GENETIC LOCI RESPONSIBLE FOR GROWTH RESTRICTION OF MOUSE-ADAPTED DENGUE VIRUSES

Project: Research project

Project Details

Description

Serial intracerebral passage of dengue type 1 or type 2 virus in mice was
shown previously to select for mouse neurovirulent mutants which also
proved to be attenuated for humans. In a similar manner a neurovirulent
mutant of DEN4 (strain H241) was selected by serial intracerebral passage
in mice. DEN4 H241 neurovirulent (N) replicated less efficiently than
its DEN4 H241 parent (P) in simian LLC-MK2 cells. An intratypic DEN4
chimera containing the C-PreM-E structural protein genes from DEN4 H241N
also exhibited marked restriction of growth in LLC-MK2 cells. Analysis
of virion proteins of DEN4 H241N or its derived C-PreM-E chimera grown
in mosquito C6/36 cells indicated that very little M was produced. There
is considerable evidence that immature PreM-containing flavivirus virions
are less infectious than the mature M-containing virus. For this reason,
we constructed a panel of chimeras that contained one or more of the
variant amino acids of the mutant C-PreM-E sequence substituting for the
corresponding sequence of the parental virus. These chimeras were
analyzed to determine if any of these mutations was responsible for
inefficient PreM cleavage. Mutants which contained two substitutions in
PreM, i. e., Lys165-Glu and Val188-Ala, with or without the Thr81-Ile
substitution in C, exhibited reduced PreM cleavage. Reduction of PreM
cleavage was also observed for chimeras which contain multiple mutations
in E, i.e., Thr434-Ile, Ser435-Pro, and Phe680-Leu, with the exception
of the chimera containing Thr434-Ile and Ser435-Pro. Efficient PreM
cleavage was required for virus to achieve high level of replication in
simian LLC-MK2 cells. Similarly, the genetic basis for the reduced
replicative efficiency of the mouse-passaged MD-1 vaccine strain in
LLC-MK2 cells compared to its parental DEN1 Hawaii strain was
investigated. A combination of three substitutions in E independently
caused restriction of replication in simian cells. A single Val207-Ala
substitution in PreM was also independently responsible for restriction
of replication of MD-1 or its derived chimera in LLC-MK2 cells. This
PreM mutation is located immediately downstream of the N-terminus of M,
which is cleaved from PreM by the host cell furin enzyme. The Val207-Ala
mutation did not affect PreM cleavage in mosquito cells, but this
mutation might possibly restrict PreM cleavage in simian cells and thus,
be responsible for growth restriction.
StatusFinished
Effective start/end date9/1/868/31/91

ASJC

  • Medicine(all)
  • Immunology and Microbiology(all)