Structural and Functional Characterizations of Cancer Targeting Nanoparticles Based on Hepatitis B Virus Capsid.
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ABSTRACT:
SUBMITTER: Heo Y
PROVIDER: S-EPMC8430771 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The hepatitis B virus (HBV) core protein (CP) forms the shell of an icosahedral nucleocapsid. In a former work, we identified 11 amino acid residues of CP exposed on the capsid surface by an alanine mutation scan as being important for capsid envelopment. We now introduced several other amino acids at six of these positions and found that almost all 27 tested point mutations at S17, K96, and I126 reproduced the phenotype of the alanine mutation (with only two exceptions): the formation of nucleocapsids and of the viral DNA genome was wild type, but capsid envelopment and virion release were strongly inhibited. This indicates that these side chains have a very specific function during nucleocapsid envelopment. We also identified several CP point mutations (e.g., F122V/S/Y and R127D/G) allowing the formation of capsids but preventing the packaging of pregenomic RNA. The envelopment of such mutant capsids was blocked. Apparently, these CP mutations hampered the recognition/packaging of the pregenome-P-protein complex by CP, a process which is still barely understood, and the mutant capsids devoid of HBV-specific nucleic acid did not express the capsid maturation signal required for envelopment.
| S-EPMC2772698 | 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:The number of synonymous mutations per synonymous site (K(s)), the number of nonsynonymous mutations per nonsynonymous site (K(a)), and the codon usage statistic (N(c)) were calculated for several hepatitis A virus (HAV) isolates. While K(s) was similar to those of poliovirus (PV) and foot-and-mouth disease virus (FMDV), K(a) was 1 order of magnitude lower. The N(c) parameter provides information on codon usage bias and decreases when bias increases. The N(c) value in HAV was about 38, while in PV and FMDV, it was about 53. The emergence of 22 rare codons in front of 8 in PV and 7 in FMDV was detected. Most of the conserved rare codons of the P1 region were strategically located at the carboxy borders of beta barrels and alpha helices, their potential function being the assurance of proper folding of the capsid proteins through a decrease in the translation speed. This strategic location was not observed for amino acids encoded by the conserved rare codons of the 3D region. The percentage of bases with low pairing number values was higher in the latter region, suggesting a role of the conserved rare codons in the maintenance of RNA structure. Many of the rare codons in HAV are among the most frequent in humans, unlike in PV or in FMDV. This fact may be explained by the lack of cellular shutoff in HAV. One hypothesis is that HAV has evolved in order to avoid competition with its host for cellular tRNAs.
| S-EPMC140588 | 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:UnlabelledHepatitis B virus (HBV) is a major human pathogen that causes serious liver disease and 600,000 deaths annually. Approved therapies for treating chronic HBV infections usually target the multifunctional viral polymerase (hPOL). Unfortunately, these therapies--broad-spectrum antivirals--are not general cures, have side effects, and cause viral resistance. While hPOL remains an attractive therapeutic target, it is notoriously difficult to express and purify in a soluble form at yields appropriate for structural studies. Thus, no empirical structural data exist for hPOL, and this impedes medicinal chemistry and rational lead discovery efforts targeting HBV. Here, we present an efficient strategy to overexpress recombinant hPOL domains in Escherichia coli, purifying them at high yield and solving their known aggregation tendencies. This allowed us to perform the first structural and biophysical characterizations of hPOL domains. Apo-hPOL domains adopt mainly α-helical structures with small amounts of β-sheet structures. Our recombinant material exhibited metal-dependent, reverse transcriptase activity in vitro, with metal binding modulating the hPOL structure. Calcomine orange 2RS, a small molecule that inhibits duck HBV POL activity, also inhibited the in vitro priming activity of recombinant hPOL. Our work paves the way for structural and biophysical characterizations of hPOL and should facilitate high-throughput lead discovery for HBV.ImportanceThe viral polymerase from human hepatitis B virus (hPOL) is a well-validated therapeutic target. However, recombinant hPOL has a well-deserved reputation for being extremely difficult to express in a soluble, active form in yields appropriate to the structural studies that usually play an important role in drug discovery programs. This has hindered the development of much-needed new antivirals for HBV. However, we have solved this problem and report here procedures for expressing recombinant hPOL domains in Escherichia coli and also methods for purifying them in soluble forms that have activity in vitro. We also present the first structural and biophysical characterizations of hPOL. Our work paves the way for new insights into hPOL structure and function, which should assist the discovery of novel antivirals for HBV.
| S-EPMC3958071 | biostudies-literature
Project description:Viruses exploit host factors and environment for their efficient replication. The virus-host interaction mechanisms for achieving an optimal hepatitis B virus (HBV) replication have been largely unknown. Here, a single cell cloning revealed that HepAD38 cells, a widely-used HBV-inducible cell line, contain cell clones with diverse permissiveness to HBV replication. The HBV permissiveness was impaired upon treatment with microtubule inhibitor nocodazole, which was identified as an HBV replication inhibitor from a pharmacological screening. In the microtubule-disrupted cells, the efficiency of HBV capsid assembly was remarkably decreased without significant change in pre-assembly process. We further found that HBV core interacted with tubulin and co-localized with microtubule-like fibriforms, but this association was abrogated upon microtubule-disassembly agents, resulting in attenuation of capsid formation. Our data thus suggest a significant role of microtubules in the efficient capsid formation during HBV replication. In line with this, a highly HBV permissive cell clone of HepAD38 cells showed a prominent association of core-microtubule and thus a high capacity to support the capsid formation. These findings provide a new aspect of virus-cell interaction for rendering efficient HBV replication.
| S-EPMC5587681 | biostudies-other
Project description:Orally-transmitted viruses have evolved in a way to resist the extreme conditions of the host's gastrointestinal environment, especially the proteolysis of their structural proteins. However, the mechanisms allowing these viruses to survive these harsh conditions remain unclear. Hepatitis E virus (HEV) is an orally-transmitted human pathogen. Its capsid protein contains three domains S, P1 and P2. The latter forms a homodimer protruding from the virus shell, making it the most exposed part. By combining biochemical and computational methods, we found the trypsin digestion sites to be highly conserved among the HEV strains. Furthermore, the constructs of the HEV capsid protein that contain an extended P2 domain were digested within the extensions leaving the P2 domain intact. The trypsinization seems to occur in three possible double cleavages at R451-R619, R460-R619 or R460-R631.The dimerization disrupts the trypsin action at three main sites in the P2 domain R542, K544 and K554. These sites are very exposed in the monomeric P2 domain constructs which makes the monomeric forms very susceptible to trypsin action. Therefore, we believe that dimerization is a structural feature that has been selected by the evolutionary forces to render the HEV capsid protein resistant to the host's proteases; an evolutionary feature that could be common to some other (if not all) orally-transmitted viruses.
| S-EPMC5788867 | biostudies-literature
Project description:Self-assembly of virus capsids is a potential target for antivirals due to its importance in the virus lifecycle. Here, we investigate the effect of phenylpropenamide derivatives B-21 and AT-130 on the assembly of hepatitis B virus (HBV) core protein. Phenylpropenamides are widely believed to yield assembly of spherical particles resembling native, empty HBV capsids. Because the details of assembly can be overlooked with ensemble measurements, we performed resistive-pulse sensing on nanofluidic devices with four pores in series to characterize the size distributions of the products in real time. With its single particle sensitivity and compatibility with typical assembly buffers, resistive-pulse sensing is well-suited for analyzing virus assembly in vitro. We observed that assembly with B-21 and AT-130 produced a large fraction of partially complete virus particles that may be on-path, off-path, or trapped. For both B-21 and AT-130, capsid assembly was more sensitive to disruption under conditions where the interprotein association energy was low at lower salt concentrations. Dilution of the reaction solutions led to the rearrangement of the incomplete particles and demonstrated that these large intermediates may be on-path, but are labile, and exist in a frustrated dynamic equilibrium. During capsid assembly, phenylpropenamide molecules modestly increase the association energy of dimers, prevent intermediates from dissociating, and lead to kinetic trapping where the formation of too many capsids has been initiated, which results in both empty and incomplete particles.
| S-EPMC6510601 | biostudies-literature
Project description:The high prevalence of hepatitis C virus (HCV) infection in the human population has triggered intensive research efforts that have led to the development of curative antiviral therapy. Moreover, HCV has become a role model to study fundamental principles that govern the replication cycle of a positive strand RNA virus. In fact, for most HCV proteins high-resolution X-ray and NMR (Nuclear Magnetic Resonance)-based structures have been established and profound insights into their biochemical and biological properties have been gained. One example is p7, a small hydrophobic protein that is dispensable for RNA replication, but crucial for the production and release of infectious HCV particles from infected cells. Owing to its ability to insert into membranes and assemble into homo-oligomeric complexes that function as minimalistic ion channels, HCV p7 is a member of the viroporin family. This review compiles the most recent findings related to the structure and dual pore/ion channel activity of p7 of different HCV genotypes. The alternative conformations and topologies proposed for HCV p7 in its monomeric and oligomeric state are described and discussed in detail. We also summarize the different roles p7 might play in the HCV replication cycle and highlight both the ion channel/pore-like function and the additional roles of p7 unrelated to its channel activity. Finally, we discuss possibilities to utilize viroporin inhibitors for antagonizing p7 ion channel/pore-like activity.
| S-EPMC4576187 | biostudies-literature
Project description:The hepatitis C virus (HCV) isolate JFH1 represents the only cloned wild-type sequence capable of efficient replication in cell culture, as well as in chimpanzees. Previous reports have pointed to the viral polymerase NS5B as a major determinant for efficient replication of this isolate. To understand the underlying mechanisms, we expressed and purified NS5B of JFH1 and of the closely related isolate J6, which replicates below the limit of detection in cell culture. The JFH1 enzyme exhibited a 5- to 10-fold-higher specific activity in vitro, consistent with the polymerase activity itself contributing to efficient replication of JFH1. The higher in vitro activity of the JFH1 enzyme was not due to increased RNA binding, elongation rate, or processivity of the polymerase but to higher initiation efficiency. By using homopolymeric and heteropolymeric templates, we found that purified JFH1 NS5B was significantly more efficient in de novo initiation of RNA synthesis than the J6 counterpart, particularly at low GTP concentrations, probably representing an important prerequisite for the rapid replication kinetics of JFH1. Furthermore, we solved the crystal structure of JFH1 NS5B, which displays a very closed conformation that is expected to facilitate de novo initiation. Structural analysis shows that this closed conformation is stabilized by a sprinkle of substitutions that together promote extra hydrophobic interactions between the subdomains "thumb" and "fingers." These analyses provide deeper insights into the initiation of HCV RNA synthesis and might help to establish more efficient cell culture models for HCV using alternative isolates.
| S-EPMC2772714 | biostudies-literature