From the Department of Biochemistry and Molecular
Biology, University of Kansas Medical Center,
Kansas City, Kansas 66160-7421 and ¶ Division of Viral
Products, Center for Biologics Evaluation and Research, Food and Drug
Administration, Rockville, Maryland 20852-1448
Received for publication, December 4, 2000, and in revised form, January 30, 2001
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ABSTRACT |
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Viral replicases of many positive-strand RNA
viruses are membrane-bound complexes of cellular and viral proteins
that include viral RNA-dependent RNA polymerase (RdRP).
The in vitro RdRP assay system that utilizes cytoplasmic
extracts from dengue viral-infected cells and exogenous RNA templates
was developed to understand the mechanism of viral replication in
vivo. Our results indicated that in vitro RNA
synthesis at the 3'-untranslated region (UTR) required the presence of
the 5'-terminal region (TR) and the two cyclization (CYC) motifs
suggesting a functional interaction between the TRs. In this study,
using a psoralen-UV cross-linking method and an in vitro
RdRP assay, we analyzed structural determinants for physical and
functional interactions. Exogenous RNA templates that were used in the
assays contained deletion mutations in the 5'-TR and substitution
mutations in the 3'-stem-loop structure including those that would
disrupt the predicted pseudoknot structure. Our results indicate that
there is physical interaction between the 5'-TR and 3'-UTR that
requires only the CYC motifs. RNA synthesis at the 3'-UTR, however,
requires long range interactions involving the 5'-UTR, CYC motifs, and
the 3'-stem-loop region that includes the tertiary pseudoknot structure.
Dengue viruses type 1-4 are members of the flavivirus family of
positive-strand RNA viruses. The diseases caused by dengue viruses
range from a simple form of dengue fever to a more complex form of
dengue hemorrhagic fever/shock syndrome, which exhibits considerable
morbidity and mortality, especially among children in the tropical and
subtropical regions of the world (for reviews see Refs. 1-3). It is
currently estimated that about 40% of the world's population living
in these areas is at risk for dengue viral diseases, and about 5% of
about one million dengue hemorrhagic/dengue shock syndrome cases are
fatal (1). Dengue virus type 2 (DEN2)1 is the most prevalent
serotype identified over a 10-year period in epidemiological studies.
DEN2 has a single-stranded RNA genome consisting of 10,723 nt (in New
Guinea-C strain) (4) which encodes a single polyprotein arranged in the
order, NH2-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-COOH. The RNA genome contains a type I cap at the 5'-end but lacks a poly(A)
tail at the 3'-end (5). The polyprotein precursor is processed in the
endoplasmic reticulum, where the action of the cellular signal
peptidase results in three structural proteins, the components of the
virion, the capsid (C), precursor membrane protein (prM), and the
envelope (E) protein (6-8). The remainder of the polyprotein codes for
at least seven nonstructural proteins that are generated by the
combination of both the cellular signal peptidase and the virally
encoded, two-component serine protease, NS2B/NS3.
The NS3 protein of flaviviruses has a serine protease catalytic triad
within the N-terminal region of 180 amino acid residues (9, 10) that
requires NS2B for protease activity (11-20). The conserved motifs
found in the NTP-binding proteins and the DEXH family of RNA helicases
were also identified within a region from amino acid residue 160 to the
C terminus of the NS3 protein (Refs. 10, 21, and 22 and for a review
see Ref. 23). The RNA helicase activity of NS3 is thought to be
required in flaviviral replication and is involved in unwinding of a
putative double-stranded RNA replicative intermediate (5, 24). In
addition, the West Nile viral and dengue viral NS3 proteins exhibit
5'-RNA triphosphatase activity
(25).2
The largest of the flaviviral proteins, NS5, contains conserved motifs
found in many viral RNA-dependent RNA polymerases (RdRP) (26, 27), suggesting a role for NS5 in viral RNA replication. In
addition to the RdRP motifs, NS5 has conserved motifs found in RNA
methyltransferases (28, 29) at the N-terminal region, although this
activity has not been experimentally demonstrated for any flaviviral
NS5. 5'-RNA triphosphatase, guanylyltransferase, and RNA
methyltransferase are the three enzyme activities required for
5'-capping (30, 31). Since NS3 and NS5 exist as a complex in dengue
virus-infected cells (32), they are likely to be the components of not
only the viral replicase but also the enzyme complex involved in
5'-capping.
Positive-strand viral RNA genomes contain recognition motifs for
specific initiation of minus- and plus-strand RNA synthesis. These
promoter elements are usually contained within the 5'- and 3'-terminal
regions (TR), especially in the conserved stem-loop (SL) structures
that are shown to be important for viral RNA replication in many
(+)-strand RNA viruses (33-45). In flavivirus RNAs, two conserved
sequences (CS1 and CS2) within the 3'-untranslated region (UTR) and
conserved SL structures and cyclization (5'-CYC and 3'-CYC) motifs
within the 3'- and 5'-TRs have been identified by sequence and
secondary structure prediction analyses (46-49). Recent studies
suggested a possible role for the 3'-SL structure in flavivirus viral
RNA replication (45). The 3'-UTR of West Nile virus is known to
interact with the translation elongation factor, eF-1 To study the mechanism of viral replication in molecular detail, we
reported the development of an in vitro assay system that utilized exogenous subgenomic RNA templates containing these conserved elements at the 5'- and 3'-TRs. A subgenomic RNA containing the 5'- and
3'-UTRs and the two CYC motifs was active as a template for RNA
synthesis in vitro. RNA synthesis at the 3'-end of the 3'-UTR in the subgenomic RNA gave rise to two labeled products. The
first was a double-stranded RNA that was resistant to RNase A under
high ionic strength and had a mobility identical in size to the
template RNA. The second product was twice the size of the input RNA
template. This product was converted to a template size RNA species
upon digestion with RNase A under high ionic strength. These results
suggested that the 2× product had a hairpin-like structure with an
RNase A-sensitive single-stranded region and that this product was
formed by a mechanism involving a snapback of the 3'-end of the
template and elongation by the viral RdRP. Moreover, when the 3'-UTR
RNA alone was used as a template, it did not form a 2× hairpin product
in the in vitro RdRP assay with infected C6/36 cell
extracts. However, addition of 5'-TR230nt RNA in
trans activated RNA synthesis from the 3'-UTR resulting in
the formation of this 2× product of the 3'-UTR in the RdRP assay.
Furthermore, it was shown that the CYC motifs are important for RNA
synthesis in vitro because if the motif in the template 3'-UTR RNA or the activator 5'-TR230nt RNA was mutated, RNA
synthesis was significantly reduced but was restored when the mutant
motifs were complementary to each other (52).
The results of our previous study supported the conclusion that a
functional interaction between the 5'-TR and 3'-UTR is required for RNA
synthesis at the 3'-end of the positive-stranded template. In this
study, we sought to determine whether there is any physical interaction
between the 5'-TR and the 3'-UTR. Moreover, the 5'-TR RNA that we had
used in our previous study contained the 5'-UTR as well as the 5'-CYC
motif. Therefore, it remained to be established whether the 5'-UTR was
required or only the 5'-CYC motif alone was sufficient for physical
interaction with the 3'-UTR as well as for RNA synthesis. It also
remained to be established whether the conserved 3'-SL structure of the
3'-UTR played any role in RNA synthesis in vitro. By using a
psoralen-UV cross-linking method, we now show that there is physical
interaction between the two ends of the dengue viral RNA that is
dependent on the two cyclization motifs. However, our results also
indicate that the interaction between the two ends alone is not
sufficient for RNA synthesis in vitro at the 3'-UTR and
that the 5'-UTR is also required for this process. For analyzing the
importance of 3'-SL structure in RNA synthesis, we used the
mutants that had previously been characterized for their replication
efficiencies in vivo in the context of full-length
infectious dengue viral RNAs (45). Some of these 3'- SL mutants were
constructed by nucleotide substitutions that either maintained or
disrupted base pairings within the dengue viral 3'-SL; others were
constructed by substituting different regions of the dengue viral 3'-SL
with corresponding regions of West Nile virus 3'-SL structures. We
engineered these 3'-SL mutations into the subgenomic RNAs. Analysis of
these mutant subgenomic RNA templates in the in vitro RdRP
assays revealed that the secondary structure as well as tertiary
pseudoknot structure (53) of the 3'-SL region were required for
efficient RNA synthesis. In general, the 3'-SL mutations that
interfered with the infectivity of the genomic RNAs in vivo
also had similar effects on the template efficiencies in the in
vitro RdRP assays. A few 3'-SL mutations that were defective in
viability in vivo or exhibited "host range" phenotype
(45) did not affect template efficiency in vitro suggesting that the defect in vivo could be at a step other than the
initial negative-strand RNA synthesis.
