Reconstitution of Enteroviral 2C ATPase Reveals Its Roles in Membrane Clustering, RNA Recruitment, and Enzymatic Activities

Concetti Chiave

The enteroviral 2C protein, a membrane-bound AAA+ ATPase, plays crucial roles in virus replication by mediating membrane clustering, recruiting double-stranded RNA, and exhibiting ATPase and RNA cleavage activities.

Sintesi

The content describes the in vitro reconstitution and functional characterization of the enteroviral 2C protein, a membrane-bound AAA+ ATPase. Key findings include:

1. The N-terminal membrane-binding domain of 2C contains two amphipathic helix regions, AH1 and AH2, where AH2 is the main mediator of 2C oligomerization and membrane binding.
2. AH1 is responsible for a novel function of 2C: clustering of membranes, as observed through cryo-electron tomography, which can be partially outcompeted by RNA.
3. 2C is sufficient to recruit RNA to membranes, with a preference for double-stranded RNA (the replicating form of the viral genome).
4. The reconstituted, membrane-bound 2C exhibits ATPase activity and ATP-independent, single-strand ribonuclease activity, but no detectable helicase activity, contrary to previous suggestions.
5. The reconstitution of functional, 2C-decorated vesicles provides a platform for further biochemical studies into this protein and its roles in enterovirus replication.

Statistiche

The study reports the following key figures and metrics:

2C contains a conserved glycine that divides its N-terminal membrane-binding domain into two amphipathic helix regions, AH1 and AH2.
AH2 is the main mediator of 2C oligomerization and membrane binding.
Cryo-electron tomography reveals that 2C-mediated membrane clustering can be partially outcompeted by RNA.
2C is sufficient to recruit RNA to membranes, with a preference for double-stranded RNA.
The reconstituted, membrane-bound 2C exhibits ATPase activity and ATP-independent, single-strand ribonuclease activity, but no detectable helicase activity.

Citazioni

"AH2 is the main mediator of 2C oligomerization, and is necessary and sufficient for its membrane binding."
"2C-mediated clustering is partially outcompeted by RNA, suggesting a way by which 2C can switch from an early role in coalescing replication organelles and lipid droplets, to a later role where 2C assists RNA replication and particle assembly."
"2C is sufficient to recruit RNA to membranes, with a preference for double-stranded RNA (the replicating form of the viral genome)."

Approfondimenti chiave tratti da

In vitro reconstitution reveals membrane clustering and RNA recruitment by the enteroviral AAA+ ATPase 2C

by Shankar,K., ... alle www.biorxiv.org 10-11-2023

https://www.biorxiv.org/content/10.1101/2023.10.11.561830v3

Domande più approfondite

How do the structural features and enzymatic activities of 2C contribute to its various roles in the viral replication cycle?

The structural features of 2C, particularly the N-terminal membrane-binding domain containing amphipathic helix regions AH1 and AH2, play crucial roles in its functions during the viral replication cycle. AH2, responsible for 2C oligomerization and membrane binding, facilitates the clustering of membranes, aiding in the coalescing of replication organelles and lipid droplets. This membrane clustering function is essential for the early stages of viral replication. Additionally, 2C's ability to recruit RNA to membranes, especially double-stranded RNA, is vital for RNA replication and particle assembly. Despite lacking detectable helicase activity, 2C exhibits ATPase activity and ATP-independent, single-strand ribonuclease activity, further contributing to its multifaceted roles in viral replication.

What are the potential implications of the observed RNA-mediated competition with membrane clustering for the temporal regulation of 2C functions during infection?

The observed RNA-mediated competition with membrane clustering suggests a dynamic regulation of 2C functions during viral infection. Initially, 2C facilitates membrane clustering to create a conducive environment for viral replication. However, as RNA, particularly double-stranded RNA, accumulates during replication, it competes with membrane clustering, potentially signaling a shift in 2C's role. This competition may serve as a temporal regulatory mechanism, allowing 2C to transition from membrane clustering to assisting RNA replication and particle assembly. The ability of RNA to outcompete membrane clustering highlights the intricate interplay between different stages of the viral replication cycle and the adaptability of 2C in response to changing intracellular conditions.

What other cellular factors or processes might interact with or be influenced by the membrane-clustering and RNA-recruitment activities of 2C?

The membrane-clustering and RNA-recruitment activities of 2C may interact with various cellular factors and processes involved in viral replication. For instance, host cell lipid metabolism pathways could be influenced by 2C-mediated membrane clustering, potentially impacting lipid droplet formation and utilization during viral replication. Additionally, the recruitment of RNA to membranes by 2C may intersect with host cell RNA processing and translation machinery, affecting cellular RNA metabolism. Furthermore, the competition between RNA and membrane clustering could modulate the localization and activity of other viral replication proteins or host factors involved in the replication cycle. Understanding the crosstalk between 2C's activities and cellular processes is essential for elucidating the intricate mechanisms underlying enterovirus replication.