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Detection of stable secondary structure at the 3′ terminus of dengue virus type 2 RNA
Gene, 108 (1991) 185-191 0
1991 Elsevier
GENE
Science
Publishers
B.V. All rights reserved.
185
0378-l 119/91/$03.50
06138
Detection of stable secondary structure at the 3’ terminus of dengue virus type 2 RNA (Recombinant
DNA;
flavivirus;
in vitro transcription;
polymerase
chain
reaction;
RNase A cleavage
sites;
RNase H
mapping)
P. Maruthi Mohan* and R. Padmanabhan Department
of Biochemistry
and Molecular
Biology, University of Kansas
Medical
Center, Kansas
City, KS 66103
(U.S.A.)
Received by J.R. Putnak: 6 November 1989 Revised/Accepted: 10 August/l4 August 1991 Received at publishers: 3 September 1991
SUMMARY
The 3’-terminal sequences of flavivirus genomes within approx. 100 nucleotides (nt) have been suggested to have a highly conserved secondary structure, as based on the known nt sequence data and free-energy calculations using computer programs. To test the existence of a secondary structure in solution, we devised a strategy to generate truncated RNA molecules from about 0.3-1.4 kb in length, having the same polarity and nt sequence as dengue virus type 2 (DEN-2) RNA (New Guinea-C strain). When these labeled RNA molecules were digested by RNase A, and analyzed by denaturing polyacrylamide-gel electrophoresis, three resistant fragments of 16, 20 and 23 nt in length were reproducibly obtained. To examine whether these RNase A-resistant (RNaseR) fragments emerged from a stable secondary structure formed in solution consisting of 3’ -terminal sequences, hybridization of the RNaseR fragments to four chemically synthesized oligodeoxyribonucleotides (oligos), complementary to nt l-24, 25-48, 49-72, and 73-96 from the 3’ terminus of DEN-2 RNA, followed by RNase H digestion were carried out. Oligos complementary to nt 25-48 and 49-72 from the 3’ end of DEN-2 RNA were sufficient to render all three RNaseR fragments susceptible to RNase H digestion. These data indicate that a stable secondary structure is formed in solution involving nt 18-67 from the 3’ terminus. The potential use ofthese unique transcripts to identify the viral and/or host proteins which might interact at the 3’ terminus of DEN-2 RNA during initiation of replication is discussed.
The family of flaviviridae consists of about 70 closely related enveloped viruses (Westaway et al., 1985) containing an ss RNA of approx. 11 kb as their genome with
a positive-stranded polarity (Russell et al., 1980). The 5’ end of the RNA has a type-1 cap structure, and a poly(A) track toward the 3’ end of mosquito-borne flaviviruses is absent. However, recently it has been reported that there are two types of TBE virus strains, one having a poly(A)
Correspondence to: Dr. R. Padmanabhan,
phates;
INTRODUCTION
and Molecular
Biology, University
Department
of Kansas
Medical
of Biochemistry Center,
39th and
Rainbow Blvd., Kansas City, KS 66103 (U.S.A.) Tel. (913)588-7018; Fax (913)588-4903. * Present
address:
Department
of Biochemistry,
Abbreviations:
bp, base pair(s); dengue
DEN-2,
cDNA,
Osmania
DNA complementary
virus type 2; dNTP,
ds, double
University,
deoxynucleoside
reaction;
strand(ed);
virus; kb, kilobase
litis virus; NGS-C, deoxyribonucleotide;
Hyderabad-500007 (A.P.) (India) Tel. (091)842-868951 (Ext. 245); Fax (91)842-869020.
RNA;
cephalitis
DTT,
JE, Japanese
en-
New Guinea strain C; nt, nucleotide(s); oligo, oligoORF, open reading frame; PCR, polymerase chain
PolIk, Klenow (large) fragment
resistance/resistant;
dithiothreitol;
or 1000 bp; MVE, Murray valley encepha-
RNaseR,
resistant
of6.
coli DNA polymerase
to RNase A digestion;
I; R,
ss, single
strand(ed); SSC, 0.15 M NaCI/O.OlS M Na, ‘citrate pH 7.6; 7’aq polymerase, DNA polymerase isolated from Thermus aquaficus YTl; TBE, to viral triphos-
tick-borne
encephalitis;
WN, West Nile virus; YF, yellow fever virus.
186 tract at the 3’ end (the prototype Neudoerfl strain), and the other lacking it similar to the mosquito-borne flaviviruses (Mandl et al., 1991). The first complete sequence of a flavivirus RNA reported was that of YF (Rice et al., 1985). Several interesting structural features in the RNA genome were established unequivocally& this study. (I ) There is a single long ORF coding for a large polyprotein precursor, which is then cleaved by cellular and/or viral encoded teases to form the mature structural and nonstructural
propro-
teins of the virus. (2) A model was proposed for the possible secondary structure in the 3’-terminal region of YF RNA (Rice et al., 1985; see also Grange et al., 1985). Since then, a number of flavivirus genomes have been cloned and sequenced, such as WN (Castle et al., 1985; 1986; Wengler and Castle, 1986; Wengler et al., 1985) DEN-4 (Zhao et al., 1986; Mackow et al., 1987), three distinct isolates of DEN-2 (Hahn et al., 1988; Duebel et al., 1986; 1988; Yaegashi et al., 1986; Irie et al., 1989), JE (Takegami et al., 1986; Sumiyoshi et al., 1987; McAda et al., 1987), Kunjin virus (Coia et al., 1988), and TBE (Mandl et al., 1988; 1989). These sequence data support the general structural features of the flavivirus genome mentioned above. Interestingly, the potential to form a stable secondary structure is highly conserved among the various flavivirus RNA molecules, as deduced from the primary sequence data and the computer-derived energy calculations (Tinoco et al., 1973). Two lines of experimental evidence suggested that such secondary structures could exist in solution. First, Brinton et al. (1986) found that nucleotides within the putative region of secondary structure formed at the 3’ terminus of WN were partially resistant to RNases. Second, Hahn et al. (1987) reported the isolation of rare cDNA clones of DEN-2 RNA (Sl/candidate vaccine strain of PR-159 isolate) as well as that of MVE RNA, which could have arisen by self-priming of 3’-terminal base-paired region during cDNA cloning using reverse transcriptase. The aim of the present study was to test the formation of such a stable secondary structure in solution, by devising a novel method to generate radiolabeled transcripts of high specific activity in vitro, which contained the authentic 3’terminal sequences of DEN-2 RNA (NGS-C strain). Formation of stable secondary structures in these labeled transcripts could then be analyzed by digestion with RNase A. To examine whether any of the RNaseR fragments were generated from the 3’-terminal region of the transcripts, we hybridized synthetic oligos of defined sequences complementary to the 3’-terminal region of DEN-2 RNA to the RNaseR fragments, and tested the sensitivities of the resultant RNA : DNA hybrids to RNase H. These experiments enabled us to map the RNaseR fragments within the 3’-terminal 96 nt region of DEN-2 RNA, and provided direct evidence for the formation of a stable secondary structure in solution involving this 3’-terminal region.
