Department of Molecular Genetics and Microbiology, School of Medicine, Stony Brook University, Stony Brook, NY 11794-5222, USA
Correspondence
Eckard Wimmer
ewimmer{at}ms.cc.sunysb.edu
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ABSTRACT |
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Published ahead of print on 14 May 2003 as DOI 10.1099/vir.0.19013-0.
Present address: US Department of Agriculture, Agricultural Research Service, Plum Island Animal Disease Center, Greenport, NY 11944, USA.
Present address: The Wharton School of the University of Pennsylvania, Philadelphia, PA, USA.
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INTRODUCTION |
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Genomes of the plus-strand RNA virus members of the family Picornaviridae are examples of relatively small replicating units (7500 nt) that are filled with structural information. The genomes of member viruses of two genera of the Picornaviridae, Enterovirus and Rhinovirus, are related more closely to each other than to other genera of the Picornaviridae and they have been studied intensely for RNA replication signals. Currently, four such signals have been identified: the 5'-terminal cloverleaf (CL), the cis-acting replication element (cre), the 3'-terminal heteropolymeric region and the 3'-terminal poly(A) tail (Agol et al., 1999
; Paul, 2002
; Xiang et al., 1997
). The IRES, located in the 5'NTR, might also be added. Apart from genetic evidence, however, there are no biochemical data to suggest that the IRES is involved directly in RNA synthesis (Agol et al., 1999
; Zhao & Wimmer, 2001
).
In this study, we have focused on the structure/function relationship of the 5'-terminal CL in poliovirus (PV) RNA synthesis. This structure (Fig. 1), first proposed by Rivera et al. (1988)
, has been shown to form a ribonucleoprotein (RNP) complex involved in genome replication (Andino et al., 1990
, 1993
). Key to the formation of the RNP is the recognition of the CL by the viral proteinase and RNA-binding protein 3CDpro (Andino et al., 1990
, 1993
). Together with either the cellular poly(C)-binding protein (PCBP2 and PCBP1) (Blyn et al., 1996
; Gamarnik & Andino, 1997
; Parsley et al., 1997
; Walter et al., 2002
) or the viral RNA-binding protein 3AB, the precursor for VPg (Harris et al., 1994
; Paul et al., 1994
; Xiang et al., 1995a
, b
), the CL3CDpro complex can also form a ternary complex. It was observed that the 3Cpro domain of 3CDpro carries the RNA recognition signal (Andino et al., 1990
; Blair et al., 1998
) and this activity could be modulated by 3Dpol (Cornell & Semler, 2002
). However, for PV, it is the precursor 3CDpro which possesses the higher affinity to the CL (Harris et al., 1994
). 3CproCL RNA complexes have been described for other picornaviruses as well, including rhinovirus and hepatitis A virus (Leong et al., 1993
; Kusov & Gaus-Muller, 1997
). Ternary complex formation and viral RNA synthesis co-vary; that is, if the CL is modified such that it cannot bind the above-mentioned proteins, there will be no viral RNA synthesis. Hence, this modification results in a lethal phenotype. Genetic and biochemical evidence suggests that PCBP2 binds to subdomain B of the CL, while 3CDpro binds to subdomain D (Andino et al., 1990
, 1993
; Gamarnik & Andino, 1997
; Parsley et al., 1997
). PV protein 3AB, on the other hand, is a non-specific RNA-binding protein (Paul et al., 1994
) and, thus, it will bind CL RNA non-specifically in the absence of competitor RNA. In the presence of as much as a 1000-fold molar excess of competitor RNA, however, 3AB will form the ternary complex CL3CDpro3AB with high specificity (Harris et al., 1994
; Xiang et al., 1995a
, b
).
