Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M412684200 on December 17, 2004

J. Biol. Chem., Vol. 280, Issue 10, 8748-8755, March 11, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/10/8748    most recent
M412684200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chetverin, A. B.
Right arrow Articles by Ugarov, V. I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chetverin, A. B.
Right arrow Articles by Ugarov, V. I.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Viral RNA-directed RNA Polymerases Use Diverse Mechanisms to Promote Recombination between RNA Molecules*

Alexander B. Chetverin{ddagger}, Damir S. Kopein, Helena V. Chetverina, Alexander A. Demidenko§, and Victor I. Ugarov

From the Institute of Protein Research of the Russian Academy of Sciences, Pushchino, Moscow Region, 142290 Russia

Received for publication, November 9, 2004 , and in revised form, December 17, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
An earlier developed purified cell-free system was used to explore the potential of two RNA-directed RNA polymerases (RdRps), Q{beta} phage replicase and the poliovirus 3Dpol protein, to promote RNA recombination through a primer extension mechanism. The substrates of recombination were fragments of complementary strands of a Q{beta} phage-derived RNA, such that if aligned at complementary 3'-termini and extended using one another as a template, they would produce replicable molecules detectable as RNA colonies grown in a Q{beta} replicase-containing agarose. The results show that while 3Dpol efficiently extends the aligned fragments to produce the expected homologous recombinant sequences, only nonhomologous recombinants are generated by Q{beta} replicase at a much lower yield and through a mechanism not involving the extension of RNA primers. It follows that the mechanisms of RNA recombination by poliovirus and Q{beta} RdRps are quite different. The data favor an RNA transesterification reaction catalyzed by a conformation acquired by Q{beta} replicase during RNA synthesis and provide a likely explanation for the very low frequency of homologous recombination in Q{beta} phage.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Recombinations (sequence exchange and rearrangements) between and within RNA molecules are rare but biologically important events contributing to the evolution and diversity of RNA viruses (1, 2) and generating defective interfering RNAs that attenuate viral infections (3). In contrast to splicing and other types of regular RNA rearrangements, recombinations occur without apparent sequence or structure specificity (1, 2). There are indications that recombination may occur between cellular RNAs (4–6), eventually resulting, by means of reverse transcription and integration, in alterations in the chromosomal DNA. Spontaneous Mg2+-catalyzed rearrangements in RNA sequences (7) might have been a mechanism for evolution in the prebiotic RNA world and might have evolved into contemporary sequence-specific ribozyme-catalyzed reactions (8, 9).

RNA recombination was discovered more than 40 years ago as an exchange of genetic markers between polioviruses (10, 11), and since then similar approaches were used to demonstrate that genomes of RNA viruses of animals, plants, and bacteria are all capable of recombination (2, 4, 12, 13). However, such in vivo experiments utilizing living cells, as well as in vitro studies that used crude cell lysates could not uncover the underlying molecular mechanisms or even definitely answer the question if recombining entities were RNA molecules themselves or their cDNA copies, because every living cell contained enzymes capable of reverse transcription and appropriate dNTP substrates. It became evident that further progress in this field depended on the availability of adequate in vitro systems whose composition and other parameters can be strictly controlled by the experimenter (2, 14).

The first example of such a sort has been the cell-free system employing purified Q{beta} replicase, RNA-directed RNA polymerase (RdRp)1 of bacteriophage Q{beta} (15). The system also includes two RNA molecules, "5' fragment" and "3' fragment," whose sequences supplement each other to the entire sequence of RQ135 RNA, an efficient Q{beta} replicase template (16), and are derived from the 5' and 3' segments of that RNA, respectively. None of the fragments alone can be amplified by Q{beta} replicase; however, fusion of their sequences in a manner as they are arranged in the original RQ RNA results in the appearance of replicable molecules (15), which are detected and counted by using the Q{beta} replicase version of the molecular colony technique (17, 18). To this end, a mixture of the fragments is seeded on a Q{beta} replicase-containing agarose layer, which is then covered with a nylon membrane impregnated with replicase substrate ribonucleoside triphosphates (rNTPs) to initiate replication. As the reaction takes place in agarose, copies of replicable RNAs concentrate around the progenitor templates in the form of RNA colonies. The colonies are detected by hybridizing the membrane with a labeled probe, and their number reflects the recombination frequency. Experiments in this system proved that recombination can occur between RNA molecules directly, without involving DNA intermediates (15). However, many features of the cell-free RNA recombination appeared to be different from those usually observed in the in vivo studies.

Studies on recombination in RNA viruses mainly revealed homologous recombination (1, 2), in which sequences surrounding the crossover site in the recombination substrates (recombining RNAs or segments of an RNA molecule) and in the product molecule are entirely or essentially identical to each other. To explain homologous recombination, a template switch (copy choice) mechanism was proposed (19), according to which the recombinant molecule is generated as a by-product of RNA synthesis by viral RdRp, which after copying a portion of the first ("donor") template, occasionally switches to another ("acceptor") template containing a sequence complementary to the growing end of the nascent strand. If that mechanism operated in the cell-free Q{beta} system, the 3' and 5' fragments would serve as donor and acceptor templates, respectively (Fig. 1A). However, no homologous recombinants were generated in this system, even though the fragments were provided with homologous foreign sequences to facilitate template switching. Most recombinant molecules contained the entire sequence of the 5' fragment fused with sequence of the 3' fragment, either intact or 5'-truncated to a various extent (Fig. 1B; Ref. 15). Most importantly, recombination was totally prevented when the 3' hydroxyl at the 5' fragment terminus was either removed by periodate oxidation or blocked by a phosphate group. The key role of the 3' hydroxyl of the acceptor template could not be accounted for by the template switch mechanism, but well conformed to a hypothesis that RNA recombination occurs as a transesterification reaction in which the 3' hydroxyl of the 5' fragment attacks phosphate groups within the 3' fragment. In the absence of any indication of the ability of RNA polymerases to promote such reactions, it was suggested that recombinants arose because of a site-nonspecific self-splicing activity of RNA molecules (15).



