J. Mol. Biol. (1976) 108, 789-798
A Tobacco Mosaic Virus Mutant with Non-functional Coat Protein and its Revertant: Relationship to the Virus Assembly Process A nitrous acid-induced coat protein-defective mutant (PM6) derived from the wild-type strain (U1) of tobacco mosaic virus (TMV) and its coat proteinfunctional revertant (PM6R) have been isolated. PM6 is a virus assembly mutant in which the coat protein cannot encapsidate viral RNA. The coat protein o f PM6 forms Lmusual wheel-like structures consisting of aggregated disks with a hehcal conformation. PM6 protein has two amino acid substitutions when compared to U1-TMV, an Ala to Thr exchange at position 105 mad an Asp to Gly exchange, probably at position 88. PM6R has the Ala to Thr exchange found in PM6 at position 105, but the Asp to Gly exchange observed in PM6 has apparently reverted to the native sequence found in UI-TMV. A high frequency of irregular protein rods is observed in electron micrographs of PM6R, suggesting that the conformation of PM6R coat protein, although functional, is altered from that of the wild-type strain. When tobacco mosaic virus (TM-V) is treated with nitrous acid, a class of "defective m u t a n t s " (Siegel et al., 1962) can be isolated, whose coat protein will no longer eneapsidate the TMV-RNA molecule. Cells infected with these m u t a n t s contain the coat protein (free of viral RNA) in either a soluble or insoluble form (Siegel et al., 1962; Parish & Zaitlin, 1966; H a r i h a r a s u b r a m a n i a n et al., 1973; Hubert, 1974) with amino acid substitutions fi'om the parental wild-type (U1) TM-V strain (Siegel, 1965). I n this paper we present evidence, derived from peptide analysis of coat protein of an HNOg-induced TMV m u t a n t (PM6), t h a t an Asp residue at position 88 is crucial for TM-V assembly. The PY[6 m u t a n t seems to be unstable, in t h a t another m u t a n t TMV strain, termed PM6R, m a y sometimes be recovered from plants infected with PM6. The coat protein of PM6R is functional, and it is able to encapsidate PI~I6RRNA, forming infectious virus particles. The Asp to Gly exchange in PM6 appears to have reverted to the native sequence found in the U1 strain of TMV, while the second exchange (Ala to Thr) found in PM6 remains unaltered. Unique PlV[6 coat protein aggregates are discussed in relation to the TMV assembly model proposed b y Butler & Klug (1971). (a) Isolation and propagation of T M V mutants P M 6 and P M 6 R PM6 was obtained by t r e a t m e n t with HNO2 of the U1 strain of TMV to 1.0% survival (Siegel, 1960) and propagated b y established methods (Siegel et al., 1962; H a r i h a r a s u b r a m a n i a n & Siegel, 1969). We observed that, in the greenhouse, PM6infected plants frequently developed systemic, stable infections in the youngest leaves. We suspected t h a t reversion of PM6 from a defective to a wild-type strain had occmTed. Several possible revertant isolates were obtained b y screening PM6-infected plants which were grown under a variety of different conditions of light and temperature. One such group of PM6-infected plants was placed in a greenhouse where the 789
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J.J.
H U B E R T , D. P. BOURQUE AND M. Z A I T L I N
highest daily temperatures reached were between 43 and 46~ Potential revertants were isolated from terminal leaves in which systemic, wild-type symptoms appeared. Revertants were propagated using leaf homogenates as an inoculum. (b) Virus purification Leaves from tobacco plants infected with U1 TM-V or PM6R for 14 days or more were frozen and thawed four or five times, and then homogenized in a Waring blender with a minimal amount of Sorenson's sodium phosphate buffer (0.066 M, p H 7.0). Purification of virus was then effected b y the NaCl/polyethylene glycol (M r ---- 6000) method of Gooding & Hebert (1967). Purified virus was resuspended in water and its concentration estimated spectrophotometrically using the extinction coefficient for U1/~1%, I cm __ 30). ~L~ 260 nm -(c) Purification of PM6 coat protein The property of pH-dependent aggregation of PI~I6 protein was exploited for its purification. PM6-infected tobacco leaves were frozen in liquid nitrogen and then ground to a fine powder with a mortar and pestle. All subsequent operations were performed in the cold. Sodium phosphate buffer was added (2 ml per gram leaf powder) and the resulting slurry was filtered through cheese-cloth. The filtrate was clarified by centrifugation at 12,000 g, for ten minutes. Solid KCI (0.1 M-KC1 final concn) was added to the supernatant, and the p H was adjusted to 4.7 with tIC1. The coat protein was allowed to aggregate for ten minutes in the cold, and then the p H was readjusted to 4.7 with HC1. After the solution was clarified by centrffugation (at 12,000 g, for 10 min), the supernatant was centrifuged (at 105,000 g, for 1 h) to pellet the aggregated coat protein. The gelatinous pellet was resuspended in 0.001 M-NaOH, the resulting solution was adjusted to p H 8.0 with HC1 to dissociate the coat protein, and insoluble material was removed by centrifugation (at 12,000 g for 10 min, then at 105,000 g for 1 h). The concentration of coat protein in the supernatant solution was estimated spectrophotometrically from the extinction coefficient of U1 coat protein (~i%. i o m __ 12"7). ~280
cm
--
(d) Purification of P M 6 R and U1 coat protein The coat protein of PM6R and the U1 strain were obtained by stripping the proteins from purified virus preparations with 67% acid (Fraenkel-Conrat, 1957). PM6R and U1 coat protein aggregated during this procedure, at p H 6.1 and 4.7, respectively. Concentrations of protein preparations were determined as for PM6 coat protein.
(e) Peptide preparation and purification PM6 and PM6R protein were digested with trypsin-TPCK (Worthington Biochem. Corp., Freehold, N. J.) by the method of Funatsu (1964). Peptide 1 was precipitated at p H 4.2 (Funatsu, 1964). The remaining soluble peptides were lyophilized, dissolved in pyridine buffer at p H 8-8, clarified b y centrifugation, and then chromatographed on Dowex-1. Tryptic peptide 8 from PM6 and PM6R was further digested with subtilisin (Nagarse, Enzyme Development Corp., New York) by the method of Funatsu et al. (1964) and the resulting peptides were also chromatographed. Peptides that required further purification were ehromatographed on paper (Woody & Knight, 1959; Funatsu & Funatsu, 1967).
