Mutations in the conserved portion of the BWYV RTD.
Sequence comparisons among members of the Luteoviridae
reveal extensive amino acid sequence similarities within the N-terminal half of the RTD (22
). Point mutations were generated in this region of the BWYV RTD at or near five positions that were conserved in all sequenced poleroviruses. In selecting the targets, preference was given to amino acids with charged side chains, as we reasoned that such residues are more likely to be exposed on the surface of the protein when it is folded into the native configuration. In the following discussion, amino acid substitutions within the RTD will be referred to by the following convention: XaZ, where X refers to the wild-type residue at position a of the RTD and Z refers to the substituted amino acid.
The positions of the different mutations in the RTD are shown in Fig. . In mutant BW5.121, the strictly conserved residue R24, which is located just downstream of the alternating proline tract, was replaced by an alanine (R24A; codon CGT replaced by GCT). In BW5.123, the strictly conserved amino acids ED(59–60) were replaced by alanines (E59A/D60A; GAG.GAC replaced by GCG.GCC). In BW5.125, KD(113–114), amino acids which are not conserved in the other poleroviruses but which lie near the conserved motif G-IAY (residues 118 to 122), were replaced by alanines (K113A/D114A; AAG.GAC replaced by GCG.GCC). Mutant BW5.127 (K200A/Y201D; AAA.TAT replaced by GCA.GAT) targets residues KY(200–201), which are adjacent to and in the first position of the conserved motif YNY (residues 201 to 203). Finally, in BW5.129, the strictly conserved doublet DE(225–226), which marks the end of the conserved region of the RTD, was substituted by two alanines (D225A/E226A; GAT.GAA replaced by GCT.GCA).
When inoculated to C. quinoa
protoplasts, viral transcripts containing the above point mutations directed synthesis of viral genomic and subgenomic RNA in amounts similar to those observed in protoplasts infected with wild-type BWYV transcript (data not shown). Proteins were extracted from the infected protoplasts and tested by Western blotting for the presence of the major coat protein (P22.5) and P74, using coat protein- and RTD-specific antibodies. The protoplasts infected with the RTD mutants were found to produce relative amounts of P22.5 and P74 similar to those observed in wild-type-infected protoplasts (Fig. ), indicating that none of the point mutations had interfered with translation suppression of the P22.5 cistron's termination codon. The efficiency of packaging of the mutant viral RNAs into virions and their stability were not tested directly. However, it has been shown previously that deletion of the entire RTD does not interfere with packaging of mutant BW6.4 RNA into stable virions (26
), making it unlikely that the point mutations in the RTD would have a dramatic effect.
FIG. 2 Immunodetection of the BWYV RT protein (P74) and major coat protein (P22.5) in transcript-infected C. quinoa protoplasts. Protein extracts of mock-inoculated protoplasts (left-hand lane) or protoplasts inoculated with the indicated BWYV transcript were (more ...) Accumulation of virus in agroinfected plants.
Full-length viral cDNAs containing the various RTD mutations were moved into the binary vector pBin19 under control of the cauliflower mosaic virus 35S promoter, and the resulting constructs were introduced into A. tumefaciens. Nicotiana clevelandii plants were inoculated with recombinant agrobacteria harboring either the wild-type cDNA (BW0) or one of the five above-described mutants. The content of virus antigen was assayed by ELISA on tissue samples from upper noninoculated leaves of the agroinoculated plants (18 to 21 plants tested for each construct) at 5 weeks postinoculation (p.i.).
The average ELISA A405
values for the plants agroinfected with BW5.121 (0.92 ± 0.07), BW5.123 (0.95 ± 0.08), BW5.125 (0.95 ± 0.05), and BW5.129 (1.03 ± 0.07) were slightly lower than the average A405
value observed in a parallel experiment for BW0
-infected plants (1.10 ± 0.07). BW5.127-infected plants, on the other hand, had a much lower ELISA titer (A405
= 0.55 ± 0.15). This is similar to the average A405
observed in parallel for plants agroinfected with mutant BW6.4 (0.53 ± 0.11), a mutant in which virtually the entire RTD is deleted (2
) and which has been shown previously to accumulate about 10-fold less virus than the wild type (4
). Measurements made 5 weeks later, however, revealed that the titer of viral antigen in the BW5.127-infected plants had risen significantly (0.93 ± 0.08), whereas the virus concentration in the BW6.4-infected plants remained low.
