Introduction
Up to 10% of the estimated 40 million individuals infected with HIV type 1 worldwide are co-infected with hepatitis B virus (HBV) [1–3] [4,5]
The reason(s) for increased progression of liver disease in HIV/HBV co-infected individuals are unclear. The host immune response is an important mediator of liver disease in chronic hepatitis B, but this cannot explain the more aggressive course seen in many HIV/HBV co-infected patients since their immune systems are suppressed. In patients receiving HAART, however, it is possible that restoration of the immune response is a factor in liver disease progression, although this is yet to be confirmed. In general, the HBV is not cytopathic; however, in immunosuppressed patients, including HIV-infected individuals, a more fulminant form of HBV has been described, fibrosing cholestatic hepatitis (FCH) [6,7]
Liver disease progression in individuals chronically infected with HBV is associated with the accumulation of numerous mutant viral genomes, in particular viruses with deletions in the core/precore and envelope genes [8–12] [10,13,14] in vitro . Replication of the mutant viruses was, however, rescued when the HBV core protein was supplied in trans [13,14]
In HIV/HBV co-infected individuals, increased HBV replication and lower rates of HBeAg seroconversion compared to HBV mono-infected individuals [15–17] [18,19]
Materials and methods
Cohort 1: full genome analysis
To enable complete HBV genome analysis, serum was initially obtained from 10 HIV-infected individuals (P1 to P10) from Australia and the USA who were chronically infected with HBV genotype A, the dominant genotype in HIV/HBV co-infected individuals in the USA and Australia [18] [18] [18] [20] Table 1 ). The complete antiretroviral drug history was obtained for seven of the ten patients.
Table 1: Clinical data prior to (pre) and during (post) lamivudine (LMV) therapy.
DNA extraction and hepatitis B virus genomic-length polymerase chain reaction
DNA was extracted from serum as described previously [20] [21]
Sequence analysis
Genomic-length amplicons from patients in cohort 1 were sequenced using the ABI platform (MicroMon, Monash University) [22] [23,24] [25]
Neighbour-joining phylogenetic trees were constructed using the Phylip Seqboot, DNADist/ProtDist, Neighbour and Consense programs (http://evolution.gs.washington.edu/phylip.html [26] [27]
Clonal analysis of mutations in cohort 1
To determine the frequency of a novel precore/core deletion mutation (termed –1G) in the HIV/HBV co-infected individuals and HBV mono-infected individuals from cohort 1, the region encompassing the mutation was amplified by PCR, cloned and sequenced. PCR was performed using either PicoMaxx high fidelity PCR (Stratagene) or Expand Hi Fi (Roche, Castle Hill, New South Wales, Australia) polymerase mixes and ten clones were sequenced from each patient.
Cohort 2: precore/core gene analysis
To determine the abundance of the –1G deletion mutation in other HIV/HBV co-infected individuals, HBV DNA sequences encompassing the mutation were amplified from an additional 39 HIV/HBV co-infected and 57 HBV mono-infected individuals infected with HBV genotype A and analysed by direct sequencing. The genotype of all HBV isolates was determined using the SeqHepB program [23] [18] [20]
Results
Hepatitis B virus sequence variability (cohort 1)
Analysis of genomic-length nucleotide sequences amplified from ten HBeAg positive HIV co-infected individuals infected with HBV genotype A (P1 to P10) identified a maximum of 1.3% nucleotide variability among all sequences prior to LMV therapy and 1.4% variability following between 9 and 74 months therapy (data not shown). HBV sequences from individual patients only varied by a maximum of 0.5% in pre and post LMV therapy samples. The sequences from five HBV-mono-infected individuals (P11 to P15) varied by up to 3.7%, with a maximum of 0.8% variability in HBV sequences from individual patients following between 34 and 47 months LMV therapy. Although the sample sizes were small, the difference in sequence variability between the ten HIV/HBV co-infected and the five HBV mono-infected individuals was statistically significant (P < 0.002).