LETTERS
TO T H E
EDITOR
791
(f) Amino avid analysis Hydrolysis of coat protein and coat protein peptides was carried out under nitrogen in 6 ~-HC1 in vacuo for 24 and 72 hours at ll0~ (Zaitlin & McCaughey, 1965) prior to chromatography on a Beckman 121 automatic amino acid analyzer. Cys was chromatographed separately after performic acid oxidation of the coat protein (Hirs, 1956) and Trp was determined speetrophotometrieally using the Tyr/Trp ratio of whole coat protein (Beaven & Holiday, 1952). (g) Electron microscopy The contents of TMV-infected leaf cells was examined on carbon-coated grids (400 mesh) in a Philips EM200 electron microscope by the leaf dip method of Hitchborn & Hills (1965). (h) Characterization of P M 6 (i) P M 6 is a defective mutant. The infectious entity in PM6-infected leaves was shown to be labile, and its biological characteristics appeared to be identical to those of the previously described defective TM-Vmutants (Siegel et al., 1962 ; Hariharasubramanian & Siegel, 1969 ; Hariharasubramanian et al., 1973). No virion was observed by electron microscopy in either leaf dips or extracts. (ii) Coat protein properties. PM6 coat protein was relatively unstable during its course of purification. Subjecting the protein to pH conditions below 4.0, temperatures above 15 to 20~ concentrations above 5 mg/ml, freezing and thawing, and agitation all resulted in denaturation as indicated by precipitation of the protein from solution. The protein was found to have a cation requirement for reversible aggregation at pH 4.7. The chlorides of Mg2+, Na + and K + were increasingly effective in maximizing the yield of protein. Absence of cations during aggregation of the protein at pH 4.7 results in slow precipitation of the protein from solution. (iii) Amino acid composition of coat protein and its peptides. Amino acid analysis showed that PM6 coat protein, relative to U1 coat protein, was high in one residue each of Thr and Gly and low in one each of Asp (or Asn) and Ala (PM6, Table 1). These data suggest that PM6 resulted from mutations yielding only two amino acid changes from the wild-type protein. Two tryptic peptides (6 and 8) isolated by ion exchange chromatography (Fig. 1 (a)) from PM6 protein showed an Asp (or Ash) to Gly and an Ala to Thr exchange, respectively, by composition (Table 1) when compared to the known composition of peptides 6 and 8 from U1 protein (Funatsu eta/., 1964). To localize the exchange in tryptic peptide 8 (residues 93 to 112) it was digested with subtilisin and the resulting peptides separated by ion exchange chromatography (Fig. l(c)). The replacement was localized by composition to a peptide (residues 104 to 112; Table 1) which contains two Ala residues, at positions 105 and 110 (Funatsu et al., 1964). This subtilisin peptide (residues 104 to 112) was hydrolyzed in dilute acid (0.03 M-HC1), which removes the Asp residue at position 109 (Tsung & leraenkelCourat, 1965) and the two resulting peptides were purified by chromatography (Fig. l(d)). From the amino acid composition of these peptides (Table 1) we conclude that the Ala to Thr exchange in PM6 protein is at position 105. (iv) Electron microscopy. Electron mierographs of leaf dips from P/~I6-infeeted leaf tissue show what appears to be PM6 coat protein aggregated into rods (Fig. 2(a)), whereas such structures are completely absent from uninfected leaf tissue. These long flexuous rods appear to be made up of units that are loosely joined together, and each 52
792
J. J. HUBERT, [
ii I
D. P. B O U R Q U E
I
~
AN]:) M. Z A I T L I N
12~
0.8
0.55
8 4 0'6
9 0"15
0.4
9
7-8
~ 0.2
i
I00
i
200
500
I00
400
r
I
200
500
(ol
400
(b) f
i
i
C (104-1121 0"8
0-6
I 0"3
O. 4
D (98 -99} 193-99}
0'2
A(IIO-II2)
A(100-103) B(I04)
i
i
I00
200
i
500
(c)
400
0-15~. ~04-108) i i i 50
Fraction number
I00
150
200
(d)
FIG. l. Ion exchange c h r o m a t o g r a p h y of PM6 and PM6R peptides. I o n exchange c h r o m a t o g r a p h y of peptides was performed on a Dowex l - X 2 acetate column (0.9 cm • 150 cm) equilibrated with p H 8.8 pyridine buffer (1% pyridine, 1% 2,4,6-trimethylpyridine a n d acetic acid to desired pH). Peptides were applied to the column in p H 8.8 pyridine buffer a n d then eluted with 250 m l p H 8-2 pyridine buffer followed b y a gradient elution ( B - l l ) according to the m e t h o d of F u n a t s u (1964); the flow-rate was 35 to 40 ml/h at room t e m p e r a t u r e ; 3.3-ml fractions were collected a n d 0.1-ml samples from eve~sr t h i r d fraction were tested for protein b y the L o ~ ' y m e t h o d ; color developed was measured at 750 nm. Soluble tryptie peptides fron~ PM6 (a) a n d PM6R (b) proteins are n u m b e r e d according to their order in TMV coat protein beginning from the N-terminal residue (Funatsu, 1964). P e a k 48contains peptides 8 a n d 4. Peptides 7-8, 8 a n d 10 precipitated in the fractions in which they occurred a n d were f u r t h e r purified b y isoelectric precipitation a t p H 3.5, 3.2 a n d 7-0, respectively. Peptides 3-4 and 7-8 represent amino acid residues 47 to 68 a n d 91 to 112, respectively. Subtilisin (c) a n d dilute acid (d) peptides from PM6 (. . . . ) and PM6R ( ) protein were chromatographed using the same elution schedule. Numbers in parentheses adjacent to lettered peaks represent residues of the peptide found in t h a t peak.
u n i t a p p e a r s t o be a s t a c k of t w o disks. Circular s t r u c t u r e s (Fig. 2(a)) were f r e q u e n t l y o b s e r v e d which a p p e a r t o be f o r m e d from p r o t e i n rods of sufficient l e n g t h to b e n d b a c k on t h e m s e l v e s a n d join end t o end to f o r m a n u n b r o k e n " w h e e l " . E a c h wheel is m a d e o f a n u m b e r o f u n i t s a n d each u n i t a p p e a r s t o h a v e t w o e l e c t r o n dense grooves on its o u t w a r d side a n d one on t h e inside (Fig. 2(b)).
