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Structural insights into HIV-1 capsid assembly, maturation and stability by cryo-electron tomography

Tan, Aaron Wai Kit

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Abstract

Human immunodeficiency virus type 1 (HIV-1) is an enveloped lentivirus from the family Retroviridae which infects CD4+ T-lymphocytes in a human host, leading to Acquired Immunodeficiency Syndrome (AIDS) if untreated. A subset of retroviruses, most notably lentiviruses such as HIV-1, are unique in their ability to infect non-dividing cells. To do this, the reverse transcribed viral genome must be trafficked across an intact nuclear membrane and integrated into the host cell genome. The viral capsid plays a central role in this process.

The first stage of capsid assembly is polymerisation of the viral polyprotein Gag via its CA (capsid) domain into a hexagonal immature lattice, forming a truncated sphere. The viral protease cleaves Gag and frees the CA domain, which rearranges to form a conical capsid around the viral genome, built from CA hexamers and pentamers. Despite advances in recent years, many open questions remain about immature Gag lattice assembly, maturation and modulation of capsid stability by host factors upon infection.

One key question that has persisted in the field is how the remarkable structural transition between the immature Gag lattice and the mature CA lattice is achieved, which involves breaking almost all of the interactions stabilising the immature lattice. To address this, I applied cryo-electron tomography (cryo-ET) and subtomogram averaging to obtain high resolution structures of immature and mature CA in a panel of HIV-1 constructs containing different combinations of proteolytic cleavage sites inactivated by mutation. Unexpectedly, proteolytic processing directly on either side of CA was sufficient for mature lattice formation at low frequencies. I also show that a beta-hairpin domain at the CA N-terminus, previously proposed to be a structural switch, is dispensable for maturation. Instead, destabilisation of a six-helix bundle between the CA C-terminus and the adjacent SP1 peptide is the main structural determinant of maturation.

Viral maturation is tightly linked to immature Gag lattice assembly, but many details such as the basic unit of lattice assembly remain unclear. The immature lattice is maintained by inter- and intra-hexamer interactions but is not a complete sphere, and the structure of Gag at discontinuous lattice edges is unknown. I implemented a new workflow to obtain Gag lattice structures by subtomogram classification of a cryo-ET data set of intact HIV-1 virions. These structures show that Gag forms novel, incomplete hexamers at lattice edges and that the CA-SP1 region forms ordered helical bun- dles in partial hexamers. Molecular dynamics simulations suggest that these partial bundles exhibit increased an tendency to unfold, suggesting a role of partial hexamer structures in initiation of maturation.

Capsid stability after cell entry is important to prevent degradation of the viral RNA genome, and is modulated by small molecules such as inositol hexakisphosphate (IP6) and host proteins, including cleavage and polyadenylation specific factor 6 (CPSF6) and nucleoporin 153 (Nup153). A combination of CA pentamers and hexamers that flex to adopt different curvatures provides many different potential interfaces for cofactor binding. I developed a workflow to routinely obtain near-atomic resolution structures of CA hexamers and pentamers, by subtomogram averaging of conical, IP6-stabilised in vitro CA assemblies. These were used to investigate CPSF6 and Nup153 binding to pentamers and the effect of lattice curvature on the common binding pocket for these factors. The structures obtained show that CPSF6 and Nup153 do not bind to pentamers at the concentrations used, and that lattice flexibility can modulate Nup153 binding to hexamers.

Item Type: Dissertation
Supervisor: Briggs, Dr. John
Place of Publication: Heidelberg
Date of thesis defense: 13 December 2019
Date Deposited: 08 Jan 2020 14:29
Date: 2020
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Service facilities > European Molecular Biology Laboratory (EMBL)
Subjects: 570 Life sciences
Uncontrolled Keywords: HIV Capsid Subtomogram averaging Cryo-ET Cryo-EM
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