View larger version (39K):
[in this window]
[in a new window]
  		FIG. 1.
Substrates of RNA recombination. The sequences originated from RQ135 RNA (16) are shown only partially by white letters on black background; homologous (or complementary) stretches of foreign sequences are shown on gray background. A, homologous recombinant RNA that would be generated from the 5'(BamHI) and 3' fragments by the template switch mechanism. Such a recombinant was produced by reverse transcriptase from avian myeloblastosis virus (15). B, the nonhomologous recombinant RNA most frequently produced from the same fragments by Q{beta} replicase in the purified cell-free system (15). C, homologous recombinant RNA that would be generated from the same fragments by the PAAE mechanism (22) provided that the 3' fragment is copied along its entire length and the copy (3'C fragment) dissociates from the template. D, a nonhomologous recombinant RNA that might be generated by the PAAE mechanism when the 3'C fragment remains base-paired to the 3' fragment and priming occurs on occasional melting of the duplex terminus. E, the pairs of opposite polarity fragments used in this work to probe the PAAE mechanism.

 
Such an RNA activity was indeed detected with the use of a modified experimental scheme, in which, before applying to the Q{beta} replicase-containing agarose, a reaction mixture was oxidized with periodate to suppress further recombination by eliminating any free 3' hydroxyls at the recombining RNA molecules (7). However, the spontaneous recombinations between RNA molecules appeared to be several orders of magnitude less frequent than in the presence of Q{beta} replicase. Moreover, they did not require free 3' hydroxyl groups, indicating that a quite different reaction chemistry was employed, most probably, a Mg2+-catalyzed RNA cleavage generating fragments with 2',3'-cyclic phosphate and 5' hydroxyl termini, which are then cross-ligated. These observations suggested that the 3' hydroxyl-dependent RNA recombinations are somehow promoted by Q{beta} replicase (7, 20).

The results obtained in the cell-free Q{beta} system stimulated attempts to detect similar phenomena in vivo, by transfecting susceptible cells with two supplementing fragments of the genome of an RNA virus, or with inactivated genome and a complementing fragment. Experiments with derivatives of poliovirus (5, 21) and bovine viral diarrhea virus (6) RNAs demonstrated that viable viruses could be rescued even if none of the recombination substrates could be translated or if translation could not result in the active viral RdRp. Similarly to the spontaneous rearrangements observed in the Q{beta} system, this nonreplicative recombination was not affected by elimination of the 3' hydroxyls. Moreover, its frequency increased when the 5' and 3' fragments bore 3' phosphoryl and 5' hydroxyl groups, respectively, suggesting an involvement of the 2',3'-cyclic phosphate intermediate (5, 21). Crossing poliovirus RNA fragments overlapping within a nonessential segment of the 5'-untranslated region resulted in the rescue of only nonhomologous recombinants (5). At the same time, only homologous and aberrant homologous recombinants that retained the translation reading frame were rescued when RNA fragments overlapped within the sequence coding for protein 3Dpol (poliovirus RdRp) (21), indicating that natural selection can considerably distort the results of in vivo experiments.

Quite different results were obtained in another type of in vivo experiments, in which an expressible cDNA clone of poliovirus capable of producing active RdRp, but incapable of replication because of two point mutations in the 5'-untranslated region, was rescued by recombination with a mutation-free 5'-untranslated region fragment (22). In these experiments, no viruses were rescued if the 5' fragment had been modified at the 3' terminus with cordycepin (3'-deoxyadenosine). Thus, as with Q{beta} replicase, RNA recombination in the presence of poliovirus RdRp appeared to be entirely dependent on the availability of the free 3' hydroxyl of the 5' fragment. However, in contrast to the Q{beta} replicase-promoted reaction (15) and RdRp-independent poliovirus RNA recombination in vivo (5), only homologous recombinants were produced, even though coding sequences were not involved. To explain their observations, the authors proposed a "primer alignment and extension" (PAAE) mechanism, in which no templates are switched. Instead, a pre-existing fragment of poliovirus RNA (in nature, this can be a fragment generated by abortive synthesis or by an enzymatic degradation exposing the 3'-OH termini) is extended by RdRp using the strand of opposite polarity as a template, to which it hybridizes by a complementary 3' terminal segment.

The striking similarity of the responses of Q{beta} and poliovirus systems to the elimination of the 3' hydroxyl of the 5' fragment, together with the fact that Q{beta} replicase is involved in the 3' hydroxyl-dependent recombination, raised the possibility that similar mechanisms operate in both systems. However, the absence of homologous recombination in the Q{beta} system remained unexplained. Whether the latter fact reflects a fundamental mechanistic difference between Q{beta} replicase and poliovirus RdRp, or the mere limitations of the respective in vitro and in vivo systems and/or sequence or structure dissimilarities of the recombination substrates remains to be answered.

To resolve these alternatives, we compared effects of the two RdRps under similar conditions and with the same RNA substrates, using the cell-free Q{beta} system to monitor RNA recombination. The results of these studies demonstrate that although ongoing RNA synthesis is required for the generation of recombinant RNAs by both Q{beta} replicase and 3Dpol, the mechanisms employed by the two enzymes are entirely different. In particular, whereas the PAAE mechanism is efficiently used by 3Dpol to generate recombinant molecules from RNA fragments of opposite polarities, it is totally rejected by Q{beta} replicase.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Enzymes—Q{beta} replicase was isolated from Escherichia coli HB101 cells transformed with plasmid pRep (23) as described (24, 25). The poliovirus RdRp (3Dpol) gene was PCR-amplified using plasmid pKKT7E-3D (26) as a template and primers that introduced an upstream NcoI site and a downstream His6-coding sequence followed by the BamHI site, cloned in pET15b between sites NcoI and BamHI and expressed in E. coli BL21(DE3) cells (27). A highly purified 3Dpol protein containing the hexahistidine tag peptide at the C terminus was isolated by chromatography on Zn2+-iminodiacetate-Sepharose CL-4B (28) from a cell lysate prepared in a buffer of 50 mM Tris-HCl, pH 8.0, 10% glycerol, 100 mM NaCl, 0.1% Nonidet P-40 (29), dialyzed against the same buffer containing 2 mM dithiothreitol, and after addition of glycerol to 50%, was stored at –20 °C. The resulting preparation was active in the poly(A)-directed synthesis of poly(U) (30) and contained no detectable activities of E. coli RNA polymerase, polynucleotide phosphorylase, or ribonucleases.

