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- Dahmane, Selma, et al.
(författare)
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A 3D view of how enteroviruses hijack autophagy
- 2023
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Ingår i: Autophagy. - : Taylor & Francis Group. - 1554-8627 .- 1554-8635. ; 19:7, s. 2156-2158
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Tidskriftsartikel (refereegranskat)abstract
- Viruses are masters at using cellular pathways to aid their replication. Cryo-electron tomography of poliovirus-infected cells revealed how it utilizes macroautophagy to its advantage. Assembly of these non-enveloped virions takes place directly on membranes and requires PIK3C3/VPS34 activity to be completed, whereas the canonical autophagy inducer ULK1 restricts virus assembly. The tomograms further revealed that enterovirus-induced autophagy is selective for RNA-loaded virions, which may help ensure maximum infectivity of the virus-laden vesicles released through secretory autophagy.
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| 2. |
- Dahmane, Selma, et al.
(författare)
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Membrane-assisted assembly and selective secretory autophagy of enteroviruses
- 2022
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 13:1
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Tidskriftsartikel (refereegranskat)abstract
- Enteroviruses are non-enveloped positive-sense RNA viruses that cause diverse diseases in humans. Their rapid multiplication depends on remodeling of cytoplasmic membranes for viral genome replication. It is unknown how virions assemble around these newly synthesized genomes and how they are then loaded into autophagic membranes for release through secretory autophagy. Here, we use cryo-electron tomography of infected cells to show that poliovirus assembles directly on replication membranes. Pharmacological untethering of capsids from membranes abrogates RNA encapsidation. Our data directly visualize a membrane-bound half-capsid as a prominent virion assembly intermediate. Assembly progression past this intermediate depends on the class III phosphatidylinositol 3-kinase VPS34, a key host-cell autophagy factor. On the other hand, the canonical autophagy initiator ULK1 is shown to restrict virion production since its inhibition leads to increased accumulation of virions in vast intracellular arrays, followed by an increased vesicular release at later time points. Finally, we identify multiple layers of selectivity in virus-induced autophagy, with a strong selection for RNA-loaded virions over empty capsids and the segregation of virions from other types of autophagosome contents. These findings provide an integrated structural framework for multiple stages of the poliovirus life cycle. Enteroviruses are non-enveloped positive-sense RNA viruses that modulate cytoplasmic membranes for replication. To enlighten how enteroviruses assemble around nascent RNA genomes and get package into autophagosomes for release, Dahmane et al. perform cryo-electron tomography of poliovirus-infected cells. They find assembly intermediates that are only present on the cytosolic side of the replication compartment and provide evidence that host factor VPS34 is involved in progression of assembly intermediates.
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| 3. |
- Knyazeva, Anastasia, 1995-, et al.
(författare)
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A chemical inhibitor of IST1-CHMP1B interaction impairs endosomal recycling and induces noncanonical LC3 lipidation
- 2024
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 121:17
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Tidskriftsartikel (refereegranskat)abstract
- The endosomal sorting complex required for transport (ESCRT) machinery constitutes multisubunit protein complexes that play an essential role in membrane remodeling and trafficking. ESCRTs regulate a wide array of cellular processes, including cytokinetic abscission, cargo sorting into multivesicular bodies (MVBs), membrane repair, and autophagy. Given the versatile functionality of ESCRTs, and the intricate organizational structure of the ESCRT machinery, the targeted modulation of distinct ESCRT complexes is considerably challenging. This study presents a pseudonatural product targeting IST1-CHMP1B within the ESCRT-III complexes. The compound specifically disrupts the interaction between IST1 and CHMP1B, thereby inhibiting the formation of IST1-CHMP1B copolymers essential for normal-topology membrane scission events. While the compound has no impact on cytokinesis, MVB sorting, or biogenesis of extracellular vesicles, it rapidly inhibits transferrin receptor recycling in cells, resulting in the accumulation of transferrin in stalled sorting endosomes. Stalled endosomes become decorated by lipidated LC3, suggesting a link between noncanonical LC3 lipidation and inhibition of the IST1-CHMP1B complex.
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| 6. |
- Shankar, Kasturika, 1993-
(författare)
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Biochemical and structural studies of proteins supporting the genome replication of enteroviruses and Giardia intestinalis
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The Enterovirus genus of the Picornaviridae family includes non-enveloped, positive-sense single-stranded RNA (ssRNA) viruses. This genus of viruses causes many diseases such as poliomyelitis by poliovirus (PV), cardiomyopathy by coxsackievirus B3 (CVB3), common cold by rhinoviruses (RVs) and meningitis by Enterovirus 71 (EV 71). The 7.5 kb enterovirus genome encodes a polyprotein, which is subsequently cleaved to yield viral proteins. These viral proteins hijack and modify the membranes of the Golgi and ER to give rise to replication organelles (ROs).In the first paper of my thesis, we showed that membranes of the ROs act as an assembly line for the assembly of enteroviruses. Using cryo-electron tomography we studied poliovirusinfected cells. The one feature that we found most interesting was that a protein was tethering viral capsid to membranes as well as two membranes together. We hypothesized that viral protein 2C was a strong candidate. Therefore, I tried to mimic the interaction between 2C and viral capsid using purified Poliovirus and 2C protein. However, after trying several biochemical conditions I could not mimic this interaction. This strongly indicated that I might need to have a membrane in the system and this led me to study the membrane binding activity of 2C in the second paper.In the second paper, I studied an Enteroviral multi-functional protein: 2C, a highly conserved AAA+ ATPase that plays an important role in the biogenesis of ROs and virus assembly. One of the most interesting features of 2C is its N-terminal membrane-binding domain consisting of 40 amino acids. Using in vitro reconstitution methods and biochemistry, I investigated the association of full-length 2C with lipid vesicles and how this affects the function of the protein. I showed that amino acids 12 to 40 are important not only for membrane binding but also for hexamerization. Moreover, truncation of the first 11 amino acids leads to loss of membrane tethering activity of the protein, which is essential for the formation of ROs. I was also able to demonstrate that the protein is sufficient to recruit RNA to the membrane as it was not previously known how RNA replication is localized to the membrane and here, we found the possible mechanism. In this realistic reconstituted system, I have shown that 2C is not a helicase but an ATP-independent RNA chaperone. Collectively, these discoveries offer a biochemical foundation for various functions of 2C in enterovirus replication. This sets up a more practical biochemical framework for future research, which could potentially contribute to the development of drugs aimed at thwarting enterovirus infections.In the third project, I studied deoxyadenosine kinase (dAK) from Giardia intestinalis, a protozoan responsible for severe diarrhea spread by the fecal-oral route. Giardia is completely dependent on the host for deoxyribonucleosides as it lacks a de novo pathway for their synthesis. dAK uses ATP as a phosphate donor to phosphorylate deoxyadenosine, this activity is essential for genome replication of the protozoan. In this project, we have biochemically and structurally characterized dAK. Here, I used cryo-electron microscopy (cryo-EM) single particle analysis to show that dAK exists as a homo-tetramer in solution. This is an important finding because this is the first example of a non-thymidine kinase1-like deoxyribonucleoside kinase with a tetrameric structure and subsequent mutagenesis analysis showed that tetramerization allows the enzyme to achieve a higher affinity for its deoxyadenosine substrate.In summary, in my thesis, I have biochemically and structurally characterized proteins supporting the genome replication of enteroviruses and Giardia intestinalis.
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