Structural roles of PCV2 capsid protein N-terminus in PCV2 particle assembly and identification of PCV2 type-specific neutralizing epitope.

Postweaning multisystemic wasting disease (PMWS) in piglets caused by porcine circovirus type 2 (PCV2) is one of the major threats to most pig farms worldwide. Among all the PCV types, PCV2 is the dominant genotype causing PMWS and associated diseases. Considerable efforts were made to study the virus-like-particle (VLP) assembly and the specific PCV2-associated epitope(s) in order to establish the solid foundation for engineered PCV2 vaccine development. Although the N-terminal fragment including Nuclear Localization Signal (NLS) sequence seems important for recombinant PCV2 capsid protein expression and VLP assembly, the detailed structural and functional information regarding this important fragment are largely unknown. In this study, we report crystal structure of PCV2 VLP assembled from N-terminal NLS truncated PCV2 capsid protein at 2.8 Å resolution and cryo-EM structure of PCV2 VLP assembled from full-length PCV2 capsid protein at 4.1Å resolution. Our in vitro PCV2 VLP assembly results show that NLS-truncated PCV2 capsid protein only forms instable VLPs which were easily disassembled in solution, whereas full-length PCV2 capsid protein forms stable VLPs due to interaction between 15PRSHLGQILRRRP27 (α-helix) and 33RHRYRWRRKN42 (NLS-B) in a repeated manner. In addition, our results also showed that N-terminal truncation of PCV2 capsid protein up to 27 residues still forms PCV2 particles in solution with similar size and immunogenicity, while N-terminal truncation of PCV2 capsid protein with more than 30 residues is not able to form stable PCV2 particles in solution, demonstrating the importance of interaction between the α-helix at N-terminal and NLS-B in PCV2 VLP formation. Moreover, we also report the cryo-EM structure of PCV2 VLP in complex with 3H11-Fab, a PCV2 type-specific neutralizing antibody, at 15 Å resolution. MAb-3H11 specifically recognizes one exposed epitope located on the VLP surface EF-loop (residues 128-143), which is further confirmed by PCV1-PCV2 epitope swapping assay. Hence, our results have revealed the structural roles of N-terminal fragment of PCV2 capsid protein in PCV2 particle assembly and pinpointed one PCV2 type-specific neutralizing epitope for the first time, which could provide clear clue for next generation PCV2 vaccine and diagnostic kits development.

Structural insights into Drosophila-C3PO complex assembly and 'Dynamic Side Port' model in substrate entry and release.

In Drosophila and human, component 3 promoter of RISC (C3PO), a heteromeric complex, enhances RISC assembly and promotes RISC activity. Here, we report crystal structure of full-length Drosophila C3PO (E126Q), an inactive C3PO mutant displaying much weaker RNA binding ability, at 2.1 Å resolution. In addition, we also report the cryo-EM structures of full-length Drosophila C3PO (E126Q), C3PO (WT) and SUMO-C3PO (WT, sumo-TRAX + Translin) particles trapped at different conformations at 12, 19.7 and 12.8 Å resolutions, respectively. Crystal structure of C3PO (E126Q) displays a half-barrel architecture consisting of two Trax/Translin heterodimers, whereas cryo-EM structures of C3PO (E126Q), C3PO (WT) and SUMO-C3PO (WT) adopt a closed football-like shape with a hollow interior cavity. Remarkably, both cryo-EM structures of Drosophila C3PO (E126Q) and Drosophila SUMO-C3PO (WT) particles contain a wide side port (∼25 Å × âˆ¼30 Å versus ∼15 Å × âˆ¼20 Å) for RNA substrate entry and release, formed by a pair of anti-parallel packed long α1 helices of TRAX subunits. Notably, cryo-EM structure of SUMO-C3PO showed that four copies of extra densities belonging to N-terminal SUMO tag are located at the outside shell of SUMO-C3PO particle, which demonstrated that the stoichiometry of TRAX/Translin for the in vitro expressed and assembled full-length Drosophila-SUMO-C3PO particle is 4:4, suggesting Drosophila C3PO is composed by TRAX/translin at a ratio of 4:4. Remarkably, the comparison of the cryo-EM structures suggests that the C3PO side ports regulated by α1 helices of TRAX molecules are highly dynamic. Hence, we propose that C3PO particles could adopt a 'Dynamic Side Port' model to capture/digest nucleic acid duplex substrate and release the digested fragments through the dynamic side ports.

