Different mutations in the HHV-6 DNA polymerase gene accounting for resistance to foscarnet.
BACKGROUND
Human herpesvirus 6 (HHV-6), which is closely related to human cytomegalovirus, is sensitive to foscarnet (PFA). Up to now, the resistance of HHV-6 to PFA has not been investigated.


OBJECTIVE
The goal of the study was to isolate and characterize PFA-resistant HHV-6 mutants in order to determine the mechanisms of resistance to the drug.


METHODS
PFA-resistant viruses, isolated in MT4 cell culture under increasing concentrations of PFA, were characterized phenotypically and genotypically. The mutations identified in the HHV-6 DNA polymerase gene were evaluated in a functional assay using recombinant mutated forms of the enzyme, and their effect on protein structure was analysed in a three-dimensional model derived from available structures of DNA polymerases.


RESULTS
Two mutants were selected and were 8- and 15-fold more resistant to PFA than the wild-type strain. Four amino acid changes were detected in the HHV-6 DNA polymerase in association with PFA resistance: T435R, H507Y, C525S, located in the deltaC conserved domain, and F292S. Either alone or in combination, these substitutions significantly decreased the inhibitory effect of PFA at the level of the polymerase, as measured by the incorporation of radiolabelled nucleotides in a DNA elongation assay. In the three-dimensional model of HHV-6 DNA polymerase structure the four changes were not located within the putative catalytic site, but they might induce either a disturbance of local conformation or a restricted access of PFA to its target site.


CONCLUSIONS
This first characterization of HHV-6 resistance to PFA highlights the role of distinct DNA polymerase gene mutations.
Bonnafous
Pascale
P
University Pierre et Marie Curie-Paris 6, EA2387, Groupe hospitalier Pitié-Salpêtrière, Paris, France.
Naesens
Lieve
L
Petrella
Stéphanie
S
Gautheret-Dejean
Agnès
A
Boutolleau
David
D
Sougakoff
Wladimir
W
Agut
Henri
H
D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000494:pharmacology*; D020134:Catalytic Domain; D002460:Cell Line; D003587:Cytomegalovirus / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D024882:Drug Resistance, Viral / Q000235:genetics*; D017245:Foscarnet / Q000494:pharmacology*; D005814:Genes, Viral; D015654:Herpesvirus 6, Human / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D016415:Sequence Alignment
D000998:Antiviral Agents; D017245:Foscarnet; D004259:DNA-Directed DNA Polymerase