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Previous Article | Next Article 
Journal of Virology, January 1999, p. 427-435, Vol. 73, No. 1
Department of Molecular, Cellular and
Developmental Biology and Howard Hughes Medical Institute, University
of Colorado, Boulder, Colorado 803091;
Department of Biochemistry, Biophysics and Genetics, University
of Colorado Health Sciences Center, Denver, Colorado
802622; and
Department of Microbiology
and Immunology, Stanford University School of Medicine, Stanford,
California 943053
Received 22 May 1998/Accepted 16 September 1998
Poliovirus RNA genomes that contained deletions in the
capsid-coding regions were synthesized in monkey kidney cells that constitutively expressed T7 RNA polymerase. These replication-competent subgenomic RNAs, or replicons (G. Kaplan and V. R. Racaniello, J. Virol. 62:1687-1696, 1988), were encapsidated in
trans by superinfecting polioviruses. When superinfecting
poliovirus resistant to the antiviral compound guanidine was used, the
RNA replication of the replicon RNAs could be inhibited independently
of the RNA replication of the guanidine-resistant helper virus.
Inhibiting the replication of the replicon RNA also profoundly
inhibited its trans-encapsidation, even though the capsid
proteins present in the cells could efficiently encapsidate the helper
virus. The observed coupling between RNA replication and RNA packaging
could account for the specificity of poliovirus RNA packaging in
infected cells and the observed effects of mutations in the coding
regions of nonstructural proteins on virion morphogenesis. It is
suggested that this coupling results from direct interactions between
the RNA replication machinery and the capsid proteins. The coupling of
RNA packaging to RNA replication and of RNA replication to translation
(J. E. Novak and K. Kirkegaard, Genes Dev. 8:1726-1737, 1994)
could serve as mechanisms for late proofreading of poliovirus RNA,
allowing only those RNA genomes capable of translating a full
complement of functional RNA replication proteins to be propagated.
The mechanism of packaging of
picornavirus RNA has not been determined, and simple questions such as
whether a packaging signal or sequence that confers specificity to the
RNA packaging reaction have not been answered. Poliovirus
positive-sense RNAs are specifically packaged in infected cells:
cellular mRNAs, rRNAs, tRNAs, and negative-sense poliovirus RNAs are
excluded from the capsids (34, 35). Sensitive
trans-encapsidation assays have shown that replicating RNA
genomes derived from poliovirus type 1 can be encapsidated by virion
proteins encoded by any of the three poliovirus serotypes but not by
capsid proteins from the related picornaviruses coxsackievirus A21 or
B3 or enterovirus 70 (45). What determines the specificity of poliovirus RNA packaging? RNA genomes that contain deletions of most
of the capsid-encoding region of the genome can be packaged in
trans by coinfecting genomes (26, 39), arguing
that a unique packaging signal, if it exists, does not lie between
nucleotides (nt) 756 and 2956. Similarly, the ability of 5' and 3'
noncoding sequences from other picornaviruses, which are not
encapsidated by poliovirus capsids during coinfections, to replace the
poliovirus 5' and 3' noncoding regions argues that nt 108 to 740 and
7385 to 7440 also do not contain specific packaging sites (1,
57).
One explanation (20, 38, 49) for the observed specificity of
RNA packaging in poliovirus-infected cells is that, as first proposed
by Baltimore (4), poliovirus RNA replication and packaging
are directly coupled and only RNA genomes that are actively being
replicated can be packaged. Several published observations are
consistent with the hypothesis that only newly replicated poliovirus
positive RNA strands are encapsidated. For example, when
poliovirus-infected cells were pulse-labeled with
[3H]uridine, radioactivity was detected in the mature
virion peak within 5 min (5). However, newly synthesized
35S-labeled proteins required 30 min on average to move
into the virion peak (5). By electron microscopy, RNA
replication complexes and virion precursors colocalize to the
membranous vesicles that proliferate during infection (40),
and after pulse labeling with [3H]uridine, virions with
the highest specific activity were found in direct association with the
membranes on which RNA replication occurs (13).
