Functional characterization of naturally occurring variants of human hepatitis B virus containing the core internal deletion mutation

Abstract

Naturally occurring variants of human hepatitis B virus (HBV) containing the core internal deletion (CID) mutation have been found frequently in HBV carriers worldwide. Despite numerous sequence analysis reports of CID variants in patients, in the past decade, CID variants have not been characterized functionally, and thus their biological significance to HBV infection remains unclear. We report here two different CID variants identified from two patients that are replication defective, most likely due to the absence of detectable core protein. In addition, we were unable to detect the presence of the precore protein and e antigen from CID variants. However, the production of polymerase appeared to be normal. The replication defect of the CID variants can be rescued in trans by complementation with wild-type core protein. The rescued CID variant particles, which utilize the wild-type core protein, presumably are enveloped properly since they can be secreted into the medium and band at a position similar to that of mature wild-type Dane particles, as determined by gradient centrifugation analysis. Our results also provide an explanation for the association of CID variants with helper or wild-type HBV in nature. The significance of CID variants in HBV infection and pathogenesis is discussed.

FIG. 1

Identification of replicating CID variants in tumor and nontumor liver tissues. (A) Demonstration of the presence of replicative forms of HBV DNA in liver samples by Southern blot analysis. Vector-free HBV DNA was used as the probe. The data from samples N109 and T109 are shown here as an example. A lambda HindIII size marker is shown in lane 3. (B and C) Core gene of HBV, from liver samples containing replicative HBV DNAs, amplified by PCR by using core gene-specific primers (27). After gel electrophoresis, the PCR-amplified DNA fragments were identified by Southern blotting with the HBV-specific oligonucleotide probe outside (HBC-7) (B) or inside (HBC-9) (C) the deletion hot spot of the core gene (65). HBC-7 (5′-CTG TGG AGT TAC TCT CTT TTT TGC-3′) contains the consensus HBV sequence in the core gene from nt 1937 to nt 1960. HBC-9 (5′-ATG TCA ATG TTA ATA TGG GCC TAA AAA TCA GA-3′) contains the HBV consensus sequence in the core gene from nt 2165 to nt 2196. The sequence of HBC-9 resides within the core internal deletion, and it does not hybridize with HBV sequences containing the core internal deletion. RC, relaxed-circle DNA; SS, single-stranded DNA.

FIG. 2

Sequence analysis of the 1-kb DNA fragments used for the construction of the HBV CID mutant dimers. The diagram illustrates the deletion regions of HBcAg of two different CID variants identified from two different hepatoma patients, T85 and T109. This deletion region does not overlap with any other HBV genes, including X, P (pol), and pre-S/S (envelope). DEL85 is missing aa 88 to 135, while DEL109 is missing aa 82 to 122.

FIG. 3

HBV CID variants are replication defective upon transfection into human hepatoma cell line Huh7. (A) Five days after transfection with wild-type (pWT) (55) or mutant HBV, viral DNAs from intracellular core particles were harvested and subjected to Southern blot analysis with the 3.1-kb full-length vector-free HBV DNA probe. (B) Encapsidation activity was assayed by primer extension using core particle-associated viral RNA from transfected cultures and a 5′-end-labeled oligonucleotide primer (nt 1980 to nt 2001), which was described in a previous report (52). (C) Twenty-five micrograms of cellular RNA from transfected cells was subjected to Northern blot analysis and probed with a 3.1-kb full-length HBV probe (top). Similar amounts of cellular RNA were used in all of the lanes, as indicated by the similar intensities of ethidium bromide staining (bottom). (D) The CAT reporter gene was fused in frame with pol sequences originating from pWT, pDEL85, and pDEL109. CAT activities of the pol-CAT fusion proteins were measured 2 days after transfection as detailed elsewhere (48). (E) The core proteins produced from pWT, pDEL85, and pDEL109 were analyzed by immunoblot assay with a rabbit anti-core antibody.

FIG. 4

The replication-defective CID variants can be rescued by trans complementation with wild-type HBcAg and can be secreted into the medium with a buoyant density similar to that of wild-type HBV. (A) Increasing doses of a wild-type HBcAg expression vector (pSVC) were cotransfected with constant amounts of pWT, pDEL85, and pDEL109. Viral DNAs from intracellular core particles were analyzed by Southern blotting as described in the legend to Fig. 3. (B) Ten micrograms of pDEL85 or pDEL109 was transfected either alone or with 10 μg of pSVC. Extracellular HBV particles from 20 ml of conditioned medium were collected 5 days after transfection via centrifugation through a 20% sucrose cushion. HBV replication activity was assayed as described in the legend to Fig. 3. (C) Conditioned medium was collected from cells transfected with 10 μg of pWT. Viral particles in the medium were purified through a 20% sucrose cushion and subjected to isopycnic centrifugation in a gradient of 20 to 50% (wt/vol) cesium chloride. Fractions were collected for the assay of HBsAg with the Abbott Auszyme EIA kit (top). To locate the fractions containing HBV genomes, Southern blot analysis was performed as described in Materials and Methods (bottom). (D) Conditioned medium from cells transfected with 10 μg of DEL85 and pSVC was assayed for HBsAg and HBV DNA as described in the legend to Fig. 4B and C. RC, relaxed-circle DNA; SS, single-stranded DNA.

FIG. 5

The internally deleted core proteins produced by the CID variants can be detected in vitro but not in vivo. The flu epitope peptide sequence (YPYDVPDYA) from the influenza virus hemagglutinin was introduced into the carboxyl termini of the wild-type and CID core proteins in an SV40 expression vector (see Materials and Methods). The wild-type core protein from pWT and the wild-type and deleted core flu fusion proteins from pSVCflu, pSV85flu, and pSV109flu were measured by immunoblot analysis with anti-core (A) or anti-hemagglutinin (B) antibody. (C) The wild-type and deleted core proteins were expressed in vitro from pSPC, pSP85, and pSP109 (see Materials and Methods) by using a rabbit reticulocyte lysate system (Promega Co.). The in vitro-synthesized proteins were analyzed on an SDS–12% polyacrylamide gel (top). pSP109ATA is a derivative of pSP109, except that the ATG initiation codon of the core gene has been changed to ATA. To control for equal amounts of RNAs used in the in vitro translation experiment, the in vitro-synthesized RNA transcripts from these plasmids were quantitated by gel electrophoresis (bottom). The beta-actin transcript, with a size of 360 nt, from pRT1 was used as a positive control and a size marker.