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Stephen Locarnini, Victorian Infectious Diseases Reference Laboratory, Victoria, Australia
Antiviral drug resistant mutants emerge as a function of at least six factors: the viral mutation frequency, the intrinsic mutability of the antiviral target site, the selective pressure exerted by the drug, the magnitude and rate of virus replication, the overall replication fitness of the mutant and the availability of replication space The mechanisms of nucleoside/nucleotide analogue resistance include steric hindrance, sub-optimal nucleophilic attack geometry and pyrophosphorolysis. The hepatitis B virus (HBV) is an ancient pathogen of man and has evolved a number of elegant strategies to avoid elimination from an infected host. Thus the emergence of antiviral drug resistance during the treatment of HBV infections was not surprising once nucleoside/nucleotide analogues were introduced as specific therapy. Because of the overlap of the reading frames of the HBV polymerase (Pol) with the frame-shifted hepatitis B surface antigen (HBsAg), drug-resistant mutations in the HBV Pol can directly impact on the nature of HBsAg and its function, including properties of viral neutralization. The clinical, pathological and public health importance of HBV drug-resistant mutants is now very clear and improved treatment strategies are urgently required to prevent their continued selection, otherwise inadequately treated patients and the wider community will suffer the subsequent disastrous consequences.
Key words: antiviral drug-resistance, cross-resistance, viral dynamics, replication fitness, replication space, molecular modelling, public health impact, compensatory mutations.
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Professor Doug Richman has recently defined an antiviral drug as one that selects for resistance . Antiviral drug resistance depends on the viral mutation frequency, intrinsic mutability of the antiviral target site, the selective pressure exerted by the drug, and the magnitude and rate of virus replication . Other factors include replication fitness and replication space  and each of these factors will be discussed in this review. A number of clinical and virological risk factors have been identified as they pertain specifically to lamivudine and chronic hepatitis B including pre-therapy serum HBV DNA and ALT level [3, 4], inadequate HBV DNA suppression , duration of therapy [6, 7], viral HBsAg serotype/viral genotype  and body mass index . These factors will be covered by Professor Fabien Zoulim.
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There has been considerable confusion with the use of different numbering systems by particular investigators for the various mutated codons in the HBV polymerase (Pol). The reason for this confusion can be found in the fact that HBV is presently classified into 7 main genotypes (A-G) (see Table 1) based on a greater than eight percent difference in nucleotide sequence over the entire genome [9, 10]. Not all HBVs are exactly the same genomic length and the Pol is likewise unequal, due to the presence of deletions or insertions within the linker or spacer domain between the terminal protein and catalytic components of the protein (see Figure 1). To overcome this confusion, a group of investigators developed a genotype - independent numbering scheme for the HBV Pol domain , where the methionine (M) in the YMDD locus of the catalytic C domain of the Pol is numbered as rtM204 rather than 539, 549, 550 or 552 (see Table 1). This numbering system will be used in this paper.
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4. Detection of Drug-Resistant Mutant HBV
There are three major methods for detecting and measuring antiviral drug-resistance: genotypic assays, phenotypic methods and virtual phenotyping.
Genotypic resistance assays use DNA sequencing methods to examine the polymerase region of the HBV genome for recognizable resistance-associated mutations [11-13]. An interpretation of sequence is required (see Table 2). The line probe assay (LiPA) is based on an oligonucleotide hybridization capture system  which probes for previously identified subsets of resistance mutations known to occur in HBV Pol. This approach does not require an interpretation of sequence data.
Phenotypic assays typically use cell culture based methods for measuring the inhibitory concentration of an antiviral drug that reduces viral replication by at least fifty-percent (IC50) and a number of systems are available. These have been recently reviewed by Delaney and colleagues . An increase in the IC50 level, an increase in resistance, or a decrease in susceptibility indicates that more drug is needed to inhibit the mutant virus in vitro relative to the reference virus. Usually a greater than ten-fold increase in IC50 is associated with "resistance" whilst a less than five-fold increase is regarded as "sensitive", and a five to ten-fold increase represents partial resistance .
Virtual phenotyping in the matching of polymerase sequences from a HBV strain of interest to HBV strains that have both identical or near identical sequences and a known antiviral drug phenotype which are held in a large database. A "virtual" phenotype for the test HBV strain can then be assigned by extrapolation and reported as the mean IC50 of all matches to the wild-type virus. An in-house system has been developed at the VIDRL (Virtual Virology: SeqHepB) similar to the analysis module used by Virco for the human immunodeficiency virus (HIV) .
