As the most stable HBV replication intermediate, HBV cccDNA exists in the hepatocyte nucleus as a minichromosome. The stable maintenance and proper functions of the cccDNA minichromosome require its interaction with both viral and host proteins.
4- Newbold J.E.
- Xin H.
- Tencza M.
- Sherman G.
- Dean J.
- Bowden S.
- Locarnini S.
The covalently closed duplex form of the hepadnavirus genome exists in situ as a heterogeneous population of viral minichromosomes.
Accumulating evidence has shown that remodeling of the minichromosome is mediated by recruitment of epigenetic regulators (such as CREB binding protein [CBP]/E1A binding protein p300 [p300], histone deacetylase 1 [HDAC1], protein arginine methyltransferase 5 [PRMT5], and sirtuin 3 [Sirt3]) and transcriptional factors (including hepatocyte nuclear factor 1-alpha [HNF1α], HNF4α, zinc fingers and homeoboxes 2 [ZHX2], and farnesoid X-activated receptor [FXR]), which are associated strongly with cccDNA transcriptional activity.
5- Zhang W.
- Chen J.
- Wu M.
- Zhang X.
- Zhang M.
- Yue L.
- Li Y.
- Liu J.
- Li B.
- Shen F.
- Wang Y.
- Bai L.
- Protzer U.
- Levrero M.
- Yuan Z.
PRMT5 restricts hepatitis B virus replication through epigenetic repression of covalently closed circular DNA transcription and interference with pregenomic RNA encapsidation.
, 6- Belloni L.
- Pollicino T.
- De Nicola F.
- Guerrieri F.
- Raffa G.
- Fanciulli M.
- Raimondo G.
- Levrero M.
Nuclear HBx binds the HBV minichromosome and modifies the epigenetic regulation of cccDNA function.
, 7- Pollicino T.
- Belloni L.
- Raffa G.
- Pediconi N.
- Squadrito G.
- Raimondo G.
- Levrero M.
Hepatitis B virus replication is regulated by the acetylation status of hepatitis B virus cccDNA-bound H3 and H4 histones.
, 8- Ren J.H.
- Hu J.L.
- Cheng S.T.
- Yu H.B.
- Wong V.K.W.
- Law B.Y.K.
- Yang Y.F.
- Huang Y.
- Liu Y.
- Chen W.X.
- Cai X.F.
- Tang H.
- Hu Y.
- Zhang W.L.
- Liu X.
- Long Q.X.
- Zhou L.
- Tao N.N.
- Zhou H.Z.
- Yang Q.X.
- Ren F.
- He L.
- Gong R.
- Huang A.L.
- Chen J.
SIRT3 restricts hepatitis B virus transcription and replication through epigenetic regulation of covalently closed circular DNA involving suppressor of variegation 3-9 homolog 1 and SET domain containing 1A histone methyltransferases.
, 9- Decorsiere A.
- Mueller H.
- van Breugel P.C.
- Abdul F.
- Gerossier L.
- Beran R.K.
- Livingston C.M.
- Niu C.
- Fletcher S.P.
- Hantz O.
- Strubin M.
Hepatitis B virus X protein identifies the Smc5/6 complex as a host restriction factor.
, 10- Xu L.
- Wu Z.
- Tan S.
- Wang Z.
- Lin Q.
- Li X.
- Song X.
- Liu Y.
- Song Y.
- Zhang J.
- Peng J.
- Gao L.
- Gong Y.
- Liang X.
- Zuo X.
- Ma C.
Tumor suppressor ZHX2 restricts hepatitis B virus replication via epigenetic and non-epigenetic manners.
Furthermore, HBV has evolved multiple strategies to hijack or antagonize host machines for viral replication. HBV X protein (HBx) is the most important viral protein for both HBV replication and minichromosome formation. Through transcriptional transactivation and recruitment of interacting proteins, HBx relieves cccDNA transcriptional repression by altering the acetylation status and methylation status of cccDNA-bound H3/H4.
6- Belloni L.
- Pollicino T.
- De Nicola F.
- Guerrieri F.
- Raffa G.
- Fanciulli M.
- Raimondo G.
- Levrero M.
Nuclear HBx binds the HBV minichromosome and modifies the epigenetic regulation of cccDNA function.
,11- Riviere L.
- Gerossier L.
- Ducroux A.
- Dion S.
- Deng Q.
- Michel M.L.
- Buendia M.A.
- Hantz O.
- Neuveut C.
HBx relieves chromatin-mediated transcriptional repression of hepatitis B viral cccDNA involving SETDB1 histone methyltransferase.
In addition, HBx activates cccDNA transcription and viral replication by hijacking damage specific DNA binding protein 1 (DDB1)-Cullin 4 (CUL4)-ring finger-containing protein 1 (ROC1) E3 ligase to degrade host proteins such as structural maintenance of chromosomes (SMC)5/6.
8- Ren J.H.
- Hu J.L.
- Cheng S.T.
- Yu H.B.
- Wong V.K.W.
- Law B.Y.K.
- Yang Y.F.
- Huang Y.
- Liu Y.
- Chen W.X.
- Cai X.F.
- Tang H.
- Hu Y.
- Zhang W.L.
- Liu X.
- Long Q.X.
- Zhou L.
- Tao N.N.
- Zhou H.Z.
- Yang Q.X.
- Ren F.
- He L.
- Gong R.
- Huang A.L.
- Chen J.
SIRT3 restricts hepatitis B virus transcription and replication through epigenetic regulation of covalently closed circular DNA involving suppressor of variegation 3-9 homolog 1 and SET domain containing 1A histone methyltransferases.
,9- Decorsiere A.
- Mueller H.
- van Breugel P.C.
- Abdul F.
- Gerossier L.
- Beran R.K.
- Livingston C.M.
- Niu C.
- Fletcher S.P.
- Hantz O.
- Strubin M.
Hepatitis B virus X protein identifies the Smc5/6 complex as a host restriction factor.
,12- Murphy C.M.
- Xu Y.
- Li F.
- Nio K.
- Reszka-Blanco N.
- Li X.
- Wu Y.
- Yu Y.
- Xiong Y.
