Multiple Pathways in Capsid Assembly

Abstract

For a three-dimensional structure to spontaneously self-assemble from many identical components, the steps on the pathway must be kinetically accessible. Many virus capsids are icosahedral and assembled from hundreds of identical proteins, but how they navigate the assembly process is poorly understood. Capsid assembly is thought to involve stepwise addition of subunits to a growing capsid fragment. Coarse-grained models suggest that the reaction occurs on a downhill energy landscape, so intermediates are expected to be fleeting. In this work, charge detection mass spectrometry (CDMS) has been used to track assembly of the hepatitis B virus (HBV) capsid in real time. The icosahedral T = 4 capsid of HBV is assembled from 120 capsid protein dimers. Our results indicate that there are multiple pathways for assembly. Under conditions that favor a modest association energy there is no accumulation of large intermediates, which indicates that available pathways include ones on a downhill energy surface. Under higher salt conditions, where subunit interactions are strengthened, around half of the products of the initial assembly reaction have masses close to the T = 4 capsid and the other half are stalled intermediates which emerge abruptly at around 90 dimers, indicating a bifurcation in the ensemble of assembly paths. When incubated at room temperature, the 90-dimer intermediates accumulate dimers and gradually shift to higher mass and merge with the capsid peak. Though free subunits are present in solution, the stalled intermediates indicate the presence of a local minima on the energy landscape. Some intermediates may result from hole closure, where the growing capsid distorts to close the hole due to the missing capsid proteins or from a species where subsequent additions are particularly labile.

Expression of quasi-equivalence and capsid dimorphism in the Hepadnaviridae

“…Capsid assembly has been proposed to begin with the formation of a trimer-of-dimers nucleus and then to proceed by the further addition of dimers [13,14]. Early intermediates consisting of up to five dimers (with species consisting of three and five dimers prominent) have been detected by charge detection mass spectrometry (CDMS) [16] but have yet to be visualized. To visualize early intermediates, we examined reaction products formed after initiating assembly under mild conditions (low ionic strength and neutral pH; see Materials and Methods) by negative stain electron microscopy.…”

“…Assembly has been proposed to begin, at least in vitro, with the formation of a nucleus, a triangular trimer-of-dimers, and then to proceed by the addition of further dimers [13,14]. Early assembly intermediates, while detected by mass spectrometry [15][16][17], have not been structurally characterized.…”

Mechanism of action of HBV capsid assembly modulators predicted from binding to early assembly intermediates

“…unlikely. Furthermore, mass spectroscopy experiments are able to detect the formation of tetramers during capsid assembly, suggesting that they are also important assembly intermediates, and are likely hexamer precursors [27][28][29] . Based on what is known about HBV capsid assembly, there are three possibilities for the nucleation event: 20,40 , suggesting that either hexamer closure or conformational change after this closure could be the bottleneck of the assembly process.…”

“…Both compounds occupy a similar space in the HAP pocket and interact with Trp102. (C) HBV capsid assembly process based on experimental data and mathematical modeling 17,[27][28][29] . mechanistic classes (see Figure S1 for structures).…”

“…However, it is possible that CAMs induce distinct structural changes in early assembly intermediates. Although these intermediates have been detected with mass spectroscopy [27][28][29] , little information is available about their structure, their dynamics and how these properties are altered upon CAM binding.…”