Sir Albert Sakzewski Virus Research Centre,
Royal Children's Hospital, and Clinical Medical Virology
Centre, University of Queensland, Brisbane, Australia
A possible role in RNA replication for interactions between
conserved complementary (cyclization) sequences in the 5'- and 3'-terminal regions of Flavivirus RNA was previously
suggested but never tested in vivo. Using the M-fold program for RNA
secondary-structure predictions, we examined for the first time the
base-pairing interactions between the covalently linked 5' genomic
region (first ~160 nucleotides) and the 3' untranslated region (last
~115 nucleotides) for a range of mosquito-borne
Flavivirus species. Base-pairing occurred as predicted for
the previously proposed conserved cyclization sequences. In order to
obtain experimental evidence of the predicted interactions, the
putative cyclization sequences (5' or 3') in the replicon RNA of the
mosquito-borne Kunjin virus were mutated either separately, to destroy
base-pairing, or simultaneously, to restore the complementarity. None
of the RNAs with separate mutations in only the 5' or only the 3'
cyclization sequences was able to replicate after transfection into BHK
cells, while replicon RNA with simultaneous compensatory mutations in
both cyclization sequences was replication competent. This was detected
by immunofluorescence for expression of the major nonstructural protein
NS3 and by Northern blot analysis for amplification and accumulation of
replicon RNA. We then used the M-fold program to analyze RNA secondary
structure of the covalently linked 5'- and 3'-terminal regions of three
tick-borne virus species and identified a previously undescribed
additional pair of conserved complementary sequences in locations
similar to those of the mosquito-borne species. They base-paired with
G values of approximately 
20 kcal, equivalent or
greater in stability than those calculated for the originally proposed
cyclization sequences. The results show that the base-pairing between
5' and 3' complementary sequences, rather than the nucleotide sequence
per se, is essential for the replication of mosquito-borne Kunjin virus
RNA and that more than one pair of cyclization sequences might be
involved in the replication of the tick-borne Flavivirus species.
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TEXT |
Despite its essential role in the
virus replication cycle as a template for the synthesis of minus-strand
RNA, the conformation of the genomic RNA of Flavivirus
species has not been defined. Particularly important is the mode of its
presentation to the RNA-dependent RNA polymerase NS5, and possibly
other components of the replicase complex (RC) (28, 51),
in order for copying to commence correctly from the 3' end. The size of
the Flavivirus genome is about 11 kb, and the complete
nucleotide sequence is available for a range of species, (7, 12,
14-16, 18, 25, 27, 29, 33, 40, 45, 54). All sequences share a
common gene order (5'-C prM E NS1 NS2A NS2B NS4A NS4B NS5-3'),
i.e., structural (C, prM, and E) followed by nonstructural (NS)
genes, and are flanked by 5' and 3' untranslated regions (UTR) of about 100 and 600 nucleotides, respectively (39). Conserved
complementary cyclization sequences (CS) of 8 nucleotides in the 5'
region of the core, or capsid, gene, and in the 3' UTR, were noted in
genomic RNA for several mosquito-borne species (20). Hahn
et al. (20) suggested that cyclization of
Flavivirus genomic RNA could "help ensure that virus RNA
molecules that are replicated are full-length RNA, if a viral replicase
were required to bind to both 5' and 3' regions simultaneously in order
to initiate RNA replication." For Kunjin virus (KUN) RNA, these CS
are located at nucleotides 137 to 144 in the 5' region and at
nucleotides 97 to 104 from the 3' terminus (see Fig. 1A). Another
important sequence in replication may be the conserved pentanucleotide
loop 5'-CACAG(A/U)-3' (49) in the upper half of
the 3'-terminal stem-loop, also described as the 3' long stable hairpin
structure (37).