RNA Fragments—The 5' and 3' fragments were synthesized by run-off transcription with T7 polymerase using corresponding plasmids digested with appropriate restriction endonucleases, and gel-purified as described (15). Plasmid for the synthesis of the 3'C fragment was prepared by a PCR templated with a pUC18-derived plasmid, in which a T7 promoter/RQ135–1(+) cDNA construct was inserted between sites HindIII and SmaI (31), using primers 5'-CTGCAGGCATGCAAGCTTAATACGACT-3', partially overlapping the sequence of the T7 promoter (bold) and containing the HindIII site (underlined), and 3'-AGTTTAGGGAGCATCTAGGAGATCTCAGCTGGACGTCCTTAAG-5', partially overlapping the sequence of the 3' fragment (bold) and containing and an identical sequence to the foreign sequence of the 5'(BamHI) fragment (italic), including the PstI site (underlined). The PCR product was digested at the HindIII site, blunt-ended by filling in the recessed 3' terminus with Klenow enzyme, digested at the PstI site, and ligated into plasmid pUC18 that had been digested at PstI and SmaI and dephosphorylated. The primary structure of the resulting plasmid was checked by sequencing.

RNA Recombination—To separate the recombination and replication steps (as in experiments of Figs. 2 and 5A), the earlier devised procedure (7) was employed. Unless indicated otherwise, the recombining fragments (3 x 1011 molecules each) were annealed in a 2 times incubation buffer (see figure legends), not including Mg2+ and rNTPs, by incubating during 2 min in a boiling bath followed by cooling to {approx}30 °C during 1 h. After incubation under specified conditions followed by the addition of EDTA to chelate all Mg2+, the reaction mixture was extracted with phenol/chloroform (32), oxidized with sodium periodate (33), desalted by passing through a Sephadex G-25 spun column, and melted (7).



View larger version (94K):
[in this window]
[in a new window]
  		FIG. 2.
Requirements of the Q{beta} replicase-promoted recombination. The annealed 5'(BamHI) and 3' fragments were incubated during 1 h at 22 °C in the presence of 10 mM Tris-HCl, pH 7.8, 100 mM NaCl, 10 mM MgCl2, 1 mM EDTA, 35 µg/ml Q{beta} replicase, and unless specified otherwise, 1 mM each of rNTPs. Then the reaction mixture was extracted with phenol/chloroform, oxidized with periodate, and desalted. A, during incubation, the GTP concentration was varied as indicated. Top panel, the reaction mixture was assayed for the presence of replicable RNAs. Each sample contained 109 molecules of each of the 3' fragments and either the native (5') or a periodate-oxidized (5'oxi)5' fragment. Bottom panel, analysis of the reaction mixtures prior to phenol/chloroform extraction by electrophoresis in a polyacrylamide gel under non-denaturing conditions (32) followed by silver staining (52). Lane M contained a mixture of the 5'(BamHI) and 3' fragments; ss, single-stranded; ds, double-stranded RNAs (cf. Ref. 35). Because of a strong binding to replicase (35), most of the single-stranded 5' fragment migrated at top of the gel (not shown). B, effects of 3'-deoxy-ATP (cordycepin triphosphate) on recombination between the 5'(BamHI) and 3' fragments (109 molecules each). The reaction mixture contained 0.2 mM ATP and, at a specified incubation time, 3'-deoxy-ATP was added to the final concentration of 4 mM; the concentration of Mg2+ was adjusted to compensate for the increased concentration of nucleotides. Before phenol/chloroform extraction, 3'-deoxy-ATP was dephosphorylated (together with other NTPs) by additionally incubating the reaction mixture during 20 min with 1 unit of calf intestine alkaline phosphatase (molecular biology grade, Roche Molecular Biochemicals).

 



View larger version (69K):
[in this window]
[in a new window]
  		FIG. 5.
Recombinations between RNA fragments of opposite polarities in the presence of poliovirus RdRp. A, fragments that had been annealed (bottom panel) or not (top panel) were incubated during 30 min at 30 °C under conditions optimal for the primer-dependent 3Dpol activity (39): 50 mM Hepes-KOH, pH 7.0, 0.8 mM MgCl2, 5 mM dithiotreitol, 0.1 mM each of rNTPs, and 30 µg/ml 3Dpol. The reaction mixtures additionally contained the following reagents introduced together with the enzyme preparation: 5 mM Tris-HCl, pH 8.0, 10 mM NaCl, 5% glycerol, and 0.01% Nonidet P-40. Before assaying replicable RNAs in aliquots containing the specified number of molecules of each of the indicated fragments, the reaction mixtures were extracted with phenol/chloroform, oxidized with periodate, and desalted. B, primary structures of RNAs generated by recombination between fragments 5'(BamHI) and 3'C(SphI). A value in parentheses indicates the number of clones sharing that sequence.

 
Detection and Sequencing of Recombinant RNAs—In the experiments of Fig. 2, RNA colonies were grown in agarose slabs (18 x 18 x 0.37 mm) as reported (15, 18). A 10-µl sample containing the specified amount of RNA was distributed over Q{beta} replicase-containing agarose and covered with a nylon membrane (Hybond N, Amersham Biosciences) containing rNTPs. In the experiments of Figs. 3 and 5A, RNA colonies were grown in round (14 mm diameter, 0.4 mm thick) polyacrylamide gels that were earlier used for growing DNA colonies (34). Pre-cast and dried gels were reconstituted by soaking in 70 µl of a solution containing the RNA sample and all the replication reaction components but rNTPs that were introduced with a nylon membrane covering the gel. As compared with agarose gels, the use of polyacrylamide gels resulted in a 5–10 times higher recovery of recombinant RNAs. In either case, the final concentrations of reaction components were: 80 mM Tris-HCl, pH 7.8, 8 mM MgCl2, 1 mM EDTA, 20% glycerol, 1 mg/ml acetylated bovine serum albumin (Sigma), 35 µg/ml Q{beta} replicase, and 1 mM each of rNTPs. After incubation during 1 h at 22 °C, the membranes were fixed (18) and hybridized with a 32P-labeled 5'(BamHI) fragment to reveal the colonies by autoradiography (15). Then RNAs were extracted from gels, cloned, and sequenced (15).



