A mechanism for initiating RNA-dependent RNA polymerization

Area of Science:

• Virology
• Structural Biology
• Molecular Biology

Background:

• Viral RNA-dependent RNA polymerases are crucial for RNA virus replication and transcription.
• Double-stranded RNA viruses utilize an enzyme within a capsid for these processes, capable of reading both single- and double-stranded RNA.
• Previous structural studies of viral capsids lacked detailed resolution of polymerase subunits.

Purpose of the Study:

• To elucidate the structure of the active polymerase subunit from the double-stranded RNA bacteriophage phi6.
• To investigate the evolutionary relationship between double-stranded RNA viruses and flaviviruses.
• To propose a mechanism for the initiation of viral RNA replication and transcription.

Main Methods:

• X-ray crystallography at 2 Å resolution to determine the structure of the phi6 polymerase subunit.
• Crystal soaking and co-crystallization techniques to study complexes with oligonucleotide and nucleoside triphosphates (NTPs).

Main Results:

• The X-ray structure of the phi6 polymerase subunit shows high similarity to the hepatitis C virus polymerase, suggesting an evolutionary link.
• Structures of complexes with NTPs suggest a mechanism for double-stranded RNA processing and template feeding into the active site.
• A model for initiation involving template strand overshoot, a specificity pocket, and engagement of two NTPs is proposed.

Conclusions:

• The study provides a high-resolution structure of a double-stranded RNA virus polymerase, revealing evolutionary connections.
• A detailed mechanism for the initiation of viral RNA replication and transcription is proposed based on structural data.
• This work offers a working model for understanding fundamental viral RNA synthesis processes.