Proteolytic Cleavage of VP1 in 'A' Particles of Coxsackievirus B3 Does Not Appear to Mediate Virus Uncoating by HeLa Cells

Abstract

SUMMARY'A' particles of Coxsackievirus B3 were generated from native virus by heating and purified by sucrose gradient centrifugation. These particles were found to be similar to 'A' particles formed by elution from cellular receptors of HeLa cells. Electrophoretic analysis of [35S]methionine-labelled 'A' particles revealed that treatment of the particles with chymotrypsin resulted in the cleavage of VP 1 and the formation of a cleavage product which migrated between VP2 and VP3. Analysis of the protease-treated material on sucrose gradients revealed a ribonuclease-sensitive particle which sedimented more slowly than an 'A' particle. This particle apparently degraded to release the viral RNA, thereby providing an in vitro model for protease-mediated uncoating of 'A' particles. The subviral particles of Coxsackievirus B3 were found to be immunoprecipitable with heterotypic Coxsackievirus group B antisera, thereby providing a method for the recovery of products produced in the cell early in infection. Infected cells which had been treated to remove unreacted virus were disrupted, and the lysates were reacted with heterotypic antisera. Analysis of the precipitated material revealed that no cleavage products were formed and no polypeptides were lost. Therefore, it appears that proteolysis is not involved in the uncoating of Coxsackievirus B3 in infected cells.

The structure of coxsackievirus B3 at 3.5 å resolution

“…The electron-density map of CVB3 shows very weak density, resembling 3-structure, beneath residues 4001-4008 of VP4 which may, by analogy to the polioviruses, be the N terminus of VP1. The interaction of the N termini of VP1 and VP4 is consistent with the observation that the N terminus of VP1 is externalized and becomes exposed to proteases upon the formation of altered ('A') particles which lack VP4 [41,42].…”

nanoDSF: In vitro Label-Free Method to Monitor Picornavirus Uncoating and Test Compounds Affecting Particle Stability

“…Heating of enteroviral particles such as poliovirus, rhinovirus, coxsackievirus B3, echovirus 1, and enterovirus 71 has been frequently used to trigger uncoating in vitro, allowing for the structural characterization of the resulting subviral particles. Dependent on the temperature (37 to 60 • C), time of treatment (minutes to hours), and buffer composition, this results in the preferential generation of A particles, a mixture of A and B particles, or almost pure B particles that are indistinguishable from those observed in vivo (Lonberg-Holm and Noble-Harvey, 1973;McGeady and Crowell, 1981;Wetz and Kucinski, 1991;Curry et al, 1996;Okun et al, 1999;Belnap et al, 2000;Shingler et al, 2013;Subirats et al, 2013;Ruokolainen et al, 2019). Adjusting the above parameters, it was also possible to isolate an intermediate conformational state of poliovirus in the process of RNA release (Bostina et al, 2011).…”

Monoclonal antibody that inhibits infection of HeLa and rhabdomyosarcoma cells by selected enteroviruses through receptor blockade

“…In brief, the early interactions of coxsackieviruses with receptors at the cell surface consist of attachment, penetration of the plasma membrane (endocytosis), and viral eclipse leading to uncoating of the viral genome. However, as many as 80% of attached virions are eluted or dissociated into the extracellular environment with loss of the low-molecular-weight virion polypeptide VP4 (11), making it difficult to identify the molecular events in virus uncoating (28).…”