Assembly of physalis mottle virus capsid protein in Escherichia coli and the role of amino and carboxy termini in the formation of the icosahedral particles.
The coat protein gene of physalis mottle tymovirus (PhMV) was over expressed in Escherichia coli using pET-3d vector. The recombinant protein was found to self assemble into capsids in vivo. The purified recombinant capsids had an apparent s value of 56.5 S and a diameter of 29(+/-2) nm. In order to establish the role of amino and carboxy-terminal regions in capsid assembly, two amino-terminal deletions clones lacking the first 11 and 26 amino acid residues and two carboxy-terminal deletions lacking the last five and ten amino acid residues were constructed and overexpressed. The proteins lacking N-terminal 11 (PhCPN1) and 26 (PhCPN2) amino acid residues self assembled into T=3 capsids in vivo, as evident from electron microscopy, ultracentrifugation and agarose gel electrophoresis. The recombinant, PhCPN1 and PhCPN2 capsids were as stable as the empty capsids formed in vivo and encapsidated a small amount of mRNA. The monoclonal antibody PA3B2, which recognizes the epitope within region 22 to 36, failed to react with PhCPN2 capsids while it recognized the recombinant and PhCPN1 capsids. Disassembly of the capsids upon treatment with urea showed that PhCPN2 capsids were most stable. These results demonstrate that the N-terminal 26 amino acid residues are not essential for T=3 capsid assembly in PhMV. In contrast, both the proteins lacking the C-terminal five and ten amino acid residues were present only in the insoluble fraction and could not assemble into capsids, suggesting that these residues are crucial for folding and assembly of the particles.
Sastri
M
M
Department of Biochemistry, Indian Institute of Science, Bangalore, -560 012, India.
Kekuda
R
R
Gopinath
K
K
Kumar
C T
CT
Jagath
J R
JR
Savithri
H S
HS
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D002213:Capsid / Q000235:genetics / Q000502:physiology*; D036022:Capsid Proteins; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011489:Protein Denaturation; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000276:immunology; D013329:Structure-Activity Relationship; D017806:Tymovirus / Q000235:genetics / Q000502:physiology*; D014508:Urea; D019065:Virus Assembly
D000911:Antibodies, Monoclonal; D036022:Capsid Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; C108931:capsid protein, Physalis mottle virus; D014508:Urea