Baculovirus gp64 gene expression: negative regulation by a minicistron

Area of Science:

• Molecular Biology
• Virology
• Gene Regulation

Background:

• Small upstream open reading frames (minicistrons) regulate translation in eukaryotic genes.
• Baculovirus genomes, including Autographa californica MNPV, contain potential minicistrons in late gene transcripts.
• The gp64 gene's minicistron is conserved across baculoviruses, suggesting a regulatory role.

Purpose of the Study:

• Investigate the regulatory function of the gp64 minicistron on gp64 late mRNA translation.
• Determine if the gp64 minicistron acts as a positive or negative translational regulator.

Main Methods:

• Generated recombinant baculoviruses with a chloramphenicol acetyltransferase (cat) reporter gene fused to the gp64 minicistron and ORF.
• Created point mutations to inactivate the minicistron initiator ATG and an upstream ATG.
• Quantitatively analyzed gp64-cat RNA and GP64-CAT protein levels to measure translational efficiency.

Main Results:

• Translation efficiency of the downstream gp64-cat reporter ORF increased when the minicistron was absent or inactivated.
• Inactivating the minicistron initiator ATG led to the use of an upstream ATG within the minicistron coding region.
• Inactivating both ATGs further increased translational efficiency of the downstream ORF.

Conclusions:

• The gp64 minicistron functions as a negative regulatory element.
• This minicistron actively decreases the translation of the downstream gp64 open reading frame on late baculovirus mRNAs.