During the initial copying of KUN genomic RNA, double-stranded RNA was
formed, and on completion, this replicative form (RF) was shown to
function as a recycling template from which progeny RNA plus strands
were copied in an asymmetric and semiconservative manner via a
replicative intermediate with an average of only one nascent RNA strand
(9, 11, 50). At least during replication of KUN replicon
RNA, no free minus-strand RNA appears to be present in the cells
(26). In order to analyze minus-strand RNA synthesis in
vitro, the putative RNA polymerase (NS5) of dengue virus type 1 (DEN-1), West Nile virus, and KUN has been expressed either in
Escherichia coli or from recombinant baculoviruses
(19, 43, 47). These purified NS5 proteins exhibited
relatively weak RNA-dependent RNA polymerase activity on RNA templates
representing the homologous RNA templates and also on nonspecific RNA
templates (19, 43, 47). Using the endogenous DEN-2 RC in
infected cell lysates and exogenous truncated viral RNA templates, You
and Padmanabhan (52) showed that self-primed RNA synthesis
occurred by elongation of the authentic 3' end of the template RNA.
Interestingly, the RNA synthesis required both 5'- and 3'-terminal
regions containing the conserved cyclization motifs, either connected
in one molecule or with the isolated (not linked) 5'-terminal region
added in trans to the 3'-terminal region. Mutation of the
conserved CS in either the 5' or 3' region blocked this elongation, but
elongation ability was restored when both regions contained
compensatory mutations that allowed interaction between them. Because
these experiments measured only 3'-terminal elongation and copy back of
the 3'-terminal plus-strand RNA to yield predominantly a
double-stranded hairpin molecule, no evidence was obtained of de novo
minus-strand RNA synthesis, the first and essential prerequisite for
enabling subsequent synthesis of progeny plus-strand RNA during the
normal replication cycle.
The most convincing evidence for an essential role of the CS must come
from analyses of Flavivirus RNA replication in cells. For
this purpose we used the KUN replicon RNA, which has a deletion of most
of the structural gene region but is still able to replicate autonomously after transfection into cells (26). The
results obtained using RNAs with wild-type and mutated CS, or RNAs with compensatory mutations in these 5' and 3' sequences, showed that complementary cyclization motifs are essential for viral RNA
replication in vivo.
Computer-generated conformation of the linked 5' and 3' regions of
KUN RNA.
We first verified that the covalently linked CS, each
flanked by longer sequences representing the 5' region (first 150 to 170 nucleotides) and part of the 3' UTR (last 110 to 120 nucleotides) of genomic RNA from four mosquito-borne Flavivirus species
and separated from each other by a stuffer poly(A) sequence, could hybridize to form a proposed panhandle RNA structure using the M-fold
program (31, 55) (Fig. 1).
Base-pairing of the proposed CS
clearly occurred for all four species of RNA. Because of base-pairing elsewhere between the 5' and 3' sequences, i.e., upstream and downstream of the CS, respectively, the conformations differ from those
obtained previously from comparable regions analyzed only as either the
5' or 3' sequence of several other mosquito-borne species. The previous
analyses of secondary structures for isolated 5'- or 3'-terminal
regions, obtained either by computer predictions or by biochemical
analyses, uniformly showed the presence of terminal stem-loops, each of
about 100 nucleotides. The 3' stem-loop was reported to be particularly
well conserved in structure (although variants were noted) in all
publications reviewed (3, 4, 17, 18, 20, 29, 30, 35-38, 40-42,
46, 49, 53). Only the upper half of the 3'-terminal stem-loop
(nucleotides 15 to 66 for KUN) in the conserved secondary structure
of the isolated 3' UTR was retained (Fig. 1). This upper half comprises
the previously reported secondary structure shown to be important for
the replication of DEN-2 (53). Pertinently, NS3 and NS5 of
Japanese encephalitis virus were shown to cooperatively bind to the
3'-terminal stem-loop by Chen et al. (8), who suggested
that this binding may facilitate the process for minus-strand RNA
synthesis. Furthermore, in gel shift assays the 3'-terminal stem-loop
of DEN-1 RNA containing the pentanucleotide loop bound to NS3
(13). The binding of NS3 to the N-terminal regions of NS5
during their translation was suggested by complementation experiments
involving deletion analyses of the KUN genome (24). Such
binding, possibly involving other components of the assembling RC, such
as NS2A (23) and/or cellular proteins (1, 2),
to the upper portion of the 3' stem-loop, e.g., the conserved
pentanucleotide loop, may facilitate the cyclization process. There
appears to be no opportunity in our M-fold-program-predicted models of
5' and 3' interactions (Fig. 1) for formation of the pseudoknot
involving the lower half of the 3'-terminal stem-loop, as was proposed
in the computer-predicted secondary structures of the isolated 3' UTR
of West Nile virus, yellow fever virus, and DEN-3 RNAs
(41).
