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Importin β Can Bind Hepatitis B Virus Core Protein and Empty Core-Like Particles and Induce Structural Changes
Chao Chen ,
Joseph Che-Yen Wang ,
Elizabeth E. Pierson,
David Z. Keifer,
Mildred Delaleau,
Lara Gallucci,
Christian Cazenave,
Michael Kann,
Martin F. Jarrold,
Adam Zlotnick
PLOS x
Published: August 12, 2016
http://dx.doi.org/10.1371/journal.ppat.1005802
Chao Chen
Contributed equally to this work with: Chao Chen, Joseph Che-Yen Wang
Address Current address: Center for Biomedical Imaging, Medical University of South Carolina, Charleston, South Carolina, United States of America
Affiliation Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana, United States of America
Joseph Che-Yen Wang
Contributed equally to this work with: Chao Chen, Joseph Che-Yen Wang
Affiliation Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana, United States of America
Elizabeth E. Pierson
Address Current address: Department of Analytical Sciences, Merck Research Laboratories, Merck & Co., Inc., Rahway, New Jersey, United States of America
Affiliation Department of Chemistry, Indiana University, Bloomington, Indiana, United States of America
David Z. Keifer
Affiliation Department of Chemistry, Indiana University, Bloomington, Indiana, United States of America
Mildred Delaleau
Address Current address: Institute for Research in Immunology and Cancer & Department of Pathology and Cell Biology, Université de Montréal, Montréal, QC, Canada
Affiliations Universite de Bordeaux, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France, CNRS, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France
Lara Gallucci
Affiliations Universite de Bordeaux, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France, CNRS, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France
Christian Cazenave
Affiliations Universite de Bordeaux, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France, CNRS, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France
Michael Kann
Affiliations Universite de Bordeaux, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France, CNRS, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France, CHU de Bordeaux, Bordeaux, France
Martin F. Jarrold
Affiliation Department of Chemistry, Indiana University, Bloomington, Indiana, United States of America
Adam Zlotnick
* E-mail: [email protected]
Affiliations Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana, United States of America, Department of Chemistry, Indiana University, Bloomington, Indiana, United States of America, Department of Biology, Indiana University, Bloomington, Indiana, United States of America
Abstract
Hepatitis B virus (HBV) capsids are found in many forms: immature single-stranded RNA-filled cores, single-stranded DNA-filled replication intermediates, mature cores with relaxed circular double-stranded DNA, and empty capsids. A capsid, the protein shell of the core, is a complex of 240 copies of core protein. Mature cores are transported to the nucleus by a complex that includes both importin α and importin β (Impα and Impβ), which bind to the core protein’s C-terminal domains (CTDs). Here we have investigated the interactions of HBV core protein with importins in vitro. Strikingly, empty capsids and free core protein can bind Impβ without Impα. Cryo-EM image reconstructions show that the CTDs, which are located inside the capsid, can extrude through the capsid to be bound by Impβ. Impβ density localized on the capsid exterior near the quasi-sixfold vertices, suggested a maximum of 30 Impβ per capsid. However, examination of complexes using single molecule charge-detection mass spectrometry indicate that some complexes include over 90 Impβ molecules. Cryo-EM of capsids incubated with excess Impβ shows a population of damaged particles and a population of “dark” particles with internal density, suggesting that Impβ is effectively swallowed by the capsids, which implies that the capsids transiently open and close and can be destabilized by Impβ. Though the in vitro complexes with great excess of Impβ are not biological, these results have implications for trafficking of empty capsids and free core protein; activities that affect the basis of chronic HBV infection.
Author Summary
Viruses take advantage of host proteins. During infection, DNA-filled Hepatitis B Virus (HBV) cores are ferried to the nucleus by a complex of importin α and importin β; importinβ alone does not bind mature cores. However, a surprisingly large amount of the HBV core protein accumulates in nuclei. Here we show that both empty cores and free core protein can bind importin β alone. Using cryo-electron microscopy reconstructions we show that importin β associates with a putative importin β-binding motif, which is located on the interior of empty cores but can extrude through pores in the particle. Single-particle mass spectrometry, electron microscopy, and biochemical studies showed the importin β can destabilize empty particles. These results together demonstrate unsuspected interactions between the HBV core protein and the nuclear transport system. These interactions may have roles in regulating stability of empty capsids and transfer of HBV core protein to the nucleus.
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发表于 2016-8-20 13:38
