Leiden Institute of Chemistry, Department of Biochemistry, Gorlaeus Laboratories, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands1
Author for correspondence: Jan van Duin. Fax +31 71 5274340. e-mail Duin.J{at}chem.leidenuniv.nl
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Abstract |
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Introduction |
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Another important potential application is the generation of hybrid phages when overlapping information derived from two different phages is divided between two plasmids. The overlap can provide an abundance of potential crossover points from which only those that lead to viable phages will survive by natural selection.
There are many reports showing the exchange of segments between eukaryotic RNA viruses (Masuta et al., 1998 ; Aaziz & Tepfer, 1999
; Worobey et al., 1999
; Yuan et al., 1999
; Canto et al., 2001
). In contrast, the coliphages sequenced thus far bear no traces of genetic exchange with other species or genera. Instead, sequence variability between strains within a species is extremely low. In this paper, we used the two-plasmid system to generate viable hybrids between different RNA phage strains and species.
We focused on the 5' untranslated leader (5' UTR) because the structure and function of this part of the genome are well known (van Meerten et al., 2001 ). Fig. 1(B)
shows that the 5' UTR of MS2 (a species I phage) folds into a hairpin (North) followed by a cloverleaf structure, i.e. three local hairpins (West, South and East), held together by a long-distance interaction (LDI). This structure precludes any translation of the maturation gene since the ShineDalgarno (SD) sequence is strongly base-paired to an upstream complementary sequence (UCS) (Groeneveld et al., 1995
). However, during its formation from the (-) strand, the RNA becomes temporarily trapped in an alternative structure (Fig. 1D
), which captures the nucleotides that pair with the SD sequence in the loop of a small hairpin (van Meerten et al., 2001
). Only during this time interval, which lasts several minutes in vitro (Poot et al., 1997
), is translation of the maturation gene possible. Thereafter, the RNA rearranges to form the closed structure. Thus, the 5' UTR of MS2 functions as a timer to allow transient translation of the maturation gene.
As can be seen from Fig. 1(C), the 5' UTR of KU1 (a species II phage) forms an equilibrium structure very similar to that of MS2, though from a largely different sequence. The KU1 sequence also enables formation of the alternative structure (Fig. 1E
). Here, we have shown that the 5' UTR of KU1 can functionally replace the MS2 counterpart in live MS2 phages.
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Methods |
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Plasmids pFR741 and pFR400 are pUC9 derivatives with fr cDNA (1741 or 1400, respectively) positioned in reverse orientation behind the lac promotor. pKU8 is a pUC9 derivative carrying KU1 cDNA from 1 to 1572 (Groeneveld, 1997 ). pUCMS2 contains the MS2 sequence from 1 to 741 and has been described earlier (van Meerten et al., 2001
). The plasmids carry the ampicillin-resistance marker.
Plasmid pMSBA, pMS
SA and pMS
NA (Groeneveld et al., 1995
) carry the complete MS2 cDNA sequence with deletions from 1 to 112, 40 to 113 and 78 to 113, respectively. The MS2 cDNA, which is preceded by 11 G residues, is present just behind the thermoinducible PL promoter. The deletion mutants are derivatives of pMS2000. This plasmid contains the complete infectious MS2 cDNA and has been described by Olsthoorn et al. (1994)
. pMS2000 and its derivatives confer kanamycin resistance.
Phage generation by in vivo recombination and phage evolution.
Double transformants harbouring the two plasmids containing overlapping and complementary information for hybrid formation were grown overnight at 28 °C in liquid cultures in the presence of antibiotics (cycle 1). Each culture was tested for phage production by plating appropriate dilutions of the supernatant on a lawn of F+ cells. Plaques (cycle 2) were counted and phages from some of these plaques were amplified overnight at 37 °C in liquid cultures of F+ cells (cycle 3). Five µl of the supernatant was then used for subsequent rounds of infection in liquid cultures and from these cultures phage RNA was extracted and purified.
RNA isolation, RTPCR and sequence analysis.
