Structures of Nucleotide-Bound Redondovirus Rep Protein Link Conformation and Function

Area of Science:

• Structural Biology
• Virology
• Biochemistry

Background:

• Circular Rep-encoding single-stranded DNA (CRESS-DNA) viruses utilize Rep proteins for replication.
• Rep proteins possess nicking, helicase, and DNA joining activities essential for viral genome replication.
• The Redondoviridae family, a newly identified group of human-associated CRESS-DNA viruses, replicates in Entamoeba gingivalis.

Purpose of the Study:

• To determine the first structures of a Rep protein from the Redondoviridae family.
• To elucidate the structural mechanisms underlying Rep protein function in viral DNA replication.
• To investigate the oligomeric states and their functional implications in CRESS-DNA virus replication.

Main Methods:

• Cryo-electron microscopy (cryo-EM) was used to determine high-resolution structures.
• Structures of Redondovirus Rep helicase were characterized in complex with ATPγS and ADP.
• Biophysical analyses were employed to study Rep protein oligomerization.

Main Results:

• Hexameric structures of Redondovirus Rep in ATP-bound (ATPγS) and post-hydrolysis (ADP) states were resolved.
• The ADP-bound Rep exhibited a staircase arrangement of DNA-binding loops, critical for SF3 helicase models.
• A head-to-tail dodecameric structure of ATPγS-bound Rep revealed ordered helicase and endonuclease domains.
• Conserved residues suggest the dodecameric assembly is functionally relevant across many CRESS-DNA viruses.

Conclusions:

• The structural and oligomeric insights into Redondovirus Rep provide a deeper understanding of CRESS-DNA virus replication.
• The positioning of endonuclease domains in the hexamer and observed oligomerization states offer new functional perspectives.
• This study lays the groundwork for further investigations into the Rep protein's role in viral lifecycle and potential therapeutic targeting.