1 UMR BGPI, CIRAD-INRA-ENSAM, TA 41/K, Campus International de Baillarguet, 34398 Montpellier Cedex 5, France
2 Friedrich Miescher Institut, PO Box 2543, CH-4002 Basel, Switzerland
Correspondence
Stéphane Blanc
blanc{at}ensam.inra.fr
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ABSTRACT |
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Present address: Equipe Ecologie et Evolution des Micro-Organismes, INSERM E 0339, Hôpital X. Bichat, 16 rue Henri Huchard, 75018 Paris, France.
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INTRODUCTION |
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CaMV is a pararetrovirus, the double-stranded DNA genome of which is replicated by reverse transcription of a pre-genomic 35S RNA. This 35S RNA is also used as a polycistronic mRNA for all virus genes [reviewed by Ryabova et al. (2002)]. In earlier work, several additional RNA species that were detected in CaMV-infected plants were suspected to originate from splicing of the 35S RNA (Hirochika et al., 1985
; Vaden & Melcher, 1990
). That splicing of the 35S RNA is an absolute requirement for virus infectivity was later demonstrated by Kiss-László et al. (1995)
. These authors characterized four additional RNA species (C1C4) that are produced by splicing between four donor sites and one unique acceptor site in the 35S RNA. One of the donor sites is located in the long 5' untranslated region (UTR) of the 35S RNA and the three others are located within ORF I; the unique acceptor site is located in the first half of ORF II at nt 1508 [numbering according to Franck et al. (1980)
]. Mutations of the consensus sequence of the splice acceptor site, designated ma1 and ma3 (changing AGG to CCG and AGA, respectively), totally abolished splicing and were lethal for CaMV. Rare second-site revertants eventually appeared upon deletion of the central region of ORF II, which allowed the use of a cryptic splice acceptor site that is located further downstream in the ORF II sequence. Several hypotheses were proposed to attribute a biological function to these splicing events, the most commonly accepted of which postulates that splicing might facilitate expression of the downstream genes (Kiss-László & Hohn, 1996
; Hull, 2001
). Indeed, in RNA species C4, in which the end of the 5' UTR is joined to the acceptor site at position 1508, ORF III is moved to the position of the first translated ORF and efficient translation of ORFs III and IV from C4 RNA was verified in transient expression experiments in protoplasts. Nevertheless, Kiss-László et al. (1995)
mentioned an alternative (but not mutually exclusive) hypothesis, i.e. that the splicing events observed might also serve to modulate expression of genes I and/or II, which can be translated only from the remaining 30 % of unspliced RNA. In this work, we reinvestigate the question of exactly what feature of the splicing events characterized by Kiss-László et al. (1995)
is vital for CaMV.
ORF II is involved in aphid transmission [for a review, see Blanc et al. (2001)] and is otherwise dispensable for host plant infection, as demonstrated by the CaMV isolate CM4-184, where a 421 bp deletion removes almost the entire coding sequence of P2, the ORF II product (Howarth et al., 1981
). Surprisingly, and in apparent contradiction, several amino acid changes in P2 are lethal for the virus (S. Blanc, unpublished results), suggesting that, under some circumstances, P2 may be toxic for virus replication and hence may require very tight regulation (Pirone & Blanc, 1996
; Blanc et al., 2001
). This led to the hypothesis that splicing of 35S RNA is required to downregulate P2 accumulation, and the prediction that the splicing event is vital only where functional P2 accumulates. To test this hypothesis, we produced a number of different CaMV mutants that were debilitated in their capacity to accumulate P2 in infected plants. As predicted, impairing 35S RNA splicing in these mutant viruses was no longer lethal. Moreover, despite disruption of the splice acceptor site at position 1508, symptom appearance and virus accumulation in most of these mutants was not modified, demonstrating that, in the absence of P2, splicing per se has no important biological function in CaMV. Hence, we conclude that the vital role of the splicing events analysed is downregulation of P2 expression.
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METHODS |
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The mutant CaMV II-S was obtained similarly, with the partially overlapping primers
IIS-Forward (5'-AATAAACTAGTATGGCTAATCTTAATC-3') and
IIS-Reverse (5'-GCCATACTAGTTTATTCTCCACAGATTTC-3') instead of Del-S-Forward and Del-S-Reverse. This construct resulted in a mutant that harboured a total deletion of ORF II, with a unique SpeI restriction site separating ORFs I and III.