Biochemical and genetic studies have suggested the possibility of
direct interactions between proteins of picornavirus replication complexes and capsid proteins. For example, the virions of
foot-and-mouth disease virus contain measurable amounts of 3D, the
viral RNA-dependent RNA polymerase (32). Poliovirus protease
3CD shows higher specificity for capsid precursors than does 3C alone,
arguing that the 3D sequences confer some binding energy to the capsid
proteins (12, 63). Finally, RNAs that bear certain mutations
in proteins 2C (28) and 3D (17) of the RNA
replication complex can program the production of virions with altered
cell entry properties.
The effects of compounds that inhibit poliovirus RNA replication and
RNA packaging have also suggested a mechanistic link between these two
processes. Low concentrations of guanidine hydrochloride specifically
inhibit the poliovirus replicative cycle; both RNA synthesis and the
formation of mature virus particles quickly cease following treatment
of infected cells with 0.1 to 2.0 mM guanidine hydrochloride
(21). Although originally it was thought that guanidine
might have some effect on morphogenesis itself, it has since become
clear that guanidine directly inhibits RNA replication (reviewed in
reference 62). For example, mutations that confer
resistance to guanidine map to the 2C region of the poliovirus genome
(42) and mutations in the 2C coding region that cause
defects in RNA replication (27) or suppress the cold sensitivity of wild-type virus (18) have been identified.
Furthermore, the addition of guanidine to cell extracts that support
poliovirus RNA replication was shown to inhibit the initiation of
negative-strand synthesis (8) and to prevent the further
packaging of positive-strand RNA (29). Therefore, either the
inhibition of RNA packaging by guanidine derives from a requirement for
nascent RNA in RNA packaging, or protein 2C is also directly involved
in virion assembly, or both. Recently, hydantoin, a compound that
inhibits the growth of both poliovirus and coxsackievirus A3, has been
shown to inhibit poliovirus morphogenesis without affecting RNA
replication (60). Mutations in the poliovirus genome that
confer resistance to hydantoin map to the 2C region of the viral genome
(60). Assuming that these mutations exert their effect
through the change in the amino acid sequence of 2C, a direct role for
2C in virion assembly is suggested. Since 2C protein is firmly embedded
in the intracellular membranes on which poliovirus RNA replication
occurs (10, 11, 30, 40, 52), a physical interaction between
poliovirus virion assembly and RNA synthesis is implied whether the
role of the 2C protein in RNA packaging is direct or indirect.
Several review articles have described previously published data
linking poliovirus RNA replication and RNA packaging (20, 49), attempts to identify RNA-packaging sites (2), and
the use of trans-encapsidation to create poliovirus vectors
(2). In the present study, we used RNase protection
experiments to demonstrate the trans-encapsidation of
poliovirus replicon RNAs in infected cells and to monitor this
trans-encapsidation when such RNAs are associated with
productive replication complexes and when they are not. Preaccumulated
RNAs are not packaged in trans by capsids made from a
coinfecting helper virus, even under conditions in which the helper
virus is actively packaging its own RNAs and the preaccumulated RNA is
stable. Therefore, only newly synthesized poliovirus RNAs are packaged.
The requirement of new RNA synthesis for RNA packaging is likely to be
due to a requirement for direct association with the RNA replication complex.
Cell lines.
The KJT7 cell line, which constitutively
expresses T7RNA polymerase, was constructed from CV-1 cells by the
stable introduction of two plasmids: pSV2-neo (54), which
confers resistance to the antibiotic G418, and pAR3126 (19),
which contains the bacteriophage T7 gene 1 that does not contain a
nuclear localization sequence. The cells were maintained in Dulbecco's
modified Eagle's medium (DMEM) supplemented with 10% calf serum, 1%
penicillin-streptomycin, and 500 µg of G418 (Geneticin; GibcoBRL) per
ml. HeLa cells were maintained as previously described (25).
Virus stocks.
Wild-type stocks of type 1 Mahoney poliovirus
were amplified from individual plaque isolates obtained from
transfection of HeLa cells with a plasmid, pPolio (17),
containing the full-length poliovirus genome (46).