RESULTS
AND
DISCUSSION
(a) Generation of transcripts containing the 3’4erminal sequences of DEN-2 RNA To examine the potential of the 3’-terminal sequences to form a secondary structure in solution, we devised a strategy to generate truncated RNA molecules having the authentic 3’ terminus of DEN-2 RNA, differing only in their lengths as their 5’ end. The parent plasmid contains an insert of 1339 bp corresponding to the 3’-terminal sequence of DEN-2 RNA cloned between X&I and KpnI sites of pGEM 7Zf’ vector (Promega, Madison, WI) (Fig. 1). Several subclones were generated from this parent plasmid by sequential digestion with Escherichiu coli exonuclease III and S 1 nuclease (Henikoff, 1984). The end points of deletion in these subclones were determined by sequence analysis (Sanger et al., 1977). Two cDNA clones containing the 3’-terminal sequences of 292 bp, and 1339 bp were linearized with XbaI, followed by digestion with S 1 nuclease. They were then used as templates for in vitro transcription using SP6 RNA polymerase in the presence of labeled rNTPs to give rise to transcripts having the same polarity, as well as the 3’-terminal sequence as DEN-2 RNA, differing only in their lengths (see Fig. 2). (b) Resistance to RNase A digestion To investigate whether labeled transcripts could form a stable secondary structure in solution, digestion by RNase A was carried out under two different salt concentrations. Since ds regions of RNA are resistant and ss regions sensitive to RNase A digestion, any secondary structure formation in RNA could be visualized as RNaseR fragments by electrophoresis on polyacrylamide/urea gels followed by autoradiography. Fig. 2 shows that, when labeled transcripts of 292 and 1339 nt in length were digested by RNase A in 0.01 x SSC, or 2 x SSC, some common RNaseR fragments were obtained. The sizes of these fragments did not change when S 1 nuclease digestion to remove the four nt from the 5’ end of the XbaI-linearized plasmid was omitted, prior to in vitro transcription (not shown). The sizes of the protected fragments were estimated to be within two clusters, the first between 23 and 16 nt, the second between 1 and 10 nt long (Fig. 2, A and B), by comparison with the ladder of 5’ -labeled oligo (5 1-mer) partially digested with snake venom phoshodiesterase (not shown). The fragments indicated by arrows in the first cluster were the predominant species, and their sizes were estimated to be about 23, 20, and 16 nt in length (Fig. 2, A and B). Other RNaseR fragments produced in the digest of 1339 nt transcript were not present in the digest of 292 nt transcript in the first cluster, suggesting that these could have been produced from the region not shared by the two transcripts. Interestingly, under the conditions of RNase A
187 promoter
Tl
JkU I’
TCTAGA AGATCT
T7 promoter
XLUI I'
T AGATC Pollk
Tl promoter
I
TCTAG AGATC
Ligation cloning
t A T
and
GGTAC C PCR fragment (1.35 kb)
Fig. 1. Strategy
for generating
transcripts
containing
the same polarity
and 3’-terminal
from DEN-2 RNA which was tailed with poly(A) using E. coli poly(A) polymerase The oligos 5’-GGACAAGTTGGTACCTATGG CAACACACC amplified
clones having the parent
(representing
to the 3’-terminal
sequence
DNA after a total of 25 cycles of denaturation,
and blunt-ended sequence
complementary
XbaI sites of pGEM7Zf’ the regenerated
of DEN-2
1.35-kb cDNA
clone having
Madison,
orientation
identical
3’-terminal
treating
WI), following (up
was digested routine
for amplification
with E. coli exonuclease
by Taq polymerase.
procedures
(Maniatis
lengths
III and S 1 nuclease
prepared
previously
toward
(Henikoff,
The PCR the KpnI
et al., 1982). Several independent
to about 120 nt from X&I site corresponding
with varying
library,
by PCR (Saiki et al., 1988).
with KpnI. It was then cloned between
in this region to those reported
sequences
RNA in vitro. A cDNA
see Irie et al., 1989), and 5’-AGAACCTGTTGATT-
were used as primers
were sequenced
DEN-2
as DEN-2
1973), was used for amplification
of DEN-2 genome;
genome
and DNA synthesis
data were found to be identical
parent clone at the SacI site, and sequentially verified by DNA sequence
annealing
vector (Promega,
XbaI site in the correct
RNA). The sequence
nt 9373-9392 of DEN-2
sequences
(Sippel,
to the 3’terminal
(Irie et al., 1989). Subclones
the 5’ end were prepared
by digesting
1984). The end points of all deletions
from the were
analysis.
digestion in 0.01 x SSC, only the 23 nt fragment was produced as the RNaseR fragment (Fig. 2A, lane 5). The low salt conditions (0.0 1 x S SC) are probably not favorable for the formation of a stable secondary structure in solution. Next, the question whether these RNaseR fragments resulted specifically from the transcripts containing the 3’-terminal sequences of DEN-2 RNA was addressed. A control transcript from the region of l-550 nt at the 5’ end of DEN-2-RNA was digested with RNase A under the same conditions of high ionic strength. Fig. 2B shows that the pattern of RNaseR fragments (lane 4) was quite different from that of 3’ -terminal transcript (lane 2).