Phylogenetic analysis of the deduced amino acid sequences of the polymerase gene showed that all HBV sequences amplified from HIV/HBV co-infected (Fig. 1 ) and HBV mono-infected individuals (results not shown) grouped within the A2 (Ae, European/North American) subtype of HBV genotype A, with the observed topology receiving strong bootstrap support. The sequences from HIV/HBV co-infected and mono-infected individuals were distributed throughout the A2 clade and there was no significant clustering of either group. Similar tree topologies were obtained using neighbour-joining and maximum likelihood algorithms, indicating the observed topologies were valid (results not shown).
Fig. 1: Unrooted neighbour-joining phylogenetic tree of the deduced HBV polymerase amino acid sequences amplified from ten HIV co-infected individuals (A to J) and 8 HBV genotype A reference sequences. The sequences grouped into the A1 and A3 subtypes respectively are circled, with sequences in subtype A2 indicated by the solid line on the right and side of the cladeogram. The accession numbers of the reference sequences are indicated. Bootstrap values (1000 replicates) at the major nodes are indicated.
Identification of a novel hepatitis B virus core/precore mutation (cohort 1)
Despite the high level of nucleotide sequence conservation, a number of important mutations were identified in HBV coding sequences and regulatory elements. Analysis of genomic-length HBV sequences from ten HIV/HBV co-infected individuals and five HBV mono-infected individuals identified a –1G deletion in the HBV precore and core genes of six HIV/HBV co-infected individuals and three HBV mono-infected individuals respectively (Table 2 ). All individuals harbouring the –1G mutation were HBeAg positive. The –1G deletion was detected in the six HIV/HBV co-infected individuals during LMV therapy and HAART and was also detected in four of these individuals prior to LMV therapy and HAART (Table 2 ). Although the presence of the –1G mutation was not associated with HIV RNA levels, CD4 cell counts, or ALT levels (result not shown), this clinical information was not available for all patients and the sample sizes were too small to permit statistical analysis. The –1G mutation was, however, associated with high HBV viral DNA concentrations (P = 0.002, see later). Of the five HBV mono-infected individuals selected for genomic-length analysis, direct sequencing showed that the –1G mutation was present at low levels in three individuals prior to LMV therapy and was not present in any individuals following LMV treatment (Table 2 ). Of these three individuals, one of them (P13) was anti-HBc negative, another had severe liver disease that later required a liver transplant (P14), and the disease status of the third individual (P15) was unknown. The amplification of sequences harbouring the –1G mutation with two different polymerase/proof-reading enzymes confirmed the mutation was not an artifact of the PCR itself (data not shown).
Table 2: Detection of –1G mutation by direct sequencing and cloning prior to (pre) and during (post) lamivudine (LMV) therapy or HAART.
The –1G deletion was located in a homopolymeric string of guanosine (G) nucleotides between HBV core nucleotides 184 and 190 (core codons 63 and 64) and introduced a frameshift that terminated the coding sequence of the HBV core and precore genes (Fig. 2 ). This mutation was located in a major CD4 T-cell epitope (Fig. 2 ) and the molecular weight of the deduced truncated core gene product was 7.5 kDa, compared to 21 kDa for the full-length core protein. Similarly, the mutation also truncated the HBV precore protein. The HBV precore protein (p25) is a 25 kDA protein that is subsequently processed into 22 kDa (p22) and 17 kDa (HBeAg) proteins, the latter being secreted from the cell. Premature termination of p25 and p22 as a result of the –1G mutation would result in cellular proteins of 10.5 kDa and 8.4 kDa, respectively. The N-terminal sequences of P22 and HBeAg are identical, suggesting that any secreted truncated peptide resulting from the –1G mutation would also be 8.4 kDa in size; however, the absence of C-terminal precore sequences important for secretion [28]
Fig. 2: The location of the –1G deletion in the deduced HBV precore and core genes. The top panel shows location of homopolymeric G sequence in relation to characterized T cell and B cell epitopes
[15–17] and the deduced gene products encoded by the HBV precore and core genes. The lower panel shows the deduced truncated precore and core gene products that would result from the –1G deletion.
