Termination of convulsion seizures by destabilizing and perturbing seizure memory engrams.

Epileptogenesis, arising from alterations in synaptic strength, shares mechanistic and phenotypic parallels with memory formation. However, direct evidence supporting the existence of seizure memory remains scarce. Leveraging a conditioned seizure memory (CSM) paradigm, we found that CSM enabled the environmental cue to trigger seizure repetitively, and activating cue-responding engram cells could generate CSM artificially. Moreover, cue exposure initiated an analogous process of memory reconsolidation driven by mammalian target of rapamycin-brain-derived neurotrophic factor signaling. Pharmacological targeting of the mammalian target of rapamycin pathway within a limited time window reduced seizures in animals and interictal epileptiform discharges in patients with refractory seizures. Our findings reveal a causal link between seizure memory engrams and seizures, which leads us to a deeper understanding of epileptogenesis and points to a promising direction for epilepsy treatment.

Internal carotid artery stenosis: hemodynamics in the ipsilateral ACA affects CT angiography manifestations.

Objective: This study aimed to evaluate whether CT angiography (CTA) manifestations in anterior cerebral artery a1 segment (A1) were related to the hemodynamics in patients with internal carotid artery stenosis (ICAS). Methods: A total of 97 cases were selected. The degree of ICAS and symmetry of A1 were evaluated by CTA examination. Hemodynamic indexes were detected by transcranial Doppler (TCD). The differences in CTA presentations of A1 and hemodynamics between the vessels on the stenotic and contralateral sides were analyzed according to the different degrees of stenosis. The degree of ICAS according to the different manifestations of A1 and the hemodynamics of A1's adjacent vessels were also analyzed. Results: In the case of unilateral ICAS, the difference in Vm of A1 between the stenotic and the contralateral side was the most significant relative to the stenosis degree. When unilateral ICAS was ≥70%, the presentation of A1 on the stenotic side was more slender or non-visualized compared to that on the contralateral side, while in cases with unilateral stenosis <70% or bilateral stenosis with a similar degree of stenosis, A1 were mainly symmetrical. When A1 on the side of ICAS was slender or non-visualized, the Vm of A1 was significantly slower than that on the contralateral side (P < 0.001). Conclusion: The CTA manifestations of A1 on the side of ICAS embodied the overall changes of the intracranial hemodynamics after ICAS. A combination of TCD and CTA examination of A1 can assist in judging the location and degree of ICAS.

Transcriptome profiling and bioinformatic analysis of the effect of ganoderic acid T prevents Sendai virus infection.

Ganoderic acid T (GA-T) is an important triterpene of Ganoderma lucidum, which is utilized to treat viral infections. Sendai virus (SeV) is widely studied to determine the molecular biological characteristics of RNA viruses and employed to elucidate the mechanisms governing the innate immune response. However, the comprehensive mechanism governing the antiviral effects of GA-T against SeV infection remains unknown. In this study, SeV-infected host cells were treated with 16.3 µM GA-T, subsequently RNA-seq analysis was performed to screen the differentially expressed genes (DEGs). The RNA-seq data showed that GA-T treatment upregulated 934 DEGs and downregulated 1283 DEGs against viral infection, in particularly, IFNGR1, IL1A, and IL1R1 were upregulated, and mTOR, SMAD3, IFNL2 and IFNL3 were decreased. GO and KEGG analysis illustrated that DEGs were clustered in mTOR and IL-17 signalling pathways. Protein-protein interaction network analysis indicated the high degree of nodes, such as CXCL8, CSF2, CXCL1 and MYD88. Our results indicated that GA-T exerted its antiviral pharmacological effects through inhibition of the mTOR signalling pathway and adjustment of innate immunity system and the inflammatory response involving the IL-17 signalling pathway. Our results may help to elucidate the potential functions and underlying mechanisms governing the antiviral effects of GA-T.

Efficient production and characterization of immunogenic HEV-PCV2 chimeric virus-like particles.

