Identification of novel HCV subgenome replicating persistently in chronic active hepatitis C patients

Abstract

In an effort to clarify the life cycle of HCV, the HCV genome in liver biopsies taken from chronic active hepatitis C patients undergoing interferon treatment was investigated. Molecular cloning by long distance reverse-transcription polymerase chain reaction (RT-PCR) revealed that the HCV genome in two patients with high viral loads in the liver had in-frame deletions of approximately 2 kb between E1 and NS2, which encode the E1-NS2 fusion protein and six other HCV proteins: core, NS3, NS4A, NS4B, NS5A, and NS5B. Among the remaining 21 chronic active hepatitis C patients, these types of deletion were found in another two patients and in two hepatocellular carcinoma patients. Out-of-frame deletions in the structural region were isolated from the other five patients, but the dominant RT-PCR products were non-truncated genomes. Retrospective analysis of a series of serum samples taken from a patient carrying the subgenome with the in-frame deletion revealed that both the subgenome and the full genome persisted through the 2-year period of investigation, with the subgenome being predominant during this period. Sequence analysis of the isolated cDNA suggested that both the subgenome and the full genome evolved independently. Western blotting analysis of HCV proteins from the HCV subgenome indicated that they were processed in the same way as those from the full genome. HCV subgenomes thus appear to be involved in the HCV life cycle.

Evidence for cellular uptake of recombinant hepatitis C virus non‐enveloped capsid‐like particles

“…Recently, defective HCV genomes lacking envelope-coding sequences have been identified to be predominant in several patients [10,11,28]. It is tempting to speculate that non-enveloped capsids might be produced under certain circumstances, containing either the defective or the full-length genome.…”

Naturally Occurring Hepatitis C Virus Subgenomic Deletion Mutants Replicate Efficiently in Huh-7 Cells and Are trans -Packaged In Vitro To Generate Infectious Defective Particles

“…Because of this huge genetic diversity, HCV is currently classified into six major genotypes and more than 80 subtypes (44). Recombination may be another mechanism exploited by HCV to increase genetic diversity: naturally occurring intergenotypic recombinant viruses that often have their recombination points in the trans-membrane domains of NS2 were recently identified (20,21,26,27,33,35).Recent publications have reported the presence of natural HCV subgenomic RNAs in serum and liver of infected patients, mostly containing large in-frame deletions from E1 up to NS2, always found together with the full-length wild-type (wt) RNAs (5,16,36,54). These mutant viral genomes persist for a long time (at least 2 years), and sequence analysis suggests that subgenomic (the predominant species during this period) and full-length HCV evolve independently (54).…”

“…Recent publications have reported the presence of natural HCV subgenomic RNAs in serum and liver of infected patients, mostly containing large in-frame deletions from E1 up to NS2, always found together with the full-length wild-type (wt) RNAs (5,16,36,54). These mutant viral genomes persist for a long time (at least 2 years), and sequence analysis suggests that subgenomic (the predominant species during this period) and full-length HCV evolve independently (54).…”

“…These mutant viral genomes persist for a long time (at least 2 years), and sequence analysis suggests that subgenomic (the predominant species during this period) and full-length HCV evolve independently (54). The relative abundance and persistence of such subgenomic RNAs in vivo suggests that (i) they are capable of autonomous replication and (ii) they can be packaged and secreted in infectious viral particles, presumably harboring the envelope proteins from wt virus coinfecting the same cell.…”

Genetic Analysis of Hepatitis C Virus with Defective Genome and Its Infectivity in Vitro

“…Most DI viruses occur spontaneously in the course of cell culture infected with a high titer of wild-type viruses. Hepatitis C virus (HCV) with a defective genome has been found in liver and serum specimens of some HCV patients (4,8,15). HCV has a plus-strand RNA genome that encodes the viral core, E1, E2, and p7 structural proteins and NS2, NS3, NS4A, NS4B, NS5A, and NS5B nonstructural proteins (10).…”