Photon Factory Activity Report 2019 #37 (2020)

BL-1A, BL-17A/2017-R15, 2018-RP31

Structures of HIV-1 reverse transcriptase with HBV-associated triple mutations incomplex with chirally-distinct nucleoside analogues, ETV-TP and 3TC-TP
Yoshiaki YASUTAKE1,2,*, Shin-ichiro HATTORI3, Noriko TAMURA1, Kouki MATSUDA3, Satoru
KOHGO3,4, Kenji MAEDA3, Hiroaki MITSUYA3,5,6
1Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology(AIST), Sapporo 062-8517, Japan
2Computational Bio Big-Data Open Innovation Laboratory, AIST, Tokyo 169-8555, Japan
3National Center for Global Health and Medicine (NCGM) Research Institute,Tokyo 162-8655, Japan
4Faculty of Pharmaceutical Sciences, Sojo University, Kumamoto 860-0082, Japan.
5National Cancer Institute, National Institutes of Health (NIH), Bethesda, MD 20892, USA.
6Kumamoto University Hospital, Kumamoto 860-8556, Japan.
1 Introduction
Hepatitis B virus (HBV) reverse transcriptase (RT) is a
crucial enzyme for viral replication, and several nucleosideanalogues RT inhibitors are currently used for anti-HBV
treatment. Chirally-distinct nucleoside analogues RT
inhibitors, entecavir (ETV) and lamivudine (3TC), aremajor anti-HBV drugs, while common drug resistantamino acid substitutions in HBV RT (M204V/I) are known
to emerge in individuals given ETV/3TC. Because thestructures of ETV and 3TC are considerably different, it isof interest how ETV/3TC are bound to HBV RT and why
the common M204V/I mutations decrease the ETV/3TCsusceptibility. We have shown that HIV-1 with HBVassociated triple mutations (Q151M/Y115F/F116Y) in its
RT is highly susceptible to both ETV/3TC [1]. In addition,
the introduction of M184V (M204V in HBV RT) renders
HIV-1Q151M/Y115F/F116Y resistant to ETV/3TC [2]. Thus, wehave undertaken the structural studies of HIV-1
RTQ151M/Y115F/F116Y with 3TC/ETV to explore the mechanismof 3TC/ETV binding and of drug resistance conferred by
M184V/I.
2 ExperimentHIV-1 RT with mutations Q151M, Y115F and F116Y
was overexpressed using Escherichia coli and purified by
Ni-affinity/ion-exchanging chromatography as previously
described [1]. The modified DNA aptamer [3] was used foraccommodation of dNTP/NRTI at the active site of RT.
After RT:DNA complex formation, the sample was further
purified by gel-filtration. HIV-1 RTQ151M/Y115F/F116Y:DNAbinary complex was crystallized using reservoir solution
containing bis-Tris-HCl pH 6.0, ammonium di-hydrogencitrate, MgCl2, PEG 6000, glycerol and sucrose. The
crystals were soaked into the cryoprotectant solution
supplemented with ETV-triphosphate (ETV-TP)/3TCtriphosphate (3TC-TP). The X-ray diffraction data werecollected at BL-1A/17A, PF. The crystals belong to therhombohedral space group H3, with unit-cell dimensions a= b = 284 and c = 98 Å. The data were processed with theprogram XDS. The model refinement was performed using
the programs REFMAC5 and Phenix.

3 Results and Discussion
The structures of HIV-1 RTQ151M/Y115F/F116Y complexedwith DNA:ETV-TP and DNA:3TC-TP were determined at2.32 Å and 2.51 Å resolution, respectively [2]. Thestructures revealed that the L-nucleoside analogue 3TC-TPbinds in an atypical tight binding conformation dissimilar
to the common D-form nucleosides ETV-TP and otherdNTPs reported to date. Moreover, the structures haveshown that both cyclopentyl methylen of ETV-TP andoxathiolane sulfur of 3TC-TP directly push the Met184
side chain. These results provide structural basis forcommon mechanism of 3TC/ETV resitance by steric clash
between methylene/oxathiolane of ETV/3TC andM184V/I side-chain (Fig. 1).

Fig. 1: HIV-1 RTQ151M/Y115F/F116Y:DNA:3TC-TP/ETV-TP
tertiary complex. (A) Overall structure. (B) Active site
structure with bound 3TC-TP/ETV-TP. Met184 (Met204in HBV) side chain is pushed backward by oxathiolane
sulfur and exocyclic methylene of 3TC-TP and ETV-TP.

Photon Factory Activity Report 2019 #37 (2020)

Acknowledgement
The authors would like to thank beamline staff at thePhoton Factory during X-ray diffraction experiments. This
study was supported by a grant from the Program on theInnovative Development and the Application of New
Drugs for Hepatitis B from the Japan Agency for Medical
Research and Development (AMED) (JP19fk0310113).
Synchrotron radiation experiments at PF were supportedby the Platform Project for Supporting Drug Discovery andLife Science Research (Basis for Supporting Innovative
Drug Discovery and Life Science Research (BINDS)) fromAMED.
References[1] Y. Yasutake et al., Sci. Rep. 8, 1624 (2018).
[2] Y. Yasutake et al., Sci. Rep.10, 3021 (2020).
[3] M. T. Miller et al., Protein Sci. 25, 46-55 (2015).
* y-yasutake@aist.go.jp