Effect of the 5′ non-translated region on self-assembly of hepatitis C virus genotype 1a structural proteins produced in insect cells

The effect of the 5' non-translated region (5'NTR) on hepatitis C virus (HCV) morphogenesis in insect cells is investigated in this study. Expression in baculovirus-infected cells of a sequence encoding the C and E1 structural proteins under the control of the very late promoter P10 (AcSLP10-C-E1) led to the synthesis of C and C-E1 complexes, essentially found in dense reticular material associated with the ER and sedimenting at a density of 1(.)24-1(.)26 g ml(-1). Addition of the 5'NTR upstream of the C-E1 sequence (AcSLP10-5'NTR-E1) prevents translation from the initiating codon, probably because of the presence of five AUG codons in this sequence. When cells were co-infected with these two viruses, virus-like particles (VLPs) were found in the cytoplasm. The size and shape of these VLPs were variable. Concomitantly, a shift in the sedimentation profile from 1(.)24-1(.)26 to 1(.)15-1(.)18 g ml(-1) was observed, suggesting an association of C/E1 with the ER membrane. A unique vector was then constructed bearing a mutated 5'NTR (mutation of the five AUGs) and the sequence encoding all of the structural proteins and part of NS2 (5'NTRm-C-E1-E2-p7-NS2Delta). Translation of structural proteins was restored and electron microscopic observation of a cytoplasmic extract showed the presence of icosahedral particles with a density of 1(.)15-1(.)18 g ml(-1).