Identification and characterization of a G-quadruplex structure in the pre-core promoter region of hepatitis B virus covalently closed circular DNA
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100589
ISSN1083-351X
AutoresVanessa Meier‐Stephenson, Maulik D. Badmalia, Tyler Mrozowich, Keith C.K. Lau, Sarah K. Schultz, Darren L. Gemmill, Carla Osiowy, Guido van Marle, Carla S. Coffin, Trushar R. Patel,
Tópico(s)RNA Interference and Gene Delivery
ResumoApproximately 250 million people worldwide are chronically infected with the hepatitis B virus (HBV) and are at increased risk of developing cirrhosis and hepatocellular carcinoma. The HBV genome persists as covalently closed circular DNA (cccDNA), which serves as the template for all HBV mRNA transcripts. Current nucleos(t)ide analogs used to treat HBV do not directly target the HBV cccDNA genome and thus cannot eradicate HBV infection. Here, we report the discovery of a unique G-quadruplex structure in the pre-core promoter region of the HBV genome that is conserved among nearly all genotypes. This region is central to critical steps in the viral life cycle, including the generation of pregenomic RNA, synthesis of core and polymerase proteins, and genome encapsidation; thus, an increased understanding of the HBV pre-core region may lead to the identification of novel anti-HBV cccDNA targets. We utilized biophysical methods (circular dichroism and small-angle X-ray scattering) to characterize the HBV G-quadruplex and the effect of three distinct G to A mutants. We also used microscale thermophoresis to quantify the binding affinity of G-quadruplex and its mutants with a known quadruplex-binding protein (DHX36). To investigate the physiological relevance of HBV G-quadruplex, we employed assays using DHX36 to pull-down cccDNA and compared HBV infection in HepG2 cells transfected with wild-type and mutant HBV plasmids by monitoring the levels of genomic DNA, pregenomic RNA, and antigens. Further evaluation of this critical host-protein interaction site in the HBV cccDNA genome may facilitate the development of novel anti-HBV therapeutics against the resilient cccDNA template. Approximately 250 million people worldwide are chronically infected with the hepatitis B virus (HBV) and are at increased risk of developing cirrhosis and hepatocellular carcinoma. The HBV genome persists as covalently closed circular DNA (cccDNA), which serves as the template for all HBV mRNA transcripts. Current nucleos(t)ide analogs used to treat HBV do not directly target the HBV cccDNA genome and thus cannot eradicate HBV infection. Here, we report the discovery of a unique G-quadruplex structure in the pre-core promoter region of the HBV genome that is conserved among nearly all genotypes. This region is central to critical steps in the viral life cycle, including the generation of pregenomic RNA, synthesis of core and polymerase proteins, and genome encapsidation; thus, an increased understanding of the HBV pre-core region may lead to the identification of novel anti-HBV cccDNA targets. We utilized biophysical methods (circular dichroism and small-angle X-ray scattering) to characterize the HBV G-quadruplex and the effect of three distinct G to A mutants. We also used microscale thermophoresis to quantify the binding affinity of G-quadruplex and its mutants with a known quadruplex-binding protein (DHX36). To investigate the physiological relevance of HBV G-quadruplex, we employed assays using DHX36 to pull-down cccDNA and compared HBV infection in HepG2 cells transfected with wild-type and mutant HBV plasmids by monitoring the levels of genomic DNA, pregenomic RNA, and antigens. Further evaluation of this critical host-protein interaction site in the HBV cccDNA genome may facilitate the development of novel anti-HBV therapeutics against the resilient cccDNA template. Approximately 250 million people worldwide are chronic hepatitis B virus (HBV) carriers and are at elevated risk of developing cirrhosis, liver failure, and hepatocellular carcinoma (HCC) (1WHOGlobal Hepatitis Report, 2017. WHO, Geneva, Switzerland2018Google Scholar, 2Beasley R.P. Hepatitis B virus. 