# Nodavirus protein A’s interdomain elbow controls RNA replication organelle formation and function

## TL;DR

This study reveals how a small segment of a nodavirus protein controls the formation and function of RNA replication organelles.

## Contribution

The paper identifies a 17-amino-acid 'elbow' segment in protein A that regulates replication organelle assembly and function.

## Key findings

- The elbow segment coordinates interactions between subunits in the proto-crown structure.
- Elbow subsegments separately activate RNA capping and polymerase domains.
- The elbow and a flexible linker control conformational changes in protein A.

## Abstract

Positive-strand [(+)RNA] viruses replicate their RNA genomes in poorly understood membrane-associated replication organelles (ROs). Cryo-electron microscopy of nodaviral ROs revealed that viral RNA replication protein A, with polymerase and RNA capping domains, forms a “crown” of two stacked 12-mer rings at the RO’s opening to the cytosol, providing powerful foundations for analyzing RO formation and function. The lower proto-crown is a ring of 12 polymerases with RNA capping domains clustered to form a central floor. The upper crown mirrors the polymerase ring but has RNA capping domains projecting radially outward. Here, we identify a critical protein A “elbow” segment of only 17 amino acids that coordinates most interactions between crown floor subunits. Our extensive mutational and genetic complementation analyses reveal that distinct elbow subsegments cooperatively support proto-crown formation and separately activate the neighboring RNA capping and distal polymerase domains. These and other findings establish the elbow as a master regulator whose separable interactions drive proto-crown assembly, license RNA polymerase and capping and, with an adjacent flexible linker, control protein A’s conformational changes. They also further localize RNA capping to the crown floor. This work illuminates (+)RNA viruses’ elegant regulation of genome replication steps and future control strategies.

Graphical Abstract