Reverse transcription PCR-based sequence analysis of hepatitis C virus replicon RNA.
Since the advent of efficient cell-culture methods for HCV replication and, more recently, infection, there has been a need to efficiently sequence the viral RNA in these systems. This need is especially urgent in light of the error-prone nature of HCV RNA replication, which leads to a variety of interesting mutations. The adaptation of hepatitis C replicons to cell culture, which greatly increased their replication capacity, and the subsequent identification of viral point mutations responsible for this adaptation are prime examples of the type of phenotype-genotype connection that viral RNA sequencing methods can provide. More recently, researchers have used similar sequencing methods to identify changes in replicons that represent viral adaptation to engineered mutations, adaptation to a variety of host cells, and viral evasion of antiviral compound susceptibility. Here, we describe the cloning and isolation of HCV replicon-bearing cells, the extraction of total RNA, the generation of cDNA, and the amplification of specific HCV replicon sequences for sequence analysis. The methods we describe permit rapid and robust determination of HCV RNA sequences from cell culture.
Tellinghuisen
Timothy L
TL
Department of Infectology, The Scripps Research Institute, Jupiter, FL, USA.
Lindenbach
Brett D
BD
D018929:Cell Culture Techniques; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D016679:Genome, Viral; D005838:Genotype; D016174:Hepacivirus / Q000235:genetics*; D007202:Indicators and Reagents; D012313:RNA / Q000235:genetics; D012367:RNA, Viral / Q000032:analysis* / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D012093:Replicon / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D048348:Reverse Transcription; D017422:Sequence Analysis, DNA / Q000379:methods*; D013045:Species Specificity; D014779:Virus Replication / Q000235:genetics*
D018076:DNA, Complementary; D007202:Indicators and Reagents; C034477:RNA primers; D012367:RNA, Viral; D012313:RNA