L E T T E R S TO T H E E D I T O R
793
TABLE 1
Amino acid composition of PM6 and PM6R1 coat 1~rotein and peptides 6 and 8 Amino acid residue
Lys Arg Asp Thr Ser Glu Pro Gly Ala Cys Val I/e Leu Tyr Phe Try
Mol amino acid/mol protein or peptide a Coat protein Peptide 6 U1 PM6 PM6R1 U1 PM6 PM6R~ 2 11 18 16b 16b 16c 8 6 14 1~ 14 94 12 4 8 3~
2.1 11.1 16"8 i6.6 16-2 16.0 8.1 7-2 13.1 0-7 14.1 9.1 12.1 4.1 8.2 3.1
1.9 10.9 17.9 16.8 15.8 16.0 7"8 6-1 12.9 0-9 13.9 8.7 12.1 3.9 7.9 3-3
1 3 2
0"9 2.2 2-1
1"0 3"0 2.3
1 1 3c
0-9 2.2 3.0
1.0 1.O 3.0
2
2.0
1.9
4 1 1
3.8 0.9 0.9
3.6 0-8 0-8
U1
Peptide 8 PM6 PM6R1
1 3o 4
0.9 3.0 4-9
0.9 3.0 5.2
4 1
3.9 0.9
3-9 0.7
3
2.0
1.9
1 2a 1
1.0 1.8 1.0
1.0 1.8 1.0
Values which differ from U1 coat protein are underlined. a Experimental values for PM6 and PM6R1 from a 24 h hydrolysis. Integral values expected for U1 coat protein taken from Hennlg & Wittmann (1972), and those for peptides 6 and 8 from Funatsu (1964). b Experimental values for PM6 and PM6R1 adjusted to those of 24 h hydrolysates of U1 protein. o Other values in column calculated on basis of U1 integral value for this residue used as a standard. d Values obtained on separate analysis after 72 h hydrolysis. e Value obtained on separate analysis of performic acid oxidized protein. Value determined speetrophotometrically. A s t r u c t u r e for t h e u n i t of t h e wheel t h a t w o u l d be c o m p a t i b l e with these observat i o n s is a 2 89 helix of s u b u n i t proteins. A model of a wheel composed of such helices (Fig. 2(c)) shows its a s y m m e t r i c n a t u r e . The o u t w a r d side of t h e helix has three t u r n s of coat p r o t e i n s u b u n i t s a n d t h e inside of t h e helix has two. Some of the p r o t e i n rods also a p p e a r to h a v e a few u n i t s t h a t are 2 89 helices (Fig. 2(d)) a n d some of t h e wheels also seem to have u n i t s t h a t are composed of disks (Fig. 2(a)), a l t h o u g h t h e model of t h e wheel p r e s e n t e d here has been c o n s t r u c t e d e n t i r e l y of 2 89 helices. T h e m a x i m u m n u m b e r of u n i t s t h a t can m a k e u p a wheel is u n k n o w n , b u t t h e m i n i m u m n u m b e r of u n i t s c o u n t e d in a wheel was 13 (Fig. 2(b)). (i) Characterization of P M 6 R P M 6 R a p p e a r e d m o s t f r e q u e n t l y i n PM6-infected p l a n t s gro~ql a t higher greenhouse t e m p e r a t u r e s (43 to 46~ I t was isolated i n 18 different i n s t a n c e s a n d identified b y its coat p r o t e i n a m i n o acid composition. U n l i k e PM6 coat protein, P M 6 R p r o t e i n is as stable as t h a t of U1 protein. I t forms aggregates a t p H 6.1, i n c o n t r a s t to U1 p r o t e i n which aggregates a t p H 5.0; it has a n isoelectric p o i n t of a b o u t 4.7, different from t h e p H 3.2 isoeleetric p o i n t d e t e r m i n e d for U1 coat p r o t e i n ( K r a m e r & W i t t m a n n , 1958). T h e composition of P M 6 R p r o t e i n indicates it has one a m i n o acid exchange,
794
J. J. HUBERT,
D. P. B O U R Q U E
A N D M. Z A I T L I N
FIe. 2. Electron micrographs of PM6 and PM6R and model of PM6 protein wheels. All micrographs from negatively stained leaf dips of PM6 or PM6R infected Samsun tobacco plants or from a purified virus preparation (Fig. l(e) only). (a) PM6 protein showing region of stacked disks in a rod and a wheel. The arrows indicate a region in a protein wheel where stacked disks are clearly visible. PM6 protein in the wheel (arrow) configuration appears to be constructed from both stacked disks and 289 helices, on the lower side and upper side, respectively. Magnification, 124,000 x . (b) The 289 helix in the PM6 protein wheel. The arrow indicates one 2~-turn helix t h a t is clearly visible. Magnification 680,000 • (c) Model o f the PM6 protein wheel. The model of the proposed wheel configuration shows the unit of construction as a 289 helix. The tubing alignment depicts the helices as t h e y would
LETTERS TO THE EDITOR
795
i.e. Ala to Thr (Table 1). The same procedures were employed to localize the exchange in PM6R as were used for PM6 protein. Ion exchange chromatography of P1K6R coat protein peptides (Fig. l(b), (c) and (d)) and amino acid analysis of these peptides (Table 1) show t h a t the Ala to Thr exchange occurs at position 105 in PM6R, as it does in PiK6 protein. In electron micrographs of PM6R leaf dips, a high frequency of irregular protein rods are observed (Fig. 2(e)) when compared to U1 TMV leaf dips, suggesting t h a t PM6R and U1 protein are not equivalent. PlV[6R protein seems to encapsidate TlVIV-RNA in a less compact manner t h a n U1 protein, resulting in P1K6R virions (Fig. 2(f)) which appear abnormal when compared to wild-type TMV virions (Caspar, 1963). (j) Origin of P M 6 and P M 6 R PM6 was obtained after treatment of the wild-type T1KV strain U1 with H-N02. Its coat protein was shown to be non-functional and to contain an Asp (or Asn) to Gly and an Ala to Thr exchange (Table 1). The position of the Asp (or Asn) to Gly exchange in PM6 coat protein has been localized to peptide 6; the exact location within this peptide is not known. Peptide 6 from U1 TM~ contains an Asn residue at position 73 and two Asp residues at positions 77 and 88 (Tsung et al., 1964). Considering the genetic code, all of these residues could be converted to Gly by I-[NO2-induced mutagenesis of TMV-RNA (~Iundry & Geirer, 1958; Schuster & Wilhelm, 1963). The possible origin of the second amino acid exchange in the PM6 coat protein (Ala to Thr at position 105) is not explained by the accepted mechanism of HN02 action (Schuster & Schramm, 1958). PM6 coat protein is not unique among defective TMV strains in which an amino acid exchange appears to be inconsistent with the mechanism of action of HN02. One of the two replacements is defective m u t a n t PM2 (Wittmann, 1965) and several replacements in two mutants with insoluble, defective coat proteins (PLY[1and PM4) are similarly unexplained (Hariharasubramanian et al., 1973). PM6 appears to revert to PI~I6R by a one-step reversion; the Gly exchange in PiK6 coat protein reverts to Asp (or Asn) but the Ala to Thr exchange remains unaltered. The resulting PM6R protein can encapsidate TMV-RNA, but may have a conformation which differs from wild-type coat protein, judging from the high frequency of irregular protein rods (Fig. 2(e)) and the abnormal nature of the PM6R virions (Fig. 2(g)) seeri in the electron microscope. (k) Localization of an amino acid residue critical for virus azaembly Because PlVI6R has a functional coat protein, we conclude t h a t the Asp (or Asn) to Gly exchange in PM6 protein is responsible for the inability of PM6 protein to aggregate correctly and encapsidate PM6-RNA to form normal virions. The change from an Asp (or Ash) to a Gly residue would engender a change in the net charge of appear in an electron micrograph of negatively stained PM6 protein wheels. Magnification, 1,500,000 x .
(d) PM6 protein rod showing possible helical region. The arrow indicates a portion of rod that m a y be composed of helical units, possible 289or 3-turn helices. Magnification, 124,000x.