Zoonotic hepatitis E virus (HEV) infection is an emerging global public health concern. It is usually transmitted to humans from domestic pigs (main host). Since virus-like particles (VLPs) exhibit unique structural and immunological characteristics that make them of momentous applications in vaccine development, the purpose of the present study was the production of immunogenic chimeric VLPs as vaccine candidates for the control of zoonotic HEV in its main host and the prevention of porcine circovirus associated disease, a multi-factorial disease with major economic repercussions on global pig industry. An immuno-informatics approach was applied for the design and screening of new chimeric antigens presenting the dominant immunogenic domains of both HEV and porcine circovirus 2 (PCV2). Then, using molecular cloning techniques, the chimeric proteins were expressed in Escherichia coli. After purification, full characterization of the physicochemical, morphological, and immunological properties of the target proteins has been conducted. The chimeric immunogens were successfully overexpressed and after the optimization of the expression conditions, 5 chimeric proteins were efficiently purified under native conditions. The purified HEV-PCV2 chimeric proteins were found thermo-stable and able to self-assemble into spherical virus-like particles. Four HEV-PCV2 chimeric proteins have displayed optimal antigenicity and immunogenicity properties, with the nPCV2cp-p166 chimeric immunogen slightly outranking the other designed proteins. In conclusion, this study reports the production of stable HEV-PCV2 chimeric VLPs that exhibited optimal antigenicity and immunogenicity and thus with potential applications in diagnostics and vaccine development. Besides, this study provides a reproducible approach for the design, assessment, and production of chimeric antigens.

Hepatitis E Virus Capsid as a Carrier of Exogenous Antigens for the Development of Chimeric Virus-Like Particles.

INTRODUCTION: Virus-like particles (VLPs), self-assembled multiprotein structures, can stimulate robust immune responses due to their structural similarity to native virions that allow the presentation of multiple copies of the target epitopes. Utilizing VLPs as vaccine platforms to present exogenous antigens is a promising and challenging approach in the vaccine development field. This study investigates the potential of the truncated hepatitis E virus (HEV) capsid as a VLP platform to present foreign antigens. METHODS: The S and M domains of the HEV capsid protein were selected as the optimal carrier (CaSM). The exogenous antigen Seq8 containing 3 neutralizing epitopes from 3 different foot-and-mouth disease virus (FMDV) strains was linked to the C-terminal of CaSM to construct a chimeric VLP (CaSM-Seq8). The chimeric particles were produced in Escherichia coli, and their morphology, physicochemical properties, antigenicity, and immunogenicity were analyzed. RESULTS: Morphological analysis showed that CaSM-Seq8 self-assembled into VLPs similar to CaSM VLPs (∼26 nm in diameter) but smaller than native HEV virions. Further, the thermal stability and the resistance to enzymatic proteolysis of Seq8 were enhanced when it was attached to the CaSM carrier. The antigenicity analysis revealed a more robust reactivity against anti-FMDV antibodies when Seq8 was presented on CaSM particles. Upon injection into mice, FMDV-specific IgGs induced by CaSM-Seq8 appeared earlier, increased faster, and maintained higher levels for a longer time than those induced by Seq8 alone or the inactivated FMDV vaccine. CONCLUSION: This study demonstrated the potential of utilizing the truncated HEV capsid as an antigen-presenting platform for the development of chimeric VLP immunogens.

Sult2b1 deficiency exacerbates ischemic stroke by promoting pro-inflammatory macrophage polarization in mice.

Rationale: Stroke is a leading causes of human death worldwide. Ischemic damage induces the sterile neuroinflammation, which directly determines the recovery of patients. Lipids, a major component of the brain, significantly altered after stroke. Cholesterol sulfate, a naturally occurring analog of cholesterol, can directly regulate immune cell activation, indicating the possible involvement of cholesterol metabolites in neuroinflammation. Sulfotransferase family 2b member 1 (Sult2b1) is the key enzyme that catalyzes the synthesis of cholesterol sulfate. This study aimed to investigate the function of Sult2b1 and cholesterol sulfate in the neuroinflammation after ischemic stroke. Methods and Results: Sult2b1-/- and wild-type mice were subjected to transient middle cerebral artery occlusion. Our data showed that Sult2b1-/- mice had larger infarction and worse neurological scores. To determine whether immune cells were involved in the worsening stroke outcome in Sult2b1-/- mice, bone marrow transplantation, immune cell depletion, and adoptive monocyte transfer were performed. Combined with CyTOF and immunofluorescence techniques, we demonstrated that after stroke, the peripheral monocyte-derived macrophages were the dominant cell type promoting the pro-inflammatory status in Sult2b1-/-mice. Using primary bone marrow-derived macrophages, we showed that cholesterol sulfate could attenuate the pro-inflammatory polarization of macrophages under both normal and oxygen-glucose deprivation conditions by regulating the levels of nicotinamide adenine dinucleotide phosphate (NADPH), reactive oxygen species (ROS), and activating the AMP-activated protein kinase (AMPK) - cAMP responsive element-binding protein (CREB) signaling pathway. Conclusions:Sult2b1-/- promoted the polarization of macrophages into pro-inflammatory status. This trend could be attenuated by adding cholesterol sulfate, which promotes the polarization of macrophages into anti-inflammatory status by metabolic regulation. In this study, we established an inflammation-metabolism axis during the macrophage polarization after ischemic stroke.

