High serum levels of pregenomic RNA reflect frequently failing reverse transcription in hepatitis B virus particles

Abstract

Background: Hepatocytes infected by hepatitis B virus (HBV) produce different HBV RNA species, including pregenomic RNA (pgRNA), which is reverse transcribed during replication. Particles containing HBV RNA are present in serum of infected individuals, and quantification of this HBV RNA could be clinically useful.

Methods: In a retrospective study of 95 patients with chronic HBV infection, we characterised HBV RNA in serum in terms of concentration, particle association and sequence. HBV RNA was detected by real-time PCR at levels almost as high as HBV DNA.

Results: The HBV RNA was protected from RNase and it was found in particles of similar density as particles containing HBV DNA after fractionation on a Nycodenz gradient. Sequencing the epsilon region of the RNA did not reveal mutations that would preclude its binding to the viral polymerase before encapsidation. Specific quantification of precore RNA and pgRNA by digital PCR showed almost seven times lower ratio of precore RNA/pgRNA in serum than in liver tissue, which corresponds to poorer encapsidation of this RNA as compared with pgRNA. The serum ratio between HBV DNA and HBV RNA was higher in genotype D as compared with other genotypes.

Conclusions: The results suggest that HBV RNA in serum is present in viral particles with failing reverse transcription activity, which are produced at almost as high rates as viral particles containing DNA. The results encourage further studies of the mechanisms by which these particles are produced, the impact of genotype, and the potential clinical utility of quantifying HBV RNA in serum.

Fig. 1

The HBV RNA level in serum correlated with HBV DNA in serum (a, n = 95), and with pgRNA (b, n = 65) in the liver. Filled circles, HBeAg+, unfilled circles HBeAg–

Fig. 2

HBV RNA in serum was present in particles with similar properties as HBV virions. a) Quantification of HBV DNA, HBV RNA and HBsAg in Nycodenz gradient fractions from HBV positive serum. b) A parallel experiment in which the serum was treated with the detergent Tween-80 before fractionation

Fig. 3

Sequences of the core promoter and precore regions of HBV identified in serum from three patients. The sequences were obtained by Sanger sequencing of products from separate amplification of DNA and RNA. The RNA sequences represent a merge of data from the 5′ end (amplified by primers 1819F and 1966R) and the 3′ end (amplified by primers 1603F and 1882R) of pgRNA. The DNA sequences were obtained from PCR using primers 1603F and 2058R. ORF, open reading frame. The priming (1863–1866 in the 5′ loop) and primer recipient sites (nt 1824–1827 in the 3′ loop) are boxed

Fig. 4

The ratio between HBV DNA and HBV RNA levels in serum was significantly higher in patients infected with HBV genotype D (n = 51) as compared with non-D genotypes (n = 44; p = 0.0001)

Fig. 5

Levels of precore RNA and pgRNA in liver tissue (a) and serum (b) as measured by digital PCR. The level of pgRNA was on average 30 times higher (1.5 log₁₀ units) than precore RNA in liver tissue, as compared with 200 times higher (2.3 log₁₀ units) in serum, indicating that precore RNA is encapsidated and secreted to serum 6.6 times less effectively than pgRNA