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1. Structure of the Hepatitis B virus capsid quasi-6-fold with a trapped C-terminal domain reveals capsid movements associated with domain exit

   https://doi.org/10.1016/j.jbc.2023.105104  

   Kim, Christine; Schlicksup, Christopher J.; Pérez-Segura, Carolina; Hadden-Perilla, Jodi A.; Wang, Joseph Che-Yen; Zlotnick, Adam (September 2023, Journal of Biological Chemistry)

   Many viruses undergo transient conformational change to surveil their environments for receptors and host factors. In Hepatitis B virus (HBV) infection, after the virus enters the cell, it is transported to the nucleus by interaction of the HBV capsid with an importin α/β complex. The interaction between virus and importins is mediated by nuclear localization signals on the capsid protein’s C-terminal domain (CTD). However, CTDs are located inside the capsid. In this study, we asked where does a CTD exit the capsid, are all quasi-equivalent CTDs created equal, and does the capsid structure deform to facilitate CTD egress from the capsid? Here, we used Impβ as a tool to trap transiently exposed CTDs and examined this complex by cryo-electron microscopy. We examined an asymmetric reconstruction of a T = 4 icosahedral capsid and a focused reconstruction of a quasi-6-fold vertex (3.8 and 4.0 Å resolution, respectively). Both approaches showed that a subset of CTDs extended through a pore in the center of the quasi-6-fold complex. CTD egress was accompanied by enlargement of the pore and subtle changes in quaternary and tertiary structure of the quasi-6-fold. When compared to molecular dynamics simulations, structural changes were within the normal range of capsid flexibility. Although pore diameter was enlarged in the Impβ-bound reconstruction, simulations indicate that CTD egress does not exclusively depend on enlarged pores. In summary, we find that HBV surveillance of its environment by transient exposure of its CTD requires only modest conformational change of the capsid. 

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2. A computational spatial whole-Cell model for hepatitis B viral infection and drug interactions

   https://doi.org/10.1038/s41598-023-45998-0  

   Ghaemi, Zhaleh; Nafiu, Oluwadara; Tajkhorshid, Emad; Gruebele, Martin; Hu, Jianming (December 2023, Scientific Reports)

   Abstract Despite a vaccine, hepatitis B virus (HBV) remains a world-wide source of infections and deaths. We develop a whole-cell computational platform combining spatial and kinetic models describing the infection cycle of HBV in a hepatocyte host. We simulate key parts of the infection cycle with this whole-cell platform for 10 min of biological time, to predict infection progression, map out virus-host and virus-drug interactions. We find that starting from an established infection, decreasing the copy number of the viral envelope proteins shifts the dominant infection pathway from capsid secretion to re-importing the capsids into the nucleus, resulting in more nuclear-localized viral covalently closed circular DNA (cccDNA) and boosting transcription. This scenario can mimic the consequence of drugs designed to manipulate viral gene expression. Mutating capsid proteins facilitates capsid destabilization and disassembly at nuclear pore complexes, resulting in an increase in cccDNA copy number. However, excessive destabilization leads to premature cytoplasmic disassembly and does not increase the cccDNA counts. Finally, our simulations can predict the best drug dosage and its administration timing to reduce the cccDNA counts. Our adaptable computational platform can be parameterized to study other viruses and identify the most central viral pathways that can be targeted by drugs. 

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3. Proxiome assembly of the plant nuclear pore reveals an essential hub for gene expression regulation

   https://doi.org/10.1038/s41477-024-01698-9  

   Tang, Yu; Yang, Xiangyun; Huang, Aobo; Seong, Kyungyong; Ye, Mao; Li, Mengting; Zhao, Qiao; Krasileva, Ksenia; Gu, Yangnan (June 2024, Nature Plants)

   The nuclear pore complex (NPC) is vital for nucleocytoplasmic communication. Recent evidence emphasizes its extensive association with proteins of diverse functions, suggesting roles beyond cargo transport. However, our understanding of NPC's composition and functionality at this extended level remains limited. Here, through proximity labeling proteomics, we uncover both local and global NPC-associated proteome in Arabidopsis, comprising over 500 unique proteins, predominantly associated with NPC's peripheral extension structures. Compositional analysis of these proteins revealed that the NPC concentrates chromatin remodelers, transcriptional regulators, and mRNA processing machineries in the nucleoplasmic region, while recruiting translation regulatory machinery on the cytoplasmic side, achieving a remarkable orchestration of the genetic information flow by coupling RNA transcription, maturation, transport, and translation regulation. Further biochemical and structural modeling analyses reveal that extensive interactions with nucleoporins, along with phase separation mediated by substantial intrinsically disordered proteins, may drive the formation of the unexpectedly large nuclear pore proteome assembly. 

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4. Highly Basic Clusters in the Herpes Simplex Virus 1 Nuclear Egress Complex Drive Membrane Budding by Inducing Lipid Ordering

   https://doi.org/10.1128/mBio.01548-21  

   Thorsen, Michael K.; Lai, Alex; Lee, Michelle W.; Hoogerheide, David P.; Wong, Gerard C.; Freed, Jack H.; Heldwein, Ekaterina E. (August 2021, mBio)

   Shenk, Thomas (Ed.)

   ABSTRACT During replication of herpesviruses, capsids escape from the nucleus into the cytoplasm by budding at the inner nuclear membrane. This unusual process is mediated by the viral nuclear egress complex (NEC) that deforms the membrane around the capsid by oligomerizing into a hexagonal, membrane-bound scaffold. Here, we found that highly basic membrane-proximal regions (MPRs) of the NEC alter lipid order by inserting into the lipid headgroups and promote negative Gaussian curvature. We also find that the electrostatic interactions between the MPRs and the membranes are essential for membrane deformation. One of the MPRs is phosphorylated by a viral kinase during infection, and the corresponding phosphomimicking mutations block capsid nuclear egress. We show that the same phosphomimicking mutations disrupt the NEC-membrane interactions and inhibit NEC-mediated budding in vitro , providing a biophysical explanation for the in vivo phenomenon. Our data suggest that the NEC generates negative membrane curvature by both lipid ordering and protein scaffolding and that phosphorylation acts as an off switch that inhibits the membrane-budding activity of the NEC to prevent capsid-less budding. IMPORTANCE Herpesviruses are large viruses that infect nearly all vertebrates and some invertebrates and cause lifelong infections in most of the world’s population. During replication, herpesviruses export their capsids from the nucleus into the cytoplasm by an unusual mechanism in which the viral nuclear egress complex (NEC) deforms the nuclear membrane around the capsid. However, how membrane deformation is achieved is unclear. Here, we show that the NEC from herpes simplex virus 1, a prototypical herpesvirus, uses clusters of positive charges to bind membranes and order membrane lipids. Reducing the positive charge or introducing negative charges weakens the membrane deforming ability of the NEC. We propose that the virus employs electrostatics to deform nuclear membrane around the capsid and can control this process by changing the NEC charge through phosphorylation. Blocking NEC-membrane interactions could be exploited as a therapeutic strategy. 

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5. Coordination of NMCP1- and NMCP2-class proteins within the plant nucleoskeleton

   https://doi.org/10.1091/mbc.E19-08-0464  

   Blunt, Endia L.; Shandler, Jason A.; Hughes, Erika J.; Sussman, Hayley; Christopherson, Rachel C.; Richards, Eric J. (December 2020, Molecular Biology of the Cell)

   Misteli, Tom (Ed.)

   Plants lack lamin proteins but contain a class of coiled-coil proteins that serve as analogues to form a laminal structure at the nuclear periphery. These nuclear matrix constituent proteins (NMCPs) play important roles in regulating nuclear morphology and are partitioned into two distinct groups. We investigated Arabidopsis NMCPs (called CRWNs) to study the interrelationship between the three NMCP1-type paralogues (CRWN1, 2, and 3) and the lone NMCP2-type paralogue, CRWN4. An examination of crwn mutants using protein immunoblots demonstrated that CRWN4 abundance depends on the presence of the NMCP1-type proteins, particularly CRWN1. The possibility that CRWN4 is coimported into the nucleus with nuclear localization signal (NLS)-bearing paralogues in the NMCP1-clade was discounted based on recovery of a crwn4-2 missense allele that disrupts a predicted NLS and lowers the abundance of CRWN4 in the nucleus. Further, a screen for mutations that suppress the effects of the crwn4-2 mutation led to the discovery of a missense allele, impa-1 G146E , in one of the nine importin-α genes in the Arabidopsis genome. Our results indicate that the CRWN4 carries a functional NLS that interacts with canonic nuclear import machinery. Once imported, the level of CRWN4 within the nucleus is modulated by the abundance of NMCP1 proteins. 

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