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The structure of all enterovirus and rhinovirus CLs is similar (Zell et al., 2002; and references therein) (Fig. 1
); they consist of four subdomains, stemloop B being the most stable (Andino et al., 1990
; Larsen et al., 1981
; Rivera et al., 1988
). Rohll et al. (1994)
have reported that substitution of subdomain D in the HRV14 CL with that of PV3(L) can rescue the replication function of the HRV14/PV3(L) chimera. This observation has led us to focus on sequence parameters of subdomain D in the HRV14 CL and relating genetically changed versions of subdomain D with the ability to form ternary complexes and replication phenotypes. Although the structure of subdomain D of the HRV14 CL loop consists only of 3 nt (the loop in the subdomains D of the other viruses have 4 nt), the formation of tetra loops in this region is still evident (Fig. 1
and see Fig. 4B
). Addition of a G residue to the loop of HRV14 subdomain D rescued weakly the ability of this CL to form a ternary complex, with either 3CDpro3AB or 3CDproPCBP2, but this characteristic was not sufficient to rescue replication in the context of the PV genome. In addition, nucleotide changes were required to yield a proliferating HRV14/PV genome.
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METHODS |
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RNA transfection, plaque assay and one-step growth curve.
RNA transfections were performed with R19 HeLa cells by the DEAEDextran method, as described elsewhere (Rieder et al., 2000). Supernatants harvested from transfected cells were subjected to plaque assays (Molla et al., 1991
). For the one-step growth curve assays, R19 HeLa cell monolayers in 35 mm plastic culture dishes were infected at an m.o.i. of 10, unless indicated otherwise. At various time-points post-infection, the cells were harvested and the titres of the viruses were determined by plaque assay.
Viral RNA isolation and RT-PCR.
Total RNA was isolated from infected cells using Trizol (Life technologies). First-strand cDNAs were synthesized using a random deoxynucleotide hexamer and Moloney murine leukaemia virus reverse transcriptase (Life Technologies). cDNAs were amplified with specific pairs of primers by standard PCR procedures and sequenced directly.
Luciferase assay.
Luciferase assays were performed using protocols described previously. Briefly, about 20 µg RNA transcript was used to transfect 106 HeLa cell monolayers. The RNA transfection was carried out in the presence of 2 U RNase inhibitor µl-1 (Boehringer Mannheim). The cells were then incubated at 32, 37 or 39·5 °C. At each time-point, the cells were washed with cold HBSS buffer and lysed with 200 µl lysis buffer (10 mM Tris/HCl pH 7·5, 1 mM EDTA, 0·5 % NP-40 and 100 mM NaCl). The suspension was centrifuged at 2000 r.p.m. in a Sorvall RC-5B centrifuge and the supernatant was collected. The relative luciferase activity was measured in an Optocomp I luminometer (MGM Instruments).
Dot blot hybridization.
Viral RNA transcripts produced in vitro (5 µg) were transfected into 35 mm dishes of R19 HeLa cells. At several time-points after transfection, total RNA was extracted from the cells using Trizol. Digoxigenin (DIG)-labelled minus-strand RNA was synthesized from a BamHI digestion of pBS-2C (a gift from T. Pfister, Cytos Biotechnology AG, Schlieren, Switzerland), corresponding to the 5'-terminal portion of the 2CATPase-encoding region (nt 41234400), using T3 RNA polymerase and a mixture containing DIG-labelled UTP (Roche). The RNA was denatured and blotted onto a nylon membrane (Boehringer Manheim) pre-soaked with 20x SSC (3 M NaCl and 0·3 M sodium citrate, pH 7·0) using a Dot Blotter (Schleicher&Schuell). The RNA was then cross-linked to the membrane in a 1800 Stratalinker. The membrane was incubated with the DIG-labelled RNA in hybridization buffer (50 % formamide, 5x SSC, 0·2 % SDS, 0·1 % sodium-lauroylsarcosine and 0·05 mg tRNA ml-1) at 68 °C overnight. Afterwards, the membrane was washed twice in 2x wash solution (2x SSC and 0·1 % SDS) at room temperature, followed by two washes in 0·5x wash solution (0·5x SSC and 0·1 % SDS) at 68 °C (15 min each). The membrane was incubated with anti-DIG alkaline phosphatase-conjugated antibody (Roche) and incubated for 5 min. To detect the chemiluminescent signal, the membrane was exposed to a Kodak MR X-ray film.
RNA binding assay.
The binding of proteins to the CL was carried out by an RNA mobility shift assay, similar to what was described before (Xiang et al., 1995b). The reaction mixture (25 µl) contained 30 000 c.p.m. [
-32P]UTP-labelled RNA probes generated by in vitro transcription, as described above. The riboprobes were incubated at 45 °C for 30 min prior to the addition to the binding reactions. Protein/RNA mixtures were incubated at 30 °C for 10 min, stopped by the addition of 5 µl 50 % glycerol and loaded on a native 0·5x TBE, 5 % polyacrylamide (40 : 1) gel containing 5 % glycerol. Purified 3AB (Lama et al., 1994
), 3CDpro and PCBP2 recombinant proteins [the latter two derived from His-tagged plasmids pET21b/3CDpro (3Cpro/H40A) and pET21b/PCBP2; Paul et al., 2000
) were used at 0·2, 0·8 and 0·3 µM, respectively. A 15 µg sample of tRNA from Baker's yeast (Boehringer Mannheim) was added as non-specific RNA competitor in each reaction.
VPg uridylylation assay.
This assay was similar to that described before (Paul et al., 2000; Rieder et al., 2000
). The standard reaction mixture (20 µl) for VPg uridylylation contained 50 mM HEPES pH 7·6, 3·5 mM MgAc2, 8 % glycerol, 2·0 µg genomic RNA template transcribed in vitro, 2 µg synthetic PV VPg, 10 µM unlabelled UTP, 1 µg (1 µM) purified 3Dpol and 1 µCi [
-32P]UTP (0·017 µM) (3000 Ci mmol-1, Dupont NEN). Unless indicated otherwise, 0·7 µg 3CDpro was added to each reaction. Samples were incubated for 1 h at 30 °C and the reactions were stopped by the addition of 5 µl gel-loading buffer (Bio-Rad) and analysed by Tris/Tricine/SDS-PAGE (Bio-Rad) with 13·5 % polyacrylamide. Gels were dried at 68 °C for 2 h and autoradiographed (Kodak Biomax MS film). Reaction products were quantified by measuring the amount of [
-32P]UMP incorporated into the VPgpU and VPgpUpU products (Molecular Dynamics PhosphoImager, Storm 860).
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RESULTS |
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To analyse the kinetics of genome replication of the PV/HRV chimeras, we used dicistronic genomes carrying the luciferase gene. The prototype (PLEP) of such a dicistronic genome [(PV)5'NTR-Luc-(EMCV)IRES-(PV)P1,2,3-3'NTR,poly(A)] (Fig. 2A) was constructed originally by Alexander et al. (1994)
. Although the RNA (PLEP) is too large for encapsidation, it provides a measure of genome replication. Plasmids of the parental construct and two replicons with exchanges of the PV CL with that of HRVs, PV/HRV2 (R2LEP) and PV/HRV14 (R14LEP) (Fig. 2A
), were transcribed and the transcript RNAs were transfected into HeLa cells. At different times, luciferase activity was monitored (Fig. 2B
). Whereas the parental RNA expresses luciferase activity as long as 30 h or more, R2LEP RNA expressed luciferase at approximately 1 % of PLEP, with a delay in reaching the peak level. Addition of 2 mM guanidine hydrochloride (GH), an inhibitor of PV RNA replication (Wimmer et al., 1993
), to R2LEP-transfected cells suppressed luciferase activity. This observation suggested that the bulk of the signal was produced by replicating RNA and not transfected RNA. In cells transfected with R14LEP RNA, only background levels of luciferase were observed, which confirmed that this RNA is unable to replicate under these conditions.
Rohll et al. (1994) made the interesting observation that a HRV14/PV3 chimeric CL in which subdomains A, B and C (the first 42 nt) originated from HRV14 and subdomain D originated from PV3(L) was capable of RNA synthesis. These authors measured PV3 RNA replication by replicon-expressed CAT assays. Making use of this HRV14/PV3(L) chimeric CL (Fig. 1D
, named here as CLC) [kindly provided by J. B. Rohll (Department of Microbiology, University of Reading, UK) and J. W. Almond (Aventis Pasteur, Lyon, France)], we constructed viral genomes with the purpose of assaying genome replication and virus production.
Replication of the dicistronic replicon carrying CLC at its 5' end (Fig. 2A, CLCLEP) was apparent, as assayed by luciferase activity (Fig. 2C
). Rescue of RNA synthesis by CLC, however, was poor, as compared to a replicon carrying the PV CL (Fig. 2C
). At 39·5 °C, the luciferase signal was especially low, an observation that suggested a temperature-sensitive (ts) phenotype (Fig. 2C
, lower panel).
The replication phenotypes observed with CLCLEP could be due, at least in part, to the complex structure of the dicistronic genomes. Therefore, we replaced the cognate CL of PV1(M) with CLC (Fig. 3A, CLCPV) to generate a proliferating chimeric genome. RNA transcripts of wt PV1(M) or CLCPV were used to transfect R19 HeLa cell monolayers and the cells were incubated at 32, 37 and 39·5 °C. At 37 °C, CPE with wt RNA-transfected cells was apparent after 20 h. In contrast, CLCPV RNA produced CPE only after 72 h, an observation indicative of impaired replication. Indeed, viable virus recovered from the cells transfected with CLCPV RNA at 37 °C yielded virus expressing a minute plaque phenotype (data not shown). In a one-step growth experiment, CLCPV produced virus at 32 and 37 °C, approximately one log lower than the wt construct at the same temperatures (Fig. 3B
). Interestingly, replication of CLCPV expressed a very strong ts phenotype at 39·5 °C, just as CLCLEP did. Indeed, the yield of CLCPV in a one-step growth experiment was <1 % of that of the wt virus (Fig. 3B
). These data confirm that the replacement of subdomain D of the HRV14 CL with that of PV3(L) can rescue CL function. The efficiency of the chimeric CL, however, is poor.
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Insertion of a G residue at position 61 (C57UAU60GG61; M1-R14PV) yielded a non-viable chimeric genome when transfected into HeLa cells (Fig. 5A). No virus was isolated even after 4 days of incubation and blind passages. We then changed the U60 residue to a C (C57UAC60GG61), since a C is found in this position in CL elements compatible with PV replication (Fig. 4B
). Indeed, in the context of the chimeric virus, this altered D loop (CUAC60GG61) rescued replication (Fig. 5A, M
2-R14PV). A third mutation in the HRV14 D loop, a substitution of C57 with U (U57UAC60GG61; M3-R14PV), had little effect on virus replication (Fig. 5A
; and see below).
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The growth characteristics of viruses M2-R14PV and M3-R14PV were investigated by performing a one-step growth experiment. HeLa R19 cells were infected with viruses and titres were determined by plaque assay at different times (Fig. 5B). Although the replication kinetics of wt and mutant chimeric viruses are similar, the yield of the mutant viruses was reduced. This apparent phenotype in virus yield was mirrored by a non-radioactive RNA hybridization assay, performed to detect plus-strand RNA. For this purpose, plus-strand RNA synthesis was monitored at different times after transfection of PV1(M), R2/PV, M2-R14PV, M3-R14PV, R14/PV or M1-R14PV RNAs into HeLa cell monolayers. The signal indicating synthesis of PV1(M), R2/PV, M2-R14PV and M3-R14PV genomes increased over time (Fig. 5C
). In contrast, no plus-strand RNA synthesis could be detected in cells transfected with either R14/PV or M1-R14PV RNAs. The reduced signal for the R2/PV, M2-R14PV and M3-R14PV genomes may be the reason for the reduced yield of the respective viruses. These results show that the lethal phenotype of R14/PV and M1-R14PV genomes is related to a defect in RNA synthesis.
A translation phenotype has been reported as the result of an insertion mutation into the CL of PV1(M) (Simoes & Sarnow, 1991). Therefore, it could be argued that the abrogation of replication of the R14/PV and M1-R14PV genomes is due to aberrant protein synthesis and/or proteolytic processing. To test this possibility, we performed RNA translation assays. Full-length PV1(M) and chimeric RNA transcripts were translated using a HeLa cell-free extract (Molla et al., 1991
). Neither the relative translation efficiencies nor the processing levels were altered due to sequence differences in the CLs (Fig. 5D
), an observation ruling out an indirect effect of polyprotein processing for the lack of replication of the R14/PV and M1-R14PV genomes.