(e) PM6R protein rods showing loose association of the disks. Magnification, 136,000• (f) PM6R virions. Magnification, 111,000 • (g) PM6 protein aggregates that appear to be in intermediate states of wheel formation. Magnification, 204,000 •
796
J. J. HUBERT, D. P. BOURQUE AND M. ZAITLAN
the protein and could affect the normal functioning of the protein by altering its conformation or ligand binding properties. Tryptic peptide 6 of the PM6 coat protein (Table 1) is the site of the Asp (or Asn) to Gly exchange and possible locations for this exchange are an Asn and two Asp residues at positions 73, 77 and 88, respectively (Tsung et al., 1964). Because a HNO2-derived TMV mutant (Ni 1103) with a functional coat protein has been found (Wittmann-Leibold & Wittmann, 1965) which has an Asn to Ser exchange at position 73, it is unlikely that the Ash residue at position 73 plays a critical role in function of the protein. Peptide 6 from PM6 is ehited from the column earlier than wild-type peptide 6 (Fig. l(a) and (b)), presumably as a result of its less acidic nature. Such elution behavior would be consistent with an Asp to Gly exchange but not with an Asn to Gly exchange. Consequently, Asn is probably still present in PM6. These data strongly imply that the exchanged residue in peptide 6 is either the Asp residue at position 77 or 88. Holmes Ribgrass, cowpea, and CGMMVcucumber TMV strains (Hennig & Wittmann, 1972; Rces & Short, 1975) all have residues other than Asp at position 77 in their coat proteins and all of these strains form infectious virions. Furthermore, residues 88 to 92 are conserved in cowpea TMV (Rees & Short, 1975) as well as in other virion-producing TMV strains (Butler & Durham, 1972). The preceding evidence suggests that position 88 is the site of the Asp to Gly exchange in PM6 coat protein and that position 88 may play a special role in TMV assembly. Durham & Butler (1975) have presented evidence that residue 88 is proximal to the RNA molecule, at about 40 A from the rod axis, whereas residue 77 is at 60/~ radius (A. Klug, personal communication). Consequently, it is suspected that the Asp residue at position 88 together with the Arg residues at positions 90 and 92 may be involved in the site for TMV-RI~/A binding to the TMV coat protein subunit (A. Durham, personal communication). These data support the concept that the amino acid sequence from residues 88 to 92 must be intact to insure correct proteinRNA interactions. However, defective mutants such as PM2, which are altered in other regions of the coat protein (Wittmann, 1965), show that interactions of two or more amino acid substitutions can also result in blocked virion assembly. (I) Relationship between P M 6 protein rods and protein wheels in vivo Durham & Klug (1971) propose that the growth of TMV protein rods takes place in vivo and in vit~v by the stacking of disks of protein (each disk is composed of two planar rings of 17 subunits each). At neutral pH the disks can be dislocated, forming two-turn helices which can interlock forming either protein rods in vitro, or virions in vivo by interacting with TMV-RNA. We have shown electron micrographic evidence that the coat protein of PM6 aggregates in vivo to form protein rods and wheels. It is interesting to speculate about the manner in which the wheels are formed and of what significance their structure may be to the theory offered by Butler & Klug (1971) for the assembly of TMV protein rods and virions. Rods of PI~I6 coat protein appear to be composed of disks that are weakly bonded, allowing them to be very flexible. Short rods of this type appear capable of bending back on themselves and annealing end to end to form wheels (Fig. 2). Disks formed from this protein show a greater tendency to dislocate in vivo into two-turn helices which have two open ends. It is then possible that single coat protein subunits can be added to these ends until steric hindrance prevents further subunit addition (Ohno et al., 1972; Okada & Ohno, 1972; Butler & Klug, 1973). Such helices then would be approximately 289 and would exist in the wheel configuration under physiological conditions. In the many wheels that have
L E T T E R S TO THE E D I T O R
797
been observed, the units comprising them appear to be both 2~-turn helices a n d disks (Fig. 2(a)). The rods also seem to have two-turn helices in regions where t h e y are bent allowing for the addition of single subunits, as evidenced by what appear to be 289 helices in these regions (Fig. 2(d)). I f units making up the wheels represent two-turn helices t h a t have been lengthened b y the addition of extra subunits, then it is possible t h a t the 289 helices observed in the wheel configuration represent a form of the lock washer or two-turn helix postulated by Butler & Klug (1971), which plays a major role in their theory on TM-~ assembly. This research was supported in part by National Science Foundation grant GB-39333 to one of us (M. Z.), by Hatch Act funds (to M. Z. and D. P. B.) allocated by the University of Arizona Agricultural Experiment Station, and by grant 5-RO1GM21242 from the National Institute of General Medical Sciences (to D.P.B.). The authors are also indebted to Ruth Smith and Angelo Longo for technical assistance. This is publication No. 2687 of the Arizona Agricultural Experiment Station. Department of Agricultural Biochemistry University of Arizona, Tucson, Ariz. 85721, U.S.A.
JEFFREY J. HUBERTt DoN P. BOURQD-E I~/IILTONZAITLIN$
Received 2 March 1976, and in revised form 7 September 1976 REFERENCES Beaven, G. H. & Holiday, E. R. (1952). Advan. Protein Chem. 7, 319-386. Butler, P. J. G. & Durham, A. C. H. (1972). J. Mol. Biol. 72, 19-24. Butler, P. J. G. & Klug, A. (1971). Nature New Biol. 229, 47-50. Butler, P. J. G. & Klug, A. (1973). Mol. Gen. Goner. 120, 91-93. Caspar, D. L. D. (1963). Advan. Protein Chem. 18, 37-121. Durham, A. C. H. & Butler, P. J. G. (1975). Eur. J. Biochem. 53, 397-404. Durham, A. C. H. & Klug, A. (1971). Nature New Biol. 229, 42-46. Fraenkel-Conrat, H. (1957). Virology, 4, 1-4. Funatsu, G. (1964). Biochemistry, 3, 1351-1355. Funatsu, G. & Funatsu, M. (1967). Agric. Biol. Chem. 31, 48-53. Funatsu, G., Tsugita, A. & Fraenkel-Conrat, H. (1964). Arch. Biochem. Biophys. 105, 35--41. Gooding, G. V., Jr & Hebert, T. T. (1967). Phytopathology, 57, 1285. Hariharasubramanian, V. & Siegel, A. (1969). Virology, 37, 203-208. Hariharasubramanian, V., Zaitlin, M. & Smith, R. C. (1973). Virology, 55, 202-210. Hennig, B. & Wittmann, H. G. (1972). In Principles & Techniques in Plant Virology (Kado, G. I. & Agrawal, H. O., eds), pp. 546-594, Van Nostrand Reinhold Co., New York. Hits, C. H. W. (1956). J. Biol. Chem. 219, 611-621. Hitchborn, J. H. & Hills, G. J. (1965). Virology, 26, 756-758. Hubert, J. 5. (1974). P h . D . dissertation, University of Arizona. Kramer, E. & Wittmann, H. G. (1958). Z. Naturforsch. 13b, 30-33. Mundry, K. W. & Gierer, A. (1958). Z. Vererbungsl. 89, 614-630. Ohno, T., Yamaura, R., Kuriyama, K., J_noue, H. & Okada, Y. (1972). Virology, 50, 76-83. Okada, Y. & Ohno, T. (1972). Mol. Gen. Genet. 114, 205-213. Parish, C. L. & Zaitlin, M. (1966). Virology, 30, 297-302. Rees, M. W. & Short, M. N. (1975). Biochem. Biophys. Acta, 393, 15-23. CPresent address: Division of Biology, California Institute of Technology, Pasadena, Calif. 91125, U.S.A. Present address: Department of Plant Pathology, Cornell University, Ithaca, N.Y. 14853, U.S.A.
