The NS3 helicase and NS5B-to-3'X regions are important for efficient hepatitis C virus strain JFH-1 replication in Huh7 cells.
The JFH-1 strain of hepatitis C virus (HCV) is a genotype 2a strain that can replicate autonomously in Huh7 cells. The J6 strain is also a genotype 2a strain, but its full genomic RNA does not replicate in Huh7 cells. However, chimeric J6/JFH-1 RNA that has J6 structural-protein-coding regions and JFH-1 nonstructural-protein-coding regions can replicate autonomously and produce infectious HCV particles. In order to determine the mechanisms underlying JFH-1 RNA replication, we constructed various J6/JFH-1 chimeras and tested their RNA replication and virus particle production abilities in Huh7 cells. Via subgenomic-RNA-replication assays, we found that both the JFH-1 NS5B-to-3'X (N5BX) and the NS3 helicase (N3H) regions are important for the replication of the J6CF replicon. We applied these results to full-length genomic RNA replication and analyzed replication using Northern blotting. We found that a chimeric J6 clone with JFH-1 N3H and N5BX could replicate autonomously but that a chimeric J6 clone with only JFH-1 N5BX had no replication ability. Finally, we tested the virus production abilities of these clones and found that a chimeric J6 clone with JFH-1 N3H and N5BX could produce infectious HCV particles. In conclusion, the JFH-1 NS3 helicase and NS5B-to-3'X regions are important for efficient replication and virus particle formation of HCV genotype 2a strains.
Murayama
Asako
A
Department of Virology II, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku, Tokyo 162-8640, Japan.
Date
Tomoko
T
Morikawa
Kenichi
K
Akazawa
Daisuke
D
Miyamoto
Michiko
M
Kaga
Minako
M
Ishii
Koji
K
Suzuki
Tetsuro
T
Kato
Takanobu
T
Mizokami
Masashi
M
Wakita
Takaji
T
D020413:3' Untranslated Regions; D002460:Cell Line; D005838:Genotype; D016174:Hepacivirus / Q000502:physiology*; D006801:Humans; D020365:RNA Helicases / Q000235:genetics / Q000502:physiology; D012367:RNA, Viral / Q000235:genetics*; D012093:Replicon; D017361:Viral Nonstructural Proteins / Q000235:genetics* / Q000502:physiology; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; D012324:RNA-Dependent RNA Polymerase
D020413:3' Untranslated Regions; D020365:RNA Helicases; D012367:RNA, Viral; 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; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase