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Journal of Virology, November 2000, p. 10359-10370, Vol. 74, No. 22
Department of Molecular Genetics and
Microbiology, State University of New York at Stony Brook, Stony
Brook, New York 11794,1 and Gorlaeus Laboratory,
Leiden University, 2300 RA Leiden, The
Netherlands2
Received 10 May 2000/Accepted 17 August 2000
The first step in the replication of the plus-stranded poliovirus
RNA is the synthesis of a complementary minus strand. This process is
initiated by the covalent attachment of UMP to the terminal protein
VPg, yielding VPgpU and VPgpUpU. We have previously shown that these
products can be made in vitro in a reaction that requires only
synthetic VPg, UTP, poly(A), purified poliovirus RNA polymerase
3Dpol, and Mg2+ (A. V. Paul, J. H. van Boom, D. Filippov, and E. Wimmer, Nature 393:280-284, 1998). Since
such a poly(A)-dependent process cannot confer sufficient specificity
to poliovirus RNA replication, we have developed a new assay to search
for a viral RNA template in conjunction with viral or cellular factors
that could provide this function. We have now discovered a small RNA
hairpin in the coding region of protein 2C as the site in PV1(M) RNA
that is used as the primary template for the in vitro uridylylation of VPg. This hairpin has recently been described in poliovirus RNA as
being an essential structure for the initiation of minus strand RNA
synthesis (I. Goodfellow, Y. Chaudhry, A. Richardson, J. Meredith, J. W. Almond, W. Barclay, and D. J. Evans, J. Virol.
74:4590-4600, 2000). The uridylylation reaction either with
transcripts of cre(2C) RNA or with full-length PV1(M) RNA
as the template is strongly stimulated by the addition of purified
viral protein 3CDpro. Deletion of the cre(2C)
RNA sequences from minigenomes eliminates their ability to serve as
template in the reaction. A similar signal in the coding region of VP1
in HRV14 RNA (K. L. McKnight and S. M. Lemon, RNA
4:1569-1584, 1998) and the poliovirus cre(2C) can be
functionally exchanged in the assay. The mechanism by which the VPgpUpU
precursor, made specifically on the cre(2C) template, might
be transferred to the site where it serves as primer for poliovirus RNA
synthesis, remains to be determined.
The Picornaviridae family
of plus-strand RNA viruses includes a large number of pathogens with
widely different host range and disease symptoms (38). At
the same time, picornaviruses show a strong similarity in their gene
organization and in the mechanism by which they replicate their genomes
(58). An unusual feature of their genomes is the presence of
a small protein VPg, covalently linked to the 5' end of the RNA. Virus
replication in the infected host cell is a two-step process, carried
out primarily by the viral RNA polymerase, in conjunction with other
viral and possibly also cellular proteins. It takes place in small
vesicles that are derived from the host's cellular membranes and with
which the nonstructural proteins of the virus are associated. First, the incoming viral RNA is transcribed into complementary minus strands
which are then used as templates for the synthesis of the progeny plus
strands. Although the basic steps of replication are well known, very
little is understood about the details of these processes and in
particular about the exact functions of the cis-acting RNA
structures contained within picornaviral RNAs (1). One of
the important unanswered questions about minus-strand synthesis is how
the viral RNA polymerase recognizes and selects only its own RNA as
template among the many polyadenylated mRNAs that are present in the
host cell (45).
Poliovirus is perhaps the best known member of the
Picornaviridae. Its RNA genome of about 7,500 nucleotides
(nt) is composed of a long 5'-nontranslated region (NTR), a single open
reading frame, a short 3'NTR, and a poly(A) tail (Fig.
1A) (32). The 5'-terminal UMP
of the viral RNA is linked to the hydroxyl group of VPg by a
phosphodiester bond (Fig. 1A) (2, 56). The 5'NTR consists of
two independent domains. The first is a cloverleaf-like structure which
is involved both in plus-strand RNA synthesis (3, 4, 24, 70)
and in the process of switching from translation to replication
(18). The second is a large and complex structure, the
internal ribosomal entry site (IRES) (28, 47), which
promotes translation of a polyprotein. This polyprotein (Fig. 1A)
contains a capsid region (P1) and two nonstructural domains (P2 and P3)
(32). The initial cleavage of the polyprotein is carried out
by proteinase 2Apro at the P1/P2 site (64). Most
other cleavages are mediated by the activities of proteinase
3Cpro and its precursor, 3CDpro (22, 23,
29, 73). The proteins of the P2 domain are predominantly involved
in inducing the biochemical and structural changes that occur in the
infected cell (8), but 2CATPase is also
essential for viral genome replication (49). Those of the P3
region are the ones most directly involved with the process of RNA
synthesis. These include the important viral RNA polymerase
3Dpol, which is both primer and template dependent
(16). The other members of the P3 domain (Fig. 1A; reviewed
in reference 68) are a small membrane-bound protein
3A, the 22-amino-acid terminal protein VPg, proteinase
3Cpro, and two multifunctional precursors 3AB and
3CDpro (3, 4, 24, 69, 70). The binding to the 5'
cloverleaf of 3CDpro, either in a complex with 3AB
(24, 70) or with the cellular protein PCBP2 (3, 4, 10,
17, 43), has an important role in plus-strand RNA synthesis. The
viral genome is terminated with a poly(A) tail (72) that is
attached to a stem-loop structure of the 3'NTR (Fig. 1A). The 3'NTR,
whose exact function is not yet known, is important (1, 37,
50-52, 71), but it appears that it is not essential (1, 57,
63) for RNA replication.
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Identification of an RNA Hairpin in Poliovirus RNA
That Serves as the Primary Template in the In Vitro Uridylylation
of VPg
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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FIG. 1.
(A) Structure of poliovirus genomic RNA. The
single-stranded RNA genome of poliovirus is shown with the terminal
protein VPg (3B) at the 5' end of the NTR (single line) and the 3'NTR
(single line) with the poly(A) tail. The 5'NTR consists of a small
cloverleaf and a large IRES element. The location of the
cre(2C) element and of SLII of the IRES are indicated. The
attachment site of the 5'-terminal UMP of the RNA to the tyrosine of
VPg is shown enlarged. The polyprotein contains structural (P1) and
nonstructural (P2 and P3) domains. The boxed region containing vertical
lines represents the polyprotein with the proteinase cleavage sites.
Processing of the P3 domain by proteinases 3C and 3CDpro is
shown enlarged. (B) Predicted secondary structures of the HRV14
cre(VP1) (35, 36) and the PV1(M)
cre(2C) RNAs (19). Conserved sequences are shown
in boldface. (C) Conserved cre sequences in the coding
regions of picornaviral RNAs (19, 34-36).
It has been generally believed that the 5'- and 3'NTRs of picornaviral RNAs contain all the cis-acting replication (cre) signals required for RNA replication (1). All natural or synthetic subgenomic picornaviral RNAs that are replication competent contain these terminal structured RNA elements (4, 30, 35, 48). Subgenomic replicons derived from poliovirus or coxsackie B3 virus are fully replication competent even if the entire P1 region is deleted (4, 67). This, however, is not the case with some other members of the Picornaviridae. It was first shown in the case of human rhinovirus 14 (HRV14) that a small RNA hairpin, located in the coding sequences of its capsid protein VP1, is involved in RNA replication (35, 36). Recently the VP2 coding sequences of mengo virus and Theiler's virus (34) were also found to contain such cis-acting RNA structures. In contrast to HRV14 and the cardioviruses, poliovirus contains a cis-replicating element not in the capsid region but in the coding region of protein 2C (19). This RNA hairpin, just like that of HRV14 (36) was found to be position independent for function and required for minus-strand RNA synthesis (19). The internally located cre elements of these different picornaviruses all consist of a relatively simple stem-loop structure but differ in location and in nucleotide sequences (Fig. 1B) (19, 34-36).
An important, but poorly understood, feature of picornaviral RNA replication is that RNA transcription is confined to the viral template and does not involve cellular mRNAs. The conserved RNA structural features in the 3'NTR were predicted to posses all the necessary recognition signals for the RNA polymerase to recognize its specific template and initiate minus-strand synthesis (1, 71). The 3'NTR preceding the poly(A) tract was therefore designated as the origin of replication (oriR) for minus-strand synthesis (51). This proposal was based on numerous genetic studies indicating the importance of the RNA structure that is maintained by "kissing" interactions (37, 50, 51). This, however, has been difficult to reconcile with subsequent genetic and biochemical data. Recently, it was shown that the exchange of the 3'NTR between poliovirus and HRV14 or CBV4 yielded virus growing with nearly wild-type (wt) kinetics (57). Even more unexpected was the finding that the 3'NTR of poliovirus can be deleted without loss of viral viability, although the deleted genome is greatly debilitated with respect to replication (63). In order to explain these conflicting results, Todd et al. (63) have proposed that the primary determinant for template selection might be the localization of viral RNAs on membrane-bound complexes leading to a high local concentration of the replication proteins and the physical proximity of the P2 or P3 gene products to the 3'NTR following translation. Alternatively, the 3'NTRs of poliovirus, CBV4, and HRV14 may all mimic a common structural signal, as yet undefined, promoting the formation of a replication complex (1, 71).
The first step in minus-strand RNA synthesis, the covalent attachment of UMP to the tyrosine residue of VPg, is expected to occur immediately following selection of the polioviral RNA template. We have previously shown that this reaction can be studied in vitro with a simple assay that requires only purified 3Dpol, synthetic VPg, a poly(A) template, UTP, and Mg2+ (45). The products are VPgpU and VPgpUpU, the precursors of VPg-linked poly(U), the 5' end of minus-strand RNA. This reaction is likely to represent the uridylylation of VPg in vivo, as it is strongly supported by genetic studies with variants of VPg and 3Dpol (A. V. Paul, J. Peters, J. Mugavero, J. H. van Boom, and E. Wimmer, manuscript in preparation). However, although uridylylation in vitro is strictly dependent on poly(A) serving presumably as template, the homopolymeric region can hardly function as the selecting cis-acting signal in vivo. Therefore, we changed the uridylylation assay such that poly(A) was replaced with poliovirus transcript RNA, which offered a direct way of testing the question of template selection. We have also searched for viral and cellular factors that might assist the RNA polymerase either in selecting its template or in the catalysis of the uridylylation reaction. We provide here evidence that in the presence of 3CDpro the primary template in PV1(M) RNA for the in vitro uridylylation of VPg is not the poly(A) tract but the cre(2C) element (19). The specificity of the reaction appears to be provided by the binding activity of 3CDpro to the cre(2C) RNA, either alone or complexed with 3Dpol/VPg. The cre(2C) RNA works in the in vitro reaction only in the context of the plus-strand sequence, suggesting that it is involved in minus-strand RNA synthesis. Mutagenesis of the cre(2C) element reduced or eliminated the ability of that RNA to serve as template in the in vitro reaction and interfered with viral growth but had no effect on the translation and processing of the polyprotein (55). We do not yet know how the VPg-linked precursors are transposed to the poly(A) tail where they might serve as primer for elongation during minus-strand synthesis.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Construction of plasmids. Poliovirus cDNA (pT7PVM) (12) nucleotide sequences listed for plasmids or oligonucleotides refer to the full-length (nt 1 to 7525) plus-strand poliovirus cDNA sequence. Mutated nucleotides are underlined in the oligonucleotide sequences.
(i) pET21b/3CD(3Cpro/H40A). The coding sequences of poliovirus Sabin 2 3CDpro(H40A) (D. W. Kim and E. Wimmer, unpublished) was amplified by PCR using primers 5'-GGGAATTCGGGCCCTGGGTTTGATTAC-3' (plus-strand sequence) and 5'-CCAAGCTTAAAAGAGTCGAGCCAACG-3' (minus-strand sequence). The fragment was digested with EcoRI and HindIII and was ligated into the same sites of pET21b (Novagen).
(ii) pET21b/PCBP2. The coding sequences of PCBP2 were amplified from plasmid pQE30-PCBP2 (gift of B. L. Semler) (43) by PCR using oligonucleotides 5'-CCGGATCCTATGGACACCGGTGTGATT-3' (plus-strand sequence) and 5'-CGGAAGCTTGTGCTCACAAGAAAAAAGGCA-3' (minus-strand sequence). The fragment was digested with BamHI and HindIII and was inserted into the BamHI/HindIII-restricted pET21b vector (Novagen).
(iii) pProEX HTb/3CD(3Cpro/H40A). The coding sequences of poliovirus Sabin 2 3CDpro (3Cpro/H40A) was amplified from plasmid pET21b/3CD(3Cpro/H40A) with oligonucleotides 5'-GGGGAATTCAAGGCCCTGGGTTTGATTACGCAGTGGC-3' (plus-strand sequence) and 5'-GGGCTGCAGTAAAATGAGTCAAGCCAACGGCGG-3' (minus-strand sequence). The resulting fragment was cut with PstI and BamHI and was ligated into the same sites of pProEX HTb (Lifetech).
(iv) pPV1(M)cre(2C). The T7 promoter and the coding sequences of protein 2C (nt 4444 to 4505) from plasmid pT7PVM (12) were inserted into the EcoRI/NaeI sites of plasmid pBR322 (55). The plasmid was digested with NheI or EcoRI and was transcribed with T7 RNA polymerase.
(v) pPV1(M)IRES/SLII. The SLII (stem loop II) sequences (nt 121 to 165) of the PV1(M) IRES were amplified from plasmid pT7PVM (12) using oligonucleotides 5'-GGGGGATCCTAATACGACTCACTATAGGCAAGTTCAATAGAAGGGG-3' (plus-strand sequence, also contains T7 promoter sequences) and 5'-CCCGAATTCCTTGTTCGTGGTGGTACTG-3' (minus-strand sequence). The resulting fragment was cut with EcoRI and BamHI and was inserted into the EcoRI/BamHI sites of pBR322. The plasmid was digested with EcoRI and was transcribed with T7 RNA polymerase.
(vi) pBS/+CL. The coding sequences of the plus cloverleaf (+CL), nt 1 to 106 of pT7PVM (12), was cloned into the SacI and XhoI sites of Bluescript II KS(+) (Stratagene). Plasmid pBS/+CL (T. Pfister and E. Wimmer, unpublished) was linearized with XhoI and transcribed by T7 RNA polymerase.
(vii) pBS/
CL.
This plasmid (Pfister and Wimmer,
unpublished) contains a T7 promoter, and the minus-cloverleaf
sequences, nt 1 to 96 of pT7PVM (12) in the SmaI
and SacI sites of pUC19 (New England BioLabs). It was
linearized with SacI and transcribed with T7 RNA polymerase.
(viii) pT7PV1(M)[no poly(A)]. This construct (K. S. Harris and E. Wimmer, unpublished) contained a SacI site immediately upstream of the poly(A) tail. The plasmid was linearized with SacI prior to transcription with T7 RNA polymerase. The transcript consisted of a full-length poliovirus RNA lacking only the poly(A) tail.
(ix) pT7PV1(M)[no 3'NTR-poly(A)]. This plasmid (Harris and Wimmer, unpublished) contained a SmaI site downstream of nt 7375. It was digested with SmaI and transcribed with T7 RNA polymerase to yield a full-length poliovirus transcript RNA without the 3'NTR and poly(A) sequences.
(x) Minigenomes I to III. Two new XhoI sites were introduced into the full-length pT7PVM (12) clone at nt 750 and 6164 and an MluI site immediately following the poly(A) tail (Kim and Wimmer, unpublished). Following digestion of the mutant plasmid with XhoI the fragment from nt 750 to 6164 was deleted and replaced by the cre(2C) fragment (nt 4444 to 4505) of pT7PVM (12) which contained XhoI linkers at both ends. Insertion of cre (2C) was either in the forward [cre(2C)F] or reverse direction [cre(2C)R]. Minigenome II lacked the cre(2C) sequences. The plasmids were linearized with MluI and were treated with mung bean nuclease (New England BioLabs) before transcription with T7 RNA polymerase.
(xi) pT7PVM (3CproR84S/I86A). Two mutations were introduced into the 3Cpro domain of pT7PVM using PCR mutagenesis. The first PCR fragment was made with oligonucleotides a (5'-GAGAAATGAAAAGTTCAGCGACGCTAGACCACATATACCTACTCAA ATCAC 3'; plus-strand sequence, nt 5671 to 5721) and b (5'-GGCCCTTTCGTCTTCAAGAATTCCGTT 3'; minus-strand sequence, nt 7520 to 7546). The second fragment was made with oligonucleotides c (5'-GGGTTGGATAGTCAACATCACCAGCC-3'; plus-strand sequence, nt 5230 to 5255) and d (5'-GAGTAGGTATATGTGGTCTAGCGTCGCTGAACTTTTCATTTCTCTTTAGAGTG-3'; minus-strand sequence, nt 5662 to 5714). The two fragments were mixed and used as template in the second PCR step with oligonucleotides b and c. The resulting fragment was digested with EcoRI and BglII and was ligated into similarly cut pT7PVM.
Enzymes. Poliovirus RNA polymerase was expressed in Escherichia coli BL21(DE3)pLysS from plasmid pT5T-3D which contains the wt 3D coding sequences, preceded by a methionine, and was purified by the method of Pata et al. (44). Poliovirus 3CDpro (3Cpro H40A), containing either an N-terminal or a C-terminal His tag was expressed in E. coli from plasmids pET21b/3CD(3CproH40A) and pProEX HTb/3CD(3Cpro/H40A), respectively. PCBP2 with C-terminal His tag was expressed in E. coli from plasmid pET21b/PCBP2. The proteins were purified by nickel column chromatography (Qiagen). Purified proteins 2C (49) and 2BC (Pfister and Wimmer, unpublished) were a gift of T. Pfister. Purified poly(A) binding protein was a gift of R. Rhoads, and 3AB was purified as described before (33). The plasmid for the expression of His-tagged PCBP2 (43) was a kind gift of Bert Semler.
Assay of RNA polymerase 3Dpol.
The synthesis of
VPgpU(pU) was measured by an assay similar to what was described before
(45). The reaction mixture (20 µl) contained 50 mM HEPES
(pH 7.5), 8% glycerol, 3.5 mM magnesium acetate, 0.5 µg of poly(A)
or transcript RNA, 2 µg of synthetic poliovirus VPg (14),
1 µg of purified poliovirus polymerase 3Dpol, 0.75 µCi
of [
-32P]UTP (3,000 Ci/mmol; DuPont-NEN), and 10 µM
unlabeled UTP. Where indicated, 0.3 to 0.5 µg of purified
3CDpro(3CproH40A), with either N-terminal or
C-terminal His tag, was added to the reaction mixtures. After 1 h
of incubation at 34°C, the reaction was stopped by the addition of 5 µl of gel loading buffer. The samples were analyzed by Tris-tricine
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Bio-Rad)
with 13.5% polyacrylamide. Gels were dried and autoradiographed, and
the reaction products were quantitated with a PhosphorImager (Molecular
Dynamics Storm 860) by measuring the amount of [32P]UMP
incorporated. VPgpU(pU) refers to the sum of VPgpU and VPgpUpU.
In vitro transcription and translation. Plasmid DNAs were linearized with EcoRI and were transcribed by using T7 RNA polymerase (66). The transcript RNAs were purified by phenol-chloroform extraction and ethanol precipitation. They were translated at 34°C in HeLa cell extracts (40).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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We have considered two possible models by which specificity might be provided in vivo to the reaction in which VPg is uridylylated. According to the first model, poly(A) would be the template and the adjacent the 3'NTR would provide the specific viral sequences needed for recognition by the RNA polymerase and/or other replication proteins (45). Alternatively, a poliovirus-specific RNA sequence or structure located somewhere else in the genome could serve both as the site of recognition for the RNA polymerase and the template for the uridylylation reaction. These models were based on the example of other viral polymerases that catalyze the covalent linkage of a nucleotide to a protein (see Fig. 9) (59). In order to distinguish between these two models we have extended our studies to include RNA templates derived from PV1(M) sequences and to search for viral and/or cellular factors that might aid the RNA polymerase either in the template selection or in the VPg-uridylylation reaction.
Uridylylation of VPg on templates derived from PV1(M) RNA.
In
our preliminary experiments with full-length PV1(M) transcript RNA as
template in the 3Dpol-catalyzed VPg-uridylylation reaction
we observed a strong stimulation by a preparation of purified
3CDpro (Fig. 2A, compare lane
1 with lane 2). As we will show later, this is in strong contrast to
the basic VPg-uridylylation reaction in which synthetic poly(A) serves
as the template for 3Dpol (see Fig. 5, compare lane 1 with
lane 2). These results suggested that when full-length PV1(M) RNA is
the template, a poliovirus-specific RNA sequence or structure is
recognized by 3CDpro and/or 3Dpol/VPg.
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Comparison of poly(A) and cre(2C) RNA as templates for
the in vitro uridylylation of VPg by 3Dpol.
The basic
assay system, that we have previously described for VPg-uridylylation,
used poly(A) as template and Mg2+ as cofactor of
3Dpol (45). The low yield of reaction products
obtained under these conditions (45) could be much improved
by replacing Mg2+ with Mn2+ (Fig.
5, lanes 1 and 3; A. V. Paul and E. Wimmer, unpublished data). The same effect has also been observed with
other DNA polymerases that catalyze nucleotidylylation of proteins,
such as the phage 
29 (15), phage PRD1 (11),
and adenovirus (54), or the reverse transcriptase of
hepatitis B virus (65). As mentioned above, the reaction
with a poly(A) template is not stimulated by 3CDpro, in the
presence of either Mg2+ (Fig. 5, lanes 1 and 2) or
Mn2+ (lanes 3 and 4). The partial inhibition of the
reaction by the 3CDpro preparation (Fig. 5, lanes 1 and 2 and lanes 3 and 4) is most likely due to an inorganic component of the
solution since a heated aliquot had the same effect (data not shown).
With cre(2C) RNA as the template and Mg2+ as the
cofactor of 3Dpol there was a >50-fold stimulation of the
reaction by 3CDpro (Fig. 5, compare lane 6 with lane 7).
The optimal yield of products with the two different templates was
about the same (Fig. 5, lanes 3 and 7). Importantly, Mn2+
did not stimulate uridylylation with cre(2C) to the same
extent as Mg2+ (Fig. 5, compare lane 7 with lane 9). Thus,
the "manganese effect" all but disappeared when cre(2C)
RNA is used as a template in the presence of 3CDpro.
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Characterization of the VPg-uridylylation reaction on a
cre(2C) template.
The optimal reaction conditions for
the uridylylation of VPg on the cre(2C) RNA (data not shown)
are essentially the same as what was found previously with the poly(A)
template (45). The exception is the metal requirement and
the stimulatory activity of 3CDpro (see above). The
concentration of 3CDpro required for optimal stimulation of
the reaction on a cre(2C) template was determined to be ca.
0.5 µM (Fig. 6A). This corresponds to a
molar ratio of 3CDpro to 3Dpol of ca. 0.5:1.
Since 3CDpro is the precursor of 3Dpol, we
tested the possibility that this protein also possesses
VPg-uridylylating activity. It can be seen from the data in Fig. 6B
that there are no products formed when 3Dpol is omitted
from the reaction (lane 2), an observation suggesting that
3CDpro only enhances the reaction catalyzed by
3Dpol. A heated aliquot of 3CDpro has no
stimulatory activity, demonstrating that the protein itself is the
active component of the enzyme preparation (Fig. 6B, lane 3). Other
purified viral proteins, GST-linked 2C (49) or 2BC (Pfister
and Wimmer, unpublished), 3AB (33), and the cellular proteins His-tagged PCBP2 (43), poly(A) binding protein
(31), or uninfected HeLa cell extracts had no stimulatory
activity in the reaction (data not shown).
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Stimulation of VPg uridylylation by translation reactions of PV1(M)
RNA.
In our search of other viral or cellular proteins that might
serve as cofactors and also enhance the uridylylation of VPg, we have
tested the effect of translation reactions of PV1(M) RNA in HeLa cell
free extracts. As shown on Fig. 7, using
either a cre(2C) RNA (compare lane 1 with lane 3) or PV1(M)
RNA (compare lane 7 with lane 9) as template, translation reactions (6 h at 34°C) have a strong stimulatory effect. The concentration of the full-length PV1(M) RNA is about 50-fold lower than that of the cre(2C) RNAs, so the yield of products obtained with these
templates is not directly comparable. Unincubated translation mixtures
(0 h) (Fig. 7, lanes 1 and 2 and lanes 7 and 8) have no effect on the
reaction, an observation indicating that the function of a viral
protein is involved in the stimulation. When using a poly(A) template,
neither the 0-h nor the 6-h translation reactions exhibit a significant
effect (Fig. 7, lanes 12 to 14). The similarity of these results to
those presented above suggests that 3CDpro is the active
component of the translation mixtures. However, we cannot rule out the
possibility that other proteins are also involved in the process, in
conjunction with 3CDpro.
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The cre(VP1) of HRV14 functions as template for VPg
uridylylation by poliovirus 3Dpol.
The
cre(2C) element of poliovirus (19) is similar in
structure to a small stem-loop RNA in the VP1 coding sequence of HRV14 which was previously described by McKnight et al. (35, 36). Although the function of these RNA structures could not be determined, McKnight, Goodfellow, and their coworkers could show a relation to
minus-strand RNA synthesis (19, 36). Mutagenesis of the HRV14 structure has shown that the top of the stem and the loop are
important for its function (36). We have noted that the loop
segment of both the cre(VP1) of HRV14 and the
cre(2C) of PV1(M) contain a conserved sequence of 5 nt
(AAACA, Fig. 1C). Otherwise the stem portions of the two RNAs are quite
different (Fig. 1B). Our results with mutant PV1(M) cre(2C)
RNAs in the in vitro assay and also in vivo led to the conclusion that
the first 2 A's in the conserved sequence of the loop and the
integrity of the top of the stem in the hairpin are important for its
function (55). Lobert et al. (34) have shown that
for the replication of minigenomic RNAs the cre(VP2) element
of Theiler's virus can be functionally replaced by that of Mengo virus
[cre(VP2)] but not with that of HRV14
[cre(VP1)]. We were interested to test whether the HRV14
cre(VP1) RNA can replace the PV1(M) cre(2C) RNA
in our in vitro assay. Our results show that the cre RNA
elements of the two viruses are both good templates for
3Dpol in the uridylylation of PV1(M) VPg (Fig.
8, compare lane 2 with lane 4). Moreover,
both reactions are stimulated by poliovirus 3CDpro (Fig. 8,
lanes 1 and 2 and lanes 3 and 4). The yield of VPg-linked products
obtained with the two cre RNAs is similar (Fig. 8, lanes 2 and 4). These results and our genetic studies (55) suggest that the first two A's of the conserved sequence in both viral cre RNAs are used by poliovirus 3Dpol as
template for the synthesis of VPgpUpU. Whether the same sequence of the
loop or the structure of the stem of the hairpin is involved in the
recognition by 3CDpro and/or 3Dpol/VPg remains
to be determined.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The experiments reported here were undertaken to obtain further
information about the mechanism by which 3Dpol selects its
template for transcription. Recognition of specific viral RNA sequences
is expected to precede the very first step in minus-strand RNA
synthesis which is the uridylylation of VPg by the RNA polymerase. Our
simple in vitro assay (45) that measured this biochemical
reaction offered a direct way to study the question of specificity. We
considered two different models by which specificity could be provided
to the reaction in which VPg is uridylylated: first, that the RNA
polymerase would be recruited to specific sequences or structures in
the 3'NTR, which is spatially close to the poly(A) tail, where
uridylylation of VPg would then occur; and second, that specific RNA
sequences or structures located somewhere else in PV1(M) RNA could
serve both as the recognition site and as the template for the RNA
polymerase. Both models were suggested to us by studies of other
nucleic acid polymerases that use protein priming for the replication
of their genomes. The first model is exemplified by adenovirus, which
possesses a linear double-stranded DNA genome and uses sequences near
the end of its genome both as a recognition site and as a template to
deoxynucleotidylylate the preterminal protein by its DNA polymerase
(Fig. 9) (25, 59). Our second
model was based on the example of the protein priming reaction carried
out by the reverse transcriptase of hepatitis B virus (Fig. 9)
(26). This enzyme binds to an internal RNA hairpin and uses
it as a template for the synthesis of a short DNA oligonucleotide that
is covalently linked to the polymerase molecule. The enzyme then
translocates close to the 3' end of the plus-strand RNA where the
oligonucleotide hybridizes to a complementary stretch and is elongated
into minus-strand DNA.
|
In order to distinguish between these possibilities, we have used full-length poliovirus transcript RNA as template for the uridylylation reaction and tested the effect of viral and cellular proteins on these reactions. Purified 3CDpro or translation reactions of poliovirus RNA in HeLa cell extracts strongly stimulated the reaction on full-length poliovirus RNA but not on a poly(A) template, an observation indicating the involvement of a specific viral RNA sequence in the reaction. This lead to the identification of the cre element in the coding region of (2C) (19), both as the primary site of recognition by 3CDpro and/or 3Dpol/VPg and as the template for the in vitro uridylylation of VPg. This conclusion is supported by several lines of evidence. Deletion of the poly(A) tail or of the 3'NTR-poly(A) from PV1(M) RNA resulted only in a small reduction in the ability of such transcripts to serve as templates both in the absence (data not shown) and in the presence of 3CDpro. Similarly, deletion of the cre(2C) sequences from minigenomes that still retain the 3'NTR-poly(A) tail are nearly totally inactive as templates. Finally, transcripts of the small cre(2C) RNA itself are excellent templates in the reaction that requires the RNA binding activity of 3CDpro. In agreement with our data, an essential interaction of 3CDpro with a viral factor of the P2-P3 domain has been recently predicted. These studies involved chimeric poliovirus replicons in which the 3CDpro domain of PV1(M) was replaced by that of coxsackievirus B3 (7).
The protein priming reactions in which the cre(2C) RNA or poly(A) serve as templates appear to be identical, except that the former reaction is greatly enhanced by the presence of 3CDpro, and Mg2+ is the best cofactor for the polymerase. Our data indicate that both of these in vitro assays can be used to test the reaction in which VPg is uridylylated in vivo. Genetic data with VPg or 3Dpol variants show a strong correlation between viral growth phenotype and yield of uridylylated products obtained in the in vitro reactions (Paul et al., in preparation). It should be noted that poliovirus 3Dpol is not unique among polymerases that are able to use in vitro either a homopolymer or their viral nucleic acid sequences as templates for the protein priming reaction (39). The exact function of 3CDpro in the reaction that uses a cre(2C) RNA template is not yet known. The data strongly suggest that the RNA binding activity of this viral proteinase is involved. A mutation in the 3Cpro(R84S) domain of 3CDpro was previously shown to eliminate the ability of the protein to bind to the 5' cloverleaf (4, 9) but not affect the processing of the polyprotein (9, 20). Translation reactions of a PV1(M) RNA [3Cpro(R84S/I86A)] exhibited no stimulatory activity of the in vitro uridylylation reaction. The most likely function of 3CDpro is to enhance the binding of 3Dpol/VPg to the template either by complexing with the other two proteins or by stabilizing the structure of the cre RNA or both. In this respect the function of 3CDpro would resemble that of Mn2+ when poly(A) is used as template. The enhanced cofactor activity of Mn2+ over that of Mg2+ during elongation of oligonucleotide primers is known to be due to a reduction in the Km value for 3Dpol binding to primer/template (5). In any case, we describe a new function of 3CDpro: the selection of a specific RNA structure in the viral genome for uridylylation of VPg by 3Dpol to occur, thereby providing all-important specificity to this event.
The cre(VP1) of HRV14 (35, 36) is functionally exchangeable in the in vitro assay with the PV1(M) cre(2C) element, an observation suggesting that although these structures are located in different regions of the viral RNA, they do have identical in vivo functions. Both of these RNAs were shown to function only in the plus-strand sequence and to be required for minus-strand RNA synthesis (19, 36). Whether or not the VPgpUpU made on the cre(2C) template is also used for plus-strand synthesis remains to be determined. It is interesting that PV1(M) transcript RNAs lacking the two initial 5'UMP residues are not only infectious but, remarkably, the virus produced during infection has regained the authentic pUpU end (21). This implies that initiation of plus-strand synthesis is independent of the presence of the two terminal AMPs of the minus strand.
It should be emphasized that we cannot rule out the possibility that at some stage during the viral life cycle poly(A) itself also serves as template in the reaction, albeit only with low efficiency compared to the cre(2C) RNA. However, although in the presence of Mn2+ poly(A) is an efficient template for the uridylylation of VPg in vitro, the cre(2C) element would be superior to it in vivo for several reasons. First, by using a specific and internal viral RNA sequence/structure both as the site of recognition and as the template for VPgpU(pU) synthesis the possibility that the wrong poly(A)-tailed mRNA would be selected by 3Dpol for transcription would be reduced. Second, the presence of another viral protein, 3CDpro, during VPg-uridylylation would provide additional specificity to the reaction. Finally, the use of Mg2+, instead of Mn2+, as cofactor of 3Dpol in this reaction would enhance the overall specificity of minus RNA synthesis. In general Mn2+ is known to relax the specificity of nucleic acid polymerases toward their substrates and templates (5, 62; Paul and Wimmer, unpublished).
The internally located cre elements of picornaviruses thus far identified all consist of a small and relatively simple hairpin structure (19, 34-36). The only conserved sequence in these RNAs (19, 34-36) is a stretch of 5 nt (AAACA; Fig. 1C) found in the loop or bulge. Studies by McKnight et al. (35, 36), Lobert et al. (34), and us (55) have shown that the first A in the sequence is essential for viral viability. Consistent with the viral viability data is our observation that in the context of either the cre(2C) RNA or the PV1(M) RNA, the first A of the AAACA sequence is indispensable for the in vitro VPg-uridylylation reaction (55). Mutagenesis of the second A reduces the yield of uridylylated products, while a change in the third A has no significant effect (55).
The loop portion of SLII of the PV1(M) IRES contains a sequence (AAACCA) (27) that is very similar to the conserved sequence in the internal cre RNAs discussed above. Deletion of these nucleotides from SLII were found to result in a defective replication phenotype (27). Despite its strong similarity to the essential sequences in cre(2C) RNA, the SLII RNA is nearly inactive as template in the VPg-uridylylation reaction either in the absence or in the presence of 3CDpro. These results provide further proof that in the presence of 3CDpro the cre(2C) RNA is a unique structure in poliovirus RNA in that it serves as an efficient and specific template for the reaction.
