Isolation and characterization of herpes simplex virus type 1 resistant to aminothiazolylphenyl-based inhibitors of the viral helicase-primase.
The aminothiazolylphenyl-containing compounds BILS 179 BS and BILS 45 BS are novel inhibitors of the herpes simplex virus helicase-primase with antiviral activity in vitro and in animal models of HSV disease. To verify the mechanism of antiviral action, resistant viruses were selected by serial passage or by single-step plaque selection of HSV-1 KOS in the presence of inhibitors. Three resistant isolates K138r3, K22r5, and K22r1 were found to be 38-, 316-, and 2500-fold resistant to BILS 22 BS, a potent analog of BILS 45 BS. All three viruses had growth properties in vitro similar to wild-type HSV-1 KOS but they were sensitive to acyclovir. Cutaneous and intra-cerebral inoculation of mice with K22r1 or K22r5 resulted in pathogenicity equivalent to that of HSV-1 KOS. Both isolates were fully competent for reactivation from latency following corneal inoculation. Helicase-primase purified from cells infected with resistant viruses showed decreased inhibition in an in vitro DNA-dependent ATPase assay that correlated well with antiviral resistance. Marker transfer experiments and DNA sequence analysis identified single base pair mutations clustered in the N-terminus of the UL5 gene that resulted in single amino acid changes in the UL5 protein. Taken together, the results indicate that helicase-primase inhibitors prevent HSV growth by inhibiting HSV helicase-primase through specific interaction with the UL5 protein.
Liuzzi
Michel
M
Boehringer Ingelheim (Canada) Ltd., Research and Development, 2100 Cunard Street, Laval, Que., H7S 2G5, Canada. mliuzzi@lav.boehringer-ingelheim.ca
Kibler
Philip
P
Bousquet
Christiane
C
Harji
Fayaz
F
Bolger
Gordon
G
Garneau
Michel
M
Lapeyre
Nicole
N
McCollum
Robert S
RS
Faucher
Anne-Marie
AM
Simoneau
Bruno
B
Cordingley
Michael G
MG
D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D004265:DNA Helicases / Q000037:antagonists & inhibitors*; D019915:DNA Primase; D004195:Disease Models, Animal; D024882:Drug Resistance, Viral; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D018259:Herpesvirus 1, Human / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification*; D051379:Mice; D008807:Mice, Inbred BALB C; D016296:Mutagenesis; D013844:Thiazoles / Q000737:chemistry* / Q000494:pharmacology; D014764:Viral Proteins
D000998:Antiviral Agents; D004791:Enzyme Inhibitors; D013844:Thiazoles; D014764:Viral Proteins; C004483:2-aminothiazole; D019915:DNA Primase; C062021:helicase-primase, Human herpesvirus 1; D004265:DNA Helicases