Strategies to Inhibit Entry of HBV and HDV Into Hepatocytes
2014; Elsevier BV; Volume: 147; Issue: 1 Linguagem: Inglês
10.1053/j.gastro.2014.04.030
ISSN1528-0012
AutoresStephan Urban, Ralf Bartenschlager, Ralf Kubitz, Fabien Zoulim,
Tópico(s)Liver Disease Diagnosis and Treatment
ResumoAlthough there has been much research into the pathogenesis and treatment of hepatitis B virus (HBV) and hepatitis D virus (HDV) infections, we still do not completely understand how these pathogens enter hepatocytes. This is because in vitro infection studies have only been performed in primary human hepatocytes. Development of a polarizable, HBV-susceptible human hepatoma cell line and studies of primary hepatocytes from Tupaia belangeri have provided important insights into the viral and cellular factors involved in virus binding and infection. The large envelope (L) protein on the surface of HBV and HDV particles has many different functions and is required for virus entry. The L protein mediates attachment of virions to heparan sulfate proteoglycans on the surface of hepatocytes. The myristoylated N-terminal preS1 domain of the L protein subsequently binds to the sodium taurocholate cotransporting polypeptide (NTCP, encoded by SLC10A1), the recently identified bona fide receptor for HBV and HDV. The receptor functions of NTCP and virus entry are blocked, in vitro and in vivo, by Myrcludex B, a synthetic N-acylated preS1 lipopeptide. Currently, the only agents available to treat chronic HBV infection target the viral polymerase, and no selective therapies are available for HDV infection. It is therefore important to study the therapeutic potential of virus entry inhibitors, especially when combined with strategies to induce immune-mediated killing of infected hepatocytes. Although there has been much research into the pathogenesis and treatment of hepatitis B virus (HBV) and hepatitis D virus (HDV) infections, we still do not completely understand how these pathogens enter hepatocytes. This is because in vitro infection studies have only been performed in primary human hepatocytes. Development of a polarizable, HBV-susceptible human hepatoma cell line and studies of primary hepatocytes from Tupaia belangeri have provided important insights into the viral and cellular factors involved in virus binding and infection. The large envelope (L) protein on the surface of HBV and HDV particles has many different functions and is required for virus entry. The L protein mediates attachment of virions to heparan sulfate proteoglycans on the surface of hepatocytes. The myristoylated N-terminal preS1 domain of the L protein subsequently binds to the sodium taurocholate cotransporting polypeptide (NTCP, encoded by SLC10A1), the recently identified bona fide receptor for HBV and HDV. The receptor functions of NTCP and virus entry are blocked, in vitro and in vivo, by Myrcludex B, a synthetic N-acylated preS1 lipopeptide. Currently, the only agents available to treat chronic HBV infection target the viral polymerase, and no selective therapies are available for HDV infection. It is therefore important to study the therapeutic potential of virus entry inhibitors, especially when combined with strategies to induce immune-mediated killing of infected hepatocytes. Chronic hepatitis B virus (HBV) infection is a major public health problem worldwide. Persistently infected people are at high risk for development of cirrhosis and hepatocellular carcinoma.1Kwon H. Lok A.S. Hepatitis B therapy.Nat Rev Gastroenterol Hepatol. 2011; 8: 275-284PubMed Google Scholar Coinfection or superinfection with hepatitis D virus (HDV), a satellite virus that requires the HBV envelope proteins for dissemination, accelerates and worsens the disease.2Rizzetto M. Current management of delta hepatitis.Liver Int. 2013; 33: 195-197Crossref PubMed Scopus (3) Google Scholar Chronic HBV infection is treated with interferon (IFN)-α (or pegylated form) and/or nucleos(t)ide analogues.3Zoulim F. Locarnini S. Hepatitis B virus resistance to nucleos(t)ide analogues.Gastroenterology. 