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H U B E R T , D. P. B O U R Q U E AND M. Z A I T L I N
Schuster, H. & Schramm, G. (1958). Z. 2Va~urforsch. 13b, 697-704. Schuster, H. & Wilhelm, R. C. (1963}. Biochem. Biophys. Acta, 68, 554-560. Siegel, A. (1960). Virology, 11, 156-15~. Siegel, A. (1965). Advan. Virus Res. 11, 25-60. Siegel, A., Zaitlm, M. & Sehgal, O. P. (1962). Proc. Na~. Acad.,~ci., U.S.A. 48, 1845-1851. Tsung, C. M. & Fraenkel-Conrat, H. (1965). Biochemistry, 4, 793-800. Tsung, C. M., Funatsu, G. & Young, J. D. (1964). Arch. Biochem. Biophys. 105, 42-50. Wittmann, H. G. (1965). Z. Vercrbungsl. 97, 297-304. Wittmann-Liebold, B. & Wittmann, H. G. (1965). Z. Vcrerbungsl. 97, 305--326. Woody, B. R. & Knight, C. A. (1959). Virology, 9, 359-374. Zaitlin, M. & MeCaughey, W. F. (1965}. Virology, 25, 500-503.
A Tobacco Mosaic Virus Mutant with Non-functional Coat Protein and its Revertant: Relationship to the Virus Assembly Process A nitrous acid-induced coat protein-defective mutant (PM6) derived from the wild-type strain (U1) of tobacco mosaic virus (TMV) and its coat proteinfunctional revertant (PM6R) have been isolated. PM6 is a virus assembly mutant in which the coat protein cannot encapsidate viral RNA. The coat protein o f PM6 forms Lmusual wheel-like structures consisting of aggregated disks with a hehcal conformation. PM6 protein has two amino acid substitutions when compared to U1-TMV, an Ala to Thr exchange at position 105 mad an Asp to Gly exchange, probably at position 88. PM6R has the Ala to Thr exchange found in PM6 at position 105, but the Asp to Gly exchange observed in PM6 has apparently reverted to the native sequence found in UI-TMV. A high frequency of irregular protein rods is observed in electron micrographs of PM6R, suggesting that the conformation of PM6R coat protein, although functional, is altered from that of the wild-type strain. When tobacco mosaic virus (TM-V) is treated with nitrous acid, a class of "defective m u t a n t s " (Siegel et al., 1962) can be isolated, whose coat protein will no longer eneapsidate the TMV-RNA molecule. Cells infected with these m u t a n t s contain the coat protein (free of viral RNA) in either a soluble or insoluble form (Siegel et al., 1962; Parish & Zaitlin, 1966; H a r i h a r a s u b r a m a n i a n et al., 1973; Hubert, 1974) with amino acid substitutions fi'om the parental wild-type (U1) TM-V strain (Siegel, 1965). I n this paper we present evidence, derived from peptide analysis of coat protein of an HNOg-induced TMV m u t a n t (PM6), t h a t an Asp residue at position 88 is crucial for TM-V assembly. The PY[6 m u t a n t seems to be unstable, in t h a t another m u t a n t TMV strain, termed PM6R, m a y sometimes be recovered from plants infected with PM6. The coat protein of PM6R is functional, and it is able to encapsidate PI~I6RRNA, forming infectious virus particles. The Asp to Gly exchange in PM6 appears to have reverted to the native sequence found in the U1 strain of TMV, while the second exchange (Ala to Thr) found in PM6 remains unaltered. Unique PlV[6 coat protein aggregates are discussed in relation to the TMV assembly model proposed b y Butler & Klug (1971). (a) Isolation and propagation of T M V mutants P M 6 and P M 6 R PM6 was obtained by t r e a t m e n t with HNO2 of the U1 strain of TMV to 1.0% survival (Siegel, 1960) and propagated b y established methods (Siegel et al., 1962; H a r i h a r a s u b r a m a n i a n & Siegel, 1969). We observed that, in the greenhouse, PM6infected plants frequently developed systemic, stable infections in the youngest leaves. We suspected t h a t reversion of PM6 from a defective to a wild-type strain had occmTed. Several possible revertant isolates were obtained b y screening PM6-infected plants which were grown under a variety of different conditions of light and temperature. One such group of PM6-infected plants was placed in a greenhouse where the 789
790
J.J.
H U B E R T , D. P. BOURQUE AND M. Z A I T L I N
highest daily temperatures reached were between 43 and 46~ Potential revertants were isolated from terminal leaves in which systemic, wild-type symptoms appeared. Revertants were propagated using leaf homogenates as an inoculum. (b) Virus purification Leaves from tobacco plants infected with U1 TM-V or PM6R for 14 days or more were frozen and thawed four or five times, and then homogenized in a Waring blender with a minimal amount of Sorenson's sodium phosphate buffer (0.066 M, p H 7.0). Purification of virus was then effected b y the NaCl/polyethylene glycol (M r ---- 6000) method of Gooding & Hebert (1967). Purified virus was resuspended in water and its concentration estimated spectrophotometrically using the extinction coefficient for U1/~1%, I cm __ 30). ~L~ 260 nm -(c) Purification of PM6 coat protein The property of pH-dependent aggregation of PI~I6 protein was exploited for its purification. PM6-infected tobacco leaves were frozen in liquid nitrogen and then ground to a fine powder with a mortar and pestle. All subsequent operations were performed in the cold. Sodium phosphate buffer was added (2 ml per gram leaf powder) and the resulting slurry was filtered through cheese-cloth. The filtrate was clarified by centrifugation at 12,000 g, for ten minutes. Solid KCI (0.1 M-KC1 final concn) was added to the supernatant, and the p H was adjusted to 4.7 with tIC1. The coat protein was allowed to aggregate for ten minutes in the cold, and then the p H was readjusted to 4.7 with HC1. After the solution was clarified by centrffugation (at 12,000 g, for 10 min), the supernatant was centrifuged (at 105,000 g, for 1 h) to pellet the aggregated coat protein. The gelatinous pellet was resuspended in 0.001 M-NaOH, the resulting solution was adjusted to p H 8.0 with HC1 to dissociate the coat protein, and insoluble material was removed by centrifugation (at 12,000 g for 10 min, then at 105,000 g for 1 h). The concentration of coat protein in the supernatant solution was estimated spectrophotometrically from the extinction coefficient of U1 coat protein (~i%. i o m __ 12"7). ~280
cm
--
(d) Purification of P M 6 R and U1 coat protein The coat protein of PM6R and the U1 strain were obtained by stripping the proteins from purified virus preparations with 67% acid (Fraenkel-Conrat, 1957). PM6R and U1 coat protein aggregated during this procedure, at p H 6.1 and 4.7, respectively. Concentrations of protein preparations were determined as for PM6 coat protein.
