2.0 Virology: viral replication and drug resistance

KEY POINTS
  • The ability of hepatitis B virus (HBV) to cause persistent infection is due to the establishment of a covalently closed circular DNA (ccc DNA), which forms a stable minichromosome in the hepatocyte nucleus.
  • In chronic hepatitis B, the ccc DNA is largely refractory to elimination by the immune response or by nucleos(t)ide analogue (NA) therapy. Upon immunosuppression or cessation of therapy, the ccc DNA can reinitiate virus replication, leading to reactivation of disease.
  • HBV has an unusual replication strategy; it employs an error-prone reverse transcription step that allows mutations to be created.
  • Under selection pressures (e.g. from vaccines or from NA therapy), escape mutants or variants conferring resistance can be readily selected.

Under normal circumstances, hepatitis B virus (HBV) infection is not cytopathic to liver cells. The liver damage associated with acute hepatitis B or with chronic hepatitis B (CHB) occurs mainly as a result of attempts by the host’s immune response to clear HBV from infected hepatocytes (1).

HBV is a member of the family Hepadnaviridae. Novel features of hepadnavirus replication are a reverse transcription step and production of excess viral coat or envelope material – the hepatitis B surface antigen (HBsAg) that circulates in the blood, in titres that often exceed 1012 particles/mL. The viral reverse transcriptase (rt) lacks a proofreading capacity; hence, it produces a population of closely related variants, known as a ‘quasispecies’. This diversity ensures the survival of HBV because, when the virus is under pressure from the immune response or the introduction of an antiviral agent, a resistant viral sub-population will already be present in the infected person’s pool of newly replicating virus. Therefore, whenever a new selection pressure is introduced, a number of immune or antiviral drug ‘escape’ (i.e. resistant) mutants of HBV can evolve to become the dominant population.

Figure 2.1 Life cycle of the hepatitis B virus from entering the cell (at the top) to exiting the cell (shown at the bottom)
ccc, covalently closed circular DNA; HBcAg, hepatitis B core antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; RC, relaxed circular; rt, reverse transcriptase

HBV is a DNA virus. The viral DNA genome is found inside the viral core structure (or hepatitis B core antigen [HBcAg]) along with the viral rt (a DNA polymerase). The core structure is surrounded by the viral envelope: HBsAg. The life cycle of HBV, shown in Figure 2.1, begins when its envelope protein attaches to the sodium taurocholate cotransporting polypeptide – the newly discovered receptor on the surface of the hepatocyte (2) – allowing the virus to enter the cell. Once inside the cell, the viral genomic DNA penetrates the nucleus and is converted into a covalently closed circular (ccc) DNA form. The HBV ccc DNA, the major transcriptional template of the virus, associates with cellular histone proteins and establishes itself as viral minichromosomes. After transcription from the ccc DNA, viral RNA is transported to the cytoplasm of the hepatocyte, where the viral structural proteins (HBcAg and HBsAg) and the replication enzyme are synthesised. The HBV rt then reverse transcribes the HBV pregenomic (pg)RNA into DNA, inside the core particle. The viral envelope proteins now coat the replicating core complexes, creating mature virions that are released from the cell to complete the life cycle. The only HBV enzyme identified to date is the viral rt, and this is the target for nucleos(t)ide analogue (NA) antiviral therapy.

Figure 2.2 The hepatitis B virus genome and the virus polymerase-surface antigen link

B:

 

 

 

 

 

 

 

A:

            

A: Shows the circular arrangement of the four overlapping but frame-shifted open reading frames (ORFs)

B: Shows how the changes observed in the HBV Pol due to emergence of drug resistance affect the hepatitis B surface antigen (HBsAg) directly for all four nucleos(t)ide analogues approved for chronic hepatitis B treatment

The HBV genome encodes only four genes, but has evolved a remarkable replication strategy. It maximises its information content by using overlapping reading frames (ORFs), the longest of which (Pol ORF) encodes the viral rt/DNA polymerase. (This is shown in Figure 2.2[A]).