Cerebral Hemodynamic Evaluation of Main Cerebral Vessels in Epileptic Patients Based on Transcranial Doppler.

Objective: To study whether there is a difference in peak and mean blood flow velocity between the left and right major cerebral vessels in patients with epilepsy. Methods: Sixteen patients with epilepsy underwent FDG18-PET-CT (PET) scan and electroencephalogram (EEG) examinations. Transcranial Doppler (TCD) was used to detect the peak flow velocity (PFV), mean flow velocity (MFV), and other hemodynamic indicators of bilateral anterior, middle, and posterior cerebral arteries in each patient. According to different patterns of the PET or interictal EEG, the differences in PFV, and MFV of corresponding vessels on both sides under different patterns were compared. Results: According to the PET of the low-metabolism region corresponding to the supplying artery, the PFV and MFV of the supplying artery in the low-metabolism region were lower than the value of the corresponding contralateral vessel. The PFV and MFV on the low metabolic side of PET were lower than that of the corresponding vessels on the opposite side. The PFV and MFV on the discharge side of interictal EEG were also lower than the PFV and MFV of the corresponding vessels on the opposite side. The MFV of posterior cerebral artery on the low metabolic side of PET or the interictal discharge side was significantly different from that of the contralateral vessels (P < 0.05). However, the other aforementioned differences in PFV and MFV did not achieve statistical significance. Conclusion: In epileptic patients, the PFV and MFV of main cerebral vessels on the PET hypometabolized side or the interictal discharge side was lower than that of corresponding vessels on the contralateral side. To some extent, the difference in the MFV of PCA between the bilateral sides can facilitate the lateral diagnosis of the epileptogenic zone.

Systematic Study of Immune Cell Diversity in ischemic postconditioning Using High-Dimensional Single-Cell Analysis with Mass Cytometry.

Ischemic postconditioning (IPostC) is a concept of ischemic stroke treatment, in which several cycles of brief reocclusion after reperfusion are repeated. It is essential to have an accurate understanding of the immune response in IPostC. By using high parametric single-cell mass cytometry, immune cell subsets and characterize their unique functions from ischemic brain and peripheral blood were identified after IPostC. This study enabled us to better understand the immune cell phenotypical and functional characteristics in ischemic brain and peripheral blood at the single-cell and protein levels. Since some cell surface markers can serve as functional markers, reflecting the degree of inflammation, the cell surface marker intensity among different groups was analyzed. The results showed that downregulation of 4E-BP1 and p38 of Microglia and MoDM in the ischemic brain was involved in IPostC-induced protection. In the peripheral blood, downregulation of P38 of CD4 T cell and Treg has also participated in IPostC-induced protection.

Chitosan Nanoparticles Loaded with Truncated ORF2 Protein as an Oral Vaccine Candidate against Hepatitis E.

In a continuous effort to develop effective vaccines against hepatitis E (HE), oral vaccine nanoparticles using the truncated capsid protein p146 (aa460-605) are formulated and characterized. To improve the immunogenicity of p146, chitosan nanoparticles (CSNPs) are used as a mucosal delivery system. Next, the physical-chemical properties, cytotoxic effects in vitro, and immunogenicity in mice of the produced NPs are analyzed. The results show that the produced CS/p146 NPs are stable and well dispersive and display a near-spherical shape with a mean size of 200-300 nm. The findings also demonstrate high encapsulation efficiency (65-73.9%) and loading capacity (27.7-67.5%) of the formulated nanoparticles. Further, the CS/p146 NPs exhibit low cytotoxicity and an obvious sustained-release effect in vitro. Immunogenicity experiments in mice indicate that CS/p146 NPs can induce antigen-specific systemic and mucosal immune responses higher than the purified p146 do. Besides, the expression levels and mRNA transcription of Interleukin (IL)-4 in spleen cells of CS/p146 NPs-immunized mice are higher than those of p146, indicating that a Th2-mediated cellular immune response is activated by the CS/p146 NPs. Overall, the synthesized CS/p146 NPs display promising properties as a potential HE oral vaccine candidate.

