Persistent infection with the hepatitis B virus (HBV) is found in up to 10% of HIV-infected patients and represents a major risk factor for the development of liver failure, cirrhosis and hepatocellular carcinoma . HIV-related immune suppression plus treatment with HBV-effective antiviral agents provide a unique milieu for the selection of unusual and potentially harmful HBV mutations .
Selection of rtV191I hepatitis B virus polymerase mutation during tenofovir therapy
We report the case of a 33-year-old male patient dually infected with HIV and HBe-antigen (HBeAg) positive hepatitis B. This patient had been discontinuously on antiretroviral therapies for 5 years, before initiating a tenofovir-containing regimen (with lamivudine, zidovudine and efavirenz; Fig. 1a). Initial sequence analysis [3,4] revealed a wild-type polymerase pattern. After 29 weeks of therapy, sequencing of the HBV polymerase uncovered the selection of a point mutation causing a valine-to-isoleucine exchange at position rt191. Interestingly, this rtV191I mutation also created a stop-codon in the overlapping surface antigen (sW182stop), deleting the last 44 amino acids of the HBsAg, which resulted in HBsAg-negativity in routine diagnostic serum tests. Serum HBV-DNA was 6.1 log copies/ml, which slowly dropped to 4.3 log copies/ml over the course of 40 weeks. Alanine aminotransferase levels normalized after 8 weeks of tenofovir therapy (Fig. 1b). Plasma HIV-RNA remained undetectable (<50 copies/ml), and CD4+ counts oscillated between 324 and 602 cells/μl.
rtV191I polymerase mutation reduces viral replication efficacy
We introduced the rtV191I mutation into replication-competent HBV vectors and assessed its replication after transient transfection of human Huh7 hepatoma cells, following a methodology previously described [5–7]. Interestingly, rtV191I mutants (with wild-type core region) showed significantly impaired replication efficacies compared to HBV-wildtype, as demonstrated by reduced transcriptional activity by Northern Blot (Fig. 1c), reduced formation of intracapsid HBV Progeny-DNA (Fig. 1d) and diminished release of HBV virions (Fig. 1e). Introduction of the stop codon in the overlapping S gene prevented secretion of full-length HBsAg in the supernatant of rtV191I constructs, whereas HBeAg was unchanged (Fig. 1f). Taken together, these assays consistently revealed that the rtV191I mutation impaired the replicative capacity of HBV, but simultaneously abolished secretion of the immunogenic HBsAg, thereby procuring escape from the humoral immune response. The HBsAg-negativity emphasizes the crucial need with respect to HBV DNA levels in the monitoring of chronically HBV-infected patients . The reduced replication is not unexpected, because mutations in the HBV polymerase usually display reduced polymerase activities and replication yields, especially if they are selected during antiviral therapy and confer drug-resistance, for example, against lamivudine or entecavir [2,8].
Precore and basal core promoter mutations restore replication efficacy of rtV191I mutants
We and others have previously reported that the concomitant presence of precore or basal core promoter (BCP) mutations is able to enhance the replication of polymerase mutants; for example, in the case of lamivudine-resistance [5,9]. Precore and BCP mutants are the most prevalent alterations found in HBeAg-negative chronic HBV infection, a condition that is increasingly found worldwide . In the case of the rtV191I mutation, combination with either precore or BCP enhanced HBV transcription (Fig. 1c), HBV Progeny-DNA formation (Fig. 1d) and virion release (Fig. 1e), thereby restoring the replicative capacity of mutants to wild-type level. Of note, rtV191I vectors with precore mutations failed to secrete HBeAg, whereas BCP combination mutants had significantly reduced HBeAg concentrations in the supernatant compared to wild-type (Fig. 1f).