Clonal analysis of –1G mutation in cohort 1
To determine the abundance of the –1G mutation relative to wild-type HBV sequence on individual virus genomes, the region was amplified by PCR and cloned, using serum-derived DNA extracted prior to and/or during lamividuine therapy. The –1G deletion was detected in clones from four of six HIV/HBV co-infected individuals prior to LMV therapy and HAART and all six individuals post LMV therapy and HAART. The –1G mutation was the dominant sequence relative to wild-type HBV in four HIV/HBV co-infected individuals, with 50 to 90% of clones harbouring the –1G mutation (Table 2 ). Clones harbouring wild-type HBV sequence were, however, always detected but usually as a minor population with the dominant –1G deletion population. No clones harbouring the –1G mutation were obtained if the mutation was not visible by direct sequencing. Although the –1G mutation was observed at low levels by direct sequencing in three HBV mono-infected individuals prior to LMV treatment, clones harbouring the mutation were only obtained from two patients. The mutation was detected in one clone from one HBV mono-infected individual and four clones from the other. Cloned DNA from the third HBV mono-infected individual did not harbour the –1G deletion, however some clones had either an eight nucleotide deletion (10%, one clone) or a 26 nucleotide insertion in the BCP (30%, three clones) that did not disrupt the HBV core gene open reading frame (data not shown).
Prevalence of the –1G mutation in additional HIV/hepatitis B virus co-infected persons (cohort 2)
To determine the abundance of the –1G mutation in additional HIV/HBV co-infected and HBV mono-infected individuals, direct sequencing of HBV genomes was performed on HBV sequences from an additional 39 HIV/HBV co-infected individuals and 57 HBV mono-infected individuals infected with HBV genotype A. The mutation was not detected in any of the additional 57 HBV mono-infected individuals. Overall, including the ten HIV/HBV co-infected and five HBV mono-infected patients analysed by genomic-length sequencing and cloning from cohort 1 (P1 to P15), the –1G deletion was detected in 34% of samples from HIV/HBV co-infected individuals and 5% of samples from HBV mono-infected individuals (P < 0.001, data not shown). In the 49 HIV/HBV co-infected individuals, the –1G mutation was detected in approximately 20% of (5/26) sequences prior to LMV therapy and 40% (20/48) of sequences during lamivudine therapy. In 62 HBV mono-infected individuals, the –1G mutation was only detected in three patients prior to LMV treatment and was not detected during LMV therapy.
Association of the –1G mutation with hepatitis B virus DNA concentration
Mann–Whitney rank sum analysis comparing the DNA concentrations in 58 samples from persons in cohorts 1 and 2 infected with –1G mutant or wild-type HBV, showed that the HBV DNA concentration was higher in patients harbouring the –1G mutation than in patients infected with wild-type HBV alone (P = 0.002) (Table 3 ). The –1G mutation was predominantly detected in individuals with a HBV DNA concentration of ≥1 × 106 IU/ml (16/22 individuals), with a maximum DNA concentration of 1 × 1010 IU/ml, although it was also detected in one individual with a HBV DNA concentration of 6.7 × 104 IU/ml and two individuals with a HBV DNA concentration of ≥ 4.5 × 105 IU/ml (not shown). In persons without the –1G mutant HBV, HBV DNA concentrations ranged from 8 × 102 to 1.7 × 1010 IU/ml (not shown).
Table 3: Mann–Whitney non-parametric analysis of hepatitis B virus (HBV) DNA concentration compared with detection of the –1G mutant.
Additional hepatitis B virus mutations identified by genomic length sequencing
In addition to previously described mutations in the HBV basal core promoter and polymerase/envelope genes in HIV/HBV co-infected individuals [18] Table 4 ). A number of these mutations were detected exclusively in the HIV/HBV co-infected individuals and included:
Table 4: Hepatitis B virus (HBV) mutations that emerged during lamivudine (LMV) therapy in 10 HIV individuals (P1 to P10) co-infected with HBV genotype A.