In silico identification of strong binders of the SARS-CoV-2 receptor-binding domain.

The world is currently witnessing the spread of the deadly severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the coronavirus disease 2019 (COVID-19). In less than three months since the first cases were reported, the World Health Organization declared it a pandemic disease. Although several treatment and prevention strategies are currently under investigation, a continuous effort to investigate and develop effective cures is urgently needed. Thus, we performed molecular docking and structure-based virtual screening of libraries of approved drugs, antivirals, inhibitors of protein-protein interactions, and one million other small molecules to identify strong binders of the SARS-CoV-2 receptor-binding domain (RBD) that might interfere with the receptor recognition process, so as to inhibit the viral cellular entry. According to our screening and selection criteria, three approved antivirals (elbasvir, grazoprevir, and sovaprevir) and 4 other drugs (hesperidin, pamaqueside, diosmin, and sitogluside) were identified as potent binders of the RBD. The binding of these molecules involved several RBD residues required for the interaction of the virus with its cellular receptor. Furthermore, this study also discussed the pharmacological action of the 4 non-antiviral drugs on hematological and neurological disorders that, in addition to inhibiting the viral entry, could be beneficial against the neurological disorders identified in COVID-19 patients. Besides, six other small-molecules were identified, with no pharmacological description so far, exhibiting strong binding affinities to the RBD that we believe worth being investigated as inhibitors of the SARS-CoV-2-receptor interaction.

Design and development of a chimeric vaccine candidate against zoonotic hepatitis E and foot-and-mouth disease.

BACKGROUND: Zoonotic hepatitis E virus (HEV) infection emerged as a serious threat in the industrialized countries. The aim of this study is exploring a new approach for the control of zoonotic HEV in its main host (swine) through the design and development of an economically interesting chimeric vaccine against HEV and against a devastating swine infection: the foot-and-mouth disease virus (FMDV) infection. RESULTS: First, we adopted a computational approach for rational and effective screening of the different HEV-FMDV chimeric proteins. Next, we further expressed and purified the selected chimeric immunogens in Escherichia coli (E. coli) using molecular cloning techniques. Finally, we assessed the antigenicity and immunogenicity profiles of the chimeric vaccine candidates. Following this methodology, we designed and successfully produced an HEV-FMDV chimeric vaccine candidate (Seq 8-P222) that was highly over-expressed in E. coli as a soluble protein and could self-assemble into virus-like particles. Moreover, the vaccine candidate was thermo-stable and exhibited optimal antigenicity and immunogenicity properties. CONCLUSION: This study provides new insights into the vaccine development technology by using bioinformatics for the selection of the best candidates from larger sets prior to experimentation. It also presents the first HEV-FMDV chimeric protein produced in E. coli as a promising chimeric vaccine candidate that could participate in reducing the transmission of zoonotic HEV to humans while preventing the highly contagious foot-and-mouth disease in swine.

Role of the GTNGTKR motif in the N-terminal receptor-binding domain of the SARS-CoV-2 spike protein.

The 2019 novel coronavirus disease (COVID-19) that emerged in China has been declared as public health emergency of international concern by the World Health Organization and the causative pathogen was named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this report, we analyzed the structural characteristics of the N-terminal domain of the S1 subunit (S1-NTD) of the SARS-CoV-2 spike protein in comparison to the SARS-CoV in particular, and to other viruses presenting similar characteristic in general. Given the severity and the wide and rapid spread of the SARS-CoV-2 infection, it is very likely that the virus recognizes other receptors/co-receptors besides the ACE2. The NTD of the SARS-CoV-2 contains a receptor-binding motif different from that of SARS-CoV, with some insertions that could confer to the new coronavirus new receptor binding abilities. In particular, motifs similar to the insertion 72GTNGTKR78 have been found in structural proteins of other viruses; and these motifs were located in putative regions involved in recognizing protein and sugar receptors, suggesting therefore that similar binding abilities could be displayed by the SARS-CoV-2 S1-NTD. Moreover, concerning the origin of these NTD insertions, our findings point towards an evolutionary acquisition rather than the hypothesis of an engineered virus.

Role of the C-terminal cysteines in virus-like particle formation and oligomerization of the hepatitis E virus ORF2 truncated proteins.

