Hirth, L.

Lebeurier, G.

Nicolaieff, A.

Richards, K.E.

PubMed

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The self-assembly of tobacco mosaic virus Influence of the viral RNA and protein components upon the assembly process

This research has been supported by grants from the National Institutes of Health (N.I.H.) (GM-1 1016) and the
American Cancer Society (NP-150) to Bruno Zimm and by an N.I.H. postdoctoral fellowship (GM 07272) to the
author.
Receivedfor publication 17 December 1979.
REFERENCES
1. Alberts, B., and L. Frey. 1970. T4 bacteriophage gene 32: a structural protein in the replication and
recombination of DNA. Nature (Lond.). 227:1313-1318.
2. Peterman, B., and C. W. Wu. 1978. Ionic strength perturbation kinetics of gene 32 protein dissociation from its
complex with single stranded DNA. Biochemistry. 17:3889-3892.
3. Epstein, I. R. 1979. Kinetics of nucleic acid-large ligand interactions: exact Monte Carlo treatment and limiting
cases of reversible binding. Biopolymers. 18:2037-2050.
THE SELF-ASSEMBLY OF TOBACCO MOSAIC VIRUS
Influence of the Viral RNA and Protein Components
upon the Assembly Process
L. Hirth, G. Lebeurier, A. Nicolaieff, and K. E. Richards, Laboratoire de
Virologie, Institut de Biologie Moleculaire et Cellulaire, 15 rue Descartes,
67000 Strasbourg, France
The initial stage in the self-assembly of tobacco mosaic virus (TMV) RNA and coat protein
into virions involves insertion of a loop ofTMV RNA between layers of a double-layered disk
ofTMV protein (containing 17 coat protein subunits per layer) via the central channel of the
disk, followed by conversion of the disk-RNA complex into a two-turn protohelix and addition
of more protein (elongation). This mechanism leads to assembling particles in which one of
the uncoated RNA tails, that containing the 5'-terminus, runs back along the central channel
of the growing rod (1, 2).
Bidirectional Elongation
The nucleotide sequence recognized during initiation is located 950 residues (- 15% total
chain length) from the 3'-end of the RNA (3). Thus, the looped-back 5'-tail of short
incompletely assembled rods is much longer than the 5'-tail. It has been suggested that the
proximity of the looped-back 5'-tail to the growing point of the particle in the 3'-direction may
hinder encapsidation of the 3'-tail (1, 4). Study of the reconstitution of nearly complete
particles shows that, for such particles at least, encapsidation can proceed simultaneously on
both tails although growth is on the average much faster in the 5' than in the 3'-sense if we
consider the whole of the assembly process. Surprisingly, the ultimate step in assembly
appears to be encapsidation of the last several hundred nucleotides of the 5'-tail, as incomplete
particles having about nine-tenths the full length (but with no visible RNA tail) may persist
for several hours after the beginning of reconstitution.
Heterologous Reconstitution and the Specificity ofInitiation
The mechanism for assuring the specificity of self-assembly is thought to reside in the
initiation stage of the reaction. The portion of the RNA chain which first binds to coat
protein during initiation has been characterized and shown to possess a hairpin loop structure
460 ASSEMBLY
EC ~ ~ a2-o
0:
KCI MOLAR-ITY
Figure I Initial rate of assembly ofTMV particles as measured turbidometrically for heterologous and
homologous reconstitutions between the RNA and protein components of vulgare and U2 strain TMV.
Assembly was performed at 20°'C in 10 mM MES, pH 7.0, plus indicated KCI concentration. Protein
concentration: 4 mg/ml; RNA concentration: 50 ug/ml. ( o) TMV RnA plus U2 protein; (- *-)
U2 RNA plus U2 protein; (--o - - -) TMV RNA plus TMV protein; (- - - - -- -) U2 RNA plus
TMV protein.
with the semirepetitive triplet sequence GAA.GAA.GUU.GUU.GAU at its summit (5, 6).
Presumably, TMV protein has a heightened affinity for such a sequence although other
features of sequence and secondary structure probably reinforce the binding. Heterologous
reconstitution experiments have confirmed, however, that the specificity of assembly is not
complete. Coat protein from TMV, vulgare strain, can distinguish between its own RNA and
that ofTMV strain U2, encapsidating the one but not the other. U2 coat protein, on the other
hand, accepts RNA from either strain and, indeed, reassembles about twice as rapidly with
vulgare strain RNA as with its own RNA (Fig. 1). U2 coat protein differs from vulgare coat
protein at 38 positions. Two of these substitutions, Gly (85) --,Asn and Ala (86)--,Ser, occur
in the proximal end of the right radial helix, a region of the coat protein subunit close to the
RNA binding site (7). Conceivably, these changes may be responsible for the diminished
specifi'city of U2 coat protein in reconstitution. Alternatively, the diminished specificity may
be a consequence of changes in distant parts of the subunit which work their effect by slightly
altering the configuration of the portion of the polypeptide chain which interacts with the
RNA.
Receivedfor publication II December 1979.
REFERENCES
1. Lebeurier, G., A. Nicolaieff, and K. E. Richards. 1977. Inside-out model for self-assembly of tobacco mosaic
virus. Proc. Natl. Acad. Sci. U.S.A. 74:149-153.
POSTER SUMMARIES 461
2. Butler, P. J. G., J. T. Finch, and D. Zimmern. 1977. Configuration of tobacco mosaic virus RNA during virus
assembly. Nature (Lond.). 265:217-219.
3. Zimmern, D. and T. M. A. Wilson. 1976. Location of the origin for viral assembly on TMV RNA and its relation
to stable fragment. FEBS Lett. 71:294-298.
4. Otsuki, Y., I. Takebe, T. Ohno, M. Fukada, and Y. Okada. 1977. Reconstitution of tobacco mosaic virus rods
occurs bidirectionally from an internal initiation region: demonstration by electron microscopic serology. Proc.
Natl. Acad. Sci. U.S.A. 74:1913-1917
5. Zimmern, D. 1977. The nucleotide sequence at the origin for assembly on tobacco mosaic virus RNA. Cell.
11:463-482.
6. Jonard, G., K. Richards, H. Guilley, and L. Hirth. 1977. Sequence from the assembly nucleation region ofTMV
RNA. Cell. 11:483-493.
7. Bloomer, A. C., J. N. Champness, G. Bricogne, R. Staden, and A. Klug. 1978. Protein disk of tobacco mosaic virus
at 2.8 A resolution showing the interactions within and between subunits. Nature (Lond.). 276:362-368.
462 ASSEMBLY


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THE SELF-ASSEMBLY OF TOBACCO MOSAIC VIRUS: INFLUENCE OF THE VIRAL RNA AND PROTEIN COMPONENTS UPON THE ASSEMBLY PROCESS

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