The N-terminal amino acid sequence is essential for foot-and-mouth disease virus replicase activity.
1. Foot-and-mouth disease virus replicase was expressed by fusing its cDNA to the OmpA signal peptide coding sequence present in the pIN-III ompA series vectors. 2. Two constructions were developed to express either a full-length or truncated enzyme lacking the 20 amino acids at the N-terminal end. Bacterial extracts expressing the recombinant proteins were submitted to SDS-PAGE and the presence of the replicase was revealed by immunoblotting. The truncated form exhibited a higher mobility and the relative positions of the proteins show that the signal peptide was removed. 3. The biological activity of these two molecules was tested using a poly(A)-dependent oligo(U)-primed poly(U)-polymerase assay. The full-length replicase is active. The aminoterminal truncated enzyme had 0.02% activity of the intact one. 4. This result indicates the importance of the twenty N-terminal amino acids for the activity of FMDV RNA-dependent RNA polymerase.
Pacheco
A B
AB
Instituto de Biofísica, Universidade Federal do Rio de Janeiro, Brasil.
Brindeiro
R M
RM
Soares
M A
MA
de-Almeida
D F
DF
Tanuri
A
A
D000595:Amino Acid Sequence; D005537:Aphthovirus / Q000201:enzymology* / Q000502:physiology; D001483:Base Sequence; D004261:DNA Replication; D004279:DNA, Viral / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000032:analysis / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids; D013329:Structure-Activity Relationship; D014779:Virus Replication
D004279:DNA, Viral; C023140:DNA replicase; D004259:DNA-Directed DNA Polymerase