Phage particles were precipitated by adding to 1 ml of lysate, 0·3 ml 40% polyethylene glycol (PEG6000) in 2 M NaCl and incubating the mixture for at least 1 h at 4 °C. The pellet was dissolved in 200 µl TE (10 mM TrisHCl pH 7·6, 0·1 mM EDTA) and extracted with the same volume of phenol:chloroform (1:1). The RNA was precipitated with ethanol and dissolved in 20 µl of distilled water.
Two µl of this solution was used for RTPCR in a total of 50 µl according to standard procedures recommended by the suppliers (SigmaAldrich and Eurogentec). The primers used were biotin-labelled BIO790 (identical to MS2/fr 117) or biotin-labelled BIO971 (identical to KU1 119) and unlabelled BIO42 (complementary to MS2 872904). PCR fragments were sequenced after separation and purification of the strands using Dynabeads (Dynal) with DUI360 (complementary to 146167 of the MS2 sequence).
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Results |
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Hybrid phages were visualized as plaques on a lawn of E. coli F+ cells (cycle 2). They were sequenced after multiplication in liquid medium (cycle 3) and also after passaging for further infection cycles.
Complementing plasmid pMSBA with pKU8
Plasmid pMSBA, lacking the first 111 nucleotides of MS2, was combined with pKU8 containing the first 1572 nucleotides from KU1. Six plaques, from two different transformations, were taken for sequence analysis. The results, presented in Fig. 3
, will first be discussed in general terms. The first two lines of Fig. 3(A)
(in a bracket) give a schematic presentation of the various structure elements present in the 5' UTR of the donor and acceptor sequences. The zigzag line shows the crossover or fusion site. Line three (kumsBA.x) presents the general structure of the initial hybrids and line four (kums4), the structure after evolution. Fig. 3(B
, C
) shows the nucleotide sequence of the initial and evolved hybrids in the context of the predicted RNA secondary structure. The shaded parts in Fig. 3(B)
were deleted upon passaging. Finally, Fig. 3(D)
shows the linear sequences of the relevant sections of donor and acceptor plasmids. MS2 sequences are shown as black on white (Fig. 3A
) and in normal letters (Fig. 3BD
), whereas KU1 sequences are white on black (Fig. 3A
) and bold letters (Fig. 3BD
). Italic letters in Fig. 3(D)
are derived from the vector.
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The sixth plaque we examined was only sequenced after cycle 5 and 10. At cycle 5 all recombination events had apparently been completed for this plaque as there were no changes in the next five cycles (cycle 10). The sequence of this plaque and the putative structure of its RNA is shown as kums1 in Fig. 5. As for kums4, the final fusion between the two phages was between the SD sequence of MS2 and the East arm of KU1, though at a slightly different point resulting in a hybrid two nucleotides shorter than kums4.
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Complementing pMSSA with pKU8 and pFR741
We then used pMSSA, lacking the three arms of the cloverleaf, for hybrid generation using pKU8 (Fig. 5A
). The titre of this plasmid combination was about the same as the previous MS2/KU1 combination (Table 1
). Six plaques were analysed and all were identical. Strikingly, the pathway of hybrid generation was the same as before. The complete 5' UTR of KU1, including the start codon, was appended to the 5' end of the defective MS2 sequence (Fig. 5A
, C
; kumsSA.3). Upon evolution of this hybrid, the redundant elements, SD (KU1), fMet (KU1), N (MS2) and UCS (MS2), were deleted resulting in kums1, a hybrid also found above as the end result of a different plasmid combination (Fig. 5B
). As before in the pMS
BA series, there was the choice of removing the SD sequence and start codon from either KU1 or MS2. It was even possible to combine the SD of KU1 with the start codon of MS2, by deleting fMet (KU1) and N, UCS and SD elements of MS2. We always observed that the SD sequence that originally belonged to the maturation gene was maintained, even though this created an LDI that derived its constituent sequences from different phages. We do not know why this should be so.
We have also complemented the pMSSA plasmid with fr sequences (pFR741) (Fig. 6
). Again, generation of hybrids proceeded along the same lines as with pKU8. That is, first, the 5' UTR of fr up to and including the East arm was appended to the very 5' end of pMS
SA (Fig. 6
). Subsequently, the redundant MS2 elements (N and UCS) were removed giving rise to frms3 (Fig. 4
), a hybrid we have obtained before as the result of another plasmid combination. The deletion may be facilitated by the common UAGG sequence (boxed in Fig. 6C
).