(c) Mapping the location of RNaseR fragments by annealing with synthetic oligos, followed by RNase H digestion To investigate further whether these RNaseR fragments contained sequences from the 3 ‘-terminal region of DEN-2 RNA, four oligos were synthesized complementary to the first 96 nt of DEN-2 RNA. Subsequent to RNase A digestion of the two transcripts containing the 3’-terminal sequences, the RNaseR fragments were annealed to each of the four oligos, and the annealed mixtures were digested with RNase H. Since RNase H digests only the RNA of the RNA : DNA hybrid, this experiment could reveal the speci-
188
A
123456
B
1234
ticity and location of the RNaseR fragments. The results of RNase A, followed by RNase H digestion in Fig. 3 indicate that oligos Nos. 1 and 4 did not have any effect (lanes 3 and 6 in Fig. 3, A and B). These results indicate that the RNaseR fragments do not form stable hybrids with oligos Nos. 1 and 4 to become susceptible to RNase H digestion, or do not contain sequences complementary to these oligos. However, when oligo No. 2 was annealed, 23 and 20 nt long RNaseR fragments became sensitive to RNase H (Fig. 3, A and B, lanes 4) indicating that these fragments resulted from the region of transcript corresponding to nt 17-39 from the 3’ terminus of DEN-2 RNA (see also Fig. 4). The digestion of 23-nt fragment appeared incomplete (Fig. 3, A and B, lanes 4), and there was no change in this pattern even when a mixture of all four oligos were used for annealing (Fig. 3, A and B, lanes 7). However, in another experiment in which 400 ng of oligo No. 2 was used for hybridization, this 23-nt band was completely susceptible to RNase H digestion (data not shown). When oligo No. 3 was annealed, RNaseR fragments of 20 and 16 nt in length became sensitive to RNase H digestion, suggesting that these fragments contained sequences complementary to nt 44-65 from the 3’ terminus of DEN-2
aliquots
at
- 70°C.
(Panel A) An aliquot
(5 x lo“-1 x lo5 cpm) was incubated (50 PI), containing
of the labeled
at 37°C
transcript
for 60 min in a buffer
RNase A (3 pg) as follows. Lanes 1
2
3
4
292 nt RNA
+ _
f _
+ _
0.01 x ssc
+
+ _
_
+
+
f _
+
+
_
+
1339 nt RNA
2 x ssc RNase A
5
6
_
_
_
+
+
+ _ + +
RNase A was removed by proteinase K (3 pg) digestion in 1 y0 SDS at 37°C for 15 min, followed by phenol: CHCl, extraction and ethanol precipitation Fig. 2. Analysis transcripts.
of RNaseR
fragments
Two cDNA clones having 3’-terminal
292 bp as inserts were sequentially Labeled
transcripts
templates
generated
by the method
was carried
sequences
of 1339 and
with XbaI, and S 1 nuclease.
of high specific activity
were prepared
in vitro transcription
treated
from the 3’-terminal
from the linearized of Melton
plasmid
et al. (1984). The
out in 25-~1 reaction
volume containing
40 mM Tris . HCl pH 7.5/6 mM MgCl,/2 mM spermidine/0.05 mM each of GTP, CTP, UTP/O.S mM ATP/one unit of Inhibit-ACE (5 Prime-3 Prime, Inc., West Chester, serum
albumin/l0
PCi
PA)/1 mM m7G(5’)ppp(5’)G/100 each
of
[c+~‘P]GTP,
CTP,
gg bovine UTP
(400 Ci/
mmole)/two units of SP6 RNA polymerase. The reaction mixture was incubated at 40°C for 60 min. The DNase I (1 unit) was then added and incubated
for a further
15 min at 37°C.
The reaction
mixture
was ex-
tracted with phenol: CHCI, and the unincorporated nt were removed on Biospin-101 column (BioRad). The labeled transcript was extracted once with phenol
: CHCI, and precipitated
with ethanol
and stored
as small
in the presence
of E. coli tRNA
(3 pg) added
The labeled RNA was collected
by centrifugation,
was
finally
small
Tris
0.089 M borate/2
dissolved
in
a
mM EDTA
volume buffer
as a carrier.
and the pelleted RNA of
7M
urea/O.089
pH 8.3, containing
phenol blue dye, heated at 90°C for 3 min and electrophoresed
M
bromoon a 7 M
urea/O.089 M Tris .0.089M borate/2 mM EDTA (20%) polyacrylamide gel until the bromophenol dye reached 21 cm. The sizes of the fragments were determined
from a ladder
of 5’-labeled
oligos generated
from a
partial snake venom phosphodiesterase digestion (Jay et al., 1974) of a 51-mer deoxyoligo. The gel was covered with plastic wrap and exposed directly
to Kodak
XAR-5 film at 70’ C. The sizes of the three fragments
shown by arrows are estimated to be 16, 20 and 23 nt. (Panel B) The transcript containing 292 nt of 3’-terminal sequence of DEN-2 RNA, and a transcript
(control
RNA)
from
a cDNA
clone
corresponding
to
nt I-550 from the S’terminus of DEN-2 RNA (5 x IO4 cpm each) were either incubated in buffer with no RNase A, or digested with RNase A under the same conditions
as in panel A, lanes 3 and 6.
189 lanes 5), whereas
it appeared
to be almost complete
when
oligo No. 2 was used (Fig. 3, A and B, lanes 4). These results suggested a possibility that there are two RNaseR fragments of 20 nt in length, both of which were sensitive to oligo No. 2, but only one of which was sensitive to oligo No. 3 (see section d, and Fig. 4, A and B). (d) Location of the RNase A cleavage sites in the putative model for stem-loop structure at the 3’ terminus of DEN-2 RNA The data presented in Figs. 2 and 3 support the formation of a stable secondary structure in solution. Hahn et al. (1988) proposed a model for the stem-loop structure from the primary sequence data and the computer-derived
Fig. 3. Mapping fic synthetic containing sequences
of the RNase A-resistant
oligos,
followed
fragments
by annealing
by RNase H digestion.
1339 (panel A), and 292 (panel B) nt of 3’-terminal were digested
in length are shown by arrows and B. The digests
and hybridized
with 100 ng of synthetic (oligo
oligos (24-mers)
No. 1); 2, nt 25-48
(oligo No. 3); 4, nt 73-96
(oligo
nt is 1. The hybridization
containing
25 mM Tris
HCl
was done in 10 ~1 reaction
pH 7.5/300 mM
mM MgCI, at 90°C for 3 min, followed
KC&‘2 mM
formamide
was stopped
by the addition
(80x)-bromophenol indicate
the RNase
blue dye. The samples H products
mM
at 37°C for 60 min.
of stop
buffer
containing
were heated
90” C for 3 min and loaded onto a 7 M urea/2Oft% polyacrylamid~ arrowheads
volume
DTTjl
by quick chilling in ice
for 5 min. RNase H (1 unit) was added and incubated The reaction
complementary
No. 2); 3, nt 49-72
(oligo No. 4) of DEN-Z RNA (see Fig. 4). The
3’-terminal EDTA/6
of E. coli tRNA
in the presence
Each of the digests was divided into aliquots,
to
1, nt l-24
of panels A
: CHCI,, and the labeled
with phenol
with ethanol
(OS pg) added as a carrier.
as in
of 23,20 and 16 nt
on the left and right margins
were treated
was precipitated
DEN-2
with RNase A under the same conditions
Fig. 2, panel A, lanes 3 and 6. The RNaseR fragments
RNA
speci-
The transcripts
at
gel. The
of 12 and 13 nt in length.
