Institut de Biologie Moléculaire des Plantes du CNRS et de l'Université Louis Pasteur, 12 Rue du Général Zimmer, 67084 Strasbourg Cedex, France
Correspondence
Odile Hemmer
odile.hemmer{at}ibmp-ulp.u-strasbg.fr
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ABSTRACT |
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INTRODUCTION |
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In contrast to the wealth of information concerning TMV, very little is known about the assembly of other rod-shaped plant viruses. There is evidence that the OAS of the potyvirus Tobacco vein mottling virus (TVMV) is situated near the 5'-extremity of the viral RNA (Wu & Shaw, 1998), and the 5'-extremity of the viral RNA is also the site of the OAS on RNA of Papaya mosaic virus (PMV) (Sit et al., 1994
). This does not, however, appear to be a general feature for Potexviridae since both the genomic and the 3'-co-terminal subgenomic RNAs of Bamboo mosaic virus-V isolate (BaMV-V) are packaged, suggesting that the OAS resides somewhere in the 3'-terminal 1000 nucleotides (nt) of these viral RNAs (Lee et al., 1998
).
In this paper we have investigated the location of RNA sequences important for the assembly of virions of Peanut clump virus (PCV; genus Pecluvirus). PCV has rigid rod-shaped virions which superficially resemble those of TMV, except that the virus particles display a bimodal length distribution (190 and 245 nm; Fritsch & Dollet, 2000). The genome of PCV is composed of two plus-sense RNAs which display little sequence homology except for the 3'-terminal 273 nt, which are 95 % identical between the two RNAs (Manohar et al., 1993
). RNA-1 is 5·9 kb in length and encodes two proteins involved in viral RNA replication (P131 and P191, a C-terminally extended form of P131 produced by translational readthrough of the P131 stop codon) (Herzog et al., 1994
), plus P15, a suppressor of post-transcriptional gene silencing (PTGS) (Dunoyer et al., 2002a
) (Fig. 1
). RNA-2 (4·5 kb) encodes five proteins: the 23 kDa viral CP, P39, a putative vector transmission factor, and the triple gene block (TGB) movement proteins P51, P14 and P17 (Manohar et al., 1993
; Herzog et al., 1994
) (Fig. 1
). P15 on RNA-1 and P51 on RNA-2 are translated from relatively abundant subgenomic RNAs. Subgenomic RNAs responsible for synthesis of P14 and P17 have not been detected. Analogy with other TGB-containing viruses, however, suggests that both proteins are probably synthesized from a low abundance subgenomic RNA with a 5' terminus upstream of the P14 gene (Verchot et al., 1998
)
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METHODS |
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Clone 2CP1, which contains a 172 nt deletion in the CP gene (nt 808982), was produced by replacing the BamHINheI fragment from pPC2 (the BamHI site is upstream of the insert in pPC2; NheI is at nt 1620 of the cDNA insert) by the corresponding fragment from the clone 2M
172 (Herzog et al., 1995
). Clone 2
CP1
39TGB was produced by digestion of 2
CP1 with SalI and SpeI, fill-in with Klenow fragment and recircularization. To produce Rep
CP2, a SalI site was created by PCR just upstream of the position corresponding to nt 5221 of RNA-1 (the novel SalI site was built in to the primer used in the PCR reaction). The resulting PCR fragment extended to the 3' terminus of the cDNA and was cleaved by SalI and MluI (the MluI site is at the 3' end of the cDNA sequence). The SalIMluI fragment was used to replace the NcoIMluI fragment of Rep-15 to produce Rep
CP2. The same approach was used to create 1
B5220 by using the SalI/MluI-cleaved PCR product to replace the BamHIMluI fragment of pPC1. The SalI site was also used to produce a SalINheI fragment (nt 52215513) of the P15 gene to replace the SalISpeI fragment of 2CP
1 to produce 2CP
1
39TGB-15Nh. The validity of all constructs was confirmed by digestion with appropriate restriction enzymes and sequence analysis.
Synthesis of transcripts and purification of viral RNA.
Capped in vitro transcripts were obtained with a Ribomax transcription kit (Promega) following the manufacturer's instructions. pPC1- and Rep-15-derived plasmids were linearized with MluI and pPC2-derived plasmids with HindIII before transcription. Viral RNA was extracted from purified PCV (isolate PCV2) (Manohar et al., 1993).
Detection of viral proteins in vitro and in vivo.
