Membrane Protein of Human Coronavirus NL63 Is Responsible for Interaction with the Adhesion Receptor.

Human coronavirus NL63 (HCoV-NL63) is a common respiratory virus that causes moderately severe infections. We have previously shown that the virus uses heparan sulfate proteoglycans (HSPGs) as the initial attachment factors, facilitating viral entry into the cell. In the present study, we show that the membrane protein (M) of HCoV-NL63 mediates this attachment. Using viruslike particles lacking the spike (S) protein, we demonstrate that binding to the cell is not S protein dependent. Furthermore, we mapped the M protein site responsible for the interaction with HSPG and confirmed its relevance using a viable virus. Importantly, in silico analysis of the region responsible for HSPG binding in different clinical isolates and the Amsterdam I strain did not exhibit any signs of cell culture adaptation.IMPORTANCE It is generally accepted that the coronaviral S protein is responsible for viral interaction with a cellular receptor. Here we show that the M protein is also an important player during early stages of HCoV-NL63 infection and that the concerted action of the two proteins (M and S) is a prerequisite for effective infection. We believe that this study broadens the understanding of HCoV-NL63 biology and may also alter the way in which we perceive the first steps of cell infection with the virus. The data presented here may also be important for future research into vaccine or drug development.

Immunological Properties of Protein-Polymer Nanoparticles.

The covalent attachment of polymers to the surface of proteins and nanoparticles has been widely employed in the development of biomedical platforms capable of delaying or diminishing immune surveillance. The most widely employed polymer for these applications has been poly(ethylene glycol) (PEG), yet recent evidence has suggested that other polymer architectures and compositions provide significantly better in vitro and in vivo properties of protein-polymer hybrid materials. Moreover, few direct comparisons of PEG to these polymers have been reported. Here we describe the assembly and characterization of a series of polymer conjugates of a representative immunogenic viruslike particle (VLP) using (poly(oligo(ethylene glycol) methacrylate), poly(methacrylamido glucopyranose), and PEG, and an investigation of their ability to shield the protein from antibody recognition as a function of polymer loading density, chain length, architecture, and conjugation site. Increasing chain length and loading density were both found to significantly diminish antibody recognition of the VLP conjugates; the conformation adopted by different polymer architectures was also found to greatly influence antibody recognition. A direct comparison of these conjugates to PEGylated VLPs in vivo showed that all formulations gave rise to similar antibody titers that were significantly diminished relative to unmodified particles. Interestingly, the quality of the antibody response was impacted by the properties of the conjugate, with differences in observed affinity and avidity suggesting a complex dependence on loading density, chain length, and architecture.

Tellurium/Bovine Serum Albumin Nanocomposites Inducing the Formation of Stress Granules in a Protein Kinase R-Dependent Manner.

The effect of nanoparticles (NPs) on cellular stress responses is important to the understanding of nanotoxicities and developing safe therapies. Although the relationship between NPs and cellular stress responses has been preliminarily investigated, stress responses to NPs remain unclear. Here, tellurium/bovine serum albumin (Te/BSA) nanocomposites were prepared using sodium tellurite, BSA, and glutathione as precursors. The as-prepared Te/BSA nanocomposites, with particle size similar to that of many viruses, are found to induce the formation of stress granules (SGs), a kind of cytoplasmic RNA granule formed under various stresses. The SGs in Te/BSA nanocomposite-treated cells are composed of T-cell internal antigen 1 (TIA1), TIA1-related protein, and eukaryotic initiation factor 3η. Using chemical inhibitors and small interfering RNA-mediated silencing, protein kinase R (PKR) is identified as the α-subunit of eukaryotic initiation factor 2 (eIF2α)-kinase activated upon Te/BSA nanocomposite incubation, which is also the dominant kinase responsible for eIF2α activation under virus infection. Mechanistically, PKR is activated in a heparin-dependent manner. This study reveals a biological effect of Te/BSA nanocomposites on stress responses, providing a preliminary basis for further research on viruslike particles and the application of NPs in biology.

Development of recombinant Yarrowia lipolytica producing virus-like particles of a fish nervous necrosis virus.

Nervous necrosis virus (NNV) causes viral encephalopathy and retinopathy, a devastating disease of many species of cultured marine fish worldwide. In this study, we used the dimorphic non-pathogenic yeast Yarrowia lipolytica as a host to express the capsid protein of red-spotted grouper nervous necrosis virus (RGNNV-CP) and evaluated its potential as a platform for vaccine production. An initial attempt was made to express the codon-optimized synthetic genes encoding intact and N-terminal truncated forms of RGNNV-CP under the strong constitutive TEF1 promoter using autonomously replicating sequence (ARS)-based vectors. The full-length recombinant capsid proteins expressed in Y. lipolytica were detected not only as monomers and but also as trimers, which is a basic unit for formation of NNV virus-like particles (VLPs). Oral immunization of mice with whole recombinant Y. lipolytica harboring the ARS-based plasmids was shown to efficiently induce the formation of IgG against RGNNV-CP. To increase the number of integrated copies of the RGNNV-CP expression cassette, a set of 26S ribosomal DNA-based multiple integrative vectors was constructed in combination with a series of defective Ylura3 with truncated promoters as selection markers, resulting in integrants harboring up to eight copies of the RGNNV-CP cassette. Sucrose gradient centrifugation and transmission electron microscopy of this high-copy integrant were carried out to confirm the expression of RGNNV-CPs as VLPs. This is the first report on efficient expression of viral capsid proteins as VLPs in Y. lipolytica, demonstrating high potential for the Y. lipolytica expression system as a platform for recombinant vaccine production based on VLPs.

Modeling Ebolavirus Budding with Virus Like Particles.

About 15 years ago, several groups initially described the release of virus like particles (VLPs) upon expression of Ebola virus VP40 in mammalian cells. Further development of the protocol later allowed for the dissection of the Ebola virus budding mechanism and for the identification of critical VP40 residues involved in this process. VLPs are now produced routinely in several laboratories as a tool to study virus entry or egress and have even been proposed as vaccine candidates against Ebola virus disease. Here we described protocols for the production and the analysis of Ebola virus VLP release.

Vaccines against norovirus: state of the art trials in children and adults.

Noroviruses (NoVs), a group of nonenveloped, single-stranded RNA viruses belonging to the Caliciviridae family, are the leading cause worldwide of acute infectious gastroenteritis. Serious and eventual fatal outcomes may be observed in at-risk populations such as the very young or older adults, especially in those with underlying diseases. NoVs are highly infectious, with a low number of virus particles causing infection, and they are highly resistant to environmental conditions. NoVs have multiple routes of transmission including faecal-oral, aerosolized vomitus, person to person and via contaminated surfaces or food and water. NoVs can cause frequent and dramatic outbreaks where people congregate in close quarters such as hospitals, long-term care facilities, cruise liners and military barracks and ships. Of the seven NoV genogroups, human disease is most frequently caused by genogroups I and II, although genogroup IV has also been associated with illness. The absence of reliable, high-yield cell culture systems or animal models has steered the development of vaccines towards nonreplicating recombinant capsid proteins including viruslike particles and the sub-virus-sized P particles. Takeda Vaccines is developing a candidate NoV vaccine formulation based on adjuvanted viruslike particles from the GI.1 genotype and a consensus GII.4 sequence derived from three natural GII.4 variants. Early clinical trial results show good tolerability and robust immune responses to both components. This approach is designed to induce broad protective immune responses in adults and children.

Construction and immunological evaluation of truncated hepatitis B core particles carrying HBsAg amino acids 119-152 in the major immunodominant region (MIR).

Hepatitis B capsid protein expressed in Escherichia coli can reassemble into icosahedral particles, which could strongly enhance the immunogenicity of foreign epitopes, especially those inserted into its major immunodominant region. Herein, we inserted the entire 'α' antigenic determinant amino acids (aa) 119-152 of HBsAg into the truncated HBc (aa 1-144), between Asp(78) and Pro(79). Prokaryotic expression showed that the mosaic HBc was mainly in the form of inclusion bodies. After denaturation with urea, it was dialyzed progressively for protein renaturation. We observed that before and after renaturation, mosaic HBc was antigenic as determined by HBsAg ELISA and a lot of viruslike particles were observed after renaturation. Thus, we further purified the mosaic viruslike particles by (NH4)2SO4 precipitation, DEAE chromatography, and Sepharose 4FF chromatography. Negative staining electron microscopy demonstrated the morphology of the viruslike particles. Immunization of Balb/c mice with mosaic particles induced the production of anti-HBs antibody and Th1 cell immune response supported by ELISPOT and CD4/CD8 proportions assay. In conclusion, we constructed mosaic hepatitis core particles displaying the entire 'α' antigenic determinant on the surface and laid a foundation for researching therapeutic hepatits B vaccines.