Structure and function of flavivirus NS5 methyltransferase

Area of Science:

• Virology
• Molecular Biology
• Structural Biology

Background:

• Flaviviruses possess a 5' cap 1 structure (m7GpppAmG) on their positive-strand RNA genome.
• Viral cap formation is crucial for flavivirus replication and involves methyltransferase activity.
• The NS5 protein's N-terminal region harbors the methyltransferase responsible for guanine N-7 and ribose 2'-OH methylations.

Purpose of the Study:

• To investigate the distinct methylation activities of flavivirus methyltransferases.
• To elucidate the structural basis for sequential N-7 and 2'-O methylations.
• To determine the essentiality of these methylation activities for West Nile virus (WNV) infectivity and therapeutic potential.

Main Methods:

• Crystal structure determination of WNV methyltransferase at 2.8 Å resolution.
• Electrostatic analysis of the WNV methyltransferase structure to identify potential RNA binding sites.
• Biochemical assays and mutagenesis to analyze N-7 and 2'-O methylation mechanisms and requirements.
• In vivo studies using WNV mutants to assess the impact of methylation defects on viral infectivity and immunogenicity.

Main Results:

• The WNV methyltransferase exhibits two distinct methylation activities (N-7 and 2'-O) but possesses a single S-adenosyl-L-methionine (SAM) binding site.
• Structural and biochemical data suggest substrate RNA repositioning and distinct catalytic mechanisms for the two methylation steps.
• N-7 methylation is essential for WNV viability, while 2'-O methylation is dispensable but confers protection against WNV challenge.

Conclusions:

• Flavivirus methyltransferase is essential for viral RNA cap formation and WNV life cycle.
• The N-7 methylation activity is critical for WNV infectivity.
• Flavivirus methyltransferase represents a promising novel therapeutic target for flavivirus infections.