Detection of Late Intermediates in Virus Capsid Assembly by Charge Detection Mass Spectrometry

Abstract

The assembly of hundreds of identical proteins into an icosahedral virus capsid is a remarkable feat of molecular engineering. How this occurs is poorly understood. Key intermediates have been anticipated at the end of the assembly reaction, but it has not been possible to detect them. In this work we have used charge detection mass spectrometry to identify trapped intermediates from late in the assembly of the hepatitis B virus T = 4 capsid, a complex of 120 protein dimers. Prominent intermediates are found with 104/105, 110/111, and 117/118 dimers. Cryo-EM observations indicate the intermediates are incomplete capsids and, hence, on the assembly pathway. On the basis of their stability and kinetic accessibility we have proposed plausible structures. The prominent trapped intermediate with 104 dimers is attributed to an icosahedron missing two neighboring facets, the 111-dimer species is assigned to an icosahedron missing a single facet, and the intermediate with 117 dimers is assigned to a capsid missing a ring of three dimers in the center of a facet.

Charge Detection Mass Spectrometry for Single Ions with an Uncertainty in the Charge Measurement of 0.65 e

“…A detailed discussion of how the charge and m/z measurements influence the mass resolution was published previously [2]. Improving the mass resolution is of great importance in studies of mixtures of high mass ions that can result, for example, in virus assembly reactions [31]. In Figure 6, we compare the mass resolution achieved in our previous work with that obtained here.…”

“…By increasing the trapping time to 129 ms, we were able to improve the accuracy of the charge measurements to 1.3 e, though only 1% of the ions were trapped for this long [2]. This configuration has now been used to probe higher order multimers of pyruvate kinase [2], the mass distribution of 24-MDa bacteriophage P22 procapsids [30] late intermediates in the assembly of hepatitis B virus capsids [31] and woodchuck hepatitis virus [32].…”

Virus Matryoshka: A Bacteriophage Particle—Guided Molecular Assembly Approach to a Monodisperse Model of the Immature Human Immunodeficiency Virus

“…Further details about signal processing can be found elsewhere. [67][68][69] Prior to CDMS, samples were buffer exchanged into 100 × 10 −3 m ammonium acetate.…”

Core protein: A pleiotropic keystone in the HBV lifecycle

“…Because HBV capsids have been the subject of numerous assembly studies (summarized in (Zlotnick and Fane, 2011)), it is important to note that capsids from E coli and assembled in vitro under mild conditions lead to complete capsids (Pierson et al, 2014; Uetrecht et al, 2008). However, as predicted by theory, when association energy is relatively strong, incomplete capsids are readily trapped (Pierson et al, 2014).…”

“…Because HBV capsids have been the subject of numerous assembly studies (summarized in (Zlotnick and Fane, 2011)), it is important to note that capsids from E coli and assembled in vitro under mild conditions lead to complete capsids (Pierson et al, 2014; Uetrecht et al, 2008). However, as predicted by theory, when association energy is relatively strong, incomplete capsids are readily trapped (Pierson et al, 2014). The capsids and dimers that are able to adopt so many structures are found to be extremely flexible based on protease sensitivity and the hydrogen-deuterium exchange rate (Bereszczak et al, 2013; Bereszczak et al, 2014; Hilmer et al, 2008).…”