By 6 weeks p.i., most of the ELISA-positive plants infected with the RTD point mutants had developed typical interveinal yellowing symptoms. When total proteins were extracted from such plants and analyzed by Western blotting both P22.5 and P74 were readily detected (Fig. A). In a previous study (4
), short in-frame deletions in the conserved portion of the RTD were shown to interfere with incorporation of RT protein into viral particles. To determine if any of the RTD point mutants were similarly affected in RT protein packaging, we purified virus from the agroinfected plants and tested their protein contents by Western blotting. All mutant virions contained the C-terminally truncated form of RT protein of about 53 kDa (P53) in amounts comparable to that observed for the wild-type virus (Fig. B).
FIG. 3 Immunodetection of the BWYV RT protein (P74 or P53) and major coat protein (P22.5) in protein extracts from agroinfected N. clevelandii (A) and in purified virus purified from agroinfected N. clevelandii (B). The plants were agroinfected with the indicated (more ...) Sequence of progeny virus in agroinfected plants.
The stability of the engineered RTD point mutations during virus multiplication in the agroinfected plants was investigated by RT-PCR. Total RNA was isolated from systemically infected leaves of two plants which had been agroinfected with each mutant, and a region of the genome encompassing the mutation was selectively amplified by RT-PCR. The amplified cDNA (nt 4006 to 4544 for BW5.121, BW5.123, and BW5.125; nt 4006 to 5327 for BW5.127; nt 4545 to 4822 for BW5.129) was then cloned, and inserts from randomly selected clones were sequenced.
All sequence alterations detected in the RT-PCR clones obtained from agroinfected N. clevelandii are summarized in Fig. A. In the cDNA clones, two types of nucleotide substitutions were observed: (i) those which changed an amino acid residue within or near the primary mutation and/or were present in several RT-PCR clones and (ii) those (either silent or resulting in an amino acid substitution) which were distant from the primary mutation site and/or were detected only one time. Mutations of the second type were observed in the RT-PCR progeny for all mutants except BW5.129. When averaged over the entire set of RT-PCR clones analyzed for the five mutants, these mutations were found to occur with a frequency of about one for every 1,800 nt sequenced, similar to the substitution frequency of one per 1,700 nt sequenced observed for RT-PCR clones derived from plants agroinfected with the wild-type construct BW0 (Fig. A). Some of these background mutations no doubt reflect errors introduced during RT-PCR, while most if not all of the others probably represent neutral sequence variants which arise naturally in the virus population. For this reason we will limit the following discussion to mutations of the first type, whose appearance is more likely to have been favored by selective pressure exerted by the primary mutations.
FIG. 4 Distribution of nucleotide mutations detected in progeny viral RNA following agroinfection of N. clevelandii with wild-type virus or an RTD mutant (A) and after aphid transmission to M. perfoliata (B). For each mutant, the horizontal line indicates the (more ...) (i) BW5.121.
For BW5.121, the original R24A mutation was maintained in each of the 12 RT-PCR clones characterized. In two clones, a second-site mutation, P32L or T34I, was present near the primary mutation site (Fig. A). For brevity, we shall refer to the wild-type sequence PVT(32–34) to which these mutations map as the PVT patch.
FIG. 5 Sequences in the vicinity of the primary mutations of progeny RT-PCR clones following agroinfection (A) or successful aphid transmission (B) with mutants BW5.121 and BW5.123. The wild-type (BW0) RTD sequence is shown at the top; the amino acid(s) targeted (more ...) (ii) BW5.123.
The primary mutations E59A and D60A were conserved in all 10 RT-PCR clones analyzed, but four of them also contained a second-site mutation, P32L (Fig. A). Interestingly, this is the same as one of the PVT patch substitutions observed in BW5.121, although in this case the PVT patch is 26 nt upstream of the primary mutation site at positions 59 and 60.
The primary mutation K113A/D114A was maintained in all 10 RT-PCR clones analyzed, and no significant second-site mutations were noted (Fig. A).
As shown above, plants agroinfected with BW5.127 initially accumulated low levels of virus, but near-wild-type levels appeared at later times. This observation suggests that the primary K200A/Y201D mutation had affected a motif in the RTD important for virus accumulation but that modified forms of the virus which overcome this defect appear later in infection. To test this hypothesis, viral RT-PCR clones were produced from RNA extracted from leaves taken either early (4 weeks) or later (7 weeks) following agroinfection. The sequence analysis revealed that both of the primary mutations (K200A and Y201D) were present in 20 of 21 RT-PCR clones from three different plants at 4 weeks p.i. (Fig. A). In the single exception, D201 had been replaced by N in a clone derived from plant 9. We also detected the mutation S206L in two other clones from plant 9 at 4 weeks p.i. (Fig. A), but the significance, if any, of this particular second-site substitution has not been further investigated.