Unfortunately, we have not yet been able to achieve efficient elongation reaction of the precursors either on a full-length PV1(M) template or on the minigenomes. The addition of purified viral proteins 2C, 2BC, and 3AB or cellular poly(A) binding protein had no effect on this process (data not shown). The same results were obtained when the reaction was supplemented with uninfected or poliovirus-infected HeLa cell extracts (data not shown). However, the small amount of polymeric product is difficult to detect under the conditions of our assay because there is always a relatively high background of such product made in a VPg-independent reaction. This is due to the terminal uridylyl transferase activity of the polymerase (53), to oligonucleotide priming by traces of degraded RNA, or to hairpin priming of nicked high-molecular-weight RNA. Our inability to obtain elongation in the in vitro reaction might be related to the fact that the mutant 3CDpro used in the assay is unable to undergo autoprocessing, possibly a prerequisite to the release of the 3Dpol-VPgpUpU complex from the cre(2C) RNA. Alternatively, this RNA element might have to be located on the same genome from which 3Dpol and/or 3CDpro were translated. This possibility is suggested by the finding that a mutation in the cre(2C) RNA cannot be complemented in trans (19, 55). It has been known for many years that in poliovirus translation and replication are coupled (1, 42, 68). Finally, our failure to achieve efficient elongation with VPgpUpU primer might be due to the lack of, or the insufficiency of, one or more cellular or viral factors that are required for complete minus-strand RNA synthesis. Barton and Flanegan (6) have concluded that in the in vitro translation-transcription system that synthesizes viable virus (40), the guanidine-sensitive ATPase activity of 2C (49), and a soluble HeLa cell factor are required for minus-strand RNA synthesis. The function of this putative cellular factor in RNA replication remains unknown.
It should be noted that free VPgpUpU has been found in the cytoplasm of poliovirus-infected cells (13). Thus, the precursor to elongation is clearly synthesized preceding and most likely independently of the elongation reaction. We do not know, however, what happens to VPgpUpU after it has been synthesized on the cre(2C) RNA. It is most likely that the precursors are translocated to the 3' end of the poly(A) tail and then elongated into full-length minus-strand RNA (Fig. 9) in a mechanism similar to what is used by the reverse transcriptase of hepatitis B virus (26, 60) (Fig. 9) or the DNA polymerase of adenovirus (25, 59) (Fig. 9). All three of these protein-primed nucleic acid synthesis systems are characterized first by the abortive synthesis of a short oligonucleotide attached to the hydroxyl group of a tyrosine or serine of a viral protein (terminal or preterminal protein, or polymerase). The template for this reaction is located either on an internal hairpin structure (poliovirus and hepatitis B virus) or on a partially opened double-stranded structure near the end of the genome (adenovirus). The second step involves the translocation of the nucleotidylylated protein-polymerase complex back to or near the 3' end of the template strand. This is followed by the elongation of the protein-linked oligonucleotide primer into minus strands by the polymerase. The last two steps take place only with low efficiency (reference 26 and see above) and probably involve a conformational change in the structure of the polymerase. The affinity of this enzyme for its template is expected to be relatively high for the initiation step but subsequently low so that it can be released and translocated and then move along its template during elongation of the primer. For optimal minus-strand synthesis adenovirus recruits three cellular proteins (NFI, NFII, and Oct I), and the viral DNA binding protein (25) and hepatitis B virus uses a chaperone complex of at least two proteins (hsp90 and p23) (26, 60). At this time one can only speculate as to how poliovirus achieves the translocation and elongation steps. One possibility is that the polymerase, with the help of additional protein(s), undergoes a structural change such as dissociation of oligomers (44) into monomers. Both the RNA binding (44) and uridylylating activity of 3Dpol (45) is enhanced under conditions that favor oligomerization of the protein, while the elongation step has no such requirement (46). Our in vitro assay that uses purified proteins to study the steps of minus-strand RNA synthesis offers a useful system for the identification of all the required components of this very complex process.
| |
ACKNOWLEDGMENTS |
|---|
We thank J. Mugavero and F. Maggiore for excellent technical
assistance. We thank Karla Kirkegaard for the generous gift of plasmid
pT5T-3D, Bert Semler for plasmid pQUE30-PCBP2, Thomas Pfister for
plasmid pBS+CL and pBSCL, and Kevin Harris for plasmid pT7PV1(M)[no
poly(A)] and pT7PV1(M)[no 3' NTR-poly(A)]. Purified poly(A) binding
protein was a gift from R. Rhoads; purified 2C and 2BC were gifts from
Thomas Pfister.
This work was supported by the National Institute of Allergy and Infectious Diseases of the NIH (5R37AI15122).
| |
FOOTNOTES |
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* Corresponding author. Mailing address: Department of Molecular Genetics and Microbiology, State University of New York at Stony Brook, Stony Brook, NY 11794. Phone: (631) 632-9777. Fax: (631) 632-8891. E-mail: apaul{at}ms.cc.sunysb.edu.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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(2001). Identification of the Amino Acid Residue Involved in Rabbit Hemorrhagic Disease Virus VPg Uridylylation. J. Biol. Chem.
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