Prolonged suppression of HBV in mice by a novel antibody that targets a unique epitope on hepatitis B surface antigen.

OBJECTIVE: This study aimed to investigate the therapeutic potential of monoclonal antibody (mAb) against HBV as a novel treatment approach to chronic hepatitis B (CHB) in mouse models. METHODS: Therapeutic effects of mAbs against various epitopes on viral surface protein were evaluated in mice mimicking persistent HBV infection. The immunological mechanisms of mAb-mediated viral clearance were systematically investigated. RESULTS: Among 11 tested mAbs, a novel mAb E6F6 exhibited the most striking therapeutic effects in several HBV-persistent mice. Single-dose administration of E6F6 could profoundly suppress the levels of hepatitis B surface antigen (HBsAg) and HBV DNA for several weeks in HBV-transgenic mice. E6F6 regimen efficiently prevented initial HBV infection, and reduced viral dissemination from infected hepatocytes in human-liver-chimeric mice. E6F6-based immunotherapy facilitated the restoration of anti-HBV T-cell response in hydrodynamic injection (HDI)-based HBV carrier mice. Immunological analyses suggested that the Fcγ receptor-dependent phagocytosis plays a predominant role in E6F6-mediated viral suppression. Molecular analyses suggested that E6F6 recognises an evolutionarily conserved epitope (GPCK(R)TCT) and only forms a smaller antibody-viral particle immune complex with limited interparticle crosslinking when it binds to viral particles. This unique binding characteristic of E6F6 to HBV was possibly associated with its effective in vivo opsonophagocytosis for viral clearance. CONCLUSIONS: These results provided new insight into understanding the therapeutic role and mechanism of antibody against persistent viral infection. The E6F6-like mAbs may provide a novel immunotherapeutic agent against human chronic HBV infection.

Large-scale production of foot-and-mouth disease virus (serotype Asia1) VLP vaccine in Escherichia coli and protection potency evaluation in cattle.

BACKGROUND: Foot-and-mouth disease (FMD) is an acute, highly contagious disease that infects cloven-hoofed animals. Vaccination is an effective means of preventing and controlling FMD. Compared to conventional inactivated FMDV vaccines, the format of FMDV virus-like particles (VLPs) as a non-replicating particulate vaccine candidate is a promising alternative. RESULTS: In this study, we have developed a co-expression system in E. coli, which drove the expression of FMDV capsid proteins (VP0, VP1, and VP3) in tandem by a single plasmid. The co-expressed FMDV capsid proteins (VP0, VP1, and VP3) were produced in large scale by fermentation at 10 L scale and the chromatographic purified capsid proteins were auto-assembled as VLPs in vitro. Cattle vaccinated with a single dose of the subunit vaccine, comprising in vitro assembled FMDV VLP and adjuvant, developed FMDV-specific antibody response (ELISA antibodies and neutralizing antibodies) with the persistent period of 6 months. Moreover, cattle vaccinated with the subunit vaccine showed the high protection potency with the 50 % bovine protective dose (PD50) reaching 11.75 PD50 per dose. CONCLUSIONS: Our data strongly suggest that in vitro assembled recombinant FMDV VLPs produced from E. coli could function as a potent FMDV vaccine candidate against FMDV Asia1 infection. Furthermore, the robust protein expression and purification approaches described here could lead to the development of industrial level large-scale production of E. coli-based VLPs against FMDV infections with different serotypes.

Insights into the inhibition of protospacer integration via direct interaction between Cas2 and AcrVA5.

Spacer acquisition step in CRISPR-Cas system involves the recognition and subsequent integration of protospacer by the Cas1-Cas2 complex in CRISPR-Cas systems. Here we report an anti-CRISPR protein, AcrVA5, and reveal the mechanisms by which it strongly inhibits protospacer integration. Our biochemical data shows that the integration by Cas1-Cas2 was abrogated in the presence of AcrVA5. AcrVA5 exhibits low binding affinity towards Cas2 and acetylates Cas2 at Lys55 on the binding interface of the Cas2 and AcrVA5 N-terminal peptide complex to inhibit the Cas2-mediated endonuclease activity. Moreover, a detailed structural comparison between our crystal structure and homolog structure shows that binding of AcrVA5 to Cas2 causes steric hindrance to the neighboring protospacer resulting in the partial disassembly of the Cas1-Cas2 and protospacer complex, as demonstrated by electrophoretic mobility shift assay. Our study focuses on this mechanism of spacer acquisition inhibition and provides insights into the biology of CRISPR-Cas systems.

