RT-PCR for the assessment of genetically heterogenous populations of the hepatitis C virus.

Abstract

AbstractSince the discovery of the Hepatitis C Virus (HCV) in 1989 (1), known to that point as the infectious agent of Non-A Non-B hepatitis (2), there has been an intensive focus on understanding the underlying biology of its disease process. Of significant interest has been the study of heterogenous populations of closely related, but genetically non-identical HCV virions, commonly termed quasispecies (3,4), and their relationship to the pathogenesis of HCV infection. Studies of HCV quasispecies have predominantly focused on two viral genetic loci, Hypervariable Region 1 (HVR 1) and the Interferon Sensitivity Determining Region (ISDR) (5) (Fig. 1). HVR 1 encodes an approx 27 amino-acid sequence at the N-terminal of envelope glycoprotein 2 (E2), and sequence variation at this locus is used to characterize HCV quasispecies. HVR 1 acts as an immune escape epitope that, through continuous alteration of its antigenicity, is believed to hinder humoral immune-mediated clearance of the virus. The ISDR encodes an approx 40 amino-acid sequence in the C-terminal half of the nonstructural 5A (NS5A) protein. Protein kinase inhibition and transcriptional activation activities have been ascribed to NS5A, both of which are known to be modulated by sequence variation within the ISDR (6,7). The current view is that although the role of the NS5A protein in HCV disease progression is important, pathologically relevant viral genomic sequence evolution may not be strictly confined to the ISDR alone. Sequence variation of other regions in NS5A, such as the V3 region (8), nuclear localization signals, and phosphorylation motifs may also play important roles (9). Structure of the HCV genome and relative location of PCR products discussed in this chapter. (A) HCV genome comprising 5′ and 3′ untranslated regions (bold lines), flanking an open reading frame (box). Nucleotide (upper, italics), and amino acid (lower, plain) scales are shown. (B) Enlarged envelope 2 (E2) and nonstructural 5A (NS5A) genes showing (bold) HVR1 and 2, and ISDR, respectively. (C) Location of PCR products (hatched areas) in relation to HVR and ISDR loci. The NS5A PCR precisely covers the NS5A gene. Diagram based on HCV-H-strain nucleotide and amino-acid sequence (5). KeywordsLuria BertaniInterferon Sensitivity Determine RegionQuasispecies PopulationThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.