View larger version (79K):
[in this window]
[in a new window]
  		FIG. 3.
Recombinations between RNA fragments of opposite polarities in the presence of Q{beta} replicase. After annealing, the indicated 5'- and 3'C fragments (108 molecules each, unless otherwise indicated) were introduced into the RNA amplification gels without preliminary incubation or any other treatment. A, correlation between the recombination frequency and the length of a complementary overlap between foreign sequences of the fragments, which was, from left to right, 24, 18, and 6 nt, and null; cf. Fig. 1E). B, effect of annealing and comparison to recombination between the same polarity fragments. C, effect of periodate oxidation of the recombination substrates.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
RNA Synthesis Is Required for the Generation of Recombinant RNAs by Q{beta} Replicase—To investigate requirements of the 3' hydroxyl-dependent recombination, we used the experimental approach earlier established for exploring the ability of RNA molecules to self-recombine (7). The same polarity 5' and 3' fragments of RQ135 RNA were incubated under chosen conditions with or without the addition of the components of the cell-free replication system (Q{beta} replicase, rNTPs, and Mg2+), and before assaying for the presence of RNAs capable of replicating in a Q{beta} replicase-containing gel, the incubation mixture was treated with sodium periodate and melted. This separated the recombination and replication events, and RNA colonies only grew if recombination had occurred before the oxidation step. Control samples, in which a pre-oxidized 5' fragment substituted for the normal one, were run in parallel to ascertain if the observed recombination was 3' hydroxyl-dependent.

We observed no recombination between the RNA fragments above the level of spontaneous reaction (7) unless the incubation mixture contained all the reagents needed for RNA synthesis, including each of the four rNTPs (not shown). Fig. 2A shows that recombination requires the same concentration of the initiating nucleotide GTP as does copying of the 3' fragment (monitored by the generation of a double-stranded product) that has inherited the initiation site of RQ135 RNA. Requirements of the 5' fragment copying are saturated at a lower GTP concentration, as reported earlier (35).

Thus, recombination between the 5' and 3' RQ135 RNA fragments is observed under conditions that provide for their copying by Q{beta} replicase. Whereas recombination was always accompanied by copying of the 3' fragment (the donor template in terms of the template switch mechanism), it was not firmly linked with copying of the 5' fragment (the acceptor template). For example, elimination of the 3' hydroxyl group of the 5' fragment by periodate oxidation prevents recombination (Fig. 2A and Ref. 15), but only slightly affects synthesis of the complementary copy (35). A reverse example is provided by the PstI and {Delta}EcoRI variants of the 5' fragment, which are hardly copied by Q{beta} replicase (Fig. 1 in Ref. 35), but are excellent recombination substrates (not shown).

Fig. 2B shows that chain terminator 3'-deoxy-ATP (cordycepin 5'-triphosphate) inhibits recombination (as well as the synthesis of full-sized complementary copies of the recombining fragments, not shown) if added to the reaction mixture at a 20:1 ratio to ATP, at which it is expected to be used by Q{beta} replicase instead of ATP at about a 20% probability (36). It follows that RNA synthesis is required for recombination, rather than merely accompanying it.

Q{beta} Replicase Does Not Utilize the Primer Alignment and Extension Mechanisms—The fact that 3' hydroxyl-dependent recombination between the 5' and 3' fragments requires RNA synthesis, in particular, copying of the 3' fragment raises a possibility that it is driven by the PAAE mechanism. In this case, the genuine substrates of recombination would be fragments of opposite polarities, i.e. the 5' fragment and the 3'C fragment (the complementary copy of the 3' fragment).

To explore the PAAE mechanism directly, we replaced the 3' fragment by its complement synthesized by run-off transcription with T7 RNA polymerase, and checked if the 5'- and 3'C fragments can serve as primers for their own extension by Q{beta} replicase using each other as a template. Furthermore, by manipulating foreign sequences, we prepared several pairs of the 5'- and 3'C fragments whose 3'-terminal sequences were capable of complementary overlapping one another to various lengths (Fig. 1E); the fragments were designated after the restriction endonucleases used to cleave the plasmid DNAs before transcription. If the PAAE mechanism operates, one should expect that: 1) pairs with longer complementary overlaps will recombine at a higher frequency; 2) mostly homologous recombinants will be generated at longer overlaps; and 3) recombination between the fragments of opposite polarities (5' and 3'C) will be more efficient than between the same polarity fragments (5' and 3'), because the 3'C fragment does not need to be synthesized by Q{beta} replicase and is not base-paired with the 3' fragment.

It turned out that recombination between fragments 5'(BamHI) and 3'C(PstI) overlapping by 18 nt (nucleotides) does occur (Fig. 3A) and is promoted by preliminary annealing of the fragments (Fig. 3B). Fragment pairs with shorter complementary overlaps, 5'(SalI) x 3'C(PstI) and 5'(PstI) x 3'C(PstI) (cf. Fig. 1E), recombine at a lower frequency (Fig. 3A). Oxidation of both the 5'- and 3'C fragments is required to suppress recombination, indicating that the 3' hydroxyl group of each of them is important (Fig. 3C). These observations are in apparent agreement with the PAAE mechanism.

However, other findings do not support this mechanism. Instead of the expected increase in recombination frequency, recombination between the fragments of opposite polarities turned out to be some 3 orders of magnitude less efficient than between the same polarity fragments (Fig. 3B). Also, fragments 5'(BamHI) and 3'C(SphI), capable of a longer complementary overlap (24 nt, cf. Fig. 1E) recombine at a 10 times lower frequency than do fragments 5'(BamHI) and 3'C(PstI) (Fig. 3, A and C). Finally, sequencing has shown that homologous recombinants are not generated (Fig. 4). Almost every recombinant molecule contains sequences originated from the full size 5'- and 3'C fragments, separated by an insert of variable length. The inserts contain nucleotide stretches complementary to the foreign sequences of the fragments (shown in bold and underlined). Their possible origin is discussed below. Thus, the above data demonstrate that, although Q{beta} replicase is capable of promoting recombination between the fragments of opposite polarities, it does it without using the PAAE mechanism.



