Role of capsid structure and membrane protein processing in determining the size and copy number of peptides displayed on the major coat protein of filamentous bacteriophage.
Filamentous bacteriophage virions can be engineered to display small foreign peptides in the N-terminal regions of all 2700 copies of the major coat protein (pVIII), but larger peptides can be accommodated only in hybrid virions, in which modified and wild-type coat protein subunits are interspersed. The copy number of peptides accepted in hybrid virions is generally believed to be related to peptide size: the larger the insert, the lower the number of modified coat protein subunits in the assembled virion. However, we show here that some large peptides can be displayed at a much higher copy number than smaller ones and that some relatively small peptides are poorly displayed, if at all, in hybrid virions. X-ray diffraction studies of a recombinant virion together with model building experiments with peptide and protein epitopes of known structure demonstrated that it is feasible to accommodate much larger structures, without perturbation of the capsid protein packing, than it has proved possible to generate in vivo. We show further that the insertion of certain peptides greatly slowed or even prevented the processing of the pVIII pro-coat by leader peptidase at the inner membrane of the Escherichia coli cell. A good correlation was found between the effect of the insert on the rate of the processing of the pro-coat, an essential step in virus assembly, and the number of the mature but modified proteins in the subsequently assembled hybrid virion. These results have important implications for the design of peptide display systems based on filamentous bacteriophage.
Malik
P
P
Department of Biochemistry University of Cambridge, UK.
Terry
T D
TD
Gowda
L R
LR
Langara
A
A
Petukhov
S A
SA
Symmons
M F
MF
Welsh
L C
LC
Marvin
D A
DA
Perham
R N
RN
D000595:Amino Acid Sequence; D001483:Base Sequence; D002213:Capsid / Q000737:chemistry* / Q000235:genetics; D002462:Cell Membrane / Q000821:virology; D010450:Endopeptidases / Q000378:metabolism; D000939:Epitopes; D004926:Escherichia coli / Q000648:ultrastructure; D017912:Inovirus / Q000737:chemistry*; D008565:Membrane Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012697:Serine Endopeptidases; D014758:Viral Core Proteins / Q000737:chemistry* / Q000276:immunology; D014763:Viral Matrix Proteins / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry*; D014771:Virion / Q000737:chemistry / Q000235:genetics; D014961:X-Ray Diffraction / Q000379:methods
D000939:Epitopes; D008565:Membrane Proteins; D010455:Peptides; D011994:Recombinant Proteins; D014758:Viral Core Proteins; D014763:Viral Matrix Proteins; D014764:Viral Proteins; D010450:Endopeptidases; D012697:Serine Endopeptidases; C026515:type I signal peptidase