The envelope ORF is located within the Pol ORF, whereas the core (C) and the X ORFs overlap it. (This is shown in Figure 2.2[B]).

The life cycle of HBV involves two key processes, as shown in Figure 2.3:

  • generation of HBV ccc DNA from genomic DNA to form a minichromosome, and subsequent processing of the minichromosome by host enzymes to produce viral RNA
  • reverse transcription of the pregenomic (pg)RNA within the viral nucleocapsid to form HBV relaxed circular (RC) or genomic DNA, thereby completing the cycle (see Figure 2.1).

As discussed, HBV uses reverse transcription to copy its genome (i.e. from RNA to DNA). This viral enzyme lacks a proofreading or editing function; hence, many ‘transcriptional mistakes’ are introduced into the numerous newly replicated HBV DNA progeny molecules, resulting in substantial diversity in the viral genome.

Figure 2.3 Major processes involved in hepatitis B virus (HBV) genome replication
Conversion of relaxed circular (RC) DNA into covalently closed circular (ccc) DNA; transcription of ccc DNA to produce pregenomic (pg)RNA; reverse transcription of pgRNA to make minus (–) HBV DNA; and HBV DNA polymerase activity to make the RC DNA, completing the cycle. The ccc DNA can only be found in the liver within the nucleus of infected hepatocytes and in the form of a viral minichromosome (3, 4)

This provides the virus with a strong survival advantage because, on a daily basis, every single nucleotide in the viral genome of 3,200 base pairs can be changed. Random mutations from copying errors can lead to phenotypic changes, which in turn may confer a selective advantage. Thus, single and double mutations associated with antiviral drug resistance are present before antiviral therapy is introduced. However, if three or four mutations in the HBV DNA were to be required to confer resistance to NA therapy, these would be unlikely to be found before therapy. This concept is the basis for the use of combination chemotherapy for chronic viral diseases such as human immunodeficiency virus (HIV) (e.g. combination anti-retroviral therapy – cART).

2.3.1 Mutations affecting hepatitis B e antigen

In addition to coding for HBcAg and HBsAg, the HBV genome encodes for hepatitis B e antigen (HBeAg). The HBeAg protein is thought to act as a tolerogen (5), and its production helps the virus avoid elimination by the host immunological response, especially during pregnancy or acute infection. Without HBeAg, it is unlikely that HBV could establish a chronic infection.

When put under the immunological pressure of HBeAg seroconversion, which is part of the natural history of CHB, the virus has a number of ways of ‘escaping’. Two major groups of mutations have been identified that result in reduced or truncated HBeAg expression. The first group of mutations affect the basal core promoter (BCP), typically at nucleotide (nt)1762 and nt1764, resulting in a reduction in transcription of the precore mRNA (6). Mutations in the BCP, such as the change of an A to a T at nt1762 (designated A1762T), and of a G to an A at nt1764 (G1764A), may be found in isolation or in conjunction with precore mutations (discussed below). The double mutation of A1762T plus G1764A results in a significant decrease in HBeAg levels, but not its absolute absence, and this mutation has been associated with an increased viral load in patients. Importantly, these BCP mutations do not affect the transcription of HBV pgRNA or the translation of the core or polymerase protein. Instead, by removing the inhibitory effect of the precore protein on HBV replication, the BCP mutations appear to enhance viral replication by suppressing precore or core mRNA relative to pgRNA (6).

The second group of mutations introduce a translational stop codon mutation at nt1896 (codon 28: TGG; tryptophan) of the HBV precore gene (7). The single base substitution (G to A) at nt1896 (G1986A) gives rise to a translational stop codon (TGG to TAG; TAG = stop codon) in the second last codon (codon 28) of the precore gene that is located within the epsilon (ε) structure of pgRNA. The ntG1896A forms a base pair with ntT1858 at the base of the stem loop (7). Patients infected with precore mutant (G1896A) HBV are typically HBeAg negative. Other mutations in the precore gene can block HBeAg production; these include a mutation that abolishes the methionine initiation codon (8).