- Su L.
Hepatitis B virus X protein promotes degradation of SMC5/6 to enhance HBV replication.
A complete understanding of host–cccDNA interactions adopted by HBx is mandatory to develop novel strategies to eradicate HBV. However, until now, the host factors responsible for functional maintenance and transcriptional regulation of cccDNA still are less known.
In the present study, by combining chimeric–intron and minicircle DNA technology,
14- Qi Z.
- Li G.
- Hu H.
- Yang C.
- Zhang X.
- Leng Q.
- Xie Y.
- Yu D.
- Zhang X.
- Gao Y.
- Lan K.
- Deng Q.
Recombinant covalently closed circular hepatitis B virus DNA induces prolonged viral persistence in immunocompetent mice.
we established a recombinant cccDNA model named minicircle HBV (MC-HBV), which supports HBV replication and mimics minichromosome bound with histone and epigenetic factors. Using biotin-labeled MC-HBV, we systemically explored the interaction profile of cccDNA–host factors in hepatocytes by pull-down and mass spectrometry (MS) analysis. Furthermore, we identified cohesin complex, composed of SMC3 and SMC1, as a novel host factor interacting with cccDNA. Mechanistically, cohesin bound and shaped cccDNA conformation to prevent RNA polymerase II (RNAPII) enrichment, leading to inhibition of HBV replication. To get persistent replication, HBx transcriptionally inhibited SMC3 expression. It recently was shown that SMC5/6 inhibits HBV replication but is targeted by the DDB1–CUL4 E3 ligase, hijacked by HBX.
9- Decorsiere A.
- Mueller H.
- van Breugel P.C.
- Abdul F.
- Gerossier L.
- Beran R.K.
- Livingston C.M.
- Niu C.
- Fletcher S.P.
- Hantz O.
- Strubin M.
Hepatitis B virus X protein identifies the Smc5/6 complex as a host restriction factor.
Although SMC5/6 and cohesin are both members of the SMC complex, they share functional differences and are regulated by distinct mechanisms.
16The Smc5/6 complex: new and old functions of the enigmatic long-distance relative.
Our findings strengthen the idea that host chromatin-organizing factors such as SMCs play crucial functions in cccDNA transcription, which different mechanisms can exert. Our data suggest the HBx–cohesin–cccDNA axis as a potential therapeutic target in HBV infection.
Discussion
Chronic HBV infection remains a critical global public health concern, and cccDNA is the major barrier to curative HBV therapy.
2HBV cccDNA: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B.
cccDNA in the nuclei of a hepatocyte exists as a minichromosome via interacting with viral and host proteins, and these interactions are crucial for HBV replication.
27- Bock C.T.
- Schwinn S.
- Locarnini S.
- Fyfe J.
- Manns M.P.
- Trautwein C.
- Zentgraf H.
Structural organization of the hepatitis B virus minichromosome.
However, the host factors involved in the functional regulation of HBV cccDNA remain poorly understood. Here, by a MC-HBV–based screen model, we identified cohesin complex as a novel host factor binding with cccDNA and restricting HBV replication. Mechanistically, with the help of the NIPBL-MAU2 complex and CTCF, cohesin occupies and shapes cccDNA to prevent the recruitment of RNAPII and the consequent transcription of cccDNA. Conversely, HBV antagonized its antiviral role via HBx-mediated SMC3 transcriptional repression, forming a negative feedback loop in HBV infection. Thus, our data link cohesin to HBV transcription/replication control and provide novel insight into HBV therapy.
The interplay between host factors and cccDNA determines minichromosome remodeling and its transcriptional activity,
4- Newbold J.E.
- Xin H.
- Tencza M.
- Sherman G.
- Dean J.
- Bowden S.
- Locarnini S.
The covalently closed duplex form of the hepadnavirus genome exists in situ as a heterogeneous population of viral minichromosomes.
but the very low cccDNA copies in HBV-infected hepatocytes and lack of a suitable cccDNA labeling method severely limit the labeling cccDNA–host interaction study. Here, we established an MC-HBV model serving as a cccDNA surrogate, supporting a high level of HBV replication and binding with epigenetic regulators in HBV-infected HepG2
NTCP cells. With the help of a biotinylated MC-HBV/pull-down/MS–based screening approach, we described the interplay profile between cccDNA and host cell nuclear proteins. A total of 306 candidates clustered mainly into DNA conformation/function regulation, and RNA metabolism regulation pathways, were identified. In particular, 134 candidates were enriched into DNA conformation and function regulation clusters, consistent with accumulating evidence showing the close association of epigenetic regulation of cccDNA with HBV transcription and replication.
5- Zhang W.
- Chen J.
- Wu M.
- Zhang X.
- Zhang M.
- Yue L.
- Li Y.
- Liu J.
- Li B.
- Shen F.
- Wang Y.
- Bai L.
- Protzer U.
- Levrero M.
- Yuan Z.
PRMT5 restricts hepatitis B virus replication through epigenetic repression of covalently closed circular DNA transcription and interference with pregenomic RNA encapsidation.
,7- Pollicino T.
- Belloni L.
- Raffa G.
- Pediconi N.
- Squadrito G.
- Raimondo G.
- Levrero M.
Hepatitis B virus replication is regulated by the acetylation status of hepatitis B virus cccDNA-bound H3 and H4 histones.
,28- Tropberger P.
- Mercier A.
- Robinson M.
- Zhong W.
- Ganem D.E.
- Holdorf M.
Mapping of histone modifications in episomal HBV cccDNA uncovers an unusual chromatin organization amenable to epigenetic manipulation.
The candidates identified in our study include some reported epigenetic regulators of cccDNA such as HDAC1, HDAC2, and KDM2A. In addition to the cohesin complex, we found several chromatin remodeling complexes in our MS data, including the minichromosome maintenance (MCM) complex, the tailless complex polypeptide 1 ring complex (TRiC)/the ATP-dependent chaperone chaperonin containing tailless complex polypeptide 1 (CCT) complex, the LCR-associated remodeling complex (LARC), the RuvB like AAA ATPase 1/2 (RUVBL1/RUVBL2) complex, and the Switch/sucrose non-fermentable (SWI-SNF) chromatin remodeling-related breast cancer type 1 susceptibility protein (BRCA1) complex. These complexes have been reported to regulate viral replication,
29De novo replication of the influenza virus RNA genome is regulated by DNA replicative helicase, MCM.
, 30- Morwitzer M.J.
- Tritsch S.R.
- Cazares L.H.
- Ward M.D.
- Nuss J.E.
- Bavari S.
- Reid S.P.
Identification of RUVBL1 and RUVBL2 as novel cellular interactors of the Ebola virus nucleoprotein.
, 31Antiviral screen identifies EV71 inhibitors and reveals camptothecin-target, DNA topoisomerase 1 as a novel EV71 host factor.
, 32- Takahashi K.
- Halfmann P.
- Oyama M.
- Kozuka-Hata H.
- Noda T.
- Kawaoka Y.
DNA topoisomerase 1 facilitates the transcription and replication of the Ebola virus genome.
but it needs to be investigated further whether they participate in cccDNA minichromosome maintenance and HBV replication.
Interestingly, some RNA-binding proteins (RBPs), such as several DEAD-box RNA helicases (DDXs) and RNA binding motif proteins (RBMs), are identified as the candidate interaction partners of cccDNA. Recent large-scale RBP ChIP sequencing analysis showed that widespread RBPs present inactive chromatin regions in the human genome, and RBPs show a strong preference for hotspots in the genome, particularly in gene promoters. RBPs show extensive co-association with transcription factors, which are important in splicing regulation and chromatin binding, DNA looping, and transcription.
33- Xiao R.
- Chen J.Y.
- Liang Z.Y.
- Luo D.J.
- Chen G.
- Lu Z.J.
- Chen Y.
- Zhou B.
- Li H.R.
- Du X.
- Yang Y.
- San M.K.
- Wei X.T.
- Liu W.
- Lecuyer E.
- Graveley B.R.
- Yeo G.W.
- Burge C.B.
- Zhang M.Q.
- Zhou Y.
- Fu X.D.
Pervasive chromatin-RNA binding protein interactions enable RNA-based regulation of transcription.
Thus, we propose that the presence of these RBPs in cccDNA might enhance the interaction network of transcription factors, regulatory RNAs, and epigenetic chromosome remodeling. Altogether, although the MC-HBV model carries bacterial dam methylation and cannot simulate the cccDNA-host interaction under HBV infection circumstances well, we provide whole cell–based interplay profiling of cccDNA with host nuclear factors, which should be beneficial for understanding the molecular mechanisms of cccDNA maintenance and function.
The other important finding is that we identified cohesin complex as a novel host restriction factor of HBV. Cohesin originally was identified and named for its role in controlling sister chromatid cohesion. Increasing evidence indicates that cohesin participates in DNA looping, transcriptional regulation, and chromosome stability.
34- Peters J.M.
- Tedeschi A.
- Schmitz J.
The cohesin complex and its roles in chromosome biology.
Interestingly, for Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) with the circular genome, cohesin is shown to regulate viral replication through mediating DNA looping of viral episome and controlling viral gene transcription.
35- Arvey A.
- Tempera I.
- Tsai K.
- Chen H.S.
- Tikhmyanova N.
- Klichinsky M.
- Leslie C.
- Lieberman P.M.
An atlas of the Epstein-Barr virus transcriptome and epigenome reveals host-virus regulatory interactions.
,36- Stedman W.
- Kang H.
- Lin S.
- Kissil J.L.
- Bartolomei M.S.
- Lieberman P.M.
Cohesins localize with CTCF at the KSHV latency control region and at cellular c-myc and H19/Igf2 insulators.
The conformation of cccDNA plays a vital role in HBV transcription.
7- Pollicino T.
- Belloni L.
- Raffa G.
- Pediconi N.
- Squadrito G.
- Raimondo G.
- Levrero M.
Hepatitis B virus replication is regulated by the acetylation status of hepatitis B virus cccDNA-bound H3 and H4 histones.
,27- Bock C.T.
- Schwinn S.
- Locarnini S.
- Fyfe J.
- Manns M.P.
- Trautwein C.
- Zentgraf H.
Structural organization of the hepatitis B virus minichromosome.
,28- Tropberger P.
- Mercier A.
- Robinson M.
- Zhong W.
- Ganem D.E.
- Holdorf M.
Mapping of histone modifications in episomal HBV cccDNA uncovers an unusual chromatin organization amenable to epigenetic manipulation.
Our data indicate that cohesin represses pgRNA transcription via compacting cccDNA to pause RNAPII recruitment on HBV cccDNA. Pull-down, ChIP, and MST assays validated the binding of cohesin with cccDNA.
Furthermore, AFM analysis observed that the closed cccDNA looping was formed from the relaxed circular formation after incubation with purified cohesin complex, suggesting that cohesin complex is an important cccDNA conformation reconstruction factor. Cohesin's loading on chromosomes depends on the NIPBL-MAU2 loader.
21- Ciosk R.
- Shirayama M.
- Shevchenko A.
- Tanaka T.
- Toth A.
- Shevchenko A.
- Nasmyth K.
Cohesin's binding to chromosomes depends on a separate complex consisting of Scc2 and Scc4 proteins.
Either NIPBL silence or MAU2 knockdown reduced the cohesin-cccDNA binding and impaired the inhibitory role of cohesin on HBV replication. The ATPase activity of cohesin also is necessary for cohesin loading and cohesion establishment.
23- Ladurner R.
- Bhaskara V.
- in't Veld P.J.H.
- Davidson I.F.
- Kreidl E.
- Petzold G.
- Peters J.M.
Cohesin's ATPase activity couples cohesin loading onto DNA with Smc3 acetylation.
SMC3-K38A mutation abolished its ATPase activity
23- Ladurner R.
- Bhaskara V.
- in't Veld P.J.H.
- Davidson I.F.
- Kreidl E.
- Petzold G.
- Peters J.M.
Cohesin's ATPase activity couples cohesin loading onto DNA with Smc3 acetylation.
and successively compromised its cccDNA loading and antiviral ability. Although another SMC complex, SMC5/6, has been identified as a novel host restriction factor of cccDNA, its antiviral mechanism and cccDNA loading regulation are unclear.
9- Decorsiere A.
- Mueller H.
- van Breugel P.C.
- Abdul F.
- Gerossier L.
- Beran R.K.
- Livingston C.M.
- Niu C.
- Fletcher S.P.
- Hantz O.
- Strubin M.
Hepatitis B virus X protein identifies the Smc5/6 complex as a host restriction factor.
Otherwise, the inhibitory role of cohesin on HBV was independent of the SMC5/6 complex. Our findings extend the understanding of cohesin complex assembly on cccDNA to form a closed conformation and suggest the conservative role of SMC complexes in determining cccDNA conformation and viral replication.
It has been well documented that CTCF is critically involved in cohesin-mediated DNA looping of both human and viral genomes.
24- Wendt K.S.
- Yoshida K.
- Itoh T.
- Bando M.
- Koch B.
- Schirghuber E.
- Tsutsumi S.
- Nagae G.
- Ishihara K.
- Mishiro T.
- Yahata K.
- Imamoto F.
- Aburatani H.
- Nakao M.
- Imamoto N.
- Maeshima K.
- Shirahige K.
- Peters J.M.
Cohesin mediates transcriptional insulation by CCCTC-binding factor.
,35- Arvey A.
- Tempera I.
- Tsai K.
- Chen H.S.
- Tikhmyanova N.
- Klichinsky M.
- Leslie C.
- Lieberman P.M.
An atlas of the Epstein-Barr virus transcriptome and epigenome reveals host-virus regulatory interactions.
,36- Stedman W.
- Kang H.
- Lin S.
- Kissil J.L.
- Bartolomei M.S.
- Lieberman P.M.
Cohesins localize with CTCF at the KSHV latency control region and at cellular c-myc and H19/Igf2 insulators.
It also has been reported that CTCF can be co-immunoprecipitated with the cohesin complex.
37- Hansen A.S.
- Pustova I.
- Cattoglio C.
- Tjian R.
- Darzacq X.
CTCF and cohesin regulate chromatin loop stability with distinct dynamics.
In this study, the reChIP and Co-IP assays confirmed that CTCF and cohesin composed a complex to co-localize to cccDNA. CTCF fostered cohesin's loading on cccDNA and is required for cohesin-mediated inhibition of HBV replication. Nevertheless, in the mammal genome, CTCF is dispensable for cohesin to load onto DNA, which mainly favors enrichment of cohesin at specific binding sites.
24- Wendt K.S.
- Yoshida K.
- Itoh T.
- Bando M.
- Koch B.
- Schirghuber E.
- Tsutsumi S.
- Nagae G.
- Ishihara K.
- Mishiro T.
- Yahata K.
- Imamoto F.
- Aburatani H.
- Nakao M.
- Imamoto N.
- Maeshima K.
- Shirahige K.
- Peters J.M.
Cohesin mediates transcriptional insulation by CCCTC-binding factor.
The small size of cccDNA may cause this difference, and CTCF is in favor of positioning cohesin. HBV mutagenesis assays found that CTCF binding sites in the HBV genome (BS1 at the enhancer I (EnhI) region and BS3 at the HBc coding region) were responsible for CTCF and cohesin's binding on cccDNA. Similarly, D'Arienzo et al
38- D'Arienzo V.
- Ferguson J.
- Giraud G.
- Chapus F.
- Harris J.M.
- Wing P.A.C.
- Claydon A.
- Begum S.
- Zhuang X.
- Balfe P.
- Testoni B.
- McKeating J.A.
- Parish J.L.
The CCCTC-binding factor CTCF represses hepatitis B virus enhancer I and regulates viral transcription.
reported that CTCF accumulated on cccDNA through binding to the BS1 to repress HBV transcription. Here, we identified an extra CTCF binding site, BS3, in the HBV genome, which is located in the HBc gene and served as another evolutionarily conserved CTCF-BS among genotype A–H strains. Functionally, CTCF is indispensable for recruiting cohesin complex to insulate viral transcription.
HBx, a multifunctional regulator, is required for the viral life cycle and can hijack host factors for sustaining viral replication.
39Hepatitis B virus X protein: a multifunctional viral regulator.
HBV manipulates HBx to degrade SMC5/6 by hijacking DDB1-containing E3 ubiquitin ligase, relieving the inhibition and allowing HBV replication.
9- Decorsiere A.
- Mueller H.
- van Breugel P.C.
- Abdul F.
- Gerossier L.
- Beran R.K.
- Livingston C.M.
- Niu C.
- Fletcher S.P.
- Hantz O.
- Strubin M.
Hepatitis B virus X protein identifies the Smc5/6 complex as a host restriction factor.
Here, we showed the HBx-mediated repression of SMC3. Moreover, HBx down-regulated SMC3 expression via transcriptional repression, and SMC3 knockdown partially rescued viral replication in the MC-HBVΔHBx model. These findings suggest a common feature of HBx-mediated functional inhibition of SMC complexes to maintain cccDNA-driven viral replication. Considering the well-known protumoral role of HBx, HBx-induced SMC3 down-regulation may be a new pathway contributing to HBV-related HCC. SMC3 deficiency triggers genomic instability and p53-dependent apoptosis,
34- Peters J.M.
- Tedeschi A.
- Schmitz J.
The cohesin complex and its roles in chromosome biology.
and mutations in genes encoding cohesin subunits recently were identified in several types of tumors.
40Cohesin in cancer: chromosome segregation and beyond.
However, further effort is needed to uncover the exact role of HBx-mediated SMC3 repression in HCC.
HBx-induced degradation of the SMC5/6 complex is one of the most important mechanisms for HBV to relieve host restriction. Here, we found that SMC3 silence also partially rescued the replication of HBVΔHBx. The cohesin and SMC5/6 complexes have been reported to interplay with each other during chromosome segregation and DNA repair. Human SMC5/6 complex promotes DNA double-strand break repair by facilitating the recruitment of cohesin to double-strand breaks.
41- Potts P.R.
- Porteus M.H.
- Yu H.
Human SMC5/6 complex promotes sister chromatid homologous recombination by recruiting the SMC1/3 cohesin complex to double-strand breaks.
In addition, cohesin retention defects also have been described in
Saccharomyces cerevisiae with
Smc5 mutation during aberrant meiotic divisions.
42- Copsey A.
- Tang S.
- Jordan P.W.
- Blitzblau H.G.
- Newcombe S.
- Chan A.C.
- Newnham L.
- Li Z.
- Gray S.
- Herbert A.D.
- Arumugam P.
- Hochwagen A.
- Hunter N.
- Hoffmann E.
Smc5/6 coordinates formation and resolution of joint molecules with chromosome morphology to ensure meiotic divisions.
More importantly, SMC5/6 dynamics are very similar to those of cohesin in time and space.
43- Gomez R.
- Jordan P.W.
- Viera A.
- Alsheimer M.
- Fukuda T.
- Jessberger R.
- Llano E.
- Pendas A.M.
- Handel M.A.
- Suja J.A.
Dynamic localization of SMC5/6 complex proteins during mammalian meiosis and mitosis suggests functions in distinct chromosome processes.
,44- Gallego-Paez L.M.
- Tanaka H.
- Bando M.
- Takahashi M.
- Nozaki N.
- Nakato R.
- Shirahige K.
- Hirota T.
Smc5/6-mediated regulation of replication progression contributes to chromosome assembly during mitosis in human cells.
In
Schizosaccharomyces pombe, SMC5/6 locations also significantly overlap with cohesin distribution, and the SMC5/6 complex is required for timely removal of cohesin from chromosome arms.
45- Outwin E.A.
- Irmisch A.
- Murray J.M.
- O'Connell M.J.
Smc5-Smc6-dependent removal of cohesin from mitotic chromosomes.
,46- Tapia-Alveal C.
- Outwin E.A.
- Trempolec N.
- Dziadkowiec D.
- Murray J.M.
- O'Connell M.J.
SMC complexes and topoisomerase II work together so that sister chromatids can work apart.
This evidence indicate the functional crosstalk of these 2 complexes in chromosomal structure maintenance, although little is known about the mechanisms by which this occurs. Both cohesin and SMC5/6 complexes occupy HBV cccDNA and restrict its transcription, suggesting that cohesin also may interplay with SMC5/6 complex on cccDNA and this interplay might further lead to the failure of the SMC5/6 complex in restricting HBVΔHBx transcription when SMC3 was knocked down. The potential functional interplay of cohesin and SMC5/6 complexes on HBV cccDNA transcription will be investigated further in future studies.
In conclusion, this study established a cccDNA-host interaction screening model and provided a comprehensive view of the cellular proteins associated with cccDNA functional regulation. The cohesin complex binds to cccDNA to restrict HBV transcription and replication in a CTCF-dependent manner. These findings expand our understanding of HBV and host interaction and provide new insights into controlling HBV replication via targeting the cohesin complex.
Materials and Methods
Regents and Cell Culture
The human HCC cell lines Huh7, HepG2, HepG2
NTCP, and Huh7
NTCP47- Wu Z.C.
- Tan S.Y.
- Xu L.Q.
- Gao L.F.
- Zhu H.Z.
- Ma C.H.
- Liang X.H.
NgAgo-gDNA system efficiently suppresses hepatitis B virus replication through accelerating decay of pregenomic RNA.
were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The HepG2.2.15 cell line stably transformed with 2 copies of the HBV genome was maintained in minimum essential medium (MEM) supplemented with 10% FBS, 200 μg/mL G418, and 2 mmol/L glutamine. The HLCZ01 cell line was kindly gifted by Professor Zhu (Hunan University) and cultured with DMEM/F12 medium supplemented with 10% FBS, 40 ng/mL dexamethasone, and 10 ng/mL epidermal growth factor (EGF) in collagen-coated tissue culture plates as described previously.
48- Yang D.
- Zuo C.
- Wang X.
- Meng X.
- Xue B.
- Liu N.
- Yu R.
- Qin Y.
- Gao Y.
- Wang Q.
- Hu J.
- Wang L.
- Zhou Z.
- Liu B.
- Tan D.
- Guan Y.
- Zhu H.
Complete replication of hepatitis B virus and hepatitis C virus in a newly developed hepatoma cell line.
HepaRG
NTCP cells
49Hepatitis B virus infection of HepaRG Cells, HepaRG-hNTCP cells, and primary human hepatocytes.
were kindly gifted by Professor Yuchen Xia (Wuhan University) and Professor Stephan Urban (University Hospital Heidelberg) and cultured with Williams' medium E, supplied with 10% FBS, 1% penicillin-streptomycin (PS), insulin, 5 μg/mL hydrocortisone, and 80 μg/mL gentamicin. Before HBV infection, HepaRG
NTCP cells were cultured with medium containing 1.8% dimethyl sulfoxide for 48 hours. PHH cells were cultured on rat tail collagen-coated plates with Advanced DMEM/F12 (Thermo Scientific, Waltham, MA) plus 500 ng/mL R-spondin1 (Peprotech, Rocky Hill, NJ), B27 (minus vitamin A), 1.25 mmol/L N-acetylcysteine (Sigma, St. Louis, MO, USA), 10 mmol/L nicotinamide (Sigma), 50 ng/mL epidermal growth factor (Peprotech), 10 nmol/L gastrin (Sigma), 3 μmol/L CHIR99021 (Sigma), 50 ng/mL hepatocyte growth factor (HGF) (Peprotech), 100 ng/mL fibroblast growth factor-7 (FGF7) (Peprotech), 100 ng/mL FGF10 (Peprotech), 20 ng/mL transforming growth factor α (Peprotech), and 2 μmol/L A83-01 (Tocris Bioscience, Bristol, UK).
The commercial antibodies used for Western blot in this study were as follows: rabbit anti-SMC3 monoclonal antibody (1:2000, 5696; Cell Signaling Technology, Danvers, MA), rabbit anti-SMC1A monoclonal antibody (1:10,000, ab109238; Abcam, Cambridge, MA), rabbit anti-CTCF polyclonal antibody (1:4000, ab188408; Abcam), rabbit anti-HBc polyclonal antibody (1:2000, B0586; Dako, Copenhagen, Denmark), rabbit anti-HBx polyclonal antibody (1:2000, ab39716; Abcam), mouse anti-PDS5B monoclonal antibody (1:50, SC-81635; Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-PDS5A monoclonal antibody (1:50, SC-515263; Santa Cruz Biotechnology), mouse anti-RAD21 monoclonal antibody (1:50, SC-271601; Santa Cruz Biotechnology), mouse anti–β-actin monoclonal antibody (1:5000, 66009-I-Ig; Proteintech, Chicago, IL), mouse anti–glyceraldehyde 3-phosphate dehydrogenase (GAPDH) monoclonal antibody (1:5000, 66004-I-Ig; Proteintech), mouse anti-Flag-tag monoclonal antibody (1:1000, M2; Sigma), and mouse anti-HA-tag monoclonal antibody (1:2000, ab130275; Abcam). The commercial antibodies used for ChIP assay in this study were as follows: anti-H3K4me3 antibody (ab8580; Abcam), anti-H3K27me3 (ab6002; Abcam), anti-H3K27ac (ab4729; Abcam), anti-H3K36me3 (ABE435; Sigma), anti-H3K122ac (ab33309; Abcam), anti-H3K9me3 (ab8898; Abcam), anti-KAT3A/CBP (ab2832; Abcam), anti-RNAPII C-terminal domain (CTD) repeat YSPTSPS (ab5095; Abcam), and anti-RNAPII antibody (05-623-Z; Sigma).
The minicircle DNA vector system was purchased from System Biosciences, LLC (Palo Alto, CA), In-Fusion HD Cloning Kit was purchased from Takara Bio, Inc (Kyoto, Japan), commercial enzyme-linked immunosorbent assay kit for HBsAg and HBeAg was purchased from Dade Behring (Marburg, Germany), Label IT nucleic acid labeling kit was purchased from Mirus Bio LLC (Madison, WI), NE-PE Nuclear and Cytoplasmic Extraction Reagents was purchased from Thermo Fisher Scientific, ChIP kit was purchased from Millipore (Merck KGaA, Darmstadt, Germany), plasmid-safe DNase was purchased from Epicentre (Madison, WI), PEG8000, 3-aminopropyltriethoxy-silane and horseradish-peroxidase–streptavidin all were purchased from Sigma, and care HBV PCR Assay V3 kits were purchased from QIAGEN Divisions (Shenzhen, China).
Establishment of MC-HBV
MC-HBV was constructed by combining minicircle DNA technology and the HBV rcccDNA model.
14- Qi Z.
- Li G.
- Hu H.
- Yang C.
- Zhang X.
- Leng Q.
- Xie Y.
- Yu D.
- Zhang X.
- Gao Y.
- Lan K.
- Deng Q.
Recombinant covalently closed circular hepatitis B virus DNA induces prolonged viral persistence in immunocompetent mice.
Briefly, a HBV 1.0 copy genome-containing chimeric intron was amplified from parent rcccDNA plasmid, and minimized minicircle vector retaining the bacterial attachment site (attB)/the phage attachment site (attP) was amplified from pCMV.MC plasmid by high-fidelity PCR. The fragments were extracted and ligated with the In-Fusion HD Cloning Kit (Takara Bio) to form a 7.3-kb pmini-HBV PP. The PP then was transformed into
E coli strain ZYCY10P3S2T and the transformed
E coli then was induced with 0.2 mg/mL arabinose (Sigma-Aldrich, St. Louis, MO) at 30°C, pH 7.0, to generate MC-HBV (
Figure 1A). The induction efficiency was estimated with EcoRI digestion and gel electrophoresis analysis. MC-HBV contains a 1.0 copy HBV genome (3.2 kb) and a chimeric intron (128 bp). As theoretically calculated, MC-HBV only produced a single band of approximately 3.3 kb after total recombination and degradation of PP plasmid (
Figure 1B). In hepatocytes, because of the alternative splicing of pgRNA, the chimeric intron was removed from nascent rcDNA and cccDNA, which can be identified by PCR amplification using P1/P2 primer spanning the chimeric intron region in
Table 1, and produced a 431-bp fragment in MC-HBV and a shorter fragment (303 bp) in rcDNA and nascent cccDNA (
Figure 1C).
Table 1siRNA and Primers in This Study
fwd, forward; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; rev, reverse.
MC-HBV Biotinylation, Pull-Down, and MS Analysis
MC-HBV was labeled with Label IT biotin reagent (Mirus Bio, LLC) at 37°C for 1 hour and purified with a G50 microspin column according to the manufacturer's protocol. A total of 10 μg biotin-MC-HBV, unlabeled MC-HBV, and biotin-linear-MC-HBV were incubated with 10 mg HepG2 cell nuclear protein at 4°C for 6 hours under constant agitation, and then incubated for another 2 hours with an extra 50 μL of Dynabeads M-280 Streptavidin (Thermo Fisher Scientific, Waltham, MA). After careful wash with phosphate-buffered saline with Tween 20 (PBST) 3 times and PBS with 0.2% Nonidet P-40 (NP40) 2 times, boiling with 25 μL 1 × sodium dodecyl sulfate loading buffer, protein samples then were sent to PTM Biolabs, Inc (Hangzhou, Zhejiang, China) for LC-MS/MS analysis.
MST
Cell lysates from Huh7 cells with SMC3-GFP, SMC1A-GFP, or CTCF-GFP overexpression were incubated with MC-HBV at different concentrations in buffer containing 20 mmol/L Tris (pH 7.5), 150 mmol/L NaCl, 0.5% Triton X-100 (Sigma, St. Louis, MO), 0.3% bovine serum albumin, and 0.5 mmol/L ATP for 20 minutes at room temperature; GFP-overexpressed cell lysates were used as control. Then, all MST measurements were performed at 27°C using Monolith NT standard capillaries (NanoTemper Technologies, Munich, Germany) and the Monolith NT.115T device (NanoTemper Technologies) with the laser being on for 5 seconds using 40% power. All curves were plotted with the MO Affinity Analysis V10 software (NanoTemper Technologies), and the thermophoresis signals were fitted with the Kd model and normalized to the fraction bound (X), where X = (Y[c] - minimum)/(maximum - minimum), error bars (SD) were normalized by SD norm = SD(c)/(maximum - minimum).