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Functional Coupling between Replication and
Packaging of Poliovirus Replicon RNA
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Plasmids. Plasmids containing the T7 R2 and T7 R3 poliovirus constructs (22) were constructed by Janet Novak by deletion of sequences 1175 to 2956 and 1175 to 2471, respectively, in the T7pGempolio plasmid (51). The Z polymorphism was engineered into the T7R2 and T7R3 plasmids by replacing the DNA sequences from nt 675 to 702 with the following heterologous sequence: GGTCGACCAAATCTCTTCGAACTTTAGAAACACCGTGTCTGCCTTCGTAAAAGGTACCAGTCGACGGG CCAGCTGGTTTAGAGAAGCTTGAAATCTTTGTGGCACAGACGGAAGCATTTTCCATGGTCAGCTGCCC
The phenotype of virus that contains the Z substitution is indistinguishable from that of wild-type virus (53a). The plasmid construct, pBSIISK+RZ, used for transcription of the probes for RNase protection contains the 68-bp Z sequence and sequences 702 to 742 from the wild-type poliovirus cDNA (47), cloned between a T3 promoter and a T7 promoter in the pBSIISK+ vector (Stratagene).DNA transfection. KJT7 cells were transfected with supercoiled plasmids as described previously (6). For the transfection mixture, 7 µg of DNA in 0.5 ml of OptiMEM was mixed with 70 µl (70 µg) of Lipofectin reagent (Gibco-BRL) in 1.5 ml of OptiMEM, allowed to sit at room temperature for 10 to 20 min, and placed on subconfluent cell monolayers (approximately 2 × 107 cells on a 100-mm plate) that had been washed twice with OptiMEM. An additional 2 ml of OptiMEM was then added, and the cells were placed in a CO2 incubator at 32.5 or 37.5°C. After 10 to 15 h, the lipofection mix was removed and 10 ml of DMEM containing 10% calf serum was added to each plate for the remainder of the incubation period.
Superinfections. Transfected or mock-transfected cells were infected with virus stocks at multiplicities of infection of 20 to 100 PFU/cell, and cytoplasmic extracts were prepared as described previously (36). Virus stocks were made by resuspending cell pellets containing ~2 × 107 cells in 0.5 ml of phosphate-buffered saline containing 0.01% CaCl2 and 0.01% MgCl2 (PBS+) and freeze-thawing the cells three times to lyse them. To make virus stocks that were free from full-length unpackaged RNAs, the virus stock was treated at room temperature with 17 µg of RNase A per ml for 20 min.
To infect HeLa cell monolayers (~2 × 107 cells) with only encapsidated RNAs, plates of cells were exposed to 100 µl of the various treated virus stocks for 30 min at 37°C, medium containing DMEM and 10% calf serum was added, and incubation was continued either at 37°C for 5 h or at 32.5°C for 10 h as indicated.Plaque assays. Plaque assays on HeLa and KJT7 cell monolayers were performed as previously described (23). The virus was placed on the cells in PBS+ in a total volume of 200 µl. The mixture was incubated for 30 min at 32.5°C to allow the virus to adsorb to the cells, and then 5 ml of 1× DMEM-10% calf serum (Gibco-BRL)-1% agar was added to each plate. For virus that was grown in the presence of 0.5 mM guanidine-HCl, the drug was added to the agar overlays. After incubation for 3 days (32.5°C), the agar overlays were removed and the cells were stained with crystal violet.
Preparation of cytoplasmic and viral RNA. To prepare RNA from frozen pellets of approximately 6 × 107 cells, the cells were thawed on ice and lysed as described previously (36). To prepare cytoplasmic RNA, 10 mM vanadate ribonucleoside complexes (New England Biolabs) was added to each cell aliquot. The lysates were clarified by centrifugation at 4°C for 8 min at 1,600 × g. The supernatants were transferred to fresh tubes, 5 µg of tRNA and sodium dodecyl sulfate (SDS) to 0.5% were added, and the lysates were extracted with buffer-saturated phenol. The aqueous phases were collected, EDTA was added to 1 mM, and the RNA was precipitated by the addition of 0.1 volume of 3 M sodium acetate and 3 volumes of ethanol, freezing on dry ice, and centrifugation.