Biochemical and structural insights into an Ochrobactrum sp. CSL1 ribose-5-phosphate isomerase A and its roles in isomerization of rare sugars.

Rare sugars have received increasing attention due to their important applications as sweeteners and building blocks. The substrate specificity and catalytic properties of ribose-5-phosphate isomerase A (RpiA) in isomerization of rare sugars have not been extensively explored. In this study, an RpiA from Ochrobactrum sp. CSL1 was cloned and expressed in Escherichia coli. The biochemical and reaction features were explored and its broad substrate specificity was identified. A higher reaction rate in isomerizing l-rhamnose to l-rhamnulose by OsRpiA, compared with OsRpiB found in the same strain indicated higher efficiency in preparing rare sugars, which was verified by kinetics study. The 2.8 Šresolution structure of OsRpiA was then solved and used in subsequent molecular dynamics experiments, providing a possible explanation for its distinct substrate specificity. The present study highlighted the unique role of microbial RpiA in preparing rare sugars, and its structural information provided a reliable reference for further reaction mechanism research and enzyme engineering work.

Structural insight into the type-specific epitope of porcine circovirus type 3.

The recently identified pathogenic Porcine circovirus type 3 (PCV3) may threaten to reduce the pig population dramatically worldwide. In our previous study, a PCV3-specific monoclonal antibody (mAb-1H11) was successfully applied in immune-histochemistry staining and ELISA, which specifically recognize PCV3 capsid protein in PCV3-positive pig tissues. In the present study, we expressed and purified the soluble sole capsid protein of PCV3. The purified capsid protein was capable of self-assembly into virus-like-particles (VLPs), which is validated by transmission electron microscopy and dynamic light scattering assays. Moreover, the epitope of mAb-1H11 was identified in the CD-loop region (a.a. 72-79) on the VLP surface, which is confirmed by PCV2-PCV3 epitope swapping assay. For the first time, we determined the cryo-EM structure of PCV3-VLP at 8.5 Å resolution that reveals the detailed structural information of PCV3-VLP. In our cryo-EM structure, PCV3-VLP is composed of 60 capsid protein subunits assembled with T = 1 icosahedral symmetry. Consistent to our bio-dot Western blot assay, the structural comparison between PCV3 and PCV2 revealed significant structural differences in the surface-exposed loops, including the CD-loop (a.a. 72-79) and the EF-loop (a.a. 109-131). Our work provides a structural framework for engineering future PCV3 vaccine and diagnosis kits development.

Structural and functional analyses of hepatitis B virus X protein BH3-like domain and Bcl-xL interaction.

Hepatitis B virus (HBV) X protein, HBx, interacts with anti-apoptotic Bcl-2 and Bcl-xL proteins through its BH3-like motif to promote HBV replication and cytotoxicity. Here we report the crystal structure of HBx BH3-like motif in complex with Bcl-xL where the BH3-like motif adopts a short α-helix to snuggle into a hydrophobic pocket in Bcl-xL via its noncanonical Trp120 residue and conserved Leu123 residue. This binding pocket is ~2 Å away from the canonical BH3-only binding pocket in structures of Bcl-xL with proapoptotic BH3-only proteins. Mutations altering Trp120 and Leu123 in HBx impair its binding to Bcl-xL in vitro and HBV replication in vivo, confirming the importance of this motif to HBV. A HBx BH3-like peptide, HBx-aa113-135, restores HBV replication from a HBx-null HBV replicon, while a shorter peptide, HBx-aa118-127, inhibits HBV replication. These results provide crucial structural and functional insights into drug designs for inhibiting HBV replication and treating HBV patients.

The Vestigial Esterase Domain of Haemagglutinin of H5N1 Avian Influenza A Virus: Antigenicity and Contribution to Viral Pathogenesis.

Initial attempts to develop monoclonal antibodies as therapeutics to resolve influenza infections focused mainly on searching for antibodies with the potential to neutralise the virus in vitro with classical haemagglutination inhibition and microneutralisation assays. This led to the identification of many antibodies that bind to the head domain of haemagglutinin (HA), which generally have potent neutralisation capabilities that block viral entry or viral membrane fusion. However, this class of antibodies has a narrow breadth of protection in that they are usually strain-specific. This led to the emphasis on stalk-targeting antibodies, which are able to bind a broad range of viral targets that span across different influenza subtypes. Recently, a third class of antibodies targeting the vestigial esterase (VE) domain have been characterised. In this review, we describe the key features of neutralising VE-targeting antibodies and compare them with head- and stalk-class antibodies.

N-terminal truncations on L1 proteins of human papillomaviruses promote their soluble expression in Escherichia coli and self-assembly in vitro.

Human papillomavirus (HPV) is the causative agent in genital warts and nearly all cervical, anogenital, and oropharyngeal cancers. Nine HPV types (6, 11, 16, 18, 31, 33, 45, 52, and 58) are associated with about 90% of cervical cancers and 90% of genital warts. HPV neutralization by vaccine-elicited neutralizing antibodies can block viral infection and prevent HPV-associated diseases. However, there is only one commercially available HPV vaccine, Gardasil 9, produced from Saccharomyces cerevisiae that covers all nine types, raising the need for microbial production of broad-spectrum HPV vaccines. Here, we investigated whether N-terminal truncations of the major HPV capsid proteins L1, improve their soluble expression in Escherichia coli. We found that N-terminal truncations promoted the soluble expression of HPV 33 (truncated by 10 amino acids [aa]), 52 (15 aa), and 58 (10 aa). The resultant HPV L1 proteins were purified in pentamer form and extensively characterized with biochemical, biophysical, and immunochemical methods. The pentamers self-assembled into virus-like particles (VLPs) in vitro, and 3D cryo-EM reconstructions revealed that all formed T = 7 icosahedral particles having 50-60-nm diameters. Moreover, we formulated a nine-valent HPV vaccine candidate with aluminum adjuvant and L1 VLPs from four genotypes used in this study and five from previous work. Immunogenicity assays in mice and non-human primates indicated that this HPV nine-valent vaccine candidate elicits neutralizing antibody titers comparable to those induced by Gardasil 9. Our study provides a method for producing a nine-valent HPV vaccine in E. coli and may inform strategies for the soluble expression of other vaccine candidates.

Production of Escherichia coli-based virus-like particle vaccine against porcine circovirus type 2 challenge in piglets: Structure characterization and protective efficacy validation.

We report the strategies leading to the large-production of soluble non-tag full-length porcine circovirus type 2 (PCV2) Cap protein in Escherichia coli. Under neutral pH condition, the purified recombinant Cap protein derived from E. coli expression self-assembles into homogenous round virus-like particle at the similar size of that of the intact PCV2 virus, which is further characterized by Cryo-EM single particle structure determined at 4.5Å. The engineered PCV2 rCap VLP was tested as a subunit vaccine for the protective efficacy against PCV2 challenge on 3-week old piglets. Similar to commercial available PCV2 vaccine, the Cap VLP-immunized piglets developed specific antibody-mediated response and were protected from the virulent SH PCV2 strain challenge. Hence, the production of E. coli based PCV2Cap-VLP could be applied as a cost-friendly and effective subunit vaccine to control PCV2 spreading in developing countries.

[Expression, purification and immunogenicity analysis of HPV type 18 virus-like particles from Escherichia coli].

Here, we presented a method to bacterially express the major structural protein L1 of Human Papillomavirus type 18 (HPV18) as soluble form. We found that the purified L1 could self-assemble to virus-like particles (VLPs). Further, we investigated the immunogenicity and the induced level of neutralizing antibody using these VLPs. First, the genome of HPV18 was cloned from a patient in Xiamen. It was used as template for PCR amplification of HPV18 L1 gene. The resultant DNA fragment was inserted into expression vector pTrxFus and expressed in Escherichia coli GI724. Second, L1 protein was purified by ammonium sulfate precipitation, ion-exchange chromatography and hydrophobic interaction chromatography; and the purified L1 was subjected to self-assembly to form VLPs with the removal of premixed reductant DTT. Finally, the size and morphology of these VLPs was investigated by Dynamic Light Scattering and Transmission Electronic Microscopy as 29.34 nm in hydrated radius and globular particles similar with native HPV18. The half effective dosage (ED50) and maximum level of neutralizing antibody elicitation were measured by vaccinations on mice, rabbit and goat using pseudovirus neutralization cell model. The results showed that the ED50 of HPV18 VLPs is 0.006 microg in mice, and the maximum titer of neutralizing antibody elicited in rabbit and goat is up to 10(7). As a conclusion, we can provide HPV18 VLPs with highly immunogenicity from prokaryote expression system, which may pave a new way for research and development of prophylactic vaccine for HPV18.