Department of Microbiology, University of Alabama at Birmingham, BBRB 373/17, 845 19th St South, Birmingham, AL 35294-2170, USA
Correspondence
L. Andrew Ball
andyb{at}uab.edu
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ABSTRACT |
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MAIN TEXT |
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The genome of the insect nodavirus Pariacoto virus (PaV) comprises a 3·0 kb RNA1 that encodes the RNA-dependent RNA polymerase and a 1·3 kb RNA2 that encodes protein alpha (Johnson et al., 2000, 2001
; Zeddam et al., 1999
). The 401 aa protein alpha is cleaved between residues 361/362 to yield the mature capsid proteins (Johnson et al., 2000
; Tang et al., 2001
). The 3 Å crystal structure of wild-type PaV particles (Tang et al., 2001
) indicates that the capsid comprises 180 chemically identical protein subunits. The N termini of the 60 subunits that surround each 5-fold axis interact extensively with icosahedrally ordered regions of the encapsidated RNAs. This N-terminal region is highly basic in both PaV and other nodaviruses (Johnson et al., 2000
; Kaesberg et al., 1990
) and has been implicated in the specificity of RNA encapsidation in FHV (Dong et al., 1998
; Marshall & Schneemann, 2001
).
We showed previously that PaV particles contained the mature capsid proteins beta and gamma and a small amount of uncleaved capsid precursor alpha. We also observed minor proteins migrating faster than beta that reacted with anti-PaV antibodies (Fig. 1 and Johnson & Ball, 2001
). This suggested that not all PaV virion proteins were chemically identical, and that at least some capsids might therefore exhibit protein asymmetry. To examine this possibility, we tested whether the second AUG of the capsid protein ORF (Fig. 1B
) initiates translation of an N-terminally truncated protein.
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The N-terminal sequences of the capsid protein species in bands B and C were analysed by direct protein sequencing. Capsid proteins from PaV were resolved by SDS-PAGE, transferred to PVDF membranes in 10 mM CAPS buffer pH 11·1, 10 % methanol, for 1 h at 400 mA and stained with Ponceau S. Bands B and C were excised and subjected to five cycles of N-terminal sequencing. The N-terminal sequence of band B (VSRTK with strong signals for V, S and T) was consistent with translation of the major PaV capsid protein initiating at the first AUG of the ORF (nt 2325 of RNA2) and subsequent removal of the initiating methionine residue (Fig. 1B). The N-terminal sequence of band C was not reliable enough to unambiguously define the sequence, so we used reverse genetics to establish the origin of the proteins.
Plasmids that express full-length infectious transcripts of RNAs 1 and 2 [called PaV1(1,0) and PaV2(0,0), respectively] have been described previously (Johnson & Ball, 2001; Johnson et al., 2000
). We used site-directed mutagenesis on PaV2(0,0) to introduce mutations designed to eliminate translation from the first or second AUG codon in the capsid protein ORF, or from both codons, as shown schematically in Fig. 1(B)
. For clarity, we will refer to the proteins initiated at these two AUG codons as the Met1 and Met25 proteins, respectively. Translation initiation from the Met25 codon was enhanced by altering the surrounding sequence (Fig. 1B
) so it was more similar to the invertebrate and vertebrate consensus sequences (Cavener & Ray, 1991
). For the wild-type and each mutant the calculated sizes for the predicted Met1 and Met25 alpha and beta proteins are shown in Fig. 1(C)
.
BSRT7/5 cells constitutively expressing cytoplasmic T7 RNA polymerase (Buchholz et al., 1999) were transfected with plasmids PaV1(1,0) and either wild-type or mutant PaV2(0,0) plasmids. In all cases, the RNAs were confirmed to replicate by metabolic labelling in the presence of actinomycin D (data not shown). Four days post-transfection (p.t.), the cells were lysed and proteins resolved by SDS-PAGE with an aliquot of the same wild-type virus sample as shown in Fig. 1(A)
for comparison. PaV-specific proteins were detected by immunoblotting using a polyclonal rabbit antiserum raised against purified PaV (Johnson & Ball, 2001
).
Wild-type RNA2 directed the synthesis of PaV-specific proteins (Fig. 1D, lane 3), which comigrated with proteins detected in PaV purified from insects (Fig. 1D
, lane 1). In order of increasing mobility these corresponded to band A, which we interpreted as uncleaved Met1 alpha; band B, which we initially interpreted as the larger of its two cleavage products, Met1 beta; and a protein with the mobility of band C in Fig. 1(A)
. Gamma is not detected using this method. Band C had an estimated molecular mass of 36 kDa and was also detected in lysates of BSRT7/5 cells transfected with non-recombinant PaV vRNA (data not shown). Proteins corresponding to band D were sometimes detected (for example Fig. 1D
, lane 1). Lysates of cells that received the 25ko plasmid contained proteins that comigrated with authentic PaV alpha and beta proteins but no detectable 36 kDa protein (Fig. 1D
, lane 5), suggesting that this protein resulted from initiation at the second AUG codon and not from degradation or cleavage of the Met1 proteins. In contrast, the 1ko25en mutant produced abundant amounts of both the 36 kDa protein and a protein that comigrated with Met1 beta (Fig. 1D
, lane 7). The calculated molecular mass for Met25 alpha differs from Met1 beta by only 1·5 kDa (Fig. 1C
), so it was likely that the two predominant capsid proteins made by 1ko25en corresponded to Met25 alpha and beta. These data also suggest that band B seen in wild-type virus most likely contained two protein species, namely Met1 beta (39·1 kDa) and Met25 alpha (40·6 kDa), which comigrated as a single band. The lack of a clear N-terminal sequence corresponding to Met25 alpha in band B suggests that the amount of Met25 alpha in band B is low. However, the comigration of these proteins obscured their relative contributions to band B.
