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  • br Financial support This study was supported

    2022-06-28


    Financial support This study was supported by Grant PH-102-PP-11, PH-103-PP-11 from the National Health Research Institutes, Taiwan.
    Conflict of interest Please refer to the accompanying ICMJE disclosure forms for further details.
    Authors’ contributions
    Acknowledgements
    Introduction Reverse transcriptase (RT) is essential for the viral replication cycle. Nucleoside analogue RT inhibitors (NRTIs) are thus important antiviral agents and are widely used for both human immunodeficiency virus type-1 (HIV-1) and human hepatitis B virus (HBV) infection [1,2]. Some NRTIs, such as lamivudine and tenofovir, are effective against HIV-1 and HBV and are approved for use in treatments (Fig. 1). Certain bulkier NRTIs are potent against either HIV-1 or HBV, but not both. For example, 4ʹ-ethynyl-2-fluoro-2ʹ-deoxyadenosine (EFdA), a powerful anti-HIV-1 agent currently under clinical trials, is not effective against HBV [[3], [4], [5]]. Conversely, the potent anti-HBV drug entecavir (ETV) is least active against HIV-1 [4]. We recently showed that certain 4ʹ-modified NRTIs, such as 4ʹ-C-cyano-2-amino-2ʹ-deoxyadenosine (CAdA), are highly potent against both HIV-1 and HBV (Fig. 1) [4]. These findings suggest that there are considerable structural differences between the nucleoside-binding sites (N-sites) of HIV-1 and HBV RT. Extensive structural studies of HIV-1 RT have been performed to elucidate the binding and inhibition mechanisms of anti-HIV-1 NRTIs [[5], [6], [7], [8], [9], [10], [11], [12], [13], [14]]. By contrast, structural studies of HBV RT have been less successful; this enzyme functions only within the nucleocapsid core particle, and obtaining the reasonable amounts of active and soluble HBV RT required for structural studies has proven extremely challenging [[15], [16], [17]]. To explore the structural mechanism of anti-HBV NRTI binding to HBV RT, we previously altered the N-site of HIV-1 RT by introducing HBV-mimicking amino Cyclophosphamide substitutions, and found that a Q151M mutation in the RT renders HIV-1 highly susceptible to ETV [18]. The results of antiviral assays combined with structural analysis of HIV-1 RT containing the Q151M substitution (RTQ151M) complexed with entecavir-triphosphate (ETV-TP) indicated that two methionine residues (Met151 and Met184) are crucial for ETV-TP binding to the HIV-1 RT N-site [18]. The single Q151M substitution in HIV-1 RT is certainly insufficient to predict the N-site structure of HBV RT. In fact, although HBV is, as mentioned above, less sensitive to EFdA (the powerful anti-HIV-1 NRTI), the HIV-1 containing RTQ151M becomes hyper-sensitive to EFdA [4,18].
    Materials and methods
    Results and discussion
    Accession numbers The atomic coordinates and structure factor amplitudes of RT7MC:DNA:dGTP and RT7MC:DNA:ETV-TP were deposited in the RCSB Protein Data Bank (http://www.rcsb.org) under accession codes 6IK9 and 6IKA, respectively.
    Conflicts of interest
    Acknowledgements The authors would like to thank the beamline staff at PF for their kind support in the X-ray diffraction experiments. This work was supported by a grant to Y.Y., Ke.M., and H.M. from the Japan Agency for Medical Research and Development (AMED; grant numbers JP16fk0310501 and JP18fk0310113). Ke.M. was also supported by a grant from the Japan Society for the Promotion of Science (JSPS-KAKENHI; grant number JP16K08826) and a grant from the National Center for Global Health and Medicine, Japan (grant number 29a-2008). The present work was also supported in part by the Intramural Research Program of the Center for Cancer Research, National Cancer Institute, National Institutes of Health, USA (H.M.). Synchrotron radiation experiments at PF were supported by the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED (grant number JP18am0101071), and were conducted under approval #2017-R15 and #2018-RP31.