(e) Peptide preparation and purification PM6 and PM6R protein were digested with trypsin-TPCK (Worthington Biochem. Corp., Freehold, N. J.) by the method of Funatsu (1964). Peptide 1 was precipitated at p H 4.2 (Funatsu, 1964). The remaining soluble peptides were lyophilized, dissolved in pyridine buffer at p H 8-8, clarified b y centrifugation, and then chromatographed on Dowex-1. Tryptic peptide 8 from PM6 and PM6R was further digested with subtilisin (Nagarse, Enzyme Development Corp., New York) by the method of Funatsu et al. (1964) and the resulting peptides were also chromatographed. Peptides that required further purification were ehromatographed on paper (Woody & Knight, 1959; Funatsu & Funatsu, 1967).
LETTERS
TO T H E
EDITOR
791
(f) Amino avid analysis Hydrolysis of coat protein and coat protein peptides was carried out under nitrogen in 6 ~-HC1 in vacuo for 24 and 72 hours at ll0~ (Zaitlin & McCaughey, 1965) prior to chromatography on a Beckman 121 automatic amino acid analyzer. Cys was chromatographed separately after performic acid oxidation of the coat protein (Hirs, 1956) and Trp was determined speetrophotometrieally using the Tyr/Trp ratio of whole coat protein (Beaven & Holiday, 1952). (g) Electron microscopy The contents of TMV-infected leaf cells was examined on carbon-coated grids (400 mesh) in a Philips EM200 electron microscope by the leaf dip method of Hitchborn & Hills (1965). (h) Characterization of P M 6 (i) P M 6 is a defective mutant. The infectious entity in PM6-infected leaves was shown to be labile, and its biological characteristics appeared to be identical to those of the previously described defective TM-Vmutants (Siegel et al., 1962 ; Hariharasubramanian & Siegel, 1969 ; Hariharasubramanian et al., 1973). No virion was observed by electron microscopy in either leaf dips or extracts. (ii) Coat protein properties. PM6 coat protein was relatively unstable during its course of purification. Subjecting the protein to pH conditions below 4.0, temperatures above 15 to 20~ concentrations above 5 mg/ml, freezing and thawing, and agitation all resulted in denaturation as indicated by precipitation of the protein from solution. The protein was found to have a cation requirement for reversible aggregation at pH 4.7. The chlorides of Mg2+, Na + and K + were increasingly effective in maximizing the yield of protein. Absence of cations during aggregation of the protein at pH 4.7 results in slow precipitation of the protein from solution. (iii) Amino acid composition of coat protein and its peptides. Amino acid analysis showed that PM6 coat protein, relative to U1 coat protein, was high in one residue each of Thr and Gly and low in one each of Asp (or Asn) and Ala (PM6, Table 1). These data suggest that PM6 resulted from mutations yielding only two amino acid changes from the wild-type protein. Two tryptic peptides (6 and 8) isolated by ion exchange chromatography (Fig. 1 (a)) from PM6 protein showed an Asp (or Ash) to Gly and an Ala to Thr exchange, respectively, by composition (Table 1) when compared to the known composition of peptides 6 and 8 from U1 protein (Funatsu eta/., 1964). To localize the exchange in tryptic peptide 8 (residues 93 to 112) it was digested with subtilisin and the resulting peptides separated by ion exchange chromatography (Fig. l(c)). The replacement was localized by composition to a peptide (residues 104 to 112; Table 1) which contains two Ala residues, at positions 105 and 110 (Funatsu et al., 1964). This subtilisin peptide (residues 104 to 112) was hydrolyzed in dilute acid (0.03 M-HC1), which removes the Asp residue at position 109 (Tsung & leraenkelCourat, 1965) and the two resulting peptides were purified by chromatography (Fig. l(d)). From the amino acid composition of these peptides (Table 1) we conclude that the Ala to Thr exchange in PM6 protein is at position 105. (iv) Electron microscopy. Electron mierographs of leaf dips from P/~I6-infeeted leaf tissue show what appears to be PM6 coat protein aggregated into rods (Fig. 2(a)), whereas such structures are completely absent from uninfected leaf tissue. These long flexuous rods appear to be made up of units that are loosely joined together, and each 52
792
J. J. HUBERT, [
ii I
D. P. B O U R Q U E
I
~
AN]:) M. Z A I T L I N
12~
0.8
0.55
8 4 0'6
9 0"15
0.4
9
7-8
~ 0.2
i
I00
i
200
500
I00
400
r
I
200
500
(ol
400
(b) f
i
i
C (104-1121 0"8
0-6
I 0"3
O. 4
D (98 -99} 193-99}
0'2
A(IIO-II2)
A(100-103) B(I04)
i
i
I00
200
i
500
(c)
400
0-15~. ~04-108) i i i 50
Fraction number
I00
150
200
(d)
FIG. l. Ion exchange c h r o m a t o g r a p h y of PM6 and PM6R peptides. I o n exchange c h r o m a t o g r a p h y of peptides was performed on a Dowex l - X 2 acetate column (0.9 cm • 150 cm) equilibrated with p H 8.8 pyridine buffer (1% pyridine, 1% 2,4,6-trimethylpyridine a n d acetic acid to desired pH). Peptides were applied to the column in p H 8.8 pyridine buffer a n d then eluted with 250 m l p H 8-2 pyridine buffer followed b y a gradient elution ( B - l l ) according to the m e t h o d of F u n a t s u (1964); the flow-rate was 35 to 40 ml/h at room t e m p e r a t u r e ; 3.3-ml fractions were collected a n d 0.1-ml samples from eve~sr t h i r d fraction were tested for protein b y the L o ~ ' y m e t h o d ; color developed was measured at 750 nm. Soluble tryptie peptides fron~ PM6 (a) a n d PM6R (b) proteins are n u m b e r e d according to their order in TMV coat protein beginning from the N-terminal residue (Funatsu, 1964). P e a k 48contains peptides 8 a n d 4. Peptides 7-8, 8 a n d 10 precipitated in the fractions in which they occurred a n d were f u r t h e r purified b y isoelectric precipitation a t p H 3.5, 3.2 a n d 7-0, respectively. Peptides 3-4 and 7-8 represent amino acid residues 47 to 68 a n d 91 to 112, respectively. Subtilisin (c) a n d dilute acid (d) peptides from PM6 (. . . . ) and PM6R ( ) protein were chromatographed using the same elution schedule. Numbers in parentheses adjacent to lettered peaks represent residues of the peptide found in t h a t peak.
u n i t a p p e a r s t o be a s t a c k of t w o disks. Circular s t r u c t u r e s (Fig. 2(a)) were f r e q u e n t l y o b s e r v e d which a p p e a r t o be f o r m e d from p r o t e i n rods of sufficient l e n g t h to b e n d b a c k on t h e m s e l v e s a n d join end t o end to f o r m a n u n b r o k e n " w h e e l " . E a c h wheel is m a d e o f a n u m b e r o f u n i t s a n d each u n i t a p p e a r s t o h a v e t w o e l e c t r o n dense grooves on its o u t w a r d side a n d one on t h e inside (Fig. 2(b)).