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FIG. 1.
Computer-generated secondary-structure analysis of
the interaction between the genomic plus-strand RNA at the 5' and 3'
ends for four mosquito-borne flaviviruses. The predicted secondary
structures of the proposed CS and some flanking sequences connected by
a poly(A) insert were produced using version 3.0 of the M-fold program
(31, 55). The conserved putative CS are boxed. The arrows
indicate insertion points for the stuffer poly(A) sequence. The AUG
initiation codon and the conserved pentanucleotide loop
[5'-CACAG(A/U)-3'] in the 3'-terminal stem-loop are shown
in bold. Nucleotides are numbered from the 5' and 3' termini. (A) KUN
(12, 25); (B) Japanese encephalitis virus
(45); (C) yellow fever virus vaccine strain 17D
(40); (D) DEN-2 (15). The relevant GenBank
accession numbers are shown in Table 1.
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|
Computer-generated secondary structures of the 5'-terminal region of
RNA of several Flavivirus species contain a terminal stem-loop incorporating a nonconserved loop and bulges
(4). Cahour et al. (5) showed that most of
several small (5- or 6-nucleotide) deletions between nucleotides 55 and
98 in the 5' UTR of DEN-4 RNA were lethal. This region varied in
secondary structure for the four viruses shown in Fig. 1. In the
present analyses, two small stem-loops appeared in the first 70 nucleotides of the 5' region, and thereafter, base-pairing occurred
with 3'-terminal sequences, interspersed with an additional one or two
stem-loops involving only the 5' nucleotide sequence. To obtain viable
KUN replicon RNA, it was essential to include the first 60 nucleotides of the core gene, which incorporates the 5' CS (26).
Overall, the patterns of structures produced by the M-fold program and shown in Fig. 1 have only limited similarity apart from the
base-pairing of the CS. Notably, all stem-loops are formed by
base-pairing only within an individual sequence of a 5' or 3' region;
none involve direct interaction between the 5' and 3' regions. The secondary structures produced by the M-fold program and shown in Fig. 1
require confirmation using analyses of the whole genome, as employed
for topological organization of picornaviral genomes (34).
However, the large size of the Flavivirus RNA (11 kb) precludes such analyses at present.
Having established in principle that base-pairing of the CS is feasible
in the KUN replicon RNA sequence, we mutated them in order to observe
the effects on conformation (using the M-fold program) and replication
in transfected cells. Figure
2A shows the relevant mutations and the designations of plasmids containing the
cDNA for transcription. The wild-type sequence copied from pC17
includes both the conserved 5' and 3' CS (20) with
flanking sequences as shown in Fig. 1A. In pC13, the 5' cyclization
motif has been deleted. In pC17-5'mut and pC17-3'mut, five mutations were introduced that left only three of the original eight
base-pairings in the CS. Both sets of mutations were combined in
pC17-5'&3'mut as compensatory mutations so as to restore the original
number of base-pairings in the CS. When the secondary-structure
analyses of the interactions between the wild-type and mutated 5' and
3' ends were compared by the M-fold program as shown in Fig. 1 and 2B,
it was found that only the sequence with combined compensatory mutations (pC17-5'&3'mut) was able to achieve the base-pairing conformation and structure comparable to those of the wild-type sequence pC17. The structures of the mutants pC17-5'mut and pC17-3'mut showed drastic changes in the M-fold pattern (Fig. 2B); the deletion mutant pC13 also obviously differed substantially in structure from the
wild type (result not shown).