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5. Patterns of Resistance and Profiles of Cross-Resistance
Six major genotypic patterns of lamivudine resistance have been described (see Table 2). In each, the rtM204 of the YMDD locus is altered. The group 2 mutation of rtM204I is the only single mutation, whilst for the other five groups other changes are detected in either the B or A domains of the HBV Pol. Many other secondary changes have been observed including rtL80V/I, rtL82M, rtT128N, rtF166L, rtL179P, rtA181T, rtT184S, rtA200V, rtV207T, rtS213T, rtS219P, rtI224S and rtL229V/M and these also are found in conjunction with the M204 V/I/S mutants [10, 15, 18].
Using transient transfection assays, antiviral cross-resistance testing for nucleoside/nucleotide analogues has been performed against the major lamivudine and famciclovir resistant mutants (see Table 3). Only adefovir and diaminopurine dioxalone (DAPD) demonstrate activity against all these mutants whilst entecavir has partial activity [15, 16, 19].
Several non-nucleoside analogue inhibitors have been developed including the phenylpropenamide derivatives AT-61 and AT-130  as well as LY 582563, a novel antiviral agent from Lilly Pharmaceuticals. All of these compounds have significant antiviral activity against drug-resistant HBV [21, 22].
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6. Viral Dynamics, Replication and Mutation Rate of the Viral Polymerase
The rate of HBV virion production in vivo can influence the production of viral genetic mutants. When compared with HIV and hepatitis C virus (HCV), the HBV production rate is considerably higher. A number of groups have made estimates of HBV production and found it to be in the order of 1011 virions per day compared with 109 for HIV and HCV [23-26]. The half-life of HBV in plasma ranges from 1 to 3 days [23-26] whilst the half-life of an infected cell is 10-100 days [23-26]. With this enormous daily virion production, errors of the replication process will inevitably occur. Viral reverse transcriptases (RTs), unlike DNA polymerases, do not encode for a proof reading or editing activity that can remove a misincorporated dNTP . The error rate of the HBV Pol has been calculated to occur at 10-4 per nucleotide per replication cycle . Therefore, on a daily basis, approximately 1014 nucleotides are replicated with potentially 1010 base-pairing errors. When a viable mutation occurs, the expansion of the virus harbouring this mutation is predicted on the functional consequences of the mutation. In the absence of an intact immune system or other selection pressure, the faster replicating mutant would be expected to outgrow the slower viruses. This seems to be the case in the setting of a newly infected liver, but as the liver becomes fully infected, the effect of selective advantage is reduced , and other factors such as viral fitness and replication space become important (see below). The recent description of a pyrophosphorolysis activity for the duck HBV Pol and its potential role on drug resistance will be discussed below .
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7. Replication Fitness, Selection Pressure & Replication Space
The pattern and kinetics of HBV resistance to antiviral agents can be understood in terms of a balance between replication fitness, replication space and selection pressure.
(i) Replication Fitness
Differences in the phenotypic expression of antiviral drug resistant mutants based on qualitative assessment of the signal densities of the viral replicative intermediates, including cytoplasmic core-associated single-strand HBV DNA as detected by Southern hybridization (ie. yield assay), have been used by a number of investigators as an indicator of "replication fitness" [31-34]. All of these studies claimed that lamivudine resistant HBV was "replication impaired" based on in vitro data generated from transient transfection systems. In the clinical situation however, these viruses become the dominant quasispecies after only a few months of antiviral treatment, and have been associated with and linked to exacerbation of liver disease in chronically infected patients . Additionally, such viruses have been shown to be associated with graft failure in transplant patients . These yield assays are not true measures of viral fitness. The Darwinian definition of fitness is the ability to produce offspring in the setting of natural selection . Clearly, there are differences in viral replication between mutant and wild-type HBV which play an important role in the replacement of "the less fit viral species" in patients undergoing antiviral therapy, but current in vitro assays used for studying HBV, such as transient transfections, are incapable of resolving them.
The relative replication fitness of viral mutants can be assessed using in vitro co-infection competition assays. For HIV, this approach is now regarded as the accepted method of measurement of "competence" or "fitness" [38, 39]. In these assays, by the process of Darwinian competition, there is selection of the "fittest" virus which becomes the predominant species after several rounds of replication. Recently, De Ronde and colleagues (2001)  have extended this approach and been able to quantify the different HIV quasispecies in vitro during one round of replication in cells using molecular beacons and real time nucleic acid sequence based amplification (NASBA). Similar studies need to be carried out for HBV and the recently described recombinant HBV baculovirus model  can be used to deliver replication competent genomes of both wild-type and mutant HBV to HepG-2 cells in a co-infection format (Locarnini S, Bowden S, Bartholomeusz A and Lewin S 2002, unpublished data) using molecular beacons and real-time PCR .
There are also in vitro assays that can describe the behaviour of HBV polymerase mutants such as the kinetics of RNA- and DNA- polymerization , enzyme processivity  and enzyme fidelity and mutation frequencies . The ability of the polymerase to catalyse the incorporation of nucleotides during synthesis of cDNA products before the enzyme dissociates from the template is described as processivity whilst the ability of the polymerase to correct base-pair the template during polymerization is defined as enzyme fidelity. There have only been limited studies that have attempted to describe these enzymatic properties in HBV. Xiong and colleagues [45, 46] have used the baculovirus expressed wild-type  and mutant HBV polymerases to study enzyme inhibition using the active forms of nucleoside/nucleotide analogues as well as natural substrates. These studies were performed in the absence of the nucleocapsid of the virus and so may not necessarily reflect viral polymerase activity in infected cells. However, enzyme processivity of wild-type HBV polymerase has been studied in recombinant HBV nucleocapsids generated by co-expression of HBV Pol and core in the baculovirus expression system [48, 49]. The lack of published studies is a reflection of the inherent limitations of HBV cell culture systems and the technical difficulties of obtaining functionally active HBV Pol in sufficient quantities and enzymatic reliability .
(ii) Selection Pressure
In clinical trials of HIV drugs, most of the beneficial effect resulting from monotherapy is largely reversed within 4 weeks after initiation of treatment . This reversal is directly associated with the emergence of drug-resistant mutants [51, 52]. These observations are also consistent with the existence of drug-resistant populations in patients before treatment  and similar populations of pre-existing lamivudine resistant HBV have been identified in drug-naive individuals . Since these mutations are rarely without cost from the virological perspective , compromising mutations tend to survive under the pressure of selection by a second compensatory mutation that partially restores the original levels of viral replication . Contrary to earlier in vitro data in which the mutant viruses were studied in the absence of selective drug pressures [33, 34, 57], Bock and colleagues  showed enhanced in vitro replication of the lamivudine resistant rtM204 V/I containing HBV Pol mutants when cultured in the presence of the lamivudine selection pressure. This was particularly noticeable when other mutations in the viral envelope (eg.: G145R or P120T) were also present on the same genome. These findings have important clinical and pathogenesis implications, since the viral load in serum would be expected to drop significantly when drug therapy was withdrawn .
(iii) Replication Space
Replication space can be understood in terms of the major transcriptional template of HBV, the covalently closed circular (ccc) DNA molecule. Using this approach, Zhang and Summers [29, 60], have defined replication space as the potential of the liver to accommodate new HBV ccc DNA molecules. The maximum capacity of ccc DNA in a liver is limited by the number of hepatocytes that can be infected and by the maximum number of ccc DNA copies per hepatocyte. In a fully infected liver, the synthesis of new ccc DNA can occur only if uninfected cells are generated by growth of the liver, hepatocyte turnover, or loss of ccc DNA from infected hepatocytes [2, 61]. In the absence of the appropriate selection pressure, an emerging virus strain can prevail over pre-existing strains via competitive out-growth only in the setting of hepatocyte turnover or ccc DNA loss . Therefore, the enrichment of one species over another implies that the expanding virus has augmented its population via an expansion of ccc DNA synthesis . However, if there is limited initiation of new infections, which can be the case in a chronically infected liver with minimal necroinflammatory activity , then the expansion of a drug-resistant mutant in the infected liver can only be possible with the creation of new replication space .
The emergence of drug-resistant viruses during antiviral therapy may correspond to the loss of the wild-type ccc DNA from the liver and their replacement by mutant viral ccc DNA, thereby allowing the mutant HBV to expand. Hepadnaviral ccc DNA, which is found in viral minichromosomes in the nucleus of infected hepatocytes [62, 63] appears to be quite stable and "relatively resistant" to nucleoside/nucleotide analogue therapy. In animal models and tissue cultures, treatment with single antiviral agents does not result in any appreciable loss of wild type ccc DNA molecules in hepatocytes [64, 65]. However, during antiviral combination chemotherapy experiments in vitro  and in vivo , the level of viral ccc DNA has been observed to fall significantly. Unfortunately, this key replicative form has never been eliminated by antiviral therapy [68, 69] although it can be controlled by an endogenous antiviral cytokine response . Some of these discrepant observations can be explained by the fact that hepadnaviral ccc DNA exists in the liver as multiple topoisomers [62, 63] which may represent different transcriptional states reflecting complex genetic regulation .
Patients with chronic hepatitis B with raised serum ALT appear to have increased hepatocyte turnover [2, 60], which provides the necessary replication space for the expansion of drug-resistant mutants. Thus, in the presence of the selection pressure, the selected resistant quasispecies in the nascent virion pool can slowly replace the existing wild-type ccc DNA .
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8. Mechanistic Considerations
The HBV DNA polymerase gene has regions of sequence homology to other viral RTs, in particular HIV . However, the multi functional nature of the HBV Pol is unique to hepadnaviruses, and can be defined by four distinct domains: a N-terminal protein that serves as the primer for RT, a spacer region of unknown function, the RT domain, and ribonuclease H . The polymerase domain has been further divided into at least five subdomains designated A through E  and for RTs, two other domains F and G can be identified (Figure 1).
The genotypic resistance patterns found in patients with drug-resistant HBVs was discussed above (Table 2). Studies of drug-resistant mutations of HIV RT have yielded much data on the mechanisms of nucleoside/nucleotide analogue resistance [74, 75], some of which can be extended to drug-resistant HBV Pols. The structure of the HIV RT has been resolved by X-ray crystallography [76, 77]. Drawing upon the sequence homology to other viral RT including HIV, several groups have developed 3 dimensional models of HBV Pol [71, 78, 79]. Each model assumes a right-handed conformation similar to other viral reverse transcriptases with a thumb, palm and finger domains [76, 77]. Domains A, C and D are mainly involved in deoxynucleoside triphosphate (dNTP) binding and catalysis and correspond to the palm domains. Domains B and E interact with the RNA template and primer regions respectively, and are also found in the palm domain of this right-handed model [74, 75]. The C domain of the HBV Pol contains the YMDD motif which is highly conserved in other RTs .
Point mutations in the HIV RT that lead to nucleoside/nucleotide analogue resistance typically cluster in the vicinity of the dNTP binding site, which has a precisely configured structure. From the crystallographic data, mutations affecting the YMDD locus typically alter the ability of the dNTP binding pocket to accommodate the nucleoside/nucleotide analogue. Thus, the primary mechanism of resistance appears to be by steric hindrance within the dNTP binding pocket [74, 75]. When the methionine (M) in the YMDD motif is replaced by valine (V) or isoleucine (I), the new amino acid side-chain projects into the dNTP binding site. Steric hindrance has been demonstrated for lamivudine when it was modelled into the dNTP binding pocket of the lamivudine-resistant mutations YIDD or YVDD [74, 75, 79]. The ? branched side-group of the V or I competes against the sulphur atom of lamivudine for the same space within the binding pocket. These alterations in the dNTP binding pocket can also hinder binding of the usual D-conformation nucleoside triphosphates.
The second mechanism whereby these mutations can cause resistance has been recently recognised following a more detailed examination of these molecular models . The catalytic efficiency of the enzyme is adversely affected by causing sub-optimal nucleophilic attack geometry . The catalysis of the incoming dNTP to the elongating DNA strand is dependent on a precise spatial arrangement of the 5'-a- phosphate and the 3'- hydroxyl group. Alterations in the geometry of the reaction constituents can greatly diminish the efficiency of catalysis. As well as the steric hindrance encountered by lamivudine in the dNTP binding site of drug-resistant [rtM204 V/I] polymerase, the low-affinity occupation of the site by lamivudine is further compounded by this sub-optimal nucleophilic attack geometry  caused by the particular amino-acid substitutions [74, 75].
Other mutations associated with lamivudine-resistent HBV Pol occur outside the dNTP binding site. For example, the B domain changes rtV173L and rtL180M can occur in parallel with the rtM204V/I mutations [78, 80] as can the A domain change rtL80V/I .
Pyrophosphorolysis and pyrophosphate exchange are two non hydrolytic HIV RT activities that can result in the removal of newly incorporated nucleotides, thereby providing a third mechanism of nucleoside/nucleotide analogue resistance [30, 81, 82]. The ability to remove newly incorporated nucleotides during replication has important biological and clinical implications, since these reactions may serve a primer unblocking function. The role of increased primer unblocking by pyrophosphorolysis in AZT-resistance has been documented for HIV RT [81, 82]. However, this does not appear to be a mechanism by which further resistance to lamivudine can be mediated, since lamivudine-resistant HIV RT demonstrates little, if any pyrophosphorolytic activity on lamivudine-terminated DNA . Similarly, drug removal by pyrophosphorolysis appears unlikely to be involved in HBV resistance to lamivudine . However, drug-resistance to other antivirals could develop because of enhanced primer unblocking by either wild-type or lamivudine-resistant HBV Pol .
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9. HBV Pol-Env Link
The genome of HBV is organised into overlapping reading frames, with the polymerase gene overlapping the envelope gene(s). As a result of this arrangement, HBV Pol gene mutations that are selected during the course of antiviral therapy do affect the envelope proteins and so may represent potential antibody neutralization-escape and or T-cell escape mutants. Therapy with lamivudine results in mutations in the polymerase gene of which a number are associated with alterations in the neuralization or "a" determinant of the HBsAg protein [84-86] (see Table 4). In a recent study exploring the link between polymerase mutations and altered envelope antibody binding, Torresi and colleagues  demonstrated markedly reduced binding of anti-HBs to the following HBV mutants exhibiting rtV173L, rtM204I, rtL180M+rtM204V, rtV207I, and rtV173L+rtL180M+rtM204V mutated polymerases (Table 4). These findings raise the possibility that the selection of lamivudine resistant HBV mutants with antigenically distinct HBsAg proteins could potentially behave as vaccine escape mutants (Figure 2).
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10. Compensatory Mutations
Compensatory mutations that partly or wholly restore the level of viral fitness have been documented during therapy for HIV infections . Similar scenarios have been described for HBV. Ogata and colleagues  first described that multiple polymerase mutations rtL80I/V (domain A) plus rtM204I (domain C) in genotype C infected patients were associated with significantly higher viral loads, increased lamivudine resistance as well as disease exacerbation.
Hadziyannis and colleagues  have documented that basal core promoter (BCP) and precore stop codon mutations in genotype D infected patients that are associated with HBeAg-negative chronic hepatitis B are also resistant to lamivudine (rtM204I), disease exacerbation and disease progression occurs whilst therapy is maintained.
These reports, and the observed enhanced replication of HBV in the presence of lamivudine , lead to the conclusion that multiple mutations that are selected for during inadequate antiviral therapy can act as compensatory mutations and have the potential to restore replication fitness.
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The transmissibility of drug-resistant HBV mutants has now emerged as a potentially important public health issue. Thibault and colleagues  recently documented the transmission of lamivudine-resistant HBV (rtL180M+rtM204V) between two male homosexuals. The incubation period and clinical presentation was typical of acute viral hepatitis B, although the viral replication levels were lower during the acute phase. The patient had not previously been vaccinated against HBV but did have an uneventful recovery. This case does raise several serious public health concerns in the context of the current immunisation program that underpin the global control program for HBV infection .
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In conclusion, the swarm of genetic variants that can be found in any patient infected with a viral pathogen such as HBV tend to represent a mixture of viruses with varying replication fitnesses and selection advantages under the changing conditions of host-cell type, immune response or pressure from drug treatment. The fitness of the predominant population changes in response to changing selection pressures . Increasing the antiviral effectiveness of therapy is accompanied by an increasing probability that drug-resistant mutants will be selected, but only up to a point [1, 92]. At high levels of antiviral activity suppressing virus replication reduces the likelihood that resistance mutations will be selected because replication is the process responsible for the emergence of the mutations [1, 92]. This key relationship can be described as a bell-shaped curve[1, 50, 93] and leads to the inevitable conclusion that no replication (NR) equates to no resistance (NR), or put another way: NR=NR .
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The author acknowledges the editorial assistance of Pam Nagle and Sandra Maunders. I am also grateful to Dr. Angeline Bartholomeusz for reviewing the manuscript and constructive comments and suggestions.
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