TY - JOUR

T1 - Assembly of Capsids from Hepatitis B Virus Core Protein Progresses through Highly Populated Intermediates in the Presence and Absence of RNA

AU - Oliver, Ryan C.

AU - Potrzebowski, Wojciech

AU - Najibi, Seyed Morteza

AU - Pedersen, Martin Nors

AU - Arleth, Lise

AU - Mahmoudi, Najet

AU - Andre, Ingemar

PY - 2020/8/25

Y1 - 2020/8/25

N2 - The genetic material of viruses is protected by protein shells that are assembled from a large number of subunits in a process that is efficient and robust. Many of the mechanistic details underpinning efficient assembly of virus capsids are still unknown. The assembly mechanism of hepatitis B capsids has been intensively researched using a truncated core protein lacking the C-terminal domain responsible for binding genomic RNA. To resolve the assembly intermediates of hepatitis B virus (HBV), we studied the formation of nucleocapsids and empty capsids from full-length hepatitis B core proteins, using time-resolved small-angle X-ray scattering. We developed a detailed structural model of the HBV capsid assembly process using a combination of analysis with multivariate curve resolution, structural modeling, and Bayesian ensemble inference. The detailed structural analysis supports an assembly pathway that proceeds through the formation of two highly populated intermediates, a trimer of dimers and a partially closed shell consisting of around 40 dimers. These intermediates are on-path, transient and efficiently convert into fully formed capsids. In the presence of an RNA oligo that binds specifically to the C-terminal domain the assembly proceeds via a similar mechanism to that in the absence of nucleic acids. Comparisons between truncated and full-length HBV capsid proteins reveal that the unstructured C-terminal domain has a significant impact on the assembly process and is required to obtain a more complete mechanistic understanding of HBV capsid formation. These results also illustrate how combining scattering information from different time-points during time-resolved experiments can be utilized to derive a structural model of protein self-assembly pathways.

AB - The genetic material of viruses is protected by protein shells that are assembled from a large number of subunits in a process that is efficient and robust. Many of the mechanistic details underpinning efficient assembly of virus capsids are still unknown. The assembly mechanism of hepatitis B capsids has been intensively researched using a truncated core protein lacking the C-terminal domain responsible for binding genomic RNA. To resolve the assembly intermediates of hepatitis B virus (HBV), we studied the formation of nucleocapsids and empty capsids from full-length hepatitis B core proteins, using time-resolved small-angle X-ray scattering. We developed a detailed structural model of the HBV capsid assembly process using a combination of analysis with multivariate curve resolution, structural modeling, and Bayesian ensemble inference. The detailed structural analysis supports an assembly pathway that proceeds through the formation of two highly populated intermediates, a trimer of dimers and a partially closed shell consisting of around 40 dimers. These intermediates are on-path, transient and efficiently convert into fully formed capsids. In the presence of an RNA oligo that binds specifically to the C-terminal domain the assembly proceeds via a similar mechanism to that in the absence of nucleic acids. Comparisons between truncated and full-length HBV capsid proteins reveal that the unstructured C-terminal domain has a significant impact on the assembly process and is required to obtain a more complete mechanistic understanding of HBV capsid formation. These results also illustrate how combining scattering information from different time-points during time-resolved experiments can be utilized to derive a structural model of protein self-assembly pathways.

KW - hepatitis B virus

KW - capsid assembly

KW - time-resolved

KW - small-angle scattering

KW - electron microscopy

KW - multivariate curve resolution

KW - Bayesian statistics

KW - SMALL-ANGLE

KW - RESOLUTION

KW - KINETICS

KW - MODEL

KW - TIME

KW - MASS

KW - SIMULATION

KW - SCATTERING

KW - BINDING

U2 - 10.1021/acsnano.0c03569

DO - 10.1021/acsnano.0c03569

M3 - Journal article

C2 - 32672447

VL - 14

SP - 10226

EP - 10238

JO - A C S Nano

JF - A C S Nano

SN - 1936-0851

IS - 8

ER -