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).
Girard
Christel
C
Station de Pathologie Comparée, INRA-CNRS, 30380 Saint-Christol-lès-Alès, France.
Ravallec
Marc
M
Station de Pathologie Comparée, INRA-CNRS, 30380 Saint-Christol-lès-Alès, France.
Mariller
Marcel
M
Station de Pathologie Comparée, INRA-CNRS, 30380 Saint-Christol-lès-Alès, France.
Bossy
Jean-Pierre
JP
Station de Pathologie Comparée, INRA-CNRS, 30380 Saint-Christol-lès-Alès, France.
Cahour
Annie
A
CERVI Laboratoire de Virologie, UPRES EA 2387 Hospital Pitié-Salpétrière, 83 boulevard de l'Hôpital, 75651 Paris Cedex 13, France.
López-Ferber
Miguel
M
Station de Pathologie Comparée, INRA-CNRS, 30380 Saint-Christol-lès-Alès, France.
Devauchelle
Gérard
G
Station de Pathologie Comparée, INRA-CNRS, 30380 Saint-Christol-lès-Alès, France.
Inchauspé
Geneviève
G
Unité Mixte CNRS-bioMérieux, École Normale Supérieure, 69000 Lyon, France.
Duonor-Cérutti
Martine
M
Station de Pathologie Comparée, INRA-CNRS, 30380 Saint-Christol-lès-Alès, France.
D020121:5' Untranslated Regions / Q000502:physiology*; D000818:Animals; D036022:Capsid Proteins / Q000737:chemistry; D005838:Genotype; D016174:Hepacivirus / Q000502:physiology*; D014176:Protein Biosynthesis; D018411:Spodoptera; D014758:Viral Core Proteins / Q000502:physiology*; D014759:Viral Envelope Proteins / Q000502:physiology*; D019065:Virus Assembly
D020121:5' Untranslated Regions; D036022:Capsid Proteins; C087035:E1 protein, Hepatitis C virus; D014758:Viral Core Proteins; D014759:Viral Envelope Proteins