FIG. 6 Sequences in the vicinity of the primary mutations of progeny RT-PCR clones following agroinfection (A) and successful aphid transmission (B) of BW5.127. The wild-type RTD sequence is shown at the top, and the amino acids targeted in the mutant are indicated (more ...)
In contrast, RT-PCR clones prepared from RNA extracted 7 weeks p.i. revealed numerous modifications at D201. Thus, of the 20 RT-PCR clones analyzed, D201 had reverted to the wild-type Y in 8 clones and had undergone a pseudoreversion to N in 9 (Fig. A). The primary mutation D201 was retained in the remaining three clones. We conclude that the D201 substitution probably impedes efficient virus accumulation and that selection for revertants or pseudorevertants that overcome this defect is responsible for the enhanced virus accumulation at later times. The K200A substitution, on the other hand, is apparently neutral with respect to virus accumulation in planta.
All of the seven RT-PCR clones examined for BW5.129 retained the primary mutations D225A/E226A and no modifications were noted elsewhere in the region sequenced (Fig. A).
Aphid transmission from extracts of virus-infected protoplasts.
Extracts of transcript-infected protoplasts were used as a virus source in some transmission experiments. The aphids were allowed a 24-h acquisition access period (AAP) on the protoplast extract. The aphids were then transferred to healthy Montia perfoliata plants for a 4-day inoculation access period (IAP), and the plants were assayed for viral infection by ELISA 3 to 4 weeks later. In such experiments, only mutant BW5.125 was efficiently transmitted (Table ). Mutant BW5.129 was weakly transmitted, infecting only 3 of 26 test plants following challenge with 30 aphids per test plant. No transmission events were recorded for BW5.121, BW5.123, or BW5.127 even though 100% transmission efficiencies were routinely observed in parallel tests with extracts of BW0-infected protoplasts (Table ).
TABLE 1 Aphid transmission of BWYV RT pointmutants Aphid transmission of virus from agroinfected plants.
In other transmission experiments, young, fully expanded leaves of agroinfected N. clevelandii (4 to 6 weeks p.i.) or a solution of purified virus prepared from agroinfected plants was used as a virus source. Nonviruliferous M. persicae nymphs were allowed a 24-h AAP on the leaves or the purified virus solution. Then, 8 or 30 aphids were transferred to healthy M. perfoliata for a 4-day IAP, and the plants were assayed for infection as described above. The tests (Table ) revealed that for both types of inoculum, mutants BW5.125 and BW5.129 were transmitted with efficiencies similar to that observed in parallel experiments with BW0. Mutants BW5.121 and BW5.127 were transmitted with intermediate efficiency. Transmission of BW5.123, on the other hand, occurred at a very low rate even though a high inoculation level (30 aphids/plant) was used; only 1 of 8 test plants became infected when BW5.123-agro-infected plants were used as the virus source, and only 2 of 34 plants became infected by aphids which had been given a high concentration (25 μg/ml) of purified virus (Table ).
Transmission by virus-microinjected aphids.
Experiments were carried out to determine if the lower transmissibility observed when the aphids were allowed to acquire BW5.121, BW5.123, and BW5.127 from leaves of agroinfected plants or by membrane feeding on purified virus was due to a defect in a step or steps of the mutant's circuit through the aphid subsequent to passage of the virions from the intestine into the hemocoel. To this end, 0.25 to 0.5 ng of purified wild-type or mutant virus was microinjected into the hemocoel of M. persicae nymphs. The nymphs were then placed on test plants (five aphids per plant) for a 4-day IAP, and infection was scored by ELISA 4 weeks later. BW5.121, BW5.123, and BW5.127 had transmission efficiencies similar to that of wild-type virus (Table ), suggesting that the RTD mutations in question interfere with the initial step of the transmission circuit, i.e., movement of virions through the intestinal epithelial cell barrier.