To prepare encapsidated RNA, cell lysates were incubated with 12 U of DNase I (Promega) per ml, 30 µg of RNase A (Amersham/USB) per ml, and 12 U of RNase T1 (Gibco-BRL) per ml for 25 to 45 min at room temperature to digest the unprotected RNA and DNA and clarified by centrifugation at 4°C and 1,600 × g for 8 min. SDS to 0.1% was then added to the supernatant, and the virion particles were collected by centrifugation through sucrose step gradients. The gradients were composed of 1 ml of 45% sucrose in PBS+ that contained 0.1% SDS, 2.5 ml of 30% sucrose in PBS+, and 0.5 to 1.0 ml of virion preparation. Following centrifugation in an SW50.1 rotor (Beckman) for 4 h 10 min at 49,000 rpm at 4°C, the virion pellets were resuspended in 1 ml of RSB (10 mM TrisHCl, 10 mM NaCl, 1.5 mM MgCl2) with 10 U of RNase T1 per ml. After incubation at room temperature for 20 to 30 min, the virions were pelleted through a second sucrose step gradient as described above. After centrifugation, the virions were resuspended in 200 µl of TNE (10 mM TrisHCl, 10 mM NaCl, 1 mM EDTA) containing 10 mM vanadate ribonucleoside complexes, 10 µg of tRNA, 0.5% SDS, and 1% Brij 35. SDS was added to 1%, and the solution was extracted with phenol taking both the supernatant and the interface. The virions were then extracted twice with phenol-chloroform-isoamyl alcohol. Finally, the RNA was precipitated by the addition of 0.1 volume of 3 M sodium acetate and 3 volumes of ethanol and collected by centrifugation. To test whether the viral RNA preparations were free from contamination by unpackaged RNAs, one pellet of ~6 × 107 mock-transfected, superinfected cells was deliberately contaminated with 31 fmol of in vitro-transcribed T7R2Z RNA before viral and cytoplasmic RNAs were prepared. RNase protection of the viral RNA from these cells shows no protected band corresponding to the R2Z sequence, indicating that the virions were successfully purified from the abundant contaminating transcript present in the cytoplasmic RNA (data not shown).RNase protection.
RNase protection assays were performed as
previously described (35). To detect positive-strand R2Z,
R3Z, wild-type poliovirus, and 3NC202 viral RNAs, the RNA probe was
transcribed from BstUI-digested pBSIISK+RZ by
bacteriophage T7 RNA polymerase (Promega). To detect negative strands,
excess positive-strand RNAs were removed by a cycle of RNase protection
in the absence of added probe (35) and then the probe was
synthesized by T3 RNA polymerase from pBSIISK+RZ.
Transcription reaction mixtures contained 570 Ci of
[
-32P]UTP per mmol of UTP. The labeled probes were
purified by electrophoresis through a 5% polyacrylamide gel and
eluted. The samples were denatured at 85°C for 5 min and hybridized
overnight at 60 to 61°C. RNase digestion was performed in an
ice-water bath for 60 min in a high-salt RNase cocktail (500 mM NaCl,
10 mM Tris-HCl [pH 7.5], 5 mM EDTA, 4.5 µg of RNase A per ml, 350 U
of RNase T1 per ml). The samples were treated with
proteinase K, extracted with phenol, precipitated with ethanol and 5 µg of tRNA, and loaded onto 8% polyacrylamide-8 M urea gels. The
radioactivity in the bands was quantitated by phosphorimager analysis.