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FIG. 2.
Nucleotide sequence and secondary-structure analysis of
wild-type and mutant KUN replicon RNAs. (A) Interaction between 5' and
3' ends of the putative cyclization motif (shown in boxes). Mutated
nucleotides are shown in bold. Dashes indicate deleted nucleotides. The
G values shown are for base-pairing of the boxed CS. (B)
Computer-generated secondary-structure analysis of the interaction
between the genomic plus-strand RNA at the 5' and 3' ends of wild-type
and mutant KUN replicon RNAs. The predicted secondary structures of the
proposed CS and some flanking sequences connected by a poly(A) insert
were produced using the M-fold program as for Fig. 1. The boxes enclose
either the conserved cyclization motifs or the relevant mutated
sequences. The arrows indicate insertion points for the stuffer poly(A)
sequence. The AUG initiation codon and the conserved pentanucleotide
loop [5'-CACAC(A/U)-3'] in the 3'-terminal stem-loop are
shown in bold. Nucleotides are numbered from the 5' and 3' termini.
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Effects of mutations in CS on RNA replication in vivo.
In
order to ascertain the effect of the mutations on replication, KUN
replicons incorporating the mutations shown in Fig. 2 into the
wild-type replicon sequence of the plasmid pC17 (derivative of
C20DXrep) were constructed (22). Briefly, cDNA fragments containing mutated KUN 5' and/or 3' regions were obtained by PCR amplification, using the appropriate primers with incorporated mutations and restriction sites, and cloned into pC17, replacing the
wild-type sequences. (Further details of plasmid construction can be
obtained from the corresponding author upon request.) Replicon RNAs
were transcribed in vitro and subsequently transfected by electroporation into BHK cells as previously described
(26). Amplification and expression of the replicon RNAs
were initially monitored by immunofluorescence (IF) using antibodies to
KUN NS3 at various time intervals as described previously
(51). Most of the cells were strongly positive by IF at
24 h after transfection with the wild-type KUN replicon pC17,
compared with only a small number of cells for the compensatory mutant
pC17-5'&3'mut (Fig. 3). However, the
number of strongly positive cells increased severalfold later in
transfection with pC17-5'&3'mut RNA, and nearly all cells were strongly
positive by 36 and 48 h after transfection. No positive cells were
observed at 48 or 72 h after transfection for any of the other
mutants.
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FIG. 3.
Detection of replication and expression of the wild-type
and mutated KUN replicon RNAs by IF analysis. BHK cells were
electroporated with ~5 to 10 µg of in vitro-transcribed wild-type
and mutated replicon RNAs as described previously (26) and
assayed for expression of the NS3 protein by IF analysis with anti-NS3
antibodies (51) at 24, 36, and 48 h after
electroporation. Panels 1 to 3 show the results of IF analysis of the
wild-type (pC17) RNA, and panels 4 to 6 show the corresponding results
for cells transfected with RNA containing simultaneous compensatory
mutations in the 5' and 3' CS (pC17-5'&3'mut). Transfection with RNAs
containing mutations only in the 5'- and 3'-terminal regions
(pC17-5'mut and pC17-3'mut, respectively) (Fig. 2A) did not result in
the detection of NS3-positive cells.