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Discussion |
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Pathway of hybrid formation
The path along which hybrids were generated is outlined in its simplest form in Fig. 9. It was basically the same for complementation with fr or KU1, in spite of the fact that sequence identity of MS2 with fr is high (
90%) whereas it is very low with KU1. The pathway also seemed insensitive to the size and position of the deletion. We always found as a first step that the 5' UTR of the acceptor plasmid became attached to the 5' end of the defective phage (the donor). It did not seem to matter very much where exactly the junction with the acceptor sequence was formed. Mostly, it included the fMet start codon of the acceptors maturation gene, but sometimes the connection was made between the East arm and SD sequence of the acceptor. It was interesting that the acceptor 5' UTR was nearly always attached to the first nucleotide of the MS2 sequence. In pMS
SA and pMS
NA this is the original 5' end (GGG). The likely purpose of this first step seems to be to create a replicable (-) strand. Addition of the 5' UTR was not needed to rescue A-protein synthesis because it is known that the three MS2 deletion mutants used here can still make this protein (see Fig. 2
legend), even though the timer function has been lost (at least in pMS
SA and pMS
BA). Because uncontrolled production of maturation protein is disadvantageous but not lethal (Poot et al., 1997
), we have ascribed lethality of the initial MS2 deletion constructs to failure to transcribe the (-) strand.
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If we had not measured the early stages of hybrid formation, but only the final outcome after passaging, one could wrongly have concluded that hybrids such as frms3, kums1 and kums4 were the result of a simple RNA or DNA crossover at regions of sequence identity such as CUAGG (frms3) and GGAGG (kums1 and kums4). However, the existence of the early hybrids shows that such a crossover is not the most likely event. Instead, the unexpected attachment of the acceptor 5' UTR to the very 5' end of the defective donor RNA from pMSNA and pMS
SA turns out to be the more probable reaction. These new RNA combinations are unlikely to be driven by nucleotide identity between donor and acceptor sequence because there is none at the initial fusion points. If recombination occurred at the DNA level it would be difficult to rationalize what preference there could be to cross over at the first nucleotide of the phage in the absence of any sequence identity. Therefore, our observations suggest that the linkage between donor and acceptor sequence occurs at the RNA level. If so, the mechanism of this acquisition could be as follows. It is known that MS2 and Q
replicase can generate the proper 5' and 3' termini from cloned cDNA copies (Taniguchi et al., 1978
; Olsthoorn et al., 1994
) and from oversized phage RNA transcripts transfected in spheroplasts (Shaklee et al., 1988
). Therefore, we would expect MS2 replicase to be able to make the (-) strand carrying the proper 3' terminus with the deletion present in pMS
NA and pMS
SA. At this point we can see two possibilities. The (-) strand is released by the enzyme and the free 3' hydroxyl attacks a phosphodiester bond of a transcript derived from the acceptor plasmid. Natural selection will subsequently preserve only those recombinants that can produce phages. This recombination by transesterification was shown to exist for Q
replicase by Chetverin et al. (1997)
. However, there is one problem. As part of the termination reaction, the replicase usually adds a 3' A residue to the strand it has just produced and we see no U preceding the GGG sequence that marks the start of the donor RNA.
The second possibility is that when the replicase has polymerized the end of the (-) strand of the donor RNA chain it does not terminate but switches to the acceptor template. This is precisely what has been found for hybrids between defective interfering (DI) RNA derived from tomato bushy stunt virus (TBSV) and cucumber necrosis virus (CNV) RNA (White & Morris, 1995 ). Also in that study, the fusion occurred between the 3' terminal nucleotide of the donor transcript and variable positions in the acceptor molecule. This mechanism was termed run off recombination (White & Morris, 1995
) and the fact that we find it in almost all of our hybrids is in agreement with its reported efficiency. We note that this mechanism does not predict the presence of the terminally added A residue, because the produced chain is still attached to the replicase and has not been terminated.
