Southern bean mosaic virus RNA remains associated with swollen virions during translation in wheat germ cell-free extracts

VIROLOGY

171,602-606

(1989)

Southern

Bean Mosaic Virus RNA Remains Associated with Swollen Virions during Translation in Wheat Germ Ceil-Free Extracts S. A. SHIELDS, M. J. BRISCO, T. M. A. WILSON, AND R. HULL’

John lnnes institute and AFRC institute of Plant Science Research, Colney Lane, Norwich,

NR4 7UH, United Kingdom

Received October 7, 1988; accepted April 3, 1989 L-[35S]Methionine-labeled translation complexes were prepared by incubating either swollen intact southern bean mosaic virus (SBMV) particles or unencapsidated SBMV RNA in a wheat germ extract. The complexes were analysed by sucrose gradient centrifugation and by electron microscopy and dot blot hybridization of fractions from these gradients. In these complexes, 80 S ribosomes appeared to be associated with intact or near intact particles, suggesting that SBMV particles disassemble only after their RNA has initiated translation. This is in contrast to some other isometric viruses, such as turnip yellow mosaic virus, which appear to release their RNA rapidly prior to translation. o 198s Academic

Press, Inc.

a wheat germ cell-free system, in which encapsidated SBMV RNA is translated more efficiently than in our rabbit reticulocyte lysate. t.-[35S]Methionine-labeled arrested translation complexes were prepared from incubations programmed with encapsidated RNA (i.e., swollen SBMV particles), or with unencapsidated SBMV RNA, and their properties were compared. SBMV (Ghana strain) was propagated and prepared as previously described (2, 8). SBMV RNA was extracted by the phenol-chloroform method (8, 9). Wheat germ cell-free extract was prepared and used as previously described (2, 10). Translation products were quantified by scintillation counting of trichloroacetic acid precipitates and analyzed by gel electrophoresis (2, 8, 10). In developing an experimental system to analyze SBMV translation complexes, the various stages of the experimental procedure were optimized.

When southern bean mosaic virus (SBMV) or certain other plant viruses are added to wheat germ or rabbit reticulocyte cell-free translation systems, virus-specific proteins are synthesised, showing that the virus particles have been disassembled (I-3). For successful translation, the particle structure must first be relaxed (i.e., the particles swollen) which, for SBMV, involves raising the pH above 7 and chelating calcium ions (4, 6). However, the precise mechanisms of RNA release and particle disassembly are not known. SBMV particles may behave in vivo like those of turnip yellow mosaic virus, which release all their RNA within minutes of contact with leaves (6) or those of belladonna mottle virus (another tymovirus), which release their RNA immediately when the pH is raised above 7 in vitro (7). Hence, the full-length RNA of both these viruses is released so rapidly that extensive translation cannot be essential to the process. Alternatively, SBMV particles may behave like particles of tobacco mosaic virus (TMV), in which the RNA is uncoated sequentially during translation (1). Electron micrographs of intermediate SBMV translation complexes prepared from rabbit reticulocyte lysate apparently showed ribosomes translating RNA as it emerged from intact virus particles (8). However, despite choosing well-spread areas on the grid, the apparent association may still have been due to a coincidental juxtaposition, as many ribosomes and virus particles were visible in the sample. In this paper we have addressed the question, does disassembly of SBMV particles occur spontaneously before translation, or does it involve cotranslational extraction of the RNA from the virus particles? We used

(a) Particle swelling: It was important to make sure that virus particles were swollen and that the RNA in them was not degraded. Particles of two independent SBMV preparations were dialyzed against 10 mMTrisHCI, ImM EGTA, pH 8.25, at 21 o for 1, 3, 5, and 24 hr. Samples from these treatments were negatively stained with 1% (w/v) uranyl acetate and examined in the electron microscope or used to program wheat germ translations or the RNA was extracted and electrophoresed in an agarose gel. Both preparations gave similar results. The samples which had been dialysed for 1 hr gave low TCA-precipitable counts when added to the wheat germ system whereas those dialyzed for 3 hr or longer gave reproducibly higher counts (data not shown). There was little sign of degradation of the RNA up to 5 hr dialysis (Fig. 1) whereas the samples

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FIG. 1. Agarose gel electrophoresis of RNA from two preparations of SBMV. A (lanes 1, 3. 5, and 7) and B (lanes 2, 4, 6, and 8), after swelling for 0 hr (1, 2), 3 hr(3,4), 5 hr (5, 6), or 24 hr (7.8). G indicates the position of genomic RNA and S, the position of a subgenomic RNA. Gel stained with ethidium bromide.

dialysed for 24 hr showed considerable RNA degradation. This was supported by electron microscopy which revealed that particle shape was retained up to 5 hr dialysis but that after 24 hr dialysis, extensive degradation had occurred. (b) Translation of the RNA: Encapsidated (after swelling of particles) or unencapsidated SBMV RNA was incubated at equivalent final RNA concentrations (57-l 00 pg/mI) for various periods of time at 28” in 450 ~1 wheat germ extract. Optimal final cation concentrations for unencapsidated RNA were 2.9 mlLl Mg*+ and 107 mM K+, and for encapsidated RNA were 2.5 mM Mg2+ and 67 m/v! Kf (2); 1 &il~l L-[35S]methionine (>l 100 Ci/mmol) was used in each reaction. Translations were terminated either before or after incubation by addition of 450 ~1 of inhibitor solution (25 mN1 cycloheximide, 25 mM MgCI,) to the wheat germ extract. At 12.5 mM (final), cycloheximide inhibits both initiation and elongation of polypeptides and preserves preformed polyribosomes (I, 1 I- 73). Magnesium also preserves polyribosomes and stabilizes SBMV capsids (14, 15). To isolate intermediate translation complexes it was necessary to terminate translation before protein synthesis was complete. Brisco et al. (2) showed that protein synthesis could be detected after 5 min incubation of swollen SBMV particles in wheat germ extracts and that the first completed products were detectable after 18 min. Thus, for our purpose, translation for 15 min was considered to be optimal.

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(c) Removal of unincorporated label: It was necessary to remove unincorporated L-[35S]methionine from the translation reactions before further analyzing the translation complexes. Pelleting the translation complexes through a 2-ml cushion of 20% (w/v) sucrose (pretreated with 0.19/o v/v diethyl pyrocarbonate) dissolved in 15 mM magnesium acetate, 20 mM KCI, 50 mMTris-HCI, pH 8.25 (MKT) (Beckman Type 40 rotor, 36,000 rpm, 4 hr, 4”) was compared with dialysis. It was found that dialysis at 4” with stirring for 1 hr against three to four changes, each of 1 liter, of 2.5 mM magnesium acetate, 20 mn/l KCI, 50 mMTris-HCI, pH 8.25 (“low MKT”; similar to the translation buffer) removed sufficient unincorporated label for it no longer to be a problem during subsequent gradient analyses. (d) Gradient centrifugation conditions: 35S-labeled translation complexes were centrifuged for 90 min through 15-30% (w/v) linear sucrose gradients containing low MKT in a Beckman SW41 rotor at 36,000 rpm for 75 or 90 min at 4”. The radioactivity in aliquots of 0.1 ml from each 0.3-ml fraction was counted by liquid scintillation. Other aliquots were analyzed as described below. In the experiment shown in Fig. 2, a wheat germ extract which had been programmed with swollen SBMV for 15 min before addition of cycloheximide was compared with three control treatments: (i) an extract to which cycloheximide and swollen SBMV had been added together at zero time; (ii) a translation mix programmed with unencapsidated SBMV RNA for 15 min before addition of cycloheximide; and (ii) a translation mix programmed with SBMV RNA for 15 min, then cycloheximide added followed by swollen SBMV. It can be seen from Fig. 2a that the simultaneous addition of cycloheximide and swollen virus to the translation mix at zero time prevented formation of any rapidly sedimenting 35S-labeled translation complexes. Programming with naked SBMV RNA gave a major peak of L[35S]methionine at fraction 28 (the position expected for monosomes) and a minor peak in the position expected for disomes (fraction 25). Addition of swollen SBMV after termination of SBMV RNA translation at 15 min did not substantially alter this gradient profile. Only the incubation programmed with swollen SBMV gave a significantly different gradient labeling pattern from the others. Here, minor peaks occurred at the positions of monosomes and disomes and a major broad peak of L-[35S]methionine was located at fractions 19-22. Using TMV and swollen SBMV particles as internal markers it was estimated that particles sedimenting in fractions 20 and 21 would have sedimentation coefficients of about 158 and 145 S, respectively. This is a faster sedimentation rate than disomes on mRNA

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FIG. 2. Sucrose gradient centrifugation of samples after in vitro translation in wheat germ extract programmed for 15 min with swollen SBMV (0) swollen SBMV to which cycloheximide was added at time 0 (0) SBMV RNA (*). or SBMV RNA with swollen SBMV added after the 15.min incubation (0). (a) Radioactivity in gradient fractions. Positions of the size markers T (TMV particles; 195 S), SS (swollen SBMV; 98 S), and R (ribosomes; 80 S) are indicated. For clarity only the points for alternate fractions are given. The inset shows data for fractions 20-26 in detail, (b) Average numbers of SBMV particlesl~m’ immune-trapped from selected fractions. (c) Amounts of SBMV RNA (ng) detected in aliquots from selected fractions by dot blot hybridization with SBMV cDNA. (d) The amounts of ribosomal RNA (ng) estimated in aliquots from selected fractions by dot blot hybridization with a ribosomal cDNA probe.

which should sediment at 113 S (= ‘w) (fraction 25) or even trisomes (139 S). It was more consistent with one SBMV particle (swollen SBMV sediments at 98 S; Ref. (76)) associated with ribosomes; a complex of one SBMV particle with one ribosome would be expected to sediment at 126 S (= ‘m) and

with two ribosomes at 149 S. The minor peaks in the positions of mono- and disomes are most likely due to released RNA fragments associated with ribosomes. To examine the particles in the various fractions, electron microscope grids were coated with antiserum to SBMV using the conditions described by Roberts

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(17). The virus, in 1O-PI aliquots from selected fractions, was adsorbed onto SBMV antiserum-coated grids which were then negatively stained with 1o/o(w/v) uranyl acetate and examined under the electron microscope. The numbers of particles in 16 areas of 1.2 x 1.35 pm on electron micrographs of each fraction were counted and averaged (Fig. 2b). Figure 2b shows a peak of particles detected in fraction 20 from the gradient of the incubation programmed with swollen SBMV particles for 15 min. This peak was not present in the two controls which also (eventually) contained swollen SBMV particles. Many of the particles in fraction 20 were penetrated by stain and appeared to have disruptions of the capsid and to have material attached to or trailing from the capsid (similar to those in Fig. 3 in Ref. (8)). In contrast, particles from fraction 24 were mainly complete. Nucleic acid was extracted with phenol from 25-p.1 aliquots of selected fractions and, after addition of 10 pg calf thymus carrier DNA, was ethanol-precipitated and resuspended in 12 ~1 H,O. Two 5-p.1aliquots of the nucleic acid from each fraction were then spotted onto separate nitrocellulose sheets, baked, and prehybridized as described by Maule et al. (18). 32P-labeled probes were made from SBMV RNA and ribosomal RNA (prepared by phenol extraction of a 100,000 g pellet of wheat germ extract) by random priming and cDNA synthesis (19). One nitrocellulose sheet was hybridized with the SBMV probe and the other with the ribosomal RNA probe as described by Maule et a/. (18). After washing in 2x SSC (0.3 11/1NaCI, 0.03 M sodium citrate) at 65”, the filters were dried and cut into squares for each fraction, and the bound 32P was counted. Control filters showed that the L-[35S]methionine did not contribute to the counts. Parallel hybridizations were made against dots of a series of dilutions of viral and ribosomal RNAs. These showed that the probes were specific and they enabled the amounts of the two nucleic acids in the assayed fractions to be quantified (Figs. 2c and 2d). It can be seen from Figs. 2c and 2d that in fraction 20 from the sucrose gradient containing the incubation programmed with swollen SBMV there were peaks of SBMV RNA and ribosomal RNA as well as the peak of SBMV particles already noted. These peaks were not present in gradients from the other incubations, notably that to which swollen SBMV was added after a conventional SBMV RNA translation. The above data suggest that the translation complexes formed when swollen SBMV is added to wheat germ extract comprise virus particles and associated ribosomes. This is consistent with our cotranslational disassembly-RNA release model for SBMV (8). However, we have not been able to eliminate the remote

possibility that the rapidly sedimenting material might be a fortuitous association of macromolecules, e.g., of nascent chains translated from prereleased SBMV RNA associating with swollen SBMV particles during the 15-min incubation. We consider this an unlikely explanation for the observations in Fig. 2, although it can be only partly eliminated by the control of adding swollen SBMV after arrest of a 15min SBMV RNA translation. Brome mosaic virus (BMV), cowpea chlorotic mottle virus (CCMV), and the bacilliform alfalfa mosaic virus (AIMV) may also, like SBMV, disassemble while their RNAs are being translated. Under normal translation conditions the particles of these viruses are swollen, with properties similar to swollen SBMV particles, i.e., susceptibility to RNase, susceptibility of the N-terminal domain of the coat protein to protease, and salt lability (20-24). The encapsidated RNA of all these viruses can be translated when swollen particles are added to wheat germ extracts (2). In the process of cotranslational disassembly the ribosome has to “find” the 5’-end of the RNA. The 5’end of TMV RNA is located at the concave end of the rod-shaped particle, and thus is probably the first part of the RNA to be released for translation (1; T. M. A. Wilson, K. W. Mundry, and P. A. C. Watkins, unpublished data). During the swelling of BMV the RNA may move toward the particle surface, and AIMV particles are permanently swollen (25, 26). The extrusion of some RNA has been suggested to explain the large increase in hydrodynamic diameter recorded during the swelling of SBMV (27). The 5’-end of SBMV RNA is bound to a small protein (VPg) (28) which might cause the 5’-end to be located near to or on the surface of the virus particle, possibly allowing its selective extrusion after swelling. It may also be significant to the need for ribosomes to locate the 5’-end of the RNA that, in all the isometric viruses which exhibit swelling, a small proportion of the coat protein exists as covalent dimers (29) and there appears to be a polarity during particle assembly. Very recently, independent studies with CCMV (30) revealed essentially similar results to those reported here. Although we did not elect to stabilize the labeled complexes by cross-linking with formaldehyde, work by the two groups is mutually supportive of the phenomenon of cotranslational disassembly. ACKNOWLEDGMENTS M.J.B. was in receipt of a Cooperative Award for Science and Technology from the Department of Education for Northern Ireland. S.A.S. was funded under the Nuffield Foundation Small Grants Scheme for Research in Science. We thank 6. Wells for assistance with electron microscopy and M. Harvey for the computer graphics.

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