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We next examined the accumulation of replicating RNA by Northern blot
assay. RNA was extracted from the transfected BHK cell cultures at 24, 36, and 48 h and assayed for viral RNA content by Northern blot
analysis (Fig. 4) as described previously
(26). No viral RNA was detected in cells transfected with
the mutated RNAs designated pC13, pC17-5'mut, and pC17-3'mut, in accord
with the IF results showing the lack of expression of NS3. A positive signal was obtained at 24 h with the compensatory mutant
pC17-5'&3'mut, although the signal was very weak. However, the signal
shown by Northern blotting from the compensatory mutant increased
dramatically by 36 and 48 h; this increase correlated well with
the increase in the number of positive cells and the intensity of IF
staining. The low signal shown by Northern blotting and the small
number of IF-positive cells at 24 h for the compensatory mutant
indicate inefficient early RNA replication leading to an extended delay in expression and attainment of the threshold of protein (NS3) required
for detection by IF. The retention of introduced mutations in both the
5' and 3' ends of pC17-5'&3'mut RNA during its amplification in
transfected cells was confirmed by restriction digest analysis with
SdaI restrictase of the DNA fragments obtained by reverse transcription-PCR amplification with appropriate primers of a total
cellular RNA isolated 48 h after transfection (data not shown).
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FIG. 4.
Northern blot showing effects of mutations in the
cyclization motifs on replication of KUN replicon RNA. BHK cells were
electroporated with ~5 to 10 µg of in vitro-transcribed wild-type
and mutated replicon RNAs as described previously (26),
and total cellular RNA was harvested with Trizol (Gibco BRL) at 24, 36, and 48 h postelectroporation. Fifteen micrograms of total cellular
RNA was separated electrophoretically in a 1% agarose gel under fully
denaturing conditions and transferred to nylon (Hybond-N; Amersham),
and the blot was probed simultaneously with two 32P-labeled
cDNA fragments encompassing either the entire KUN 3' UTR or 291 nucleotides of human  -actin sequence. The upper panel was exposed to
X-ray film for 22 h; the arrow indicates the position of RNA of
ca. 9 kb. The lower panel was exposed for 2 h, and it indicates
the relative abundance of the -actin transcript in each RNA
sample.
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In earlier in vivo experiments, deletions of as many as 352 nucleotides
after the stop codon in the KUN replicon, which left the 3'-terminal
272 nucleotides (including the 3' CS) intact, partially inhibited RNA
replication but were not lethal (26). Deletions in the
DEN-4 3' UTR of 30 to 262 nucleotides, including all but the 113 3'-terminal nucleotides, still permitted the recovery of progeny virus
(32). However, a deletion of an additional 30 nucleotides
was lethal; this region included the DEN-4 3' CS (3'-terminal
nucleotides 92 to 99) equivalent to those shown in Fig. 1 for other
mosquito-borne viruses.
Comparisons with possible cyclization motifs in RNA of
Flavivirus species not transmitted by mosquitoes.
The
genus Flavivirus includes a total of about 60 species
(21). In addition to the numerous mosquito-borne
Flavivirus species, there are a number of tick-borne viruses
and some other species with no known vector. The conserved cyclization
motifs first reported by Hahn et al. (20) for
mosquito-borne viruses are absent in the other reported sequences.
Putative CS were proposed in tick-borne encephalitis (TBE) virus RNA
located at nucleotides 114 to 124 (5' region) and at nucleotides 11061 to 11071 in the 3' UTR (29). As successive deletions in
the 3' UTR progressed downstream from the variable region (nucleotides
10376 to 10795) into the core element, a deletion terminating at
nucleotide 10919 severely impaired replication and a further deletion
extending to nucleotide 10994 was lethal (29). In this
last deletion mutant the proposed 3' CS was still retained in the
terminal sequence from nucleotides 10995 to 11141. We therefore scanned
the TBE virus RNA sequence between nucleotides 10795 and 10994 and
continued to the 3'-terminal nucleotide 11141 using the M-fold program
as used for Fig. 1 for any base-pairing with the 5' region (Fig.
5). In addition to base-pairing in the
CS, proposed by Mandl et al. (30) and shown here as CS "A," interactions were also observed between nucleotides 164 to 174 (5' region) and nucleotides 10949 to 10958 (3' UTR), shown as CS
"B" in Fig. 5. These complementary sequences were conserved at
corresponding locations in several TBE virus strains as well as in
Powassan (POW) and louping ill viruses (Table
1) (18, 29, 48).
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FIG. 5.