Functionality of the hybrids
Evidently, the foreign sequence (fr or KU1) must be able to perform in the new context and it is interesting to see if this can be rationalized on the basis of what is known about the functioning of the 5' UTR. Let us consider the hybrids kums1 and kums4, in which the KU1 cloverleaf structure replaced that of MS2 [the titre of the plasmid combination was 105 p.f.u./ml, but after passaging the titre of the evolved hybrid phage increased to approximately wild-type level (1011 p.f.u./ml)].
A major function of the cloverleaf is to ensure transient translation of the maturation gene by the metastable structure shown in Fig. 1(D, E
). This metastable intermediate could still be formed in kums1 and kums4. In fact, we now had the KU1 metastable structure in the MS2 context. The long-distance interaction between the SD sequence and its upstream complement (UCS) was actually of a hybrid character since the UCS was a KU1 sequence, whereas MS2 provided the SD box. As in both phages the SD box contained the sequence GGAGG, the LDI could be formed in kums1 and kums4, though the stability of the interaction may have been slightly different from wild-type. In summary, in the hybrids one structure module has been replaced by another. Still, it could be argued that an even better kums hybrid might be made if the deletion that matures e.g. kumsNA.3 (Fig. 7
) was shifted so as to keep the SD box from KU1 but discard the MS2 SD box. Then an LDI with only KU1 sequences would be present. It may be that we do not find this because there is no sequence identity here from which the deletion may profit. Alternatively, for unknown reasons, it may be that the SD box cannot be uncoupled from the coding region.
Another function of the 5' UTR is that it encodes the 3' end of the (-) strand, which must interact with the MS2 replicase. Almost nothing is known about this subject. We can only conclude that MS2 replicase can function with a KU1 3' end in the (-) strand.
Hybrids cannot compete with wild-type
The family of Leviviridae has two genera; the Leviviruses such as MS2 and KU1 and the Alloleviviruses like Q and SP. In each genus there are two species, each having its distinct sequence with limited similarity to the other one. Within the species, all characterized strains cluster in a narrow zone around the master sequence (>95% identity) (Inokuchi et al., 1986
, 1988
; Adhin, 1989
; Beekwilder, 1996
; Olsthoorn, 1996
; Groeneveld, 1997
). Hybrids between species have never been found. This is possibly a consequence of the fact that sampling was mostly performed in city sewage systems, which represent enormously large pools of RNA phages with continuous input (Furuse, 1987
). Therefore, it is not difficult to imagine that competition in such an environment is strong, and that aberrant phages, such as hybrids, will quickly be outgrown by wild-type. Indeed, when we mixed hybrid kums4 in a 10:1 ratio with wild-type MS2 and the mixture was passaged, it took only five infection cycles before the hybrid was completely outgrown by wild-type and could no longer be detected by RTPCR. When mixed in a 100:1 ratio this hybrid was no longer detectable at cycle 10. Eukaryotic viruses, on the other hand, may develop in relative isolation within a single organism, where hybrids or other aberrant viruses may multiply and evolve in the absence of strong competition.
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Acknowledgments |
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References |
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Adhin, M. R. (1989). A comparative analysis of RNA coliphages. Structural and regulatory features. PhD thesis, Leiden University, The Netherlands.
Beekwilder, M. J. (1996). Secondary structure of the RNA genome of bacteriophage Q. PhD thesis, Leiden University, The Netherlands.
Biebricher, C. K. & Luce, R. (1992). In vitro recombination and terminal elongation of RNA by Q replicase. EMBO Journal 11, 5129-5135.[Abstract]
Canto, T., Choi, S. K. & Palukaitis, P. (2001). A subpopulation of RNA 1 of cucumber mosaic virus contains 3' termini originating from RNAs 2 or 3. Journal of General Virology 82, 941-945.
Chetverin, A. B., Chetverina, H. V., Demidenko, A. A. & Ugarov, V. I. (1997). Nonhomologous RNA recombination in a cell-free system: evidence for a transesterfication mechanism guided by secondary structure. Cell 88, 503-513.[Medline]
Furuse, K. (1987). Distribution of coliphages in the environment: general considerations. In Phage Ecology , pp. 87-124. Edited by S. M. Goyal. New York:John Wiley & Sons.
Groeneveld, H. (1997). Secondary structure of bacteriophage MS2 RNA. PhD thesis, Leiden University, The Netherlands.
