Assessing the contribution of the herpes simplex virus DNA polymerase to spontaneous mutations.
BACKGROUND
The thymidine kinase (tk) mutagenesis assay is often utilized to determine the frequency of herpes simplex virus (HSV) replication-mediated mutations. Using this assay, clinical and laboratory HSV-2 isolates were shown to have a 10- to 80-fold higher frequency of spontaneous mutations compared to HSV-1.


METHODS
A panel of HSV-1 and HSV-2, along with polymerase-recombinant viruses expressing type 2 polymerase (Pol) within a type 1 genome, were evaluated using the tk and non-HSV DNA mutagenesis assays to measure HSV replication-dependent errors and determine whether the higher mutation frequency of HSV-2 is a distinct property of type 2 polymerases.


RESULTS
Although HSV-2 have mutation frequencies higher than HSV-1 in the tk assay, these errors are assay-specific. In fact, wild type HSV-1 and the antimutator HSV-1 PAAr5 exhibited a 2-4 fold higher frequency than HSV-2 in the non-HSV DNA mutatagenesis assay. Furthermore, regardless of assay, HSV-1 recombinants expressing HSV-2 Pol had error rates similar to HSV-1, whereas the high mutator virus, HSV-2 6757, consistently showed significant errors. Additionally, plasmid DNA containing the HSV-2 tk gene, but not type 1 tk or LacZ DNA, was shown to form an anisomorphic DNA structure.


CONCLUSIONS
This study suggests that the Pol is not solely responsible for the virus-type specific differences in mutation frequency. Accordingly, it is possible that (a) mutations may be modulated by other viral polypeptides cooperating with Pol, and (b) the localized secondary structure of the viral genome may partially account for the apparently enhanced error frequency of HSV-2.
Duffy
Karen E
KE
Department of Host Defense, The Antimicrobial and Host Defense Center of Excellence for Drug Discovery, 1250 South Collegeville Road, Collegeville, Pennsylvania 19426, USA. karen_e_duffy@gsk.com
Quail
Matthew R
MR
Nguyen
Tammy T
TT
Wittrock
Robert J
RJ
Bartus
Joan O
JO
Halsey
Wendy M
WM
Leary
Jeffry J
JJ
Bacon
Teresa H
TH
Sarisky
Robert T
RT
D000818:Animals; D001681:Biological Assay; D002460:Cell Line; D002522:Chlorocebus aethiops; D004257:DNA Polymerase II / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication / Q000187:drug effects / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D016679:Genome, Viral; D018259:Herpesvirus 1, Human / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D018258:Herpesvirus 2, Human / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D016296:Mutagenesis / Q000187:drug effects / Q000235:genetics; D009154:Mutation / Q000187:drug effects / Q000235:genetics*; D009690:Nucleic Acid Conformation / Q000187:drug effects; D010957:Plasmids / Q000096:biosynthesis / Q000235:genetics; D013937:Thymidine Kinase / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014709:Vero Cells / Q000737:chemistry / Q000378:metabolism; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*
D004274:DNA, Recombinant; D004279:DNA, Viral; D014764:Viral Proteins; D013937:Thymidine Kinase; D004257:DNA Polymerase II; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; C043417:DNA polymerase, Simplexvirus