Complete translation of the hepatitis C virus genome in vitro: membranes play a critical role in the maturation of all virus proteins except for NS3.
We developed an in vitro translation extract from Krebs-2 cells that translates the entire open reading frame of the hepatitis C virus (HCV) strain H77 and properly processes the viral protein precursors when supplemented with canine microsomal membranes (CMMs). Translation of the C-terminal portion of the viral polyprotein in this system is documented by the synthesis of NS5B. Evidence for posttranslational modification of the viral proteins, the N-terminal glycosylation of E1 and the E2 precursor (E2-p7), and phosphorylation of NS5A is presented. With the exception of NS3, efficient generation of all virus-specific proteins is CMM dependent. A time course of the appearance of HCV products indicates that the viral polyprotein is cleaved cotranslationally. A competitive inhibitor of the NS3 protease inhibited accumulation of NS3, NS4B, NS5A, and NS5B, but not that of NS2 or structural proteins. CMMs also stabilized HCV mRNA during translation. Finally, the formyl-[35S]methionyl moiety of the initiator tRNA(Met) was incorporated exclusively into the core protein portion of the polyprotein, demonstrating that translation initiation in this system occurs with high fidelity.
Svitkin
Yuri V
YV
Department of Biochemistry, McGill University, 3655 Promenade Sir William Osler, Montreal, Quebec, Canada H3G 1Y6. yuri.svitkine@mcgill.ca
Pause
Arnim
A
Lopez-Lastra
Marcelo
M
Perreault
Sandra
S
Sonenberg
Nahum
N
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002474:Cell-Free System; D004279:DNA, Viral / Q000235:genetics; D004285:Dogs; D016679:Genome, Viral; D006031:Glycosylation; D006367:HeLa Cells; D016174:Hepacivirus / Q000235:genetics / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D051379:Mice; D008861:Microsomes / Q000378:metabolism; D010766:Phosphorylation; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D020871:RNA Stability; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012358:RNA, Transfer, Met / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis* / Q000235:genetics*; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D004279:DNA, Viral; D012333:RNA, Messenger; D012358:RNA, Transfer, Met; D012367:RNA, Viral; D015842:Serine Proteinase Inhibitors; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases