Photon Factory Activity Report 2018 #36 (2019)

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

Structures of HIV-1 reverse transcriptase with HBV-associated septuple mutations incomplex with DNA and ETV-TPYoshiaki YASUTAKE1,2,*, Shin-ichiro HATTORI3, Noriko TAMURA1, Kouki MATSUDA3, Satoru
KOHGO3,4, Kenji MAEDA3, Hiroaki MITSUYA3,5,61
Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology(AIST), Sapporo 062-8517, Japan
2
Computational Bio Big-Data Open Innovation Laboratory, AIST, Sapporo 062-8517, Japan
3
National Center for Global Health and Medicine (NCGM) Research Institute,Tokyo 162-8655, Japan
4
Faculty 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 an
essential enzyme for viral life cycle, and all of theapproved direct-acting chemotherapeutics for anti-HBV
treatment to date are HBV RT inhibitors. Nevertheless, thethree-dimensional structure of HBV RT has not been
determined due to its notorious insoluble property, and thus
the mechanisms of anti-HBV drug binding to HBV RT andof drug resistance by mutations in HBV RT are less clear.
We recently showed that HBV-associated Q151M
mutation in human immunodeficiency virus type I (HIV-1)
RT renders HIV-1 highly sensitive to entecavir (ETV), apotent anti-HBV agent. This finding indicated that theHIV-1 RT with HBV-mimicking mutations at the active
site might be useful to explore putative structure of HBV
RT active site in the complex state with HBV RT inhibitors.
In this study, we have newly designed HIV-1 RT mutant
carrying up to seven amino-acid substitutions so as tocontain complete reproduction of HBV RT sequences at
the active site region. Unfortunately, such septuple aminoacid substitutions impair viral replication and thus the antiviral assay could not be performed, whereas therecombinant HIV-1 RT mutants could be stably producedwith retaining RT activity.
2 ExperimentHIV-1 RT carrying mutations Q151M, G112S, D113A,
Y115F, F116Y, F160L and I159L (RT7MC) wasoverexpressed using Escherichia coli BL21(DE3)-RIL,and purified by Ni-affinity and ion-exchanging
chromatography as previously described [1]. The hairpin
template-primer DNA aptamer [2] was used foraccommodation of dNTP/NRTI at the active site. After
RT:DNA complex formation, the sample was further
purified by gel-filtration chromatography [1]. HIV-1
RT7MC:DNA binary complex was crystallized by hangingdrop vapor-diffusion technique at 20°C using reservoir
solution containing bis-Tris-HCl pH 6.0, ammonium dihydrogen citrate, MgCl2, PEG 6000, glycerol and sucrose.
Prior to X-ray diffraction experiments, the crystals weresoaked into the cryoprotectant solution supplemented with
ETV-triphosphate (ETV-TP)/dGTP. The X-ray diffraction

data were collected at the beamline BL-1A, PF. Thecrystals belong to the rhombohedral space group H3, with
unit-cell dimensions a = b = 284 and c = 98 Å. The data
were processed with the programs XDS and AIMLESS.
The model refinement was performed using the programs
REFMAC5 and Phenix.
3 Results and Discussion
The structures of HIV-1 RT7MC in complex with
DNA:dGTP and DNA:ETV-TP were determined at 2.43 Åand 2.60 Å resolution, respectively [3]. Two RT moleculesin the asymmetric unit are well superimposed with eachother with main chain rmsd of below 0.7 Å. Simulated
annealing omit map for the bound substrate dGTP andETV-TP was obvious (Fig. 1), and as shown in theprevious structural study of HIV-1 RTQ151M, ETV-TP isbound to the active site of RT7MC with its exocyclic
methylen pushing directly the side-chain of Met184
backward. The Met184 of HIV-1 RT corresponds toMet204 in HBV RT, and the M204I/V is known as a major
ETV resistant mutation in HBV RT. The present structureof HIV-1 RT7MC strongly suggests that the M204I/V
mutation might disrupt the fitness for the methylen groupof ETV-TP at the active site of HBV RT.

Fig. 1: Simulated annealing omit map for the bounddGTP (A) and ETV-TP (B) at the active site of HIV-1
RT7MC. The maps were contoured at 2.2σ level. The
side-chain of Met184 moved backward by exocyclic
methylene of ETV-TP as shown by red arrow.

Photon Factory Activity Report 2018 #36 (2019)

In addition, structural comparison with previously
determined wild-type and Q151M mutant of HIV-1 RT
showed that slight but significant conformational
rearrangements of the amino-acid side chain occurred at
the active site of RT7MC (Fig. 2). This structural feature of
the RT7MC active site plausibly explain the observed
difference between HIV-1 and HBV susceptibility to 4ʹmodified nucleoside analogues. EFdA, a strong anti-HIV
4ʹ-ethynyl nucleoside analogue, is not potent against HBV.
The theoretical modeling of 4ʹ-ethynyl at the active site ofRT7MC suggests that the sever steric clash could occur
between Leu160 side chain and 4ʹ-ethynyl group. Incontrast, 4ʹ-cyano nucleoside analogues such as CAdA areknown to be potent against both HBV and HIV-1. The
modeling of the cyano group indicates that the expected
position of cyano nitrogen would be compatible with both
Leu/Phe160 without sever steric clashes. Theseobservations suggest that the structure of the RT7MC active
site could experimentally simulate the structure of HBV
RT active site.

Fig. 2: Conformational rearrangements observed in theactive site structure of RT7MC. The movements of theresidues 160, 115, and 157 side-chains and the bound
ETVTP are represented by red arrows. The expected
direction of the C4ʹ-ethynyl/cyano group is also indicatedby blue arrow.
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 the
Innovative Development and the Application of New
Drugs for Hepatitis B from the Japan Agency for Medical
Research and Development (AMED).
References[1] Y. Yasutake et al., Sci. Rep. 8, 1624 (2018).
[2] M. T. Miller et al., Protein Sci. 25, 46-55 (2015).
[3] Y. Yasutake et al., Biochem. Biophys. Res. Commun.
509, 943-948.
* y-yasutake@aist.go.jp