2009; 137: 1593-1608Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar IFN drugs induce a sustained virologic response, in which serum levels of HBV DNA remain 50% of PHH form clonal islands with accurate cell architecture. HBV infection kinetics in these animals is slow but finally results in up to 100% of infected transplanted cells. HBV titers, as well as HBsAg and hepatitis B e antigen (HBeAg) levels in serum, compare with those of infected patients. uPA-SCID mice with PHH xenografts have been used to study the dynamics of hepatocyte turnover, including the stability of covalently closed circular DNA (cccDNA),30Lutgehetmann M. Volz T. Kopke A. et al.In vivo proliferation of hepadnavirus-infected hepatocytes induces loss of covalently closed circular DNA in mice.Hepatology. 2010; 52: 16-24Crossref PubMed Scopus (23) Google Scholar and the mechanisms of action of drugs.31Lutgehetmann M. Bornscheuer T. Volz T. et al.Hepatitis B virus limits response of human hepatocytes to interferon-alpha in chimeric mice.Gastroenterology. 2011; 140: 2074-2083Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar These mice were used to show the ability of the NTCP inhibitor Myrcludex B to block HBV and HDV entry.32Lutgehetmann M. Mancke L.V. Volz T. et al.Humanized chimeric uPA mouse model for the study of hepatitis B and D virus interactions and preclinical drug evaluation.Hepatology. 2012; 55: 685-694Crossref PubMed Scopus (38) Google Scholar, 33Volz T. Allweiss L. Ben MBarek M. et al.The entry inhibitor Myrcludex-B efficiently blocks intrahepatic virus spreading in humanized mice previously infected with hepatitis B virus.J Hepatol. 2013; 58: 861-867Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 34Petersen J. Dandri M. Mier W. et al.Prevention of hepatitis B virus infection in vivo by entry inhibitors derived from the large envelope protein.Nat Biotechnol. 2008; 26: 335-341Crossref PubMed Scopus (126) Google Scholar Transfection of hepatoma cells with HBV-encoding plasmids resulted in transcription of viral RNAs, HBV genome replication, and virus production. Likewise, HBV transgenic mice produce virions and have been used to study the immune response to and pathogenesis of HBV infection. However, these mice are immune tolerant and are therefore not an ideal model of infected patients. Moreover, hepatocytes of these mice do not support de novo entry of virions or produce detectable amounts of cccDNA, the template for transcription of viral messenger RNA (mRNA).35Chisari F.V. Hepatitis B virus transgenic mice: models of viral immunobiology and pathogenesis.Curr Top Microbiol Immunol. 1996; 206: 149-173Crossref PubMed Google Scholar These animals are therefore inappropriate for studying early infection events, the dynamics of virus spread, or mechanisms that affect the regulation and transcriptional activity of cccDNA. One alternative to overcome these limitations is hydrodynamic injection of HBV DNA into mice.36Yang P.L. Althage A. Chung J. et al.Hydrodynamic injection of viral DNA: a mouse model of acute hepatitis B virus infection.Proc Natl Acad Sci U S A. 2002; 99: 13825-13830Crossref PubMed Scopus (177) Google Scholar This results in long-term in vivo transfection of hepatocytes, resulting in viral gene expression and replication. The technique is used to simulate acute infection for investigation of immune responses and evaluation of antiviral drugs.36Yang P.L. Althage A. Chung J. et al.Hydrodynamic injection of viral DNA: a mouse model of acute hepatitis B virus infection.Proc Natl Acad Sci U S A. 2002; 99: 13825-13830Crossref PubMed Scopus (177) Google Scholar, 37Ketzinel-Gilad M. Zauberman A. Nussbaum O. et al.The use of the hydrodynamic HBV animal model to study HBV biology and anti-viral therapy.Hepatol Res. 2006; 34: 228-237Crossref PubMed Scopus (11) Google Scholar An alternative to hydrodynamic transfer of HBV-encoding plasmids involves transfer of HBV genomes via adenoviral vectors. This simulates acute infection and, when low levels of adenovirus vector are transferred, persistent HBV infection.38Huang L.R. Gabel Y.A. Graf S. et al.Transfer of HBV genomes using low doses of adenovirus vectors leads to persistent infection in immune competent mice.Gastroenterology. 2012; 142: 1447-1450Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar, 39Sprinzl M.F. Oberwinkler H. Schaller H. et al.Transfer of hepatitis B virus genome by adenovirus vectors into cultured cells and mice: crossing the species barrier.J Virol. 