Aa. Khromykh et Eg. Westaway, SUBGENOMIC REPLICONS OF THE FLAVIVIRUS KUNJIN - CONSTRUCTION AND APPLICATIONS, Journal of virology, 71(2), 1997, pp. 1497-1505
Several Kunjin virus (KUN) subgenomic replicons containing large delet
ions in the structural region (C-prM-E) and in the 3' untranslated reg
ion (3'UTR) of the genome have been constructed. Replicon RNA Delta ME
with 1,987 nucleotides deleted (from nucleotide 417 [in codon 108] in
the C gene to nucleotide 2403 near the carboxy terminus of the E gene
, inclusive) and replicon RNA C20rep with 2,247 nucleotides deleted (f
rom nucleotide 157 [in codon 20] in C to nucleotide 2403) replicated e
fficiently in electroporated BHK21 cells. A further deletion from C20r
ep of 53 nucleotides, reducing the coding sequence in core protein to
two codons (C2rep RNA), resulted in abolishment of RNA replication. Re
plicon Delta ME/76 with a deletion of 76 nucleotides in the 3'UTR of D
elta ME RNA (nucleotides 10423 to 10498) replicated efficiently, where
as replicon Delta ME/352 with a larger deletion of 352 nucleotides (nu
cleotides 10423 to 10774), including two conserved sequences RCS3 and
CS3, was significantly inhibited in RNA replication. To explore the po
ssibility of using a reporter gene assay to monitor synthesis of the p
ositive strand and the negative strand of KUN RNA, we inserted a chlor
amphenicol acetyltransferase (CAT) gene into the 3'UTR of Delta ME/76
RNA under control of the internal ribosomal entry site (IRES) of encep
halomyelocarditis virus RNA in both plus (Delta ME/76CAT[+])- and minu
s (Delta ME/76CAT[-])-sense orientations. Although insertion of the IR
ES-CAT cassette in the plus-sense orientation resulted in a significan
t (10- to 20-fold) reduction of RNA replication compared to that of th
e parental Delta ME/76 RNA, CAT expression was readily detected in ele
ctroporated BHK cells. No CAT expression was detected after electropor
ation of RNA containing the IRES-CAT cassette inserted in the minus-se
nse orientation despite its apparently more efficient replication (sim
ilar to that of Delta ME/76 RNA); this result indicated that KUN negat
ive-strand RNA was probably not released from its template after synth
esis. Replacement of the CAT gene in the Delta ME/76CAT(+) RNA with th
e neomycin gene (Neo) enabled selection and recovery of a BHK cell cul
ture in which the majority of cells were continuously expressing the r
eplicon RNA for 41 days (nine passages) without apparent cytopathic ef
fect. The constructed KUN replicons should provide valuable tools to s
tudy flavivirus RNA replication as well as providing possible vectors
for a long-lasting and noncytopathic RNA virus expression system.