Viruses modify the host environment to suit their own requirements and complete the life cycle. Viruses control the cellular gene transcription machinery either to activate genes that help them or to repress those that are hostile. Acetylation is an important phenomenon that controls gene transcription; hence it is speculated that viruses control this signaling pathway (Caron et al., 2003). Several viral proteins interact with histone acetyltransferases and histone deacetyltransferases to disrupt cellular acetylation signaling pathways (Caron et al., 2003). Similarly, acetylation of α-tubulin is linked to early fusion of HIV (Jeng at al., 2015). HDAC6 inhibits influenza virus replication by negatively regulating the assembly of viral components at the site of replication via acetylated microtubules (Husain et al., 2014). For efficient infection, as well as for early steps of HCV infection, a dynamic microtubule structure is required (Roohvand et al., 2009).
To determine the acetylation status of α-tubulin during the HBV life cycle, we examined α-tubulin acetylation in HBV-replicating HCC cells (i.e., stable HBV-expressing HepAD38 (Ladner et al., 1997) and HepG2.2.15 (Sells et al., 1987) cells), and in Huh7 cells transiently transfected with HBV WT in which replicative intermediate (RI) DNAs, including HBV RC,
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stranded linear (DL), and single-stranded (SS) DNAs, are synthesized (Fig.
1A, bottom panel, lanes 2, 4, and 6). Acetylation of α-tubulin in cells containing replicating HBV was lower than that in respective non-replicating or mock controls (Fig. 1A, top panel, lane 1 vs. 2, lane 3 vs. 4, and lane 5 vs.
6). Of note, the α-tubulin level was constant (Fig. 1A, second panel). Since Sirt2 is a cytoplasmic α-tubulin deacetylase (North et al., 2003), endogenous Sirt2 mRNA and protein expressions were examined by Northern and Western blot analyses, respectively. The results showed that Sirt2 mRNA and protein expressions were upregulated when HBV was replicating (Fig. 1A, third and seventh panels, lanes 2, 4, and 6,).
To determine whether α-tubulin deacetylation and upregulated Sirt2 expression are linked to HBV DNA synthesis or HBV expression, we transfected Huh7 cells with HBV WT or replication-deficient TP-Y65F or RT-YMHA mutants. HBV TP-Y65F, a protein-priming reaction-deficient mutant, initiates minus-strand DNA without a primer, thereby synthesizing the short oligomers TGAA or GAA (the nascent minus-strand DNA); however, oligomer translocation and HBV minus-strand DNA elongation cannot occur, making the virus replication-deficient (Kim et al., 2004). Presence of HBV RT-YMHA, the conserved reverse transcriptase (RT) active YMDD motif in YMHA mutants, results in a RT-deficient, dead virus that does not support HBV DNA synthesis in vivo (Kim et al., 2004, Chang et al., 1990). Hence, TP-Y65F and RT-YMHA mutants show HBV RNA (Fig. 1B, eighth panel) and HBc protein expression (Fig. 1B, fourth panel), core particle formation (Fig. 1B, fifth panel), and pregenomic RNA (pgRNA) encapsidation (Fig. 1B, sixth panel), but cannot synthesize HBV RI DNAs (Fig. 1B, seventh panel, compare lane 2 vs. 3 and 4) (Kim et al., 2004). It should be mentioned here
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that HBV pgRNAs of WT and replication-deficient mutants are expressed under the control of cytomegalovirus immediate early (CMV IE) promoter and subgenomic mRNAs are expressed under the control of their authentic promoters (Fig. 1B, eighth panel) (Kim et al., 2004). Consistent with Figure 1A (third panel), Sirt2 mRNA and protein expressions were upregulated markedly when HBV DNA was synthesized in HBV WT-transfected cells (Fig. 1B, third and bottom panels, lane 2); however, Sirt2 mRNA and protein levels were downregulated markedly in HBV replication-deficient mutant-transfected cells (Fig. 1B, third and bottom panels, lanes 3 and 4).
Accordingly, α-tubulin was deacetylated in HBV WT-transfected cells when HBV DNA was synthesized (Fig. 1B, top panel, lane 2), whereas α-tubulin was deacetylated to a lesser extent in HBV replication-deficient mutant-transfected cells than in mock-mutant-transfected cells (Fig. 1B, top panel, lane 1 vs.
3 and 4). Of particular note, replication-deficient mutant-transfected cells have more decreased S mRNA levels than that of HBV WT-transfected cells (Fig.
1B, eighth panel, compare lane 2 vs. 3 and 4).
To further strengthen the above observation that α-tubulin deacetylation and upregulated Sirt2 expression are associated with HBV DNA synthesis, 1.3mer HBV WT-transfected HepG2 cells were treated with RT inhibitor, entecavir (1 µM) or lamivudine (5 µM), to inhibit HBV DNA synthesis from 24 to 72 h post-transfection (Fig. 1C, bottom panel, compare lane 2 vs. 3 and 4) (Doong et al., 1991, Abdelhamed et al., 2003). The results of an MTT (3-[4,5-dimethylththiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay has revealed that neither entecavir nor lamivudine was cytotoxic (data not shown). Consistent with Figure 1B, Sirt2 expression was markedly
downregulated and α-tubulin was deacetylated to a lesser extent in entecavir-
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or lamivudine-treated cells than in mock-transfected cells (Fig. 1C, top panel and third panels, lane 1 vs. 3 and 4). This demonstrates that HBV replication increases Sirt2 expression, leading to α-tubulin deacetylation; however, HBV expression or RT inhibitor treatment without DNA synthesis inhibits Sirt2 expression, leading to α-tubulin acetylation.
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Fig. 1. Sirt2 is upregulated and α-tubulin is deacetylated in HBV WT-replicating cells but not in HBV replication-deficient mutant-expressing cells. (A) Association between α-tubulin deacetylation and Sirt2 expression in HBV-replicating stable HepAD38, HepG2.2.15, and HBV WT (4 µg)-transfected Huh7 cells. HepAD38 and tetracycline-removed HepAD38 cells (lanes 1 and 2), newly plated HepG2 and HepG2.2.15 cells (lanes 3 and 4), and transiently mock- and HBV WT-transfected Huh7 cells (lanes 5 and 6) were cultured for 72 h. (B) α-tubulin deacetylation and Sirt2 expression in HBV WT- and replication-deficient mutant-transfected Huh7 cells. At 72 h post-transfection, lysates were prepared from mock- (lane 1), HBV WT- (lane 2), TP-Y65F mutant- (lane 3), and RT-YMHA mutant-transfected Huh7 cells (lane 4). (C) α-tubulin deacetylation and Sirt2 expression in RT inhibitor-treated HBV WT-transfected HepG2 cells. 1.3mer HBV WT transfected HepG2 cells were treated with mock- (lane 2), 1 µM of entecavir (lane 3), or 5 µM of lamivudine (lane 4) at 24h post-transfection for 48 h. Lane 1 was mock-transfected HepG2 cell. SDS-PAGE and Western blotting (first–fourth panels), and native agarose gel electrophoresis and Western blotting (fifth panel), were performed to detect proteins and core particle formation, respectively (A–C). In situ nucleic acid blotting (B and C, sixth panel) was performed to detect all of the nucleic acids inside core particles. Southern and Northern blot analyses were performed to reveal HBV DNA synthesis (A–C, bottom, seventh, and bottom panels, respectively), HBV total RNA (B, eighth and bottom panels), and Sirt2 RNA level (Fig. A, seventh and bottom panels, lanes 5 and 6), respectively. Acetylated α-tubulin and α-tubulin, endogenous Sirt2, and HBc proteins were detected using monoclonal anti-acetylated tubulin clone 6-11B-1 [1:1,000] (Sigma-Aldrich #T 6793), mouse monoclonal
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anti-α-tubulin (TU-02) [1:5,000] (Santa Cruz #sc-8035), polyclonal rabbit Sirt2 (H-95) [1:1,000] (Santa Cruz #sc-20966), and polyclonal rabbit anti-HBc [1:1,000] (Jung et al., 2012) antibodies, respectively. Tubulin was used as a loading control. For core particle formation, core particles on PVDF membranes were incubated with a polyclonal rabbit anti-HBc [1:1,000] (Jung et al., 2012) antibody. For Southern blotting, HBV DNA extracted from isolated core particles was separated, transferred to a nylon membrane, hybridized with a random-primed 32P-labeled full length HBV-specific probe, and subjected to autoradiography. HBV replicative intermediate, single-stranded, double-stranded linear, and partially double-stranded relaxed circular DNAs are marked as HBV RI DNA, SS, DL, and RC, respectively.
For in situ nucleic acid blotting, isolated core particles on PVDF membranes were treated with 0.2 N sodium hydroxide, hybridized, and subjected to autoradiography. For Northern blotting, 20 µg of total RNA was separated by 1% formaldehyde gel electrophoresis, transferred to nylon membranes, hybridized, and subjected to autoradiography as described above for Southern blotting. The 3.5 kb pgRNA and the 2.1 and 2.4 kb mRNAs encoding the S protein are indicated. For Sirt2 mRNA level, RT-PCR was performed.
Relative levels of acetylated α-tubulin, endogenous Sirt2, and 2.4 and 2.1kb S mRNA were measured using ImageJ. 1.46r. Data represent the mean level of acetylated α-tubulin, Sirt2, and S mRNA from three independent experiments.
Statistical significance was evaluated using Student’s t-test. *p < 0.05 and **p
< 0.005, relative to control.
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