Structure of the Varicella Zoster Virus Thymidylate Synthase Establishes Functional and Structural Similarities as the Human Enzyme and Potentiates Itself as a Target of Brivudine

Varicella zoster virus (VZV) is a highly infectious human herpesvirus that is the causative agent for chicken pox and shingles. VZV encodes a functional thymidylate synthase (TS), which is the sole enzyme that produces dTMP from dUMP de novo. To study substrate binding, the complex structure of TS(VZV) with dUMP was determined to a resolution of 2.9 Å. In the absence of a folate co-substrate, dUMP binds in the conserved TS active site and is coordinated similarly as in the human encoded TS (TS(HS)) in an open conformation. The interactions between TS(VZV) with dUMP and a cofactor analog, raltitrexed, were also studied using differential scanning fluorimetry (DSF), suggesting that TS(VZV) binds dUMP and raltitrexed in a sequential binding mode like other TS. The DSF also revealed interactions between TS(VZV) and in vitro phosphorylated brivudine (BVDU(P)), a highly potent anti-herpesvirus drug against VZV infections. The binding of BVDU(P) to TS(VZV) was further confirmed by the complex structure of TS(VZV) and BVDU(P) solved at a resolution of 2.9 Å. BVDU(P) binds similarly as dUMP in the TS(HS) but it induces a closed conformation of the active site. The structure supports that the 5-bromovinyl substituent on BVDU(P) is likely to inhibit TS(VZV) by preventing the transfer of a methylene group from its cofactor and the subsequent formation of dTMP. The interactions between TS(VZV) and BVDU(P) are consistent with that TS(VZV) is indeed a target of brivudine in vivo. The work also provided the structural basis for rational design of more specific TS(VZV) inhibitors.