2.3.2 Envelope gene mutations

During both HBeAg-positive and HBeAg-negative CHB, some patients have been found to be infected with HBV containing deletions, insertions and mutations in the viral envelope region that result in reduced or truncated viral secretion (9). Because the envelope region overlaps the polymerase region, envelope mutations can also produce a change in the overlapping polymerase, and vice versa (Figures 2.2[A] and 2.2[B)]. Hence, substitutions in the HBsAg can result in changes in the polymerase that may confer antiviral resistance; conversely, changes induced by antiviral resistance may encode changes in the HBsAg, leading to potential vaccine escape mutants.

The current hepatitis B vaccine contains recombinant HBsAg. The subsequent immune response to the major hydrophilic region (MHR) of HBsAg, located from amino acid residues 99 to 170, induces protective immunity in the form of anti-HBs. Mutations within the MHR have been selected during vaccination (10), and following treatment of liver transplant recipients with hepatitis B immune globulin (HBIg) prophylaxis (11). Most vaccine-HBIg escape isolates have an amino acid change from glycine to arginine at residue 145 of HBsAg (sG145R), or aspartate to glutamic acid at residue 144 (sD144E). The sG145R mutation has been associated with vaccine failure (10); it has also been shown to be transmitted, establish persistent infection and cause disease.

2.3.3 Polymerase mutations: antiviral drug resistance

Antiviral drug resistance in clinical practice is discussed further in Chapter 7. The treatment of CHB has advanced significantly during the past 15 years as a result of the development of safe and efficacious orally available antiviral NAs. Two synthetic NAs with an unnatural L-conformation – lamivudine and telbivudine – are widely available but are not commonly used in Australia. Adefovir is a prodrug for the acyclic dAMP analogue, adefovir. Adefovir gained approval in 2002, but has largely been replaced by its congener, tenofovir, which is now commonly used as a first-line agent. Tenofovir lacks the potential nephrotoxicity of adefovir; consequently, a higher dose can be used (300 mg/day vs. 10 mg/day for adefovir), which may explain its greater efficacy in vivo. The most potent anti-HBV drug discovered to date is the deoxyguanosine analogue, entecavir (12), which is now also widely used as a first-line agent for treating CHB.

2.3.3.1 Lamivudine and other L-nucleoside analogues

Antiviral resistance to lamivudine is conferred by mutations that result in replacement of methionine at amino acid position 204 in the tyrosine-methionine-aspartate (YMDD) catalytic site motif (C-domain) of the rt by valine (rtM204V) or leucine (rtM204I) (Figure 2.4) (13). In a few cases, the B-domain change at rtA181T is also responsible for primary resistance to lamivudine. For other L-nucleosides such as telbivudine, the B-domain (rtA181T/V) and C-domain (rtM204I) changes are the main substitutions associated with the development of resistance (Figure 2.4).

Figure 2.4 Location of the major primary antiviral drug-resistant mutations associated with resistance in the major catalytic domains of the hepatitis B virus polymerase gene
ADV, adefovir; LdT, telbivudine; LAM, lamivudine; POL/RT, polymerase reverse transcriptase gene; rt, reverse transcriptase; TDF, tenofovir

Lamivudine resistance increases progressively during treatment at rates between 14% and 32% annually. At year four of therapy, rates of lamivudine resistance reach over 70% in HBV mono-infection, and exceed 90% in HBV–HIV co-infection (14, 15). Factors that increase the risk of development of resistance include high pretherapy serum HBV DNA and alanine transaminase (ALT) levels, and incomplete suppression of viral replication (14, 16). Lamivudine resistance does not usually confer cross-resistance to adefovir or tenofovir unless rtA181T is selected; however, the presence of rtM204I/V confers cross-resistance to the other L-nucleoside analogues, including telbivudine and, to a lesser extent, entecavir (Table 2.1).

