Defective hepatitis B virus DNA is not associated with disease status but is reduced by polymerase mutations associated with drug resistance

Abstract

Defective hepatitis B virus DNA (dDNA) is reverse-transcribed from spliced hepatitis B virus (HBV) pregenomic messenger RNA (pgRNA) and has been identified in patients with chronic HBV (CH-B). The major 2.2-kb spliced pgRNA encodes a novel HBV gene product, the hepatitis B splice protein (HBSP) via a deletion and frame shift within the polymerase. Although spliced RNA and HBSP expression have been associated with increased HBV DNA levels and liver fibrosis, the role of dDNA in HBV-associated disease is largely undefined. Our aims were to (1) compare the relative proportions of dDNA (% dDNA) in a range of HBV-infected serum samples, including patients with human immunodeficiency virus (HIV)/HBV coinfection and HBV-monoinfected persons with differing severities of liver disease, and (2) determine the effect of mutations associated with drug resistance on defective DNA production. Defective DNA was detected in 90% of persons with CH-B. There was no significant difference in the relative abundance of dDNA between the monoinfected and HIV/HBV-coinfected groups. We also found no association between the % dDNA and alanine aminotransferase, hepatitis B e antigen status, HBV DNA levels, fibrosis levels, compensated or decompensated liver cirrhosis, genotype, or drug treatment. However, the % dDNA was significantly lower in individuals infected with lamivudine-resistant (LMV-R) HBV compared with wild-type HBV (P < 0.0001), indicating that antiviral drug resistance alters the balance between defective and genomic length DNA in circulation. Experiments in vitro using HBV encoding LMV-R mutations confirmed these results.

Conclusion: Our results identified no association between dDNA and parameters associated with disease status and suggested that the relative abundance of dDNA is largely dependent on the integrity of the HBV polymerase and is unrelated to the severity of liver disease.

Fig. 1

The relative abundance of defective DNA in persons infected with lamivudine-sensitive (LMV-S) and resistant (LMV-R) HBV. Samples were separated into discrete % dDNA ranges, and the frequency with which each range was represented in the population (y axis) was plotted against the midpoint of the range (x axis). The ranges used were 0%, >0%, and <1%, >1% and <%5, >5% and <10%, and >10%. Eighty-nine percent of samples with >5% dDNA were LMV-S, compared with 70% of samples being LMV-S overall.

Fig. 2

The relative abundance (y axis) of defective DNA and spliced pgRNA in HBV nucleocapsids and extracellular virions, harvested 5 days after transient transfection with HBV infectious clones encoding polymerase mutants (x axis). Error bars represent 1 standard deviation, n = 3. *Statistically significant differences from 1.3D WT.

Fig. 3

Box and whisker plot of % dDNA (y axis) distributions for all LMVr samples without the rtV173L mutation compared with a population of LMVr samples with the rtV173L compensatory mutation. The horizontal line inside the box represents the median values, with the box enclosing values between the 25% percentile value and the 75% percentile value. Error bars enclose 90% of values, and dot points represent outliers beyond the 90^th percentile.