Open AccessHSV-1 DNA polymerase 3′-5′ exonuclease-deficient mutant D368A exhibits severely reduced viral DNA synthesis and polymerase expression
Author(s) -
Jessica L. Lawler,
Donald M. Coen
Publication year - 2018
Publication title -
journal of general virology
Language(s) - Uncategorized
Resource type - Journals
SCImago Journal Rank - 1.55
H-Index - 167
eISSN - 1465-2099
pISSN - 0022-1317
DOI - 10.1099/jgv.0.001138
Subject(s) - biology , dna polymerase , exonuclease , polymerase , virology , dna polymerase ii , mutant , microbiology and biotechnology , dna , dna clamp , polymerase chain reaction , genetics , gene , reverse transcriptase
Herpesviruses, including herpes simplex virus-1, encode and express a DNA polymerase that is required for replication of their dsDNA genomes. The catalytic subunit of this enzyme contains a 3'-5' exonuclease that is involved in proofreading during replication. Although certain mutations that severely impair exonuclease activity are not lethal to the virus, it was reported that virus containing the substitution of alanine for aspartate 368 (D368A), which ablates exonuclease activity, could not be recovered, raising the possibility that this activity is essential for viral replication. To investigate this issue, we produced virus containing this mutation (D368A Pol) using a complementing cell line. D368A Pol virus was unable to form plaques on non-complementing cells. Viral DNA synthesis and polymerase activity were severely inhibited in D368A-infected cells, as was expression of the enzyme, suggesting that effects on polymerase expression rather than on exonuclease activity per se largely explain the lethal phenotype of this mutation.