Ternary complexes formed between 3CDpro3AB and 3CDproPCBP proteins with wt and mutant HRV14 CLs
To investigate to what extent wt or mutant HRV14 CL elements are capable of RNP formation with 3CDpro and 3AB or 3CDpro and PCBP2, [-32P]UTP-labelled CL probes were synthesized in vitro. Recombinant PCBP2, 3AB and 3CDpro, alone or in combination, were incubated with plus-strand riboprobes in a binding reaction containing a 1000-fold molar excess of tRNA. The proteinRNA complexes were then examined in non-denaturing gels by EMSA (Fig. 6
). Both 3CDpro3AB and 3CDproPCBP2 formed ternary complexes efficiently with the CLs of PV1, HRV2, M2-R14PV and M3-R14PV (Fig. 6
, lanes 4, 5, 10, 11, 28, 29, 34 and 35, respectively). Binding was also observed with either 3CDpro or PCBP2 alone upon incubation with these probes (Fig. 6
, lanes 2, 3, 8, 9, 20, 21, 26, 27, 32 and 33, respectively). In contrast, neither 3CDpro nor 3CDpro3AB expressed any significant binding affinity to the HRV14 CL (Fig. 6
, lanes 14 and 16), while PCBP2 and 3CDproPCBP bound weakly to the HRV14 CL (Fig. 6
, lanes 15 and 17). 3AB alone, although an efficient non-specific RNA-binding protein (Fig. 3C
, lanes 79), was unable to shift the probes in the presence of competitor tRNA (Fig. 6
, lanes 1, 7, 13, 19, 25 and 31), as expected (Paul et al., 1994
).
It is noteworthy that 3CDpro could bind quite efficiently the M1-R14PV CL probe (Fig. 6, lane 20), although this binding did not result in an efficient formation of the ternary complexes CL3CDpro3AB or CL3CDproPCBP (Fig. 6
, lanes 22 and 23, respectively). Thus, whereas 3CDpro binding to the newly formed tetra loop can be restored partially, the interaction between CLCDpro and the third protein binding partner is impaired (30 % of the riboprobe has been shifted by the formation of this complex; Fig. 6
, lane 22). We suggest that this observation may explain why the M1-R14PV CL element is unable to support genome replication. In contrast, the transition of U60
C (C57UAC60GG61) in the M2-R14PV CL stimulated ternary complex formation (Fig. 6
, lanes 28 and 29), which co-varied with the rescue of replication. Protein binding was stimulated further in M3-R14PV CL (U57UAC60GG61), although this transition at position 57 had little effect on replication (Fig. 6
, lanes 34 and 35). The latter is not surprising, since the D loop of M3-R14PV CL is identical to that of the CL of HRV2 (Fig. 4B
). As we have reported previously, the replacement of the cognate PV1(M) CL with that of HRV2 CL also yields a replication phenotype (Xiang et al., 1995b
). We conclude that the consensus sequence in the D loop, as required for function in the context of PV proteins, is YYRCGG (the sequence underlined represents a tetra loop).
CL and PVcre(2C) RNA signals appear to function independently from each other
Previous studies have suggested that CL and PVcre(2C), a cis replication signal encoded in the PV P2 region (Gamarnik & Andino, 1998; Goodfellow et al., 2000
; Paul et al., 2000
; Rieder et al., 2000
), are essential for minus-strand RNA synthesis in PV replication. As pointed out before, available evidence suggests that these RNA elements accomplish their function via the formation of RNP complexes, both depending stringently upon 3CDpro. Does the CLRNP cross-talk with the creRNP complex in VPg uridylylation? To answer this question, we examined VPg uridylylation in vitro in the context of the PV chimera R14PV. Specifically, we analysed whether the inability of the HRV14 CL in R14PV to form an RNP with 3CDpro3AB or 3CDproPCBP2 has any effect on the template function provided by PVcre(2C). In vitro VPg-uridylylation reactions using 3Dpol, 3CDpro, UTP/Mg2+ and VPg with full-length viral RNA templates were carried out as described previously (Paul et al., 2000
; Rieder et al., 2000
). PVcre(2C)-dependent VPg uridylylation with chimeric RNAs as templates was as efficient as that with wt PV1(M) RNA and it was dependent strictly on the presence of 3CDpro (Fig. 7
). Thus, the replacement of CL elements at the 5'NTR by competent or non-functional structures did not result in any appreciable change in the overall PVcre(2C) activity as a template in the uridylylation reaction of VPg. These results suggest that the CL and PVcre(2C) elements function as two independent domains in their role in virus RNA replication.
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DISCUSSION |
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Based on genetic and biochemical analyses, a function of the 5'-terminal CL in PV RNA replication was proposed first by Andino et al. (1993). These authors found that the CL can form an RNP with PV 3CDpro and a host factor, identified later as PCBP2 (Blyn et al., 1996
; Gamarnik & Andino, 1997
; Parsley et al., 1997
). Originally, it was suggested that the 5'-terminal CL RNP plays a role in the initiation of plus-strand RNA synthesis (Andino et al., 1993
). Recently, however, the importance of this complex has been shifted to the initiation of minus-strand RNA synthesis via the formation of a circular plus-strand template (Barton et al., 2001
; Herold & Andino, 2001
; Teterina et al., 2001
; Paul, 2002
). Circularization has been proposed to be mediated through the affinity of the poly(A)-associated poly(A)-binding protein (PABP) to the CL-bound 3CDpro and PCBP. Whatever the role of the CL, the viral proteinase 3CDpro, which is also an RNA-binding protein (Andino et al., 1993
; Harris et al., 1994
), is the key in the specific recognition of the 5'-terminal RNA structure.
Whereas the cellular protein PCBP2 forms a specific ternary complex with CL3CDpro (Gamarnik & Andino, 1997, 2000
; Parsley et al., 1997
; Walter et al., 2002
), it can be replaced with the virus-specific protein 3AB (Harris et al., 1994
; Xiang et al., 1995a
, b
), a non-specific RNA-binding protein (Paul et al., 1994
) and the precursor for VPg. Indeed, in the presence of a 1000-fold molar excess of tRNA, the formation of the ternary complex CL3CDpro3AB is highly specific. Its significance has been supported by genetic and biochemical analyses that included different CL elements (Xiang et al., 1995b
) and numerous mutants of 3AB (Xiang et al., 1995a
). Importantly, a failure to form the CL3CDpro3AB complex in vitro co-varies in all cases with a lethal replication phenotype in vivo (Xiang et al., 1995a
, b
).
An exchange of the cognate CL of PV1(M) with that of HRV2 yields a virus with a replication phenotype (Xiang et al., 1995b). The HRV14 CL, on the other hand, cannot substitute for the PV CL (Rohll et al., 1994
; Xiang et al., 1995b
). Superficially, the three CL elements have similar structures (Fig. 1A
C). There are, however, many differences, as, for example, the deletions in the HRV CLs relative to the PV CL. However, in the context of PV replication, the integrity of the short stem (D1) at the bottom of subdomain D (Fig. 1
, compare A with B and C) cannot be essential because the HRV2/PV1(M) chimera can replicate and produce virus (Fig. 2B
) (Xiang et al., 1995b
). Similarly, the deletion of two nucleotides in the loop of subdomain B of the HRV14 CL cannot have a lethal effect in the context of a replicating PV genome. This is because a chimeric CL consisting of subdomains A, B and C of HRV14 and subdomain D of PV3(L) (Fig. 1D
), when placed to the 5' end of a PV3(L) replicon (Rohll et al., 1994
) or PV genome, promoted RNA synthesis, albeit poorly (Figs 2 and 3
). Unexpectedly, both the dicistronic and the monocistronic PV1(M) genomes carrying the chimeric CLC were not only debilitated in replication at 37 °C but also expressed a strong ts phenotype. The reason for this phenotype is obscure. A ts phenotype has been observed for some other 5' chimeras before (Johnson & Semler, 1988
; Zell et al., 1995
).
To determine the molecular basis for the failure of the HRV14 CL to substitute for the PV CL, we have changed the sequence of the HRV14 CL by site-directed mutagenesis. Based on the data described above, it was highly likely that the subdomain D within the HRV14 CL is the culprit of misfunction. A comparison of different PV and HRV subdomain D structures (Fig. 4A) revealed a consensus sequence in the loop (Fig. 4B
) that was highly likely to play a major role in CL function. This loop consensus sequence, YYRCGG (the underlined residues forming a tetra loop; Fig. 1
), is identical to that proposed recently by Zell et al. (2002)
. In contrast, the loop sequence in subdomain D of HRV14 is shorter by one residue (CUAU
G) (Fig. 4B
). Insertion of a G residue to yield the sequence CUAUGG, however, was not sufficient to rescue the genome replication of the HRV14/PV chimera (M1-R14PV). According to the studies of Zell et al. (2002)
, the mere presence of a tetra loop in subdomain D (as indicated by the underlined sequence in CUAUGG), but not its sequence, is sufficient to form a complex between CL and the viral RNA-binding protein 3Cpro. However, binding of 3CDpro alone is not sufficient to restore the replication competence of M1-R14PV and this issue is discussed below. Further mutagenesis of the loop region of the HRV14 subdomain D to yield CUACGG generated a chimeric genome (M2-R14PV) that replicated efficiently, albeit slightly slower than wt PV1(M). An additional mutation to UUACGG (M3-R14PV), leading to a sequence identical to that of the Sabin strain of PV2 [PV2(S)], did not improve virus replication. It is likely that the replication phenotypes of M2-R14PV and M3-R14PV are related to the sequences (and structures) of the HRV14 CL in stemloops AC. These subdomains are quite different from that of PV. This hypothesis is supported by the observation that the chimeric HRV2/PV genome, having the identical loop sequence in subdomain D of its HRV2 CL as PV1(S), also expresses a replication phenotype (Xiang et al., 1995b
).
The likely reason for the lethal phenotype of the HRV14/PV chimera R14/PV is the failure of the HRV14 CL to form ternary complexes with either 3CDpro3AB or 3CDproPCBP2. Fittingly, mutagenesis of the loop region of the HRV14 subdomain D that led to the rescue of replication also generated CL structures capable of interacting with the RNA-binding proteins (Fig. 6). However, the generation of a tetra loop in subdomain D of M1-R14PV did produce only patterns of very weak protein binding (Fig. 6
, lanes 2023). Significantly, M1-R14PV failed to generate viable virus. Thus, a tetra loop per se in the HRV14 CL subdomain D is not sufficient for replication function in this system. Although Zell et al. (2002)
elegantly showed that subdomain D of the HRV14 CL carrying an engineered tetra loop could bind to CVB3 3Cpro, the RNA-binding moiety of 3CDpro, binding of a protein to this domain is not sufficient for the replication function of the CL element. An additional mutation in the tetra loop of M1-R14PV was necessary to effect significant ternary complex formation, that is, recruit the third binding partner 3AB or PCBP2 (Fig. 6
, lanes 2629). Accordingly, this CL promotes replication of M2-R14PV.
Tetra loops formed by single-strand RNAs are exceptionally stable, presumably because they form complex higher order structures. These structures vary with the sequence of the tetra loop (Nowakowski & Tinoco, 1997). There are several tetra loop families known, of which one has the sequence UNCG, the sequence in subdomain D of PV1(M). In this tetra loop, the C base forms an important hydrogen bond with the phosphate on the 3' side of the U nucleotide (Allain & Varani, 1995
). In M1-R14PV, this requirement is not fulfilled. However, a tetra loop with the sequence UNCG has been generated in M2-R14PV (CUAUGG
CUACGG), the C residue 5' adjacent to the G residue being essential. Thus, in variance of the conclusions by Zell et al. (2002)
, we propose that the sequence and, consequently the structure, of the loop in domain D of the CL plays an additional important role in signalling initiation of RNA synthesis. Of course, the tetra loop (CNCG) in the CL of PV2(L) and PV3(L) (Fig. 4B
) is slightly different than the tetra loop of PV1(M) (UNCG), yet it functions efficiently, even in serotype chimeras (for example, in recombinants between serotypes). Moreover, tetra loops in subdomain D of CL elements are clearly not essential for some members of the genus Rhinovirus, as, for example, HRV14. The HRV14 replication proteins are perfectly happy with the small loop in subdomain D of their CL. It is noteworthy that the HRV14 replication proteins are also perfectly happy with a PV1(M) CL carrying a tetra loop. Todd et al. (1997)
reported that a chimeric genome in which the entire 5'-terminal NTR of PV was spliced onto the ORF and 3'NTR of HRV14 replicated as efficiently as the parental HRV14. These considerations underline the complexity of the structure/function relationships in PV RNA replication (Agol et al., 1999
).