Groeneveld, H., Thimon, H. & van Duin, J. (1995). Translational control of maturation-protein synthesis in phage MS2: a role for the kinetics of RNA folding? RNA 1, 79-88.[Abstract]
Groeneveld, H., Oudot, F. & van Duin, J. (1996). RNA phage KU1 has an insertion of 18 nucleotides in the start codon of its lysis gene. Virology 218, 141-147.[Medline]
Inokuchi, Y., Takahashi, R., Hirose, T., Inayama, S., Jacobson, A. B. & Hirashima, A. (1986). The complete nucleotide sequence of group II RNA coliphage GA. Journal of Biochemistry (Tokyo) 99, 1169-1180.[Abstract]
Inokuchi, Y., Jacobson, A. B., Hirose, T., Inayama, S. & Hirashima, A. (1988). Analysis of the complete nucleotide sequence of the group IV coliphage SP. Nucleic Acids Research 16, 6205-6221.[Abstract]
Klovins, J., Tsareva, N. V., de Smit, M. H., Berzins, V. & van Duin, J. (1997). Rapid evolution of translational control mechanisms in RNA genomes. Journal of Molecular Biology 265, 372-384.[Medline]
Klovins, J., Berzins, V. & van Duin, J. (1998). A long-range interaction in Q RNA that bridges the thousand nucleotides between the M-site and the 3' end is required for replication. RNA 4, 948-957.
Masuta, C., Ueda, S., Suzuki, M. & Uyeda, I. (1998). Evolution of a quadripartite hybrid virus by interspecific exchange and recombination between replicase components of two related tripartite RNA viruses. Proceedings of the National Academy of Sciences, USA 95, 10487-10492.
Miller, J. H., Ganem, D., Lu, P. & Schmitz, A. (1977). Genetic studies of the lac repressor. Journal of Molecular Biology 109, 275-301.[Medline]
Olsthoorn, R. C. L. (1996). Structure and evolution of RNA phages. PhD thesis, Leiden University, The Netherlands.
Olsthoorn, R. C. L., Licis, N. & van Duin, J. (1994). Leeway and constraints in the forced evolution of a regulatory RNA helix. EMBO Journal 13, 2660-2668.[Abstract]
Poot, R. A., Tsareva, N. V., Boni, I. V. & van Duin, J. (1997). RNA folding kinetics regulates translation of phage MS2 maturation gene. Proceedings of the National Academy of Sciences, USA 94, 10110-10115.
Remaut, E., Stanssens, P. & Fiers, W. (1981). Plasmid vectors for high efficiency expression controlled by the PL promoter of coliphage lambda. Gene 15, 81-93.[Medline]
Shaklee, P. N., Miglietta, J. J., Palmenberg, A. C. & Kaesberg, P. (1988). Infectious positive- and negative-strand transcript RNAs from bacteriophage Q cDNA clones. Virology 163, 209-213.[Medline]
Taniguchi, T., Palmieri, M. & Weissmann, C. (1978). Q DNA-containing hybrid plasmids give rise to Q
phage formation in the bacterial host. Nature 274, 223-228.[Medline]
van Meerten, D., Zelwer, M., Régnier, P. & van Duin, J. (1999). In vivo oligo(A) insertions in phage MS2: role of Escherichia coli poly(A) polymerase. Nucleic Acids Research 27, 3891-3898.
van Meerten, D., Girard, G. & van Duin, J. (2001). Translational control by delayed RNA folding: identification of the kinetic trap. RNA 7, 483-494.
White, A. K. & Morris, J. T. (1995). RNA determinants of junction site selection in RNA virus recombinants and defective interfering RNAs. RNA 1, 1029-1040.[Abstract]
Worobey, M., Rambaut, A. & Holmes, E. C. (1999). Widespread intra-serotype recombination in natural populations of dengue virus. Proceedings of the National Academy of Sciences, USA 96, 7352-7357.
Yuan, S., Nelsen, C. J., Murtaugh, M. P., Schmitt, B. J. & Faaberg, K. S. (1999). Recombination between North American strains of porcine reproductive and respiratory syndrome virus. Virus Research 61, 87-98.[Medline]
Received 26 September 2001;
accepted 21 December 2001.