The number of moles of RNA represented was determined from the
protection signals of known amounts of RNA included in standard curves.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Packaging and superinfection assays. To measure encapsidation of one poliovirus RNA genome in trans by proteins encoded by another poliovirus genome, the RNA genomes diagrammed in Fig. 1A were used. R2Z and R3Z are RNA molecules which encode all of the viral proteins required for poliovirus RNA synthesis but contain, within their capsid coding regions, in-frame deletions of 1,791 and 1,296 nt, respectively (22). These replicon RNAs thus cannot be packaged by capsid proteins encoded by their own genomes and can be packaged only in trans by coinfecting virus. trans-encapsidation of replicon RNAs by capsid proteins expressed from recombinant vaccinia virus has been reported previously (3, 44). In the present study, however, the capsid proteins are provided by superinfecting poliovirus, more closely resembling the circumstance of naturally occurring defective interfering particle RNAs (15, 26, 39). To allow quantitative discrimination between replicon and helper viral RNAs, both R2Z and R3Z replicon RNAs bear a 68-nt insertion in their 5' noncoding regions of the RNA (Z in Fig. 1A) that does not alter the phenotype of viruses or replicons that contain it (37).
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R2Z and R3Z RNAs are encapsidated in trans. To determine whether R2Z and R3Z RNAs could be encapsidated in trans by the capsid proteins provided by wild-type poliovirus, KJT7 cells transfected with plasmids encoding T7R2Z and T7R3Z were incubated at 32.5°C for 24 h, superinfected with wild-type poliovirus, and incubated for an additional 7 h at 32.5°C (Fig. 1B). Cytoplasmic extracts were prepared, and RNase protection was performed to display the total amounts of replicon and wild-type positive-strand RNAs in the cells (Fig. 3, lanes 6 to 10). A single RNA probe was used to detect both the replicon RNAs and the superinfecting viral RNAs. This RNA probe contained 76 nt of wild-type poliovirus sequence and the 68-nt Z insert present in the replicon RNAs (Fig. 1A). R2Z and R3Z replicon RNAs each protected 138 nt of the radioactive RNA probe, whereas RNAs from superinfecting viruses that did not contain the insertion protected only 76 nt of the labeled probe. The smaller size of the RNA protected by the full-length viral RNAs also allowed the use of hybridization conditions that were somewhat destabilizing to the 76-bp RNA duplex but not to the 138-bp duplex: this allowed the visualization of both the replicon RNAs, present in only the transfected cells, and the more abundant viral RNAs, present in all the cells in the population, in a single experiment. As shown in Fig. 3, R2Z and R3Z replicon RNAs accumulated in cells in both the presence and absence of superinfecting wild-type poliovirus RNA (lanes 6 to 10).
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Sensitivity of R3Z RNA replication to 0.5 mM guanidine is not efficiently rescued by superinfection with guanidine-resistant 3NC-202guaR virus. To test the effect of ongoing replication of replicon RNAs on their ability to be packaged in trans, we needed a method to inhibit the replication of R3Z replicon RNA but not to inhibit the replication of superinfecting helper virus RNA. Mutations that confer resistance to guanidine have been characterized (41): most of them fall within the coding region for nonstructural protein 2C, and, curiously, many of them are cis dominant (24, 58, 59). That is, in coinfections between guanidine-sensitive wild-type viruses and certain guanidine-resistant mutant viruses, the wild-type virus is not rescued and the guanidine-resistant virus is not inhibited by the presence of guanidine. The guanidine-resistant allele used in the present study, present in the virus 3NC-202guaR, has been previously shown to be cis dominant in viral plaque assays (24). The 3NC-202guaR virus contains two mutations in the 2C coding region that together confer both guanidine resistance and temperature sensitivity (Fig. 1A) (24); an 8-nt insertion in the 3' noncoding region also confers a temperature-sensitive phenotype (50).