For mutants CaMV Top-S and Tart-S, similar chimeric PCR fragments were again amplified from the template pCa37. For the first round of PCR, completely overlapping primers were used for both constructs. These primers spanned nt 13431387 and 13401375 for CaMV Top-S and Tart-S, respectively. For CaMV Top-S, the complementary overlapping primers contained a mutation that altered codon 6 of ORF II from CAA to TAA, thus introducing an early termination codon. For CaMV Tart-S, the complementary overlapping primers contained a mutation that altered the ATG of ORF II to AAG, thus abolishing initiation of translation. The first and second rounds of PCR amplification were performed as above with primers RKpn and FBsiW and the amplified mutated fragments were introduced into pCa37 at the BsiWI and KpnI restriction sites. To introduce the splicing acceptor mutations ma1 or ma3 into Tart-S, the same procedure was carried out on the plasmid templates pCa37ma1 and pCa37ma3 (Kiss-László et al., 1995).
CaMV-IISthio was produced by introducing the Escherichia coli thioredoxin gene, PCR-amplified from the plasmid pTrxFus (Invitrogen) and flanked with SpeI restriction sites, into the corresponding cloning site in CaMV-
IIS.
CaMV mut-S was constructed by amplifying the CaMV Cabb-S region between the unique XhoI and KpnI sites with primers F-mut-S and Rkpn. The 73 nt F-mut-S primer overlapped the XhoI site and the downstream codon that corresponds to aa 116 of P2, which was modified to encode an aspartic acid, instead of the original isoleucine. The PCR fragment obtained was introduced into the XhoI and KpnI restriction sites of pCa37. To combine mutations ma3 and mut-S, we simply replaced the XhoI/KpnI fragment of plasmid pCa37ma3 (Kiss-László et al., 1995) with that of mut-S. All modified plasmids created were verified by sequencing.
Infectivity of CaMV mutants and stability of mutations.
Infectivity testing was performed by mechanical inoculation of plasmid DNA into turnip hosts (Brassica rapa cv. Just Right) as described by Melcher et al. (1986) and symptom appearance was assessed visually daily for over 3 months. When symptoms were fully developed in a host plant, the virus was transferred mechanically to a new series of host plants by sap inoculation, with serial passages being repeated three times. After each passage, viral DNA was purified from systemically infected hosts as described by Gardner & Shepherd (1980)
. The region that contained the mutation of interest was PCR-amplified and sequenced. Whenever the introduced mutation was maintained and no second-site revertant was detected for at least one passage, the corresponding mutant was declared to be infectious.
Protein analysis.
To test for accumulation of virus and P2 (ORF II product) in infected plant cells, we collected leaves with fully developed symptoms and extracted the protein contents. Leaf material (200 mg) was ground in 0·5 ml extraction buffer (200 mM Tris/HCl, pH 7·6; 100 mM EGTA; 50 mM MgCl2; 0·1 % SDS) and centrifuged at 10 000 g for 10 min. The supernatant was then submitted to 12 % SDS-PAGE according to Laemmli (1970) and separated proteins were transferred onto nitrocellulose membranes. Detection of P2 and coat protein was performed with specific antisera that were described by Blanc et al. (1993)
and Karsies et al. (2002)
, respectively.
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RESULTS AND DISCUSSION |
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Complete deletion of ORF II resulted in a 35S RNA with ORF III being preceded solely by two ORFs (VII and I), a situation comparable to that described in spliced RNA species C1, C2 and C3 (Kiss-László et al., 1995), where ORF III is preceded by ORF VII and an ORF III fusion product. Thus, infectivity of
II-S might be enabled simply by the reduction in the number of ORFs that precede ORF III. To rule out this possibility, we increased the number of ORFs again by including a bacterial thioredoxin gene (330 bp) at the original position of ORF II. This modified 35S RNA is very unlikely to be spliced in a manner comparable to that of wild-type Cabb-S RNA, due to the absence of ORF II and, thus, of the splice acceptor site at position 1508. Nevertheless, CaMV
II-Sthio proved to be infectious. This result indicates that the presence of three fully constituted ORFs between the 5' UTR and ORF III in a 35S RNA that is impaired in its splicing capacity is not in itself detrimental to CaMV replication. It further suggests that it is specifically the ORF II sequence itself that is lethal in splicing-impaired 35S RNA. Expression of a foreign gene from viable CaMV mutants (DHFR or
interferon at the position of gene II) has been described (Brisson et al., 1984
; De Zoeten et al., 1989
) but, in those cases, and in contrast to
II-Sthio, the ORF II sequences had not been totally removed.
All three mutants CaMV Del-S, II-S and
II-Sthio were delayed in symptom appearance (Table 1
). We verified that the modified sequences were stable and that no additional gene rearrangements or second-site reversions were induced. Other than the absence of splicing, a reason for the delay might be that ORF II includes one of the two G-rich priming sites that are used for + strand DNA synthesis (Bonneville & Hohn, 1993
). The fact that replication is delayed but not abolished is probably due to the presence of an additional priming site for + strand DNA synthesis located within ORF V, which is functionally redundant with that in ORF II.
Preventing translation of P2 renders the splice acceptor site in ORF II non-essential
To test the hypothesis that splicing in wild-type CaMV is required to downregulate P2, we constructed CaMV derivatives in which either the start codon of ORF II was mutated from ATG to AAG (designated CaMV Tart-S) or a premature stop codon was introduced at aa 6 of P2 (designated CaMV Top-S). The aim of this experiment was to disable accumulation of P2 with minimal changes to the CaMV genome sequence. Tart-S was fully infectious, although symptom appearance was slightly delayed (Table 1). In contrast, Top-S was not infectious without acquisition of second-site reversions. In such cases, symptoms appeared after a considerable delay (68 weeks) and the stop codon that was introduced had reverted to a new amino acid codon (see one example in Fig. 2
). Sequencing and protein analysis controls carried out in parallel (Fig. 2
) demonstrated that no reversion had occurred in Tart-S at this time. As wild-type CaMV Tart-S and Top-S theoretically produce the same spliced RNA species, no differences in symptom development would be expected in the mutant, compared to wild-type CaMV. The observed delay in symptom appearance in the case of Tart-S is an indication that the unspliced 35S RNA is used, at least partially, for translation of genes downstream of ORF II, with the long non-coding sequence (481 nt) that separates ORFs I and III, and/or the short ORFs that are present in this region in other reading frames, presumably interfering with the polycistronic mode of CaMV translation (Ryabova et al., 2002
). In agreement with this latter explanation, we eventually observed a second-site revertant after several successive passages of Tart-S in host plants, with a new initiation codon (ATG) being created at aa 4 of the native ORF II (data not shown). This restores ORF II, thus masking the small ORFs that are present in other reading frames.
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Mutations ma1 or ma3, which suppressed splicing totally by disrupting the splice acceptor site at nucleotide position 1508 and were lethal for wild-type CaMV Cabb-S (Kiss-László et al., 1995), were introduced in the Tart-S mutant background to generate CaMV Tart-S-ma1 and Tart-S-ma3. Interestingly, both proved to be infectious, with no detectable delay in symptom appearance compared to Tart-S (Table 1
). These results confirm that, when P2 cannot be translated and thus does not accumulate in infected tissues (Fig. 2
), disruption of the splice acceptor site at position 1508 no longer interferes with the CaMV infection cycle and has little or no effect on the timing of infection. We checked the virus progeny carefully by sequencing the whole ORF II region and observed neither direct reversion of ma1 or ma3, nor second-site reversions of any sort.
CaMV mutants expressing unstable P2 are not affected by their splicing capability
Amino acid changes in the C-terminal -helix 1 of P2 resulted in the complete absence of P2 accumulation in CaMV-infected plants by serendipity (Hébrard et al., 2001
), which was explained by induced conformational changes leading to protein instability. Accordingly, we assumed that the mutation I116D in P2 (CaMV mut-S) would also prevent P2 accumulation, as position 116 falls into the hydrophobic interface that is responsible for the formation of coiled-coil structures that lead to P2 self-interaction (Hébrard et al., 2001
). This additional construct is of particular interest. It was created in order to yield a mutant as comparable as possible to wild-type CaMV, where all genuine ORFs are present and can be translated normally, but where the ORF II gene product P2 would nevertheless be absent. As mut-S has no modification that is predicted to affect genome translation or replication, in contrast to all other mutants described above, we expected this mutant to be infectious without any noticeable delay in symptom appearance when compared with wild-type Cabb-S.
We first verified that mut-S was viable and displayed no delays in symptom appearance (Table 1), then verified the absence of P2 in infected tissues (Fig. 2
). Mutation ma3 was then introduced in mut-S to yield CaMV mut-S-ma3, which was assessed for infectivity. Table 1
shows that it is indeed infectious, with very short delays (if any) in symptom appearance, further confirming that the splice acceptor site at position 1508 of CaMV Cabb-S is not essential in the absence of P2 accumulation in infected plants. Moreover, the very short delays that were recorded in disease development on infection of plants with CaMV mut-S-ma3, together with normal virus accumulation in infected tissues (Fig. 2
), suggest that splicing of the 35S RNA of CaMV (Kiss-László et al., 1995
) has no major biological function other than regulating expression of P2. However, as very short delays did sometimes occur, we could not totally exclude a secondary, minor, dispensable effect that was caused either by enhancement of translation of downstream ORFs or modulation of P1 expression, or both.
Three of the four spliced RNA species C1C4 contain in-frame fusions between ORFs I and II, the putative function of which was questioned by Kiss-László et al. (1995). As demonstrated here, the presence or absence of such P1P2 fusions (mutants with an unmodified acceptor site at position 1508, and any mutant ma1 or ma3 where the acceptor site is disrupted, respectively) is not critical for CaMV viability. We therefore believe that these putative fusion proteins have no function, at least in the absence of P2, and are rather unstable, underlined by the fact that they were barely detectable in infected tissues (Kiss-László et al., 1995
). We propose that the P1P2 fusions in RNA species C2, C3 and C4 are in-frame in order to prevent the deleterious appearance of many small ORFs that would result from out-of-frame fusions. This idea is supported by the lack of infectivity of our mutant Top-S, demonstrating that a series of small ORFs located between coding ORFs is deleterious for the CaMV life cycle.
Spliced RNA species are extremely difficult to detect in CaMV-infected plants, due to the presence of many partially digested virus RNA molecules that are released during reverse transcription (Kiss-László et al., 1995). For this reason, we did not analyse putative alternative spliced RNA species in our various mutants, but designed the latter to allow solid conclusions from simple testing of infectivity, timing of symptom appearance and virus and P2 accumulation. The logic underlying the design of the mutants used is discussed above, together with the results in each case, but we draw attention particularly to the comparison of mutants mut-S and mut-S-ma3. The change in mut-S is restricted to a single amino acid change in the last third of the protein. Whilst it results in the total disappearance of P2, it affects neither virus fitness, as judged by timing of infection (Table 1
), nor virus accumulation (Fig. 2
). The ma3 disruption of the splice acceptor site at position 1508 has virtually no effect on mut-S (Table 1
; Fig. 2
), whilst it kills wild-type Cabb-S. The only difference between the two is the presence/absence of P2; thus, the most logical conclusion is that P2 aborts CaMV infection in the absence of splicing. Indeed, other hypotheses that invoke putative alternative splicing events that could compensate for the ma3 mutation in mut-S are very unlikely, as these alternative splicing events obviously do not occur in the wild-type Cabb-S.
Pirone & Blanc (1996) first suggested the hypothetical requirement for tight regulation of P2 expression and accumulation. They speculated that the properties of P2 that are required for aphid transmission might, in turn, be toxic for the virus replication cycle or cell-to-cell movement. Thus, precise mechanisms of regulation may exist to harmonize the different steps of the CaMV life cycle: plant invasion versus aphid transmission. Here, we provide the first indirect experimental evidence for such toxic properties by showing that, whilst P2 can be removed totally without abolishing host plant infection, its accumulation can be lethal in the absence of splicing of the 35S RNA. At this point, we can speculate that some properties of P2 may potentially interfere with CaMV infection. The affinity of P2 for the microtubule network of the host cell (Blanc et al., 1996
) or its association with the virionP3 complex for aphid transmission (Schmidt et al., 1994
; Leh et al., 1999
, 2001
) appear to be good candidates. Both properties might interfere with replication or cell-to-cell movement and unregulated binding to the P3virion complex could, for instance, mask the nuclear localization signal motif of the coat protein and prevent nuclear targeting of the virions. It was shown that P3 is also required for cell-to-cell movement (Kobayashi et al., 2002
) and that P3 virions interact with the movement protein (L. Stavolone, personal communication). P2 could inhibit such an interaction and, consequently, cell-to-cell movement. Surprisingly, it was demonstrated recently that, in infected plant cells, P2 is not massively bound either to virionP3 complexes or to the microtubule network. Instead, it is stored entirely within specific, electron-lucent inclusion bodies (ELIBs) as P2P3 aggregates (Drucker et al., 2002
). The mechanisms that sequester P2 in these ELIBs remain to be discovered, but P3 may play a pivotal role. In this sense, when P3 is targeted to the ELIB together with P2, one could postulate that the P3/P2 ratio is important and might be regulated by the splicing events that are discussed here. Whilst P3 is probably expressed from both spliced and unspliced viral RNAs, P2 can be derived only from unspliced 35S species.
More generally, alternative RNA splicing is commonly interpreted as producing new RNA species that allow (or enhance) the expression of a protein with a specific biological function (Huang et al., 2000; Kondrashov & Koonin, 2003
; Kriventseva et al., 2003
; Lu & Cullen, 2003
). To the best of our knowledge, the work presented here is the first example of a splicing event of which the primary purpose appears to be prevention of excess accumulation of a specific gene product that would otherwise be produced from the unspliced RNA.
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ACKNOWLEDGEMENTS |
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Received 11 February 2004;
accepted 6 May 2004.