2001; 75: 5108-5118Crossref PubMed Scopus (75) Google Scholar No high-throughput approaches have been undertaken to identify host factors required for HBV or HDV infections. Host pattern recognition factors recognize HBV, as described in a recent review.40Chang J. Block T.M. Guo J.T. The innate immune response to hepatitis B virus infection: implications for pathogenesis and therapy.Antiviral Res. 2012; 96: 405-413Crossref PubMed Scopus (8) Google Scholar Factors that regulate transcription have been identified and characterized through analyses of specific DNA motifs within the viral cccDNA.41Quasdorff M. Protzer U. Control of hepatitis B virus at the level of transcription.J Viral Hepat. 2010; 17: 527-536Crossref PubMed Scopus (31) Google Scholar Additionally, cellular factors have been identified that participate in the maturation, assembly, and egress of virions and subviral particles (SVPs).42Prange R. Host factors involved in hepatitis B virus maturation, assembly, and egress.Med Microbiol Immunol. 2012; 201: 449-461Crossref PubMed Scopus (13) Google Scholar Until recently, almost nothing was known about host factors involved in early infection events, including the specific receptor(s) or proteins involved in transport of nucleocapsids to the nucleus or enzymes that convert (repair) relaxed circular DNA to cccDNA (for a comprehensive illustration of the HBV replication cycle, see Urban et al43Urban S. Schulze A. Dandri M. et al.The replication cycle of hepatitis B virus.J Hepatol. 2010; 52: 282-284Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Identification of these factors is crucial for our understanding of viral replication, the development of small animal models, and the identification of drug targets. Recently developed cell culture systems (notably HepG2hNTCP cells) might be used to identify these factors.22Ni Y. Lempp F.A. Mehrle S. et al.Hepatitis B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes.Gastroenterology. 2014; 146: 1070-1083Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar The identical envelope protein composition of the genetically dissimilar HBV and HDV particles allows for discrimination between host factors involved in prefusion and postfusion events of each virus (Figure 1). It is assumed that both viruses enter hepatocytes by similar or even identical mechanisms.44Sureau C. The use of hepatocytes to investigate HDV infection: the HDV/HepaRG model.Methods Mol Biol. 2010; 640: 463-473Crossref PubMed Scopus (9) Google Scholar This assumption is based on the finding that HBV and HDV use the same receptor21Schulze A. Gripon P. Urban S. Hepatitis B virus infection initiates with a large surface protein-dependent binding to heparan sulfate proteoglycans.Hepatology. 2007; 46: 1759-1768Crossref PubMed Scopus (80) Google Scholar, 22Ni Y. Lempp F.A. Mehrle S. et al.Hepatitis B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes.Gastroenterology. 2014; 146: 1070-1083Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar and are sensitive to peptide entry inhibitors, suramin, or neutralizing antibodies.45Taylor J.M. Virus entry mediated by hepatitis B virus envelope proteins.World J Gastroenterol. 2013; 19: 6730-6734Crossref PubMed Scopus (1) Google Scholar However, on release into the cytoplasm, HBV nucleocapsids or the HDV-ribonucleoprotein complex (RNP) follow different paths and require different host factors. HDV is less restricted by liver-specific postentry factors than HBV and even replicates in nonhepatoma cells from nonhuman species.46Taylor J.M. Hepatitis delta virus.Virology. 2006; 344: 71-76Crossref PubMed Scopus (100) Google Scholar Therefore, during early stages of infection, both viruses use the same host factors,20Gripon P. Diot C. Corlu A. et al.Regulation by dimethylsulfoxide, insulin, and corticosteroids of hepatitis B virus replication in a transfected human hepatoma cell line.J Med Virol. 1989; 28: 193-199Crossref PubMed Google Scholar, 47Lamas L.O. Schmidt T.T. Schoneweis K. et al.Proteoglycans act as cellular hepatitis delta virus attachment receptors.PLoS One. 2013; 8: e58340Crossref PubMed Scopus (6) Google Scholar, 48Leistner C.M. Gruen-Bernhard S. Glebe D. Role of glycosaminoglycans for binding and infection of hepatitis B virus.Cell Microbiol. 2008; 10: 122-133PubMed Google Scholar, 49Sureau C. Salisse J. A conformational heparan sulfate-binding site essential to infectivity overlaps with the conserved hepatitis B virus a-determinant.Hepatology. 