Preparation of DEN2-infected Cell Lysates
Aedes albopictus cells (C6/36 cells) were cultured as
described (54, 55). LLC-MK2 cells were cultured at 37 °C in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum. Cells were infected by DEN2 (New Guinea C-strain) for 3 days using ~5 plaque-forming units/cell. Cell lysates from infected
C6/36 or LLC-MK2 cells were prepared as described (52). The viral proteins, NS3 and NS5, were detected in the cell lysates by Western blotting using rabbit polyclonal antibodies, followed by a
chemiluminescence detection system (ECL system from Amersham Pharmacia
Biotech) (16, 56).
Plasmid Constructs
Plasmids Containing cDNAs Encoding Subgenomic RNAs
with Mutant CYC Motifs--
The preparation of pSY-2 plasmid
containing the wild type 5'-CYC and 3'-CYC motifs, TCAATATGC and
GCATATTGA, respectively, and the PCR fragment containing the
5'-TR230nt-mutCYC or the 3'-UTR373nt mutCYC
motif was as described (52). The PCR fragments were phosphorylated by
polynucleotide kinase and cloned into pSP64 vector (Promega, Madison,
WI) that was digested with SmaI and PvuII and
dephosphorylated at the blunt ends to prevent religation. The pSY-1
plasmid (52) was digested with two sets of double digestions; for
cloning the 5'-CYC motif, the plasmid was digested with
EcoRI + SalI, and for cloning the 3'-CYC motif,
the plasmid was digested with NcoI + PflM1. For
construction of subgenomic RNA containing the 5'-CYCmut motif, the
pSP64 intermediate plasmid was digested with EcoRI and
SalI, and the 200-nt fragment containing the 5'-CYC was
isolated; from the EcoRI + SalI digest of pSY1
plasmid, the Sal fragment (950 nt) containing the wild type
3'-CYC motif and the EcoRI-SalI vector fragment
(2.9 kb) were also isolated and ligated with the 200-nt fragment to
yield the pSY-1-5'-CYCmut plasmid. Similarly, the NcoI-PflM1
fragment (225 nt) containing the 3'-CYCmut motif was isolated from the
pSP64 intermediate plasmid clone and was ligated to the two fragments
derived from the NcoI + PflM1 digest of the pSY-1
plasmid, NcoI fragment (430 nt) and the vector fragment (3.3 kb). In both ligation reactions, the vector fragments of 2.9 and 3.3 kb
were dephosphorylated prior to ligation. The pSY-2 plasmid derivatives
(containing the cDNA coding for the 770-nt-long subgenomic RNA)
were obtained from the pSY-1 plasmid constructs by digesting the
respective plasmids with XmnI and Bsu36I,
followed by treatment with the Escherichia coli Klenow
fragment and ligation of the blunt ends. The subgenomic RNA construct
containing both 5'- and 3'-CYCmut motifs was constructed by digesting
the respective pSY-2 construct containing a single mutant motif with
AlwN1 and NcoI. The 1.2-kb fragment containing
the 5'-CYCmut motif was ligated to the vector fragment generated from
the pSY-2 plasmid containing the 3'-CYCmut motif to yield the
pSY-2-5'-3'-CYCmut plasmid.
Plasmid Constructs Containing cDNAs Encoding 3'-SL Mutant
Subgenomic RNAs--
In a previous study, several substitution
mutations in the 3'-SL structure of DEN2 were generated by
repositioning of the DEN2 sequences within the 3'-stem either without
disrupting base pairing (D2-SL(a) and -(b) mutants) or with disruption
of base pairing within the top half of the long stem (D2-SL(c) mutant) as well as by replacing different segments of 3'-SL with corresponding regions from West Nile virus (WN) 3'-SL. The effects of these mutations
in the context of DEN2-infectious clones were analyzed by their
replication efficiencies and infectivities in C6/36 and LLC-MK2 cells
(45). To analyze the effects of these 3'-SL mutations on the exogenous
RNA template efficiencies in the in vitro RdRP assays,
plasmids encoding the 3'-SL mutant 3'-UTR RNAs and subgenomic RNAs
containing the 3'-SL mutations were constructed by PCR. The 3'-SL
mutants of the 3'-UTR were first constructed by using the 5'-primer
consisting of the T7 promoter sequence (underlined) upstream of
the 3'-UTR start site,
5'-TAATACGACTCACTATAGAAGGCAAAACTAACATGAAAC-3', and a specific 3'-primer depending on the mutant. Four different 3'-end
primers used for PCR were as follows: D2 3', 5'-AGAACCTGTTGATTCAA-3'; WN 3', 5'-AGATCCTGT GTTCTC-3'; mutE 3', 5'-AGAACCCTGTGTTCTC-3'; and mutF 3', 5'-AGATCCGTTGATTCAA-3'. To amplify the wild type D2-SL, D2/WN-SL(mutA), -SL(mutD), D2-SL(a), -SL(b), -SL(c), and D2-SL(C71G) mutants, the 5'- primer and D2 3'-primer were used. For the
D2/WN-SL, D2/WN-SL(mutB), and -SL(mutC) mutants, the WN 3'-primer was
used. To amplify the D2/WN-SL(mutE) and -SL(mutF) mutants, the mutE 3'-
and mutF 3'-primers were used, respectively. PCR was for 30 cycles in
the following order: 94 °C for 1.5 min, 40 °C for 1.5 min, and
72 °C for 2 min. To increase the efficiency of in vitro
transcription, a second PCR was carried out with a different 5'-primer
containing the T7 promoter (underlined),
5'-TTACGAATTCGAGCTCGCCCTAATACGACTCACTATAG-3', and the same 3'-primer that was used for the first PCR of each mutant 3'-SL. The sequences upstream of the T7 promoter were found to
increase the efficiency of in vitro transcription probably by increasing the stability of T7 RNA polymerase interaction with the promoter.
The amplified PCR products were purified from agarose gel by Qiagen
gel extraction kit. Plasmids encoding the subgenomic RNAs containing
3'-SL mutations were constructed by cloning the PCR products as
follows. The 5'-end of PCR products was phosphorylated by T4
polynucleotide kinase and subcloned into the SmaI-digested and dephosphorylated pSP64 vector. Clones that contained the 3'-UTR inserts upstream of the SP6 promoter were selected (series pUPT7-X clones where "X" is a specific 3'-SL mutation). Each intermediate clone was cleaved with ApaI and XbaI, and the
~200-nt fragment was cloned into the
ApaI/XbaI-digested pSY-2 plasmid (52). The regions of the 3'-SL structure replacements in the subgenomic RNA
constructs were confirmed by DNA sequencing.
Plasmid Constructs Containing the Pseudoknot Mutation in the
3'-SL--
In order to disrupt the potential pseudoknot structure in
WN-3-SL structure (WN-3'SL(C73G)), a site-directed mutagenesis was performed by PCR. Two PCR products were generated with overlapping sequences using two sets of primers. PCR1 was obtained using the 5'-primer (T7-3'UTR), 5'-
TAATACGACTCACTATAGAAGGCAAAACTAACATGAAAC-3' that corresponds
to the T7 promoter (underlined) followed by the first 21 nt of 3'-UTR,
and 3'-primer (C73G(A)),
5'-TTGTGCAGAGCACAAGATCTCCTAG-3'. PCR2 was
produced using the 5'-primer (C73G(B)),
5'-CTAGGAGATCTTGTGCTCTGCACAA-3' and 3'-WN
primer, 5'-AGATCCTGTGTTCTC-3'. The underlined complementary nucleotides
in bold letters in C73G(A/B) primers represent the mutated pseudoknot
nucleotide C-G. The two products, PCR1 and PCR2, were mixed and used
for a third PCR in the presence of the primer set T7-3'-UTR and 3'-WN
primers. The final PCR product was purified and used as template for
the second PCR as described above. The PCR product was treated with T4
polynucleotide kinase. This PCR fragment was first subcloned into
pSP64-SmaI/PvuII cut plasmid. The intermediate
plasmid was digested by ApaI/XbaI and subcloned
into the ApaI/XbaI-digested pSY-2 plasmid to
yield the plasmid encoding the subgenomic RNA containing the pseudoknot mutation. The region of the mutation was confirmed by DNA sequencing.
Preparation of DNA Fragments for Use as Templates in the
in Vitro RNA Transcription by T7 RNA Polymerase
PCR Products Encoding the Subgenomic RNA770nt (WT and CYC
Mutants)--
To generate the subgenomic RNA770nt WT as
well as the CYC motif mutants without any additional 3'-terminal
nucleotides, PCR was performed using a plasmid construct encoding a
specific subgenomic RNA as a template and two primers as follows: the
5'-primer containing the EcoRI site,
5'-AGCTATGACCATGATTACGAATTC-3', that corresponds to the sequences
upstream of the T7 promoter, and the pSP64 vector sequence in each
plasmid and the 3'-primer, 5'-AGAACCTGTTGATTCAACAGCACC-3', that anneals
with the 3'-end region of the 3'-UTR. PCR was performed for 35 cycles
of each step as follows: 94 °C for 1.5 min, 66 °C for 1.5 min,
and 72 °C for 2 min.
PCR Product Encoding the Subgenomic RNA674nt (without
5'-UTR)--
The DNA template for preparation of the subgenomic
RNA674nt that lacks the 5'-UTR (96 nt) but is otherwise
identical to the subgenomic RNA770nt was prepared by PCR
using pSY-2 as the template and the two primers as follows: the
5'-primer,
5'-TAATACGACTCACTATAGATGAATAACCAACGAAAAAAGGCG-3', containing the T7 promoter (underlined), and the region beginning with the downstream capsid coding sequence. The 3'-primer contains the
complementary sequence of the 3'-end of the 3'-UTR. A second PCR was
performed using the 5'-primer described under "Plasmid Constructs"
and the same 3'-primer as mentioned above.
PCR Product Encoding the 5'-TR230nt RNA--
PCR was
carried out with the pSY-2 as a template and two primers: the
5'-primer, same as under "Preparation of DNA Fragments for Use as
Templates in the in Vitro RNA Transcription by T7 RNA Polymerase,"
and the 3'-primer, 5'-TGAGGTCCTCGTCCTG-3' (the underlined sequence is in the vicinity of Bsu36I site).
PCR Fragment for the 5'-TR180nt RNA Transcript--
PCR
was performed with the same 5'-primer and the 3'-primer,
5'-CTGTTGTACAGTCGACACGCG-3'.
PCR Fragment for the 5'-TR130nt RNA Transcript--
To
generate 5'-TR130nt RNA without the 5'-UTR, PCR was
performed as described for the production of the 5'-TR230nt
PCR fragment except that the 5'-primer used for making the subgenomic
RNA674nt (see under "PCR Product Encoding the Subgenomic
RNA674nt (without 5'-UTR)") was employed.
Production of PCR Products Containing Mutations in the 3'-SL
Region--
To generate the subgenomic RNA templates with various
mutations in the 3'-SL region, the corresponding plasmid construct
described above was used as a template for PCR using the same 5'-primer as used for the subgenomic RNA770nt described above along
with an appropriate 3'-primer. Again four different 3'-end primers (see
"Plasmid Constructs Containing cDNAs Encoding 3'-SL Mutant Subgenomic RNAs") were used for PCR. All PCR products were purified from an agarose gel using the Qiagen gel extraction kit.
Preparation of in Vitro RNA Transcripts
In vitro transcription was performed at 37 °C for
3 h in a 100-µl reaction volume as described by the manufacturer
(Promega, Madison, WI) (see also Ref. 52 except 40 units of T7 RNA
polymerase was used). The in vitro transcripts were
quantitated spectrophotometrically, and their integrity was verified by
electrophoresis on 3.5% polyacrylamide, 7 M urea gel
followed by staining with acridine orange.
In Vitro RdRP Assay with Exogenous RNA Transcripts
The RdRP assays using the DEN2-infected A. albopictus
(C6/36) or the LLC-MK2 cell extracts and the various exogenous RNA
templates were carried out as described (52). In the RdRP assays, where the various 5'-TR RNAs were added in trans to the reaction
mixtures containing the 3'-UTR454nt RNA template (3 µg),
the RNAs were added in equimolar amounts. The template efficiencies of
the various 3'-UTR454nt RNAs containing different 3'-SL
mutations were assayed by incubating 3'-UTR454nt RNAs (3 µg each) with 1.2 µg of the 5'-TR180nt RNA. The
reactions were terminated by phenol extraction (phenol:chloroform, 5:1,
pH 4.5), followed by chloroform extraction (chloroform:isoamyl alcohol,
24:1), and the unincorporated radiolabeled CTP was removed by passage
through a G-30 Spin Column (Bio-Rad). After ethanol precipitation, the
RdRP assay products were analyzed on a 1.5% agarose-formaldehyde gel
followed by autoradiography (52). In the RdRP assays where the
subgenomic RNA770nt containing the various 3'-SL mutations
was used as templates, 3 µg of each of the RNAs were used per assay.
Analysis of the Polarity of the RdRP Product by RNase A Digestion
followed by RNase H Mapping
The RdRP reaction was first carried out under standard
conditions at 30 °C for 1.5 h using the infected C6/36
extracts, and the RNA products were purified by
phenol:CHCl3 extraction and ethanol precipitation. RNA
pellet was then resuspended in 20 µl of H2O and purified
by using the G-30 Spin Column as described above. An aliquot of RNA was
digested with RNase A under high ionic strength conditions as described
(52) except in a 50-µl reaction mixture containing 300 ng of RNase A. The RNase A-digested sample was phenol:CHCl3-extracted and
precipitated with ethanol. This step, expected to remove the
single-stranded loop region of the hairpin, was found to be necessary
before annealing with the oligodeoxynucleotides followed by RNase H
digestion. Aliquots of RNA sample were resuspended in RNase H reaction
mixture (29 µl) containing 50 mM Tris-HCl, pH 8, 0.1 M NaCl, 2 mM MgCl2, 1 mM dithiothreitol, 6.6 µM
oligodeoxynucleotide of either positive- or negative-strand polarity,
and formamide (30% final concentration). The reaction mixture was
heated at 95 °C for 2 min and slowly cooled down to 65 °C by
turning off the heating source. It was then chilled in ice, and 3 units
of RNase H was added and incubated at 37 °C for 30 min. The reaction
was terminated by phenol:CHCl3 extraction followed by
ethanol precipitation. RNA products were analyzed by denaturing
formaldehyde/agarose gel electrophoresis (52). The sequences of
oligodeoxynucleotides were selected from a 19-nt region (between 231 and 250 nt from the 3'-end of 3'-UTR) for annealing. RNase H digestion
would generate two fragments, 530 and 230 nt in length.
RNA:RNA Interaction Analysis
Preparation of a Radiolabeled RNA Template--
The various
5'-TR RNAs were radiolabeled during in vitro transcription.
DNA template (1 µg) was mixed with 20 µl of 1× reaction buffer for
in vitro transcription as mentioned above, except containing 12.5 µM CTP, 30 µCi of [ Psoralen/UV Cross-linking--
To study the physical interaction
between the 5'-TR RNA and 3'-UTR RNA, the psoralen/UV cross-linking
method was used (57). Briefly, the radiolabeled 5'-TR RNA (0.3 × 104 cpm) and 5 µg of the unlabeled 3'-UTR RNA were
denatured at 90 °C for 2 min and chilled quickly on ice. Then, 25 µl of annealing buffer (50 mM sodium cacodylate, 300 mM KCl) was added, and the mixture was incubated at
70 °C for 20 min. The samples were supplemented with 5 mM MgCl2, and were incubated at 37 °C for an
additional 15 min. The samples were kept on ice until 2 µl of
AMT-psoralen (4'-aminomethyl-4',5',8'-trimethylpsoralen; 1 mg/ml in
RNase-free water; Sigma) was added, followed by further incubation on
ice for 15 min in the dark. The samples were transferred to a 96-well flat bottom plate on ice. The plate was exposed to UV irradiation (365 nm) at a distance of 2-3 cm for 30 min. The samples were transferred
back to microcentrifuge tubes, followed by phenol extraction and
ethanol precipitation. The precipitated and cross-linked RNA samples
were analyzed on a formaldehyde (2.2 M)-agarose (1.5%) gel. The gel was dried under vacuum and was subjected to autoradiography.
Secondary Structure Modeling of RNA
The predicted secondary structures of the mutant subgenomic RNAs
were analyzed by using the RNA structure algorithm developed by Zuker
et al. (58). Among the predicted structures for each RNA,
the most stable forms based on Analysis of the Polarity of the RdRP Product by RNase H
Digestion--
In a previous study (52), we reported the first
in vitro RdRP assay that utilizes exogenous subgenomic DEN2
RNA templates and membrane-bound cytoplasmic extracts from the
DEN2-infected mosquito (C6/36) cells. By using a subgenomic RNA
template that contained wild type 5'- and 3'-UTRs and CYC motifs in
this assay system, we found that two RNA products were formed as
follows: one had the same mobility and size as the input RNA template, and the second product had a faster mobility on a partially denaturing polyacrylamide, 7 M urea gel but a mobility that was
consistent with a size twice that of the template RNA on a completely
denaturing formaldehyde-agarose gel. The RNA product that was twice the
size of the input RNA (2×) was converted to a product with the same mobility as the template RNA (1×) upon digestion with RNase A. These
results suggested that 2× product was a double-stranded hairpin RNA
formed via a snapback mechanism by the 3'-end elongation of the input
RNA template. The 1× RNA species was also resistant to RNase A
suggesting that this double-stranded RNA product was formed either by
de novo initiation of RNA synthesis by RdRP at the 3'-end of
the template RNA or it was formed by a cleavage of the 2× hairpin
product by a nuclease present in the cytoplasmic extract at the
single-stranded loop region. Since the predominant RNA product that was
formed in the in vitro RdRP assay was the 2× hairpin
product, the structural requirements of the template for the formation
of the 2× product were analyzed in this study.
First, the polarity of the radiolabeled RNA formed in the RdRP reaction
carried out using the subgenomic RNA770nt template of
(+)-polarity was determined by RNase H mapping. The
oligodeoxynucleotides were designed to anneal with the region 231-250
nt from the 3'-end of the genomic RNA so that RNase H digestion would
be expected to yield fragments of 520 and 230 nt in length. However,
denaturation of the covalently linked double-stranded RNA hairpin
followed by annealing with an oligodeoxynucleotide of either polarity
followed by RNase H digestion did not produce any discrete RNA products suggesting that complete denaturation of the highly structured RNA was
not attained under these conditions. Therefore, the 2× RNA product
formed in the standard RdRP reaction was first digested with RNase A
under high salt conditions in order to remove the single-stranded loop
region that connects the unlabeled input RNA with the newly synthesized
complementary RNA strand. RNase A treatment would produce a
double-strand RNA not covalently linked which would facilitate
denaturation and annealing with the oligodeoxynucleotides (Fig.
1, panel A). Inclusion of 30%
formamide in the annealing reaction was also found to be necessary for
optimum annealing of RNA-DNA hybrids and that under these conditions
RNase H activity was not affected.
The RdRP product (Fig. 1, panel B, lane 1) was digested by
RNase A under high salt conditions. Equal aliquots were digested with
RNase H after annealing with oligodeoxynucleotide of either (+)- or
( 5'-TR RNA and 3'-UTR RNA Physically Interact via the Cyclization
Motif--
In a previous study, we showed that in vitro RNA
synthesis by the cytoplasmic extract from dengue virus-infected cells
that required specifically the 5'- and 3'-TRs of the dengue virus RNA be present in the same template RNA molecule (in cis) or
that the 5'-TR230nt RNA be added in trans to the
3'-UTR373nt RNA. However, the 3'-UTR373nt RNA
alone was not active to form the 2× RNA product. The results of that
study suggested that there is a functional interaction between the 5'-
and 3'-TRs that conferred to the inactive 3'-UTR373nt RNA
the ability to serve as an efficient template for RNA synthesis
resulting in the formation of the 2× product in vitro.
Similar results were also obtained when the full-length 3'-UTR454nt and the 5'-TR230nt RNA were both
present in the RdRP assay (data not shown).
In this study, we investigated whether there is any physical
interaction between the 5'-TR and 3'-UTR RNA by using the psoralen-UV cross-linking method. Psoralen intercalates between base pairs, and
upon UV treatment it cross-links opposite strands especially at
pyrimidine bases. The psoralen-UV method has been used extensively to
study RNA-RNA and RNA-protein interactions (57, 59-64). To determine
whether there is a physical interaction between the two ends of DEN2
RNA, the 5'-TR230nt RNA was radiolabeled with 32P in the in vitro transcription reaction and
purified from the polyacrylamide 7 M urea gel. It was then
mixed with unlabeled 3'-TR454nt RNA and treated with
psoralen, followed by UV irradiation. The cross-linked products were
analyzed by formaldehyde-agarose gel electrophoresis and
autoradiography. The results shown in Fig.
2, panel A indicate that the
formation of cross-linked products was observed only when the
5'-TR230nt and the 3'-UTR454nt RNAs containing
the wild type or mutant CYC motifs, which were complementary to each
other, were present in the psoralen/UV cross-linking reaction (lanes 3 and 10). The cross-linked products were
significantly reduced under conditions in which one of the CYC motifs
was mutated which would disrupt complementarity between the two CYC
motifs (lanes 5 and 8). No cross-linked products
were formed in control reactions omitting the psoralen (lanes 2, 4, and 9) or reactions containing only the
5'-TR230nt RNA but treated with psoralen/UV (lanes
1 and 6). In the reaction mixtures containing only the labeled 5'-TR RNAs, treatment with psoralen/UV resulted in loss of
radioactivity probably due to degradation, and the loss was greater for
5'-TR230nt RNA than for 5'-TR130nt RNA
(lanes 1 and 6, panel A
versus B). These results taken together indicated
that there was a physical interaction between the 5'-TR and 3'-UTRs in
the viral subgenomic RNA and that the CYC motifs played an important
role in facilitating this interaction. Moreover, this interaction still
occurred even if CYC motifs were mutated as long as the complementarity
between them was maintained.
Next, we investigated whether a shorter RNA from the 5'-TR containing
the CYC motifs but without the 5'-UTR could still physically interact
with the 3'-UTR454nt RNA. The results of the psoralen/UV cross-linking experiment shown in Fig. 2, panel B
(lane 3) indicated that the 5'-TR130nt RNA
containing the 5'-CYC motif alone was sufficient for interaction with
the 3'-UTR454nt RNA. Again, substitution mutations in the
5'-CYC motif did not abolish interaction as long as the complementary
mutations were introduced into the 3'-CYC motif (lane 10).
In control experiments, it was found that omission of either
3'-UTR454nt RNA (lanes 1 and 6) or
psoralen/UV treatment (lanes 2, 4, 7, and 9) did
not give any cross-linked products. Substitution mutations of either of
the CYC motifs in the two RNAs significantly reduced the ability to
form cross-linked products although they did not abolish cross-linking
completely (see lanes 5 and 8). In both
experiments shown in Fig. 2, panels A and B, there were two major species of cross-linked products formed suggesting that psoralen cross-linking occurred at more than one site. The results
presented thus far indicated that the interaction between the 5'- and
3'-TR was facilitated by the CYC motifs, and that the 130-nt region
containing the CYC motif alone in the absence of the 5'-UTR was
sufficient for physical interaction with the 3'-UTR454nt
RNA. The 5'-UTR96nt RNA alone, lacking the 5'-CYC motif,
did not interact with the 3'-UTR454nt RNA to form the
psoralen cross-linked product (data not shown).
5'-UTR Is Crucial for RdRP to Initiate RNA Synthesis--
Next, we
sought to determine whether this 5'-TR130nt RNA can
transactivate the 3'-UTR454nt RNA in the in
vitro RdRP assay. To address this question, in addition to the
5'-TR230nt RNA, we used three different RNA templates in
the in vitro assays as follows: the 5'-TR180nt
RNA containing 5'-UTR and the CYC motif but shorter at the 3'-end, the
5'-TR130nt RNA lacking the 5'-UTR region, and the
5'-UTR96nt lacking the CYC motif. The
3'-UTR454nt RNA alone was inactive in the formation of the
2× product in the RdRP assay as reported earlier (Fig.
3, lane 1). However, a labeled
RNA species of the same size as the input template RNA (1×) was formed
(lane 1). The 3'-UTR454nt RNA alone in the
absence of 5'-TR was radiolabeled with varied efficiency in the RdRP
assays with different infected C6/36 extracts or with uninfected cell
lysates (data not shown; see also Ref. 52). The formation of this
labeled (1×) RNA species is likely due to addition of a few
nucleotides to the 3'-end of the template strand by the host terminal
nucleotidyltransferase (a membrane-bound enzyme), and this 1× product
was sensitive to RNase A digestion under high ionic strength
conditions; however, the 1× RNA product formed from the 3'-UTR RNA
template in the presence of 5'-TR230nt RNA or the
subgenomic RNA template was resistant to RNase A (52). Furthermore, the
results shown in Fig. 3 indicate that the 5'-TR230nt and
the 5'-TR180nt RNA could both transactivate the
3'-UTR454nt RNA (lanes 7 and 9), but
the 5'-TR130nt and the 5'-UTR96nt RNAs were
essentially inactive in conferring RNA synthesis at the 3'-end of the
3'-UTR454nt RNA to form the 2× product (lanes 3 and 5). In control RdRP assays containing only 5'-TR RNA
species as templates, the labeled 1 and 2× products of the 5'-TR RNAs
(lanes 2, 4, 6, and 8) were also formed, and the
former was more predominant than the latter species. This observation
was consistent with our previous study (52) which also reported that
blocking the 3'-end of the 5'-TR230nt RNA abolished its
template activity but not its ability to transactivate the
3'-UTR454nt RNA template for 3'-end elongation. However,
the presence of 3'-UTR454nt and 5'-TR RNAs together in the
RdRP assay resulted in significant suppression of RNA synthesis at the
5'-TR RNA templates for reasons unknown (lanes 2 and
4 versus 3 and 5). Our
results shown in Fig. 3, taken together, indicated that although the
5'-TR RNA130nt containing the CYC motif alone was sufficient for physical interaction with the 3'-UTR454nt
(Fig. 2, panel B), it was not sufficient for activation of
RNA synthesis at the 3'-end of 3'-UTR454nt RNA template,
and both the 5'-UTR and the 5'-CYC motifs are required.
In the previous study, we demonstrated a functional interaction between
the 5'-TR and the 3'-UTR for RNA synthesis at the 3'-end when both
regions are in the same RNA template such as the subgenomic
RNA770nt that includes the CYC motifs. To confirm further
the role of 5'-UTR and CYC motifs, four mutant subgenomic RNA770nt templates were used in the RdRP assays as follows:
subgenomic RNA674nt with a deletion of the 5'-UTR, two
subgenomic RNAs770nt containing the same substitution
mutations as described before within either the 5'-CYC or the 3'-CYC
motif which would disrupt base pairings, and the subgenomic
RNA770nt in which both CYC motifs were mutated such that
the complementarity was restored (Fig. 4).
The template efficiencies of these mutant subgenomic RNAs were analyzed
using the in vitro RdRP assay. The results indicated that
the subgenomic RNA674nt (without the 5'-UTR) had
significantly reduced template activity for RNA synthesis (Fig. 4,
panel A). These results support the conclusion reached from
the transactivation assays that the 5'-UTR is necessary for the viral
RdRP to initiate RNA synthesis at the 3'-end of the RNA template (Fig.
3).
Next, the contribution of the two CYC motifs for RNA synthesis at the
3'-end of subgenomic RNA templates was examined. The subgenomic
RNA770nt, containing either of the CYC motifs mutated or
both mutated, were used as templates in RdRP assays. The results indicate that the double mutant subgenomic RNA containing complementary mutant CYC motifs was an equally efficient template as the wild type
RNA for RNA synthesis (Fig. 4, panel B, lanes 1 and
4). Furthermore, the subgenomic RNA with the mutated 5'-CYC
motif showed a dramatic reduction in template efficiency compared with
the wild type (lane 2). These results are consistent with
the conclusion reached in the previous study based on the
transactivation assays (52). However, the subgenomic RNA with the
3'-CYC mutation showed a reduced template activity (about 60% of the
wild type) (lane 3 versus lane 1). But
this result was contrary to the results obtained in the transactivation
assays in that the 5'-TR with wild type CYC motif was essentially
inactive in promoting RNA synthesis from the 3'-UTR RNA containing the
mutant 3'-CYC motif (52). This experiment was repeated four times, and
similar results were obtained. One possible explanation for this
difference is that the interaction between the 5'-TR containing the
wild type 5'-CYC motif with the 3'-UTR RNA containing the mutant 3'-CYC
motif is intramolecular and may be more favorable as the two ends are
in the same molecule compared with the scenario in which the
interaction between the two terminal regions of RNAs is intermolecular
as is the case with 5'-TR230nt/wtCYC and the
3'-UTR373nt/mutCYC used in the transactivation assays.
Importance of 3'-Stem-loop Structure in the 3'-UTR for RNA
Synthesis in Vitro by RdRP--
The 3'-most 90-100 nucleotides of the
flavivirus RNA genomes have been predicted to form a stable stem-loop
(SL) structure (Fig. 5, panels
A and B) (40, 45, 46, 48, 53, 65-68). Although the
primary sequences of the region are less homologous, the 3'-SL
structures are highly conserved throughout the flavivirus family. This
implies that the conserved 3'-SL structure might have functional
importance for viral replication.
Recently, Zeng et al. (45) reported the functional role of
the 3'-SL structure in DEN2 viral replication and infectivity. They
constructed several substitution mutations within the DEN2 3'-SL either
by rearrangement of base pairings that would disrupt or maintain
secondary structure or by replacing all or part of the 3'-SL of the
DEN2 genome with the corresponding regions of the West Nile 3'-SL,
without disrupting the overall secondary structure. These substitution
mutations were cloned into the full-length DEN2 infectious clone
(diagrammatically shown in Fig. 5, panel C). In that study,
monkey kidney (LLC-MK2) cells were electroporated with the genome
length RNA transcript from each mutant clone, and virus production and
the spread of infection was followed over time by immunofluorescence
assay (45). The ability of the viable mutant viruses to replicate in
the mosquito (C6/36) cells and monkey kidney cells (LLC-MK2) was also
examined. From the analysis of infectivity of these RNA transcripts, it
was concluded that the stem structure of the top half and the
nucleotide sequence of the 11 base-paired region in the uppermost
portion of the bottom half of the 3'-SL are important for efficient
viral replication (45). Furthermore, a point mutation that would
disrupt the predicted pseudoknot structure within the 3'-SL (53) in the
context of infectious clone was lethal for viral replication and
infectivity in
vivo3 (C71G; see Fig. 5,
panel A). Four of the first 6 nt of the WN small loop region
also had the potential to form a pseudoknot structure with nucleotides
71-74 in the long stem (53), suggesting an interesting possibility
that disruption of this tertiary structure interaction could account
for the loss of replication potential of the chimeric mutants in this
part of the viral genome.
To study the effects of these substitutions within the DEN2 3'-SL
structures on their template efficiencies in the in vitro RdRP assay system, we subcloned these 3'-SL mutations into the plasmid
coding for the subgenomic RNA. RNAs from these constructs were produced
by in vitro transcription and were used as templates in the
in vitro RdRP assays using DEN2-infected mosquito (C6/36) as
well as monkey kidney (LLC-MK2) cell lysates. The rationale for using
these two cell lysates was to examine whether there are any differences
in host restriction for RNA synthesis in vitro as was found
previously with one mutant (mutF) in the in vivo infectivity
assays (45).
As shown in Fig. 6, the 3'-SL mutant
subgenomic RNAs showed template efficiency at different levels.
Subgenomic RNAs with the D2/WN-SL(mutA), D2-SL(a), D2-SL(b), D2/WN-SL,
D2/WN-SL(mutB), and D2/WN-SL(mutF) mutations exhibited template
efficiency either close to or only slightly reduced compared with the
wild type template in the in vitro RdRP assays that utilized
either C6/36 or LLC-MK2 cell extracts for the source of viral replicase
(Fig. 6, panels B and C, lanes
1, 3, 4, 7, 8, and 11 versus 12).
Of these mutants, mutagenesis of the top half of the DEN2-3'-SL either completely or partially, as in D2/WN-SL(mutA), D2-SL(a), and D2-SL(b) (see Fig. 5, panel C), had only modest effects on
virus growth compared with the wild type DEN2 genome (45). The in
vitro template efficiencies also support this finding (Fig. 6,
panels B and C, lanes 1, 3 and
4). In contrast, D2/WN-SL and D2/WN-SL (mutB) behaved like
the wild type in the in vitro assays, whereas they were
severely affected for growth in vivo (45). Moreover, the
mutants D2/WN-SL(mutD) and D2/WN-SL(mutF) exhibited similar template
activities with the C6/36 extract which were closer to that of the wild
type template (Fig. 6, panel B, lanes 2 and 11 versus lane 12). On the other hand, with the
LLC-MK2 cell extract, the -(mutD) was less active than -(mutF), but
both mutants were less active than the wild type template in the
in vitro assay (Fig. 6, panel C, lane 2 versus 11; compare lanes 2 and
11 with lane 12). The -(mutF) exhibited a
"host-range" phenotype in vivo in that it was severely
restricted for replication in C6/36 cells but grew like the wild type
in LLC-MK2 cells (45). In contrast, the results of the in
vitro RdRP assays showed little difference in template
efficiencies with the extracts from LLC-MK2- or C6/36-infected
cells.
Template efficiency of the subgenomic RNAs containing D2/WN-SL(mutC)
and -(mutE) mutations exhibited a reduced template efficiency for RNA
synthesis using either the LLC-MK2- or the C6/36-infected cell extract
(Fig. 6, panels B and C, lanes 9 and
10). Consistent with these results, these mutants exhibited
either no DEN-positive antigens (not viable) or only 10% of the cells
were positive for viral antigen (barely viable) as visualized by
immunofluorescence of DEN2-transfected LLC-MK2 cells (45). In -(mutC)
and -(mutE) mutants, the mutations were introduced at the bottom half
of the 3'-stem (Fig. 5, panel C). In addition, the mutant
subgenomic RNAs containing the D2-SL(c) and D2-SL(C71G) mutations lost
their template activities dramatically when either of the two infected cell extracts was used in the in vitro assays (Fig. 6,
panels B and C, lanes 5 and
6) which are consistent with their lethal phenotypes
in vivo. In the D2-SL(c) mutant, the lethal phenotype was
attributed to the disruption of the top half of the 3'-SL (45). The
loss of template activity of the D2-SL(C71G) mutant is particularly
interesting because this subgenomic RNA contains a mutation that would
disrupt the predicted pseudoknot structure based on the comparison to
the homologous structure in the West Nile viral RNA 3'-SL (53) (see
Fig. 5, panels A and B). When the
mutant genomic RNA was electroporated into LLC-MK2 cells, the mutation
reverted to wild type, and only the latter grew out of the transfected
cells suggesting that this pseudoknot mutant was lethal in
vivo.4 In order to prove
that the tertiary interaction involving a pseudoknot structure in the
3'-SL is important for template efficiency for RNA synthesis in
vitro, we engineered the C73G mutation to disrupt the pseudoknot
base pairing in the D2/WN-SL subgenomic RNA mutant in which the entire
D2-SL was replaced with WN-SL. These subgenomic RNA templates
containing the wild type or mutant pseudoknot base pairs within
3'-D2-SL or 3'-WN-SL were analyzed for RNA synthesis in
vitro. The results from Fig. 7
indicate that the templates harboring a single base pair change that
would disrupt the pseudoknot structure within either of the two 3'-SLs
significantly affected the template efficiency for RNA synthesis
in vitro. Based on these results, the strong template
activity of D2/WN-SL(mutB) with either of the two extracts could be
explained by the possibility that the pseudoknot structure of the West
Nile 3'-SL is not affected in this mutant. These results presented in
this study, taken together, support the conclusion that RNA synthesis
in vitro at the 3'-end of the 3'-UTR is not only dependent
on the conserved CYC motifs and the 5'-UTR but also, more importantly,
on the determinants of the conserved 3'-SL structures of the subgenomic
RNA template that are influenced by the conserved elements at the 5'-TR
of the viral genome.
Functional Interaction between the 5'-TR and 3'-UTR Required for
RNA Synthesis in Vitro--
In this study, we have further established
that the newly synthesized RNA from the subgenomic RNA template is of
(
A number of studies have employed computer algorithms that are capable
of analyzing predictive secondary structures as a guide or as a prelude
for chemical and/or enzymatic probing to understand RNA-RNA
interactions. Therefore, we sought to compare the predictive secondary
structures of the subgenomic RNAs having wild type and mutant CYC
motifs using the software described by Zuker et al. (58)
(version 3.0). Among several structures that were produced from each
RNA molecule, the most stable structures were selected for comparison
and correlation with its in vitro template efficiency (Fig.
8). As shown in Fig. 8, the subgenomic
RNAs containing the CYCwt motifs and the complementary,
double-mutant CYC (5'3'CYCmut) motifs seem to maintain a similar
predicted secondary structure (panels A and D).
Interestingly, the subgenomic RNA/3'CYCmut also seems to maintain the
same overall predicted secondary structures of the above two subgenomic
RNAs (Fig. 8, panel C), although there is no predicted base
pairing between the wild type 5'-CYC and the mutated 3'-CYC motifs. On
the other hand, the predicted structures of the subgenomic RNA/5'CYCmut
and the subgenomic RNA674nt (without 5'-UTR) are quite
different from that of the wild type (panels E and
B versus panel A). Thus the predictive
analyses of RNA secondary structure suggest that mutations in the
conserved 3'-CYC motif are tolerated, and the template efficiency in
RNA synthesis is not affected because the overall RNA structure is
maintained. Moreover, the overall conformation of RNA influenced by the
interaction of the 5'- and 3'-terminal SL structures seems more
important for RNA synthesis than the CYC motifs which seem only to
facilitate this interaction. Further work using chemical and/or
enzymatic RNA structure probing methods and by site-directed
mutagenesis is required to understand the RNA structure-template
activities of these subgenomic RNA molecules using our in
vitro RdRP assay.
The important role of the 3'-SL structure of the viral RNA genome in
dengue viral replication in vivo was analyzed in an earlier study by Zeng et al. (45). The chimeric virus D2/WN-SL,
which had the 96-nt sequence of the WN 3'-SL as a substitute for the 93-nt DEN2 3'SL, showed a phenotype severely defective but not lethal
in monkey kidney LLC-MK2 cells (defined as "sublethal" in Ref. 45).
It was suggested that the possible mechanisms involving RNA/RNA or
protein/RNA interaction might have been affected which could have
slowed down the replication of the transfected genome compared with
that of the wild type (45).
There was a good correlation between the template efficiency of these
3'-SL mutants in the in vitro assays and the in
vivo viabilities in the context of full-length infectious clones
(45) for lethal mutations, D2-SL(c), D2/WN-SL(mutC), and D2-SL(C71G), as well as for the sublethal mutant, D2/WN-SL(mutE). Moreover, this
correlation could also be extended for the mutants D2-SL(a) and
D2-SL(b) and the D2/WN-SL(mutA) in which substitutions were made within
the upper half of the 3'-SL (Fig. 5, panel C). The good
accord between the in vivo and the in vitro
results with regard to the latter three mutants support the previous
conclusion that the secondary structure rather than DEN2-specific
nucleotide sequence of the top half of the 3'-SL is a major determinant
for viral replication.
Notable exceptions to this good correlation are the two mutants,
D2/WN-SL(mutB) and the D2/WN-SL (mutB and WN in Fig. 6, panel A), which exhibited significant template activity in
vitro, but were lethal or sublethal, respectively, in
vivo (45). It was previously reported that the 3'-SL structures of
the DEN and WN viral genome contain a pseudoknot structure between the
"G" in the loop of the small SL and the "C" in the bulge of the
bottom half of the 3'-SL although the length of the base-paired regions involved in the 3'-SL structures of the DEN2 and WN RNA genomes are
different (53) (illustrated in Fig. 5, panels A and
B). Our study clearly indicates that the template efficiency
of the pseudoknot mutant D2-SL(C71G) with a single nucleotide
substitution was significantly reduced, suggesting that the pseudoknot
structure within the 3'-SL could play an important role in RNA
synthesis in vitro. This result also suggests that perhaps
the near-wild type template activities of the mutants, D2/WN-SL(mutB)
and the D2/WN-SL, might be due to the possibility that the potential
pseudoknot structure of the West Nile 3'-SL could be preserved in these
mutants. This hypothesis was confirmed by analysis of the C73G mutation in the D2/WN-SL RNA, which would be expected to disrupt this pseudoknot structure, and the resultant mutant subgenomic RNA had significantly reduced template efficiency for RNA synthesis in vitro. The
same explanation could be considered for the mutant subgenomic RNA with
D2/WN-SL(mutF); the regions containing the DEN2 sequences are
presumably involved in the pseudoknot structure although the bottommost
half of the 3'-SL is from the West Nile RNA. Thus, this -(mutF)
subgenomic RNA template had significant retention of template
efficiency in vitro. There is good correlation between the
template efficiency in vitro and the in vivo
viability of the mutant D2/WN-SL(mutD) which replicated well in both
LLC-MK2 and C6/36 cells (45). However, the template efficiency of this -(mutD) mutant RNA was greater in the extracts from the infected C6/36
cells compared with the LLC-MK2 cells. Although our in vitro results are consistent with the notion that tertiary pseudoknot interaction might play an important role for RNA synthesis, they do not
explain why mutants, D2/WN-SL(mutB), D2/WN-SL, and D2/WN-SL(mutF), that
are active as templates in vitro are defective in
vivo. One possible explanation for the disparity is that other
steps in the viral life cycle that contribute to the overall
viability such as defects in re-initiation of positive-strand RNA
synthesis from the double-stranded RNA intermediate or in viral
assembly might be affected in vivo.
All of the 3'-SL mutant RNAs were also able to interact physically with
the 5'-TR180nt RNA after psoralen/UV treatment (data not
shown), suggesting that substitutions within the 3'-SL do not affect
the ability of the 5'-TR RNA to interact with the 3'-UTR. These results
indicated that although both 5'-TR and 3'-UTR RNAs could interact with
each other, possibly mediated by the wild type CYC motifs that are
upstream of the 3'-SL, the mutations at the 3'-SL domains of the 3'-UTR
RNAs per se must have contributed to their differences in
template efficiency for RNA synthesis in vitro. These
results also emphasize that the overall conformation of the 3'-SL is
more important for RNA synthesis than the CYC motifs alone.
Possible Role of Cross-talk between the 5'- and 3'-Terminal Regions
of the Viral RNA Genome in RNA Synthesis--
There is increasing
evidence from studies of other positive single-strand RNA viruses that
the 3'-end of the viral genome has to maintain a certain secondary and
tertiary structure for RNA synthesis (69-74). The 3'-UTR of brome
mosaic virus (BMV) RNA-3 is predicted to have five pseudoknots at the
upstream end of the 3'-end tRNA-like structure (70). The deletion
mutants of RNA-3 within these regions replicated poorly, yielding no
detectable RNA-3 or RNA-4 progeny. This result suggested that the
pseudoknot regions of the RNA-3 at the 3'-UTR contributed significantly
to the overall replication of the BMV genome. Deiman et al.
(72) also reported that the disruption of the stable pseudoknot
structure at the 3'-end of the Turnip yellow mosaic virus genome gave
rise to a drop in transcription efficiency to about 50%, indicating that the stable pseudoknot structure is crucial for initiation of
negative-strand RNA synthesis.
Our results suggest that the cross-talk between the 5'-TR and
3'-UTR of the viral genome might define a precise structure at the
3'-end involving tertiary pseudoknot interaction such that the
initiation of negative-strand RNA synthesis could be carried out by the
viral replicase complex. In fact, possible cross-talk between specific
regions of the viral genome of RNA viruses has been suggested to
participate in the viral replication of Q
Proteins interacting with the flavivirus terminal regions of the RNA
genome remain to be studied. The results of this study indicate that
using our in vitro RdRP assay and the wild type and mutant
subgenomic RNA templates, it is possible to study the higher order RNA
structural interactions and identify the viral and cellular factors
that contribute to the stability and functional long range interactions
between the ends of the viral genome.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(50) although
the biological significance of this interaction is not clear. In
addition, the 3'-UTR of the Japanese encephalitis viral RNA was found
to interact with NS3 and NS5, the putative components of the viral
replicase (51) that exist as a heteromeric complex in infected cells
(32, 51).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]CTP (800 Ci/mmol), and 10 units of T7 RNA polymerase. The reaction mixture was
incubated at 37 °C for 1 h. Then, DNase I (1 unit) was added,
and the reaction was incubated at 37 °C for 15 min to digest the DNA
template. Labeled RNA was purified by phenol:chloroform extraction and
by using a Micro-spin P-30 column (Bio-Rad) followed by ethanol
precipitation. The radiolabeled RNA transcript was run on a
polyacrylamide (4%), 7 M urea gel and eluted from the gel
as described (52). The radioactivity of the eluted RNA was measured by
a scintillation counter.
G values were chosen for comparisons.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Analysis of the polarity of the RdRP products
by RNase H digestion. Panel A, the overall scheme of
RNase H analysis. Polarity of the oligodeoxynucleotides are depicted as
(+)/( ) within boxes. A solid line indicates the
unlabeled input RNA of (+)-polarity. A dotted line indicates
the radiolabeled RNA strand of (
)-polarity synthesized in
vitro by RdRP. Panel B, the RdRP product from
(+)-polarity of RNA template was indicated in lane 1. Lane
2, the RdRP product after RNase A digestion under high ionic
strength, followed by RNase H digestion in the absence of an
oligodeoxynucleotide (oligo). Lane 3, the RdRP
product after RNase A digestion followed by annealing with
(+)-oligodeoxynucleotide and RNase H digestion. Lane 4, the
RdRP product after RNase A digestion followed by annealing with
(
)-oligodeoxynucleotide and RNase H digestion. Prior digestion of the
2× hairpin product with RNase A to remove the single-stranded loop
region and addition of formamide in the annealing reaction (as
described under "Experimental Procedures") were necessary for
digestion of the RNA:DNA hybrid by RNase H.
)-polarity or in the absence of any oligodeoxynucleotide as a
negative control. The RNA product without the addition of any
oligodeoxynucleotide was not susceptible to RNase H (Fig. 1,
panel B, lane 2). However, the RNA product upon digestion
with RNase H gave rise to essentially two distinct fragments of 520 and
230 nt after annealing with oligodeoxynucleotide of (+)-polarity (panel B, lane 3) but not with (
)-oligodeoxynucleotide
(panel B, lane 4). These results indicated that the labeled
strand from the RdRP reaction was of (
)-polarity, which is
complementary to the input (+)-strand RNA template. The radiolabeled
(+)-RNA annealed with the (
)-oligodeoxynucleotide served as a
positive control for RNase H digestion (data not shown).
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Fig. 2.
Analysis of RNA/RNA interaction.
Physical interaction between two RNAs was analyzed by the psoralen/UV
cross-linking method which is described under "Experimental
Procedures." The 3'-UTR454nt RNA (thick dotted
line) which had either wild type (W) or mutant
(M) 3'-CYC motif was unlabeled. The 5'-TR230nt
RNA is indicated by a thick solid line with a solid
rectangular box denoting the 5'-UTR. Thick rectangular
vertical bars in 3'-UTR and 5'-TR RNAs denote CYC motifs. An
X indicates the mutant CYC motif. An asterisk
denotes a radiolabeled RNA used for the cross-linking experiment.
Panel A, interaction between *5'-TR230nt RNA and
3'-UTR454nt RNA. Lanes 1 and 6, *5'-TR230nt wt and 5'-TR230nt/cycMUT,
respectively, after psoralen/UV treatment. Lanes 2 and
3, *5'-TR230nt/wt + 3'-UTR454nt/wt
without and with psoralen/UV treatment, respectively. Lanes
4 and 5, *5'-TR230nt/wt + 3'-UTR454nt/cycMUT without and with psoralen/UV treatment,
respectively. Lanes 7 and 8, *5'-TR230nt/cycMUT + 3'-UTR454nt/wt without and
with psoralen/UV treatment, respectively. Lanes 9 and
10, *5'-TR230nt/cycMUT + 3'-UTR454nt/cycMUT without and with psoralen/UV treatment,
respectively. Although the same amount of labeled 5'-TR RNA was used
for each experiment, treatment of labeled 5'-TR RNA alone with
psoralen/UV seemed to result in loss of labeled RNA which might be
attributable to degradation (lanes 1 and 6; see
also panel B). Panel B, interaction between
5'-TR130nt RNA and 3'-UTR454nt. The experiments
were carried out as described in panel A except that labeled
5'-TR130nt RNA (*) was used. The order of samples loaded
was the same as in panel A.
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Fig. 3.
Transactivation activities of various 5'-TR
RNAs. In order to map the minimum region of 5'-TR RNA which is
necessary for transactivation of the 3'-UTR454nt for
in vitro RNA synthesis, three truncated mutant 5'-TR RNAs
were constructed. The RdRP products were analyzed on a
formaldehyde-agarose gel (1.8%), followed by autoradiography.
Lane 1, 3'-UTR454nt RNA alone. Lanes 2, 4, 6, and 8, 5'-UTR96nt,
5'-TR130nt, 5'-TR180nt, and
5'-TR230nt RNAs alone, respectively. Lanes 3, 5, 7, and 9, 3'-UTR454nt RNA in the presence
of the 5'-UTR96nt, 5'-TR130nt,
5'-TR180nt, and 5'-TR230nt RNA, respectively.
The labeled 1 and 2× products produced from the unlabeled
3'-UTR454nt RNA are indicated by arrows. The
unlabeled 5'-TR RNAs also served as templates in the in
vitro RdRP assay, and the 1 and 2× products are indicated as
closed and open dots, respectively. In lane
7, 1× of 3'-UTR454nt and the 2× of
5'-TR180nt were separated, whereas in lane 9, the 3'-UTR454nt RNA band was not well resolved from the 2×
product of 5'-TR230nt RNA. The presence of 3'-UTR RNA
significantly inhibited the template activity of the 5'-TRs (see
lanes 3, 5, 7, and 9).
View larger version (26K):
[in a new window]
Fig. 4.
In vitro RdRP assay with various
subgenomic mutant RNA templates. RNA templates were produced by
in vitro transcription as described under "Experimental
Procedures." The integrity of RNAs were verified by formaldehyde,
1.8% agarose gel electrophoresis and visualized by staining with
acridine orange. Panel A, analysis of the template
efficiency of the subgenomic RNA674nt (without the 5'-UTR).
Lane 1, subgenomic RNA770nt WT. Lane
2, subgenomic RNA674nt containing the deletion of
5'-UTR. Panel B, analysis of the template efficiency of the
subgenomic RNA770nt WT, 5'cyc MUT, 3'cycMUT, and 5'3'double
cycMUT (lanes 1-4, respectively).
View larger version (30K):
[in a new window]
Fig. 5.
A schematic diagram of the 3'-SL mutations in
the 3'-UTR of DEN2. Adapted from Ref. 45. Panel A, the
secondary structure of the 93-nt region from the 3'-end of the DEN2
RNA. Panel B, the secondary structure of the 96-nt region
from the 3'-end of the West Nile virus RNA. Pseudoknot G. C base
pairing interactions within the DEN2 3'-SL and the WN-3'-SL are also
indicated as dotted circles. Panel C, diagram of
the mutations within the 3'-SL of the DEN2 RNA genome. The
boldface regions in the 1st 2 rows indicate the
substituted regions from WN RNA. The bars from D2-SL(a) and
D2-SL(b) indicate the exchanged sequences. The boldface
region from the D2-SL(c) shows the disrupted base pairings. D2-SL
(C71G) is the pseudoknot mutant (indicated by a filled
circle). The horizontal dotted line denotes the border
between upper and lower halves of the 3'-SL structure.
View larger version (60K):
[in a new window]
Fig. 6.
Comparison of template efficiencies of the
subgenomic RNAs with specific 3'-SL mutations using the in
vitro RdRP assays. Each 3'-SL mutation was subcloned
into the plasmid encoding the subgenomic RNA770nt. In
vitro transcribed 3'-SL mutant subgenomic RNA templates were
analyzed on formaldehyde, 1.8% agarose gels, followed by staining with
acridine orange prior to their use in the in vitro RdRP
assays. Panel A, in the previous study, the vivo
viabilities of the 3'-SL mutations in the monkey kidney
(LLC-MK2) cells in the context of full-length DEN2 RNA
genome were determined by immunofluorescence assays and quantitated as
the percent of DEN antigen-positive cells (45). These semiquantitative
values are presented here together with the template efficiencies of
the 3'-SL mutant subgenomic RNAs in the in vitro RdRP assay
carried out using infected LLC-MK2 extracts (see panel C).
Panel B, DEN2-infected C6/36 cell lysates were used for the
in vitro RdRP assay. Panel C, DEN2-infected
LLC-MK2 cell lysates were used for the in vitro RdRP assay.
Panel D, RNA templates used stained by acridine
orange.
View larger version (51K):
[in a new window]
Fig. 7.
Importance of predicted pseudoknot
interactions for in vitro RNA synthesis by mutational
analysis. C73G mutagenesis was carried out by PCR in the plasmid
containing the D2/WN-SL in which the entire 3'-SL of DEN2 (D2) was
replaced the WN-3'-SL as described under "Experimental Procedures."
Template efficiency of the wild type and mutant 3'-SL RNA templates was
determined using the C6/36-infected cell extract for the in
vitro RdRP assays. Lane 1, D2 3'-SL WT; lane
2, D2-SL(C71G) mutant; lane 3, D2/WN 3'-SL WT;
lane 4, D2/WN 3'-SL(C73G) mutant.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
)-polarity. Furthermore, the results of analyses of the structural
requirements for RNA synthesis indicate that both 5'-UTR and 3'-SL
structures contribute to template efficiency in addition to the
conserved CYC motifs. The results of psoralen-UV cross-linking
experiments further establish that there is physical interaction
between the 5'- and 3'-TRs that require complementarity between the CYC motifs.
View larger version (47K):
[in a new window]
Fig. 8.
Predicted secondary structure analysis of the
subgenomic RNA templates. RNA structure software (version 3) by
Zuker et al. (58) was used for secondary structure analysis.
Panel A, WT770nt; panel B, subgenomic
5'-MUT; panel C, subgenomic 3'-MUT; panel D,
subgenomic RNA 5'-3'-MUT; panel E, subgenomic
RNA674nt.
virus (75), BMV (76),
rhinovirus type 14 (77), poliovirus (78), hepatitis C virus (79, 80),
and tobacco etch virus (81).
![]() |
FOOTNOTES |
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* This research was supported in part by National Institutes of Health Grants AI-32078 and AI-45623.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported in part by the Biomedical Research Scholar Training Program of the University of Kansas Medical Center. Present address: Laboratory of Virology and Infectious Diseases, Rockefeller University, 1230 York Ave., New York, NY 10021. E-mail: yous@rockefeller.edu.
To whom correspondence should be addressed: Dept. of
Biochemistry and Molecular Biology, University of Kansas Medical
Center, 3901 Rainbow Blvd., Kansas City, KS 66160-7421. Tel.:
913-588-7018; Fax: 913-588-7440; E-mail: rpadmana@kumc.edu.
Published, JBC Papers in Press, February 5, 2001, DOI 10.1074/jbc.M010923200
2 G. Bartelma, B. Winter, and R. Padmanabhan, unpublished results.
3 L. Zeng, B. Falgout, and L. Markoff, personal communication.
4 L. Zeng, B. Falgout, and L. Markoff, unpublished results.
![]() |
ABBREVIATIONS |
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The abbreviations used are: DEN2, dengue virus type 2; CYC, cyclization; NS, nonstructural; RdRP, RNA-dependent RNA polymerase; SL, stem-loop; TR, terminal region; WT, wild type; UTR, untranslated region; kb, kilobase pair; BMV, brome mosaic virus.
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