To be useful in trans-encapsidation experiments, we needed a guaR allele that did not rescue the guanidine sensitivity of R3Z RNA replication in RNase protection assays or in the plaquing-efficiency experiments performed previously (24). To test whether R3Z RNA replication in the presence of guanidine was rescued by superinfecting 3NC-202guaR virus, KJT7 cells were transfected with T7R3Z DNA and incubated at 32.5°C for 24 h to allow R3Z RNA to accumulate in the absence of drug. Subsequently, 0.5 mM guanidine was added and incubation at 32.5°C was continued for an additional 13 h. RNase protection (35) was used to monitor the accumulation of R3Z-positive and -negative strands. Similar to its effect on wild-type virus, the presence of 0.5 mM guanidine inhibits the accumulation of R3Z RNA (Fig. 4). Even in the presence of superinfecting 3NC-202guaR virus, guanidine significantly inhibited the accumulation of both positive-strand (Fig. 4A) and negative-strand (Fig. 4B) R3Z RNA, although a small increase in the amount of both R3Z RNA species was observed. Therefore, in the presence of 0.5 mM guanidine, conditions could be achieved in which a large amount of previously synthesized R3Z RNA was present but not efficiently replicating in the cytoplasm of cells in which the 3NC-202guaR virus was undergoing active RNA replication.
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Testing the trans encapsidation of R3Z RNA in the presence and absence of guanidine. To test whether preaccumulated R3Z RNAs could be packaged when they are not being actively replicated, KJT7 cells were transfected with T7R3Z DNA and incubated at 32.5°C for 24 h to accumulate R3Z RNA. They were then superinfected with 3NC-202guaR virus and incubated at 32.5°C for 13 h in the absence or presence of 0.5 mM guanidine. To determine the amounts of total R3Z and 3NC-202guaR RNA present in the KJT7 cells, RNase protection was performed on the total cytoplasmic extracts (Fig. 5A). Accumulated R3Z RNA was easily detectable in cytoplasmic extracts from guanidine-treated cells whether or not they had been superinfected with 3NC-202guaR virus (Fig. 5A).
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Encapsidation of poliovirus RNAs in trans has been frequently documented as the mechanism by which defective interfering particles are propagated (15, 26, 33), as a technique to test the specificity of packaging (39, 45), and as a tool to propagate poliovirus-derived RNAs that contain exogenous sequences (39, 44). Poliovirus genomes can be trans-encapsidated by capsids of the same serotype provided by helper viruses, by other serotypes of poliovirus, and by capsid proteins expressed by other viruses such as vaccinia virus (reviewed in references 2 and 20). Here, we used a trans-encapsidation system to test the effect of active RNA replication on the ability of subgenomic poliovirus RNAs to be packaged.
trans encapsidation of poliovirus replicon R2Z and R3Z genomes, which contain deletions of nt 1175 to 2470 and 1175 to 2956, respectively, in the capsid-coding region (22), was readily observed (Fig. 3; Table 1). When RNA replication of R3Z replicon RNA in KJT7 cells was inhibited by the addition of guanidine, preaccumulated R3Z RNA remained in the cytoplasm for several hours (Fig. 5A). When these cells were superinfected with 3NC-202guaR virus, whose RNA replication was not inhibited by guanidine, little rescue of either positive- or negative-strand R3Z RNA synthesis was seen (Fig. 4), even though the replication and packaging of the 3NC-202guaR virus continued unabated (Fig. 5B). Therefore, the guanidine-inhibited replication complex associated with the R3Z RNA is not a trans-dominant inhibitor of 3NC-202guaR RNA packaging.
The two populations of poliovirus-derived RNAs were apparently recognized quite differently by the viral capsid proteins encoded by the 3NC-202guaR virus. The 3NC-202guaR viral RNAs, associated with functional replication complexes, were efficiently packaged, but the R3Z replicon RNAs, either free in the cytoplasm or associated with guanidine-inhibited RNA replication complexes, were not (Fig. 5; Table 2). Why would the ability of these RNAs to be packaged be so different? The coupling between RNA replication and RNA packaging could arise by several possible mechanisms.
If some transient physical property of newly synthesized RNA, such as a short-lived RNA structure or linkage to VPg, were required for packaging, only newly synthesized RNAs in the immediate vicinity of the replication complex would be packaged. However, no evidence exists for such a transient structure or linkage, and this explanation would not account for the effect of mutations in 2C (28, 60) and 3D (17) on virion function.
