Nodavirus RNA replication crown architecture reveals proto-crown precursor and viral protein A conformational switching

Proc Natl Acad Sci U S A. 2023 Jan 31;120(5):e2217412120. doi: 10.1073/pnas.2217412120. Epub 2023 Jan 24.

Abstract

Positive-strand RNA viruses replicate their genomes in virus-induced membrane vesicles, and the resulting RNA replication complexes are a major target for virus control. Nodavirus studies first revealed viral RNA replication proteins forming a 12-fold symmetric "crown" at the vesicle opening to the cytosol, an arrangement recently confirmed to extend to distantly related alphaviruses. Using cryoelectron microscopy (cryo-EM), we show that mature nodavirus crowns comprise two stacked 12-mer rings of multidomain viral RNA replication protein A. Each ring contains an ~19 nm circle of C-proximal polymerase domains, differentiated by strikingly diverged positions of N-proximal RNA capping/membrane binding domains. The lower ring is a "proto-crown" precursor that assembles prior to RNA template recruitment, RNA synthesis, and replication vesicle formation. In this proto-crown, the N-proximal segments interact to form a toroidal central floor, whose 3.1 Å resolution structure reveals many mechanistic details of the RNA capping/membrane binding domains. In the upper ring, cryo-EM fitting indicates that the N-proximal domains extend radially outside the polymerases, forming separated, membrane-binding "legs." The polymerase and N-proximal domains are connected by a long linker accommodating the conformational switch between the two rings and possibly also polymerase movements associated with RNA synthesis and nonsymmetric electron density in the lower center of mature crowns. The results reveal remarkable viral protein multifunctionality, conformational flexibility, and evolutionary plasticity and insights into (+)RNA virus replication and control.

Keywords: RNA replication complex; RNA replication crown; cryo-EM; nodavirus; positive-strand RNA virus.

MeSH terms

  • Cryoelectron Microscopy
  • RNA Replication
  • RNA Viruses* / genetics
  • RNA, Viral / genetics
  • RNA, Viral / metabolism
  • Viral Proteins* / genetics
  • Viral Proteins* / metabolism
  • Virus Replication / genetics

Substances

  • Viral Proteins
  • RNA, Viral