Bioorthogonal dissection of the replicase assembly of hepatitis C virus

Abstract

Positive-strand RNA viruses such as hepatitis C virus (HCV), flaviviruses, and coronaviruses are medically important. Assembly of replicase on host membranes is a conserved replication strategy and an attractive antiviral target. The mechanisms of replicase assembly are largely unknown, due to the technical difficulties in purifying the replicase and carrying out structural studies. Here, with an HCV replicase assembly surrogate system, we employed a bioorthogonal system to introduce the photolabile unnatural amino into each residue in the cytosolic regions of NS4B and the amphipathic helix (AH) of NS5A. Photocrosslinking enabled visualization of NS4B oligomerization and NS5A dimerization at pinpointed interacting residues and identifying contacting sites among the replicase components. Characterization of the interacting sites revealed hub elements in replicase assembly by docking replicase components to prompt protein-protein interactions. The results provide information about the molecular architecture of the replicase, advancing understanding of the mechanism of replicase assembly.

Identification and characterization of key residues in Zika virus envelope protein for virus assembly and entry

“…Thus, we sought to explore the effect of the assembly-dead mutants on E multimerization during virion assembly. We recently used a bioorthogonal system [ 54 ] to visualize oligomerization and dimerization of hepatitis C virus proteins in vivo [ 38 ]. This bioorthogonal system uses the orthogonal suppressor tRNA/aminoacyl-tRNA synthetase (aaRS) pair for the photolabile unnatural amino acid (UAA) p-azido-L-phenylalanine (AZF).…”

“…Three-Alanine (AAA) mutations were introduced into E in the plasmid phCMV-CprM-E HA by fusion PCR-mediated mutagenesis to get the plasmids phCMV-CprM-E HA -Mutants. The suppressor tRNA plasmid (pSVB.Yam) and the amino-acyl tRNA synthetase plasmid for p-azido-L-phenylalanine (pcDNA.RS) were kindly gifted by Thomas P. Sakmar (Rockefeller University) and have been described previously [ 38 ]. The TAG was introduced into E in the plasmid phCMV-CprM-E HA by fusion PCR-mediated mutagenesis.…”

“…Photo crosslinking was carried out as previously described [ 38 ]. Briefly, 7.5 × 10 5 Vero cells on 6-cm dish were cotransfected with 4.3 μg CprM-E expressing plasmids, 4.3 μg pSVB.Yam and 1.2 μg pcDNA.RS by TransIT-LT1 (Mirus) at a ratio of 1:3 (plasmid: reagent).…”

From Discovery to Cure, A Great Journey of the Hepatitis C Virus Study

“…NS3, NS4A, NS4B, NS5A, and NS5B assemble the viral replicase on the endoplasmic reticulum, forming double-membrane vesicles. [8,9] Within double-membrane vesicles, the viral genome undergoes amplification and is then assembled into virions. Finally, virions are released, together with host lipoproteins [Figure 1B].…”

“…NS5B is the viral RNA-dependent RNA polymerase [Figure 2] (see [7] for a review). NS3, NS4A, NS4B, NS5A, and NS5B assemble the viral replicase on the endoplasmic reticulum, forming double-membrane vesicles [8,9] . Within double-membrane vesicles, the viral genome undergoes amplification and is then assembled into virions.…”