TABLE 2 Transmission of BWYV RT point mutants followingmicroinjectiona
An alternative explanation for the above observations is that the inefficient transmission of BW5.121, BW5.123, and BW5.127 following membrane feeding was not due to a defect in virus acquisition from the intestine but rather to lower stability of these mutant virions in the hemolymph, and that the high concentrations of virus introduced by microinjection masked this effect. Although it is difficult to strictly rule out this possibility at present, we regard it as unlikely because symbionin, which is believed to stabilize virions in the hemolymph (32
), has similar affinities for BW5.121, BW5.123, and BW5.127 as for wild-type virus in an in vitro binding assay (J. F. J. M. van den Heuvel, personal communication).
Analysis of progeny virus after aphid transmission.
The foregoing experiments illustrate that except for BW5.125 (which is readily transmitted from both sources), transmission of the RTD mutants is more efficient following acquisition access to virus from agroinfected plants than it is when the virus is offered as an extract of transcript-infected protoplasts. A plausible explanation for this difference is that the mutant virus is defective in one or more steps of the transmission process but reversions or compensatory mutations which permit transmission have the time to appear during the 4- to 6-week duration of an agroinfection experiment. Furthermore, selective pressure for appearance of compensatory mutations in whole plants could also be exerted by a requirement for a functional RTD for efficient virus movement in planta.
To detect possible compensatory mutations, we sequenced progeny virus following successful aphid transmission of the RTD mutants when the inoculum was provided as purified virus from agroinfected N. clevelandii. The aphids were allowed to acquire the purified virus by membrane feeding or were rendered viruliferous by microinjection of the virus. The aphids were then given a 4-day IAP on M. perfoliata, and total RNA was extracted from infected leaves 4 weeks p.i. Viral cDNA sequences were amplified by RT-PCR, and regions encompassing each of the RTD mutations were cloned and sequenced as before.
Figure B summarizes of all sequence alterations detected in the RT-PCR clones obtained following aphid transmission. Again, we will consider only those nucleotide changes which provoke an amino acid substitution within or near the primary mutation site and which were observed in several RT-PCR clones. As observed for the progeny RT-PCR clones following agroinfection, such modifications (see below) were accompanied by background mutations scattered along the cloned cDNA. In the case of BW5.121, BW5.123, BW5.125, and BW5.127, these mutations occurred at frequencies similar to or below that observed among the RT-PCR clones obtained from plants infected with BW0 (data not shown). The frequency of accumulation of background mutations in BW5.129, however, was almost three times higher than for BW0, suggesting that for unknown reasons, the BW5.129 primary mutation promotes genetic instability. The sequence variants which we regard as significant for each mutant are discussed separately below.
The primary mutation (R24A) was strictly conserved in the aphid-infected plants, but proximal second-site amino acid substitutions were detected in all but one of the 14 RT-PCR clones examined (Fig. B). These second-site mutations (P32L in seven clones; T34I in six clones) were the same PVT patch mutations encountered as minor components of the sequence population in the agroinfected plants (Fig. A). We conclude that strong selective pressure for appearance of the PVT patch mutations is exerted during aphid transmission of the virus.
The foregoing observations led us to introduce one of the PVT patch mutations, P32L, directly into BW5.121 and into the wild-type virus by site-directed mutagenesis to produce BW5.121-P32L and BW0-P32L, respectively. Both mutants accumulated to near-wild-type levels in the systemic leaves of agroinoculated N. clevelandii and were readily transmitted to M. perfoliata by aphids that were allowed to feed on the agroinfected leaves (Table ). The stability of the mutants in both the agroinfected plants and the target plants after aphid transmission was verified by sequence analysis of 10 or more RT-PCR clones of the progeny RNA. In every case, both the primary mutation (in BW5.121) and the P32L second-site modification were conserved, and no significant sequence variations were observed elsewhere in the region sequenced (data not shown). We conclude that the P32L second-site mutation favors aphid transmission in the BW5.121 background and does not interfere with virus accumulation or transmission in the wild-type background.
TABLE 3 Virus accumulation in agroinfected plants and aphid transmission of BWYV RT secondary pointmutants (ii) BW5.123.
The situation with BW5.123 was more complex. Here, analysis of RT-PCR clones obtained from two plants inoculated with aphids which had been membrane-fed purified virus revealed that one of the primary mutations had undergone a modification: the first A in the mutant sequence AA(59–60) had been replaced by T in plant 2 or had reverted to the wild-type residue E59 in plant 1 (Fig. B). To determine if the partial reversion EA(59–60) found in plant 1 restored full transmissibility of the virus, this plant was used as virus source for a second round of aphid transmission. Transmission to eight new M. perfoliata plants occurred with 100% efficiency with both 8 and 30 aphids per test plant. The progeny viral RNA was extracted from two of these plants, and nine RT-PCR clones were sequenced. EA(59–60) was present in all progeny clones, and no mutations elsewhere were noted (data not shown).