Lanes I
2
3
4
5
6
7
+ -
+
-
+
-
+ +
-
+
Oligo 1
-
-
+ f
+ -
Oligo 2
-
-
-
+
+ _
Oligo 3 Oligo 4
-
-
-
-
+ _
RNase
H
+
+
RNA (Fig. 3, A and B, lanes 5). It should be noted again that the digestion of 20-nt fragment appeared incomplete when oligo No. 3 was used for annealing (Fig. 3, A and B,
energy calculations (Fig. 4A). The primary sequences are identical for DEN-2 RNA of New Guinea, Jamaica strain, and Puerto Rico-l 59/S 1 (PR- 159/S 1) vaccine strain, except at nt 42, which is a G for PR-159/Sl strain (Hahn et al., 1988; Deubel et al., 1988; Irie et al., 1989). We examined the putative RNase A cleavage sites based on this model (Fig. 4A). Since oligos Nos. 2 and 3 but not oligos Nos. 1 and 4 render the RNaseR fragments susceptible to RNase H, the RNase A cleavage sites are most likely located in the ss-loop region in the proposed model shown in Fig. 4A. Since RNase A cleaves next to pyrimidines, its putative cleavage sites in the secondary structure model can be tentatively assigned as follows. For example, the cteavages occurring at sites 1 and 3, and 1 and 2 would give rise to a 23, and a 20 nt fragment, respectively, which would be susceptible to oligo No. 2 t RNase H treatments (see also Fig. 4B). It appears that the sites 1 and 3 are the most readily cleaved, as the amount of 23 nt fragment is the predominant species obtained (see Fig. 3, A and B, lanes 3). Similarly, cleavages occurring at sites 5 and 6 would give rise to a 16 nt fragment which would become susceptible to oligo No. 3 + RNase H treatment (Fig. 3, A and B, lanes 5). The generation of 20 nt fragment(s) by RNase A cieavage which become susceptible to both oligos Nos. 2 or 3 in the presence of RNase H is difficult to explain from this putative model. One possibility is that cleavages at sites 4 and 8 in the secondary structure model shown in Fig. 4A would also result in a 20 nt fragment susceptible to oligo No. 3. A second possibility is that two 20 nt fr~ments could be generated from alternate secondary structures formed at the 3’-terminal region of DEN-2 transcript in solution. It is possible that the stem-loop structure proposed by Hahn et al. (1988) could be stabilized by interaction with proteins in vivo, but in the absence of stabilizing protein, the RNA could assume alternate stable structures in solution. It is noteworthy that smaller fragments of approx. 12-13 nt in length were also generated after RNase H digestion (as shown by arrowheads in Fig. 3, A and B, lanes 4). The origin of these bands could not be mapped at present.
190
B A A U-A G-C G-C A-U A-U U-A 24- G - C G U U-A
III GGC I ACA 48
3'..UGUCGUGGUAAGGUIIIIAAGA oligo 3’......
23 nt
I
I 30 50 I
3’. oligo
6
60 I 16
CGGACCUUACUACGAC
Fig. 4. A model for the putative proposed
secondary
minal sequences ary structure numbered Guinea, strain,
20 nt
GACC I II
A I A
CUGG
GA
G -96
sites in the previously
to form a stable second-
et al., 1988). The 3’ terminus
strain,
are identical
and Puerto
of DEN-2
for DEN-2
Rico-159/Sl
RNA is
RNA
of New
(PR-159/Sl)
vaccine
except at nt 42, which is a G for PR-159/S 1 strain (Hahn
putative AG
of DEN-2 RNA has a potential
Jamaica
et al., 1988; Irie et al., 1989). The arrows
sites of cleavage
fragments. - A
RNase A-sensitive
for DEN-2 RNA at 3’ end. (A) The 3’-ter-
as nt 1. Sequences
1988; Deubel
C-G
structure
(Hahn
nt
I I I I I I I I I I I I I I CTGGAATGATGCTGAGGAGACAGC
.
#3
CACG&XXUUACUACGACUC
G - C\ A-U 8 A C U U C-G A-U A C C - G -72 A-U G-C G-C U U U-A
I
UGUCGUGG20
U-A G-CI'
C A
TTTCB I I I I I I I I I I I I I I I
a
G-C C-G
3'-U
#2
20 nt
RNaseR
fragments
underlined. fragment,
of possible
hybrids
that
with synthetic
Base pairing
substrates
would
between
for RNase H digestion.
be formed
oligos
in the annealing
of
Nos. 2 and 3 are shown
as
the oligo No. 2 and 23-nt
and oligo No. 3 and 16-nt RNaseR
respectively)
the
by RNase A that would give rise to the RNaseR
(B) Structures
The RNA: DNA
et al.,
indicate
are shown by short vertical
fragment
RNaseR
(15 and 14 bp,
lines.
G
u u
5’
(e) Conclusions Our results support the existence of base-paired region and a general stem-loop structure proposed by Hahn et al. (1988) between nt 18-62 from the 3’ terminus of DEN-2 RNA. The region between nt 1-17, or 63-96 did not give any significant resistance to RNase A under our test conditions. This highly conserved secondary structure has been proposed to be important for replication of viral RNA (Hahn et al., 1987; Brinton et al., 1986; Takegami et al., 1986). Therefore, it will be interesting to test whether such a secondary structure is stabilized by binding of proteins in vivo. Our method to generate labeled transcripts having the same polarity and the 3’-terminal sequences of DEN-2 RNA might be useful for such studies.
(DAMD17-86-G-6038) to Dr. Fred Sampson, and by Biomedical Research Support Grant/Shared Instrumentation Facility. The views, opinions, and/or findings in this report are those of the authors only and should not be construed as an official Department of the Army position, policy, or decision unless so designated by other documentation.
