Tài liệu Báo cáo khóa học: Mutations in the hydrophobic core and in the protein–RNA interface affect

Mutations in the hydrophobic core and in the protein–RNA interface

affect the packing and stability of icosahedral viruses

Sheila M. B. Lima1

, David S. Peabody2

, Jerson L. Silva1 and Andre´ a C. de Oliveira1

1

Departamento de Bioquı´mica Me´dica, Instituto de Cieˆncias Biome´dicas and Centro Nacional de Ressonaˆncia Magne´tica Nuclear de

Macromole´culas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; 2

Department of Molecular Genetics and

Microbiology and Cancer Research and Treatment Center, University of New Mexico School of Medicine, Albuquerque, NM, USA

The information required for successful assembly of an

icosahedral virus is encoded in the native conformation of

the capsid protein and in its interaction with the nucleic acid.

Here we investigated how the packing and stability of virus

capsids are sensitive to single amino acid substitutions in the

coat protein. Tryptophan fluorescence, bis-8-anilinonaph￾thalene-1-sulfonate fluorescence, CD and light scattering

were employed to measure urea- and pressure-induced

effects on MS2 bacteriophage and temperature sensitive

mutants. M88V and T45S particles were less stable than the

wild-type forms and completely dissociated at 3.0 kbar of

pressure. M88V and T45S mutants also had lower stability

in the presence of urea.We propose that the lower stability of

M8 8 V particles is related to an increase in the cavity of

the hydrophobic core. Bis-8-anilinonaphthalene-1-sulfonate

fluorescence increased for the pressure-dissociated mutants

but not for the urea-denatured samples, indicating that the

final products were different. To verify reassembly of the

particles, gel filtration chromatography and infectivity

assays were performed. The phage titer was reduced dra￾matically when particles were treated with a high concen￾tration of urea. In contrast, the phage titer recovered after

high-pressure treatment. Thus, after pressure-induced dis￾sociation of the virus, information for correct reassembly

was preserved. In contrast to M88V and T45S, the D11N

mutant virus particle was more stable than the wild-type

virus, in spite of it also possessing a temperature sensitive

growth phenotype. Overall, our data show how point sub￾stitutions in the capsid protein, which affect either the

packing or the interaction at the protein–RNA interface,

result in changes in virus stability.

Keywords: hydrostatic pressure; MS2 bacteriophage; tem￾perature-sensitive mutants; urea; fluorescence.

The protein shells of viruses generally have several key

functions, including shielding of the nucleic acid, particle

maturation and conferring the ability to penetrate the host

cell and undergo disassembly. The coat proteins are usually

arranged in a shell with an icosahedral shape [1]. The

information required for successful assembly of a virus

particle is encoded in the native conformation of a capsid

protein subunit. Structural and thermodynamic approaches

have been employed to identify the general rules that govern

virus assembly [2–7].

The MS2 bacteriophage is an RNA virus of the

family Leviviridae, a group of single-stranded RNA

bacteriophages that infect F+ Escherichia coli cells. The

icosahedral shell of the MS2 virus particle has a

diameter of 260 A˚ and is made up of 180 copies of

the coat protein subunit (Mr 13.7 · 103

) in a T¼3

surface lattice. Each virion also contains one copy of

the maturase protein, which is responsible for attach￾ment of the phage to E. coli F-pili. The coat protein

has two functions in the viral life cycle. First, it acts as

a translational repressor of the replicase gene. A coat

protein dimer binds specifically to an RNA stem–loop

structure (known as the translational operator) and

prevents initiation of replicase translation [8–10]. Second,

coat protein serves as the major virus structural protein,

forming the shell in which the RNA genome is

contained [11,12].

The tertiary structure and topology of the MS2 coat

protein is different from those of other simple icosahedral

viruses [13]. The main chain of the protein subunit folds

into a five stranded antiparallel b-sheet (strands bC–bG)

facing the interior of the phage particle, with an

N-terminal hairpin (strands bA and bB) and two a-helices

(aA and aB) shielding most of the upper surface of the

b-sheet from the environment. Upon dimerization, exten￾sive contacts are formed between the subunits so that

the b-sheet becomes extended to form a continuous

10-stranded sheet. The two polypeptide chains are so

intimately intertwined that it seems clear that the dimer

must be the basic unit of coat protein folding; each

subunit depends on the other for acquisition of its native

fold. The 3D structure of the MS2 bacteriophage has

been determined at 2.8A˚ (Fig. 1A) [14]. Even so, the

mechanism of assembly and nucleic acid recognition are

still far from completely understood.

Correspondence to A. C. de Oliveira, Avenida Bauhinia,

400 - CCS/ICB/Bl E, sl. 08, Cidade Universita´ria,

CEP 21941-590, Rio de Janeiro, RJ, Brazil.

Fax: + 55 21 2270 8647, Tel.: + 55 21 2562 6756,

E-mail: [email protected]

Abbreviations: bis-ANS, bis-8-anilinonaphthalene-1-sulfonate; LB,

Luria–Bertani; p.f.u., plaque-forming units; ts, temperature sensitive;

WT, wild-type.

(Received 26 September 2003, accepted 7 November 2003)

Eur. J. Biochem. 271, 135–145 (2004) FEBS 2003 doi:10.1046/j.1432-1033.2003.03911.x