Ca2+/Calmodulin-Dependent Protein Kinase II Inhibits Hepatitis B Virus Replication from cccDNA via AMPK Activation and AKT/mTOR Suppression

Abstract

Ca2+/calmodulin-dependent protein kinase II (CaMKII), which is involved in the calcium signaling pathway, is an important regulator of cancer cell proliferation, motility, growth, and metastasis. The effects of CaMKII on hepatitis B virus (HBV) replication have never been evaluated. Here, we found that phosphorylated, active CaMKII is reduced during HBV replication. Similar to other members of the AMPK/AKT/mTOR signaling pathway associated with HBV replication, CaMKII, which is associated with this pathway, was found to be a novel regulator of HBV replication. Overexpression of CaMKII reduced the expression of covalently closed circular DNA (cccDNA), HBV RNAs, and replicative intermediate (RI) DNAs while activating AMPK and inhibiting the AKT/mTOR signaling pathway. Findings in HBx-deficient mutant-transfected HepG2 cells showed that the CaMKII-mediated AMPK/AKT/mTOR signaling pathway was independent of HBx. Moreover, AMPK overexpression reduced HBV cccDNA, RNAs, and RI DNAs through CaMKII activation. Although AMPK acts downstream of CaMKII, AMPK overexpression altered CaMKII phosphorylation, suggesting that CaMKII and AMPK form a positive feedback loop. These results demonstrate that HBV replication suppresses CaMKII activity, and that CaMKII upregulation suppresses HBV replication from cccDNA via AMPK and the AKT/mTOR signaling pathway. Thus, activation or overexpression of CaMKII may be a new therapeutic target against HBV infection.

Pretreatment with Kahweol Attenuates Sepsis‐Induced Acute Lung Injury via Improving Mitochondrial Homeostasis in a CaMKKII/AMPK‐Dependent Pathway

“…[ 31 ] However, one study revealed that CaMKKII and AMPK form a positive feedback loop, where AMPK overexpression increased CaMKII activity, and AMPK inhibitors also blocked CaMKII phosphorylation. [ 45 ] In our present study, we used a CaMKKII inhibitor (STO‐609) to investigate whether the activation of CaMKKII phosphorylation is mainly due to the direct activating effect of Kahweol or the AMPK‐CaMKKII positive feedback loop. The results showed that STO‐609 significantly inhibited the phosphorylation activation of AMPK by Kahweol.…”

Peptidyl-prolyl cis/trans isomerase Pin1 interacts with hepatitis B virus core particle, but not with HBc protein, to promote HBV replication

“…Then, HepG2 or HEK293T cells were transduced with supernatant containing pseudoviral particles and shRNAs (shPIN1 or shPIN4) or control shRNA, as mentioned above, and then selected with puromycin. Stable HepG2-hNTCP-C9 cells were generated as described previously using lentiviral expression vector pCDH-CMV-hNTCP-C9-EF1-Puro ( Kim et al., 2022 ). HepG2-hNTCP-C9-derived PIN1 knockdown cells were generated as described above.…”

“…HBV particles were prepared from culture supernatants of HepAD38-pCDH, HepAD38-Pin1, and HBV-infected HepG2-hNTCP-C9 cells as described previously ( Watashi et al., 2013 ; Ko et al., 2014 ; Kim et al., 2022 ), with minor modifications. Culture supernatants comprising F-12 DMEM (11320-033, Gibco) supplemented with 10% FBS, 1% penicillin/streptomycin, 5 μg/ml insulin (#I9278, Sigma-Aldrich), and 50 μM hydrocortisone hemisuccinate (#1319002, Sigma-Aldrich) were collected 4 days after tetracycline removal, centrifuged briefly, and filtered through a 0.45 μm bottle top filter to remove cell debris.…”

“…The HBV inoculum used for infection was harvested from the culture supernatant of HepAD38 cells as described previously, with minor modifications ( Watashi et al., 2013 ; Ko et al., 2014 ; Kim et al., 2022 ). Briefly, the culture supernatants were collected every 2-3 days, centrifuged, and filtered as described above.…”

HBV promotes its replication by up-regulating RAD51C gene expression

“…The persistence of HBV is attributable to its covalently closed circular DNA (cccDNA). cccDNA acts as a transcription template on a chromosome in the infected nucleus, encodes a pregenomic RNA (pgRNA) encapsulated by HBV polymerase, pgRNA is reverse transcribed by HBV DNA polymerase into partially double-stranded relaxed circular DNA (rcDNA) 1 , 2 . The viral genome has four overlapping open reading frames encoding the HBV core protein (HBc), the envelope protein, viral polymerase, reverse transcriptase, and regulatory HBX which is regarded as an oncoprotein.…”