Successful transmission of BW5.123 following microinjection of aphids with virus was associated with a different set of compensatory mutations. RT-PCR clones obtained from two such plants retained the original E59A/D60A mutation but exhibited the same PVT patch second-site mutations (P32L or T34I) observed in the BW5.121 progeny (Fig. B). The fact that these mutations were encountered only in plants infected by microinjected aphids indicates that at least in the BW5.123 background, the sequence signals permitting virus movement through the ASG are distinct from those which allow transit from the intestine into the hemocoel. To test this point directly, the double mutant BW5.123-P32L was constructed and agroinoculated to N. clevelandii. Virus accumulated to near-wild-type levels in the plants but could not be aphid transmitted to M. perfoliata (Table ). Aphids rendered viruliferous with BW5.123-P32L by microinjection, on the other hand, were efficient virus transmitters (Table ). We conclude that the A59E reversion and, probably, the A59T pseudoreversion permit movement of the mutant virus across both the intestine/hemocoel interface and through the ASG but that at least in the BW5.123 background, the PVT patch mutation operates only at the second barrier.
The primary mutation (K113A/D114A) was conserved in each of the 12 clones analyzed, and no second-site amino acid mutations were noted elsewhere in the region sequenced (Fig. B). We conclude that the particular motif targeted in BW5.125 is not required for efficient aphid acquisition or transmission.
For mutant BW5.127 (K200A/Y201D), sequence analysis of progeny RT-PCR products following transmission revealed that the K200A primary mutation was conserved in 14 of the 18 clones. The four exceptions, with a V at position 200, all derived from the same plant (Fig. B). We conclude that K200 is dispensable for aphid transmission. At position 201, on the other hand, an amino acid with a phenolic side chain (F or Y) was present in all but one of the progeny clones. The fact that only 1 of 18 RT-PCR clones examined after aphid transmission contained N201 even though this substitution was relatively abundant in BW5.127-agroinfected plants (Fig. A) suggests that selective pressure against retention of the N201 variant is applied during transmission.
Following aphid transmission, pseudorevertants with an F at position 201 were abundant in the progeny even though this substitution was not detected among the RT-PCR clones obtained from plants agroinfected with BW5.127 (Fig. A). To test the effect of F201 directly, the substitution was introduced into the BW5.127 background to yield BW5.127-D201F. The mutant multiplied to near-wild-type levels following agroinoculation and was efficiently aphid transmitted (Table ). Sequence analysis of 16 RT-PCR clones obtained following aphid transmission revealed that the D201F mutation was conserved in every case. These observations suggest that the low level of the F201 variant observed following agroinfection with BW5.127 does not reflect unfitness but rather indicates that conversion of D201 to F requires two nucleotide transversions (GAU to UUU) while conversion of D201 to Y or to N requires only one transversion or transition, respectively. Indeed, the finding that the F201 pseudorevertant is rather efficiently selected during aphid transmission from a state of low abundance in the BW5.127-agroinfected plants suggests that at least in the BW5.127 background, it has a selective advantage over the true revertant, Y201.
It was shown above that BW5.129 was transmitted poorly when the virus was supplied to the aphids as an extract of transcript-infected protoplasts but that transmission was efficient when aphids were allowed to acquire virus from agroinfected leaves or by membrane feeding of purified virus (Table ). We anticipated that the efficient transmission observed in the latter situation would be associated with reversion or compensatory mutations in the BW5.129 genome. However, when the progeny RNA following successful transmission was characterized by RT-PCR, the original D225A/E226A mutations were retained in each of the 11 clones examined (Fig. B). Furthermore, although several second-site amino acid substitutions or silent mutations were observed in eight of the progeny RT-PCR clones, none were present more than once except for a silent mutation replacing T(4472) by C, which was detected twice (Fig. ). Thus, although RTD second-site mutations were relatively frequent in the aphid-transmitted progeny of BW5.129, there was no obvious candidate for a compensatory mutation which could restore efficient transmissibility, as was observed after transmission of BW5.121 and BW5.123.