Department of Chemistry and Biomedical Sciences, University of Kalmar, S-391 82 Kalmar, Sweden1
Author for correspondence: Charlotta Polacek. Fax +46 480 446262. e-mail charlotta.polacek{at}hik.se
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Abstract |
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Introduction |
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Although enteroviruses and rhinoviruses have similar 5'UTRs, the 3'UTR is more divergent in terms of its length and structure. Within the enterovirus genus, the 3'UTR is known to fold into two main structures: a double hairpin structure (poliovirus-like viruses) with domains X and Y, and a triple hairpin structure (coxsackievirus B-like viruses) with domains X, Y and Z. Both types of structural folds are joined together by an S domain consisting of parts of the poly(A) tail and a U-rich region upstream of the stop codon. Together, these domains constitute an intramolecular secondary and tertiary structure that participates in the initiation of negative-strand RNA synthesis and is referred to as oriR (Melchers et al., 1997 ; Mirmomeni et al., 1997
; Pilipenko et al., 1992
, 1996
; Wang et al., 1999
). Negative-strand RNA is synthesized by the primer-dependent viral RNA polymerase 3D, which uses a uridylylated form of the genome-linked protein VPg as its primer (Paul et al., 1998
; Toyoda et al., 1987
). It has been suggested recently that nucleotide structures within the polyprotein-coding regions of poliovirus and rhinovirus might also be a part of the replication machinery (Goodfellow et al., 2000
; McKnight & Lemon, 1996
).
In this study, we investigated the genetic characteristics of the 3'UTR of CVB2O. Previously, we generated an infectious clone of CVB2O by RTPCR using CVB3-derived primers (Lindberg et al., 1997 ). However, subsequent determination of the wild-type (wt) sequence (Polacek et al., 1999
) showed the presence of point mutations and a five nucleotide extension at the 3'UTR prior to the poly(A) tail compared with the constructed clone and other enteroviruses. In order to investigate the importance of these additional nucleotides, virus derived from the cDNA clone was propagated in cell culture for a number of passages and the 3'UTR genotype of the virus progeny was analysed. Our results show that the clone-derived virus progeny compensate for the 5 nt deletion and the point mutations that are found in the original clone by forming more energetically favourable secondary structures at the 3'UTR, as predicted by the calculation of conformational free energy. In addition, we observed that, during the first round of passages in cell culture, the poly(A) tail of the cDNA clone was extended from the initial 17 A residues to more than 40 A residues in the virus progeny. During further passages, the poly(A) tail increased to about 100 A residues; this seems to be a more favourable length for CVB2O under these conditions.
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Methods |
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One-step growth curves and plaque assay.
Uncloned CVB2O and viruses from passage seven (LAD7) were analysed for growth properties by infecting GMK cells with 0·1 TCID50 per cell (in duplicate). Virus was allowed to attach to the cell surface for 1 h. Unbound virus was then removed and cells were washed twice with Dulbeccos modified Eagles medium (DMEM) supplemented with penicillin, streptomycin and L-glutamine. Serum-free medium was then added and samples were incubated at 37 °C in 7·5% CO2 for 0, 6, 12, 24, 48 and 72 h prior to freezing at -20 °C. All samples were titrated on GMK cells after three cycles of freezethawing. The plaque morphology of uncloned CVB2O and clone-derived virus propagated for seven passages was determined using plaque assays stained with crystal violet (Hierholzer & Killington, 1996 ).
Rapid amplification of cDNA endspoly(A) test (RACE-PAT).
The length of the viral poly(A) tail from viruses from different passage number was estimated by PCR amplification of the proximal 3' end using the RACEPAT method (Sallés et al., 1999 ). RACEPAT is based on PCR amplification using an oligo(dT) primer and a target-specific primer situated close to the poly(A) tail. Synthesized cDNA from LB0LB3 and LB7 was amplified by PCR with the primer pair CB-44 and NotdT25. pCVB2O-8 containing the 17 A residue poly(A) tail was used as the control. PCR amplicons were analysed on a 2·5% agarose gel.
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Results |
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Nucleotide analyses show the evolution of a clone-derived virus with an unfavourable structure into a virus population with structurally ameliorated revertants. Growth properties of these revertants (LAD7) were determined in comparison to the wt strain by a one-step growth curve over a 72 h period (Fig. 4). Plaque assay comparisons show that the revertants produced the same plaque size phenotype as the wt (data not shown). The overall growth rates in GMK cells were similar for the wt and the four revertant virus populations. This indicates that the compensatory mutations described in LAD7 passages produce wt-like viruses.
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Extension of the pCVB2O-8 poly(A) tail
An additional observation made during the sequence analysis of the adapted revertants was the increase in the length of the poly(A) tail during subsequent passages. In the mutant clone pCVB2O-8, the poly(A) tail was immediately extended from 17 to about 40 A residues, as detected in L0. During sequence analysis of the passages, a continual increase in the poly(A) tail was detected. Since DNA sequencing of long homologous sequences has its limitations, the RACEPAT method was used to estimate the approximate length of the tail from each of the different passages (Sallés et al., 1999 ). The last 240 nt of the 3'UTR together with the poly(A) tail from viruses LB03 and LB7 were amplified by RACEPAT using pCVB2O-8 as a control (Fig. 5
). The amplified clone appears as a sharp band, while the amplicons derived from progeny viruses appear as broad bands, indicating an increasing poly(A) tail. It can be concluded that the broadened bands arise from a heterogeneous population with different poly(A) tail lengths and, in part, from unspecific binding throughout the poly(A) tail. It is clear, however, that an increase in the poly(A) tail length is already detected in lipofected cells (L0) and that this increase proceeds until the poly(A) tail approaches optimal length, which is estimated in this study to be about 100 nt (Fig. 5
).