The possible direct physical interaction between the RNA replication complex and the assembling virion particles is shown schematically in Fig. 6. If the RNA replication complex needed to be actively replicating RNA to allow a productive association with virion precursors, a good explanation for the coupling between RNA synthesis and RNA packaging would be provided. The direct involvement of viral proteins 2C and 3D in this interaction would rationalize the effect of mutations in these coding regions on the capsid structure (17, 28, 60). If specific interactions between the proteins of the RNA replication complex and capsid proteins were required for successful RNA packaging, it is possible that they would even be sufficient for the specificity of RNA packaging (20, 38, 49). That is, even though interactions between the capsid proteins and specific RNA sequences may yet be identified, they would not be required for the specificity of RNA encapsidation if the coupling of RNA replication and RNA packaging is accomplished by specific protein-protein interactions (Fig. 5).
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Direct coupling between genome replication and packaging has been documented for a few other viruses that are not closely related to poliovirus. When synthesis of the single-stranded DNA genome of minute virus of mice (a parvovirus) is inhibited, assembly of the virus ceases immediately (48). Furthermore, no free single-stranded DNA can be found in infected cells (56). It has been proposed that the viral DNA is either stripped from its template by the process of encapsidation or driven off the template by new DNA synthesis and directly incorporated into empty capsids (reviewed in reference 16). For hepatitis B virus, a ribonucleoprotein complex that includes the viral reverse transcriptase is required for specific encapsidation of the pregenomic RNA (7, 14, 43, 61). Thus, the polymerase protein is a key participant in both reverse transcription and genome packaging, and although its role in packaging is not thought to involve its enzymatic activity (7), this role may help ensure that it is included in the newly formed capsids for the subsequent reverse transcription. Although poliovirus has not been reported to encapsidate its polymerase, detectable amounts of the RNA-dependent RNA polymerase of foot-and-mouth disease virus have been reported to copurify with virions (32).
Direct transfer of RNA between cytoplasmic macromolecular complexes has been documented in the case of the "channeling" of cellular tRNAs. Specifically, attempts to detect or create a free cytoplasmic pool of aminoacylated tRNAs have not been successful. Instead, there appears to be direct transfer of charged tRNAs from aminoacyl synthetases to the A site of the ribosome (31) and from the P site back to the synthetases (55). As we have suggested for the transfer of nascent poliovirus positive-strand RNA from the replication complex to the capsid precursors, the protein-protein interactions may help provide some of the specificity to the recognition of charged amino-acylated tRNAs by the ribosome.
For an RNA genome with a mutation rate estimated to be as high as
6 × 10
4 mutations/nucleotide synthesized (reviewed
in reference 62), coupling of RNA encapsidation to
RNA synthesis and of RNA synthesis to translation (36) could
be of evolutionary significance. Specifically, a requirement for
successful translation of the poliovirus genome before it is used as a
template for RNA synthesis should prevent the replication of RNA
genomes that contain frameshift and nonsense mutations (36).
Then the coupling between RNA synthesis and RNA packaging imposes a
requirement that RNAs to be packaged must emerge from active
replication complexes, ensuring that the templates for those RNAs were
competent to form such complexes. Thus, the coupling between
translation and RNA replication and the coupling between RNA
replication and RNA packaging can impose a form of late proofreading to
the error-prone RNA genome of poliovirus.
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ACKNOWLEDGMENTS |
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We thank Eric Simoes for providing viruses and plasmids containing insertions in the 5' noncoding region, Janet Novak for assistance with RNase protection experiments, Dana Dodd for critical reading of the manuscript, and John Lyle for help with phosphorimager quantitation.
This work was supported by NIH grant AI25166 to K.K., NIH grant AI25105 to P.S., and a fellowship from the Colorado Institute for Research in Biotechnology to C.I.N.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology and Immunology, Stanford University School of Medicine, Sherman Fairchild Science Bldg., 299 Campus Dr., Stanford, CA 94305-5124. Phone: (650) 498-7075. Fax: (650) 498-7147. E-mail: karlak{at}leland.stanford.edu.
Present address: Department of Molecular and Human Genetics, Baylor
College of Medicine, Houston, TX 77030.
Present address: Department of Microbiology, University of
Alabama, Birmingham, AL 35294.
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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