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In our prior analysis of the HBV promoter region using the HBV genome database (HBVdB: https://hbvdb.ibcp.fr), we noted a highly G-rich region in the pre-core/core promoter region in all HBV genomes except the HBV G genotype (21Meier-Stephenson V. Bremner W.T.R. Dalton C.S. Van Marle G. Coffin C.S. Patel T.R. Comprehensive analysis of hepatitis B virus promoter region mutations.Viruses. 2018; 10: 603Crossref Scopus (6) Google Scholar). This region would be overlooked if the rigid formula was used when searching for putative quadruplex sequences, G3-5 N1-7 G3-5 N1-7 G3-5 N1-7 G3-5, (i.e., where G’s are guanosines present in groups of three to five, and N’s are any nucleotide present in one to seven sequences separating these groups of guanosines), but exceptions exist (47Todd A.K. Johnston M. Neidle S. Highly prevalent putative quadruplex sequence motifs in human DNA.Nucleic Acids Res. 2005; 33: 2901-2907Crossref PubMed Scopus (720) Google Scholar). This region was of significant interest given the fact that it also binds to host specificity protein 1 (Sp1), a host transcription factor already known to bind G-quadruplexes, further supporting the link with the secondary structure in this region. The analysis of single-nucleotide mutation frequencies in this region was computed similarly to our previous study (21Meier-Stephenson V. Bremner W.T.R. Dalton C.S. Van Marle G. Coffin C.S. Patel T.R. Comprehensive analysis of hepatitis B virus promoter region mutations.Viruses. 2018; 10: 603Crossref Scopus (6) Google Scholar), but with a focus on all available basal core promoter sequences in the HBV genome database (9939 sequences, Fig. 1A), demonstrating remarkable conservation of the guanosine groups. To determine the ability of the HBV pre-core/core promoter region to form a quadruplex, we utilized a 23-mer oligomer of the wild-type (wt) promoter region for use in multiple biophysical assays (Fig. 1B). We also designed a mutant (G1748A) oligomer based on prior studies showing loss of Sp1 binding with the single-nucleotide mutation of G to A substitution at position 1748 (48Li J. Ou J.H. Differential regulation of hepatitis B virus gene expression by the Sp1 transcription factor.J. Virol. 2001; 75: 8400-8406Crossref PubMed Scopus (47) Google Scholar). Additional mutants were also included (i.e., G1738A and G1738/1748A) to determine the nature and necessity of these putative G-quadruplex-disrupting mutations. HBV genome frequency analysis using the HBV database (www.hbvdb.ibcp.fr) showed >99.8% conservation across all genotypes, hence supporting the use of these mutants for our proposed studies (Fig. 1). Oligomers were solubilized in an appropriate buffer, followed by a heat-cooled step to allow for G-quadruplex formation. Samples were purified using SEC prior to performing all experiments to ensure that they were free of aggregation and showed a single peak eluted for subsequent collection and concentration (Fig. 2A). First, we performed circular dichroism spectropolarimetry (CD) experiments to investigate whether the wild-type (wt) and mutant oligomers form a G-quadruplex structure in solution. We observed a peak in ellipticity upon CD analysis at λ≈263 nm and a minimum at λ≈242 nm (as detailed in Table S1, Fig. 2B), suggesting that the wt oligomer adopts a parallel G-quadruplex structure in solution (49Vorlickova M. Kejnovska I. Sagi J. Renciuk D. Bednarova K. Motlova J. Kypr J. Circular dichroism and guanine quadruplexes.Methods (San Diego, Calif.). 2012; 57: 64-75Crossref PubMed Scopus (274) Google Scholar, 50Carvalho J. Queiroz J.A. Cruz C. Circular dichroism of G-quadruplex: A Laboratory experiment for the study of topology and ligand binding.J. Chem. 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The CD spectra were collected for all the samples, and no considerable changes were observed in CD spectra collected for G-quadruplexes in Li+ and K+, suggesting that the DNA sequence can form G-quadruplex despite possessing two mutations irrespective of the centrally coordinated monovalent cation. Therefore, we decided to collect MST and SAXS data for G quadruplexes in the presence of K+ only. To visualize and verify the G-quadruplex formation, we employed the HPLC-SAXS (52Meier M. Moya-Torres A. Krahn N.J. McDougall M.D. Orriss G.L. McRae E.K.S. Booy E.P. McEleney K. Patel T.R. McKenna S.A. Stetefeld J. Structure and hydrodynamics of a DNA G-quadruplex with a cytosine bulge.Nucleic Acids Res. 2018; 46: 5319-5331Crossref PubMed Scopus (32) Google Scholar, 53Meier M. Patel T.R. Booy E.P. Marushchak O. Okun N. Deo S. Howard R. McEleney K. Harding S.E. Stetefeld J. McKenna S.A. Binding of G-quadruplexes to the N-terminal recognition domain of the RNA helicase associated with AU-rich element (RHAU).J. Biol. Chem. 2013; 288: 35014-35027Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). This enabled selection of the data set from a monodispersed region for further analysis. The absence of upturned profile at lower momentum transfer (q) values in Guinier analysis of SAXS data for wt and mutant oligomers implies that all samples are monodispersed and aggregation free (Fig. 3A, panel i and ii). Next, the dimensionless Kratky analysis (54Rambo R.P. Tainer J.A. Characterizing flexible and intrinsically unstructured biological macromolecules by SAS using the Porod-Debye law.Biopolymers. 2011; 95: 559-571Crossref PubMed Scopus (325) Google Scholar) of SAXS data for all samples was performed to assess their flexibility and compactness. This analysis displays a Gaussian curve suggesting that all samples were folded (Fig. 3A, panel iii). The SAXS data were then converted to the electron pair–distance distribution plots ((P(r) function) (Fig. 3A, panel iv) using the GNOM software program (55Svergun D. Determination of the regularization parameter in indirect-transform methods using perceptual criteria.J. Appl. Crystallogr. 1992; 25: 495-503Crossref Scopus (2755) Google Scholar). The characteristic Gaussian-shaped pattern observed in P(r) for the wt sample indicates that it adopts a compact structure in solution, whereas the G1738A, G1748A, and G1738/1748A mutant oligomers, despite the same nucleotide length (23 nucleotides), have an increasingly extended structure in solution (Fig. 3A, panel iv). The radius of gyration (Rg) for wt was calculated to be 18.37 Å, while the G1738A, G1748A, and G1738/1748A mutants yield increasing values of 19.10, 20.10, and 20.34 Å, respectively, which are in agreement with those obtained from the Guinier analysis (Table 1). The P(r) function also allows the determination of a maximum particle dimension (Dmax) of macromolecules. Based on this analysis, we obtained the Dmax of 46.85 Å for the wt, while the G1738A, G1748A, and G1738/1748A mutants were 53.14 Å, 60.00 Å, and 60.44 Å respectively, indicating that each mutation leads to a degree of alteration of the G-quadruplex structure (Fig. 3). To further investigate the structural differences between the samples, we used the P(r) data in the DAMMIF program that allows low-resolution structure determination. We calculated ten independent low-resolution structures for each sample that provided X values ranging from 1.2 to 1.8 indicating the good quality of our models (Table 1). Subsequently, we averaged and filtered ten models using DAMAVER program to obtain a representative low-resolution structure for each sample. We obtained the normalized spatial discrepancy (NSD) values of 0.55, 0.58, 0.58 and 0.59 for wt, G1738A, G1748A, and G1738/1748A mutant samples, respectively, indicating that the ten independent low-resolution structures are highly similar to each other in all cases. The low-resolution structures presented in Figure 3B demonstrate the compact quadruplex structure of the wt sample, while each of the mutations displays a relatively extended structure in solution. It is noteworthy that the increase in the size of mutants is also consistent with observed elution volumes from SEC where we observed that the wt displayed the highest elution volume (Fig. 2A), which progressively increased in the same order as the increase in Dmax i.e., wt < G1738A < G1748A < G1738/1748A.Table 1Analysis of small-angle X-ray scattering data for HBV wild-type (wt) and mutant (mut) core promoter oligomer samplesParameterswtG1738 AG1748AG1738/1748AMw (kDa)7.307.287.287.26I(0)aObtained from Guinier analysis (91).0.0035 ± 1.1 x 10-50.0041 ± 1.3 x 10-50.0031 ± 1.6 x 10-50.0037 ± 2.1 x 10-5q.Rg range0.24–1.300.23–1.300.23–1.300.23–1.29Rg (Å)aObtained from Guinier analysis (91).18.37 ± 0.1119.10 ± 0.1120.10 ± 0.1920.34 ± 0.22I(0)aObtained from Guinier analysis (91
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