Precore mutations may positively influence HBV polymerase activity by stabilizing the ε-structure of the pregenomic RNA to which the polymerase binds during the replication cycle  and by reducing inhibitory effects of the precore protein . BCP mutations are thought to modulate HBV replication by an altered transcription factor binding to the promoter, mutations in the overlapping HBx protein and changes in the ratio of precore to pregenomic RNA [2,12]. As HIV-positive or otherwise immune-suppressed patients generally require long-term administration of antiviral therapy, there is a high likelihood of acquiring precore or BCP mutations resulting in HBeAg-negative hepatitis B [13,14]. Selection of precore or BCP mutations in the case of the rtV191I polymerase mutation therefore carries the risk of a virological and clinical flare as it enhances replication of rtV191I HBV mutants.
rtV191I polymerase mutants are associated with lamivudine, but not with tenofovir resistance
Selection of the rtV191I mutation indicates a specific advantage of the mutant despite its reduced replicative capacity, which could be related to the escape from anti-HBs antibodies due to its HBsAg-negativity  and/or to reduced susceptibility toward antiviral drugs. The rtV191I mutation occurred during an HBV-effective regimen containing lamivudine and tenofovir. Tenofovir, although initially developed for the treatment of HIV, was found to have very potent antiviral activity against wildtype or lamivudine-resistant HBV strains [14,15]. Up to now, only the rtA194T nucleotide exchange in the HBV polymerase has been associated with tenofovir-drug resistance . We therefore tested the drug sensitivity against lamivudine and tenofovir, using previously described methodology [3,5]. Phenotypic analyses revealed that rtV191I clones were resistant to lamivudine (Fig. 1g), but remained susceptible to tenofovir (Fig. 1h).
The polymerase mutation rtV191I has been previously documented in a patient undergoing adefovir therapy, but phenotypic analyses did not confirm reduced susceptibility to adefovir . It has also been described sporadically after monotherapy with famciclovir  and lamivudine . Our present data show that the rtV191I mutation is associated with lamivudine resistance, but that this change does not confer tenofovir resistance. The rtV191I thereby differs considerably from the rtA194T nucleotide exchange in the HBV polymerase, which was also identified in two HBV-HIV-coinfected patients during tenofovir administration and conferred tenofovir-drug resistance both in vitro and in vivo .
The rtV191I HBV polymerase mutation simultaneously provokes lamivudine resistance and HBsAg-negativity, thereby promoting escape from endogenous (immune system), as well as exogeneous (drugs) antiviral mechanisms. The rtV191I mutation impaired the replication efficacy, which, however, could be restored by acquisition of precore or BCP mutations as found in HBeAg-negative hepatitis B, making HBeAg seroconversion a considerable risk in this unusual mutant. Despite its selection during tenofovir therapy, rtV191I mutants remained susceptible to tenofovir in vitro, but proved resistant to lamivudine. Mutants such as rtV191I, which carry drug and potential vaccine resistance, may represent a considerable individual risk for the patient, but also a public health concern due to the risk of transmission.
The authors thank Mrs Aline Müller for her excellent technical assistance. This work was supported by the EU grant of Virgil (to J.S., C.T., and F.T.), the German Research Foundation (DFG Ta434/2-1 to F.T.), the Interdisciplinary Centre for Clinical Research ‘BIOMAT’ within the faculty of Medicine at the RWTH Aachen University (to F.T.) and the Iranian Ministry of Health (KH/16716 to S.A.). Tenofovir was kindly provided by Gilead Sciences. C.T. and F.T. have received research grants from Novartis and Bristol-Myers Squibb.
S.A. performed the phenotypic experiments, analyzed the data and cloned the HBeAg negative constructs, J.S. sequenced the patient sample and cloned the constructs, T.L. identified the patient and provided clinical data, C.T. provided tools and helped in data analysis, F.T. designed the study, analysed the data and wrote the manuscript.
1. Soriano V, Puoti M, Peters M, Benhamou Y, Sulkowski M, Zoulim F, et al
. Care of HIV patients with chronic hepatitis B: updated recommendations from the HIV-Hepatitis B Virus International Panel. AIDS 2008; 22:1399–1410.
2. Tacke F, Manns MP, Trautwein C. Influence of mutations in the Hepatitis B Virus genome on virus replication and drug resistance: implications for novel antiviral strategies. Curr Med Chem 2004; 11:2667–2777.
