Improved Method for Rapid and Efficient Determination of Genome Replication and Protein Expression of Clinical Hepatitis B Virus Isolates

Abstract

Different hepatitis B virus (HBV) genotypes and variants are associated with different clinical outcomesand/or response to antiviral therapy, yet the comparison of the in vitro replication capacity of a large number of clinical isolates remains technically challenging and time-consuming. Although the full-length HBV genome can be amplified from high-titer blood samples by PCR using High Fidelity plus DNA polymerase and primers targeting the conserved precore region, the HBV clones thus generated are replication deficient due to the inability to generate the terminally redundant pregenomic RNA essential for genome replication. The transfection experiment is further complicated by PCR errors and the presence of viral quasispecies. A previous study found that the precise removal of non-HBV sequence by SapI digestion led to HBV replication in transfected cells, possibly due to low-level genome circularization by a cellular enzyme. We released HBV genome from the cloning vector using BspQI, an inexpensive isoschizomer of SapI, and increased the efficiency of genome replication by an extra step of in vitro DNA ligation. The uncut plasmid DNA can be used for transfection if the sole purpose is to study envelope protein expression. We found significant PCR errors associated with the High Fidelity plus DNA polymerase, which could be greatly diminished using Phusion DNA polymerase or masked by the use of a clone pool. The reduced PCR error and modified enzymatic steps prior to transfection should facilitate a more widespread functional characterization of clinical HBV isolates, while the clone pool approach is useful for samples with significant sequence heterogeneity.Hepatitis B virus (HBV) can be classified into eight genotypes with a minimum sequence divergence of 8% at the nucleotide level. These genotypes circulate in different parts of the world and are associated with different courses of infection and response to therapy (5,20,21,23). Furthermore, genetic variants can be selected at the late stage of chronic infection (such as precore and core promoter mutants), by vaccination (immune escape mutant), or following therapy with nucleoside analogues (drug-resistant mutants) (3,4,22,34). From a clinical point of view, certain HBV isolates have been associated with fulminant hepatitis with a high mortality rate (19), whereas other strains are linked to occult HBV infection (25). Understanding the molecular basis for diverse outcomes of HBV infection requires the cloning of the 3.2-kb genome from clinical samples, followed by its functional characterization through transfection experiments. In this regard, blood is a more accessible source of the HBV genome than the liver. Virion-associated HBV DNA consists of a full-length minus strand and a plus strand of variable lengths, and it has a relaxed circular configuration (Fig. 1). Fifteen years ago, Günther and colleagues developed a method to amplify the full-length HBV genome using a primer pair targeting the highly conserved precore region, which happens to be present a...

Hepatitis B Virus Genotype C Isolates with Wild-Type Core Promoter Sequence Replicate Less Efficiently than Genotype B Isolates but Possess Higher Virion Secretion Capacity

“…Intracellular envelope proteins were detected by direct Western blot analysis (8,9,32). Briefly, 50 g protein from cell lysate was separated overnight in an SDS-12% polyacrylamide gel and transferred to polyvinylidene difluoride membranes.…”

“…For the Chinese samples, circularized genomes rather than SphI dimers were employed for transfection experiments. Briefly, pUC18 DNA with an altered SapI site was subjected to two rounds of digestion with HindIII and SacI, followed by digestion with SphI to minimize vector self-ligation (32). The vector DNA was ligated with HindIII/SacI-digested PCR products and transformed to MAX Efficiency DH5␣ competent cells (Invitrogen), followed by growth of the entire transformation product in 100 ml of LB broth containing ampicillin.…”

“…Detailed procedures for transfection of Huh7 cells and subsequent detection of genome replication, protein expression, protein secretion, and virion secretion have been described previously (8,9,32,44). Huh7 cells seeded at 6 ϫ 10 5 /wells in six-well plates were transfected with 1.5 g of circularized HBV DNA or 1 g of SphI dimer DNA using TransIT-LT1 reagent (Mirus).…”

Recombinant Covalently Closed Circular Hepatitis B Virus DNA Induces Prolonged Viral Persistence in Immunocompetent Mice

“…However, the HBV replicon-based hydrodynamic injection induces only transient HBV viremia resembling an acute infection in immunocompetent mice. In this regard, a recent model of HBV persistence generated by injection of a pAAV-HBV1.2 plasmid might reflect a role of the adeno-associated virus vector in developing immune tolerance (10).A double-stranded HBV circular genome can be generated in vitro by PCR amplification with subsequent ligation (13,14). However, this approach does not generate substantial cccDNA supercoils with high efficiency.…”

Enhanced hepatitis B virus (HBV) pre-genomic RNA levels and higher transcription efficiency of defective HBV genomes

“…Digestion of the WT and dHBV constructs with BspQI released unit-length genomes, and once inside the cell the transfected linear genome is circularized by host enzymes to form HBV cccDNA. The formation of HBV cccDNA is an essential step in the formation of HBV pgRNA and for HBV replication when using linear unit-length constructs (Günther et al, 1995;Qin et al, 2011b;Vivekanandan et al, 2009Vivekanandan et al, , 2010. Each WT construct and its corresponding dHBV construct were transfected separately into Huh7 cells in a 24-well plate.…”