ß-L-1-[5-(E-2-bromovinyl)-2-(hydroxymethyl)-1,3-(dioxolan-4-yl)] uracil (L-BHDU) prevents varicella-zoster virus (VZV) replication in cultured cells and in vivo. Its mechanism of action was investigated by evaluating its activity against related herpesviruses and by analyzing resistant VZV strains. L-BHDU was effective against herpes simplex virus type 1 (HSV-1) with an EC50 of 0.22 µM in human foreskin fibroblast (HFF) cells. L-BHDU also inhibited HSV-2 and simian varicella virus (SVV) to a lesser extent. VZV mutants resistant to L-BHDU and other antiviral compounds were obtained by serial passage of the wild type VZV pOka and VZV Ellen strains in the presence of increasing drug concentrations. VZV strains resistant to L-BHDU (L-BHDU R ) were cross-resistant to acyclovir (ACV) and brivudin (BVdU) but not to foscarnet (PFA) and cidofovir (CDV). Conversely, ACV-resistant strains were also resistant to L-BHDU. Whole genome sequencing of L-BHDU R strains identified mutations in ATP-binding (G22R) and nucleoside binding (R130Q) domains of VZV thymidine kinase (TK). The wild type and mutant forms of VZV TK were cloned as GST fusion proteins and expressed in E. coli. The partially purified TK G22R -GST and TK R130Q -GST proteins failed to convert thymidine to thymidine monophosphate whereas the wild type TK-GST protein was enzymatically active. Similarly, L-BHDU R virus TK did not phosphorylate the drug. As expected, wild type VZV converted L-BHDU to L-BHDU monophosphate and diphosphate forms. In conclusion, L-BHDU effectiveness against VZV and HSV-1 depends on thymidine kinase activity.Parental Oka (POka, Accession number: AB097933) strain and VZV Ellen, a standard laboratory strain passaged more than 100 times since its isolation (Accession number: JQ972913.1), were propagated in HFFs. Simian varicella virus (SVV, Delta herpesvirus strain), kindly provided by Dr. Ravi Mahalingam (University of Colorado School of Medicine, Denver), was propagated in Vero cells and virus stock was prepared as described previously (Mahalingam et al., 1992). Wild type HSV-1 KOS and TK mutant HSV-1 KOS (LTRZ1) were a kind gift from Dr. Donald Coen (Harvard University). All the above HSV-1 strains were propagated and quantified in Vero cells according to standard protocol (Blaho et al., 2005). The E-377 and G strains of HSV-1 and HSV-2 respectively were obtained from American Type Culture Collection (ATCC, Manassas, VA) and characterized as reported previously (Prichard et al., 2009). The HCMV (AD169) was also obtained from ATCC. The HSV-1 F strain expressing luciferase (R8411) under the control of the ICP27 promoter was a gift from Dr. Bernard Roizman (University of Chicago). CompoundsL-BHDU was synthesized as described before . Acyclovir (ACV, A669, Sigma), (E)-5-(2-bromovinyl)-20-deoxyuridine (BVdU, B9647, Sigma) and sodium phosphonoformate tribasic hexahydrate (PFA, P6801, Sigma) were purchased from Sigma Aldrich, St. Louis, MO. Cidofovir (CDV) was kindly provided by Southern Research Institute, Birmingham, AL, USA. Stock solutions of ...
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