The hepatitis E virus (HEV) ORF2 truncated recombinant proteins can self-assemble into virus-like particles (VLPs) and were used as models to investigate the HEV capsid assembly. However, the structural function of the ORF2 C-terminal domain (C52aa from aa 608 to aa 660) remains unclear. Herein, by analyzing a set of ORF2 truncated proteins expressed in Escherichia coli, we found that the highly conserved C-terminal cysteines play a crucial role in the oligomerization of the truncated ORF2 proteins and in their assembly into VLPs, through the formation of dimer-dimer disulfide bonds; and the treatment of native HEV particles with dithiothreitol (DTT) induced the disassembly of the viral capsid, suggesting that the disulfide bonding is required for stabilizing the native HEV capsid. The present study sheds light on the structural role of the C-terminal region of the HEV capsid protein and contributes to the full understating of the viral capsid assembly process.

Design and immunogenicity analysis of the combined vaccine against zoonotic hepatitis E and foot-and-mouth disease.

AIM: Design and immunogenicity assessment of the combined vaccine candidate against zoonotic hepatitis E virus (HEV) and foot-and-mouth disease virus (FMDV). METHODS: Using the molecular cloning technology, we produced and purified 9 HEV ORF2-truncated proteins (HEV genotype 4). Then, we compared their thermal stability, antigenicity, and immunogenicity to select the best HEV immunogen. Next, we used the adjuvant Montanide ISA-206 to prepare different formulations of HEV vaccine alone, FMDV vaccine alone and HEV-FMDV combined vaccine. The formulations were injected into mice and the induced humoral immune responses were monitored up 12 weeks post-immunization. RESULTS: The HEV p222 protein could self-assemble into VLPs (∼34 nm) and showed higher stability and better antigenicity/immunogenicity than the other HEV antigens, thus it was selected as the best HEV immunogen. Mice immunization with the FMDV vaccine alone induced high FMDV-specific antibody titers in a dose-dependent manner; the HEV p222 protein also induced high levels of anti-HEV antibodies but in a dose-independent manner. The HEV-FMDV combination induced anti-FMDV antibody titers 7-16-fold higher than the titers induced by the FMDV vaccine alone, and HEV-specific antibody titers 2.4-fold higher than those induced by the HEV p222 antigen alone. CONCLUSION: Herein, we proposed a new approach for the control of zoonotic HEV infection through its control in its main host (pig). We also designed the first HEV-FMDV combined vaccine and the preliminary analyses revealed a synergistic effect on the immunogenicity of both HEV and FMDV antigens.

Comprehensive analysis of genetic and evolutionary features of the hepatitis E virus.

BACKGROUND: The hepatitis E virus (HEV) is the causative pathogen of hepatitis E, a global public health concern. HEV comprises 8 genotypes with a wide host range and geographic distribution. This study aims to determine the genetic factors influencing the molecular adaptive changes of HEV open reading frames (ORFs) and estimate the HEV origin and evolutionary history. RESULTS: Sequences of HEV strains isolated between 1982 and 2017 were retrieved and multiple analyses were performed to determine overall codon usage patterns, effects of natural selection and/or mutation pressure and host influence on the evolution of HEV ORFs. Besides, Bayesian Coalescent Markov Chain Monte Carlo (MCMC) Analysis was performed to estimate the spatial-temporal evolution of HEV. The results indicated an A/C nucleotide bias and ORF-dependent codon usage bias affected mainly by natural selection. The adaptation of HEV ORFs to their hosts was also ORF-dependent, with ORF1 and ORF2 sharing an almost similar adaptation profile to the different hosts. The discriminant analysis based on the adaptation index suggested that ORF1 and ORF3 could play a pivotal role in viral host tropism. CONCLUSION: In this study, we estimate that the common ancestor of the modern HEV strains emerged ~ 6000 years ago, in the period following the domestication of pigs. Then, natural selection played the major role in the evolution of the codon usage of HEV ORFs. The significant adaptation of ORF1 of genotype 1 to humans, makes ORF1 an evolutionary indicator of HEV host speciation, and could explain the epidemic character of genotype 1 strains in humans.

DGKζ Plays Crucial Roles in the Proliferation and Tumorigenicity of Human glioblastoma.

