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ANTIVIRAL ACTIVITY AND MECHANISM OF ACTION OF A NOVEL URACIL ANALOG FOR VARICELLA-ZOSTER VIRUS
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2015
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Abstract
The alphaherpesvirus varicella-zoster virus (VZV) is widespread in humans. VZV causes
primary and recurrent diseases that are preventable with live vaccines and are treatable
with antiviral drugs. New antiviral drugs for varicella-zoster virus (VZV) with increased
potency are needed, especially to prevent post-herpetic neuralgia. The purpose of this
project was to evaluate β-L-1-[5-(E-2-Bromovinyl)-2-(hydroxymethyl)-1,3-dioxolan-4-
yl)] uracil (L-BHDU) and 5′-O-valyl-L-BHDU for efficacy, safety, resistance, and
mechanism of action in three models of VZV replication: primary human foreskin
fibroblasts (HFFs), skin organ culture (SOC) and in SCID-Hu mice with skin xenografts.
We found that L-BHDU and valyl-L-BHDU were safe and effective against VZV in
culture and in a mouse model. Herpes simplex virus Type 1 was also sensitive to LBHDU in cultured cells. The mechanism of action of L-BHDU and its effect on drugdrug interactions were not known. Given its similar structure to brivudine (BVdU), we
addressed whether L-BHDU, like BVdU, inhibits 5-fluorouracil (5-FU) metabolism. LBHDU did not interfere with 5FU metabolism, indicating that L-BHDU is a safer drug
than BVdU. However, L-BHDU antagonized the activity of acyclovir (ACV), BVdU and
foscarnet (PFA) in cultured cells, which was due to competition for phosphorylation by
VZV thymidine kinase (TK). The mechanism of action of L-BHDU was studied by
evaluating its activity against related α-herpesviruses and by analyzing resistant VZV
viii
strains. VZV strains resistant to L-BHDU (L-BHDUR
) were cross-resistant to ACV and
BVdU but not to PFA and cidofovir (CDV). Whole genome sequencing of L-BHDUR
strains identified mutations in ATP-binding (G22R) and nucleoside binding (R130Q)
domains of VZV TK. The purified L-BHDUR TKs were enzymatically inactive and failed
to phosphorylate the drug. In wild type VZV- infected cells, L-BHDU was converted to
L-BHDU mono- and diphosphate forms; cells infected with L-BHDUR
virus did not
phosphorylate the drug. We also investigated whether addition of nucleosides reversed LBHDU inhibition of VZV in dividing and quiescent HFFs. Excess thymidine and uridine,
but not purines, in proportion to L-BHDU restored VZV replication only in dividing
cells, suggesting that the active form of L-BHDU interfered with pyrimidine
biosynthesis. Like other herpesviruses, VZV infection induced thymidine triphosphate
(dTTP) in confluent cells while L-BHDU treatment decreased the dTTP pool. Some
herpesviruses raise dNTP pools by inducing cellular enzymes. However, VZV infection
did not increase cellular thymidylate synthase (TS) expression to facilitate viral
replication. Furthermore, the active form of L-BHDU did not interfere with cellular
metabolism, suggesting a viral target. Further studies are required to identify the target(s)
of L-BHDU active form(s).