The hepatitis B virus core protein intradimer interface modulates capsid assembly and stability.
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ABSTRACT:
SUBMITTER: Selzer L
PROVIDER: S-EPMC4151697 | biostudies-literature | 2014 Sep
REPOSITORIES: biostudies-literature
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Project description:During the hepatitis B virus lifecycle, 120 copies of homodimeric capsid protein assemble around a copy of reverse transcriptase and viral RNA and go on to produce an infectious virion. Assembly needs to be tightly regulated by protein conformational change to ensure symmetry, fidelity, and reproducibility. Here, we show that structures at the intradimer interface regulate conformational changes at the distal interdimer interface and so regulate assembly. A pair of interacting charged residues, D78 from each monomer, conspicuously located at the top of a four-helix bundle that forms the intradimer interface, were mutated to serine to disrupt communication between the two monomers. The mutation slowed assembly and destabilized the dimer to thermal and chemical denaturation. Mutant dimers showed evidence of transient partial unfolding based on the appearance of new proteolytically sensitive sites. Though the mutant dimer was less stable, the resulting capsids were as stable as the wildtype, based on assembly and thermal denaturation studies. Cryo-EM image reconstructions of capsid indicated that the subunits adopted an "open" state more usually associated with a free dimer and that the spike tips were either disordered or highly flexible. Molecular dynamics simulations provide mechanistic explanations for these results, suggesting that D78 stabilizes helix 4a, which forms part of the intradimer interface, by capping its N-terminus and hydrogen-bonding to nearby residues, whereas the D78S mutation disrupts these interactions, leading to partial unwinding of helix 4a. This in turn weakens the connection from helix 4 and the intradimer interface to helix 5, which forms the interdimer interface.
| S-EPMC8809082 | biostudies-literature
Project description:The hepatitis E virus (HEV), a non enveloped RNA virus, causes viral hepatitis. The viral open reading frame 2 (ORF2) protein represents the capsid protein of HEV which is known to cause endoplasmic reticulum stress in ORF2 expressing cells. The initiation of endoplasmic reticulum stress induced apoptosis mainly involves the transcriptional activation of pro-apoptotic gene CHOP which will further trigger the major apoptotic pathways. However, the activation of CHOP by ORF2 protein in this study does not induce apoptotic markers such as Bax translocation to mitochondria. We have used the Affymetrix microarray platform to screen the pro-apoptotic effects induced by the expression of ORF2 protein in human hepatic cell lines (Huh7). The Huh7 cells were transduced either with recombinant adenovirus encoding the HEV ORF2 (Ad-ORF2) or an adenovirus encoding the green fluorescent protein (Ad-GFP). The array results consistently showed an ORF2 specific induction of mRNA corresponding to the chaperones Hsp72, Hsp70B’ and co-chaperone Hsp40. These studies provide further mechanisms of the ER stress mediated pro apoptotic effects caused by the ORF2 protein and its potential role for the activation of anti-apoptotic activity of the host cell. We used microarray to screen the host genes were regulated by the expression of the hepatitis E virus capsid protein. Huh7 cells transduced with Ad-GFP (control) or with Ad-HEV ORF2.
2012-12-01 | E-GEOD-29061 | biostudies-arrayexpress
Project description:Hepatitis B virus core protein has 183 amino acids divided into an assembly domain and an arginine-rich C-terminal domain (CTD) that regulates essential functions including genome packaging, reverse transcription, and intracellular trafficking. Here, we investigated the CTD in empty hepatitis B virus (HBV) T=4 capsids. We examined wild-type core protein (Cp183-WT) and a mutant core protein (Cp183-EEE), in which three CTD serines are replaced with glutamate to mimic phosphorylated protein. We found that Cp183-WT capsids were less stable than Cp183-EEE capsids. When we tested CTD sensitivity to trypsin, we detected two different populations of CTDs differentiated by their rate of trypsin cleavage. Interestingly, CTDs from Cp183-EEE capsids exhibited a much slower rate of proteolytic cleavage when compared with CTDs of Cp183-WT capsids. Cryo-electron microscopy studies of trypsin-digested capsids show that CTDs at five-fold symmetry vertices are most protected. We hypothesize that electrostatic interactions between glutamates and arginines in Cp183-EEE, particularly at five-fold, increase capsid stability and reduce CTD exposure. Our studies show that quasi-equivalent CTDs exhibit different rates of exposure and thus might perform distinct functions during the hepatitis B virus lifecycle. Our results demonstrate a structural role for CTD phosphorylation and indicate crosstalk between CTDs within a capsid particle.
| S-EPMC4653712 | biostudies-literature
Project description:Hepatitis C virus (HCV) core protein is directed to the surface of lipid droplets (LD), a step that is essential for infectious virus production. However, the process by which core is recruited from LD into nascent virus particles is not well understood. To investigate the kinetics of core trafficking, we developed methods to image functional core protein in live, virus-producing cells. During the peak of virus assembly, core formed polarized caps on large, immotile LDs, adjacent to putative sites of assembly. In addition, LD-independent, motile puncta of core were found to traffic along microtubules. Importantly, core was recruited from LDs into these puncta, and interaction between the viral NS2 and NS3-4A proteins was essential for this recruitment process. These data reveal new aspects of core trafficking and identify a novel role for viral nonstructural proteins in virus particle assembly.
| S-EPMC3197604 | biostudies-literature
Project description:The hepatitis E virus (HEV), a non enveloped RNA virus, causes viral hepatitis. The viral open reading frame 2 (ORF2) protein represents the capsid protein of HEV which is known to cause endoplasmic reticulum stress in ORF2 expressing cells. The initiation of endoplasmic reticulum stress induced apoptosis mainly involves the transcriptional activation of pro-apoptotic gene CHOP which will further trigger the major apoptotic pathways. However, the activation of CHOP by ORF2 protein in this study does not induce apoptotic markers such as Bax translocation to mitochondria. We have used the Affymetrix microarray platform to screen the pro-apoptotic effects induced by the expression of ORF2 protein in human hepatic cell lines (Huh7). The Huh7 cells were transduced either with recombinant adenovirus encoding the HEV ORF2 (Ad-ORF2) or an adenovirus encoding the green fluorescent protein (Ad-GFP). The array results consistently showed an ORF2 specific induction of mRNA corresponding to the chaperones Hsp72, Hsp70B’ and co-chaperone Hsp40. These studies provide further mechanisms of the ER stress mediated pro apoptotic effects caused by the ORF2 protein and its potential role for the activation of anti-apoptotic activity of the host cell.
2012-12-01 | GSE29061 | GEO
Project description:Hepatitis B virus (HBV) is a hepadnavirus, a small DNA virus that infects liver tissue, with some unusual replication steps that share similarities to retroviruses. HBV infection can lead to chronic hepatitis B (CHB), a life-long infection associated with significant risks of liver disease, especially if untreated. HBV is a significant global health problem, with hundreds of millions currently living with CHB. Currently approved strategies to prevent or inhibit HBV are highly effective, however, a cure for CHB has remained elusive. To achieve a cure, elimination of the functionally integrated HBV covalently closed chromosomal DNA (cccDNA) genome is required. The capsid core is an essential component of HBV replication, serving roles when establishing infection and in creating new virions. Over the last two and a half decades, significant efforts have been made to find and characterize antivirals that target the capsid, specifically the HBV core protein (Cp). The antivirals that interfere with the kinetics and morphology of the capsid, termed capsid assembly modulators (CAMs), are extremely potent, and clinical investigations indicate they are well tolerated and highly effective. Several CAMs offer the potential to cure CHB by decreasing the cccDNA pools. Here, we review the biology of the HBV capsid, focused on Cp, and the development of inhibitors that target it.
| S-EPMC10461335 | biostudies-literature
Project description:UnlabelledHepatitis B virus (HBV) capsid proteins (Cps) assemble around the pregenomic RNA (pgRNA) and viral reverse transcriptase (P). pgRNA is then reverse transcribed to double-stranded DNA (dsDNA) within the capsid. The Cp assembly domain, which forms the shell of the capsid, regulates assembly kinetics and capsid stability. The Cp, via its nucleic acid-binding C-terminal domain, also affects nucleic acid organization. We hypothesize that the structure of the capsid may also have a direct effect on nucleic acid processing. Using structure-guided design, we made a series of mutations at the interface between Cp subunits that change capsid assembly kinetics and thermodynamics in a predictable manner. Assembly in cell culture mirrored in vitro activity. However, all of these mutations led to defects in pgRNA packaging. The amount of first-strand DNA synthesized was roughly proportional to the amount of RNA packaged. However, the synthesis of second-strand DNA, which requires two template switches, was not supported by any of the substitutions. These data demonstrate that the HBV capsid is far more than an inert container, as mutations in the assembly domain, distant from packaged nucleic acid, affect reverse transcription. We suggest that capsid molecular motion plays a role in regulating genome replication.ImportanceThe hepatitis B virus (HBV) capsid plays a central role in the virus life cycle and has been studied as a potential antiviral target. The capsid protein (Cp) packages the viral pregenomic RNA (pgRNA) and polymerase to form the HBV core. The role of the capsid in subsequent nucleic acid metabolism is unknown. Here, guided by the structure of the capsid with bound antiviral molecules, we designed Cp mutants that enhanced or attenuated the assembly of purified Cp in vitro. In cell culture, assembly of mutants was consistent with their in vitro biophysical properties. However, all of these mutations inhibited HBV replication. Specifically, changing the biophysical chemistry of Cp caused defects in pgRNA packaging and synthesis of the second strand of DNA. These results suggest that the HBV Cp assembly domain potentially regulates reverse transcription, extending the activities of the capsid protein beyond its presumed role as an inert compartment.
| S-EPMC4337549 | biostudies-literature
Project description:Hepatitis B virus (HBV) core protein (HBc) serves pivotal roles in the viral life cycle, particularly serving as the basic unit for capsid assembly, and is closely associated with HBV genome replication and progeny virion production. Previous studies have demonstrated that HBc has at least two functional interfaces; two HBc monomers form a homodimer via an intradimer interface, and then 90 or 120 homodimers form an icosahedral capsid via a dimer?dimer interface. In the present study, the role of the HBc dimer?dimer interface in HBV replication was investigated. A panel of residues located at the dimer?dimer interface were identified based on the crystal structure of HBc. Native gel electrophoresis and western blotting revealed that, despite mutations in the dimer?dimer interface, HBc formed a capsid?like structure, whereas mutations at amino acid residues 23?39 completely disrupted capsid assembly. Using denaturing gel electrophoresis, Southern and Northern blotting, and quantitative polymerase chain reaction, it was demonstrated that none of the mutations in the dimer?dimer interface supported pregenomic RNA encapsidation or DNA replication. In addition, these mutants interacted with the wild-type (WT) HBc monomer and inhibited WT genome replication and virion production in a dose?dependent manner. However, the quantity of covalently closed circular DNA in the nucleus was not affected. The present study highlighted the importance of the HBc dimer?dimer interface for normal capsid function and demonstrated that the HBc dimer?dimer interface may be a novel antiviral target.
| S-EPMC6297743 | biostudies-literature
Project description:The dynamic interplay between virus and host proteins is critical for establishing efficient viral replication and virus-induced pathogenesis. Phosphorylation-dependent prolyl isomerization by Pin1 provides a unique mechanism of molecular switching to control both protein function and stability. We demonstrate here that Pin1 binds and stabilizes hepatitis B virus core protein (HBc) in a phosphorylation-dependent manner, and promotes the efficient viral propagation. Phos-tag gel electrophoresis with various site-directed mutants of HBc revealed that Thr160 and Ser162 residues within the C terminal arginine-rich domain are phosphorylated concomitantly. GST pull-down assay and co-immunoprecipitation analysis demonstrated that Pin1 associated with phosphorylated HBc at the Thr160-Pro and Ser162-Pro motifs. Chemical or genetic inhibition of Pin1 significantly accelerated the rapid degradation of HBc via a lysosome-dependent pathway. Furthermore, we found that the pyruvate dehydrogenase phosphatase catalytic subunit 2 (PDP2) could dephosphorylate HBc at the Pin1-binding sites, thereby suppressing Pin1-mediated HBc stabilization. Our findings reveal an important regulatory mechanism of HBc stability catalyzed by Pin1 and may facilitate the development of new antiviral therapeutics targeting Pin1 function.
| S-EPMC7005485 | biostudies-literature
Project description:While there is an effective vaccine for Human Hepatitis B Virus (HBV), 257 million people have chronic infections for which there is no cure. The assembly process for the viral capsid is a potential therapeutic target. In order to understand the capsid assembly process, we investigated the dimeric building blocks of the capsid. To understand what blocks assembly, we took advantage of an assembly incompetent mutant dimer, Cp149-Y132A, located in the interdimer interface. This mutation leads to changes in protein dynamics throughout the structure of the dimer as measured by hydrogen-deuterium exchange mass spectrometry (HDX-MS). To further understand how the HBV capsid assembles, the homologue woodchuck HBV (WHV) capsid protein dimer (Cp) was used. WHV is more stable than HBV in HDX-MS and native mass spectrometry experiments. Because the WHV Cp assembles more rapidly into viral capsids than HBV, it was suspected that an increase in stability of the intradimer interface and/or in the contact region leads to increased assembly rates. The differences in dynamics when comparing HBV and human Cp149-Y132A as well as the differences in dynamics when comparing the HBV and WHV Cps allowed us to map an allosteric network within the HBV dimer. Through a careful comparison of structure, stability, and dynamics using four different capsid protein dimers, we conclude that protein subunit dynamics regulate HBV capsid assembly.
| S-EPMC8011827 | biostudies-literature