3. Sheldon J, Camino N, Rodes B, Bartholomeusz A, Kuiper M, Tacke F, et al
. Selection of hepatitis B virus polymerase mutations in HIV-coinfected patients treated with tenofovir. Antivir Ther 2005; 10:727–734.
4. Henke-Gendo C, Amini-Bavil-Olyaee S, Challapalli D, Trautwein C, Deppe H, Schulz TF, et al
. Symptomatic Hepatitis B reactivation despite reduced viral fitness is associated with HBV test and immune escape mutations in an HIV-coinfected patient. J Infect Dis 2008; 198:1620–1624.
5. Tacke F, Gehrke C, Luedde T, Heim A, Manns MP, Trautwein C. Basal core promoter and precore mutations in the hepatitis B virus genome enhance replication efficacy of lamivudine-resistant mutants. J Virol 2004; 78:8524–8535.
6. Tacke F, Liedtke C, Bocklage S, Manns MP, Trautwein C. CREB/PKA sensitive signalling pathways activate and maintain expression levels of the hepatitis B virus pre-S2/S promoter. Gut 2005; 54:1309–1317.
7. Tacke F, Amini-Bavil-Olyaee S, Heim A, Luedde T, Manns MP, Trautwein C. Acute hepatitis B virus infection by genotype F despite successful vaccination in an immune-competent German patient. J Clin Virol 2007; 38:353–357.
8. Baldick CJ, Tenney DJ, Mazzucco CE, Eggers BJ, Rose RE, Pokornowski KA, et al
. Comprehensive evaluation of hepatitis B virus reverse transcriptase substitutions associated with entecavir resistance. Hepatology 2008; 47:1473–1482.
9. Chen RY, Edwards R, Shaw T, Colledge D, Delaney WE, Isom H, et al
. Effect of the G1896A precore mutation on drug sensitivity and replication yield of lamivudine-resistant HBV in vitro. Hepatology 2003; 37:27–35.
10. Jeong JK, Yoon GS, Ryu WS. Evidence that the 5′-end cap structure is essential for encapsidation of hepatitis B virus pregenomic RNA. J Virol 2000; 74:5502–5508.
11. Guidotti LG, Matzke B, Pasquinelli C, Shoenberger JM, Rogler CE, Chisari FV. The hepatitis B virus (HBV) precore protein inhibits HBV replication in transgenic mice. J Virol 1996; 70:7056–7061.
12. Parekh S, Zoulim F, Ahn SH, Tsai A, Li J, Kawai S, et al
. Genome replication, virion secretion, and e antigen expression of naturally occurring hepatitis B virus core promoter mutants. J Virol 2003; 77:6601–6612.
13. Quiros-Roldan E, Calabresi A, Lapadula G, Tirelli V, Costarelli S, Cologni G, et al
. Evidence of long-term suppression of hepatitis B virus DNA by tenofovir as rescue treatment in patients coinfected by HIV. Antivir Ther 2008; 13:341–348.
14. Hoofnagle JH, Doo E, Liang TJ, Fleischer R, Lok AS. Management of hepatitis B: summary of a clinical research workshop. Hepatology 2007; 45:1056–1075.
15. Mauss S, Wedemeyer H. Treatment of chronic hepatitis B and the implications of viral resistance to therapy. Expert Rev Anti Infect Ther 2008; 6:191–199.
16. Yang H, Westland CE, Delaney WE, Heathcote EJ, Ho V, Fry J, et al
. Resistance surveillance in chronic hepatitis B patients treated with adefovir dipivoxil for up to 60 weeks. Hepatology 2002; 36:464–473.
17. Seigneres B, Pichoud C, Ahmed SS, Hantz O, Trepo C, Zoulim F. Evolution of hepatitis B virus polymerase gene sequence during famciclovir therapy for chronic hepatitis B. J Infect Dis 2000; 181:1221–1233.
18. Wakil SM, Kazim SN, Khan LA, Raisuddin S, Parvez MK, Guptan RC, et al
. Prevalence and profile of mutations associated with lamivudine therapy in Indian patients with chronic hepatitis B in the surface and polymerase genes of hepatitis B virus. J Med Virol 2002; 68:311–318.