Glioblastoma is one of the most malignant brain cancers in adults, and it is a fatal disease because of its untimely pathogenetic location detection, infiltrative growth, and unfavorable prognosis. Unfortunately, multimodal treatment with maximal safe resection, chemotherapy and radiation has not increased the survival rate of patients with glioblastoma. Gene- and molecular-targeted therapy is considered to be a promising anticancer strategy for glioblastoma. The identification of novel potential targets in glioblastoma is of high importance. In this study, we found that both the mRNA and protein levels of diacylglycerol kinase ζ (DGKζ) were significantly higher in glioblastoma tissues than in precancerous lesions. The silencing of DGKζ by lentivirus-delivered shRNA reduced glioblastoma cell proliferation and induced G0/G1 phase arrest. Moreover, knockdown of DGKζ expression in U251 cells markedly reduced in vitro colony formation and in vivo tumorigenic capability. Further study showed that DGKζ inhibition resulted in decreases in cyclin D1, p-AKT and p-mTOR. Moreover, the rescue or overexpression of DGKζ in glioblastoma cells demonstrated the oncogenic function of DGKζ. In conclusion, these studies suggest that the suppression of DGKζ may inhibit the tumor growth of glioblastoma cells with high DGKζ expression. Thus, DGKζ might be a potential therapeutic target in malignant glioblastoma.

Establishment of competitive binding assay to detect and differentiate hepatitis E virus infection.

INTRODUCTION AND OBJECTIVES: This study was undertaken to demonstrate a promising approach for detection and differentiation the serum immunoglobulin G (IgG) against hepatitis E virus (anti-HEV IgG) using a competitive binding assay established with known genotype-specific monoclonal antibodies (mAbs) 2B1 and 4C5. MATERIALS AND METHODS: The mAb 2B1 derived from genotype 1 hepatitis E virus (HEV) antigen and specifically reacted with genotype 1, 2 antigens; 4C5 induced by genotype 4 HEV antigen was specific to genotypes 3, 4 antigens. The 2B1 and 4C5 were labeled with Horseradish peroxidase (HRP), respectively. Subsequently, the titers of coated antigens and HRP-conjugated mAbs for establishment of competitive binding assay were determined by enzyme linked immunosorbent assay (ELISA). And then, the competitive binding assay was performed to assess the inhibition percentage of mAbs binding to antigens inhibited by different genotypes anti-HEV IgG. RESULTS: The results of competitive binding assay revealed that genotype 1 anti-HEV IgG could inhibit the binding of mAb 2B1 to genotype 1 antigen more strongly than that of mAb 4C5 to genotype 4 antigen. Whereas, the genotype 3 or 4 anti-HEV IgG could inhibit the binding of mAb 4C5 to genotype 4 antigen more remarkably than that of mAb 2B1 to genotype 1 antigen. CONCLUSIONS: These findings provided us a valuable approach for detection and differentiation the HEV infection derived from genotypes 1, 2 (human) or genotypes 3, 4 (zoonosis).

Author Correction: Dimerization: a structural feature for the protection of hepatitis E virus capsid protein against trypsinization.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Dimerization: a structural feature for the protection of hepatitis E virus capsid protein against trypsinization.

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.

Effects of mRNA secondary structure on the expression of HEV ORF2 proteins in Escherichia coli.

BACKGROUND: Viral protein expression in Escherichia coli (E. coli) is a powerful tool for structural/functional studies as well as for vaccine and diagnostics development. However, numerous factors such as codon bias, mRNA secondary structure and nucleotides distribution, have been indentified to hamper this heterologous expression. RESULTS: In this study, we combined computational and biochemical methods to analyze the influence of these factors on the expression of different segments of hepatitis E virus (HEV) ORF 2 protein and hepatitis B virus surface antigen (HBsAg). Three out of five HEV antigens were expressed while all three HBsAg fragments were not. The computational analysis revealed a significant difference in nucleotide distribution between expressed and non-expressed genes; and all these non-expressing constructs shared similar stable 5'-end mRNA secondary structures that affected the accessibility of both Shine-Dalgarno (SD) sequence and start codon AUG. By modifying the 5'-end of HEV and HBV non-expressed genes, there was a significant increase in the total free energy of the mRNA secondary structures that permitted the exposure of the SD sequence and the start codon, which in turn, led to the successful expression of these genes in E. coli. CONCLUSIONS: This study demonstrates that the mRNA secondary structure near the start codon is the key limiting factor for an efficient expression of HEV ORF2 proteins in E. coli. It describes also a simple and effective strategy for the production of viral proteins of different lengths for immunogenicity/antigenicity comparative studies during vaccine and diagnostics development.