A fish encephalitis virus that differs from other nodaviruses by its capsid protein processing

Area of Science:

• Virology
• Molecular Biology
• Biochemistry

Background:

• Dicenthrarchus labrax encephalitis virus (DIEV) is a fish nodavirus causing seabass encephalitis.
• DIEV RNA2 genome segment encodes capsid proteins.
• Insect nodaviruses typically yield capsid proteins via autocatalytic proteolysis.

Purpose of the Study:

• To elucidate the synthesis mechanism of DIEV capsid proteins.
• To investigate the role of the D-75 residue and potential cleavage sites in DIEV capsid protein formation.
• To determine if DIEV capsid proteins are produced through autocatalytic cleavage, alternative initiation, frameshifting, or other mechanisms.

Main Methods:

• Cloning and sequencing of DIEV RNA2.
• Expression of wild-type and mutagenized DIEV RNA2 in cell-free translation systems and transfected cells.
• SDS-PAGE analysis of mature virions and translation products.
• Kinetics analysis of capsid protein synthesis.

Main Results:

• DIEV RNA2 contains a single open reading frame (ORF) with a D-75 residue but lacks a typical autocatalytic cleavage site.
• Mature DIEV virions contain a 43-45 kDa protein doublet.
• The DIEV capsid protein doublet is not formed by autocatalytic cleavage, alternative initiation codon usage, -1 ribosomal frameshifting, proteolytic cleavage, or degradation.
• Kinetics studies show the two capsid proteins are cosynthesized.
• The DIEV capsid protein doublet likely results from cotranslational modification(s) of the ORF-encoded protein.

Conclusions:

• The synthesis pathway for DIEV capsid proteins differs from that of insect nodaviruses.
• Autocatalytic proteolysis is not the mechanism for generating the DIEV capsid protein doublet.
• Cotranslational modification is the most probable mechanism for the synthesis of the DIEV capsid protein doublet.