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1991 Elsevier
GENE
Science
Publishers
B.V. All rights reserved.
185
0378-l 119/91/$03.50
06138
Detection of stable secondary structure at the 3’ terminus of dengue virus type 2 RNA (Recombinant
DNA;
flavivirus;
in vitro transcription;
polymerase
chain
reaction;
RNase A cleavage
sites;
RNase H
mapping)
P. Maruthi Mohan* and R. Padmanabhan Department
of Biochemistry
and Molecular
Biology, University of Kansas
Medical
Center, Kansas
City, KS 66103
(U.S.A.)
Received by J.R. Putnak: 6 November 1989 Revised/Accepted: 10 August/l4 August 1991 Received at publishers: 3 September 1991
SUMMARY
The 3’-terminal sequences of flavivirus genomes within approx. 100 nucleotides (nt) have been suggested to have a highly conserved secondary structure, as based on the known nt sequence data and free-energy calculations using computer programs. To test the existence of a secondary structure in solution, we devised a strategy to generate truncated RNA molecules from about 0.3-1.4 kb in length, having the same polarity and nt sequence as dengue virus type 2 (DEN-2) RNA (New Guinea-C strain). When these labeled RNA molecules were digested by RNase A, and analyzed by denaturing polyacrylamide-gel electrophoresis, three resistant fragments of 16, 20 and 23 nt in length were reproducibly obtained. To examine whether these RNase A-resistant (RNaseR) fragments emerged from a stable secondary structure formed in solution consisting of 3’ -terminal sequences, hybridization of the RNaseR fragments to four chemically synthesized oligodeoxyribonucleotides (oligos), complementary to nt l-24, 25-48, 49-72, and 73-96 from the 3’ terminus of DEN-2 RNA, followed by RNase H digestion were carried out. Oligos complementary to nt 25-48 and 49-72 from the 3’ end of DEN-2 RNA were sufficient to render all three RNaseR fragments susceptible to RNase H digestion. These data indicate that a stable secondary structure is formed in solution involving nt 18-67 from the 3’ terminus. The potential use ofthese unique transcripts to identify the viral and/or host proteins which might interact at the 3’ terminus of DEN-2 RNA during initiation of replication is discussed.
The family of flaviviridae consists of about 70 closely related enveloped viruses (Westaway et al., 1985) containing an ss RNA of approx. 11 kb as their genome with
a positive-stranded polarity (Russell et al., 1980). The 5’ end of the RNA has a type-1 cap structure, and a poly(A) track toward the 3’ end of mosquito-borne flaviviruses is absent. However, recently it has been reported that there are two types of TBE virus strains, one having a poly(A)
Correspondence to: Dr. R. Padmanabhan,
phates;
INTRODUCTION
and Molecular
Biology, University
Department
of Kansas
Medical
of Biochemistry Center,
39th and
Rainbow Blvd., Kansas City, KS 66103 (U.S.A.) Tel. (913)588-7018; Fax (913)588-4903. * Present
address:
Department
of Biochemistry,
Abbreviations:
bp, base pair(s); dengue
DEN-2,
cDNA,
Osmania
DNA complementary
virus type 2; dNTP,
ds, double
University,
deoxynucleoside
reaction;
strand(ed);
virus; kb, kilobase
litis virus; NGS-C, deoxyribonucleotide;
Hyderabad-500007 (A.P.) (India) Tel. (091)842-868951 (Ext. 245); Fax (91)842-869020.
RNA;
cephalitis
DTT,
JE, Japanese
en-
New Guinea strain C; nt, nucleotide(s); oligo, oligoORF, open reading frame; PCR, polymerase chain
PolIk, Klenow (large) fragment
resistance/resistant;
dithiothreitol;
or 1000 bp; MVE, Murray valley encepha-
RNaseR,
resistant
of6.
coli DNA polymerase
to RNase A digestion;
I; R,
ss, single
strand(ed); SSC, 0.15 M NaCI/O.OlS M Na, ‘citrate pH 7.6; 7’aq polymerase, DNA polymerase isolated from Thermus aquaficus YTl; TBE, to viral triphos-
tick-borne
encephalitis;
WN, West Nile virus; YF, yellow fever virus.
186 tract at the 3’ end (the prototype Neudoerfl strain), and the other lacking it similar to the mosquito-borne flaviviruses (Mandl et al., 1991). The first complete sequence of a flavivirus RNA reported was that of YF (Rice et al., 1985). Several interesting structural features in the RNA genome were established unequivocally& this study. (I ) There is a single long ORF coding for a large polyprotein precursor, which is then cleaved by cellular and/or viral encoded teases to form the mature structural and nonstructural
propro-
teins of the virus. (2) A model was proposed for the possible secondary structure in the 3’-terminal region of YF RNA (Rice et al., 1985; see also Grange et al., 1985). Since then, a number of flavivirus genomes have been cloned and sequenced, such as WN (Castle et al., 1985; 1986; Wengler and Castle, 1986; Wengler et al., 1985) DEN-4 (Zhao et al., 1986; Mackow et al., 1987), three distinct isolates of DEN-2 (Hahn et al., 1988; Duebel et al., 1986; 1988; Yaegashi et al., 1986; Irie et al., 1989), JE (Takegami et al., 1986; Sumiyoshi et al., 1987; McAda et al., 1987), Kunjin virus (Coia et al., 1988), and TBE (Mandl et al., 1988; 1989). These sequence data support the general structural features of the flavivirus genome mentioned above. Interestingly, the potential to form a stable secondary structure is highly conserved among the various flavivirus RNA molecules, as deduced from the primary sequence data and the computer-derived energy calculations (Tinoco et al., 1973). Two lines of experimental evidence suggested that such secondary structures could exist in solution. First, Brinton et al. (1986) found that nucleotides within the putative region of secondary structure formed at the 3’ terminus of WN were partially resistant to RNases. Second, Hahn et al. (1987) reported the isolation of rare cDNA clones of DEN-2 RNA (Sl/candidate vaccine strain of PR-159 isolate) as well as that of MVE RNA, which could have arisen by self-priming of 3’-terminal base-paired region during cDNA cloning using reverse transcriptase. The aim of the present study was to test the formation of such a stable secondary structure in solution, by devising a novel method to generate radiolabeled transcripts of high specific activity in vitro, which contained the authentic 3’terminal sequences of DEN-2 RNA (NGS-C strain). Formation of stable secondary structures in these labeled transcripts could then be analyzed by digestion with RNase A. To examine whether any of the RNaseR fragments were generated from the 3’-terminal region of the transcripts, we hybridized synthetic oligos of defined sequences complementary to the 3’-terminal region of DEN-2 RNA to the RNaseR fragments, and tested the sensitivities of the resultant RNA : DNA hybrids to RNase H. These experiments enabled us to map the RNaseR fragments within the 3’-terminal 96 nt region of DEN-2 RNA, and provided direct evidence for the formation of a stable secondary structure in solution involving this 3’-terminal region.