View larger version (128K):
[in this window]
[in a new window]
  		FIG. 4.
Primary structures of recombinants generated by Q{beta} replicase from RNA fragments of opposite polarities. Square brackets indicate RNA termini that were deprived of 3' hydroxyls by periodate oxidation. Values in parentheses indicate the number of clones whose sequences were identical. For other explanations, see "Results" and the legend to Fig. 1.

 
Poliovirus RdRp Uses the Primer Alignment and Extension Mechanism to Produce Homologous Recombinants from Q{beta}-specific RNAs—Fig. 5A demonstrates generation of replicable RNAs from the 5'- and 3'C fragments by poliovirus RdRp. To keep the effects of 3Dpol undistorted by Q{beta} replicase, we incubated the RNA fragments in the presence of rNTPs and 3Dpol and, before applying to the Q{beta} replicase-containing agarose, extracted the reaction mixture with phenol/chloroform to remove 3Dpol and oxidized with sodium periodate to eliminate the free 3' hydroxyls at the RNA molecules. It is seen that fragment pairs 5'(BamHI) x 3'C(PstI) and 5'(BamHI) x 3'C(SphI), overlapping by 18 nt and 24 nt complementary sequences, respectively, recombine at the same frequency that increases upon annealing of the fragments and is 4–5 orders of magnitude higher than in the presence of Q{beta} replicase (cf. Fig. 3). The frequency falls considerably when the length of a complementary overlap decreases to 6 nt (pair 5'(SalI) x 3'C(PstI)) and is not detectable with fragments 5'(PstI) and 3'C(PstI) whose termini do not show a potential for significant complementary interactions.

Sequencing of the replicable RNAs obtained showed that only homologous recombinants were produced. Moreover, because there was a G:U opposition at the 3' end of the 3'C(SphI) aligned with the 5'(BamHI) fragment (Fig. 1E), it was possible to distinguish between recombinants produced by extending the 5'(BamHI) fragment and those produced by extending the 3'C(SphI) fragment. It is seen that both the fragments were extended, with the 5'(BamHI) fragment producing a canonical 3' terminal opposition (C:G) being extended at a higher frequency (Fig. 5B). Thus, 3Dpol enzyme manifests a primer-dependent template-directed RNA polymerization activity with Q{beta}-specific RNAs, in accord with observations made on poliovirus-derived sequences (37–39).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Mechanistic Differences between Q{beta} Replicase and Poliovirus RNA Polymerase—This article presents results of the first comparative study of intermolecular RNA recombination promoted by RdRps of two different RNA viruses under similar physicochemical conditions, using the same recombination substrates and the same amplification system. Therefore, any effects that might influence the results, such as effects of the primary or a higher RNA structure, or of a selective amplification of some sort of recombinant molecules, are eliminated. The results clearly demonstrate that the fact that only nonhomologous recombinants are generated in the cell-free Q{beta} system, whereas mainly homologous recombination is observed in poliovirus, reflects a fundamental difference in the mechanisms employed by Q{beta} and poliovirus 3Dpol, rather than artifacts of either the in vivo or in vitro systems.

The puzzling observation that recombination between the 5' and 3' fragments of an RQ RNA was entirely suppressed by eliminating the free 3' hydroxyl of the 5' fragment constituted the main argument against the template switch mechanism and in favor of a transesterification mechanism in the cell-free Q{beta} system (15). This conclusion was later challenged by Pierangeli et al. (22) who made a similar observation in a poliovirus system in vivo and argued that the 3' hydroxyl requirement might also indicate that the 5' fragment served as a primer for its own extension using the 3'C fragment as a template. Our finding that the Q{beta} replicase-promoted recombination requires an ongoing RNA synthesis (Fig. 2) apparently supported their PAAE mechanism. However, it should be noted that this mechanism had problems in explaining why the newly synthesized 3'C fragment was not used as a primer with the 5' fragment as a template, as evidenced by the total suppression of recombination in the cell-free Q{beta} system upon elimination of the free 3' hydroxyl at the 5' fragment (15). One might argue that, as far as the very 5' terminus of the 3' fragment was not homologous to the 5' fragment, an end-to-end copying of the 3' fragment would result in a 3'C fragment whose 3' end would lack complementarity to the 5' fragment, making its extension on the 5' fragment impossible (Fig. 1C). However, such an argument would only be valid if the Q{beta} replicase-promoted recombination was homologous, which is opposite to what is observed in the experiment. In principle, generation of nonhomologous recombinants by the PAAE mechanism could be conceived by taking into account the facts that the newly synthesized 3'C fragment remains base-paired to the 3' fragment (35), and that Q{beta} replicase cannot unwind the RNA duplex (40, 41). Under these circumstances, priming might occasionally occur at sites of local complementarity when the duplex spontaneously unwinds a short distance away from its terminus as a result of thermal motion (Fig. 1D). Of course, such a process would be very inefficient, but the observed recombination is not efficient either. It should be, however, noted that in this case extension of the 5' fragment would have no obvious preference over extension of the 3'C fragment, and the 100% inhibition of recombination by the oxidation of the 5' fragment would remain unexplained.

The results of this study show that, even when experimental conditions are most favorable for the PAAE mechanism, Q{beta} replicase denies using it. The reluctance of Q{beta} replicase to extend RNA primers is unexpected in view of an earlier report that it employs short oligoribonucleotides to by-pass the normal GTP-dependent initiation on homopolymeric templates (42). In contrast, under similar conditions and with the same RNA substrates this mechanism is readily used by the poliovirus RdRp, even though the extended primers and the templates are heterologous to poliovirus. Although unexpected, these results are in accord with the in vivo observations that homologous recombination in the Q{beta} phage is a million times less frequent than in poliovirus (20, 43, 44).

Possible Mechanism of RNA Recombination by Q{beta} Replicase—Two important features are seen in most sequences resulting from Q{beta} replicase-promoted recombination between the 5'- and 3'C fragments: 1) both the fragments donate their entire sequences, and 2) an extra sequence is inserted between them (Fig. 4). The insert contains a stretch of nucleotides complementary to the foreign sequences of the fragments, suggesting that it has been generated by partial copying of a fragment. The fact that the primary structure of the insert depends on which of the fragments was pre-oxidized with periodate suggests that such a copying occurred after the synthesis of fragments by T7 RNA polymerase, i.e. it was performed by Q{beta} replicase.

Recently, we have shown that Q{beta} replicase can copy derivatives of a 3'-truncated RQ RNA in a GTP-independent mode (35). There can be either de novo initiation of complementary copies, with a template being copied along the entire length irrespective of its 3' terminal sequence, or a 3'-terminal elongation of the template, including a snapback RNA synthesis producing a hairpin in which the template and the complementary copy make up opposite sides of the stem. Formation of similar hairpins as a result of the 3'-terminal elongation of replicable RNAs by Q{beta} replicase was reported earlier (45). Taking into account that the 5'- and 3'C fragments are 3'-truncated derivatives of the (–) and (+) strands of the RQ135 RNA, respectively (16), one can imagine the following scenarios leading to the generation of a replicable RNA, i.e. one in which proper polarity and order of the fragments are observed. Generation of recombinant RNA with the longest insert (the uppermost sequence of Fig. 4A) is considered as an example (Fig. 6A).



View larger version (57K):
[in this window]
[in a new window]
  		FIG. 6.
Possible mechanism of the Q{beta} replicase-promoted RNA recombination. Thin arrows indicate directions of strand extension; thick arrows indicate directions of nucleophilic attacks of 3' hydroxyls on phosphorus atoms. For other explanations, see "Discussion" and the legend to Fig. 1. A, conceivable scenarios of the generation from the fragments of opposite polarities, 5'(BamHI) and 3'C(SphI), of the uppermost recombinant sequence as depicted in Fig. 4A. B, the main site of the proposed attack of the 3' fragment by the 3' hydroxyl of the same polarity 5' fragment (15).

 
In Mechanism 1, recombinant RNA is generated by a transesterification reaction in which the 5' fragment attacks an internucleotide phosphate in the 3'C fragment that has been 3' terminally extended producing a hairpin twice the size of the template. Because this scenario requires that each of the fragments possesses the free 3' hydroxyl group, it should be rejected because this particular recombinant was generated in an experiment employing the periodate-oxidized 5' fragment, which lacks such a group.

In Mechanism 2, also of a transesterification type, the 3' terminal hydroxyl of the 3'C fragment, which has been partially extended beyond its 3' end in a snapback manner, attacks the {alpha}-phosphate at the 5' terminus of the full-length complementary copy of the 5' fragment. Finally, in Mechanism 3, the 3'C fragment, which has been 3' terminally extended as in Mechanism 2, is further extended using the 5' fragment as a template. In other words, Mechanism 3 employs extension of a primer not aligned with a template; a similar mechanism was proposed to explain the synthesis of RNA longer than the template by RdRps of bovine viral diarrhea virus and some plant viruses (46). Both Mechanisms 2 and 3 can operate with the oxidized 5' fragment, because oxidation does not prevent fragment copying (35). However, the transesterification reaction (Mechanism 2) seems to be preferable for the following reasons.

In Mechanism 2, the 5' fragment is provided in a double-stranded form that protects internucleotide phosphates of its complementary copy from attack by the 3' terminal hydroxyl of the extended 3'C fragment, and this could explain why the 5' fragment almost always donates its entire sequence to the resulting recombinant (Fig. 4). In Mechanism 3, the 5' fragment is provided in a single-stranded form, and therefore the absence of internal priming, which would result in recombinants with a 3' terminally truncated 5' fragment sequence, remains unexplained. This consideration is further strengthened by the sequences of recombinants generated from the same polarity fragments, 5' and 3', which include the entire sequence of the 5' fragment and a variably truncated 3' fragment (15). In that case, the situation is reversed: the 3' fragment would be provided single-stranded in a transesterification mechanism (Fig. 6B) and double-stranded in a primer extension mechanism (Fig. 1D) and, again, the data conform to the transesterification mechanism.

The same conclusion can be drawn from comparison of recombination frequencies observed with fragments of the same (5' and 3') and opposite (5'- and 3'C) polarities, on assumption that the same mechanism operates in both cases. For a primer extension mechanism, the immediate substrates are fragments of opposite polarities, whereas for a transesterification mechanism the immediate substrates are the same polarity fragments. Hence, for any type of a primer extension mechanism, the fragments of opposite polarities should recombine at a higher frequency than the same polarity fragments, but in reality they recombine at about a 1000-fold lower frequency, in agreement with the transesterification mechanism.

If the transesterification mechanism operates, what is then the role of the ongoing RNA synthesis? The answer is not obvious for the recombination between the same polarity fragments, in which case both the recombination substrates are ready for use from the very beginning. One possibility might be that Q{beta} replicase occasionally catalyzes transesterification reactions between RNA molecules while being in a special conformation that the enzyme only acquires when it synthesizes RNA (cf. Ref. 35). In this regard, it should be noted that the proposed transesterification reaction, comprising an attack of the 3' terminal hydroxyl of one RNA molecule on an internucleotide phosphate of another, is chemically analogous to the attack of the leading 3' hydroxyl of a nascent strand on the {alpha}-phosphate of a nucleotide to be added next.

Diversity of Replicative Mechanisms for RNA Recombination—Until recently, it was generally accepted that viral RNA recombination involves viral RdRp that eventually switches between templates during RNA synthesis (1, 2, 13, 14, 19, 47), with "template switch" and "replicative mechanism" being used as synonymous terms, as opposed to recently discovered "nonreplicative" transesterification mechanisms (5–7, 21). In the classical template switch (copy choice) model, the following steps can be distinguished (e.g. Ref. 2): 1) pausing of RdRp (e.g. at sites of secondary structure); 2) dissociation of RdRp carrying the nascent strand from the first (donor) template; 3) binding of the RdRp-nascent strand complex to the second (acceptor) template; 4) elongation of the nascent strand on the second template. In a modified "processive" model (48), RdRp switches to the second template without leaving the first template. The PAAE mechanism (22) differs from the classical template switch in that the first two steps are omitted; yet, as in the template switch model, the recombinant RNA is generated through a template-directed elongation of an RNA primer by stepwise addition of monomer nucleotides to its 3' terminus. Our data obtained with poliovirus RdRp perfectly agree with the PAAE model and further experiments are needed to ascertain whether 3Dpol can play the complete template switch scenario with natural heteropolymeric RNAs, as it was suggested from the results of in vitro studies employing homopolyribonucleotides (49).

A quite different result has been obtained with Q{beta} replicase. As for the template switch mechanism, ongoing RNA synthesis is also needed in this case and, for that reason, the recombination should be regarded as a replicative one. However, this recombination does not seem to result from elongation of an RNA primer. The available data indicate that the recombinant molecule might be generated by an RdRp-catalyzed transesterification reaction, i.e. by adding to the 3' terminus of an RNA (a piece of) another RNA, rather than a mononucleotide. Besides Q{beta} and related phages, such a mechanism might also operate in other viral systems manifesting a very low rate of homologous recombination, e.g. in alphaviruses (50, 51).

Whatever is the precise molecular mechanism used by each of these enzymes, the very fact that the two viral RdRps studied here behave so differently when confronted with the same RNA substrates under similar conditions suggests that there exist more than one type of replicative mechanism for RNA recombination.


   FOOTNOTES
 
* This work was supported by the program "Molecular and Cell Biology" of the Russian Academy of Sciences, Russian Foundation for Basic Research Grant 02-04-48320, International Association for the promotion of co-operation with scientists from the New Independent States of the former Soviet Union Grant 01-2012, a grant from the Ministry of Industry, Science and Technology of the Russian Federation, and an International Research Scholar's award from the Howard Hughes Medical Institute (to A. B. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Present address: University of Chicago, Chicago, IL 60637. Back

{ddagger} To whom correspondence should be addressed: Institute of Protein Research, Pushchino, Moscow Region, Russia 142290. Tel.: 7-0967-73-2524; Fax: 7-095-924-0493; E-mail: alexch{at}vega.protres.ru.

1 The abbreviations used are: RdRp, RNA-directed RNA polymerase; rNTPs, ribonucleoside triphosphates; PAAE mechanism, primer alignment and extension mechanism; 3Dpol, poliovirus RdRp; RQ RNA, replicable by Q{beta} replicase, a non-genomic RNA capable of exponential amplification by Q{beta} replicase; 3'C fragment, the complementary copy of the 3' fragment; nt, nucleotide(s). Back


   ACKNOWLEDGMENTS
 
We thank Dr. Steve Schultz (University of Colorado, Boulder) for plasmid pKKT7E-3D; Alexander Simonenko, Dmitry Lesnyak, Zakir Tnimov, Nadezhda Androsova, Tatiana Popkova, and Larissa Shutova for technical assistance; and Dr. Vadim Agol for critical reading of the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. King, A. M. Q. (1988) in RNA Genetics (Domingo, E., Holland, J. J., and Ahlquist, P., eds) Vol. II, pp. 149–165, Chemical Rubber Company Press, Boca Raton, FL
  2. Lai, M. M. (1992) Microbiol. Rev. 56, 61–79[Abstract/Free Full Text]
  3. Schlesinger, S. (1988) in RNA Genetics (Domingo, E., Holland, J. J, and Ahlquist, P., eds) Vol. II, pp. 167–185, Chemical Rubber Company Press, Boca Raton, FL
  4. Chetverin, A. B. (1997) Semin. Virol. 8, 121–129[CrossRef]
  5. Gmyl, A. P., Belousov, E. V., Maslova, S. V., Khitrina, E. V., Chetverin, A. B., and Agol, V. I. (1999) J. Virol. 73, 8958–8965[Abstract/Free Full Text]
  6. Gallei, A., Pankraz, A., Thiel, H-J., and Becher, P. (2004) J. Virol. 78, 6271–6281[Abstract/Free Full Text]
  7. Chetverina, H. V., Demidenko, A. A., Ugarov, V. I., and Chetverin, A. B. (1999) FEBS Lett. 450, 89–94[CrossRef][Medline] [Order article via Infotrieve]
  8. Spirin, A. S. (2002) FEBS Lett. 530, 4–8[CrossRef][Medline] [Order article via Infotrieve]
  9. Chetverin, A. B. (2004) FEBS Lett. 567, 35–41[Medline] [Order article via Infotrieve]
  10. Hirst, G. K. (1962) Cold Spring Harbor Symp. Quant. Biol. 27, 303–309[Abstract/Free Full Text]
  11. Ledinko, N. (1963) Virology 20, 107–119[CrossRef][Medline] [Order article via Infotrieve]
  12. Bujarski, J. (1996) Semin. Virol. 7, 361–362[CrossRef]
  13. Nagy, P. D., and Simon, A. (1997) Virology 235, 1–9[CrossRef][Medline] [Order article via Infotrieve]
  14. Agol, V. I. (1997) Semin. Virol. 8, 77–84[CrossRef]
  15. Chetverin, A. B., Chetverina, H. V., Demidenko, A. A., and Ugarov, V. I. (1997) Cell 88, 503–513[CrossRef][Medline] [Order article via Infotrieve]
  16. Munishkin, A. V., Voronin, L. A., Ugarov, V. I., Bondareva, L. A., Chetverina, H. V., and Chetverin, A. B. (1991) J. Mol. Biol. 221, 463–472[CrossRef][Medline] [Order article via Infotrieve]
  17. Chetverin, A. B., Chetverina, H. V., and Munishkin, A. V. (1991) J. Mol. Biol. 222, 3–9[CrossRef][Medline] [Order article via Infotrieve]
  18. Chetverina, H. V., and Chetverin, A. B. (1993) Nucleic Acids Res. 21, 2349–2353[Abstract/Free Full Text]
  19. Cooper, P. D., Steiner-Pryor, S., Scotti, P. D., and Delong, D. (1974) J. Gen. Virol. 23, 41–49[Abstract/Free Full Text]
  20. Chetverin, A. B. (1999) FEBS Lett. 460, 1–5[CrossRef][Medline] [Order article via Infotrieve]
  21. Gmyl, A. P., Korshenko, S. A., Belousov, E. V., Khitrina, E. V., and Agol, V. I. (2003) RNA (N. Y.) 9, 1221–1223
  22. Pierangeli, A., Bucci, M., Forzan, M., Pagnotti, P., Equestre, M., and Pérez Bercoff, R. (1999) J. Gen. Virol. 80, 1889–1897[Abstract/Free Full Text]
  23. Shaklee, P. N., Miglietta, J. J., Palmenberg, A. C., and Kaesberg, P. (1988) Virology 163, 209–213[CrossRef][Medline] [Order article via Infotrieve]
  24. Blumenthal, T. (1979) Methods Enzymol. 60, 628–638[Medline] [Order article via Infotrieve]
  25. Berestowskaya, N. H., Vasiliev, V. D., Volkov, A. A., and Chetverin, A. B. (1988) FEBS Lett. 228, 263–267[CrossRef][Medline] [Order article via Infotrieve]
  26. Hansen, J. L., Long, A. M., and Schultz, S. C. (1997) Structure 5, 1109–1122[Medline] [Order article via Infotrieve]
  27. Studier, F. W., Rosenberg, A. H., Dunn, J. J., and Dubendorf, J. W. (1990) Methods Enzymol. 185, 60–89[Medline] [Order article via Infotrieve]
  28. Lindner, P., Guth, B., Wülfing, C., Krebber, C., Steipe, B., Müller, F., and Plükthun, A. (1992) Methods 4, 41–56[Medline] [Order article via Infotrieve]
  29. Neufeld, K. L., Richards, O. C., and Ehrenfeld, E. (1991) J. Biol. Chem. 266, 24212–24219[Abstract/Free Full Text]
  30. Flanegan, J. B., and van Dyke, T. A. (1979) J. Virol. 32, 155–161[Abstract/Free Full Text]
  31. Morozov, I. Yu., Ugarov, V. I., Chetverin, A. B., and Spirin, A. S. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 9325–9329[Abstract/Free Full Text]
  32. Sambrook, J., and Russell, D. W. (2001) Molecular Cloning, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  33. Steinschneider, A., and Fraenkel-Conrat, H. (1966) Biochemistry 5, 2729–2734[CrossRef][Medline] [Order article via Infotrieve]
  34. Chetverina, H. V., Samatov, T. R., Ugarov, V. I., and Chetverin, A. B. (2002) BioTechniques 33, 150–156[Medline] [Order article via Infotrieve]
  35. Ugarov, V. I., Demidenko, A. A., and Chetverin, A. B. (2003) J. Biol. Chem. 278, 44139–44146[Abstract/Free Full Text]
  36. Kramer, F. R., and Mills, D. R. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 5334–5338[Abstract/Free Full Text]
  37. Richards, O. C., and Ehrenfeld, E. (1998) J. Biol. Chem. 273, 12832–12840[Abstract/Free Full Text]
  38. Arnold, J. J., Ghosh, S. K. B., and Cameron, C. E. (1999) J. Biol. Chem. 274, 37060–37069[Abstract/Free Full Text]
  39. Rodriguez-Wells, V., Plotch, S. J., and DeStefano, J. J. (2001) Nucleic Acids Res. 29, 2715–2724[Abstract/Free Full Text]
  40. Weissmann, C., Feix, G., Slor, H., and Pollet, R. (1967) Proc. Natl. Acad. Sci. U. S. A. 57, 1870–1877[Free Full Text]
  41. Biebricher, C. K., Diekmann, S., and Luce, R. (1982) J. Mol. Biol. 154, 629–648[CrossRef][Medline] [Order article via Infotrieve]
  42. Feix, G., and Hake, H. (1975) Biochem. Biophys. Res. Commun. 65, 503–509[Medline] [Order article via Infotrieve]
  43. Palasingam, K., and Shaklee, P. N. (1992) J. Virol. 66, 2435–2442[Abstract/Free Full Text]
  44. Olsthoorn, R. C., and van Duin, J. (1996) J. Virol. 70, 729–736[Abstract]
  45. Biebricher, C. K., and Luce, R. (1992) EMBO J. 11, 5129–5135[Medline] [Order article via Infotrieve]
  46. Kim, M. J., and Kao, C. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 4972–4977[Abstract/Free Full Text]
  47. Kirkegaard, K., and Baltimore, D. (1986) Cell 47, 433–443[CrossRef][Medline] [Order article via Infotrieve]
  48. Jarvis, T. C., and Kirkegaard, K. (1991) Trends Genet. 7, 186–191[Medline] [Order article via Infotrieve]
  49. Arnold, J. J., and Cameron, C. E. (1999) J. Biol. Chem. 274, 2706–2716[Abstract/Free Full Text]
  50. Weiss, B. G., and Schlesinger, S. (1991) J. Virol. 65, 4017–4025[Abstract/Free Full Text]
  51. Raju, R., Subramaniam, S. V., and Hajjou, M. (1995) J. Virol. 69, 7391–7401[Abstract]
  52. Igloi, G. L. (1983) Anal. Biochem. 134, 184–188[Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Virol.Home page
R. Wierzchoslawski, A. Urbanowicz, A. Dzianott, M. Figlerowicz, and J. J. Bujarski
Characterization of a Novel 5' Subgenomic RNA3a Derived from RNA3 of Brome Mosaic Bromovirus
J. Virol., December 15, 2006; 80(24): 12357 - 12366.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/10/8748    most recent
M412684200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chetverin, A. B.
Right arrow Articles by Ugarov, V. I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chetverin, A. B.
Right arrow Articles by Ugarov, V. I.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2005 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement