Roles of cysteines Cys115 and Cys201 in the assembly and thermostability of grouper betanodavirus particles

The virus-like particle (VLP) assembled from capsid subunits of the dragon grouper nervous necrosis virus (DGNNV) is very similar to its native T = 3 virion. In order to investigate the effects of four cysteine residues in the capsid polypeptide on the assembly/dissociation pathways of DGNNV virions, we recombinantly cloned mutant VLPs by mutating each cysteine to destroy the specific disulfide linkage as compared with thiol reduction to destroy all S–S bonds. The mutant VLPs of C187A and C331A mutations were similar to wild-type VLPs (WT-VLPs); hence, the effects of Cys187 and Cys331 on the particle formation and thermostability were presumably negligible. Electron microscopy showed that either C115A or C201A mutation disrupted de novo VLP formation significantly. As shown in micrographs and thermal decay curves, β-mercaptoethanol-treated WT-VLPs remained intact, merely resulting in lower tolerance to thermal disruption than native WT-VLPs. This thiol reduction broke disulfide linkages inside the pre-fabricated VLPs, but it did not disrupt the appearance of icosahedrons. Small dissociated capsomers from EGTA-treated VLPs were able to reassemble back to icosahedrons in the presence of calcium ions, but additional treatment with β-mercaptoethanol during EGTA dissociation resulted in inability of the capsomers to reassemble into the icosahedral form. These results indicated that Cys115 and Cys201 were essential for capsid formation of DGNNV icosahedron structure in de novo assembly and reassembly pathways, as well as for the thermal stability of pre-fabricated particles

High Cooperativity of the SV40 Major Capsid Protein VP1 in Virus Assembly

SV40 is a small, non enveloped DNA virus with an icosahedral capsid of 45 nm. The outer shell is composed of pentamers of the major capsid protein, VP1, linked via their flexible carboxy-terminal arms. Its morphogenesis occurs by assembly of capsomers around the viral minichromosome. However the steps leading to the formation of mature virus are poorly understood. Intermediates of the assembly reaction could not be isolated from cells infected with wt SV40. Here we have used recombinant VP1 produced in insect cells for in vitro assembly studies around supercoiled heterologous plasmid DNA carrying a reporter gene. This strategy yields infective nanoparticles, affording a simple quantitative transduction assay. We show that VP1 assembles under physiological conditions into uniform nanoparticles of the same shape, size and CsCl density as the wild type virus. The stoichiometry is one DNA molecule per capsid. VP1 deleted in the C-arm, which is unable to assemble but can bind DNA, was inactive indicating genuine assembly rather than non-specific DNA-binding. The reaction requires host enzymatic activities, consistent with the participation of chaperones, as recently shown. Our results demonstrate dramatic cooperativity of VP1, with a Hill coefficient of ∼6. These findings suggest that assembly may be a concerted reaction. We propose that concerted assembly is facilitated by simultaneous binding of multiple capsomers to a single DNA molecule, as we have recently reported, thus increasing their local concentration. Emerging principles of SV40 assembly may help understanding assembly of other complex systems. In addition, the SV40-based nanoparticles described here are potential gene therapy vectors that combine efficient gene delivery with safety and flexibility

Chemical evidence for the origin of the cold water belt along the northeastern coast of Hokkaido

In the southwestern Okhotsk Sea, the cold water belt (CWB) is frequently observed on satellite images offshore of the Soya Warm Current flowing along the northeastern coast of Hokkaido, Japan, during summertime. It has been speculated that the CWB is upwelling cold water that originates from either subsurface water of the Japan Sea off Sakhalin or bottom water of the Okhotsk Sea. Hydrographic and chemical observations (nutrients, humic-type fluorescence intensity, and iron) were conducted in the northern Japan Sea and southwestern Okhotsk Sea in early summer 2011 to clarify the origin of the CWB. Temperature-salinity relationships, vertical distributions of chemical components, profiles of chemical components against density, and the (NO3 + NO2)/PO4 relationship confirm that water in the CWB predominantly originates from Japan Sea subsurface water

Encapsulation of DNA and non-viral protein changes the structure of murine polyomavirus virus-like particles

Asymmetrical-flow field flow fractionation with multiple-angle light scattering (AFFFF-MALS) was, for the first time, used to characterize the size of murine polyomavirus virus-like particles (MPV VLPs) packaged with either insect cell genomic DNA or non-viral protein. Encapsidation of both genomic DNA and non-viral protein were found to cause a contraction in VLP radii of gyration by approximately 1 nm. Non-viral protein packaged into VLPs consisted of a series of glutathione-S-transferase, His and S tags attached to the N-terminal end of the MPV structural protein VP2 (M (r) = 67108). Transmission electron microscopy analysis of MPV VLPs packaging non-viral protein suggested that VLPs grew in diameter by approximately 5 nm, highlighting the differences between this invasive technique and the relatively non-invasive AFFFF-MALS technique. Encapsulation of non-viral protein into MPV VLPs was found to prevent co-encapsidation of genomic DNA. Further investigation into why this occurred led to the discovery that encapsulation of non-viral protein alters the nuclear localization of MPV VLPs during in vivo assembly. VLPs were relocated away from the ring zone and the nuclear membrane towards the centre of the nucleus amongst the virogenic stroma. The change in nuclear localization away from the site where VLP assembly usually occurs is a likely reason why encapsidation of genomic DNA did not take place

Mechanisms of disease: aquaporin-4 antibodies in neuromyelitis optica.

Neuromyelitis optica (NMO) is a rare CNS inflammatory disorder that predominantly affects the optic nerves and spinal cord. Recent serological findings strongly suggest that NMO is a distinct disease rather than a subtype of multiple sclerosis. In NMO, serum antibodies, collectively known as NMO-IgG, characteristically bind to cerebral microvessels, pia mater and Virchow-Robin spaces. The main target antigen for this immunoreactivity has been identified as aquaporin-4 (AQP4). The antibodies are highly specific for NMO, and they are also found in patients with longitudinally extensive transverse myelitis without optic neuritis, which is thought to be a precursor to NMO in some cases. An antibody-mediated pathogenesis for NMO is supported by several observations, including the characteristics of the AQP4 antibodies, the distinct NMO pathology--which includes IgG and complement deposition and loss of AQP4 from spinal cord lesions--and emerging evidence of the beneficial effects of B-cell depletion and plasma exchange. Many aspects of the pathogenesis, however, remain unclear