Computer-generated secondary structures of the
interaction between the RNA at the 5' and 3' ends of TBE virus
(29) and of CFA (6). The predicted secondary
structures of the proposed CS and some flanking sequences connected by
a poly(A) insert were produced using the M-fold program as for Fig. 1.
The putative CS are boxed. The arrows indicate insertion points for the
stuffer poly(A) sequence. The AUG initiation codon and the conserved
pentanucleotide loop [5'-CACAG(A/U)-3'] in the 3'-terminal
stem-loop are shown in bold. Nucleotides are numbered from the 5' and
3' termini.
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Cell fusing agent (CFA) was isolated from a mosquito cell line
(44) and is classified as a tentative
Flavivirus species (21). In the CFA genome,
three sequences of 6, 7, and 12 nucleotides (designated A, B, and C,
respectively) in the 5' region, which are complementary to three
sequences in the 3' UTR, were noted by Cammisa-Parks et al.
(6). Of these putative CS, two (CS "A" and CS "B")
were base-paired in the 5' region within the first 45 nucleotides,
unlike all other CS examined; the 3' component of the third (CS
"C") commenced at nucleotide 471, nearly 200 bases upstream of
all those described above (Table 1). However, other possible CS (now
described as CS "D") were base-paired at nucleotides 169 to 179 (5') and 10563 to 10572 (134 to 125 in 3' end) as shown in
Fig. 5, in locations similar to those in the CS of the mosquito-borne
viruses, especially in the 3' UTR (commencing in the region from 99
to 112 [Table 1]).
In all secondary structures of Flavivirus RNA generated by
the M-fold program which showed base-pairing of the proposed CS, the
upper half of the 3'-terminal stem-loop and the conserved pentanucleotide loop were retained. The latter is uniformly located for
all species, viz., at 46 to 48 (mosquito borne), 49 (tick borne),
or 45 (CFA) nucleotides from the 3' terminus. However, the CFA
pentanucleotide loop is changed at the fourth base (C instead of A).
Table 1 summarizes the proposed CS and their relative locations within
the nucleotide sequences of genomic RNA for the range of
Flavivirus species for which data are available. There is
remarkable uniformity among the nine mosquito-borne viruses. The
conserved 5' CS always commence 37 to 40 nucleotides downstream from
the start of the initiation codon. The 3' CS always commence 99 to 112 nucleotides before the 3' terminus. The corresponding locations in CFA
virus RNA that appear to best match the mosquito-borne viruses
according to the M-fold program (Fig. 5) are newly defined CS "D"
rather than CS "C" (6). However, the CS show no
resemblance to the order of nucleotides in the mosquito-borne species.
For the 5' CS of the tick-borne species, the reference locations for CS
"A," as proposed by Mandl et al. (29), for both the
TBE and POW viruses commence 18 to 20 nucleotides before the initiation codon, whereas those of the mosquito-borne viruses commence about 40 nucleotides downstream of this codon, as noted above. The location of
the proposed 3' CS of the tick-borne viruses appears to be anomalous;
each of these is located within the lower half of the 3'-terminal
conserved stem-loop predicted when the 3' UTR is analyzed in isolation
from the remainder of the genome (29, 37). In contrast,
the newly proposed 3' CS "B" for both viruses is located further
upstream, as for the mosquito-borne viruses. For louping ill virus, the
CS and their locations conform to those described above for the TBE and
POW viruses (Table 1). Whether or not our proposed CS in RNA of the
tick-borne viruses and the CFA virus (Fig. 5; Table 1) are essential
for replication remains to be tested experimentally.
The G values for the conserved base paired CS for the
mosquito-borne viruses, as cited by Hahn et al. (20),
increase to as much as 33 kcal when four to six base pairs upstream
are included, but those for DEN-2 remain at 12 kcal. The
G values of CS for the other viruses in Table 1 are
22.9 and 24.2 kcal for CS "C" and CS "D," respectively, of
the CFA virus and 19.8 and 30.9 kcal for CS "A" and CS "B,"
respectively, of tick-borne viruses. G values for the
five or six-base pairs in Table 1 preceding the mismatch or bulge
region in CS "B" of tick-borne viruses are 19.2 or 24.0 kcal,
respectively. These values compare favorably with those of the
mosquito-borne viruses for stability of the CS.
