Unlike wild-type DEN4 or other DEN4 del etion 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. (C) 1995 Academic Pre ss, Inc.