In vitro translation in wheat germ (WG) extract and rabbit reticulocyte lysate (RRL) were as described (Hemmer et al., 1993; Herzog et al., 1995
) using [35S]methionine to radiolabel newly synthesized proteins. The 35S-labelled translation products were detected by autoradiography following electrophoresis through a 10 % polyacrylamide gel in denaturing conditions (SDS-PAGE; Hemmer et al., 1993
).
Total protein extracts from protoplasts were separated by SDS-PAGE and transferred to Immobilon-P membrane (Millipore). Western blotting procedures and the antiserum directed against the PCV P51 protein have been described (Erhardt et al., 1999; Dunoyer et al., 2001
, 2002b
).
Inoculation of plants and protoplasts.
Inoculation of Chenopodium quinoa with virion RNA (1·25 µg in 50 µl for each of two leaves) was as described (Herzog et al., 1998). Protoplasts (106 protoplasts in 0·5 ml) from BY-2 tobacco cells (Nagata et al., 1992
) were prepared and inoculated with transcripts (Dunoyer et al., 2001
). Five µg of each transcript was employed per inoculation unless a transcript smaller than
2000 nt was to be included in the inoculum mix. In this case, the shorter transcript strongly interfered with productive replication of the longer transcripts, resulting in accumulation of only very low amounts of progeny RNA-1 and -2. Therefore, preliminary tests were carried out using 5 µg each of pPC1 and pPC2 transcript plus a range of concentrations of each smaller transcript to determine the maximum amount of the latter which could be added to the inoculum mix without greatly inhibiting replication of the larger RNAs. Amounts of the smaller transcripts ranging from 0·05 µg (Rep-15 and its derivatives) to 0·20·5 µg (short pPC2-derived transcripts such as 2
NcoSty, 2
39TGB etc.) were found to meet this requirement and were used in subsequent experiments.
Northern blot analysis of viral RNA.
Total RNA was purified from inoculated leaves (0·2 g) of infected C. quinoa plants 11 days post-inoculation (p.i.) (Herzog et al., 1998) and from 106 inoculated BY-2 protoplasts 48 h p.i. by phenol/chloroform extraction (Hemmer et al., 1993
; Dunoyer et al., 2001
). For analysis of encapsidated RNA, the protoplasts (106) were collected by centrifugation for 2 min at 100 g in a swinging bucket rotor and the pellet was resuspended in 600 µl of 50 mM Tris/HCl, pH 7·5, 10 mM MgCl2 in a 1·5 ml microtube. The protoplasts were disrupted by three cycles of 10 s ultrasonication followed by 30 s in ice. Ultrasonication was carried out with a Vibracell Ultrasonicator (Sonic & Materials, Danbury, Conn., USA) equipped with a microprobe and operated at position 5 (70 W theoretical power output). After the third cycle, the disrupted protoplast extract was incubated at 37 °C for 30 min to degrade nonencapsidated viral RNAs, followed by low-speed centrifugation to remove cell debris. Encapsidated RNA was then obtained by phenol/chloroform extraction as above. The encapsidated RNA corresponding to 250 000 protoplasts and the total RNA corresponding to 80 000 protoplasts was then subjected to electrophoresis through a 1 % agarose/formaldehyde gel, followed by capillary transfer to Hybond membrane (Amersham). Viral RNAs were detected by hybridization with specific in vitro-transcribed 32P-labelled antisense riboprobes. Riboprobe 1 was complementary to nt 51935515 of RNA-1, riboprobe 2a to nt 3901624 of RNA-2 and riboprobe 2b to nt 35934024 of RNA-2. Riboprobe 1+2 was complementary to the 3'-terminal 124 nt of RNA-1 and RNA-2, which are identical in sequence in this region (Matsuda et al., 2000
). Radioactive signals on the blot were detected by autoradiography.
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RESULTS AND DISCUSSION |
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We have now carried out in vitro translation experiments with purified virion RNA in WG extract. 35S-Labelled P39, P131 and P191 translation products corresponding to the species observed in RRL (Fig. 2, lane 1) were also obtained in the WG extract (Fig. 2
, lane 3), although P39 accumulated less abundantly than in RRL. The relatively low expression of P39 in WG extract could indicate that the leaky scanning mechanism which provides access to the P39 initiation codon is less efficient in this system. CP (which contains only one methionine residue: at the N terminus) was not observed in translations using either the WG extract or RRL (Fig. 2
, lanes 1 and 3). It was readily detected, however, in both translation systems when [3H]leucine rather than [35S]methionine was used as the radioactive tracer amino acid (Herzog et al., 1995
; and data not shown). In addition to the species expected to be translated from full-length viral RNA-1 and -2, the WG extract contained an abundant
51 kDa translation product (Fig. 2
, lane 3) which was also detected in smaller amounts in the RRL (Fig. 2
, lane 1). This species co-migrated with the major product of translation of T51 (Fig. 2
, lane 2), an RNA transcript which contains the 3'-terminal half of RNA-2 starting just upstream of the gene for P51. The
51 kDa species was immuno-detected by a P51-specific antiserum from WG extract programmed with T51 or total viral RNA (Fig. 2
, lanes 5 and 6), and from a total protein extract of tobacco BY-2 protoplasts transfected with the full-length PCV RNA-1 and -2 transcripts T1+T2 (Fig. 2
, lane 7).
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When analysed by Northern blot with riboprobe 2b, which is specific for the 3' region of RNA-2 (Fig. 3A, top), a band of the size expected for subg2a was detected in both the T RNA and E RNA samples (Fig. 3A
, lanes 3 and 4), consistent with the conclusion that the subgenomic RNA is encapsidated. No such band was visible in either the T or E samples when the blot was hybridized with riboprobe 2a, which is specific for the 5'-terminal half of RNA-2 (Fig. 3A
, lanes 1 and 2), confirming that the band in question originates from the 3'-terminal half of RNA-2.
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Deletion mapping of sequences important for virion assembly on RNA-1
To obtain additional information about sequences important for assembly of virions containing PCV RNA-1, long deletions extending from a BamHI site at nt 1448 to different downstream sites were produced in a full-length cDNA corresponding to RNA-1 (Fig. 4A). In designing the deletion mutants, the 5'- and 3'-noncoding regions were left intact since sequences required in cis for viral RNA replication are likely to reside there. Encapsidation of the mutants in vivo was assayed as follows. In vitro run-off transcripts obtained from the cloned deleted cDNAs were inoculated to tobacco BY-2 cell protoplasts along with full-length run-off transcripts of RNA-1 and -2. T and E RNA fractions were prepared from the protoplasts 48 h p.i. as described in Methods and the RNAs were analysed by Northern blot. The analysis revealed that the RNA-1 constructs 1
B5220 and 1
BS, in which the deletion stopped at the beginning of the P15 gene (1
B5220) or further upstream (1
BS), were protected, as the deleted species was present in both T and E fractions (Fig. 4A
, lanes 14). In contrast, 1
BN, in which the sequence extending from nt 1453 to nt 5513 (84 nt upstream of the 3' end of the P15 gene) had been deleted, was present in the T RNA fraction (Fig. 4A
, lane 5) but not in the E fraction (Fig. 4A
, lane 6). Consequently, we conclude that this deleted form of RNA-1 can replicate but does not undergo significant encapsidation. These observations suggest that the 5'-terminal region of the P15 gene contains an OAS sequence.
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Direct evidence that the 5' portion of the P15 gene contains an OAS was provided by experiments using a deleted, encapsidation-defective form of PCV RNA-2. As will be discussed more fully below, deletion of most of the central portion of RNA-2 interferes with its assembly into virions. Thus, the RNA-2 mutant 2CP1
39TGB, which contains a short internal deletion in the CP gene plus a deletion of almost all of the P39 gene and the TGB (Fig. 4A
), replicates but is not protected from RNase degradation in extracts of protoplasts co-inoculated with the transcript plus transcripts T1 and T2 (Fig. 4A
, lanes 7 and 8). However, addition of a sequence containing the first 293 nt of the P15 gene from RNA-1 into the aforesaid RNA-2 deletion mutant to produce 2CP
1
39TGB-15Nh (Fig. 4A
) renders the mutant RNA resistant to RNase in the E fraction (Fig. 4A
, lanes 9 and 10), indicating that the P15 gene sequence in question is sufficient to drive virion assembly in the absence of other RNA-1 sequences.
The foregoing observations map a cis-acting assembly sequence to the 5'-proximal part of the P15 gene of RNA-1. At present, we do not know why subg1, which would contain this putative OAS, is not encapsidated during a normal virus infection. Possibly, subg1 is very actively translated and the translating ribosomes shield the OAS from interaction with CP. Another possibility is that the position of the OAS in the RNA molecule is important. Thus, although the 5' extremity of subg1 has not been mapped, the electrophoretic mobility of the subgenomic RNA in denaturing agarose gel indicates that subg1 contains approx. 750 nt, which would place its 5' extremity about 80 nt upstream of the beginning of the P15 gene. In the short encapsidated deletion mutants such as RepCP2 and 2CP
1
39TGB-15Nh, the sequence upstream of the P15 gene is much longer, 402 nt and 866 nt, respectively. Perhaps a certain minimum length of sequence upstream of the OAS may be required for productive assembly initiation to occur.
Encapsidation of RNA-2 containing internal deletions
We have shown above that subg2a is encapsidated during a normal PCV infection, suggesting that an OAS resides somewhere within the 3'-terminal half of RNA-2. To test this idea directly, deletions were introduced in different parts of a cDNA corresponding to full-length PCV RNA-2, beginning with the 5'-proximal CP and P39 genes (see Fig. 5 for mutant structures). In vitro transcripts obtained from the deleted cDNAs were inoculated to BY-2 protoplasts along with full-length transcripts of RNA-1 and -2. In the case of constructs containing short deletions in the CP gene (2CP
1 and 2CP
2), transcript of Rep-15 rather than full-length RNA-2 was included in the inoculum as source of CP. T and E RNA fractions were then prepared from the protoplasts and analysed by Northern blot. The results illustrate that part (2CP
1 and 2CP
2) or all (2
CP39) of the CP gene can be eliminated without interfering with assembly of virions containing the rest of the RNA-2 sequence (Fig. 5
, lanes 58 and 1112). Similarly, the P39 gene can be deleted (mutants 2
39, 2
CP39 and 2
NcoEco) without inhibiting virion assembly (Fig. 5
, lanes 914).
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Additional deletions in RNA-2 identify a second OAS
Replication/encapsidation assays were also carried out with a series of RNA-2 deletion mutants in which different internal sequences had been removed without eliminating the CP gene. Together, these mutations eliminated part or all the RNA-2 sequence between the beginning of the P39 gene and nt 4142 near the 3' terminus of the gene encoding the 3'-proximal TGB protein P17 (Fig. 6). The various RNA-2 transcripts were inoculated to BY-2 protoplasts along with RNA-1 transcript and viral RNAs in the T and E fractions were analysed by Northern blot as above. All of the RNA-2 deletion mutants were encapsidated (Fig. 6
). Interestingly, the encapsidated mutants included constructs such as 2
5, 2
5b, 2
TGB and 2
39TGB (Fig. 6
) in which the region between nt 3370 and 4142 (identified above as the locus of a 3'-proximal OAS) was removed. These experiments thus indicate that there is a second OAS located within the CP gene.
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Taken at face value, our results suggest that PCV RNA-2 contains two OAS sequences: a 5'-proximal OAS in the CP gene and a 3'-proximal OAS in the central part of the TGB. It is however, extremely unlikely that assembly of viral nucleoprotein helices would initiate at two distant loci on the same viral RNA since this would present serious steric problems in properly encapsidating the RNA at the point where the two growing helices meet. We suggest rather that only one of the two observed OAS sequences is active on full-length RNA-2 and the second sequence is a pseudo-OAS which is disfavoured kinetically for assembly initiation compared to the authentic OAS. Significant assembly-initiation activity at the pseudo-OAS would be detected only in those mutants in which the authentic OAS has been eliminated by deletion. Thus, if the authentic OAS was present in the TGB coding region, for example, and a pseudo-OAS was in the CP gene (or vice versa), this would explain why mutants such as 2CP39 (Fig. 5
) and 2
39TGB (Fig. 6
) are both encapsidated while the mutant 2
CP39TGB (Fig. 7
), which would lack both the authentic and pseudo-OAS, is not. It is interesting to note that RNA of the TMV common strain also contains a pseudo-OAS, located in the CP cistron (Guilley et al., 1975
). It has been suggested that this pseudo-OAS, which is no longer functional on full-length TMV common strain RNA but displays structure and sequence homology with the authentic OAS, may be the ancestral assembly origin and that the common strain functional OAS derived from its duplication (Zimmern, 1977
). The pseudo-OAS is in fact used to initiate assembly on viral RNA of some strains of TMV (Fukuda et al., 1980
), however, raising the possibility that different strains of PCV could similarly employ one or the other of the RNA-2 sequences mapped in this paper as functional OAS.
Wherever the authentic OAS of RNA-2 is located it is likely to share critical features of sequence and structure with the OAS of RNA-1. Thus, a fuller characterization of the RNA-1 OAS will provide a useful template for identifying similar motifs on RNA-2, which can then be more fully tested for their capacity to initiate assembly by substitution into assembly-defective transcripts of RNA-1.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 21 March 2003;
accepted 23 May 2003.
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