Hepatitis B Virus Virions Produced Under Nucleos(t)ide Analogue Treatment Are Mainly Not Infectious Because of Irreversible DNA Chain Termination

Abstract

Nucleos(t)ide analogues (NAs) have been widely used for the treatment of chronic hepatitis B (CHB). Because viral DNA polymerase lacks proofreading function (3′ exonuclease activity), theoretically, the incorporated NAs would irreversibly terminate viral DNA synthesis. This study explored the natures of nascent hepatitis B virus (HBV) DNA and infectivity of progeny virions produced under NA treatment. HBV infectivity was determined by infection of HepG2‐NTCP cells and primary human hepatocytes (PHHs). Biochemical properties of HBV DNA in the progeny virions were investigated by qPCR, northern blotting, or Southern blotting hybridization, sucrose gradient centrifugation, and in vitro endogenous DNA polymerase assay. Progeny HBV virions produced under NA treatment were mainly not infectious to HepG2‐NTCP cells or PHHs. Biochemical analysis revealed that under NA treatment, HBV DNA in nucleaocapsids or virions were predominantly short minus‐strand DNA with irreversible termination. This finding was supported by the observation of first disappearance of relaxed circular DNA and then the proportional decline of HBV‐DNA levels corresponding to the regions of PreC/C, S, and X genes in serial sera of patients receiving NA treatment. Conclusion: HBV virions produced under NA treatment are predominantly replication deficient because the viral genomes are truncated and elongation of DNA chains is irreversibly terminated. Clinically, our results suggest that the viral loads of CHB patients under NA therapy vary with the different regions of genome being detected by qPCR assays. Our findings also imply that NA prevention of perinatal and sexual HBV transmission as well as infection of transplanted livers works not only by reducing viral loads, but also by producing noninfectious virions.

Biogenesis and molecular characteristics of serum hepatitis B virus RNA

“…Due to the technical impracticality of separating RNA-virions from DNA-virions, we first examined the infectivity of total viral particles and 3TC-arrested RNA-containing viral particles collected from HepAD38 cells, and the total naked capsids and 3TC-arrested RNA-containing naked capsids collected from HepDES19 cells, in HepG2-NTCP cells. As shown in S9 Fig , while the DNA-virion successfully infected HepG2-NTCP cells as evidenced by HBc immunofluorescence and HBeAg ELISA, the naked capsids, without viral envelope for NTCP binding, failed to establish infection; Consistent with a previous report [ 26 ], the 3TC-arrested RNA-virion was noninfectious due to the irreversible DNA chain termination activity of the incorporated nucleotide analogue.…”

“…Due to the similar protein composition and density between HBV RNA-virion and DNA-virion, it is technically challenging to purify RNA virions. Though inhibition of HBV polymerase could selectively enrich the production of RNA-virions in cell cultures ( Fig 1 and S1 Fig ), the 3TC- or entecavir-arrested RNA virions are replication-incompetent and therefore noninfectious due to the irreversible DNA chain termination effect of drugs ( Fig 8 and S9 and S10 Figs) [ 24 , 26 ]. In this study, we used L-FMAU, a thymidine analogue that inhibits HBV DNA replication mechanistically distinct from other NAs [ 39 ], to produce RNA virions.…”

Amino acid residues at core protein dimer-dimer interface modulate multiple steps of hepatitis B virus replication and HBeAg biogenesis

“…M610A) at 37˚C for 30 min and followed by proteinase K (200 μg/mL) digestion at 45˚C for 1 h. For quantification of HBV DNA, a SYBR Green Real-time PCR method were performed by Light-Cycler 480 II Real-time PCR Detection System (Roche, Mannheim, Germany). The primers used to detect HBV viral load is as follows: F (303-322 nt): 5 0 -TGGCCAAAATTCGCAGTCCC-3 0 , R (448-425 nt): 5 0 -GAAGAACCAACAAGAAGATGAGGC-3 0 [74]. The serial dilutions of pHBV1.3 plasmid were used as standards of quantification.…”

Maternal antiviral treatment safeguards infants from hepatitis B transmission in contingencies of delayed immunoprophylaxis

“…The actual biological mechanism of maternal antiviral prophylaxis merits further investigation. One possible explanation might be that maternal antiviral prophylaxis during late pregnancy could not only reduce viral loads, but also result in defective HBV virions owing to irreversible DNA chain termination 37,38 …”