To what extent the structure of the stem domains of subdomain D in the HRV14 CL play a role in protein binding remains to be seen (Walker et al., 1995). In the context of the PV1(M) replication machinery, these domains appear to play a minor role.
It should be noted that the CL in natural plus-strand template RNAs is linked covalently at the 5' end to VPg (VPgpUpU) (Lee et al., 1977; Wimmer et al., 1993
). In PV protein synthesis, all viral (genome-length) mRNA has been separated from VPg by a cellular phosphodiesterase (Gulevich et al., 2002
; Hewlett et al., 1976
; Nomoto et al., 1976
) and, thus, has a 5'-terminal pUU. It is not clear whether the incoming, infecting genomic RNA undergoes this modification also. RNA free of VPg is infectious, but the specific infectivity of such RNA, for example, transcript RNAs derived from plasmids, is lower by as much as one order of magnitude as compared to VPg-linked virion RNA. This is true even if the transcript RNA, synthesized with phage T7 RNA polymerase (van der Werf et al., 1986
), carries a ribozyme moiety at the 5' end, thereby yielding mostly a pUU terminus (Herold & Andino, 2000
; D. W. Kim & E. Wimmer, unpublished results). All biochemical studies of ternary complex formation have been carried out with CL RNAs synthesized with T7 RNA polymerase. Thus, these riboprobes are not covalently linked to VPg. Consider that the C-terminal VPg portion of 3AB (3B=VPg) is responsible for the RNA-binding activity of 3AB (Xiang et al., 1995a
). It is not known to what extent the covalent attachment of VPg to the CL influences ternary complex formation. Could it be that VPg-linked CLs can form complexes with 3CDpro, for example, 3CDproVPg-CL, which would be necessary and sufficient to promote RNA replication? If so, the formation of CL3CDproPCBP2 or CL3CDpro3AB complexes may be important only during a specific phase in replication, perhaps in the very initial phase. Currently, this important problem cannot be solved because sufficient quantities of VPg-linked CL cannot be synthesized in vitro. We have ignored this fascinating question in this study also.
The first step in PV RNA synthesis is the uridylylation of VPg by 3Dpol, a reaction that provides the primer for the RNA polymerase. The reaction requires a cis-acting replication signal, termed cre, and has been identified in all picornaviral RNAs analysed so far (Gerber et al., 2001; Goodfellow et al., 2000
; Lobert et al., 1999
; Mason et al., 2002
; McKnight & Lemon, 1996
, 1998
). Interestingly, cre elements in PV RNA are located at vastly different positions within the viral genome. In PV, the cre(2C) maps to the coding region of the viral protein 2CATPase (Goodfellow et al., 2000
). Functional cre elements can be moved within the PV genome to very different locations, even between the CL and the IRES (Yin et al., 2003
). Available evidence suggests that the sole function of the cre element is to serve as specific template in the uridylylation of VPg (Paul et al., 2000
; Rieder et al., 2000
). Importantly, cre-dependent uridylylation requires 3CDpro as a co-factor, just like the function of the 5'-terminal CL. It is interesting to point out that our in vitro experiments of VPg uridylylation contrast with some studies reported by Lyons et al. (2001)
using the translation/replication system. Those results indicate that the CL is important for VPg uridylylation and minus-strand RNA synthesis. Considering the proposed, complex circular RNA structure involved in the initiation of PV RNA synthesis, it seems possible that the protein complex formed in cre-dependent uridylylation may interact functionally with the RNP formed at the 5'-terminal CL. The data presented here suggest that cre and CL function independently from each other.
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ACKNOWLEDGEMENTS |
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Received 27 November 2002;
accepted 15 April 2003.