Table 2.1 Summary of cross-resistance profiles
 LAMADV/
TDF
LdTETV
LAM-R χ χ Reduced 
ADV-R χ
LdT-R χ χ
ETV-R χ χ χ

ADV, adefovir; ETV, entecavir; LAM, lamivudine; LdT, telbivudine; R, resistant; TDF, tenofovir

χ, resistant; √, sensitive

The rtM204I change can occur in isolation, but rtM204V is found only in association with other substitutions – predominantly with rtL180M, and occasionally with both rtL180M and rtV173L. These additional changes partly compensate for the loss of replication fitness that can be associated with the development of drug resistance (17).

High maternal viral loads have been shown to be a risk factor for neonatal immunoprophylaxis failure, and lamivudine has been used during the third trimester to reduce the risk of perinatal transmission. Recent evidence, obtained using next-
generation sequencing, has shown that viral variants conferring lamivudine resistance are not only present at baseline, but (more importantly) are rapidly selected by the time of delivery (18). The presence of such variants could potentially complicate future clinical management; hence, alternative NAs (e.g. tenofovir) are usually recommended.

2.3.3.2 Adefovir and tenofovir

Resistance to adefovir has been associated with changes in the B (rtA181T/V) and D (N236T)-domains of the rt (19) (Figure 2.4). HBV resistance to adefovir occurs less frequently (about 2% after 2 years, 4% after 3 years and 18% after 4 years) than resistance to lamivudine. The rtN236T change does not significantly affect sensitivity to lamivudine (19), but the rtA181T/V change confers partial cross-resistance to lamivudine. Currently, there is little evidence for the occurrence of primary resistance to tenofovir; however, clinically, it has been shown that the presence of adefovir resistance impairs tenofovir efficacy (20).

2.3.3.3 Entecavir

Resistance to entecavir in patients naive to therapy is rare, and apparently occurs almost exclusively in patients who had already developed lamivudine-resistant HBV (21). Entecavir resistance appears to require the initial presence of rtM204V/I, followed by mutations that encode at least one additional entecavir ‘signature’ substitution at rtI169T or rtT184G (B-domain), rtS202I (C-domain) or rtM250V (E-domain) of HBV Pol (Figure 2.4).

2.3.3.4 Multidrug resistance

Multidrug-resistant (MDR) HBV has been reported in patients who received sequential treatment with different NA monotherapy, and rtA181T should be considered a MDR variant (21-26). The development of multidrug resistance will influence the efficacy of rescue therapy, as in the case of MDR HIV (27, 28). Successive evolution of different patterns of resistance mutations have been reported during long-term lamivudine monotherapy (29, 30). The isolates of HBV with these initial mutations appear to be associated with decreased replication fitness compared with wild-type HBV; however, additional mutations that can restore replication fitness are frequently detected as treatment is continued (31, 32).

2.3.3.5 Public health issues: Pol-env overlap

The polymerase gene overlaps with the envelope gene (see Figure 2.2[B]), and changes in the polymerase gene that confer antiviral resistance can cause concomitant changes to the ORFs of the envelope gene. Thus, the major resistance mutations associated with lamivudine, adefovir, entecavir and telbivudine failure also have the potential of altering the C-terminal region of HBsAg. For example, changes associated with lamivudine and entecavir resistance, such as the rtM204V, result in a change at sI195M in the HBsAg. Similarly, the rtM204I change that is associated with lamivudine and telbivudine is linked to three possible HBsAg changes: sW196S, sW196L or a termination codon. The effect of the main lamivudine resistance mutations on the altered antigenicity of HBsAg have been examined in vitro (33), and animal models suggest that these findings have public health relevance (34). In particular, one of the common HBV quasispecies selected during lamivudine treatment is rtV173L + rtL180M + rtM204V, which results in change in the HBsAg at sE164D + sI195M, and thus has the potential to escape vaccine-induced anti-HBs. About 20% of people with HIV/HBV co-infection (15) and 10% of those with mono-infection treated with lamivudine encode this ‘triple polymerase mutant’ (31). In binding assays, HBsAg expressing these lamivudine-resistant associated residues had reduced anti-HBs binding (33). This reduction was similar to the classical vaccine escape mutant, sG145R, and has been confirmed as a significant variant by infecting vaccinated chimpanzees.

