Functional analysis of the amino terminus of Epstein-Barr virus deoxyribonuclease.
A cDNA coding for the Epstein-Barr virus (EBV) alkaline deoxyribonuclease (DNase) was expressed in Escherichia coli using the T7 phage system designed to allow expression of potentially lethal proteins. Induction of protein synthesis from the gene yielded a peptide with a molecular weight of approximately 52 kDa, consistent with the predicted open reading frame of EBV BGLF5. A high level of nuclease activity was detected in crude cell extracts, and the activity was neutralized by sera from nasopharyngeal carcinoma patients with high titers of anti-DNase antibodies. A series of deletion clones truncated at the amino or carboxyl terminus was constructed and expressed to define the regions responsible for the nuclease activity of EBV DNase. All the mutated molecules lost their activities even though their expression levels were comparable to that of the full-length DNase. To determine the exact role of the amino terminus of EBV DNase, mutants with small deletions were expressed. While three mutants with deletions of amino acid residues 11-30, 16-28, or 23-28 failed to show any detectable nuclease activities, one mutant in which the first 8 amino acids were replaced by the first 12 amino acid residues of the T7 major capsid protein contributed by the vector was enzymatically active. To further define the importance of the amino-terminal region, full-length DNase with point mutations was generated among residues 23-29 by site-directed mutagenesis and expressed in the same system. Assays of the DNase activity of these mutants revealed that the mutation of residue 29 was fully active, and mutations in 24-27 retained 50% activity. Nevertheless, the mutation at residue 23 resulted in a complete loss of activity and the mutation at residue 28 resulted in loss of 70% activity. These results suggest the biological importance of the amino terminus of the EBV DNase, especially residues 23-28.
Lin
S F
SF
Graduate Institute of Microbiology, College of Medicine, National Taiwan University, Taipei, Republic of China.
Lin
S W
SW
Hsu
T Y
TY
Liu
M Y
MY
Chen
J Y
JY
Yang
C S
CS
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D003851:Deoxyribonucleases / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D004926:Escherichia coli; D004854:Herpesvirus 4, Human / Q000201:enzymology*; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009500:Neutralization Tests
D017931:DNA Primers; D003851:Deoxyribonucleases