2013; 57: 985-994Crossref PubMed Scopus (19) Google Scholar whereas in later events, viruses use different host factors. HBV is a small enveloped virus. It contains a partially double-stranded DNA genome of approximately 3.2 kilobases, which is packaged into an icosahedral nucleocapsid.50Seeger C. Mason W.S. Hepatitis B virus biology.Microbiol Mol Biol Rev. 2000; 64: 51-68Crossref PubMed Google Scholar In contrast to large enveloped viruses, the mature nucleocapsid of HBV induces an ordered arrangement of the envelope proteins through defined interactions among proteins during envelopment (Figure 1A).51Seitz S. Urban S. Antoni C. et al.Cryo-electron microscopy of hepatitis B virions reveals variability in envelope capsid interactions.EMBO J. 2007; 26: 4160-4167Crossref PubMed Scopus (30) Google Scholar The in-frame coded proteins incorporated into the virus shell are called the large (L), middle (M), and small (S) envelope proteins (Figure 2B). One peculiarity of HBV is the ability of the envelope proteins to form noninfectious, spherical and filamentous SVPs with a diameter of 22 nm; these are the major constituents of the clinically relevant HBsAg (Figure 1B and C). In the presence of nucleocapsids, specific interaction with the L protein results in envelopment and release of mature virions.52Bruss V. Hepatitis B virus morphogenesis.World J Gastroenterol. 2007; 13: 65-73Crossref PubMed Google Scholar Virions and SVPs contain different ratios of L, M, and S proteins, with the highest L protein content found in the virus particle. L, M, and S proteins share the C-terminal S domain, which contains 4 putative membrane-spanning helices. The 2 N-terminal extensions (preS2 and preS1/2) of M and L proteins have diverse functions, most importantly binding of the nucleocapsid during envelopment52Bruss V. Hepatitis B virus morphogenesis.World J Gastroenterol. 2007; 13: 65-73Crossref PubMed Google Scholar and receptor binding during entry.53Barrera A. Guerra B. Notvall L. et al.Mapping of the hepatitis B virus pre-S1 domain involved in receptor recognition.J Virol. 2005; 79: 9786-9798Crossref PubMed Scopus (50) Google Scholar, 54Engelke M. Mills K. Seitz S. et al.Characterization of a hepatitis B and hepatitis delta virus receptor binding site.Hepatology. 2006; 43: 750-760Crossref PubMed Scopus (68) Google Scholar, 55Glebe D. Aliakbari M. Krass P. et al.Pre-s1 antigen-dependent infection of Tupaia hepatocyte cultures with human hepatitis B virus.J Virol. 2003; 77: 9511-9521Crossref PubMed Scopus (78) Google Scholar, 56Glebe D. Urban S. Knoop E.V. et al.Mapping of the hepatitis B virus attachment site by use of infection-inhibiting preS1 lipopeptides and tupaia hepatocytes.Gastroenterology. 2005; 129: 234-245Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 57Gripon P. Cannie I. Urban S. Efficient inhibition of hepatitis B virus infection by acylated peptides derived from the large viral surface protein.J Virol. 2005; 79: 1613-1622Crossref PubMed Scopus (111) Google Scholar, 58LeSeyec J. Chouteau P. Cannie I. et al.Infection process of the hepatitis B virus depends on the presence of a defined sequence in the pre-S1 domain.J Virol. 1999; 73: 2052-2057PubMed Google Scholar, 59Meier A. Mehrle S. Weiss T.S. et al.The myristoylated preS1-domain of the hepatitis B virus L-protein mediates specific binding to differentiated hepatocytes.Hepatology. 2013; 58: 31-42Crossref PubMed Scopus (22) Google Scholar The L protein is not synthesized with a signal sequence but is inserted into the membrane via the transmembrane (TM)-1 domain. Thus, the preS1 domain initially faces the cytoplasm and resides in the interior of the virus particle. The very N-terminus of the preS1 domain is modified by myristic acid.60Persing D.H. Varmus H.E. Ganem D. The preS1 protein of hepatitis B virus is acylated at its amino terminus with myristic acid.J Virol. 1987; 61: 1672-1677Crossref PubMed Google Scholar Subsequently, the preS1 domain undergoes a complex posttranslational translocation process,61Lambert C. Prange R. Dual topology of the hepatitis B virus large envelope protein: determinants influencing post-translational pre-S translocation.J Biol Chem. 2001; 276: 22265-22272Crossref PubMed Scopus (65) Google Scholar resulting in its exposure on the virion surface.52Bruss V. Hepatitis B virus morphogenesis.World J Gastroenterol. 2007; 13: 65-73Crossref PubMed Google Scholar The envelope of HDV is structurally related to that of HBV, as HDV exploits the HBV su
Referência(s)