Stability and Morphology Comparisons of Self-AssembledVirus-Like Particles from Wild-Type and Mutant Human Hepatitis B VirusCapsidProteins

Abstract

Instead of displaying the wild-type selective export of virions containing mature genomes, human hepatitis B virus (HBV) mutant I97L, changing from an isoleucine to a leucine at amino acid 97 of HBV core antigen (HBcAg), lost the high stringency of selectivity in genome maturity during virion export. To understand the structural basis of this so-called "immature secretion" phenomenon, we compared the stability and morphology of self-assembled capsid particles from the wild-type and mutant I97L HBV, in either full-length (HBcAg1-183) or truncated core protein contexts (HBcAg1-149 and HBcAg1-140). Using negative staining and electron microscopy, full-length particles appear as "thick-walled" spherical particles with little interior space, whereas truncated particles appear as "thin-walled" spherical particles with a much larger inner space. We found no significant differences in capsid stability between wild-type and mutant I97L particles under denaturing pH and temperature in either full-length or truncated core protein contexts. In general, HBV capsid particles (HBcAg1-183, HBcAg1-149, and HBcAg1-140) are very robust but will dissociate at pH 2 or 14, at temperatures higher than 75°C, or in 0.1% sodium dodecyl sulfate (SDS). An unexpected upshift banding pattern of the SDS-treated full-length particles during agarose gel electrophoresis is most likely caused by disulfide bonding of the last cysteine of HBcAg. HBV capsids are known to exist in natural infection as dimorphic T‫3؍‬ or T‫4؍‬ icosahedral particles. No difference in the ratio between T‫3؍‬ (78%) and T‫4؍‬ particles (20.3%) are found between wild-type HBV and mutant I97L in the context of HBcAg1-140. In addition, we found no difference in capsid stability between T‫3؍‬ and T‫4؍‬ particles successfully separated by using a novel agarose gel electrophoresis procedure.

Hepatitis B Virus Capsid Assembly Is Enhanced by Naturally Occurring Mutation F97L

“…Multiangle laser light scattering data were consistent with assignment of the two peaks as the 4-MDa capsid and 34-kDa dimer (data not shown). In agreement with other reports (19), Cp149-F97L and -wt capsids were indistinguishable by negative-stain electron microscopy and on sucrose gradients (data not shown).…”

Plant-expressed Hepatitis B core antigen virus-like particles: Characterization and investigation of their stability in simulated and pig gastro-intestinal fluids

“…C), a clear band could be seen in the both the Coomassie-and ethidium bromide-stained gels. The two signals had the same electrophoretic mobility, suggesting that the VLP protein core, stained by the Coomassie, encapsidated nucleic acids, stained by ethidium bromide, consistent with previous reports (Newman et al, 2003). The presence of intact VLPs was further confirmed by TEM images (Figure 1.…”

“…This result is in accordance with Newman et al (2003): in that case, it was shown that bacterially expressed HBcAg incubated at pH 2.0 for only 30 minutes was unstable and it remained blocked in the well (Newman et al, 2003). Overall, in the current study, the native agarose gel electrophoresis revealed that this complete loss of the physical stability upon incubation of HBcAg for two hours in acid and further neutralisation occurs at pH values below 3 to 3.5 (Figure 3).…”

Genetic Assembly of Double‐Layered Fluorescent Protein Nanoparticles for Cancer Targeting and Imaging

“…This is well supported by Figure 4b, showing that the fastest decrease of fluorescent intensity was observed in tumor cells treated with mC out ‐HBVC, while the slowest was with mC in ‐HBVC. It seems that the low‐pH‐degradable mCardinal11 was effectively protected when it was encapsulated within the HBVC that is known to be stable over a wide range of pH (4–10) 21…”