AFM Imaging
For typical AFM imaging, muscovite mica was freshly cleaved and pretreated with volatile 3-aminopropyltriethoxy-silane in a closed tank for 30 minutes to enhance adhesive capacity. A total of 100 ng MC-HBV was mixed with purified cohesin complex on ice in PBS buffer with 112 mmol/L MgCl2, and incubated for 30 minutes at 32°C in the presence of 0.5 mmol/L ATP, with IgG purified product as control. A 50-μL sample was deposited onto the mica for 10 minutes and the sample area then was washed with 200 μL 1 × Tris-EDTA (TE) buffer with 112 mmol/L MgCl2 5 times. Commercial silicon nitride cantilevers with integrated sharpened tips (SNL-10; Bruker Corporation, Santa, Barbara, CA) were used. The topographic images were captured by peak force tapping mode experiments on a Multimode VIII system (Bruker Corporation) in liquid.
MS, Enriched Ontology Clusters. and Protein–Protein Interaction Network Analysis
Identified proteins by MS in the biotin-MC-HBV group were first compared with those in the unlabeled MC-HBV group and the biotin-linear-MC-HBV group to exclude the nonspecific binding proteins, then the remaining proteins in 2 repeats were overlaid and their peptides/scores were averaged (
Supplementary Table 1). We then identified all statistically enriched terms based on GO terms, the Kyoto Encyclopedia of Genes and Genomes (KEGG) terms, and canonical analysis with Metascape (
http://metascape.org), accumulative hypergeometric
P values and enrichment factors were calculated and used for filtering. The top 20 clusters were listed with their representative enriched terms. Count refers to the number of genes in the user-provided lists with membership in the given ontology term. Percent refers to the percentage of all the user-provided genes that are found in the given ontology term. Log
10(P) refers to the
P value in log base 10. Log
10(q) refers to the multitest adjusted
P value in log base 10.
The remaining significant terms then were clustered hierarchically into a tree based on κ statistical similarities among their gene memberships. Then, a 0.3 κ score was applied as the threshold to cast the tree into enriched term clusters. Afterward, we selected a subset of representative terms from this cluster and converted them into a network layout. More specifically, the circle nodes represent enriched terms analyzed based on GO terms and KEGG terms, where their size is proportional to the number of input genes that fall into that term, and the color represents its cluster identity (nodes of the same color belong to the same cluster). Terms with a similarity score >0.3 are linked by an edge (the thickness of the edge represents the similarity score). The network is visualized with Cytoscape (v3.1.2) with force-directed layout and with the edge bundled for clarity. One term from each cluster is selected to have its term description shown as the label.
The protein–protein interaction network was analyzed with MCODE in Cytoscape to identify neighborhoods where proteins are densely connected as previously described.
50An automated method for finding molecular complexes in large protein interaction networks.
Each MCODE network is assigned a unique color. GO enrichment analysis was applied to each MCODE network to assign meanings to the network component. Each MCODE network is assigned a unique color.
Dot-Blot Assay
Biotin-MC-HBV (10 ng) was spotted onto a nitrocellulose membrane with positive charge at the center of the grid, the membrane was allowed to dry, and then blocked by soaking in 5% bovine serum albumin in Tris Buffered Saline with Tween 20 (TBST) for 1 hour. Then, it was incubated with horseradish-peroxidase–streptavidin for 30 minutes and exposed using the enhanced chemiluminescence (ECL) reagent to detect the biotinylation of MC-HBV.
Enzyme-Linked Immunosorbent Assay
HBsAg and HBeAg secreted into cell culture supernatant were measured using commercially available HBsAg or HBeAg Enzyme-Linked Immunosorbent Assay kits (InTec, Inc, Xiamen, China) as protocol. The antigen levels were quantitated in triplicates and represented with OD450/630 optical density.
ChIP and ReChIP Assay
Huh7 cells were transfected with MC-HBV alone or with other plasmids for 72 hours, HBV cccDNA ChIP assay then was performed as previously described.
10- Xu L.
- Wu Z.
- Tan S.
- Wang Z.
- Lin Q.
- Li X.
- Song X.
- Liu Y.
- Song Y.
- Zhang J.
- Peng J.
- Gao L.
- Gong Y.
- Liang X.
- Zuo X.
- Ma C.
Tumor suppressor ZHX2 restricts hepatitis B virus replication via epigenetic and non-epigenetic manners.
Briefly, cells were fixed with 1% formaldehyde for 10 minutes at room temperature quenched by 0.125 mol/L glycine. The cell nucleus was isolated and sonicated at 25% amplitude, 10 seconds on, 10 seconds off, for 14 cycles to shear DNA. The protein–DNA complexes were immunoprecipitated with the indicated antibodies or control IgG. For ReChIP, bound material in the first ChIP with anti-Flag antibody was eluted with 50 μL 0.1 mg/mL Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (DDDDK) peptide, 0.1% sodium dodecyl sulfate, and 30 mmol/L dithiothreitol in TE for 1 hour at 37°C under constant agitation. The eluent was adjusted to 1.2 mL with dilution buffer and subjected to a second round of ChIP with anti-SMC3 antibody and eluted as described earlier. The retrieved DNA of ChIP and ReChIP were analyzed by quantitative PCR (qPCR) with the cccDNA detecting primers (cccDNA-fwd/rev in
Table 1). The qPCR results were presented as relative enrichment fold changes as control.
Cohesin Complex Purification
Huh7 cells (3 × 107) were harvested to lyse with RIPA buffer and then incubated with 4 μg SMC1A antibody (ab109238; Abcam) for 8 hours at 4°C under constant agitation. A total of 20 μL protein A/G magnetic beads was added for another 2 hours of incubation and washed 4 times with PBST buffer. Cohesin/SMC1A antibody/protein A/G beads were treated with 20 μL 0.1 mol/L glycine-HCl (pH 3.0) for 15 seconds to dissociate the binding of antibody and protein A/G and rapidly neutralized with 0.5 μL 1 mol/L Tris-HCl (pH 9.0), protein A/G magnetic beads then were discarded and the purified cohesin complex was stored at -80°C. The purification effect of the cohesin complex was evaluated through measuring subunits of cohesin using Western blot.
Immunohistochemical Staining
Immunohistochemical staining was performed in the adjacent nontumor sections from 44 HCC patients (
Table 2) who underwent surgery between January 1, 2013, and May 1, 2014, at Qilu Hospital, Shandong University. None of the patients were positive for hepatitis C virus or human immunodeficiency virus. The study was approved by the Shandong University Medical Ethics Committee in accordance with the Declaration of Helsinki. Immunohistochemical staining using anti-SMC3 antibody (5696; Cell Signaling Technology) and anti-HBc antibody (B0586; Dako) was performed, scored, and analyzed as described previously.
10- Xu L.
- Wu Z.
- Tan S.
- Wang Z.
- Lin Q.
- Li X.
- Song X.
- Liu Y.
- Song Y.
- Zhang J.
- Peng J.
- Gao L.
- Gong Y.
- Liang X.
- Zuo X.
- Ma C.
Tumor suppressor ZHX2 restricts hepatitis B virus replication via epigenetic and non-epigenetic manners.
,51- Wang L.
- Sun Y.
- Song X.
- Wang Z.
- Zhang Y.
- Zhao Y.
- Peng X.
- Zhang X.
- Li C.
- Gao C.
- Li N.
- Gao L.
- Liang X.
- Wu Z.
- Ma C.
Hepatitis B virus evades immune recognition via RNA adenosine deaminase ADAR1-mediated viral RNA editing in hepatocytes.
Briefly, 8 fields of approximately 1000 cells from each section were counted and HBc and SMC3 staining were reported separately according to the German semiquantitative scoring system. Each sample was scored according to staining intensity (no staining, 0; weak staining, 1; moderate staining, 2; and strong staining, 3) and the number of stained cells (0%, 0; 1%–25%, 1; 26%–50%, 2; 51%–75%, 3; and 76%–100%, 4). Final immunoreactive scores were determined by multiplying the staining intensity by the number of stained cells, with minimum and maximum scores of 0 and 12, respectively.
Table 2Clinical Characteristics of Enrolled Subjects
HBcAb, antibodies to HBc; HBeAb, antibodies to HBeAg; HBsAb, antibodies to HBsAg.
RNA Interference, Transfection, and RT-qPCR
siRNAs targeting indicated genes were synthesized chemically from GenePharma (Shanghai, China) (
Table 1). Cells grown overnight were transfected with siRNA using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer's protocol. Total RNA was extracted from cells using TRIzol reagent (Thermo Fisher Scientific), and 1 μg RNA was used to synthesize complementary DNA using a PrimeScript RT Reagent Kit with genomic DNA Eraser (Takara, Kyoto, Japan). RT-qPCR analysis of gene expression was performed using SYBR Premix Ex Taq (Takara) according to the manufacturer's protocol by the indicated primers (
Table 1). Glyceraldehyde-3-phosphate dehydrogenase was used as an internal control. Relative gene expression was normalized to glyceraldehyde-3-phosphate dehydrogenase using the 2 to the power of minus delta delta Ct (2–
ΔΔCt) method.
Statistical Analysis
All data are presented as the means ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism 8.0 software (GraphPad Software, San Diego, CA). Statistical differences between groups were assessed using the Student unpaired 2-tailed t test, 1-way analysis of variance, or 2-way analysis of variance. P values <.05 were considered significant.
Acknowledgments
The authors thank professor Qiang Deng (Institute Pasteur of Shanghai, Chinese Academy of Sciences) for the Cre/parent rcccDNA system. The authors thank the Translational Medicine Core Facility of Shandong University for consultation and instrument availability that supported this work.
CRediT Authorship Contributions
Chunhong Ma (Conceptualization: Lead; Supervision: Lead; Writing – review & editing: Lead)
Zhuanchang Wu (Conceptualization: Lead; Methodology: Lead; Resources: Lead; Validation: Lead; Writing – original draft: Lead; Writing – review & editing: Equal)
Liyuan Wang (Methodology: Equal)
Xin Wang (Methodology: Equal; Resources: Equal)
Yang Sun (Methodology: Equal; Project administration: Equal)
Haoran Lo (Investigation: Equal; Methodology: Equal)
Zhaoying Zhang (Methodology: Equal; Project administration: Equal)
Jinghui Lu (Formal analysis: Equal)
Leiqi Xu (Validation: Equal)
Xuetian Yue (Formal analysis: Equal; Writing – review & editing: Supporting)
Yue Hong (Methodology: Equal; Visualization: Supporting)
Qiang Li (Methodology: Supporting; Resources: Supporting)
Haizhen Zhu (Resources: Equal)
Chenjiang Gao (Resources: Equal)
Yaoqin Gong (Writing – review & editing: Supporting)
Lifen Gao (Writing – review & editing: Supporting)
Xiaohong Liang (Writing – review & editing: Supporting)
Caiyue Ren (Investigation: Supporting)
Shuangjie Li (Formal analysis: Supporting)
Huili Hu (Resources: Supporting)
Xiaohui Zhang (Investigation: Supporting; Methodology: Supporting)
Data Availability Statement
The data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Article info
Publication history
Published online: August 17, 2022
Accepted:
August 10,
2022
Received:
January 8,
2022
Footnotes
Conflicts of interest The authors disclose no conflicts.
Funding This study was funded by grants from the National Key Research and Development Program (2021YFC2300603), the National Science Foundation of China (Key Program 81830017, 81902051, and 32170157), Taishan Scholarship (tspd20181201), Major Basic Research Project of Shandong Natural Science Foundation (ZR2020ZD12), Collaborative Innovation Center of Technology and Equipment for Biological Diagnosis and Therapy in Universities of Shandong, and the Key Research and Development Program of Shandong (2019GSF108238).
Copyright
© 2022 The Authors. Published by Elsevier Inc. on behalf of the AGA Institute.