Functional analysis of complex hepatitis B virus variants associated with development of liver cirrhosis.
OBJECTIVE
Development of cirrhosis in renal transplant recipients with chronic hepatitis B is associated with the accumulation of complex hepatitis B virus (HBV) variants carrying deletions in the C gene and/or preS region and deletions/insertions in the core promoter. Here, we characterized for the first time the phenotype of these complex HBV variants.


METHODS
Representative full-length genomes of the HBV variants that were isolated and cloned from serum and liver of an immunosuppressed renal transplant recipient before and during end-stage liver disease were transfected into the human hepatoma cell line HuH7 and functionally analyzed.


RESULTS
The variant genomes showed considerably reduced levels of precore and surface messenger RNA (mRNA) and of the major spliced pregenomic RNA, an increased level of pregenomic RNA, and a partial or complete defect in hepatitis B e antigen, core, and surface protein expression/secretion. Very low amounts of variant core protein with internal deletion were detectable. Reduced hepatitis B surface antigen secretion of some variants correlated with aberrant localization of surface proteins in endoplasmic reticulum. Despite the defects in viral protein expression, enhanced replication and enrichment in competition to wild-type HBV were observed. Enhanced reverse transcription and possibly increased levels of pregenomic RNA seem to be responsible for this effect.


CONCLUSIONS
Development of cirrhosis is associated with accumulation of complex variants, which exhibit a drastically altered phenotype combining enhanced replication with defects in protein expression. This phenotype appears to be based on the major mutations in the core promoter and C gene but is considerably influenced by additional mutations throughout the genome.
Märschenz
Stefanie
S
Institut für Virologie (Helmut-Ruska-Haus), Charité-Universitätsmedizin Berlin, Campus Mitte, Berlin, Germany.
Endres
Anne-Sophie
AS
Brinckmann
Anja
A
Heise
Tilman
T
Kristiansen
Glen
G
Nürnberg
Peter
P
Krüger
Detlev H
DH
Günther
Stephan
S
Meisel
Helga
H
D015152:Blotting, Northern; D015139:Blotting, Southern; D004797:Enzyme-Linked Immunosorbent Assay; D016679:Genome, Viral; D006513:Hepatitis B e Antigens / Q000276:immunology / Q000378:metabolism; D006515:Hepatitis B virus / Q000235:genetics* / Q000276:immunology; D019694:Hepatitis B, Chronic / Q000150:complications / Q000473:pathology / Q000821:virology*; D006801:Humans; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D008103:Liver Cirrhosis / Q000209:etiology* / Q000473:pathology; D009154:Mutation; D010641:Phenotype; D012367:RNA, Viral / Q000235:genetics* / Q000502:physiology; D012189:Retrospective Studies; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D014779:Virus Replication
D006513:Hepatitis B e Antigens; D012367:RNA, Viral