Mutations in the UL97 ORF of ganciclovir-resistant clinical cytomegalovirus isolates differentially affect GCV phosphorylation as determined in a recombinant vaccinia virus system.
Mutations in the human cytomegalovirus (HCMV) UL97 phosphotransferase have been associated with ganciclovir (GCV) resistance due to an impairment of GCV monophosphorylation. Vaccinia virus recombinants (rVV) were generated that encoded different HCMV UL97 proteins (pUL97) with mutations previously detected in resistant HCMV clinical isolates at codons 460, 520, 592, 594, 595, 598 and 607. These rVVs allowed quantification of GCV phosphorylation catalyzed by the different mutated pUL97s. When compared to rVV-UL97 wild type, mean levels of residual intracellular GCV phosphorylation differed by a factor of 10 for the mutated UL97 proteins ranging from 5.2 to 51.8%. Mutations M460V (located in a UL97 region homologous to domain VIb of protein kinases) and H520Q (located in a cytomegalovirus-specific, functionally critical domain) were responsible for the lowest levels of residual GCV phosphorylation (9.3 and 5.2%). Mutations in a region homologous to the domain IX had a lower impact on GCV phosphorylation (15.8-51.8%). The relevance of pUL97 mutation G598S in inducing GCV resistance was demonstrated for the first time.
Baldanti
Fausto
F
Servizio di Virologia, IRCCS Policlinico S. Matteo, 27100 Pavia, Italy.
Michel
Detlef
D
Simoncini
Lavinia
L
Heuschmid
Maria
M
Zimmermann
Albert
A
Minisini
Rosalba
R
Schaarschmidt
Peter
P
Schmid
Thomas
T
Gerna
Giuseppe
G
Mertens
Thomas
T
D000818:Animals; D000998:Antiviral Agents / Q000378:metabolism*; D002460:Cell Line; D002522:Chlorocebus aethiops; D003587:Cytomegalovirus / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003586:Cytomegalovirus Infections / Q000821:virology; D024882:Drug Resistance, Viral; D015774:Ganciclovir / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D016296:Mutagenesis; D016366:Open Reading Frames; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D014616:Vaccinia virus
D000998:Antiviral Agents; D017853:Phosphotransferases (Alcohol Group Acceptor); C075365:ganciclovir kinase; D015774:Ganciclovir