Core An cL60I T-cell epitope mutation emerged during LMV therapy in the HBV core gene of one HIV/HBV co-infected individual and an cE77Q mutation emerged in two others. These mutations were also observed in HBV mono-infected individuals following LMV therapy, however a cI59V core gene mutation was detected exclusively in two HIV/HBV co-infected individuals prior to and during LMV treatment. The cI59V and cL60I mutations were located in the CD4 T-cell epitope that harboured the –1G deletion (Fig. 2 ).HBV polymerase, HBx and regulatory sequences . An RNaseH rhT119S polymerase mutation detected in one HIV/HBV co-infected individual (P2) also altered HBx at xD48E. A number of mutations were also identified in key HBV regulatory elements, including the basal core promoter (BCP), preS1 promoter, and the X promoter. BCP A1762T/G1764A mutations were detected prior to LMV in one HIV/HBV co-infected individual (P6) and four HBV mono-infected individuals (P11, P12, P14, P15) and following LMV therapy these mutations were detected in two HIV/HBV co-infected individuals (P2, P6) and three HBV mono-infected individuals (P12, P14, P15). HBV encoding G1786T, C1788G, and A1792T BCP mutations emerged during LMV therapy in one individual (P4) and these mutations also altered HBx at xR138M, xH139G and xK140I respectively.
The possible role of LMV or other antiretroviral drugs in the emergence of these mutations requires additional investigation.
Discussion
We have identified a novel –1G mutation in the HBV core and precore genes that was more abundant in HIV/HBV co-infected individuals than patients with HBV mono-infection. The –1G mutant HBV was always detected in the presence of wild-type HBV and was the dominant virus in some HIV/HBV co-infected individuals. The –1G mutation was associated with high HBV DNA concentrations, suggesting it may have a high replication phenotype similar to HBV core mutants identified in some immunosuppressed HBV mono-infected individuals [13,14] [29–31] [18] [32]
The –1G deletion mutation resulted in premature termination of the deduced HBV precore and core genes and from all the samples analysed by direct sequencing from the different cohorts, was present in 34% of HIV/HBV co-infected individuals and 5% of HBV mono-infected individuals. This mutation leads to a premature stop codon at amino acid 64 that could alter HBV pathogenesis through one of two mechanisms. First, this altered protein may lead to differences in virulence of the virus, as the absence of a complete core gene means the virus could only replicate in the presence of wild-type HBV. This is analogous to core deletion mutant viruses detected in renal transplant recipients, which are replication defective, but can be ‘rescued’ by supplying the HBV core protein in trans [13,14] [33]
The –1G mutation was identified in 5% (three patients) of HBV mono-infected individuals, although the mutation was only detected at low levels prior to LMV therapy. Although we detected the –1G mutation prior to and during LMV therapy in HIV/HBV co-infected individuals and prior to therapy in HBV mono-infected individuals, the small sample size precluded statistical analysis of any possible role for LMV in selection of this mutation. Mutations in homopolymer nucleotide stretches have been identified previously in T4 bacteriophage, polyomavirus and drug resistant isolates of herpes simplex virus (HSV) [34–37] [8–10]
The HBV precore gene is the precursor of the secreted HBeAg [38,39] [40]
In addition to mutations detected in the HBV precore/core genes, mutations also emerged in the HBx, envelope and the polymerase genes of some HIV/HBV co-infected individuals. A number of mutations also emerged in key HBV regulatory regions, including in the transcription regulators of the HBx, core and envelope genes. One individual harboured three BCP mutations at position 1786, 1788 and 1792 that altered the overlapping HBx gene and a second patient harboured an RNaseH mutation that altered the overlapping HBx promoter sequence. HBx is a modest transactivating protein associated with liver disease progression and hepatocellular carcinoma, although its role in HBV replication and pathogenesis remains contentious (reviewed in [41]
The identification of HBV encoding novel precore/core deletion mutants, as well as the emergence of drug resistant viruses [18,19] in vitro and quantifying the HBeAg status in HIV/HBV co-infected and HBV mono-infected individuals. This will tell us whether the –1G mutant has a high replication phenotype, similar to viruses encoding the precore G1896A stop-codon mutuation [42]
Acknowledgements
This work was supported by NIH grant no AIO60449. We thank Tim Shaw for assistance with the statistical analysis.
This work was supported by NIH grant no AIO60449 (C.L.T., P.A.R. and S.A.L.). Some of the data presented in this manuscript was collected by the Multicenter AIDS Cohort Study (MACS), USA, funded by the National Institute of Allergy and Infectious Diseases, the National Cancer Institute and the National Heart, Lung and Blood Institute.
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