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Discussion |
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In this study, we have used a combined 3'UTR point/deletion mutant of CVB2O (pCVB2O-8) with a predicted unfavourable structure and containing two mismatches in the X-stem. Propagation of pCVB2O-8 in cell culture generated revertants with more favourable RNA secondary structures compared with the initial genotype. The adapted revertants contained mutations located in the X-stem and could be divided in two types: (i) type 1 compensates for the point mutation and (ii) type 2 compensates for the structural effect of the deletion. These results indicate that the stability of the X-stem is essential for CVB2O replication, since the virus restores either of the two structurally unfavourable mutations in all cases. However, no viruses were isolated that compensated for both of the mismatches in the X-stem. Among the revertants, no preference could be observed for the restoration of either the lower or the upper part of the X-stem, since both unfavourable positions were compensated for equally. In fact, the two genotypes were simultaneously maintained in equal amounts in one of the lipofection experiments (LB). Wt CVB2O has a perfectly matching X-stem but revertants contain a single mismatch in this structure, as seen for coxsackievirus B3 and B4, where a single mismatch is present in the X-stem (Melchers et al., 1997 ; Pilipenko et al., 1992
). The mismatches or internal loops reported are always symmetrical and hence exert a relatively mild destabilizing effect. Compared with our data, this could indicate that a certain level of stability is required for virus function but that a lower match throughout the X-stem is accepted and may even be favourable. The single 7395CG7403 base pair present at the top of the X-stem in both the clone and the revertant type 2 (Fig. 3
B
, D
) might be formed by coaxial stacking of the X and K domains (Melchers et al., 2000
). Our data suggest that CVB2 does not accept more than one internal loop and that the position of the loop within the stem is of secondary importance.
The first revertant populations occurred after two passages in cell culture and, after seven passages, a predominant revertant genotype was present showing growth rates and plaque formation equivalent to the wt. The more advantageous predicted secondary structure of the 3'UTR seems to be favoured, as the emerging virus population with compensatory mutations rapidly out-competes the initial clone-derived population.
The second introduced point mutation disrupted a sequence that is completely conserved among human enteroviruses. This sequence, 7383GUAAA7387, is present as a bridge between the X and Y stems (Fig. 2). These nucleotides appear to be rather exposed in the predicted three-dimensional structure of coxsackievirus B3 (Melchers et al., 1997
) but they have not yet been reported to interact with RNA or proteins. The introduced GGAAA mutation was compensated for in only 1 of 20 individually analysed virus clones from passage seven (R5) (Table 3
) and it is not until further passage that a detectable population compensating for this mutation emerges. Since the GUAAA motif is completely conserved among human enteroviruses and partly in the bovine and porcine enteroviruses (Fig. 2
), it is surprising that restoration of this highly conserved sequence seems to be less important than the other introduced mutations.
Mutational changes in the 3'UTR may also induce single compensatory coding changes in the active site of the viral 3D polymerases, as has been shown for rhinovirus and poliovirus (Meredith et al., 1999 ). In this experimental system, no sequence alterations in the active-site cleft of 3D (LAD7) were observed, indicating that the deletions/mutations introduced at the 3'UTR are not involved in the immediate interaction with the active site cleft of 3D in CVB2O (data not shown).
The increase in the size of the CVB2O 3'UTR might be considered to be only four nucleotides, since the first of the five residues reported in this study is an A residue, as in the following poly(A) tail. However, since it is followed by additional nucleotides in both CVB2O and CVA9, we chose to consider it as a genomic extension present prior to the poly(A) tail.
During our study of the 3'UTR, we observed an increase in the length of the poly(A) tail of the virus progeny after lipofection of the cDNA clone. In our experiments, we used a cDNA clone containing a 3' terminus with 17 A residues: this is in the lower range of the number of A residues considered to be necessary for picornavirus infectivity (Cui & Porter, 1995 ; Spector & Baltimore, 1974
). Sequencing of the virus progeny from the lipofected cells (L0) revealed an increase in the length of the poly(A) tail from 17 A residues to more than 40. Using a PCR-based method of analysis, the poly(A) tail was shown to increase further during the first number of passages to a maximal length of about 100120 residues, which was then maintained during subsequent passages; this corresponds to the previously reported poly(A) tail length of polioviruses (Spector & Baltimore, 1975
; Yogo & Wimmer, 1972
). The poly(A) tail is required for infectivity and is also involved in the spatial organization of the 3'UTR by partly forming a portion of the S-stem and therefore constitutes an important part of the genome. Since no cellular adenylation signals (Zhao et al., 1999
) were found in the 3'UTR, it must be concluded that picornaviruses might use an alternative polyadenylation mechanism to the one that is known today.
The terminal sequence and folding of the wt CVB2O 3'UTR seems to form a rigid structure involving the five additional nucleotides. The variable base pairing in the S-stem seems to be adequate for the secondary structure of this domain and no mutations emerged in this region, under our experimental conditions. Instead, the main differences were observed in the X-stem. Introduced mutations in the CVB2O 3'UTR were neutralized by compensatory mutations that restored at least part of the X-stem. These compensations are, in this experimental model, point mutations of nucleotides in the X-stem or nucleotides constituting the first part of the poly(A) tail. Revertants (adapted and with wt-like propagation properties) used only one of these compensatory strategies. As the 3'UTR is believed to be involved in the synthesis of negative-strand RNA, further studies of the CVB2 3'UTR should be related to the efficiency of replication of the complementary RNA strand.
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Acknowledgments |
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References |
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Received 23 October 2000;
accepted 28 February 2001.
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