The kinesin KIF4 mediates HBV/HDV entry through regulation of surface NTCP localization and can be targeted by RXR agonists in vitro

Intracellular transport via microtubule-based dynein and kinesin family motors plays a key role in viral reproduction and transmission. We show here that Kinesin Family Member 4 (KIF4) plays an important role in HBV/HDV infection. We intended to explore host factors impacting the HBV life cycle that can be therapeutically addressed using siRNA library transfection and HBV/NLuc (HBV/NL) reporter virus infection in HepG2-hNTCP C4 cells. KIF4 silencing resulted in a 3-fold reduction in luciferase activity following HBV/NL infection and suppressed both wild-type HBV and HDV infection. Transient KIF4 depletion reduced surface and raised intracellular NTCP (HBV/HDV entry receptor) levels, according to both cellular fractionation and immunofluorescence analysis (IF). Overexpression of wild-type KIF4 but not ATPase-null KIF4 regains the surface localization of NTCP in these cells. Furthermore, immunofluorescence (IF) revealed KIF4 and NTCP colocalization across microtubule filaments, and a co-immunoprecipitation study showed that KIF4 physically binds to NTCP. KIF4 expression is regulated by FOXM1. Interestingly, we discovered that RXR agonists (Bexarotene, and Alitretinoin) down-regulated KIF4 expression via FOXM1-mediated suppression, resulting in a substantial decrease in HBV-Pre-S1 protein attachment to HepG2-hNTCP cell surface and subsequent suppression of HBV infection in HepG2-hNTCP and primary human hepatocytes (PXB) (Bexarotene, IC50 1.89 ± 0.98 μM). Overall, our findings show that human KIF4 is a critical regulator of NTCP surface transport and localization, which is required for NTCP to function as a receptor for HBV/HDV entry. Furthermore, small molecules that suppress or alleviate KIF4 expression would be potential antiviral candidates that target HBV and HDV entry phases. Author Summary Understanding HBV/HDV entry machinery and the mechanism by which NTCP (HBV/HDV entry receptor) surface expression is regulated is crucial to develop antiviral entry inhibitors. We found that NTCP surface transport is mainly controlled by the motor kinesin KIF4. Surprisingly, KIF4 was negatively regulated by RXR receptors through FOXM1-mediated suppression This study not only mechanistically correlated the role of RXR receptors in regulating HBV/HDV entry but also suggested a novel approach to develop therapeutic rexinoids for preventing HBV and/or HDV infections in important clinical situations, such as in patients undergoing liver transplantation or those who are at a high risk of HBV infection and unresponsive to HBV vaccination.

chromatin to alter spindle length and control cytokinesis (11). KIF4A has previously been 96 shown to improve the transport of HIV and adenovirus capsids early in infection (12, 13).

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KIF4 is a proviral host factor required for the early stages of HBV infection 115 We previously used HBV particles containing a chimeric HBV genome (HBV/NL), in 116 which HBV core region is substituted by NanoLuc (NL) gene, to infect HepG2-hNTCP

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KIF4 regulates HBV and HDV entry into host cells 9 for the interaction of HBV and surface NTCP. Using a luciferase reporter system for the 168 various HBV promoters, we discovered that KIF4 expression did not influence the 169 transcriptional activity of these promoters (Fig. 3B). Furthermore, utilizing HBV replicon 170 cells, HepAD38.7-Tet off, which produce HBV pgRNA following tetracycline 171 withdrawal, we discovered that suppressing KIF4 expression did not influence  180 We investigated the influence of KIF4 on total and subcellular (both surface and 181 cytoplasmic) NTCP expression after discovering that it is necessary for HBV entrance via 182 regulating the interaction between HBV preS1 and NTCP. Silencing of KIF4 expression 183 did not affect total cellular NTCP protein levels, as shown in figure 4A, but IF 184 examination revealed that silencing of KIF4 disrupted NTCP surface localization and 185 encouraged its accumulation in the cytoplasm (Fig. 4B). This conclusion was supported 186 by biochemical investigation, which indicated that silencing KIF4 dramatically decreased 187 surface NTCP in membranous fraction (Fig. 4C) while increasing intracellular NTCP 188 protein levels in the cytoplasmic fraction (Fig. 4D). Figure 4E  5C, lower panels). The si-KIF4 3′ UTR exhibited a substantial reduction of KIF4 by real-210 time RT-PCR and no cellular cytotoxicity as determined by the XTT assay ( Fig. 5D and 211 E). Overall, our findings indicated that KIF4 ATPase (motor) activity is necessary for 212 NTCP surface expression (transport). 214 We hypothesized that direct contact between KIF4 and NTCP across the microtubules 215 is necessary for KIF4 to transport NTCP to the cell surface. We transfected HepG2-216 hNTCP cells with Halo-tagged KIF4 and examined their intracellular colocalization.

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Interestingly, IF analysis revealed a significant colocalization between KIF4, NTCP, and 218 α-tubulin (a microtubule marker) (Fig. 6A upper panels). Two distinct cross-sectional 219 lines were constructed (Fig. 6A, center panels), and the colocalization signal intensities 220 were also displayed along these regions of interest ( Fig. 6A    Bexarotene was not harmful to primary human hepatocyte cultures over a wide range of 260 doses, with a 50% cytotoxic concentration (CC50) of more than 50 M. (Fig. 8C).

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Bexarotene's selectivity index (CC50/IC50 ratio) was found to be > 26. We then changed   The HBV/NL reporter system has previously been described to reflect the early phases anterograde transport function, we discovered that inhibiting the ATPase motor activity 307 of KIF4 substantially reduced surface and raised cytoplasmic NTCP levels (Fig. 5). We 308 also used immunoprecipitation to validate the physical contact of KIF4 and NTCP, as 309 well as their colocalization on microtubules (Fig. 6). These findings demonstrated that 310 KIF4 controlled the anterograde transport of NTCP to the cell surface, influencing its 311 availability as a receptor for HBV/HDV entry on the hepatocyte surface.

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Because the FOXM1 transcription factor is known to influence KIF4 expression (14) 313 it is predicted to affect surface NTCP expression and HBV internalization into     Indirect immunofluorescence assay 449 Immunofluorescence assay was basically performed as described previously (43).