Conclusions.
All previous analyses of the conformation and
possible role of 5'- and 3'-terminal nucleotide sequences of a range of
Flavivirus species defined a variety of stem-loop structures
(37), including a pseudoknot formed about 90 nucleotides
from the 3' terminus (41). However, all these analyses
examined the structure of each 5'- or 3'-terminal region in isolation,
and hence the possible interactions or base-pairing between the
proposed CS when covalently linked with flanking 5' and 3' sequences
have not hitherto been examined, either by the M-fold program or in
infectivity testing of genomes with mutated CS. Our results with the
KUN replicon establish the essential role of both the 5' and 3' CS in
replication. Other functions of the CS in addition to the role in
replication proposed by Hahn et al. (20) may be
postulated. Cyclization of genomic RNA during or immediately after
formation of the RC on, e.g., the 3'-terminal loop may allow ribosomes
involved in translation to complete their traverse of the genomic RNA
and their subsequent release but prevent their reattachment at the 5'
terminus for reinitiation. Assuming that a short delay occurs during
cyclization and assembly of the RC before its commencement of copying,
the risk of collision between the ribosome and RC moving along the
template towards each other would be eliminated. It was proposed
previously that during the initial assembly of the RC on the 3' UTR of
the plus-strand RNA template, it is transported to the membrane site of
replication by the affinity of hydrophobic regions of components of the
RC (24). Such binding may provide a sequestered
environment that prevents the reattachment of ribosomes when the
base-pairing of the CS is disrupted during the copying of the RNA
template. In this scenario, cyclization of the RNA template is required
only for minus-strand RNA synthesis, which is a relatively infrequent
event throughout infection compared to synthesis of plus-strand RNA.
There is a continuing major need for genomic RNA to function early and
late as mRNA for synthesis of components of the RC and of the
structural proteins, respectively. Hence, the M-fold-program-predicted
structures shown in Fig. 1, 2, and 5 cannot be representative of the
total viral RNA population, but they may be essential for providing the
appropriate template conformation for minus-strand RNA synthesis. The
presumably rigid structure of the double-stranded RNA in RF would not
allow cyclization to occur when RF is being converted to the
replicative intermediate during the initiation of synthesis of progeny
RNA plus strands (9, 10). Proutski et al.
(37) suggested that the essential role of cyclization of
the Flavivirus genome may be in virion packaging rather than
in RNA replication. However, the latter role is strongly supported by
our results with the KUN replicon RNA which has a deletion in the
structural genes, and hence, effects on replication do not involve packaging.
We believe that the results obtained by mutation analysis with the KUN
replicon unequivocally establish the essential requirement of
complementary CS in the 5' region and 3' UTR for replication in vivo.
Because of the conservation of these motifs, the results can be
extrapolated to other mosquito-borne flaviviruses. Clearly the results
obtained with the inactive pC17-5'mut and the pC17-3'mut RNAs show that
a single 5' or 3' cyclization motif is inadequate for replication.
Results with the compensatory mutant pC17-5'&3'mut, show that base
pairing of the CS provides the essential element rather than the
nucleotide sequences per se. The slow initial amplification of the
double mutant pC17-5'&3'mut, may indicate that the wild-type sequence
confers some early advantage in replication, but this is only
transient. Further definition of the role of CS in
Flavivirus RNA replication will be possible when efficient systems are established for in vitro assays of Flavivirus
RNA synthesis using specific RNA templates and the purified RC.
This work was supported by grant no. 981442 from the National
Health and Medical Research Council of Australia.
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Abstract
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Publication no. 132 from the Sir Albert Sakzewski Virus Research Centre.
Present address: Queensland Agricultural Biotechnology Centre,
University of Queensland, Brisbane, Australia.