RESULTS
AND
DISCUSSION
(a) Generation of transcripts containing the 3’4erminal sequences of DEN-2 RNA To examine the potential of the 3’-terminal sequences to form a secondary structure in solution, we devised a strategy to generate truncated RNA molecules having the authentic 3’ terminus of DEN-2 RNA, differing only in their lengths as their 5’ end. The parent plasmid contains an insert of 1339 bp corresponding to the 3’-terminal sequence of DEN-2 RNA cloned between X&I and KpnI sites of pGEM 7Zf’ vector (Promega, Madison, WI) (Fig. 1). Several subclones were generated from this parent plasmid by sequential digestion with Escherichiu coli exonuclease III and S 1 nuclease (Henikoff, 1984). The end points of deletion in these subclones were determined by sequence analysis (Sanger et al., 1977). Two cDNA clones containing the 3’-terminal sequences of 292 bp, and 1339 bp were linearized with XbaI, followed by digestion with S 1 nuclease. They were then used as templates for in vitro transcription using SP6 RNA polymerase in the presence of labeled rNTPs to give rise to transcripts having the same polarity, as well as the 3’-terminal sequence as DEN-2 RNA, differing only in their lengths (see Fig. 2). (b) Resistance to RNase A digestion To investigate whether labeled transcripts could form a stable secondary structure in solution, digestion by RNase A was carried out under two different salt concentrations. Since ds regions of RNA are resistant and ss regions sensitive to RNase A digestion, any secondary structure formation in RNA could be visualized as RNaseR fragments by electrophoresis on polyacrylamide/urea gels followed by autoradiography. Fig. 2 shows that, when labeled transcripts of 292 and 1339 nt in length were digested by RNase A in 0.01 x SSC, or 2 x SSC, some common RNaseR fragments were obtained. The sizes of these fragments did not change when S 1 nuclease digestion to remove the four nt from the 5’ end of the XbaI-linearized plasmid was omitted, prior to in vitro transcription (not shown). The sizes of the protected fragments were estimated to be within two clusters, the first between 23 and 16 nt, the second between 1 and 10 nt long (Fig. 2, A and B), by comparison with the ladder of 5’ -labeled oligo (5 1-mer) partially digested with snake venom phoshodiesterase (not shown). The fragments indicated by arrows in the first cluster were the predominant species, and their sizes were estimated to be about 23, 20, and 16 nt in length (Fig. 2, A and B). Other RNaseR fragments produced in the digest of 1339 nt transcript were not present in the digest of 292 nt transcript in the first cluster, suggesting that these could have been produced from the region not shared by the two transcripts. Interestingly, under the conditions of RNase A
187 promoter
Tl
JkU I’
TCTAGA AGATCT
T7 promoter
XLUI I'
T AGATC Pollk
Tl promoter
I
TCTAG AGATC
Ligation cloning
t A T
and
GGTAC C PCR fragment (1.35 kb)
Fig. 1. Strategy
for generating
transcripts
containing
the same polarity
and 3’-terminal
from DEN-2 RNA which was tailed with poly(A) using E. coli poly(A) polymerase The oligos 5’-GGACAAGTTGGTACCTATGG CAACACACC amplified
clones having the parent
(representing
to the 3’-terminal
sequence
DNA after a total of 25 cycles of denaturation,
and blunt-ended sequence
complementary
XbaI sites of pGEM7Zf’ the regenerated
of DEN-2
1.35-kb cDNA
clone having
Madison,
orientation
identical
3’-terminal
treating
WI), following (up
was digested routine
for amplification
with E. coli exonuclease
by Taq polymerase.
procedures
(Maniatis
lengths
III and S 1 nuclease
prepared
previously
toward
(Henikoff,
The PCR the KpnI
et al., 1982). Several independent
to about 120 nt from X&I site corresponding
with varying
library,
by PCR (Saiki et al., 1988).
with KpnI. It was then cloned between
in this region to those reported
sequences
RNA in vitro. A cDNA
see Irie et al., 1989), and 5’-AGAACCTGTTGATT-
were used as primers
were sequenced
DEN-2
as DEN-2
1973), was used for amplification
of DEN-2 genome;
genome
and DNA synthesis
data were found to be identical
parent clone at the SacI site, and sequentially verified by DNA sequence
annealing
vector (Promega,
XbaI site in the correct
RNA). The sequence
nt 9373-9392 of DEN-2
sequences
(Sippel,
to the 3’terminal
(Irie et al., 1989). Subclones
the 5’ end were prepared
by digesting
1984). The end points of all deletions
from the were
analysis.
digestion in 0.01 x SSC, only the 23 nt fragment was produced as the RNaseR fragment (Fig. 2A, lane 5). The low salt conditions (0.0 1 x S SC) are probably not favorable for the formation of a stable secondary structure in solution. Next, the question whether these RNaseR fragments resulted specifically from the transcripts containing the 3’-terminal sequences of DEN-2 RNA was addressed. A control transcript from the region of l-550 nt at the 5’ end of DEN-2-RNA was digested with RNase A under the same conditions of high ionic strength. Fig. 2B shows that the pattern of RNaseR fragments (lane 4) was quite different from that of 3’ -terminal transcript (lane 2).
