In vitro papillomavirus capsid assembly analyzed by light scattering

Abstract

Pentamers of the L1 major capsid protein of human papillomavirus (HPV type 11) were purified after expression in E. coli and analyzed for the kinetics of in vitro capsid self-assembly using multi-angle light scattering (MALS). Pentamers self-assembled into capsid-like structures at a rate that was a function of protein concentration. The kinetics of capsid formation were sigmoidal with a concentration-dependent lag phase, followed by a rapid increase in polymerization. Nucleation size and the rate order of subsequent subunit addition were calculated from the concentration dependence of the extent of capsid formation and the rate of the fast phase, respectively. Assembly was second order with a nucleation size of two pentamers. Thus, we suggest that dimers of pentamers are the nucleus for L1 assembly into capsid-like structures, with rapid sequential addition of single pentamers to the growing shell. Although studied in vitro without accessory factors that may be present in vivo, these data are in contrast with the "five-around-one" assembly nucleus previously proposed for polyomaviruses.

Controlling viral capsid assembly with templating

“…We show in appendix A that the average timescales of these phases for an individual capsid can be described by τ = τ nuc + τ elong , with and , where f is the subunit-subunit binding rate constant, C S is the concentration of free subunits, n nuc is the number of subunits in the nucleus. Because elongation requires N − n nuc assembly events, it introduces a minimum timescale for the overall assembly process, which is primarily responsible for the lag time in assembly kinetics reported in experiments [9,12,53], theory [21,48], and simulations [26,28,80], and results in a distribution of assembly times for an individual capsid that cannot be fit with a sum of pure exponential functions [81]. The observed assembly rate constant, f, can be considered an average quantity, since computational models [26,28] suggest that it varies for different intermediates and decreases due to excluded volume constraints as assembly nears completion.…”

“…For fast adsorption, nucleation takes place at an effective surface concentration of c surf with a timescale (9) where we define the surface assembly rate constant f surf = f D C /D, with D and D C the diffusion constants for free and adsorbed subunits, respectively. We assume that the association rate is proportional to the frequency of subunit collisions because the reaction follows second-order kinetics.…”

Purification and protective efficacy of monomeric and modified Yersinia pestis capsular F1-V antigen fusion proteins for vaccination against plague

“…Size-exclusion chromatography coupled to multiangle laser light scattering (SEC-MALLS) was applied as previously reported for F1-V [6], but with modifications as published [14,15]. The HPLC pumps were Rainin HPXL 10 ml/min pumps (Varian, Walnut, CA) run at 0.4 ml/ min.…”

The Viral Etiology of AIDS‐Associated Malignancies

“…High resolution cryoelectron microscopic structures of bovine papillomavirus (BPV) have been achieved (Baker et al, 1991); less is known about HPV structure, assembly, and uncoating. L1 expression is sufficient to allow self-assembly of virus-like particles (VLPs) in the absence of other viral components (Casini et al, 2004). However, L2, when coexpressed with L1, intercalates into L1 VLPs and appears to be required for virion infectivity (Kawana et al, 2001;Stauffer et al, 1998).…”