L E T T E R S TO T H E E D I T O R
793
TABLE 1
Amino acid composition of PM6 and PM6R1 coat 1~rotein and peptides 6 and 8 Amino acid residue
Lys Arg Asp Thr Ser Glu Pro Gly Ala Cys Val I/e Leu Tyr Phe Try
Mol amino acid/mol protein or peptide a Coat protein Peptide 6 U1 PM6 PM6R1 U1 PM6 PM6R~ 2 11 18 16b 16b 16c 8 6 14 1~ 14 94 12 4 8 3~
2.1 11.1 16"8 i6.6 16-2 16.0 8.1 7-2 13.1 0-7 14.1 9.1 12.1 4.1 8.2 3.1
1.9 10.9 17.9 16.8 15.8 16.0 7"8 6-1 12.9 0-9 13.9 8.7 12.1 3.9 7.9 3-3
1 3 2
0"9 2.2 2-1
1"0 3"0 2.3
1 1 3c
0-9 2.2 3.0
1.0 1.O 3.0
2
2.0
1.9
4 1 1
3.8 0.9 0.9
3.6 0-8 0-8
U1
Peptide 8 PM6 PM6R1
1 3o 4
0.9 3.0 4-9
0.9 3.0 5.2
4 1
3.9 0.9
3-9 0.7
3
2.0
1.9
1 2a 1
1.0 1.8 1.0
1.0 1.8 1.0
Values which differ from U1 coat protein are underlined. a Experimental values for PM6 and PM6R1 from a 24 h hydrolysis. Integral values expected for U1 coat protein taken from Hennlg & Wittmann (1972), and those for peptides 6 and 8 from Funatsu (1964). b Experimental values for PM6 and PM6R1 adjusted to those of 24 h hydrolysates of U1 protein. o Other values in column calculated on basis of U1 integral value for this residue used as a standard. d Values obtained on separate analysis after 72 h hydrolysis. e Value obtained on separate analysis of performic acid oxidized protein. Value determined speetrophotometrically. A s t r u c t u r e for t h e u n i t of t h e wheel t h a t w o u l d be c o m p a t i b l e with these observat i o n s is a 2 89 helix of s u b u n i t proteins. A model of a wheel composed of such helices (Fig. 2(c)) shows its a s y m m e t r i c n a t u r e . The o u t w a r d side of t h e helix has three t u r n s of coat p r o t e i n s u b u n i t s a n d t h e inside of t h e helix has two. Some of the p r o t e i n rods also a p p e a r to h a v e a few u n i t s t h a t are 2 89 helices (Fig. 2(d)) a n d some of t h e wheels also seem to have u n i t s t h a t are composed of disks (Fig. 2(a)), a l t h o u g h t h e model of t h e wheel p r e s e n t e d here has been c o n s t r u c t e d e n t i r e l y of 2 89 helices. T h e m a x i m u m n u m b e r of u n i t s t h a t can m a k e u p a wheel is u n k n o w n , b u t t h e m i n i m u m n u m b e r of u n i t s c o u n t e d in a wheel was 13 (Fig. 2(b)). (i) Characterization of P M 6 R P M 6 R a p p e a r e d m o s t f r e q u e n t l y i n PM6-infected p l a n t s gro~ql a t higher greenhouse t e m p e r a t u r e s (43 to 46~ I t was isolated i n 18 different i n s t a n c e s a n d identified b y its coat p r o t e i n a m i n o acid composition. U n l i k e PM6 coat protein, P M 6 R p r o t e i n is as stable as t h a t of U1 protein. I t forms aggregates a t p H 6.1, i n c o n t r a s t to U1 p r o t e i n which aggregates a t p H 5.0; it has a n isoelectric p o i n t of a b o u t 4.7, different from t h e p H 3.2 isoeleetric p o i n t d e t e r m i n e d for U1 coat p r o t e i n ( K r a m e r & W i t t m a n n , 1958). T h e composition of P M 6 R p r o t e i n indicates it has one a m i n o acid exchange,
794
J. J. HUBERT,
D. P. B O U R Q U E
A N D M. Z A I T L I N
FIe. 2. Electron micrographs of PM6 and PM6R and model of PM6 protein wheels. All micrographs from negatively stained leaf dips of PM6 or PM6R infected Samsun tobacco plants or from a purified virus preparation (Fig. l(e) only). (a) PM6 protein showing region of stacked disks in a rod and a wheel. The arrows indicate a region in a protein wheel where stacked disks are clearly visible. PM6 protein in the wheel (arrow) configuration appears to be constructed from both stacked disks and 289 helices, on the lower side and upper side, respectively. Magnification, 124,000 x . (b) The 289 helix in the PM6 protein wheel. The arrow indicates one 2~-turn helix t h a t is clearly visible. Magnification 680,000 • (c) Model o f the PM6 protein wheel. The model of the proposed wheel configuration shows the unit of construction as a 289 helix. The tubing alignment depicts the helices as t h e y would
LETTERS TO THE EDITOR
795
i.e. Ala to Thr (Table 1). The same procedures were employed to localize the exchange in PM6R as were used for PM6 protein. Ion exchange chromatography of P1K6R coat protein peptides (Fig. l(b), (c) and (d)) and amino acid analysis of these peptides (Table 1) show t h a t the Ala to Thr exchange occurs at position 105 in PM6R, as it does in PiK6 protein. In electron micrographs of PM6R leaf dips, a high frequency of irregular protein rods are observed (Fig. 2(e)) when compared to U1 TMV leaf dips, suggesting t h a t PM6R and U1 protein are not equivalent. PlV[6R protein seems to encapsidate TlVIV-RNA in a less compact manner t h a n U1 protein, resulting in P1K6R virions (Fig. 2(f)) which appear abnormal when compared to wild-type TMV virions (Caspar, 1963). (j) Origin of P M 6 and P M 6 R PM6 was obtained after treatment of the wild-type T1KV strain U1 with H-N02. Its coat protein was shown to be non-functional and to contain an Asp (or Asn) to Gly and an Ala to Thr exchange (Table 1). The position of the Asp (or Asn) to Gly exchange in PM6 coat protein has been localized to peptide 6; the exact location within this peptide is not known. Peptide 6 from U1 TM~ contains an Asn residue at position 73 and two Asp residues at positions 77 and 88 (Tsung et al., 1964). Considering the genetic code, all of these residues could be converted to Gly by I-[NO2-induced mutagenesis of TMV-RNA (~Iundry & Geirer, 1958; Schuster & Wilhelm, 1963). The possible origin of the second amino acid exchange in the PM6 coat protein (Ala to Thr at position 105) is not explained by the accepted mechanism of HN02 action (Schuster & Schramm, 1958). PM6 coat protein is not unique among defective TMV strains in which an amino acid exchange appears to be inconsistent with the mechanism of action of HN02. One of the two replacements is defective m u t a n t PM2 (Wittmann, 1965) and several replacements in two mutants with insoluble, defective coat proteins (PLY[1and PM4) are similarly unexplained (Hariharasubramanian et al., 1973). PM6 appears to revert to PI~I6R by a one-step reversion; the Gly exchange in PiK6 coat protein reverts to Asp (or Asn) but the Ala to Thr exchange remains unaltered. The resulting PM6R protein can encapsidate TMV-RNA, but may have a conformation which differs from wild-type coat protein, judging from the high frequency of irregular protein rods (Fig. 2(e)) and the abnormal nature of the PM6R virions (Fig. 2(g)) seeri in the electron microscope. (k) Localization of an amino acid residue critical for virus azaembly Because PlVI6R has a functional coat protein, we conclude t h a t the Asp (or Asn) to Gly exchange in PM6 protein is responsible for the inability of PM6 protein to aggregate correctly and encapsidate PM6-RNA to form normal virions. The change from an Asp (or Ash) to a Gly residue would engender a change in the net charge of appear in an electron micrograph of negatively stained PM6 protein wheels. Magnification, 1,500,000 x .
(d) PM6 protein rod showing possible helical region. The arrow indicates a portion of rod that m a y be composed of helical units, possible 289or 3-turn helices. Magnification, 124,000x.
(e) PM6R protein rods showing loose association of the disks. Magnification, 136,000• (f) PM6R virions. Magnification, 111,000 • (g) PM6 protein aggregates that appear to be in intermediate states of wheel formation. Magnification, 204,000 •
796
J. J. HUBERT, D. P. BOURQUE AND M. ZAITLAN
the protein and could affect the normal functioning of the protein by altering its conformation or ligand binding properties. Tryptic peptide 6 of the PM6 coat protein (Table 1) is the site of the Asp (or Asn) to Gly exchange and possible locations for this exchange are an Asn and two Asp residues at positions 73, 77 and 88, respectively (Tsung et al., 1964). Because a HNO2-derived TMV mutant (Ni 1103) with a functional coat protein has been found (Wittmann-Leibold & Wittmann, 1965) which has an Asn to Ser exchange at position 73, it is unlikely that the Ash residue at position 73 plays a critical role in function of the protein. Peptide 6 from PM6 is ehited from the column earlier than wild-type peptide 6 (Fig. l(a) and (b)), presumably as a result of its less acidic nature. Such elution behavior would be consistent with an Asp to Gly exchange but not with an Asn to Gly exchange. Consequently, Asn is probably still present in PM6. These data strongly imply that the exchanged residue in peptide 6 is either the Asp residue at position 77 or 88. Holmes Ribgrass, cowpea, and CGMMVcucumber TMV strains (Hennig & Wittmann, 1972; Rces & Short, 1975) all have residues other than Asp at position 77 in their coat proteins and all of these strains form infectious virions. Furthermore, residues 88 to 92 are conserved in cowpea TMV (Rees & Short, 1975) as well as in other virion-producing TMV strains (Butler & Durham, 1972). The preceding evidence suggests that position 88 is the site of the Asp to Gly exchange in PM6 coat protein and that position 88 may play a special role in TMV assembly. Durham & Butler (1975) have presented evidence that residue 88 is proximal to the RNA molecule, at about 40 A from the rod axis, whereas residue 77 is at 60/~ radius (A. Klug, personal communication). Consequently, it is suspected that the Asp residue at position 88 together with the Arg residues at positions 90 and 92 may be involved in the site for TMV-RI~/A binding to the TMV coat protein subunit (A. Durham, personal communication). These data support the concept that the amino acid sequence from residues 88 to 92 must be intact to insure correct proteinRNA interactions. However, defective mutants such as PM2, which are altered in other regions of the coat protein (Wittmann, 1965), show that interactions of two or more amino acid substitutions can also result in blocked virion assembly. (I) Relationship between P M 6 protein rods and protein wheels in vivo Durham & Klug (1971) propose that the growth of TMV protein rods takes place in vivo and in vit~v by the stacking of disks of protein (each disk is composed of two planar rings of 17 subunits each). At neutral pH the disks can be dislocated, forming two-turn helices which can interlock forming either protein rods in vitro, or virions in vivo by interacting with TMV-RNA. We have shown electron micrographic evidence that the coat protein of PM6 aggregates in vivo to form protein rods and wheels. It is interesting to speculate about the manner in which the wheels are formed and of what significance their structure may be to the theory offered by Butler & Klug (1971) for the assembly of TMV protein rods and virions. Rods of PI~I6 coat protein appear to be composed of disks that are weakly bonded, allowing them to be very flexible. Short rods of this type appear capable of bending back on themselves and annealing end to end to form wheels (Fig. 2). Disks formed from this protein show a greater tendency to dislocate in vivo into two-turn helices which have two open ends. It is then possible that single coat protein subunits can be added to these ends until steric hindrance prevents further subunit addition (Ohno et al., 1972; Okada & Ohno, 1972; Butler & Klug, 1973). Such helices then would be approximately 289 and would exist in the wheel configuration under physiological conditions. In the many wheels that have
L E T T E R S TO THE E D I T O R
797
been observed, the units comprising them appear to be both 2~-turn helices a n d disks (Fig. 2(a)). The rods also seem to have two-turn helices in regions where t h e y are bent allowing for the addition of single subunits, as evidenced by what appear to be 289 helices in these regions (Fig. 2(d)). I f units making up the wheels represent two-turn helices t h a t have been lengthened b y the addition of extra subunits, then it is possible t h a t the 289 helices observed in the wheel configuration represent a form of the lock washer or two-turn helix postulated by Butler & Klug (1971), which plays a major role in their theory on TM-~ assembly. This research was supported in part by National Science Foundation grant GB-39333 to one of us (M. Z.), by Hatch Act funds (to M. Z. and D. P. B.) allocated by the University of Arizona Agricultural Experiment Station, and by grant 5-RO1GM21242 from the National Institute of General Medical Sciences (to D.P.B.). The authors are also indebted to Ruth Smith and Angelo Longo for technical assistance. This is publication No. 2687 of the Arizona Agricultural Experiment Station. Department of Agricultural Biochemistry University of Arizona, Tucson, Ariz. 85721, U.S.A.
JEFFREY J. HUBERTt DoN P. BOURQD-E I~/IILTONZAITLIN$
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