(c) Mapping the location of RNaseR fragments by annealing with synthetic oligos, followed by RNase H digestion To investigate further whether these RNaseR fragments contained sequences from the 3 ‘-terminal region of DEN-2 RNA, four oligos were synthesized complementary to the first 96 nt of DEN-2 RNA. Subsequent to RNase A digestion of the two transcripts containing the 3’-terminal sequences, the RNaseR fragments were annealed to each of the four oligos, and the annealed mixtures were digested with RNase H. Since RNase H digests only the RNA of the RNA : DNA hybrid, this experiment could reveal the speci-
188
A
123456
B
1234
ticity and location of the RNaseR fragments. The results of RNase A, followed by RNase H digestion in Fig. 3 indicate that oligos Nos. 1 and 4 did not have any effect (lanes 3 and 6 in Fig. 3, A and B). These results indicate that the RNaseR fragments do not form stable hybrids with oligos Nos. 1 and 4 to become susceptible to RNase H digestion, or do not contain sequences complementary to these oligos. However, when oligo No. 2 was annealed, 23 and 20 nt long RNaseR fragments became sensitive to RNase H (Fig. 3, A and B, lanes 4) indicating that these fragments resulted from the region of transcript corresponding to nt 17-39 from the 3’ terminus of DEN-2 RNA (see also Fig. 4). The digestion of 23-nt fragment appeared incomplete (Fig. 3, A and B, lanes 4), and there was no change in this pattern even when a mixture of all four oligos were used for annealing (Fig. 3, A and B, lanes 7). However, in another experiment in which 400 ng of oligo No. 2 was used for hybridization, this 23-nt band was completely susceptible to RNase H digestion (data not shown). When oligo No. 3 was annealed, RNaseR fragments of 20 and 16 nt in length became sensitive to RNase H digestion, suggesting that these fragments contained sequences complementary to nt 44-65 from the 3’ terminus of DEN-2
aliquots
at
- 70°C.
(Panel A) An aliquot
(5 x lo“-1 x lo5 cpm) was incubated (50 PI), containing
of the labeled
at 37°C
transcript
for 60 min in a buffer
RNase A (3 pg) as follows. Lanes 1
2
3
4
292 nt RNA
+ _
f _
+ _
0.01 x ssc
+
+ _
_
+
+
f _
+
+
_
+
1339 nt RNA
2 x ssc RNase A
5
6
_
_
_
+
+
+ _ + +
RNase A was removed by proteinase K (3 pg) digestion in 1 y0 SDS at 37°C for 15 min, followed by phenol: CHCl, extraction and ethanol precipitation Fig. 2. Analysis transcripts.
of RNaseR
fragments
Two cDNA clones having 3’-terminal
292 bp as inserts were sequentially Labeled
transcripts
templates
generated
by the method
was carried
sequences
of 1339 and
with XbaI, and S 1 nuclease.
of high specific activity
were prepared
in vitro transcription
treated
from the 3’-terminal
from the linearized of Melton
plasmid
et al. (1984). The
out in 25-~1 reaction
volume containing
40 mM Tris . HCl pH 7.5/6 mM MgCl,/2 mM spermidine/0.05 mM each of GTP, CTP, UTP/O.S mM ATP/one unit of Inhibit-ACE (5 Prime-3 Prime, Inc., West Chester, serum
albumin/l0
PCi
PA)/1 mM m7G(5’)ppp(5’)G/100 each
of
[c+~‘P]GTP,
CTP,
gg bovine UTP
(400 Ci/
mmole)/two units of SP6 RNA polymerase. The reaction mixture was incubated at 40°C for 60 min. The DNase I (1 unit) was then added and incubated
for a further
15 min at 37°C.
The reaction
mixture
was ex-
tracted with phenol: CHCI, and the unincorporated nt were removed on Biospin-101 column (BioRad). The labeled transcript was extracted once with phenol
: CHCI, and precipitated
with ethanol
and stored
as small
in the presence
of E. coli tRNA
(3 pg) added
The labeled RNA was collected
by centrifugation,
was
finally
small
Tris
0.089 M borate/2
dissolved
in
a
mM EDTA
volume buffer
as a carrier.
and the pelleted RNA of
7M
urea/O.089
pH 8.3, containing
phenol blue dye, heated at 90°C for 3 min and electrophoresed
M
bromoon a 7 M
urea/O.089 M Tris .0.089M borate/2 mM EDTA (20%) polyacrylamide gel until the bromophenol dye reached 21 cm. The sizes of the fragments were determined
from a ladder
of 5’-labeled
oligos generated
from a
partial snake venom phosphodiesterase digestion (Jay et al., 1974) of a 51-mer deoxyoligo. The gel was covered with plastic wrap and exposed directly
to Kodak
XAR-5 film at 70’ C. The sizes of the three fragments
shown by arrows are estimated to be 16, 20 and 23 nt. (Panel B) The transcript containing 292 nt of 3’-terminal sequence of DEN-2 RNA, and a transcript
(control
RNA)
from
a cDNA
clone
corresponding
to
nt I-550 from the S’terminus of DEN-2 RNA (5 x IO4 cpm each) were either incubated in buffer with no RNase A, or digested with RNase A under the same conditions
as in panel A, lanes 3 and 6.
189 lanes 5), whereas
it appeared
to be almost complete
when
oligo No. 2 was used (Fig. 3, A and B, lanes 4). These results suggested a possibility that there are two RNaseR fragments of 20 nt in length, both of which were sensitive to oligo No. 2, but only one of which was sensitive to oligo No. 3 (see section d, and Fig. 4, A and B). (d) Location of the RNase A cleavage sites in the putative model for stem-loop structure at the 3’ terminus of DEN-2 RNA The data presented in Figs. 2 and 3 support the formation of a stable secondary structure in solution. Hahn et al. (1988) proposed a model for the stem-loop structure from the primary sequence data and the computer-derived
Fig. 3. Mapping fic synthetic containing sequences
of the RNase A-resistant
oligos,
followed
fragments
by annealing
by RNase H digestion.
1339 (panel A), and 292 (panel B) nt of 3’-terminal were digested
in length are shown by arrows and B. The digests
and hybridized
with 100 ng of synthetic (oligo
oligos (24-mers)
No. 1); 2, nt 25-48
(oligo No. 3); 4, nt 73-96
(oligo
nt is 1. The hybridization
containing
25 mM Tris
HCl
was done in 10 ~1 reaction
pH 7.5/300 mM
mM MgCI, at 90°C for 3 min, followed
KC&‘2 mM
formamide
was stopped
by the addition
(80x)-bromophenol indicate
the RNase
blue dye. The samples H products
mM
at 37°C for 60 min.
of stop
buffer
containing
were heated
90” C for 3 min and loaded onto a 7 M urea/2Oft% polyacrylamid~ arrowheads
volume
DTTjl
by quick chilling in ice
for 5 min. RNase H (1 unit) was added and incubated The reaction
complementary
No. 2); 3, nt 49-72
(oligo No. 4) of DEN-Z RNA (see Fig. 4). The
3’-terminal EDTA/6
of E. coli tRNA
in the presence
Each of the digests was divided into aliquots,
to
1, nt l-24
of panels A
: CHCI,, and the labeled
with phenol
with ethanol
(OS pg) added as a carrier.
as in
of 23,20 and 16 nt
on the left and right margins
were treated
was precipitated
DEN-2
with RNase A under the same conditions
Fig. 2, panel A, lanes 3 and 6. The RNaseR fragments
RNA
speci-
The transcripts
at
gel. The
of 12 and 13 nt in length.