The adefovir resistance substitution rtN236T does not affect the envelope gene, and overlaps with the stop codon at the end of the region encoding the surface antigen. The rtA181T mutation selected by adefovir and lamivudine results in a stop codon mutation at sW172*. The adefovir resistant mutation at rtA181V results in a change at sL173F. HBV with mutations that result in a stop codon in the envelope gene, such as those for lamivudine and adefovir, would be present in association with a low percentage of wild-type HBV, to enable rescue for viral assembly and release.

The entecavir resistance-associated changes at rtI169T, rtS184G and rtS202I also affect HBsAg, and result in changes at sF161L, sL/V176G and sV194F. The rtM250V is located after the end of HBsAg. The sF161L is located within the region that was defined as the ‘a’ determinant or MHR, which includes amino acids 90–170 of the HBsAg (35). This region is a highly conformational epitope, characterised by multiple disulfide bonds formed from sets of cysteine residues at amino acids 107–138, 137–149 and 139–147 (35). Since distal substitutions such as sE164D strongly affect anti-HBs binding (33), the influence of other changes to HBsAg driven by NA resistance, such as sF161L, needs investigation in order to determine the effect on the envelope structure and subsequent anti-HBsAg binding.

Although evidence for the spread of transmission of antiviral-resistant HBV is limited, there has been at least one report of the transmission of lamivudine-resistant HBV to an HIV patient undergoing lamivudine as part of antiretroviral therapy (36). In addition, HBV encoding lamivudine-resistant mutations were also found in a cohort of dialysis patients with occult HBV (37). Both primary and compensatory antiviral-resistance mutations may result in associated changes to the viral envelope that could have substantial public health relevance (although, in the era of potent first-line therapies such as tenofovir and entecavir, this has yet to be demonstrated).

2.3.3.6 Alternative antiviral therapy – interferon

The development of longer-acting pegylated forms of interferon (IFN) has stimulated renewed interest in treating patients with immunomodulatory agents. However, the drawbacks of pegylated IFN (PEG-IFN) treatment are similar to that of conventional interferon, with low efficacy and high toxicity. One advantage of the pegylated form, however, is that treatment is finite: usually 48 weeks. Furthermore, IFN therapy is effective against NA-resistant HBV, and is not associated with any changes in the HBV polymerase region (38). Recent research has used the quantification of serum HBsAg as a clinical biomarker to identify the patient subgroup most likely to respond to PEG-IFN treatment (39, 40). This may allow the use of response guided therapy for CHB and provide higher rates of HBsAg seroclearance.

Resistance may continue to be an important issue in the management of patients with CHB because long-term (probably life-long) therapy with NAs will be required in most patients. Moreover, in many countries with a high prevalence of CHB, the NAs most compromised by viral resistance (lamivudine, telbivudine) are cheap and commonly used. One of the important lessons learned from the HIV paradigm is that resistance is likely to occur if viral replication is present during treatment, as occurs with some existing monotherapy regimens that use L-nucleosides (41). Currently, however, first-line therapies with potent antivirals such as tenofovir or entecavir show little evidence of significant resistance development. It is not clear whether combination therapy for CHB – either as an initial strategy or in selected groups of patients with either MDR HBV or inadequate response to monotherapy – will be required in the future, and clinical trials are currently underway to investigate combination treatment strategies. Theoretically, combination therapy can reduce not only the viral load and quasispecies pool, but also the risk of selecting resistance, provided that the antiviral agents used do not select for mutual cross-resistance. Further, because of the overlap between the polymerase and envelope genes, the selection of drug-resistant HBV may have important clinical, diagnostic and public health implications. Envelope changes in HBV have been detected with lamivudine, adefovir, entecavir and telbivudine usage. The significance of these changes warrants further investigation to determine what effect they may have on the natural history of drug-resistant HBV and its possible transmissibility in the hepatitis B-vaccinated community at large.

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Scott Bowden & Stephen Locarnini, Victorian Infectious Diseases Reference Laboratory, North Melbourne, VIC