To examine the relationship of these proteins further, aspartic acid 68 in the capsid protein ORF was mutated to asparagine (D68N). This mutation was designed to abrogate the cleavage of alpha, because in the 3-D structure of PaV (Tang et al., 2001) the side-chain carboxylate of D68 lies close to the scissile peptide bond between beta and gamma, and occupies an equivalent position to that of residue D75 in FHV which mediates the autocatalytic cleavage of FHV alpha (Wery et al., 1994
; Zlotnick et al., 1994
). Accordingly, we interpret the two major bands detected in cells expressing the D68N mutant (Fig. 1D
, lane 4) as the uncleaved alpha proteins from Met1 and Met25. In agreement with this interpretation, only one major capsid-specific band was detected in cells that expressed 25ko/D68N (lane 6) or 1ko25en/D68N (lane 8), and in each case the single band comigrated with the putative alpha protein directed by the corresponding cleavage-competent version. A third minor band migrating slightly faster than the Met25 beta protein was detected in cells expressing the D68N mutant (Fig. 1D
, lane 4). It may represent an additional minor capsid protein or indicate that cleavage was not completely blocked by the D68N mutation. Since Met1 beta and Met25 alpha comigrate, if cleavage is not completely blocked by the D68N mutation, we cannot rule out the possibility that the D68N mutant band we interpret as Met25 alpha contains small amounts of Met1 beta. The 1ko25ko construct that had both Met1 and Met25 altered made no detectable capsid protein (Fig. 1D
, lane 9). Taken together, these results indicate that PaV particles contain a minor capsid protein which is initiated from the second AUG of the capsid ORF. This protein is initiated from Met25 and is cleaved to liberate a 36 kDa beta protein (band C) that was detected in wild-type particles both by staining and immunoblot analysis.
Since cleavage of alpha occurs after nodavirus assembly (Gallagher & Rueckert, 1988), cleavage of the mutant capsid proteins observed in Fig. 1(D)
suggested that both the longer and shorter versions of the capsid protein could self-assemble into virions. To produce enough virus particles for analysis we developed a two-step transfection procedure using BSRT7/5 cells which are refractive to infection with PaV, thereby reducing the possibility of selection for revertants. BSRT7/5 cells in 35 mm wells were transfected with 2·5 µg of plasmid PaV1(1,0) and 2·5 µg of either PaV2(0,0), 1ko25en or 25ko. Two days p.t. total cellular RNA was extracted and 15 % of the RNA was used to transfect a second culture of BSRT7/5 cells in 100 mm dishes. Four days p.t. the cells were lysed by addition of 0·1 % NP40, the lysates clarified by centrifugation (9000 g, 15 min, 4 °C), and virus was pelleted through a 30 % sucrose cushion in 50 mM sodium phosphate buffer, pH 7·2 (100 000 g, 3 h, 10 °C). Resuspended virus was layered onto 1545 % sucrose gradients and centrifuged for 2·25 h at 200 000 g, 10 °C. The gradients were collected on a Biocomp piston gradient fractionator and the fractions containing virus particles were pooled and concentrated in Microcon YM-30 concentrators (Millipore).
Proteins that assembled into purified virus particles were resolved by SDS-PAGE and visualized by staining (Fig. 2A). Wild-type virions purified from infected larvae or from transfected BSRT7/5 cells yielded the same pattern of proteins (Fig. 2A
, lanes 2 and 3). However, particles purified from BSRT7/5 cells appeared to contain about twice as much of the Met25 beta protein as those prepared in insects. Particles derived from the 25ko and 1ko25en mutant capsid proteins each contained two proteins with the mobilities expected for Met1 alpha and beta, and Met25 alpha and beta, respectively. Less cleavage of alpha was evident in 1ko25en particles compared to those assembled from the Met1 protein (Fig. 2A
, lanes 5 and 4). Particle morphology was examined by electron microscopy of negatively stained preparations (Fig. 2B
). Although the yields of particles recovered from both mutants were reduced relative to wild-type virus, wild-type and mutant particles were morphologically similar at this level of resolution.
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Wild-type, 25ko and 1ko25en viruses (1x1010 particles) were injected into the haemocoel of G. mellonella; 7 days p.i. larvae were sacrificed and virus was gradient-purified as described previously (Johnson & Ball, 2001). Both wild-type and 25ko viruses replicated well in larvae and produced large quantities of particles as assayed by SDS-PAGE analysis, and the input RNA sequences remained stable (data not shown). In contrast, very little 1ko25en virus was recovered from inoculated larvae, indicating that this mutant virus was debilitated. Analysis by Krishna et al. (2003)
of an analogous FHV mutant that lacked the first 31 aa of the capsid protein showed that less RNA2 was encapsidated. At lower m.o.i.s in FB33 cells less RNA2 was detected for the 1ko25en mutant as compared to wild-type PaV (Fig. 3
, compare lanes 3 and 11), suggesting that particles made from the truncated PaV capsid protein may be less infectious because of a similar phenomenon.
In summary, wild-type PaV particles contain a small amount of a protein that initiates at the second AUG codon of the capsid protein ORF. Mutant particles that lacked this protein were infectious in both insect cell culture and larvae, where the mutations remained stable during passage. Evidently any selection pressure that may have existed in these experiments was insufficient to restore expression of the truncated capsid protein, suggesting that it exerts at most a subtle effect on the virus phenotype under these conditions. Although a biological role for the truncated capsid protein has yet to be identified, it may nevertheless provide an experimental opportunity to introduce structural asymmetry into the T=3 icosahedral virions for analytical or technological purposes.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 11 June 2003;
accepted 11 July 2003.