Photon Factory Activity Report 2017 #35 (2018)

BL-1A, BL-17A/2015R-19, 2016R-09, 2017R-15

HIV-1 reverse transcriptase complexed with DNA and ETV-TP, an anti-HBV drug
Yoshiaki Yasutake1,*, Shin-ichiro Hattori2, Hironori Hayashi2, Kouki Matsuda2, Noriko Tamura1,Satoru Kohgo2,3, Kenji Maeda2, Hiroaki Mitsuya2,4,51
Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology(AIST), Sapporo 062-8517, Japan
2
National Center for Global Health and Medicine (NCGM) Research Institute,Tokyo 162-8655, Japan
3
Faculty of Pharmaceutical Sciences, Sojo University, Kumamoto 860-0082, Japan.
4National Cancer Institute, National Institutes of Health (NIH), Bethesda, MD 20892, USA.
5Kumamoto University Hospital, Kumamoto 860-8556, Japan.
1 Introduction
Hepatitis B virus (HBV) affecting approximately 400
million people worldwide causes acute and chronic
hepatitis, resulting in approximately one million deaths per
year [1]. HBV reverse transcriptase (RT) is a vital protein
for viral replication and is thus an important target protein
for anti-HBV drug development. Nonetheless, obtaining
soluble and catalytically active recombinant HBV RT for
structural studies remains extremely challenging [2]. Incontrast, human immunodeficiency virus type-1 (HIV-1)
RT has been known as a stable protein, and the structural
analyses of HIV-1 RT have been extensively investigatedto date. Although amino-acid sequence similarity between
HBV and HIV-1 RTs are very low (~8%), the residues
creating the dNTP/drug-binding site (N-site) aremoderately conserved. It is thus considered that there are
structural and mechanistic analogies between HBV andHIV-1 RT N-site. We have created various HIV-1 RTmutants that mimics HBV-RT N-site, and we found that
Q151M mutation in RT becomes HIV-1 highly susceptible
to entecavir (ETV), one of the potent anti-HBV RT
nucleoside analogue inhibitors. In this study, we havedetermined the X-ray structure of HIV-1 RT Q151M:DNAin complex with ETV-triphosphate (ETV-TP) or dGTP.
The structures provide insights into the mechanism ofETV-TP binding to the RT N-site, and clues to develop newdrugs to overcome the reported ETV-resistance of HBV RT.
2 ExperimentHIV-1 RT Q151M was overexpressed by Escherichiacoli, and purified by Ni-affinity and ion-exchanging
chromatography as described [3]. After RT:DNA complex
formation, the sample was further purified by gel-filtration
chromatography [4]. HIV-1 RT Q151M:DNA binary
complex was crystallized by hanging-drop vapor-diffusion
method. Prior to X-ray diffraction experiments, the crystals
were soaked into the cryoprotectant solution supplemented
with ETV-TP/dGTP. The X-ray diffraction data sets werecollected at the beamline BL-1A or BL-17A of PF. Thecrystals belong to the rhombohedral space group R3, withunit-cell parameters a = b = 284 and c = 98 Å. The data
were indexed and scaled with the program XDS. The
structures were determined by molecular replacement withthe program MOLREP, using the previously reported
RT:DNA binary complex as a search model (PDB code,5d3g) [5]. The atomic coordinates and temperature factors
were refined with the program Phenix.
3 Results and Discussion
The structures of HIV-1 RT Q151M:DNA in ligand-free, incomplex with dGTP or ETV-TP were determined at aresolution of 2.60, 2.38 and 2.45 Å, respectively (Fig. 1).

Fig. 1: Overall structure of HIV-1 RT
Q151M:DNA:ETV-TP ternary complex
There are two p66-p51 RT heterodimers in the asymmetricunit of the rhombohedral R3 lattice. The final refinedmodel contains amino acid residues 3–553 for chains A/C(p66 subunit), 4–427 for chains B/D (p51 subunit) except
214–230 owing to poor electron density, and two DNAchains (nucleotides from −1 to 33 for chain E and from −4
to 33 for chain F).
The structures revealed that the postion and orientationof the bound ETV-TP/dGTP are similar to those in thepreviously reported HIV-1 RT:DNA:dNTP complex,
while there are slight but significant structural differencesat the N-site occupied by ETV-TP. First, the riboseanalogous cyclopentane ring of the bound ETV-TP lies
slightly apart from the 3′-end nucleotide of the primer
DNA. Second, relocation of the side-chain of Met184
occurs by pressing with the protruded exocyclic methylenegroup of the ETV-TP (Fig. 2). The structures explain theputative ETV-TP binding mechanism to RT, and also thePhoton Factory Activity Report 2017 #35 (2018)

reasoning for why M204V of HBV RT (M184V in HIV-1RT) enables ETV resistant: the loss of hydrophobic
interactions between the methylene group of ETV-TP andthe Met184 side-chain could significantly decrease thebinding affinity of ETV-TP at the RT N-site. Based onthese results, we propose that modification of the ETV
methylene moiety may represent a strategic candidate todevelop new anti-viral agents for overcoming ETVresistance.

Fig. 2: N-site structure of HIV-1 RT Q151M with bound
ETV-TP
Acknowledgement
The authors would like to thank beamline staff at thePhoton Factory during X-ray diffraction experiments. Thisstudy 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).
References[1] D. Lavanchy, J. Viral. Hepat. 11, 97-107 (2004).
[2] J. Voros et al., J. Virol. 88, 2584-2599 (2013).
[3] A. Nakamura et al., Acta Crystallogr. Sect. F, 71, 13841390 (2015).
[4] Y. Yasutake et al., Sci. Rep. 8, 1624 (2018).
[5] M. T. Miller et al., Protein Sci. 25, 46-55 (2015).
*y-yasutake@aist.go.jp