Lanes I
2
3
4
5
6
7
+ -
+
-
+
-
+ +
-
+
Oligo 1
-
-
+ f
+ -
Oligo 2
-
-
-
+
+ _
Oligo 3 Oligo 4
-
-
-
-
+ _
RNase
H
+
+
RNA (Fig. 3, A and B, lanes 5). It should be noted again that the digestion of 20-nt fragment appeared incomplete when oligo No. 3 was used for annealing (Fig. 3, A and B,
energy calculations (Fig. 4A). The primary sequences are identical for DEN-2 RNA of New Guinea, Jamaica strain, and Puerto Rico-l 59/S 1 (PR- 159/S 1) vaccine strain, except at nt 42, which is a G for PR-159/Sl strain (Hahn et al., 1988; Deubel et al., 1988; Irie et al., 1989). We examined the putative RNase A cleavage sites based on this model (Fig. 4A). Since oligos Nos. 2 and 3 but not oligos Nos. 1 and 4 render the RNaseR fragments susceptible to RNase H, the RNase A cleavage sites are most likely located in the ss-loop region in the proposed model shown in Fig. 4A. Since RNase A cleaves next to pyrimidines, its putative cleavage sites in the secondary structure model can be tentatively assigned as follows. For example, the cteavages occurring at sites 1 and 3, and 1 and 2 would give rise to a 23, and a 20 nt fragment, respectively, which would be susceptible to oligo No. 2 t RNase H treatments (see also Fig. 4B). It appears that the sites 1 and 3 are the most readily cleaved, as the amount of 23 nt fragment is the predominant species obtained (see Fig. 3, A and B, lanes 3). Similarly, cleavages occurring at sites 5 and 6 would give rise to a 16 nt fragment which would become susceptible to oligo No. 3 + RNase H treatment (Fig. 3, A and B, lanes 5). The generation of 20 nt fragment(s) by RNase A cieavage which become susceptible to both oligos Nos. 2 or 3 in the presence of RNase H is difficult to explain from this putative model. One possibility is that cleavages at sites 4 and 8 in the secondary structure model shown in Fig. 4A would also result in a 20 nt fragment susceptible to oligo No. 3. A second possibility is that two 20 nt fr~ments could be generated from alternate secondary structures formed at the 3’-terminal region of DEN-2 transcript in solution. It is possible that the stem-loop structure proposed by Hahn et al. (1988) could be stabilized by interaction with proteins in vivo, but in the absence of stabilizing protein, the RNA could assume alternate stable structures in solution. It is noteworthy that smaller fragments of approx. 12-13 nt in length were also generated after RNase H digestion (as shown by arrowheads in Fig. 3, A and B, lanes 4). The origin of these bands could not be mapped at present.
190
B A A U-A G-C G-C A-U A-U U-A 24- G - C G U U-A
III GGC I ACA 48
3'..UGUCGUGGUAAGGUIIIIAAGA oligo 3’......
23 nt
I
I 30 50 I
3’. oligo
6
60 I 16
CGGACCUUACUACGAC
Fig. 4. A model for the putative proposed
secondary
minal sequences ary structure numbered Guinea, strain,
20 nt
GACC I II
A I A
CUGG
GA
G -96
sites in the previously
to form a stable second-
et al., 1988). The 3’ terminus
strain,
are identical
and Puerto
of DEN-2
for DEN-2
Rico-159/Sl
RNA is
RNA
of New
(PR-159/Sl)
vaccine
except at nt 42, which is a G for PR-159/S 1 strain (Hahn
putative AG
of DEN-2 RNA has a potential
Jamaica
et al., 1988; Irie et al., 1989). The arrows
sites of cleavage
fragments. - A
RNase A-sensitive
for DEN-2 RNA at 3’ end. (A) The 3’-ter-
as nt 1. Sequences
1988; Deubel
C-G
structure
(Hahn
nt
I I I I I I I I I I I I I I CTGGAATGATGCTGAGGAGACAGC
.
#3
CACG&XXUUACUACGACUC
G - C\ A-U 8 A C U U C-G A-U A C C - G -72 A-U G-C G-C U U U-A
I
UGUCGUGG20
U-A G-CI'
C A
TTTCB I I I I I I I I I I I I I I I
a
G-C C-G
3'-U
#2
20 nt
RNaseR
fragments
underlined. fragment,
of possible
hybrids
that
with synthetic
Base pairing
substrates
would
between
for RNase H digestion.
be formed
oligos
in the annealing
of
Nos. 2 and 3 are shown
as
the oligo No. 2 and 23-nt
and oligo No. 3 and 16-nt RNaseR
respectively)
the
by RNase A that would give rise to the RNaseR
(B) Structures
The RNA: DNA
et al.,
indicate
are shown by short vertical
fragment
RNaseR
(15 and 14 bp,
lines.
G
u u
5’
(e) Conclusions Our results support the existence of base-paired region and a general stem-loop structure proposed by Hahn et al. (1988) between nt 18-62 from the 3’ terminus of DEN-2 RNA. The region between nt 1-17, or 63-96 did not give any significant resistance to RNase A under our test conditions. This highly conserved secondary structure has been proposed to be important for replication of viral RNA (Hahn et al., 1987; Brinton et al., 1986; Takegami et al., 1986). Therefore, it will be interesting to test whether such a secondary structure is stabilized by binding of proteins in vivo. Our method to generate labeled transcripts having the same polarity and the 3’-terminal sequences of DEN-2 RNA might be useful for such studies.
(DAMD17-86-G-6038) to Dr. Fred Sampson, and by Biomedical Research Support Grant/Shared Instrumentation Facility. The views, opinions, and/or findings in this report are those of the authors only and should not be construed as an official Department of the Army position, policy, or decision unless so designated by other documentation.
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